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Antidiarrheal effects of a thermostable protein fraction obtained from the larvae of Musca domestica
Biomedicine & Pharmacotherapy 115 (2019) 108813
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Antidiarrheal effects of a thermostable protein fraction obtained from the larvae of Musca domestica
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Juan Shena, Jiali Chena, Depo Yanga, Zhimin Zhaoa, Cailin Tanga, Rongfei Zhanga, ⁎ ⁎ Wenzhe Yangb, ,1, Yi Niua, ,1 a b
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, PR China Guangdong Magtech Biotechnology Co., Ltd, Dongguan, 523808, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Musca domestica Thermostable protein fraction Antidiarrhea Folium Sennae Anti-inflammation
Our objective was to investigate whether a thermostable protein fraction (TPF) obtained from the larvae of Musca domestica, which contains cecropin family AMPs, is effective in treating senna leaf (Folium Sennae)induced diarrhea in mice and its possible underlying mechanism. We did the experiments both in vitro and in vivo. Firstly, lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 macrophages. The expression level of nitric oxide (NO) and tumor necrosis factor (TNF)-α was assessed using kits and immunofluorescence assay. A mice model of total diarrhea was established using a decoction of Folium Sennae. Levels of interleukin (IL)-6 and IL-1β in mice serum and of TNF-α in the supernatant of jejunal tissue homogenate were measured using commercially available ELISA kits. Hematoxylin–eosin staining was employed to evaluate pathological lesions, and immunohistochemistry was used for determining IL-1β, IL-6, and TNF-α expression levels. Results display that TPF markedly inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages in vitro. Moreover, TPF significantly lowered the diarrhea index (DI) in diarrheic mice; when TPF was administered at a high dose (120 mg/kg) to mice, in comparison with mice in the model group, DI was markedly reduced. TPF could also decrease the expression levels of some pro-inflammatory factors, high dose TPF treated mice were with the reduction of (202.29 ± 18.58) pg/ml (tumor necrosis factor alpha, TNF-α), (53.69 ± 7.83) pg/ml (interleukin (IL)-1β, IL-1β), (48.44 ± 3.77) pg/ml (IL-6I, L-6) to the model separately. In comparison with berberine hydrochloride, which was used as the positive control in this study, TPF could confer better intestinal protection. To conclude, our results demonstrate that TPF has potent anti-inflammatory activities in vitro and antidiarrheal effects on mice with Folium Sennae-induced diarrhea.
1. Introduction Diarrhea is one of the most common gastrointestinal disorders worldwide. Nearly 1.7 billion cases of diarrheal disease are recorded each year [1]. It is also the second leading cause of death in children under 5 years of age and is responsible for the death of approximately 76,000 children annually [1]. Oral rehydration therapy (ORT) and pharmacological intervention, such as using antibiotics and anti-inflammatory drugs, are frequently utilized for treating diarrhea. ORT has the ability to treat > 90% cases of dehydration and it thus has substantial implications for the clinical treatment of diarrhea [2]. However, although the oral solution is both safe and effective, it can neither
shorten the duration of illness nor reduce the rate of stool loss; therefore, the solution is ineffective for treating people in rural or economically underdeveloped regions in which the mortality rates from diarrhea are the highest [3]. The use of antibiotics or anti-inflammatory drugs is common in economically underdeveloped regions; however, irrational use of antibiotics provokes adverse effects and also increases the risk of developing drug resistance in microorganisms. Furthermore, antibiotics can disrupt normal microbial balance in the gastrointestinal tract and even cause antibiotic-associated diarrhea [4]. Adverse effects and recrudescence considerably restrict the application of anti-inflammatory drugs [5]. Consequently, further research to develop new therapies and/or drugs for treating diarrhea is urgently needed.
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Corresponding authors. E-mail addresses: [email protected] (J. Shen), [email protected] (J. Chen), [email protected] (D. Yang), [email protected] (Z. Zhao), [email protected] (C. Tang), [email protected] (R. Zhang), [email protected] (W. Yang), [email protected] (Y. Niu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.108813 Received 20 January 2019; Received in revised form 9 March 2019; Accepted 26 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2. Material and methods
Musca domestica Linnaeus (Diptera: Muscidae) is a type of holometabolous insect with four morphologically distinct stages: egg, larval, pupal, and adult [6]. The larvae or maggots are called “wuguchong” in traditional Chinese medicine (TCM), which has been used for hundreds of years in China for treating various gastrointestinal ailments, including damp-heat diarrhea, dysentery, vomiting, and abdominal cramps [7]. These maggots rear in filthy and decaying niches but are rarely infected by viruses or bacteria themselves; therefore, several studies have been conducted to deduce the activities of proteins isolated from maggots. For example, some previous studies have investigated the effects of their antioxidant [8], antibacterial, antifungal [9], and in vitro antitumor properties successively [10]. In addition, Chu et al. (2013) reported that the anti-inflammatory effective parts of housefly larvae (low-molecular-weight parts) possess anti-atherosclerosis activity in mouse [11]. Although maggots have been used in TCM for many years in China, modern pharmacological studies are yet to comprehensively study their antidiarrheal effects. Senna leaf (Folium Sennae) is widely used in TCM to cure malnutritional stagnation [12]. This type of medicine can improve intestinal movement and reduce reabsorption of water and electrolytes, which causes diarrhea. Excessive intake of Folium Sennae can damage intestinal epithelial cells, leading to inflammatory bowel disease. Therefore, Folium Sennae is often used to induce diarrhea in animal models [13,14]. In case of some inflammatory bowel disorders such as Crohn’s disease and ulcerative colitis, stimulation of intestinal epithelial cells leads to the inappropriate and ongoing activation of the mucosal immune system. Macrophages produce a potent mix of broadly active inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. The activation of central immune cell populations is eventually accompanied by the production of various nonspecific mediators of inflammation [15]. Thus, inflammatory bowel disorders can be diagnosed by detecting the expression levels of relevant inflammatory cytokines. Berberine, a TCM herb, is isolated from Coptis chinensis Franch and is extensively used as a non-prescription drug to treat diarrhea in China. Berberine can reportedly enhance the absorption of Na+ and water from the intestinal lumen to relieve diarrhea [16]. It has also been reported to exert anti-inflammatory effects both in vitro and in vivo [17]. Thus, treatment using berberine might serve as an effective positive control in related studies. In the present study, we assessed the antidiarrheal effects of a thermostable protein fraction (TPF), which was prepared using the larvae of M. domestica, in vivo. A mice model of total diarrhea was established using a decoction of Folium Sennae, and the antidiarrheal effects of TPF were determined via macroscopic diarrhea index (DI), histological analysis, and determination of the levels of the following inflammation markers: TNF-α, IL-1β, and IL-6. Meanwhile, anti-inflammatory activities were detected in vitro using an immunofluorescence assay and detection of nitric oxide (NO), IL-1β, IL-6, and TNF-α.
2.1. Breeding and quality control of experimental maggots M. domestica was captured from the wild in Guangzhou, China and identified by Professor Hong Pang of the Department of Life Sciences, Sun Yat-sen University (Guangzhou). Wild houseflies were domesticated for three generations for stable fecundity. Every 5000 adults were reared in one 50 × 50 × 50 cm3 mesh cage (0.2 mm pore size) with a stocking density of approximately 2.8 cm3 per fly. Housefly colonies were maintained on a diet of sugar powder, dried milk, and hen egg (2:2:1, measured in grams) at 25 °C with a photoperiod of 12 h of light and 12 h of darkness. Small white bags filled with soggy wheat bran and brown sugar (4:1) were placed in the cages as oviposition medium. Concentrated eggs were collected every 12 h and subsequently inoculated into culture media with an inoculum density of 1.5 g/kg dried wheat bran. The culture media consisted of wheat bran with moisture levels of 60%–80% at 25 °C and pH of 6–7. Once the eggs hatched, the culture medium was evenly stirred every 6 h to ensure proper ventilation. After rearing for 4 days, the third instar larvae were harvested for use. We controlled the quality of experimental maggots through single larval weight (by means of weighing 10 fresh maggots and subsequently deducing the even weight of each larva) and nutrient ingredient analysis. Fresh third instar larvae were placed into boiling water to ensure their instant death, dried in an oven at 60 °C for 24 h, and weighed to assess water content. The method of Kjeldahl nitrogen determination was used to assess the protein content of larva; KJELTEC 8400 Auto Kjeldahl Nitrogen Analysis (FOSS, Denmark) was used in our study [18]. Oil was extracted using the Soxhlet extraction method with nhexane for 8 h and concentrated under vacuum using a rotary evaporator at 50 °C. The oil concentrate was then incubated at 80 °C for 2 h to remove moisture and finally weighed to determine the oil content. Moreover, fiber and ash contents were determined by acid and alkali digestion methods using Fiberter apparatus (FOSS). These were essentially the residue left after sequential hot digestion with H2SO4 and NaOH. The carbohydrate content was that left after getting rid of the aforementioned contents.
2.2. TPF preparation The third instar larvae were removed from the cultured media, washed, and sterilized. Approximately 100 g of M. domestica larvae were adequately homogenated with 300 ml of 50 mM PBS [containing 1 mM EDTA, 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol, and 5% (v/ v) glycerol, pH 6.8] precooled at 4 °C. Subsequently, the homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The floating fat cake was removed through vacuum aspiration. The supernatant was collected, heated in a boiling water bath for 2 min to 70 °C, cooled rapidly, and then centrifuged at 8000 rpm for 10 min. The resulting supernatant
Fig. 1. Preparation of thermostable protein fraction (TPF) and Folium Sennae decoction. A, TPF preparation; B, Folium Sennae decoction preparation. 2
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was considered to be TPF, which was filtered through a membrane with a pore size of 0.22 μm, and the filtrate was stored at −80 °C. The desalted freeze-dried powder was stored at −20 °C for future use. The complete process is depicted in Fig. 1A.
containing 0.01% formic acid (buffer A) and 80% acetonitrile containing 0.01% formic acid (buffer B); the flow rate was 300 nl/min at 25 °C. The gradient elution profile was as follows: 0–5 min, 5% buffer B; 5–45 min, 5% → 50% buffer B; 45–50 min, 50% → 95% buffer B; and 50–55 min, 95% buffer B. The analysis time was 65 min and injection volume was 8 μl. A 0.22-μm nylon membrane filter (Tianjin Jinteng Experiment Equipment Co. Ltd., Tianjin, China) was used to filter the mobile phase prior to use. The separated peptide segment was directly detected online by Thermo Scientific Q-Exactive mass spectrometer. MS parameters were as follows: primary MS, resolution 70,000; AGC target3e6; maximum IT-40 ms; scan range 350–1800 m/z; secondary MS, resolution 17,500; AGC target-1e5; maximum IT-60 ms; TopN-20; NCE/ stepped NCE 27.
