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Euphorbia kansui roots induced-diarrhea in mice correlates with inflammatory response
Chinese Journal of Natural Medicines 2013, 11(3): 0231−0239
Chinese Journal of Natural Medicines
Euphorbia kansui roots induced-diarrhea in mice correlates with inflammatory response CHAI Yu-Shuang 1, HU Jun 1Δ, WANG Xiu-Kun 1, WANG Yu-Gang 1, XIAO Xin-Yue 2, CHENG Xian-Long 2, HUA Lei1, LEI Fan 1, XING Dong-Ming 1, DU Li-Jun 1* 1 MOE Key Laboratory of Protein Sciences, Laboratory of Molecular Pharmacology and Pharmaceutical Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China; 2 National Institutes for Food and Drug Control, Beijing 100050, China
Available online 20 May 2013 [ABSTRACT] AIM: Euphorbia kansui (E. KS) is a traditional medicine used in China for thousands of years with the effect of propulsion in the gastrointestines. However, there is no reported study of E. KS on gastrointestinal motility until now. The aim of this work is to study the effect of E. KS on the propulsion of gastrointestines, and to elucidate the possible mechanism of action. METHODS: E.KS was prepared as a 30% ethanol extract and used for the experiment of small and large intestines of mice by oral administration with three different dosages (1.2, 0.6 and 0.3 g·kg–1). The feces were observed in vivo. The morphology was carried out to detect if there are any changes in the intestines after the extract of E. KS administration. The assays of mRNA and protein expression were employed to observe IL-1β, TNFα and caspase 3. RESULTS: It was shown that the extract of E.KS promoted diarrhea in mouse feces after administration, inhibited the contraction of smooth muscle of mouse small intestine and caused the inflammatory exudation on the mucosa of the intestines, enhanced the expression of both mRNA and the protein levels of IL-1β and TNFα in the small or large intestines. CONCLUSION: The results showed that the extract of E. KS acted on the intestinal smooth muscle with propulsion of feces involving the irritation of the intestines with acute inflammatory reactions. [KEY WORDS] Euphorbia kansui; Intestines; Inflammation
[CLC Number] R965
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[Document code] A
[Article ID] 1672-3651(2013)03-0231-09
Introduction
Euphorbia kansui Liou ex. S. B. Ho (Euphorbiaceae) (E. KS) is a commonly used herb in China for thousands of years. It belongs to the cathartic (releasing water from the body by purge the stool) category as mentioned in the Chinese medical literature [1]. Since its action of purging feces was been recorded, E. KS is thought of as toxic with vigorous purging of feces, requiring careful use in clinical practice. Now in the
[Received on] 30-Apr.-2012 [Research funding] The project was supported partly by the National Natural Science Foundation of China (Nos. 30801523, 30973896, and 81073092), the National Key New Drug R&D Program for the 12th Five-year Plan of China (Nos. 2011ZX09101-002-11, 2012ZX09103- 201-041, and 2012ZX09102201-008), and the Drug Safety Evaluation Project for the 11th Five-year Plan of China (No. 2006BAI1 4B01). [*Corresponding author] DU Li-Jun: Prof., Tel/Fax: 86-10-6277-3630, E-mail: lijundu@mail. tsinghua.edu.cn Δ Co-first author. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved 2013 年 5 月
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clinic, it is often used to treat pancreatitis, intestinal obstruction, hydrothorax and ascitic fluid, and cirrhotic ascites, etc [2-3] . Although it was used as a cathartic in the clinic for a long time, few studies were reported regarding the pharmacologic effects of E. KS on the gastrointestines, as well as the mechanism of propulsion of intestines. A study of the pharmacological effects of E. KS on gastrointestines will benefit the understanding of the effects of E.KS in clinical practice. In a recent study, anti-tumor [4-5], cytotoxic, and anti-viral [6-8] effects were noted regarding its toxicity in biological studies. It was also reported that E. KS could promote cell division [9-10] , activate TrkA and TrkB [11] and Stat3 [12]. Through chemical study, E. KS was shown to contain as the major chemicals, diterpene esters of the ingenol-type [13-16] which are prevalent in Euphorbia. In addition, steroids, coumarins and polysaccharides were reported [17-19]. Commonly, diterpenoids are thought of as the toxic components [20] contained in the genus Euphorbia. Euphol is one of the distinctive and active terpenoids in this herb, with cytotoxicity [21], inhibition of cellular proliferation [22], inhibition of monoacylglycerol lipase [23], anti-tumor [24], etc. The goal of this research was to observe the effects of E. Chin J Nat Med May 2013
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KS on gastrointestinal propulsion. It was hypothesized that the propulsion of the intestines by E. KS is complex in terms of the pharmacology and toxicology.