2.3. Preparation of Folium Sennae decoction Folium Sennae was derived from the lobules of Cassia angustifolia Vahl, which was purchased from Qing Ping Chinese herb market (Guangzhou) and identified by Professor Depo Yang of the School of Pharmaceutical Sciences, Sun Yat-sen University. Folium Sennae (100 g) was powdered, extracted twice with 1200 ml of water at 70 °C using an ultrasonic technique for 40 min, and filtered. The obtained filtrate was concentrated to 0.3 g/ml under vacuum using a rotary evaporator at 50 °C. The concentrate was then stored in a refrigerator at 4 °C for further use. The complete process is depicted in Fig. 1B.
2.6. Anti-inflammatory activities of TPF in vitro 2.6.1. Cell culture The mouse macrophage cell line RAW 264.7 was purchased from Cell Bank, Laboratory Animal Center, Sun Yat-sen University. The cells were cultured at 37 °C in a humidified incubator containing 5% CO2 and 95% air in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 500 μg/ml geneticin.
2.4. Animals and grouping Healthy Kunming mice (17–22 g) were obtained from the Laboratory Animal Center, Sun Yat-sen University. The mice were housed individually under controlled conditions (25 °C ± 2 °C, 12-h light/dark cycle, 55% ± 5% humidity) with free access to standard pelleted feed and water ad libitum. Animals were fasted overnight with free access to water prior to the experiments. All animals received humane care, and the present study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals, as well as the guidelines for animal experimentation issued by the Administrative Committee of Laboratory Animals in Sun Yat-sen University. The animals were randomly divided into six groups (n = 8). Diarrhea was induced in five groups using the method described in section 2.6.1. The remaining group was the control group, in which mice were administered distilled water. After Folium Sennae administration for 6 days, both the control group (hereafter referred to as CONT) and model group (hereafter referred to as MODEL) received 10 mM PBS solution (0.2 ml/10 g) orally. The positive group (hereafter referred to as POSI) received 50 mg/kg body weight (BW) of berberine hydrochloride. The three remaining groups were administered TPF solution at 30 (TPFL, low-dose group), 60 (TPFM, middle-dose group), and 120 mg/kg (TPFH, high-dose group) BW at the same volume through gavage. After 1 h, all animals, except those in the normal group, were administered 0.5 ml Folium Sennae decoction. Subsequently, the animals were housed in individual cages, each of which had a piece of wire mesh (0.8 cm pore size) with a vertical distance of 4 cm to the bottom. A piece of a filter paper was placed at the bottom of the cage to collect animal feces. The frequency and morphology of stools were recorded for 6 h after Folium Sennae administration, and the treatment regimen was continued for 7 days.
2.6.2. Inhibition of NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages The anti-inflammatory activities of TPF were evaluated by assessing the inhibition rate of NO and TNF-α production. NO production was indirectly assessed by measuring nitrite levels in the cultured media; to achieve this, we used a colorimetric method based on the Griess reaction [19]. Approximately 5.5 × 104/well of RAW 264.7 macrophages were plated in 96-well plates, followed by incubation for 24 h. The cells were subsequently incubated at 37 °C with a series of test samples (20, 10, 5, 2.5, and 1.25 μg) in the presence of LPS (1 μg/ml) for 24 h. Approximately 50 μl of supernatant from each well was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 5% phosphoric acid) and transferred to a new 96-well plate; the plate was then incubated at room temperature (RT) for 10 min. Absorbance was measured at 540 nm using a microplate reader. The inhibition rate of NO expression was determined using the following equation: inhibition rate = [1– (ODsample – ODblank) / (ODLPS – ODblank)] × 100% (OD, optical density). Cell viability was determined using the MTT assay in the effect of TPF. TNF-α levels in the supernatant were measured using a commercially available ELISA kit (Elabscience Mouse Elisa Kits, Wuhan), according to manufacturer’s instructions. 2.6.3. Immunofluorescence microscopy RAW 264.7 macrophages were grown for 24 h in a 12-well dish (2 × 105 cells/well). The cells were stimulated with or without 1 μg/ml LPS and also treated with PBS or TPF separately for 24 h. After washing with PBS three times, the cells were fixed at RT in 4% paraformaldehyde for 15 min. Subsequently, the cells were permeabilized with 0.5% Triton-X100 (37 °C) for 15 min; after washing with PBS three times, a blocking step was performed using bovine serum albumin (37 °C) for 1 h. The cells were then incubated overnight at 4 °C with the primary antibody TNF-α (D2D4) XP® Rabbit mAb (mouse specific, 1:200, CST11984, USA). On the next day, the cells were washed three times with PBS for 5 min and then incubated with the secondary antibody IFKine Red AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:400, Abbkine, A24421, Wuhan) at RT in the dark for 40 min; the cells were again washed three times with PBS for 10 min. The nucleus was stained by DAPI (Beyotime, China) for 10 min in the dark at RT. The samples were then immediately visualized on a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss, Germany).
2.5. Compositional analysis of TPF 2.5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) The protein content of the crude extract was measured using the Coomassie brilliant blue assay system. SDS-PAGE was performed using 12% separating and 5% stacking gels. The gels were stained with 0.1% Coomassie brilliant blue R250 and destained using a solution containing 10% glacial acetic acid, 45% ethanol, and 45% water. 2.5.2. Liquid chromatography with tandem mass spectrometry (LC–MS/ MS) LC–MS/MS was used to assess the total protein content of TPF. An Acclaim PepMap C18 column (150 mm × 0.75 mm, 3 μm; Thermo, 160232, USA) equipped with a guard cartridge (5 mm × 3 mm i.d., Acclaim PepMap RSLC C18, Thermo, 160454) was used for chromatographic separation. The mobile phase was composed of water 3
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Fig. 2. Quality control of experimental maggots and SDS-PAGE analysis of thermostable protein fraction (TPF). A, Compositional analysis of TPF; B: SDSPAGE analysis of TPF.
available ELISA kit, according to manufacturer’s instructions. Within 30 min, OD of each sample was measured using a microplate reader (450 nm).
2.7. Antidiarrheal effects of TPF in vivo 2.7.1. Total diarrhea model A mice model of total diarrhea was established using a decoction of Folium Sennae, according to a previously reported method [13]. Briefly, the mice were administered 0.5 ml of Folium Sennae decoction once a day. When the diarrhea rate reached 100% after 6 days, the model was considered to be established.
2.8. Statistical analysis Statistical Package for the Social Sciences v13.0 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis. All data are expressed as mean ± standard deviation. Unless otherwise stated, each sample for each assay was assayed in triplicates. Statistical significance was determined using one-way ANOVA. P ≤ 0.05 was used to indicate statistical significance.
2.7.2. DI calculation and BW changes The severity of diarrhea was defined using three indices: loose stool incidence rate (LSIR), average loose stool grade (ALSG), and DI. LSIR is the ratio of the number of loose stools to the total stools within an animal. LSG describes the number of loose stools based on the diameter of the stool on the filter paper; it was classified into four grades according to the diameter of loose stools: grade 1 (< 1 cm), 2 (1–1.9 cm), 3 (2–3 cm), and 4 (> 3 cm). ALSG is the ratio of the sum of LSG of each loose stool to the total number of loose stools within an animal. DI was calculated by multiplying the results of LSIR with ALSG. Moreover, the weight of all experimental mice was measured using an electronic balance every 2 days before administering the decoction.
3. Results 3.1. Quality control of experimental maggots Maggots were cultured under the conditions described in Section 2.1. All batches of maggots had individual BWs of 12–15 mg, with 15.01% ± 0.55% protein and 3.76% ± 0.51% fat content. The compositional analysis of TPF is shown in Fig. 2A.
2.7.3. Sample collection Whole blood was collected from all groups 2 h after the last administration from the orbit and centrifuged for serum isolation. The mice were anesthetized and sacrificed through cervical dislocation immediately after blood collection. Jejunal segments were excised, freed of adherent adipose tissue, and rinsed using 4 °C PBS to remove any fecal residue. The portion of jejunal segments that was 2–3 cm proximal to the duodenum was collected and fixed in 4% paraformaldehyde for hematoxylin–eosin (H&E) staining and immunohistochemistry. The remaining jejunal tissues were stored at −80 °C for further use.
3.2. SDS-PAGE and LC–MS/MS analysis of TPF
2.7.4. Histological and immunohistochemical analyses Jejunal tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and finally sectioned into 4 μm-thick sections. These were subsequently subjected to H&E staining and immunohistochemical analysis in accordance with the standard procedures for histological evaluation. To evaluate pathological changes, an arbitrary scope was given to each microscopic field viewed at a magnification of 40–200 × . At least three fields of liver sections were obtained for the mean value.
TPF could inhibit NO production (Fig. 4A); the inhibition rate increased with an increase in TPF concentration, indicating a good dosedependent relationship. MTT assay results showed that TPF had no inhibitory effects on control RAW 264.7 macrophages at concentrations of 0–200 μg/ml. At a high concentration (200 μg/ml), the survival rate of TPF-treated macrophages was 99.3% after 24-h cultivation. Furthermore, TPF suppressed TNF-α production (Fig. 4B); pretreatment with 200 μg/ml TPF markedly decreased the release of TNF-α in LPS-stimulated RAW 264.7 macrophages. Immunofluorescence staining (Fig. 4C) showed that TNF-α in nonstimulated RAW 264.7 macrophages was distributed less than in LPSstimulated ones, and it can be reduced by treatment of TPF at a concentration of 200 μg/ml. These results indicated that TPF could prevent
SDS-PAGE analysis showed that proteins present in TPF mostly had a molecular weight of 10–80 kDa (Fig. 2B). In comparison with the crude protein extract, the relative content of low-molecular weight proteins was significantly higher. Through LC–MS/MS analysis, 63 proteins were identified; proteins (Q2EF92, A0A1I8MYU6, A0A1I8M5N4) belonging to the cecropin family were of significant interest to us (Fig. 3). 3.3. Anti-inflammatory activities of TPF in vitro
2.7.5. Effect of TPF on proinflammatory cytokines Levels of IL-6 and IL-1β in the serum and TNF-α in the supernatant of jejunal tissue homogenate were measured using a commercially 4
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Fig. 3. Liquid chromatography with tandem mass spectrometry of thermostable protein fraction. A, Chromatogram; B, C, D: Mass spectrum of proteins A0A1I8M5N4, A0A1I8MYU6, and Q2EF92, respectively.
the activation of inflammatory.
treatment groups was significantly different from that of CONT (P < 0.05). Therefore, these groups were successfully modeled. DI of POSI and each dosage treatment groups was considerably lower than that of MODEL (Fig. 4A). Furthermore, DI of POSI, TPFH, and TPFM showed a significant difference from that of MODEL (P < 0.05). Meanwhile, with regard to BW, we speculate that TPF helps in
3.4. Macroscopic evaluation of the antidiarrheal effects of TPF Macroscopic evaluation of the antidiarrheal effects of TPF was performed by assessing changes in DI and BW. DI of MODEL and 5
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Fig. 5. Macroscopic evaluation of the antidiarrheal effects of thermostable protein fraction (TPF). A: Diarrhea index of mice in each experimental group: low-dose group (TPFL), 30 mg/kg body weight (BW); middle-dose group (TPFM), 60 mg/kg BW; high-dose group (TPFH), 120 mg/kg BW; and positive group (POSI), 50 mg/kg BW. Data are expressed as means ± standard error of mean. #P < 0.05 vs. control group (CONT); *P < 0.05 vs. model group (MODEL); B: Changes in BW; C: Intestines of mice; D: Histological sections of the jejunum obtained from experimental mice. 5D-1 and -2: CONT; 5D-5 and -6: TPFL; 5D-7 and -8: TPFM; 5D-9 and -10: TPFH; 5D-11 and -12: POSI; 5D-3 and -4: MODEL. 5D-1, -3, -5, -7, -9, and -11: H&E staining; original magnification, 20 × . 5D-2, -4, -6, -8, -10, -12: H&E staining, original magnification 40×.
Fig. 4. Anti-inflammatory activities of thermostable protein fraction (TPF) in vitro. A: Nitric oxide inhibition rate of TPF in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages; B: Effect of TPF on tumor necrosis factor (TNF)-α levels in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with various concentrations of TPF for 1 h before treatment with or without LPS (1 μg/ml) for 6 h. TNF-α expression level was measured using an ELISA assay. Values are presented as mean ± standard deviation of three independent experiments. #P < 0.01 compared with the control group; **P < 0.01 compared with the LPS-only group, *P < 0.05 compared with the LPS-only group; C: Immunofluorescence analysis. Cells were stimulated with or without LPS (1 μg/ml) and also treated with PBS or TPF separately for 24 h before being tested.
markedly decreased, particularly in TPFH and TPFM: the small intestinal mucosa was slightly swollen, the villi showed almost no shedding, and the structure were intact (Fig. 5D-5, -7, -9, -11 and 5D-6, -8, -10, -12). 3.6. Effects of TPF on the secretion of cytokines in mice
improving the weight loss induced by Folium Sennae decoction in mice (Fig. 4B), thereby serving as a supplementary medicine to treat diarrhea. Intestinal tissues obtained from the animals were observed under an anatomic microscope (Fig. 5C). The intestines of mice in CONT showed clear folds, were pink in color, and had a thin wall; however, in MODEL, the intestines appeared to be congestive, swollen, and had a thick wall. Both TPF and berberine hydrochloride could alleviate this congestion and swelling. To summarize, TPF at a high dose (120 mg/kg BW) and berberine hydrochloride showed nearly the same effects on promoting the recovery of colonic lesions that appeared upon administering Folium Sennae decoction.
Immunohistochemical analyses indicated that TPF significantly alleviated TNF-α, IL-1β, and IL-6 production against inflammatory reactions as well as the blockade of inflammatory process at least in part (Fig. 6A–C). Further, ELISA results showed that the levels of IL-6 (Fig. 6D), IL-1β (Fig. 6E) (in mice serum), and TNF-α (Fig. 6F) (in the supernatant of mice jejunal tissue homogenate) in MODEL and treatment groups were significantly augmented compared with those in CONT (P < 0.01). Conversely, in comparison with MODEL, the levels of TNF-α, IL-1β, and IL-6 were significantly reduced in treatment groups as well as in POSI on day 7 (P < 0.05). In summary, TPF could decrease cytokine expression level in a dose-dependent manner, and the effects of TPF at a high dose were better than those of berberine hydrochloride.
3.5. Effects of TPF on the histological changes in the small intestine of mice Histological sections of the jejunum obtained from experimental mice were studied (Fig. 5D). H&E staining showed that the intestinal structure in CONT was intact, and the mucosal arrangement was structurally normal. The cytoplasm of villous epithelial cells was abundant, adjacent cells showed tight connection on the top, and number of goblet cells was less (Fig. 5D-1 and -2). The jejunum of mice in MODEL showed mucosal swelling and villi shedding, and the normal structure of the small intestine was damaged. The epithelial cells were edematous, and the structure of the connection between cells was defected. The number of goblet cells significantly increased (Fig. 5D-3 and -4). However, damage to the jejunum in the treatment groups was
4. Discussion Diarrhea is a common disease that seriously threatens human health, particularly in economically underdeveloped areas. Identifying inexpensive, accessible, and effective therapies to tackle diarrhea is thus urgently needed. Insects are a large, unexplored, and unexploited source of useful compounds for modern medicine [20]. Maggots have been used for treating gastrointestinal ailments in China for over hundreds of years [21]. In recent years, many studies have published findings on their preservation [22], environmental protection [23], and 6
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Fig. 6. Effects of different doses of thermostable protein fraction (TPF) and positive group (POSI) on expression levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in mice with Folium Sennaeinduced diarrhea. 6A-, B-, C-1: control group (CONT); 6A-, B-, C-2: model group (MODEL); 6A-, B-, C-3: positive group [50 mg/kg body weight (BW), POSI]; 6A-, B-, C-4: low-dose group (30 mg/kg BW, TPFL); 6A-, B-, C-5: middle-dose group (60 mg/kg BW, TPFM); 6A-, B-, C-6: high-dose group (120 mg/kg BW, TPFH). 6A-, B-, C-1, 2, 3, 4, 5, 6: Immunohistochemical analyses, original magnification 20 × . 6A-, B-, C-7 and 6D-, E-, F-: data are expressed as means ± standard error of mean. ## P < 0.01 vs. CONT, *P < 0.05 vs. MODEL, **P < 0.01 vs. MODEL.
electrolyte reabsorption, causing diarrhea in mice; moreover, intestinal epithelial cells can be damaged, leading to inflammatory bowel disorder, which can stimulate the typical course of diarrhea. According to our results, all mice administered with Folium Sennae decoction showed obvious symptoms of diarrhea. Macroscopic DI and proinflammatory cytokine levels in MODEL were significantly higher than those in the normal group (P < 0.01). Based on the results of histological analyses, the small intestines of mice in MODEL were evidently damaged; hence, the total diarrhea model established in this study can be considered to be steady and credible. We believe that macroscopic DI is the most intuitive method to evaluate the antidiarrheal effects of TPF. Our results revealed that the DI of TPFM, TPFH, and POSI was significantly lower than that of MODEL (P < 0.01). Even though the DI of TPFL was lower than that of MODEL, the difference was not significant. TPF showed potent antidiarrheal effects in mice with Folium Sennae-induced diarrhea, and the effect was highly dose-dependent. This same trend was observed in histological analyses: TPF could effectively reduce damages to the small intestines in mice with diarrhea, and TPF could provide better intestinal protection than berberine hydrochloride. This could partly be due to the major ingredients of TPF (proteins and polypeptides) and its low stimulation, good affinity, and safety. TNF-α, IL-1β, and IL-6 are important cytokines produced by activated immune cells during the intestinal inflammatory process. They regulate and amplify the immune response, induce tissue injury, and mediate complications of the inflammatory response, such as diarrhea and fibrosis [27]. When tissues are injured, the transcription levels of proinflammatory genes are stimulated via the activation of nuclear factor kappaB pathways to increase the production of the aforementioned proinflammatory cytokines [28]. In this study, the levels of TNFα, IL-1β, and IL-6 in MODEL were significantly higher than those in the normal group (P < 0.01). Moreover, in comparison with MODEL, animals in TPF-treated groups and POSI showed significantly lower proinflammatory cytokine levels. Therefore, TPF seems to influence proinflammatory cytokine production and arrest immune inflammation. These results concur with previously reported findings. The mechanism underlying the antidiarrheal effects of TPF still remains unclear. We suspect that the antidiarrheal effects of TPF are relevant to the antibacterial [24] and anti-inflammatory activities of maggot proteins. TPF seem to regulate the intestinal flora to optimize the intestinal environment in diarrheal mice. Furthermore, its anti-
bactericidal activity [24]; however, only few modern pharmacological studies have explored their antidiarrheal effects. In this study, we for the first time report the antidiarrheal effects of TPF obtained from the larvae of M. domestica. To ensure that maggots were stable and clean, we cultured the houseflies in our laboratory rather than obtaining them from the wild. Under the culturing conditions used in this study, maggots of different patches showed consistent qualities. The weight of an individual maggot remained constant at 12–15 mg, and the nutrient content hardly changed, as deduced via compositional analyses. Therefore, the results reported herein should be highly reliable. SDS-PAGE analysis showed that the proteins present in TPF mostly had a molecular weight of 10–80 kDa; the relative content of the lowmolecular weight proteins was remarkably higher. Majority of insect proteins with antibacterial and antiphlogistic activities have low molecular weights, such as bee venom [12], cecropins [25], lysozyme (14 kDa) [26], and some antimicrobial peptides (AMPs; 5–15 kDa). Through LC–MS/MS analysis, AMPs belonging to the cecropin family were identified in TPF. It has been previously reported that the cecropin family shows strong anti-inflammatory activities in LPS-stimulated macrophages by interacting with LPS in the cecropia moth Hyalophora cecropia, black fly Simulium bannaense, horsefly Tabanus yao, and swallowtail butterfly Papilio xuthus; this finding explains the antidiarrheal effects of TPF. To evaluate the pharmacological properties of TPF, researchers have assessed the anti-inflammatory activities of TPF in vitro. Excessive NO produced by inducible NO synthase plays a critical role in inflammatory diseases, and suppression of its production pathways may satisfy the unmet need for control of the inflammatory process [19]. In the current study, we investigated the effects of TPF on NO formation in RAW 264.7 macrophages stimulated with LPS (1 μg/ml) and determined cell viability using an MTT assay. TPF could inhibit the excessive production of NO in a dose-dependent manner. Furthermore, at the effective dosage, cell viabilities showed no significant inhibition, as per MTT assay results. TPF showed potent anti-inflammatory effects. Thus, in addition to the use of maggots for treating gastrointestinal ailments in TCM, our results present the antidiarrheal effects and anti-inflammation activities of TPF obtained from M. domestica. In this study, the total diarrhea model was established by administering a decoction of Folium Sennae to mice. Administration of such a decoction can aggravate intestinal movement and decrease water and 7
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inflammatory activities apparently weaken the inflammatory reactions, reducing tissue damage to consequently avoid the occurrence of diarrhea. It remains uncertain whether TPF enhances the reabsorption of water and electrolytes in the mice intestine, but according to our results, this seems plausible. Further studies are thus warranted.