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Materials and Methods
2.1
Animals Male and female ICR mice weighing 18–22 g were provided by Vital River Laboratories (Beijing, China). The animals were housed in temperature- and humidity-controlled rooms, kept on a 12 h light/dark cycle, and provided with unrestricted amount of rodent chow and drinkable water. All experiments were approved by the Institutional Animal Care & Use Committee of Tsinghua University and the Animal Welfare & Ethics Committee of Tsinghua University. 2.2 Plant extract Euphorbia kansui (E. KS) was identified by Prof. XIAO Xin-Yue. A voucher specimen was deposited in the herbarium (No. 080201) of Laboratory of Chinese Medicine, National Institutes for Food and Drug Control. This plant was dried in the shade and powdered for the extraction. Dried powder of E. kansui roots was refluxed with 30% ethanol three times, each time for 1 hour. The extract solution was collected and evaporated in vacuum at 40 ºC. The yield of E. kansui ethanol extract (EKEE) was 19.0% (W/W), and the content of euphol, the specific component of E.KS, was 1.48%, as determined by HPLC assay [25]. 2.3 Experimental 2.3.1 Experiment 1: Groups and dosages Mice were acclimated for one week and were fasted prior to dosing. EKEE, dissolved in saline solution, was administered to mice by gastric gavage in a volume of 20 mL·kg–1 body weight. Based on prior experiments, the animals were divided into four groups (n = 4), control group and three different dosage groups. Each group was consisting of six mice (n = 6). The three different dosages of EKEE were 1.2, 0.6 and 0.3 g·kg–1. Animals of vehicle groups (control group) received the normal saline solution alone in the same volume. 2.3.2 Experiment 2: Feces of mice The mice were separated to four groups randomly and the EKEE administered by intragastric (IG) oral administration at the different dosages (1.2, 0.6 and 0.3 g·kg–1). The mice in the normal control group were administered normal saline. The potentiation times were recorded from oral administration to the first appearance of feces with charcoal [26]. The feces in two hours were collected and dried at 56 ºC temperature for weighing. Mice were fasted for 12 hours before the experiment and the water was supplemented during the experiment. Each mouse was put into one clear cage during the experiment. 2.3.3 Experiment 3: Propulsion of small intestine ICR mice were separated into four groups randomly (control and three different dosages of EKEE, 1.2, 0.6 and 232 Chin J Nat Med May 2013 Vol. 11 No. 3
0.3 g·kg–1). Upper gastrointestinal transit was measured using a slightly modified Charcoal Meal Test [27-28]. Briefly, food was removed 12 hours prior to the experiment, but animals had free access to water until 20–30 min before the start of the experiment. In order to prevent coprophagy during fasting, each cage was refreshed at the time of food removal. On the day of the experiment, all animals received a charcoal (Fluka Chemika, Switzerland) meal by gavage (0.2 mL of charcoal suspension). Fifteen min after receiving the meal all mice were euthanized. The small intestine was excised, and the distance traveled by the black color marker along the gut from the pyloric sphincter was measured. Small bowel propulsion was evaluated by calculating the ratio between the distance traveled by the meal and the total length of the small bowel for each mouse [29]. Ratio of propulsion of gastrointestinal transit (= length of charcoal moving/length of small intestine × 100%). 2.3.4 Experiment 4: Contraction of smooth muscle of mouse small intestine in vitro The experiment was that described by Kirschstein [34] and Statoh [35], with some modification. ICR mice, fasting through the night before the experiment, were lightly anaesthetized with diethyl ether and then stunned by a blow on the head and bled via carotid arteries. Segments of the duodenum, jejunum and ileum were removed and placed in Krebs solution consisting of (mmol·L–1): NaCl 118.5, KCl 4.8, MgSO4 1.2, KH2PO4, NaHCO3 25, glucose 11.1, and CaCl2 2.5 at pH 7.4. The contents of the three excised segments were gently flushed out with Krebs solution. Whole segments of each intestinal region, except the ileum, were used. The segments, 2–3 cm in length, were excised from the central part of the ileum. Intestinal segments were suspended in an organ bath filled with Krebs solution aerated with 95% O2 and 5% CO2 and maintained at 37 ºC for 15–20 min until the stable contractile activity was spontaneous. The data of contraction before the extract of E. KS were recorded for 5 min. Normal saline was added for control and the extracts were added for the contraction observation. After the agents were added, the contraction of smooth muscle was recorded for 20 min. Lastly, 50 mmol·L–1 of KCl was added to check the integrity of the smooth muscle for use as the internal standard contraction. The extract was added to the organ bath in volumes of less than 1.0% of the bathing solution and the final concentration of 250 μg·mL–1 set. These volumes of normal saline did not affect either spontaneous contractile activity or muscle tone. For the contraction of the smooth muscle of the mouse small intestine in vivo, segments of the duodenum, jejunum and ileum of the mice were removed 6 h after oral administration of EKEE 1.2 g·kg–1, and the spontaneous contractile activity or muscle tone were observed as described previously. Data are shown as the percentage of the contraction by KCl (= Contraction of EKEE/Contraction of KCl × 100%). Isometric contractions and relaxations of the smooth muscle of mouse small intestine were measured in the perfu2013 年 5 月 第 11 卷 第 3 期
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sion chamber (Kent Science Corporation, USA) by force transducers (JZJ01, Chengdu Instruments, China), recorded with a bridge amplifier (SWF-2W, Chengdu Instruments, China) connected to an analog-to-digital converter (RM6240BD, Chengdu Instruments, China) and analyzed by the CAI software (Chengdu Instruments, China). 2.4 Morphology The mice were separated into four groups as previously described (control and three different dosages of EKEE, 1.2, 0.6 and 0.3 g·kg–1). Each group comprised six mice. After 6 h administration of EKEE, mice were sacrificed for morphological tests. All of the tissues were separated into two parts. One part of the intestinal tissues was placed at –80 ºC for mRNA and protein assays. The remainder of the intestinal tissue was fixed in 12% buffered formalin. Sections were prepared and evaluated by hematoxylin-eosin (HE) staining assay [30]. The histopathological diagnoses were made by two different pathologists of this laboratory. 2.5 Real-time PCR Each 50 mg sample of fresh intestinal tissue was mixed with 1 mL Trizol (Bio Basic Inc., Canada) for testing caspase 3 for apoptosis and TNFα and IL-1β for inflammation. Reverse transcription-polymerase chain reaction assay for mRNA expression of caspase 3, TNFα and IL-1β was that referenced in [31-33]. Total RNA was extracted with Trizol reagent (Bio Basic Inc., Canada) according to the manufacturer's recommended procedures. About 1 μg of total RNA was reverse-transcribed using an M-MuLV First Strand cDNA Synthesis Kit (Bio Basic Inc., Canada). PCR was performed using a real time PCR Kit, RealMater Mix (SYBR Green) (Tiangen Biotech Co., Ltd., China). PCR products were tested by Lightcycler 480II (Roche). All primer sequences used in these analyses are generally produced by Shanghai Shenggong Biotech Company. Cycling conditions were: 94 ºC for 3 min; 40 cycles of 94 ºC for 10 sec, 54 ºC for 10 sec, 95 ºC for 10 sec; 72 ºC for 10 min and cooled to 4 ºC. The data were dealt with by the software of Light Cycler 480 SW1.5. All PCR was performed in tandem with β-actin primers as an internal control. DNA sequences are as follows: caspase 3: sense:5’-CTGGACTGTGGCATTGAGACA-3’, antisense:5’-GCCTCCACC GGTATCTTCTG-3’. TNFα: sense: 5’-TCAGCCTCTCATTCCTGC-3’, antisense: 5’-TTGGTG GTTTGCTACGACGTG-3’. IL1β: sense: 5’-GCAA CTGTTCCTGAACTC-3’, antisense: 5’- C TCGGAGCCTGTAGTGCA-3’. β-actin sense: 5’-AGCCATGTACGTAGCCATCC-3’, antisense: 5’-CTCTCAGCTGTG GTGGTGAA-3’. 2.6 Western blotting Total protein was isolated from mice intestines with 3% SDS. Protein concentration was measured using a Bradford Protein Assay Kit (Sangon, China). Protein (10 μg) was loaded onto 5% to 12% SDS-PAGE gels, and transferred onto nitrocellulose membranes after electrophoresis. The membrane was blocked with 5% bovine serum albumin in 0.1% PBST for 2 h, and incubated with primary antibody in 2013 年 5 月
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0.1% PBST for 2 h in room temperature. The labeled membrane was washed three times (10 min each) with PBST and then incubated with secondary antibody in PBST for 1.5 h at room temperature. The membrane was again washed three times (10 min each) with PBST. The targeted proteins were visualized with the super signal ECL Western blot Substrate (Pierce, China) and the intensity of the visualized bands was measured using Quantity One software (Bio-Rad). The primary antibodies from rabbit for anti-mice (β-actin, Caspase3, IL-1β and TNFα) were purchased from Zhongshanqiao Biotech. Company (Beijing, China). The secondary antibody from goal for anti-rabbit was purchased also from Zhongshanqiao Biotech. Company (Beijing, China). The data are showed as the ratio to that of β-actin. 2.7 Data analysis All values were expressed as x ± s. Statistical analysis was carried out using Microsoft Excel Version 2007 (Microsoft, USA). Data were statistically analyzed by the one-way analysis of variance (ANOVA) with F value determination. Student t-test was performed to compare EKEE effects with the control group. P below 0.05 was considered statistically significant.
3
Results
3.1
Feces of mice and propulsion of intestines After oral administration of EKEE, the mice showed different responses in both feces and anus. For EKEE with large dosage 1.2 g·kg–1, the mice responded with a loose stool and an unclean anus. While the normal reaction with the fecal mass and clean anus was observed in the normal control. The potential time for feces of EKEE with three different dosages (1.2, 0.6 and 0.3 g·kg–1) were shorter, showing the stool passed through the intestines quickly (Fig. 1A). The quantity of feces of EKEE was not larger than that of normal mice (Fig. 1B), except for the feces produced with 0.3 g·kg–1 of EKEE. EKEE at a large dosage (1.2 g·kg–1) promoted the propulsion of mice small intestine significantly, but had no significant effect on the propulsion of the small intestine of mice using the medium and low doses (0.6 and 0.3 g·kg–1) (Fig. 1C). When the mice were euthanized for intestines for the pathological test, most of the animals with EKEE treatment showed severe gastro-intestinal distension with a lot of fluid accumulation, especially at the higher dosage 1.2 g·kg–1 (Fig. 2). There was no distension observed in the normal mice. The mice receivning 0.6 and 0.3 g·kg–1 of EKEE showed nearly the same appearance as normal mice. To detect the function of such distension of the intestines, the small intestinal segment was taken out and placed in an organ bath filled with Krebs solution for its physiological movement. In the group of EKEE with large dose (1.2 g·kg–1) in vivo, the segment lost the spontaneous contraction itself (Fig. 3A) in comparison with the contraction of the small intestine of normal mouse (Fig. 3B). The extract of E.KS inhibited the contraction of normal small intestinal segment nearly completely with a final concentration of 250 μg·mL–1 (Fig. 3C) in vitro. Chin J Nat Med May 2013