12.005. [7] S. Li 1518-1593, X. Luo (Eds.), Compendium of Materia Medica: Bencao Gangmu/ Compiled by Li Shizhen; Translated and Annotated by Luo Xiwen, Foreign Languages Press, Beijing, China, 2003. [8] H. Ai, F. Wang, N. Zhang, L. Zhang, C. Lei, Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica, J. Insect Sci. 112 (2013) 1–16, https://doi.org/10.1673/ 031.013.11201. [9] Q.S. Ge, H.M. Zhang, X. Liu, S.Y. Wang, D.C. Lv, X.D. Li, Crude extract of maggots: antibacterial effects against Escherichia coli, underlying mechanisms, separation and purification, World J. Gastroenterol. 5 (2015) 1510, https://doi.org/10.3748/ wjg.v21.i5.1510. [10] X. Cao, Z. Huo, M. Lu, D. Mao, Q. Zhao, C. Xu, C. Wang, B. Zeng, Purification of lectin from larvae of the fly, Musca domestica, and in vitro anti-tumor activity in MCF-7 cells, J. Insect Sci. 164 (2010) 164, https://doi.org/10.1673/031.010. 14124. [11] F.J. Chu, X.B. Jin, Y.Y. Xu, Y. Ma, X.B. Li, X.M. Lu, W.B. Liu, J.Y. Zhu, Inflammatory regulation effect and action mechanism of anti-inflammatory effective parts of housefly (Musca domestica) larvae on atherosclerosis, Evid. Complement. Alternat. Med. 2013 (2013) 1–10, https://doi.org/10.1155/2013/340267. [12] N.S. Surendra, G.N. Jayaram, M.S. Reddy, Antimicrobial activity of crude venom extracts in honeybees (Apis cerana, Apis dorsata, Apis florea) tested against selected pathogens, Afr. J. Microbiol. Res. 5 (2011) 2765–2772, https://doi.org/10.5897/ AJMR11.593. [13] G.N. Zhou, Z.H. Hu, Y.X. Wang, K.X. Zhang, Preparation of mice diarrhea model and application of the diarrhea index, Chin. Tradit. Herbal Drugs 4 (1994) 195–196, https://doi.org/10.1007/BF02007173. [14] W. Liu, D.Y. Zhang, The method for establishing an animal model of spleen deficiency caused by Senna leaf, Chin. J. Integr. Tradit. Western Med. Digestion 4 (1998) 231–232. [15] R. Balfour Sartor, Cytokines in intestinal inflammation: pathophysiological and clinical considerations, Gastroenterology 106 (2013) 533–539, https://doi.org/10. 1016/0016-5085(94)90614-9. [16] Y. Zhang, X. Wang, S. Sha, S. Liang, L. Zhao, L. Liu, N. Chai, H. Wang, K. Wu. Berberine increases the expression of NHE3 and AQP4 in sennosideA-induced diarrhoea model, Fitoterapia 6 (2012) 1014–1022, https://doi.org/10.1016/j. fitote.2012.05.015. [17] C.L. Kuo, C.W. Chi, T.Y. Liu, The anti-inflammatory potential of berberine in vitro and in vivo, Cancer Lett. 2 (2004) 127–137, https://doi.org/10.1016/j.canlet.2003. 09.002. [18] H.A. McKenzie, H.S. Wallace, The Kjeldahl determination of nitrogen: a critical study of digestion conditions—temperature, catalyst, and oxidizing agent, Aust. J. Chem. 7 (1954) 55–70, https://doi.org/10.1071/CH9540055. [19] G.J. Huang, H.Y. Chang, M.J. Sheu, C.H. Yang, T.C. Lu, Y.S. Chang, W.H. Peng, S.S. Huang, Analgesic effects and the mechanisms of anti-inflammation of hispolon in mice, Evid. Complement. Alternat. Med. 17 (2011) 9112–9119, https://doi.org/ 10.1093/ecam/nep027. [20] R.W. Pemberton, Insects and other arthropods used as drugs in Korean traditional medicine, J. Ethnopharmacol. 65 (1999) 207–216, https://doi.org/10.1016/S03788741(98)00209-8. [21] N. Tsuneo, M.Y. Hua, I. Kenji, Insect derived crude drugs in the chinese song dynasty, J. Ethnopharmacol. 24 (1988) 247–285, https://doi.org/10.1016/03788741(88)90157-2. [22] Y. Wang, X. Dang, X. Zheng, J. Wang, W. Zhang, Effect of extracted housefly pupae peptide mixture on chilled pork preservation, J. Food Sci. 75 (2010) 6, https://doi. org/10.1111/j.1750-3841.2010.01694.x. [23] H. Čičková, M. Kozánek, I. Morávek, P. Takáč, A behavioral method for separation of house fly (Diptera: Muscidae) larvae from processed pig manure, J. Econ. Entomol. 105 (2012) 62–66, https://doi.org/10.1603/EC11202. [24] X. Lu, J. Shen, X. Jin, Y. Ma, Y. Huang, H. Mei, F. Chu, J. Zhu, Bactericidal activity of Musca domestica cecropin (Mdc) on multidrug-resistant clinical isolate of Escherichia coli, Appl. Microbiol. Biotechnol. 95 (2012) 939–945, https://doi.org/ 10.1007/s00253-011-3793-2. [25] J. Andrä, O. Berninghausen, M. Leippe, Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans, Med. Microbiol. Immunol. 189 (2000) 169–173, https://doi.org/10.1007/s430-0018025-x. [26] D. Nayduch, C. Joyner, Expression of lysozyme in the life history of the house fly (Musca domestica L.), J. Med. Entomol. 50 (2013) 847–852, https://doi.org/10. 1603/ME12167. [27] R.B. Sartor, Cytokines in intestinal inflammation: pathophysiological and clinical considerations, Gastroenterology 106 (1994) 533–539, https://doi.org/10.1016/ 0016-5085(94)90614-9. [28] M. Rosenbaum, Ulcerative colitis, Psychosomatics 24 (1983) 528–529, https://doi. org/10.1016/S0033-3182(83)73183-X.
5. Conclusions In the present study, our aim was to identify a new potential treatment for diarrhea. We used the larvae of M. domestica to obtain TPF, which could effectively inhibit diarrhea, reduce damage to the small intestines of diarrheic mice, and arrest immune inflammation both in vivo and in vitro. M. domestica has many advantages; for example, this species shows high reproductive ability, fast growth, and short lifecycle. M. domestica can typically survive for 7–8 generations per year, and for even up to 20 generations under proper conditions. Year-round breeding is achievable via artificial feeding. Furthermore, breeding maggots is simple and economical, as agricultural waste can be used for culturing them. Thus, using maggots to develop new antidiarrheal drugs should be highly encouraged. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgements We thank the Animal Laboratory of Sun Yat-sen University for providing us with technical assistance in performing experiments to assess antidiarrheal effects. We thank Guangdong Magotech Biotechnology Co., Ltd for providing guidance on how to rear Musca domestica. We are grateful to everyone who contributed to this research. Juan Shen conducted the pharmacological experiments and data analysis, and also assisted with writing the manuscript. Jiali Chen assisted with the pharmacological experiments and data analysis. Depo Yang designed the pharmacological experiments and assisted with writing the manuscript. Wenzhe Yang provided the maggots, bred them, and performed quality control of experimental maggots. Cailin Tang conducted LC–MS/MS and SDS-PAGE analyses. Rongfei Zhang (Email: [email protected]) prepared TPF and Folium Sennae decoction. Zhimin Zhao contributed to polishing the language of this manuscript. Yi Niu designed the experiments and further polished this manuscript. All authors have discussed, edited, and approved the final version of the manuscript. References [1] WHO, Diarrhoeal Disease, (2013) http://www.who.int/mediacentre/factsheets/ fs330/en/. [2] M. Claeson, M.H. Merson, Global progress in the control of diarrheal diseases, Pediatr. Infect. Dis. J. 9 (1990) 345–355, https://doi.org/10.1097/00006454199005000-00008. [3] J.T. Boerma, A.E. Sommerfelt, S.O. Rutstein, Childhood Morbidity and Treatment Patterns, Aug Institute for Resource Development. Macro International, Columbia, Maryland, 1991https://www.popline.org/node/315425. [4] J. Bartlett, Clinical practice. Antibiotic-associated diarrhea, N. Engl. J. Med. (2002), https://doi.org/10.1056/NEJMcp011603. [5] D.M. Abdallah, N.R. Ismael, Resveratrol abrogates adhesion molecules and protects against TNBS-induced ulcerative colitis in rats, Can. J. Physiol. Pharmacol. 89 (2011) 811–818, https://doi.org/10.1139/y11-080. [6] R.D. Moon, Muscid Flies (Muscidae), (2002), https://doi.org/10.1016/j.tele.2015.