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Fig. 1 Alteration of mice intestines after oral administration of Euphorbia kansui ethanol extract (EKEE). (A) the potentiation time for feces of mice after oral administration of EKEE. F (3, 44) = 72.809, **P < 0.01 vs the control. (B) the feces of mice after oral administration of EKEE. F (3, 44) = 10.05, **P < 0.01 vs the control. (C) the rate of propulsion of small intestine of mice after oral administration of EKEE. F (3, 44) = 4.927, **P < 0.01 vs the control. Normal saline was used as the vehicle control. EKEE was dosed of 1.2, 0.6 and 0.3 g·kg–1. x ± s, n = 12
Fig. 2 Observation of the gastro-intestinal tract of mice after oral administration of Euphorbia kansui ethanol extract (EKEE). (A) Normal mouse. (B) Severe appearance of distension observed after the administration of EKEE (1.2 g·kg–1). The obvious distension of the stomach and intestine can be seen. (C) Mild distension appearance after the extract with dosage of 0.6 g·kg–1. (D) Nearly normal appearance after the extract with dosage of 0.3 g·kg–1
Fig. 3 Contraction of the smooth muscle of mouse small intestines after oral administration of Euphorbia kansui ethanol extract (EKEE). (A) Normal mice as control without any attenuated contraction of small intestines. (B) The contraction of smooth muscle of small intestine at 6 h after oral administration of EKEE (1.2 g·kg–1). The concentration of KCl was 50 mmol·L–1; (C) The contraction of smooth muscle in vitro. Arrow of “EKEE” showed that with final concentration of EKEE 250 μg·mL–1, immediately the contraction of small intestines was inhibited; (D) The statistical results between normal control (A) and EKEE of 1.2 g·kg–1 administered in vivo (B). The data showed the ratio to the contraction of KCl (50 mmol·L–1). F (1, 10) = 6.285, *P < 0.05 vs the control (E). The statistical results between normal control (A) and the extract 250 μg·mL–1 in vitro (C). F (1, 10) = 109.51, **P < 0.01 vs the control. The data showed the ratio to the contraction of NaCl. x ± s, n = 6
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Fig. 4 Histogram of mice small and large intestine after oral administration of Euphorbia kansui ethanol extract (EKEE). Control: normal control in A and B. C and D represent the injury in small and large intestinal mucosa after oral administration of EKEE (1.2 g·kg–1) for 6 h (magnification 100 ×). E and F represent small and large intestines after EKEE (0.6 g·kg–1) administration (magnification 100 ×). G and H represent small and large intestines after EKEE (0.3 g·kg–1) administration without any pathological appearance in mucosa (magnification 100 ×)
3.2
Morphology of small and large intestines The abnormal appearance was found by light microscopic observation. In normal mice, the mucosa of the small and large intestines appeared normal without any pathological changes (Figs. 4A & B). After 6 h of the oral administration of EKEE, the mucosa of both small and large intestines showed necrosis with acute inflammatory injury. In the small intestine, there appeared mucosal hyperemia, intestinal villus scaling, parietal relaxing, cavity enlargement with much secretion (Fig. 4C). In the large intestine, relaxation, secretion and hyperemia were noted the same as in small intestine, except for the intestinal glands with cellular swelling (Fig. 4D). In the medium dosage of 0.6 g·kg–1 of EKEE, the necrosis and inflammation were not apparent, but secretion, relaxation and mucosal stimulation in both small and large intestines were observed (Figs. 4E&F). There were no distinct pathologic changes in the intestines in the group dosed with 0.3 g·kg–1 of EKEE, except a little mucosa stimulation in the large intestine (Figs. 4G & H). 3.3 mRNA express of Caspase 3, TNFα and IL-1β in mice intestines Using real time PCR (qPCR), the mRNA expression of 2013 年 5 月
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the factors of inflammation, TNFα and IL-1β, and the factor of apoptosis, caspase 3, were expressed to detect the promotion of feces and the distention of gastrointestines. As shown in Fig. 5, the mRNA expression of TNFα and IL-1β in the group of 1.2 g·kg–1 of EKEE were up-expressed markedly in both the small and large intestines, implying that an inflammatory reaction was occurring (Fig. 5). However, there were negative differences between the control and EKEE of mRNA express of caspase 3 in both small and large intestines, indicating there is no apoptosis reaction with the administration of 1.2 g·kg–1 EKEE. There were no positive response of TNFα and IL-1β with the 0.6 and 0.3 g·kg–1 doses of EKEE. 3.4 Protein expression of Caspase 3, TNFα and IL-1β in mice intestines In Fig. 6, the protein of TNFα and IL-1β expressed up in the small and large intestines. However, IL-1β was up regulated apparently in the small intestines with the different dosages of 1.2 and 0.6 g·kg–1. And the expression of TNFα was increased distinctly in the large intestines with EKEE at the three different dosages (1.2, 0.6 and 0.3 g·kg–1). In the small intestines, only TNFα with the EKEE medium dosage (0.6 g·kg–1) was up regulated. There was no positive reaction of Caspase 3 in the small and large intestines (Figs. 6A & B), the same as that that observed for mRNA expression. The differential expression of TNFα and IL-1β in the small and large intestines implied that there were somewhat different pathologic changes produced in the intestines, inflammation and necrosis.