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Antidiarrheal effects of a thermostable protein fraction obtained from the larvae of Musca domestica
T
Juan Shena, Jiali Chena, Depo Yanga, Zhimin Zhaoa, Cailin Tanga, Rongfei Zhanga, ⁎ ⁎ Wenzhe Yangb, ,1, Yi Niua, ,1 a b
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, PR China Guangdong Magtech Biotechnology Co., Ltd, Dongguan, 523808, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Musca domestica Thermostable protein fraction Antidiarrhea Folium Sennae Anti-inflammation
Our objective was to investigate whether a thermostable protein fraction (TPF) obtained from the larvae of Musca domestica, which contains cecropin family AMPs, is effective in treating senna leaf (Folium Sennae)induced diarrhea in mice and its possible underlying mechanism. We did the experiments both in vitro and in vivo. Firstly, lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 macrophages. The expression level of nitric oxide (NO) and tumor necrosis factor (TNF)-α was assessed using kits and immunofluorescence assay. A mice model of total diarrhea was established using a decoction of Folium Sennae. Levels of interleukin (IL)-6 and IL-1β in mice serum and of TNF-α in the supernatant of jejunal tissue homogenate were measured using commercially available ELISA kits. Hematoxylin–eosin staining was employed to evaluate pathological lesions, and immunohistochemistry was used for determining IL-1β, IL-6, and TNF-α expression levels. Results display that TPF markedly inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages in vitro. Moreover, TPF significantly lowered the diarrhea index (DI) in diarrheic mice; when TPF was administered at a high dose (120 mg/kg) to mice, in comparison with mice in the model group, DI was markedly reduced. TPF could also decrease the expression levels of some pro-inflammatory factors, high dose TPF treated mice were with the reduction of (202.29 ± 18.58) pg/ml (tumor necrosis factor alpha, TNF-α), (53.69 ± 7.83) pg/ml (interleukin (IL)-1β, IL-1β), (48.44 ± 3.77) pg/ml (IL-6I, L-6) to the model separately. In comparison with berberine hydrochloride, which was used as the positive control in this study, TPF could confer better intestinal protection. To conclude, our results demonstrate that TPF has potent anti-inflammatory activities in vitro and antidiarrheal effects on mice with Folium Sennae-induced diarrhea.
1. Introduction Diarrhea is one of the most common gastrointestinal disorders worldwide. Nearly 1.7 billion cases of diarrheal disease are recorded each year [1]. It is also the second leading cause of death in children under 5 years of age and is responsible for the death of approximately 76,000 children annually [1]. Oral rehydration therapy (ORT) and pharmacological intervention, such as using antibiotics and anti-inflammatory drugs, are frequently utilized for treating diarrhea. ORT has the ability to treat > 90% cases of dehydration and it thus has substantial implications for the clinical treatment of diarrhea [2]. However, although the oral solution is both safe and effective, it can neither
shorten the duration of illness nor reduce the rate of stool loss; therefore, the solution is ineffective for treating people in rural or economically underdeveloped regions in which the mortality rates from diarrhea are the highest [3]. The use of antibiotics or anti-inflammatory drugs is common in economically underdeveloped regions; however, irrational use of antibiotics provokes adverse effects and also increases the risk of developing drug resistance in microorganisms. Furthermore, antibiotics can disrupt normal microbial balance in the gastrointestinal tract and even cause antibiotic-associated diarrhea [4]. Adverse effects and recrudescence considerably restrict the application of anti-inflammatory drugs [5]. Consequently, further research to develop new therapies and/or drugs for treating diarrhea is urgently needed.
⁎
Corresponding authors. E-mail addresses: [email protected] (J. Shen), [email protected] (J. Chen), [email protected] (D. Yang), [email protected] (Z. Zhao), [email protected] (C. Tang), [email protected] (R. Zhang), [email protected] (W. Yang), [email protected] (Y. Niu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.108813 Received 20 January 2019; Received in revised form 9 March 2019; Accepted 26 March 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2. Material and methods
Musca domestica Linnaeus (Diptera: Muscidae) is a type of holometabolous insect with four morphologically distinct stages: egg, larval, pupal, and adult [6]. The larvae or maggots are called “wuguchong” in traditional Chinese medicine (TCM), which has been used for hundreds of years in China for treating various gastrointestinal ailments, including damp-heat diarrhea, dysentery, vomiting, and abdominal cramps [7]. These maggots rear in filthy and decaying niches but are rarely infected by viruses or bacteria themselves; therefore, several studies have been conducted to deduce the activities of proteins isolated from maggots. For example, some previous studies have investigated the effects of their antioxidant [8], antibacterial, antifungal [9], and in vitro antitumor properties successively [10]. In addition, Chu et al. (2013) reported that the anti-inflammatory effective parts of housefly larvae (low-molecular-weight parts) possess anti-atherosclerosis activity in mouse [11]. Although maggots have been used in TCM for many years in China, modern pharmacological studies are yet to comprehensively study their antidiarrheal effects. Senna leaf (Folium Sennae) is widely used in TCM to cure malnutritional stagnation [12]. This type of medicine can improve intestinal movement and reduce reabsorption of water and electrolytes, which causes diarrhea. Excessive intake of Folium Sennae can damage intestinal epithelial cells, leading to inflammatory bowel disease. Therefore, Folium Sennae is often used to induce diarrhea in animal models [13,14]. In case of some inflammatory bowel disorders such as Crohn’s disease and ulcerative colitis, stimulation of intestinal epithelial cells leads to the inappropriate and ongoing activation of the mucosal immune system. Macrophages produce a potent mix of broadly active inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. The activation of central immune cell populations is eventually accompanied by the production of various nonspecific mediators of inflammation [15]. Thus, inflammatory bowel disorders can be diagnosed by detecting the expression levels of relevant inflammatory cytokines. Berberine, a TCM herb, is isolated from Coptis chinensis Franch and is extensively used as a non-prescription drug to treat diarrhea in China. Berberine can reportedly enhance the absorption of Na+ and water from the intestinal lumen to relieve diarrhea [16]. It has also been reported to exert anti-inflammatory effects both in vitro and in vivo [17]. Thus, treatment using berberine might serve as an effective positive control in related studies. In the present study, we assessed the antidiarrheal effects of a thermostable protein fraction (TPF), which was prepared using the larvae of M. domestica, in vivo. A mice model of total diarrhea was established using a decoction of Folium Sennae, and the antidiarrheal effects of TPF were determined via macroscopic diarrhea index (DI), histological analysis, and determination of the levels of the following inflammation markers: TNF-α, IL-1β, and IL-6. Meanwhile, anti-inflammatory activities were detected in vitro using an immunofluorescence assay and detection of nitric oxide (NO), IL-1β, IL-6, and TNF-α.
2.1. Breeding and quality control of experimental maggots M. domestica was captured from the wild in Guangzhou, China and identified by Professor Hong Pang of the Department of Life Sciences, Sun Yat-sen University (Guangzhou). Wild houseflies were domesticated for three generations for stable fecundity. Every 5000 adults were reared in one 50 × 50 × 50 cm3 mesh cage (0.2 mm pore size) with a stocking density of approximately 2.8 cm3 per fly. Housefly colonies were maintained on a diet of sugar powder, dried milk, and hen egg (2:2:1, measured in grams) at 25 °C with a photoperiod of 12 h of light and 12 h of darkness. Small white bags filled with soggy wheat bran and brown sugar (4:1) were placed in the cages as oviposition medium. Concentrated eggs were collected every 12 h and subsequently inoculated into culture media with an inoculum density of 1.5 g/kg dried wheat bran. The culture media consisted of wheat bran with moisture levels of 60%–80% at 25 °C and pH of 6–7. Once the eggs hatched, the culture medium was evenly stirred every 6 h to ensure proper ventilation. After rearing for 4 days, the third instar larvae were harvested for use. We controlled the quality of experimental maggots through single larval weight (by means of weighing 10 fresh maggots and subsequently deducing the even weight of each larva) and nutrient ingredient analysis. Fresh third instar larvae were placed into boiling water to ensure their instant death, dried in an oven at 60 °C for 24 h, and weighed to assess water content. The method of Kjeldahl nitrogen determination was used to assess the protein content of larva; KJELTEC 8400 Auto Kjeldahl Nitrogen Analysis (FOSS, Denmark) was used in our study [18]. Oil was extracted using the Soxhlet extraction method with nhexane for 8 h and concentrated under vacuum using a rotary evaporator at 50 °C. The oil concentrate was then incubated at 80 °C for 2 h to remove moisture and finally weighed to determine the oil content. Moreover, fiber and ash contents were determined by acid and alkali digestion methods using Fiberter apparatus (FOSS). These were essentially the residue left after sequential hot digestion with H2SO4 and NaOH. The carbohydrate content was that left after getting rid of the aforementioned contents.
2.2. TPF preparation The third instar larvae were removed from the cultured media, washed, and sterilized. Approximately 100 g of M. domestica larvae were adequately homogenated with 300 ml of 50 mM PBS [containing 1 mM EDTA, 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol, and 5% (v/ v) glycerol, pH 6.8] precooled at 4 °C. Subsequently, the homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The floating fat cake was removed through vacuum aspiration. The supernatant was collected, heated in a boiling water bath for 2 min to 70 °C, cooled rapidly, and then centrifuged at 8000 rpm for 10 min. The resulting supernatant
Fig. 1. Preparation of thermostable protein fraction (TPF) and Folium Sennae decoction. A, TPF preparation; B, Folium Sennae decoction preparation. 2
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was considered to be TPF, which was filtered through a membrane with a pore size of 0.22 μm, and the filtrate was stored at −80 °C. The desalted freeze-dried powder was stored at −20 °C for future use. The complete process is depicted in Fig. 1A.
containing 0.01% formic acid (buffer A) and 80% acetonitrile containing 0.01% formic acid (buffer B); the flow rate was 300 nl/min at 25 °C. The gradient elution profile was as follows: 0–5 min, 5% buffer B; 5–45 min, 5% → 50% buffer B; 45–50 min, 50% → 95% buffer B; and 50–55 min, 95% buffer B. The analysis time was 65 min and injection volume was 8 μl. A 0.22-μm nylon membrane filter (Tianjin Jinteng Experiment Equipment Co. Ltd., Tianjin, China) was used to filter the mobile phase prior to use. The separated peptide segment was directly detected online by Thermo Scientific Q-Exactive mass spectrometer. MS parameters were as follows: primary MS, resolution 70,000; AGC target3e6; maximum IT-40 ms; scan range 350–1800 m/z; secondary MS, resolution 17,500; AGC target-1e5; maximum IT-60 ms; TopN-20; NCE/ stepped NCE 27.