4
Discussion
Based on the literature of Euphorbia kansui (E. KS) in clinical practice, the research was conducted in order to evaluate the toxicity and pharmacological effect of E.KS, respectively. It was noted that EKEE could promote the feces, but in the form of diarrhea and loose stool. Morphologically, the mucosa of the intestines showed injury with liquid secretion. The inflammatory factors, IL-1β in the small intestines and TNFα in large intestines, were up-regulated. It was noted that after the extract the mice showed diarrhea and loose stool in the anus in the groups dosed with 1.2 and 0.6 g·kg–1 of EKEE. Although the potentiation time of feces was shorter than that of normal mice, the quantity of fecal mass of EKEE was no more than that of normal mice. The propulsion of small intestine of mice after oral administration showed that EKEE at a dosage of 1.2 g·kg–1 promoted the movement of mice small intestines. It was suggested that the short stool time of EKEE was caused probably by a heavier secretion from the intestines. To prove this hypothesis, the experiment on the smooth muscle contraction in vivo and in vitro was conducted. When EKEE was used at the concentration of 250 μg·mL–1, the contraction of smooth muscle of mouse small intestine was completely inhibited in vitro. Meanwhile, the spontaneous contraction of the smooth muscle of mouse small intestines nearly disappeared after 6 h Chin J Nat Med May 2013
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of extract administration (1.2 g·kg–1) in vivo. It was suggested that what the fecal stool did rapidly after the administration of EKEE might not be caused by the propulsion of smooth muscle of intestines, but by secretion of mucosa by the intestines. To confirm the pathological injury further, a morphological study was conducted to answer the question of what caused the secretion of the intestines by EKEE. In the experiment, the small and large intestines were studied histologically. Under the microscope, injury to the intestines was found. In Fig. 4, the specimen of E.KS extract at a dosage of 1.2 g·kg–1 showed the injury of hyperemia, intestinal villus scaling, parietal relaxation, and cavity enlarging with much secretion, indicating the appearance of an acute inflammatory injury and exudation. Although the injury from the extract doses of 0.6 and 0.3 g·kg–1 was not as marked as that of the dose of 1.2 g·kg–1, the pathological irritation was also observed in both small and large intestines. Therefore, it was concluded that EKEE caused an acute toxic injury with inflammatory reaction in the intestinal mucosa after its oral administration in a dose-dependent
manner. TNFα and IL-1β reflect the inflammatory reaction , while caspase 3 reflects the apoptotic reaction in the intestines. In both the small and large intestines at the dose of 1.2 g·kg–1, TNFα and IL-1β, in both mRNA and proteins, was up-expressed significantly, which showed that this reaction of the intestines to the administration of EKEE might correlate with necrosis of inflammation. In both mRNA and protein expression, caspase 3 was not up-regulated apparently in the intestines, suggesting there is no occurrence of apoptosis. Combined with inflammatory factor expressions, the damage in the mucus of mouse small and large intestines after oral administration of EKEE was elucidated with inflammatory hyperemia, intestinal villus scaling, and enlarged cavity with heavier exudation. The results suggested that the morphological appearance was a toxic reaction. Shu’s report proved partly this conclusion regarding the toxic inflammatory reaction induced by E. KS [40] in models of inflammation using exoteric mice splenic lymphocytes (SPL) and rat peritoneal macrophages (PMphi) in vitro. Thus, we may draw the conclusion that the diarrhea produced by EKEE was caused by the inflammatory exuda[36-38]
Fig. 5 Expression of mRNA of caspase 3, TNFα and IL-1β in mice small intestines after oral administration of Euphorbia kansui ethanol extract (EKEE) with the assay of qPCR. E.KS extract was used at three dosages of 1.2, 0.6 and 0.3 g·kg–1 by oral administration. TNFα of small intestines, F (3, 20) = 6.89, *P < 0.05 vs control. TNFα of large intestines, F (3, 20) = 8.997, *P < 0.05 vs control. IL-1β of small intestines, F (3, 20) = 5.812, *P < 0.05 vs control. IL1β of large intestines, F (3, 20) = 4.851, *P < 0.05 vs control. x ± s, n = 6