2.3. Preparation of Folium Sennae decoction Folium Sennae was derived from the lobules of Cassia angustifolia Vahl, which was purchased from Qing Ping Chinese herb market (Guangzhou) and identified by Professor Depo Yang of the School of Pharmaceutical Sciences, Sun Yat-sen University. Folium Sennae (100 g) was powdered, extracted twice with 1200 ml of water at 70 °C using an ultrasonic technique for 40 min, and filtered. The obtained filtrate was concentrated to 0.3 g/ml under vacuum using a rotary evaporator at 50 °C. The concentrate was then stored in a refrigerator at 4 °C for further use. The complete process is depicted in Fig. 1B.
2.6. Anti-inflammatory activities of TPF in vitro 2.6.1. Cell culture The mouse macrophage cell line RAW 264.7 was purchased from Cell Bank, Laboratory Animal Center, Sun Yat-sen University. The cells were cultured at 37 °C in a humidified incubator containing 5% CO2 and 95% air in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 500 μg/ml geneticin.
2.4. Animals and grouping Healthy Kunming mice (17–22 g) were obtained from the Laboratory Animal Center, Sun Yat-sen University. The mice were housed individually under controlled conditions (25 °C ± 2 °C, 12-h light/dark cycle, 55% ± 5% humidity) with free access to standard pelleted feed and water ad libitum. Animals were fasted overnight with free access to water prior to the experiments. All animals received humane care, and the present study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals, as well as the guidelines for animal experimentation issued by the Administrative Committee of Laboratory Animals in Sun Yat-sen University. The animals were randomly divided into six groups (n = 8). Diarrhea was induced in five groups using the method described in section 2.6.1. The remaining group was the control group, in which mice were administered distilled water. After Folium Sennae administration for 6 days, both the control group (hereafter referred to as CONT) and model group (hereafter referred to as MODEL) received 10 mM PBS solution (0.2 ml/10 g) orally. The positive group (hereafter referred to as POSI) received 50 mg/kg body weight (BW) of berberine hydrochloride. The three remaining groups were administered TPF solution at 30 (TPFL, low-dose group), 60 (TPFM, middle-dose group), and 120 mg/kg (TPFH, high-dose group) BW at the same volume through gavage. After 1 h, all animals, except those in the normal group, were administered 0.5 ml Folium Sennae decoction. Subsequently, the animals were housed in individual cages, each of which had a piece of wire mesh (0.8 cm pore size) with a vertical distance of 4 cm to the bottom. A piece of a filter paper was placed at the bottom of the cage to collect animal feces. The frequency and morphology of stools were recorded for 6 h after Folium Sennae administration, and the treatment regimen was continued for 7 days.
2.6.2. Inhibition of NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages The anti-inflammatory activities of TPF were evaluated by assessing the inhibition rate of NO and TNF-α production. NO production was indirectly assessed by measuring nitrite levels in the cultured media; to achieve this, we used a colorimetric method based on the Griess reaction [19]. Approximately 5.5 × 104/well of RAW 264.7 macrophages were plated in 96-well plates, followed by incubation for 24 h. The cells were subsequently incubated at 37 °C with a series of test samples (20, 10, 5, 2.5, and 1.25 μg) in the presence of LPS (1 μg/ml) for 24 h. Approximately 50 μl of supernatant from each well was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 5% phosphoric acid) and transferred to a new 96-well plate; the plate was then incubated at room temperature (RT) for 10 min. Absorbance was measured at 540 nm using a microplate reader. The inhibition rate of NO expression was determined using the following equation: inhibition rate = [1– (ODsample – ODblank) / (ODLPS – ODblank)] × 100% (OD, optical density). Cell viability was determined using the MTT assay in the effect of TPF. TNF-α levels in the supernatant were measured using a commercially available ELISA kit (Elabscience Mouse Elisa Kits, Wuhan), according to manufacturer’s instructions. 2.6.3. Immunofluorescence microscopy RAW 264.7 macrophages were grown for 24 h in a 12-well dish (2 × 105 cells/well). The cells were stimulated with or without 1 μg/ml LPS and also treated with PBS or TPF separately for 24 h. After washing with PBS three times, the cells were fixed at RT in 4% paraformaldehyde for 15 min. Subsequently, the cells were permeabilized with 0.5% Triton-X100 (37 °C) for 15 min; after washing with PBS three times, a blocking step was performed using bovine serum albumin (37 °C) for 1 h. The cells were then incubated overnight at 4 °C with the primary antibody TNF-α (D2D4) XP® Rabbit mAb (mouse specific, 1:200, CST11984, USA). On the next day, the cells were washed three times with PBS for 5 min and then incubated with the secondary antibody IFKine Red AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:400, Abbkine, A24421, Wuhan) at RT in the dark for 40 min; the cells were again washed three times with PBS for 10 min. The nucleus was stained by DAPI (Beyotime, China) for 10 min in the dark at RT. The samples were then immediately visualized on a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss, Germany).
2.5. Compositional analysis of TPF 2.5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) The protein content of the crude extract was measured using the Coomassie brilliant blue assay system. SDS-PAGE was performed using 12% separating and 5% stacking gels. The gels were stained with 0.1% Coomassie brilliant blue R250 and destained using a solution containing 10% glacial acetic acid, 45% ethanol, and 45% water. 2.5.2. Liquid chromatography with tandem mass spectrometry (LC–MS/ MS) LC–MS/MS was used to assess the total protein content of TPF. An Acclaim PepMap C18 column (150 mm × 0.75 mm, 3 μm; Thermo, 160232, USA) equipped with a guard cartridge (5 mm × 3 mm i.d., Acclaim PepMap RSLC C18, Thermo, 160454) was used for chromatographic separation. The mobile phase was composed of water 3
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Fig. 2. Quality control of experimental maggots and SDS-PAGE analysis of thermostable protein fraction (TPF). A, Compositional analysis of TPF; B: SDSPAGE analysis of TPF.
available ELISA kit, according to manufacturer’s instructions. Within 30 min, OD of each sample was measured using a microplate reader (450 nm).
2.7. Antidiarrheal effects of TPF in vivo 2.7.1. Total diarrhea model A mice model of total diarrhea was established using a decoction of Folium Sennae, according to a previously reported method [13]. Briefly, the mice were administered 0.5 ml of Folium Sennae decoction once a day. When the diarrhea rate reached 100% after 6 days, the model was considered to be established.
2.8. Statistical analysis Statistical Package for the Social Sciences v13.0 for Windows (SPSS, Chicago, IL, USA) was used for statistical analysis. All data are expressed as mean ± standard deviation. Unless otherwise stated, each sample for each assay was assayed in triplicates. Statistical significance was determined using one-way ANOVA. P ≤ 0.05 was used to indicate statistical significance.
2.7.2. DI calculation and BW changes The severity of diarrhea was defined using three indices: loose stool incidence rate (LSIR), average loose stool grade (ALSG), and DI. LSIR is the ratio of the number of loose stools to the total stools within an animal. LSG describes the number of loose stools based on the diameter of the stool on the filter paper; it was classified into four grades according to the diameter of loose stools: grade 1 (< 1 cm), 2 (1–1.9 cm), 3 (2–3 cm), and 4 (> 3 cm). ALSG is the ratio of the sum of LSG of each loose stool to the total number of loose stools within an animal. DI was calculated by multiplying the results of LSIR with ALSG. Moreover, the weight of all experimental mice was measured using an electronic balance every 2 days before administering the decoction.
3. Results 3.1. Quality control of experimental maggots Maggots were cultured under the conditions described in Section 2.1. All batches of maggots had individual BWs of 12–15 mg, with 15.01% ± 0.55% protein and 3.76% ± 0.51% fat content. The compositional analysis of TPF is shown in Fig. 2A.
2.7.3. Sample collection Whole blood was collected from all groups 2 h after the last administration from the orbit and centrifuged for serum isolation. The mice were anesthetized and sacrificed through cervical dislocation immediately after blood collection. Jejunal segments were excised, freed of adherent adipose tissue, and rinsed using 4 °C PBS to remove any fecal residue. The portion of jejunal segments that was 2–3 cm proximal to the duodenum was collected and fixed in 4% paraformaldehyde for hematoxylin–eosin (H&E) staining and immunohistochemistry. The remaining jejunal tissues were stored at −80 °C for further use.
3.2. SDS-PAGE and LC–MS/MS analysis of TPF
2.7.4. Histological and immunohistochemical analyses Jejunal tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and finally sectioned into 4 μm-thick sections. These were subsequently subjected to H&E staining and immunohistochemical analysis in accordance with the standard procedures for histological evaluation. To evaluate pathological changes, an arbitrary scope was given to each microscopic field viewed at a magnification of 40–200 × . At least three fields of liver sections were obtained for the mean value.
TPF could inhibit NO production (Fig. 4A); the inhibition rate increased with an increase in TPF concentration, indicating a good dosedependent relationship. MTT assay results showed that TPF had no inhibitory effects on control RAW 264.7 macrophages at concentrations of 0–200 μg/ml. At a high concentration (200 μg/ml), the survival rate of TPF-treated macrophages was 99.3% after 24-h cultivation. Furthermore, TPF suppressed TNF-α production (Fig. 4B); pretreatment with 200 μg/ml TPF markedly decreased the release of TNF-α in LPS-stimulated RAW 264.7 macrophages. Immunofluorescence staining (Fig. 4C) showed that TNF-α in nonstimulated RAW 264.7 macrophages was distributed less than in LPSstimulated ones, and it can be reduced by treatment of TPF at a concentration of 200 μg/ml. These results indicated that TPF could prevent
SDS-PAGE analysis showed that proteins present in TPF mostly had a molecular weight of 10–80 kDa (Fig. 2B). In comparison with the crude protein extract, the relative content of low-molecular weight proteins was significantly higher. Through LC–MS/MS analysis, 63 proteins were identified; proteins (Q2EF92, A0A1I8MYU6, A0A1I8M5N4) belonging to the cecropin family were of significant interest to us (Fig. 3). 3.3. Anti-inflammatory activities of TPF in vitro
2.7.5. Effect of TPF on proinflammatory cytokines Levels of IL-6 and IL-1β in the serum and TNF-α in the supernatant of jejunal tissue homogenate were measured using a commercially 4
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Fig. 3. Liquid chromatography with tandem mass spectrometry of thermostable protein fraction. A, Chromatogram; B, C, D: Mass spectrum of proteins A0A1I8M5N4, A0A1I8MYU6, and Q2EF92, respectively.
the activation of inflammatory.
treatment groups was significantly different from that of CONT (P < 0.05). Therefore, these groups were successfully modeled. DI of POSI and each dosage treatment groups was considerably lower than that of MODEL (Fig. 4A). Furthermore, DI of POSI, TPFH, and TPFM showed a significant difference from that of MODEL (P < 0.05). Meanwhile, with regard to BW, we speculate that TPF helps in
3.4. Macroscopic evaluation of the antidiarrheal effects of TPF Macroscopic evaluation of the antidiarrheal effects of TPF was performed by assessing changes in DI and BW. DI of MODEL and 5
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Fig. 5. Macroscopic evaluation of the antidiarrheal effects of thermostable protein fraction (TPF). A: Diarrhea index of mice in each experimental group: low-dose group (TPFL), 30 mg/kg body weight (BW); middle-dose group (TPFM), 60 mg/kg BW; high-dose group (TPFH), 120 mg/kg BW; and positive group (POSI), 50 mg/kg BW. Data are expressed as means ± standard error of mean. #P < 0.05 vs. control group (CONT); *P < 0.05 vs. model group (MODEL); B: Changes in BW; C: Intestines of mice; D: Histological sections of the jejunum obtained from experimental mice. 5D-1 and -2: CONT; 5D-5 and -6: TPFL; 5D-7 and -8: TPFM; 5D-9 and -10: TPFH; 5D-11 and -12: POSI; 5D-3 and -4: MODEL. 5D-1, -3, -5, -7, -9, and -11: H&E staining; original magnification, 20 × . 5D-2, -4, -6, -8, -10, -12: H&E staining, original magnification 40×.
Fig. 4. Anti-inflammatory activities of thermostable protein fraction (TPF) in vitro. A: Nitric oxide inhibition rate of TPF in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages; B: Effect of TPF on tumor necrosis factor (TNF)-α levels in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with various concentrations of TPF for 1 h before treatment with or without LPS (1 μg/ml) for 6 h. TNF-α expression level was measured using an ELISA assay. Values are presented as mean ± standard deviation of three independent experiments. #P < 0.01 compared with the control group; **P < 0.01 compared with the LPS-only group, *P < 0.05 compared with the LPS-only group; C: Immunofluorescence analysis. Cells were stimulated with or without LPS (1 μg/ml) and also treated with PBS or TPF separately for 24 h before being tested.
markedly decreased, particularly in TPFH and TPFM: the small intestinal mucosa was slightly swollen, the villi showed almost no shedding, and the structure were intact (Fig. 5D-5, -7, -9, -11 and 5D-6, -8, -10, -12). 3.6. Effects of TPF on the secretion of cytokines in mice
improving the weight loss induced by Folium Sennae decoction in mice (Fig. 4B), thereby serving as a supplementary medicine to treat diarrhea. Intestinal tissues obtained from the animals were observed under an anatomic microscope (Fig. 5C). The intestines of mice in CONT showed clear folds, were pink in color, and had a thin wall; however, in MODEL, the intestines appeared to be congestive, swollen, and had a thick wall. Both TPF and berberine hydrochloride could alleviate this congestion and swelling. To summarize, TPF at a high dose (120 mg/kg BW) and berberine hydrochloride showed nearly the same effects on promoting the recovery of colonic lesions that appeared upon administering Folium Sennae decoction.
Immunohistochemical analyses indicated that TPF significantly alleviated TNF-α, IL-1β, and IL-6 production against inflammatory reactions as well as the blockade of inflammatory process at least in part (Fig. 6A–C). Further, ELISA results showed that the levels of IL-6 (Fig. 6D), IL-1β (Fig. 6E) (in mice serum), and TNF-α (Fig. 6F) (in the supernatant of mice jejunal tissue homogenate) in MODEL and treatment groups were significantly augmented compared with those in CONT (P < 0.01). Conversely, in comparison with MODEL, the levels of TNF-α, IL-1β, and IL-6 were significantly reduced in treatment groups as well as in POSI on day 7 (P < 0.05). In summary, TPF could decrease cytokine expression level in a dose-dependent manner, and the effects of TPF at a high dose were better than those of berberine hydrochloride.
3.5. Effects of TPF on the histological changes in the small intestine of mice Histological sections of the jejunum obtained from experimental mice were studied (Fig. 5D). H&E staining showed that the intestinal structure in CONT was intact, and the mucosal arrangement was structurally normal. The cytoplasm of villous epithelial cells was abundant, adjacent cells showed tight connection on the top, and number of goblet cells was less (Fig. 5D-1 and -2). The jejunum of mice in MODEL showed mucosal swelling and villi shedding, and the normal structure of the small intestine was damaged. The epithelial cells were edematous, and the structure of the connection between cells was defected. The number of goblet cells significantly increased (Fig. 5D-3 and -4). However, damage to the jejunum in the treatment groups was
4. Discussion Diarrhea is a common disease that seriously threatens human health, particularly in economically underdeveloped areas. Identifying inexpensive, accessible, and effective therapies to tackle diarrhea is thus urgently needed. Insects are a large, unexplored, and unexploited source of useful compounds for modern medicine [20]. Maggots have been used for treating gastrointestinal ailments in China for over hundreds of years [21]. In recent years, many studies have published findings on their preservation [22], environmental protection [23], and 6
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Fig. 6. Effects of different doses of thermostable protein fraction (TPF) and positive group (POSI) on expression levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in mice with Folium Sennaeinduced diarrhea. 6A-, B-, C-1: control group (CONT); 6A-, B-, C-2: model group (MODEL); 6A-, B-, C-3: positive group [50 mg/kg body weight (BW), POSI]; 6A-, B-, C-4: low-dose group (30 mg/kg BW, TPFL); 6A-, B-, C-5: middle-dose group (60 mg/kg BW, TPFM); 6A-, B-, C-6: high-dose group (120 mg/kg BW, TPFH). 6A-, B-, C-1, 2, 3, 4, 5, 6: Immunohistochemical analyses, original magnification 20 × . 6A-, B-, C-7 and 6D-, E-, F-: data are expressed as means ± standard error of mean. ## P < 0.01 vs. CONT, *P < 0.05 vs. MODEL, **P < 0.01 vs. MODEL.
electrolyte reabsorption, causing diarrhea in mice; moreover, intestinal epithelial cells can be damaged, leading to inflammatory bowel disorder, which can stimulate the typical course of diarrhea. According to our results, all mice administered with Folium Sennae decoction showed obvious symptoms of diarrhea. Macroscopic DI and proinflammatory cytokine levels in MODEL were significantly higher than those in the normal group (P < 0.01). Based on the results of histological analyses, the small intestines of mice in MODEL were evidently damaged; hence, the total diarrhea model established in this study can be considered to be steady and credible. We believe that macroscopic DI is the most intuitive method to evaluate the antidiarrheal effects of TPF. Our results revealed that the DI of TPFM, TPFH, and POSI was significantly lower than that of MODEL (P < 0.01). Even though the DI of TPFL was lower than that of MODEL, the difference was not significant. TPF showed potent antidiarrheal effects in mice with Folium Sennae-induced diarrhea, and the effect was highly dose-dependent. This same trend was observed in histological analyses: TPF could effectively reduce damages to the small intestines in mice with diarrhea, and TPF could provide better intestinal protection than berberine hydrochloride. This could partly be due to the major ingredients of TPF (proteins and polypeptides) and its low stimulation, good affinity, and safety. TNF-α, IL-1β, and IL-6 are important cytokines produced by activated immune cells during the intestinal inflammatory process. They regulate and amplify the immune response, induce tissue injury, and mediate complications of the inflammatory response, such as diarrhea and fibrosis [27]. When tissues are injured, the transcription levels of proinflammatory genes are stimulated via the activation of nuclear factor kappaB pathways to increase the production of the aforementioned proinflammatory cytokines [28]. In this study, the levels of TNFα, IL-1β, and IL-6 in MODEL were significantly higher than those in the normal group (P < 0.01). Moreover, in comparison with MODEL, animals in TPF-treated groups and POSI showed significantly lower proinflammatory cytokine levels. Therefore, TPF seems to influence proinflammatory cytokine production and arrest immune inflammation. These results concur with previously reported findings. The mechanism underlying the antidiarrheal effects of TPF still remains unclear. We suspect that the antidiarrheal effects of TPF are relevant to the antibacterial [24] and anti-inflammatory activities of maggot proteins. TPF seem to regulate the intestinal flora to optimize the intestinal environment in diarrheal mice. Furthermore, its anti-
bactericidal activity [24]; however, only few modern pharmacological studies have explored their antidiarrheal effects. In this study, we for the first time report the antidiarrheal effects of TPF obtained from the larvae of M. domestica. To ensure that maggots were stable and clean, we cultured the houseflies in our laboratory rather than obtaining them from the wild. Under the culturing conditions used in this study, maggots of different patches showed consistent qualities. The weight of an individual maggot remained constant at 12–15 mg, and the nutrient content hardly changed, as deduced via compositional analyses. Therefore, the results reported herein should be highly reliable. SDS-PAGE analysis showed that the proteins present in TPF mostly had a molecular weight of 10–80 kDa; the relative content of the lowmolecular weight proteins was remarkably higher. Majority of insect proteins with antibacterial and antiphlogistic activities have low molecular weights, such as bee venom [12], cecropins [25], lysozyme (14 kDa) [26], and some antimicrobial peptides (AMPs; 5–15 kDa). Through LC–MS/MS analysis, AMPs belonging to the cecropin family were identified in TPF. It has been previously reported that the cecropin family shows strong anti-inflammatory activities in LPS-stimulated macrophages by interacting with LPS in the cecropia moth Hyalophora cecropia, black fly Simulium bannaense, horsefly Tabanus yao, and swallowtail butterfly Papilio xuthus; this finding explains the antidiarrheal effects of TPF. To evaluate the pharmacological properties of TPF, researchers have assessed the anti-inflammatory activities of TPF in vitro. Excessive NO produced by inducible NO synthase plays a critical role in inflammatory diseases, and suppression of its production pathways may satisfy the unmet need for control of the inflammatory process [19]. In the current study, we investigated the effects of TPF on NO formation in RAW 264.7 macrophages stimulated with LPS (1 μg/ml) and determined cell viability using an MTT assay. TPF could inhibit the excessive production of NO in a dose-dependent manner. Furthermore, at the effective dosage, cell viabilities showed no significant inhibition, as per MTT assay results. TPF showed potent anti-inflammatory effects. Thus, in addition to the use of maggots for treating gastrointestinal ailments in TCM, our results present the antidiarrheal effects and anti-inflammation activities of TPF obtained from M. domestica. In this study, the total diarrhea model was established by administering a decoction of Folium Sennae to mice. Administration of such a decoction can aggravate intestinal movement and decrease water and 7
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inflammatory activities apparently weaken the inflammatory reactions, reducing tissue damage to consequently avoid the occurrence of diarrhea. It remains uncertain whether TPF enhances the reabsorption of water and electrolytes in the mice intestine, but according to our results, this seems plausible. Further studies are thus warranted.