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with the first reaction of abdominal pain, and then loose stool . In these experiments, the same observation was made as that in the clinic. The loose stool of the mice appeared ahead of the charcoal feces observation after oral administration of EKEE. Although the potentiation time of the charcoal feces was shorter than that of the control, the quantity of fecal mass of EKEE was not increased compared with that of the control. Meanwhile, the small intestinal contraction was inhibited by the extract both in vivo and in vitro, and the intestines showed relaxation in morphology. It was suggested that this kind of loose stool in clinical practice was positively related with heavier exudation in the intestines due to the irritation caused of EKEE. This study showed that the promotion of feces by the extract of E. KS occurred because of inflammatory irritation and caused exudation, which is the typical characteristic of E. KS on gastrointetines. This partly explains the usage of E. KS for the inner water expulsion in traditional Chinese medicine. However, several questions still remain. Why the inner water (water retention) is dispelled just by the inflammatory exudation and how is the retained water retention dispelled? What is the molecular mechanism in pharmacology? To answer these questions further research is required. This recent work showed that EKEE could reduce inner water of water-loaded mice, accompanied with IL-1β and TNFα increasing in the kidney [43]. That means there is a complex mechanism operating to be evaluated for this kind of toxic inflammation of E. KS. In conclusion, this is the first study to evaluate the extract of Euphorbia kansui (E. KS) on mice gastrointestines concerning inflammatory injury in vivo. Comprehensive and interesting results showed that EKEE could promote a loose stool in mice, inhibit the contraction of smooth muscle of mouse small intestine, and irritate the acute inflammatory injury on mucosa of intestines with heavier exudation. It was demonstrated that the effect of E. KS on the gastrointestines is correlated with irritation of inflammatory exudation. Care must be taken in the use of E. KS because of its acute toxicity in the intestinal mucosa by direct irritation. It was proposed that the function of E. KS maybe for the fecal mass, and for the body liquid by the exudation and the indication of E. KS, relieving constipation and for other pathological changes in the body, such as pancreatitis, ascites due to cirrhosis, etc. [41-42]
Fig. 6 Expression of protein of Caspase 3, TNFα and IL1β in mice small (A) and large (B) intestines after oral administration of Euphorbia kansui ethanol extract (EKEE) using Western blotting. The extract of E.KS was used at the three dosages of 1.2, 0.6 and 0.3 g·kg–1 by oral administration. IL-1β of small intestines, F (3, 20) = 8.07, *P < 0.05 vs the control. TNFα of large intestines, F (3, 20) = 51.04, **P < 0.01 vs the control. x ± s, n = 6
tion. E. KS contains diterpenoids such as euphol, kansuinin A, kansuinin B, etc., which are thought of as toxic components [20] , and which maybe the inducer for the inflammatory injury. Yu reported that they used the inflammatory irritant action of the ethanol extract of E. KS on the peritoneal macrophage cells harvested from SD rats and evaluated the fruit of Ziziphus jujuba for a protecting effect from the inflammatory irritation of the ethanol extract of E. KS [39]. That also suggests that the components of the ethanol extract of E. KS could irritate the intestines with the inflammatory reaction and exudation. As an original indication from the Chinese medical literature, the plant was classed in the category of “Jun-Xia-Zhu-Shui” (dispelling the inner water by promoting feces vigorously). So, the Chinese doctors often use E.KS not only for the feces, but also for water retention in the body, such as ascites due to cirrhosis. This is an interesting practical experience. During the treatment with E. KS in modern Chinese clinical practice, the E. KS reaction was recorded 2013 年 5 月
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kansuiphorins A and B, two novel antileukemic diterpene esters from Euphorbia kansui [J]. J Nat Prod, 1991, 54(3): 823-829. Whelan LC, Ryan MF. Ethanolic extracts of Euphorbia and other ethnobotanical species as inhibitors of human tumour cell growth [J]. Phytomedicine, 2003, 10(1): 53-58. Akihisa T, Ogihara J, Kato J, et al. Inhibitory effects of triterpenoids and sterols on human immunodeficiency virus-1 reverse transcriptase [J]. Lipids, 2001, 36(5): 507-512. Cho HJ, Yu FQ, Sun R, et al. Lytic induction of Kaposi’s sarcoma-associated herpesvirus in primary effusion lymphoma cells with natural products identified by a cell-based fluorescence moderate-throughput screening [J]. Arch Virol, 2008, 153(8): 1517-1525. Ehrman TM, Barlow DJ, Hylands PJ. Virtual screening of Chinese herbs with random forest[J]. J Chem Inf Model, 2007, 47(2): 264-278. Wang LY, Wang NL, Yao XS, et al. Euphane and tirucallane triterpenes from the roots of Euphorbia kansui and their in vitro effects on the cell division of xenopus [J]. J Nat Prod, 2003, 66(5): 630-633. Wang LY, Wang NL, Yao XS, et al. Studies on the bioactive constituents in Euphorbiaceae. Part 3. Diterpenes from the roots of Euphorbia kansui and their in vitro effects on the cell division of xenopus [J]. Chem Pharm Bull, 2003, 51(8): 935-941. Pan Q, Ip FCF, Ip NY, et al. Activity of macrocyclic jatrophane diterpenes from Euphorbia kansui in a TrkA fibroblast survival assay [J]. J Nat Prod, 2004, 67(9): 1548-1551. Chang JS, Lee SW, Park MH, et al. Kansuinine A and kansuinine B from Euphorbia kansui L. inhibit IL-6-induced Stat3 activation [J]. Planta Med, 2010, 76(14): 1544-1549. Shimokawa K, Takamura H, Uemura D. Concise synthesis of a highly functionalized cyclopentane segment: toward the total synthesis of kansuinine A [J]. Tetrahedron Lett, 2007, 48(32): 5623-5625. Pan Q, Min ZD. Studies on ingenol-type diterpene esters in root tuber of Euphorbia kansui [J]. Chin Tradit Herb Drugs, 2003, 34(6): 489-492. Liu Y, Liu ZQ, Li HL, et al. Studies on diterpenoids constituents from Euphorbia kansui by electro-spray ionization multi-stage tandem mass spectrometry [J]. Chem J Chin Univ, 2008, 29(9): 1727-1735. Guo J, Fang X, Di YT, et al. Kansuinine J, a new macrocyclic diterpenoid from the roots of Euphorbia kansui [J]. Chin Chem Lett, 2010, 21(8): 943-946. Itokawa H, Morris-Natschke SL, Akiyama T, et al. Plant-derived natural product research aimed at new drug discovery [J]. J Nat Med, 2008, 62(3): 263-280. Shi QW, Su XH, Kiyota H. Chemical and pharmacological research of the plants in genus Euphorbia [J]. Chem Rev, 2008, 108(10): 4295-4327. Wang YB, Li YY, Wang HB, et al. Chemical constituents from the roots of Euphorbia kansui [J]. Chin J Nat Med, 2007, 5(3): 182-185. Li CF, Wang JH, Cong Y, et al. A new diterpene from the processed roots of Euphorbia kansui [J]. J Asian Nat Prod Res, 2008, 10(2): 101-104. Mazoir N, Benharref A, Bailén M, et al. Bioactive triterpene
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derivatives from latex of two Euphorbia species [J]. Phytochemistry, 2008, 69(6): 1328-1338. [22] Miyata S, Wang LY, Yoshida C, et al. Inhibition of cellular proliferation by diterpenes, topoisomerase II inhibitor [J]. Bioorg Med Chem, 2006, 14(6): 2048-2051. [23] King AR, Dotsey EY, Lodola A, et al. Discovery of potent and reversible monoacylglycerol lipase inhibitors [J]. Chem Biol, 2009, 16(10):1045-1052. [24] Yasukawa K, Akihisa T, Yoshida ZY, et al. Inhibitory effect of euphol, a triterpene alcohol from the roots of Euphorbia kansui, on tumour promotion by 12-O-tetradecanoylphorbol13-acetate in two-stage carcinogenesis in mouse skin [J]. J Pharm Pharmacol, 2000, 52(1): 119-124. [25] Cheng XL, Xiao XY, Li GH, et al. HPLC simultaneous determination of euphol and tirucallol in Radix Kansui [J]. Chin J Pharm Anal, 2009, 29(9): 1444-1447. [26] Speroni E, Schwaiger S, Egger P, et al. In vivo efficacy of different extracts of Edelweiss (Leontopodium alpinum Cass.) in animal models [J]. J Ethnopharmacol, 2006, 105(3): 421-426. [27] Broccardo M, Improta G, Tabacco A. Central effect of SNC 80, a selective and systemically active δ-opioid receptor agonist, on gastrointestinal propulsion in the mouse [J]. Eur J Pharmacol, 1998, 342(2-3): 247-251. [28] Satoh K, Kase Y, Yuzurihara M, et al. Effect of Dai-kenchu-to (Da-Jian-Zhong-Tang) on the delayed intestinal propulsion induced by chlorpromazine in mice [J]. J Ethnopharmacol, 2003, 86(1): 37-44. [29] Radzievsky AA, Cowan A, Byrd C, et al. Single millimeter wave treatment does not impair gastrointestinal transit in mice [J]. Life Sci, 2002, 71(15): 1763-1770. [30] Jiang L, Ji N, Zhou Y, et al. CAL27 is an oral adenosquamous carcinoma cell line [J]. Oral Oncol, 2009, 45: e204-e207. [31] Kolaczkowska E, Koziol A, Plytycz B, et al. Altered apoptosis of inflammatory neutrophils in MMP-9-deficient mice is due to lower expression and activity of caspase-3[J]. Immunol Lett, 2009, 126(1-2): 73-82. [32] Shen J, Zhang HY, Lin H, et al. Brazilein protects the brain against focal cerebral ischemia reperfusion injury correlating to inflammatory response suppression [J]. Eur J Pharmacol, 2007, 558(1-3): 88-95. [33] Li HY, Hu J, Ma L, et al. Comprehensive study of baicalin down-regulating NOD2 receptor expression of neurons with oxygen-glucose deprivation in vitro and cerebral ischemia-reperfusion in vivo [J]. Eur J Pharmacol, 2010, 649(1-3): 92-99. [34] Kirschstein T, Dammann F, Klostermann J, et al. Dopamine induces contraction in the proximal, but relaxation in the distal rat isolated small intestine [J]. Neurosci Lett, 2009, 465(1): 21-26. [35] Satoh Y, Uchida M, Fujita A, et al. Possible role of orexin A in nonadrenergic, noncholinergic inhibitory response of muscle of the mouse small intestine [J]. Eur J Pharmacol, 2001, 428(3): 337-342. [36] Crisafulli C, Galuppo M, Cuzzocrea S. Effects of genetic and pharmacological inhibition of TNF-α in the regulation of inflammation in macrophages [J]. Pharmacol Res, 2009, 60(4): 332-340. [37] Feldmeyer L, Werner S, French LE, et al. Interleukin-1, inflammasomes and the skin [J]. Eur J Cell Biol, 2010, 89(9): 2013 年 5 月 第 11 卷 第 3 期
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kansui [J]. J Nat Med, 2010, 64(1): 98-103. [41] Hu WB. Enema of Euphorbia kansui for treatment of epidemic hemorrhage fever in oliguric stage [J]. Henan Tradit Chin Med, 2003, 23(8): 78. [42] Chen GM. Application of Euphorbia kansui in medical treatment [J]. Sichuan Tradit Chin Med, 1999, 17(5): 21-22. [43] Li HY, Lei F, Wang YG, et al. Comprehensive study of Euphorbia kansui on urination and kidney AQP2, IL-1β and TNFα mRNA expression of mice injected with normal saline [J]. China J Chin Mater Med, 2012, 37(5): 606-610.