12.005. [7] S. Li 1518-1593, X. Luo (Eds.), Compendium of Materia Medica: Bencao Gangmu/ Compiled by Li Shizhen; Translated and Annotated by Luo Xiwen, Foreign Languages Press, Beijing, China, 2003. [8] H. Ai, F. Wang, N. Zhang, L. Zhang, C. Lei, Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica, J. Insect Sci. 112 (2013) 1–16, https://doi.org/10.1673/ 031.013.11201. [9] Q.S. Ge, H.M. Zhang, X. Liu, S.Y. Wang, D.C. Lv, X.D. Li, Crude extract of maggots: antibacterial effects against Escherichia coli, underlying mechanisms, separation and purification, World J. Gastroenterol. 5 (2015) 1510, https://doi.org/10.3748/ wjg.v21.i5.1510. [10] X. Cao, Z. Huo, M. Lu, D. Mao, Q. Zhao, C. Xu, C. Wang, B. Zeng, Purification of lectin from larvae of the fly, Musca domestica, and in vitro anti-tumor activity in MCF-7 cells, J. Insect Sci. 164 (2010) 164, https://doi.org/10.1673/031.010. 14124. [11] F.J. Chu, X.B. Jin, Y.Y. Xu, Y. Ma, X.B. Li, X.M. Lu, W.B. Liu, J.Y. Zhu, Inflammatory regulation effect and action mechanism of anti-inflammatory effective parts of housefly (Musca domestica) larvae on atherosclerosis, Evid. Complement. Alternat. Med. 2013 (2013) 1–10, https://doi.org/10.1155/2013/340267. [12] N.S. Surendra, G.N. Jayaram, M.S. Reddy, Antimicrobial activity of crude venom extracts in honeybees (Apis cerana, Apis dorsata, Apis florea) tested against selected pathogens, Afr. J. Microbiol. Res. 5 (2011) 2765–2772, https://doi.org/10.5897/ AJMR11.593. [13] G.N. Zhou, Z.H. Hu, Y.X. Wang, K.X. Zhang, Preparation of mice diarrhea model and application of the diarrhea index, Chin. Tradit. Herbal Drugs 4 (1994) 195–196, https://doi.org/10.1007/BF02007173. [14] W. Liu, D.Y. Zhang, The method for establishing an animal model of spleen deficiency caused by Senna leaf, Chin. J. Integr. Tradit. Western Med. Digestion 4 (1998) 231–232. [15] R. Balfour Sartor, Cytokines in intestinal inflammation: pathophysiological and clinical considerations, Gastroenterology 106 (2013) 533–539, https://doi.org/10. 1016/0016-5085(94)90614-9. [16] Y. Zhang, X. Wang, S. Sha, S. Liang, L. Zhao, L. Liu, N. Chai, H. Wang, K. Wu. Berberine increases the expression of NHE3 and AQP4 in sennosideA-induced diarrhoea model, Fitoterapia 6 (2012) 1014–1022, https://doi.org/10.1016/j. fitote.2012.05.015. [17] C.L. Kuo, C.W. Chi, T.Y. Liu, The anti-inflammatory potential of berberine in vitro and in vivo, Cancer Lett. 2 (2004) 127–137, https://doi.org/10.1016/j.canlet.2003. 09.002. [18] H.A. McKenzie, H.S. Wallace, The Kjeldahl determination of nitrogen: a critical study of digestion conditions—temperature, catalyst, and oxidizing agent, Aust. J. Chem. 7 (1954) 55–70, https://doi.org/10.1071/CH9540055. [19] G.J. Huang, H.Y. Chang, M.J. Sheu, C.H. Yang, T.C. Lu, Y.S. Chang, W.H. Peng, S.S. Huang, Analgesic effects and the mechanisms of anti-inflammation of hispolon in mice, Evid. Complement. Alternat. Med. 17 (2011) 9112–9119, https://doi.org/ 10.1093/ecam/nep027. [20] R.W. Pemberton, Insects and other arthropods used as drugs in Korean traditional medicine, J. Ethnopharmacol. 65 (1999) 207–216, https://doi.org/10.1016/S03788741(98)00209-8. [21] N. Tsuneo, M.Y. Hua, I. Kenji, Insect derived crude drugs in the chinese song dynasty, J. Ethnopharmacol. 24 (1988) 247–285, https://doi.org/10.1016/03788741(88)90157-2. [22] Y. Wang, X. Dang, X. Zheng, J. Wang, W. Zhang, Effect of extracted housefly pupae peptide mixture on chilled pork preservation, J. Food Sci. 75 (2010) 6, https://doi. org/10.1111/j.1750-3841.2010.01694.x. [23] H. Čičková, M. Kozánek, I. Morávek, P. Takáč, A behavioral method for separation of house fly (Diptera: Muscidae) larvae from processed pig manure, J. Econ. Entomol. 105 (2012) 62–66, https://doi.org/10.1603/EC11202. [24] X. Lu, J. Shen, X. Jin, Y. Ma, Y. Huang, H. Mei, F. Chu, J. Zhu, Bactericidal activity of Musca domestica cecropin (Mdc) on multidrug-resistant clinical isolate of Escherichia coli, Appl. Microbiol. Biotechnol. 95 (2012) 939–945, https://doi.org/ 10.1007/s00253-011-3793-2. [25] J. Andrä, O. Berninghausen, M. Leippe, Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans, Med. Microbiol. Immunol. 189 (2000) 169–173, https://doi.org/10.1007/s430-0018025-x. [26] D. Nayduch, C. Joyner, Expression of lysozyme in the life history of the house fly (Musca domestica L.), J. Med. Entomol. 50 (2013) 847–852, https://doi.org/10. 1603/ME12167. [27] R.B. Sartor, Cytokines in intestinal inflammation: pathophysiological and clinical considerations, Gastroenterology 106 (1994) 533–539, https://doi.org/10.1016/ 0016-5085(94)90614-9. [28] M. Rosenbaum, Ulcerative colitis, Psychosomatics 24 (1983) 528–529, https://doi. org/10.1016/S0033-3182(83)73183-X.
5. Conclusions In the present study, our aim was to identify a new potential treatment for diarrhea. We used the larvae of M. domestica to obtain TPF, which could effectively inhibit diarrhea, reduce damage to the small intestines of diarrheic mice, and arrest immune inflammation both in vivo and in vitro. M. domestica has many advantages; for example, this species shows high reproductive ability, fast growth, and short lifecycle. M. domestica can typically survive for 7–8 generations per year, and for even up to 20 generations under proper conditions. Year-round breeding is achievable via artificial feeding. Furthermore, breeding maggots is simple and economical, as agricultural waste can be used for culturing them. Thus, using maggots to develop new antidiarrheal drugs should be highly encouraged. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgements We thank the Animal Laboratory of Sun Yat-sen University for providing us with technical assistance in performing experiments to assess antidiarrheal effects. We thank Guangdong Magotech Biotechnology Co., Ltd for providing guidance on how to rear Musca domestica. We are grateful to everyone who contributed to this research. Juan Shen conducted the pharmacological experiments and data analysis, and also assisted with writing the manuscript. Jiali Chen assisted with the pharmacological experiments and data analysis. Depo Yang designed the pharmacological experiments and assisted with writing the manuscript. Wenzhe Yang provided the maggots, bred them, and performed quality control of experimental maggots. Cailin Tang conducted LC–MS/MS and SDS-PAGE analyses. Rongfei Zhang (Email: [email protected]) prepared TPF and Folium Sennae decoction. Zhimin Zhao contributed to polishing the language of this manuscript. Yi Niu designed the experiments and further polished this manuscript. All authors have discussed, edited, and approved the final version of the manuscript. References [1] WHO, Diarrhoeal Disease, (2013) http://www.who.int/mediacentre/factsheets/ fs330/en/. [2] M. Claeson, M.H. Merson, Global progress in the control of diarrheal diseases, Pediatr. Infect. Dis. J. 9 (1990) 345–355, https://doi.org/10.1097/00006454199005000-00008. [3] J.T. Boerma, A.E. Sommerfelt, S.O. Rutstein, Childhood Morbidity and Treatment Patterns, Aug Institute for Resource Development. Macro International, Columbia, Maryland, 1991https://www.popline.org/node/315425. [4] J. Bartlett, Clinical practice. Antibiotic-associated diarrhea, N. Engl. J. Med. (2002), https://doi.org/10.1056/NEJMcp011603. [5] D.M. Abdallah, N.R. Ismael, Resveratrol abrogates adhesion molecules and protects against TNBS-induced ulcerative colitis in rats, Can. J. Physiol. Pharmacol. 89 (2011) 811–818, https://doi.org/10.1139/y11-080. [6] R.D. Moon, Muscid Flies (Muscidae), (2002), https://doi.org/10.1016/j.tele.2015.
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