甘遂致小鼠稀便与炎性分泌相关 柴玉爽 1, 胡 珺 1, 王秀坤 1, 王玉刚 1, 肖新月 2, 程宪隆 2, 花 雷 1, 雷 帆 1, 邢东明 1, 杜力军 1* 1
清华大学教育部蛋白质科学重点实验室, 生命科学学院医学院 分子药物药理研究室, 北京 100084;
2
国家食品药品检定研究院, 北京 100050
【摘 要】 目的：甘遂在我国有着几千年的应用历史, 主要用于促进肠运动。通过实验观察甘遂通便促进肠推进同时是否 伴有病理变化。方法：应用 30%甘遂提取物给小鼠灌胃后排便及其大小肠的影响, 进行组织学检查。同时观察大小肠 IL-1β, TNFα 和 Caspase 3 的 mRNA 和蛋白表达的变化。结果：甘遂能够明显促进小鼠排稀便, 抑制肠平滑肌收缩, 肠粘膜出现明显炎性损伤, 并伴有 IL-1β 和 TNFα 表达的上调。结论：甘遂促进排便与刺激肠粘膜分泌和炎性反应有关。 【关键词】 甘遂; 肠; 炎症; 小鼠
【基金项目】 国家自然科学基金资助项目(Nos. 30801523, 30973896, 81073092), 国家“重大新药创制”科技重大专项“十二五” 计划资助项目(No. 2011ZX09101-002-11, 2012ZX09103-201-041, 2012ZX09102-201-008), “十一五” 国家科技支撑项目(No. 2006BAI1 4B01)
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Euphorbia kansui roots induced-diarrhea in mice correlates with inflammatory response CHAI Yu-Shuang 1, HU Jun 1Δ, WANG Xiu-Kun 1, WANG Yu-Gang 1, XIAO Xin-Yue 2, CHENG Xian-Long 2, HUA Lei1, LEI Fan 1, XING Dong-Ming 1, DU Li-Jun 1* 1 MOE Key Laboratory of Protein Sciences, Laboratory of Molecular Pharmacology and Pharmaceutical Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China; 2 National Institutes for Food and Drug Control, Beijing 100050, China
Available online 20 May 2013 [ABSTRACT] AIM: Euphorbia kansui (E. KS) is a traditional medicine used in China for thousands of years with the effect of propulsion in the gastrointestines. However, there is no reported study of E. KS on gastrointestinal motility until now. The aim of this work is to study the effect of E. KS on the propulsion of gastrointestines, and to elucidate the possible mechanism of action. METHODS: E.KS was prepared as a 30% ethanol extract and used for the experiment of small and large intestines of mice by oral administration with three different dosages (1.2, 0.6 and 0.3 g·kg–1). The feces were observed in vivo. The morphology was carried out to detect if there are any changes in the intestines after the extract of E. KS administration. The assays of mRNA and protein expression were employed to observe IL-1β, TNFα and caspase 3. RESULTS: It was shown that the extract of E.KS promoted diarrhea in mouse feces after administration, inhibited the contraction of smooth muscle of mouse small intestine and caused the inflammatory exudation on the mucosa of the intestines, enhanced the expression of both mRNA and the protein levels of IL-1β and TNFα in the small or large intestines. CONCLUSION: The results showed that the extract of E. KS acted on the intestinal smooth muscle with propulsion of feces involving the irritation of the intestines with acute inflammatory reactions. [KEY WORDS] Euphorbia kansui; Intestines; Inflammation
[CLC Number] R965
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[Document code] A
[Article ID] 1672-3651(2013)03-0231-09
Introduction
Euphorbia kansui Liou ex. S. B. Ho (Euphorbiaceae) (E. KS) is a commonly used herb in China for thousands of years. It belongs to the cathartic (releasing water from the body by purge the stool) category as mentioned in the Chinese medical literature [1]. Since its action of purging feces was been recorded, E. KS is thought of as toxic with vigorous purging of feces, requiring careful use in clinical practice. Now in the
[Received on] 30-Apr.-2012 [Research funding] The project was supported partly by the National Natural Science Foundation of China (Nos. 30801523, 30973896, and 81073092), the National Key New Drug R&D Program for the 12th Five-year Plan of China (Nos. 2011ZX09101-002-11, 2012ZX09103- 201-041, and 2012ZX09102201-008), and the Drug Safety Evaluation Project for the 11th Five-year Plan of China (No. 2006BAI1 4B01). [*Corresponding author] DU Li-Jun: Prof., Tel/Fax: 86-10-6277-3630, E-mail: lijundu@mail. tsinghua.edu.cn Δ Co-first author. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved 2013 年 5 月
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clinic, it is often used to treat pancreatitis, intestinal obstruction, hydrothorax and ascitic fluid, and cirrhotic ascites, etc [2-3] . Although it was used as a cathartic in the clinic for a long time, few studies were reported regarding the pharmacologic effects of E. KS on the gastrointestines, as well as the mechanism of propulsion of intestines. A study of the pharmacological effects of E. KS on gastrointestines will benefit the understanding of the effects of E.KS in clinical practice. In a recent study, anti-tumor [4-5], cytotoxic, and anti-viral [6-8] effects were noted regarding its toxicity in biological studies. It was also reported that E. KS could promote cell division [9-10] , activate TrkA and TrkB [11] and Stat3 [12]. Through chemical study, E. KS was shown to contain as the major chemicals, diterpene esters of the ingenol-type [13-16] which are prevalent in Euphorbia. In addition, steroids, coumarins and polysaccharides were reported [17-19]. Commonly, diterpenoids are thought of as the toxic components [20] contained in the genus Euphorbia. Euphol is one of the distinctive and active terpenoids in this herb, with cytotoxicity [21], inhibition of cellular proliferation [22], inhibition of monoacylglycerol lipase [23], anti-tumor [24], etc. The goal of this research was to observe the effects of E. Chin J Nat Med May 2013
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KS on gastrointestinal propulsion. It was hypothesized that the propulsion of the intestines by E. KS is complex in terms of the pharmacology and toxicology.
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Materials and Methods
2.1
Animals Male and female ICR mice weighing 18–22 g were provided by Vital River Laboratories (Beijing, China). The animals were housed in temperature- and humidity-controlled rooms, kept on a 12 h light/dark cycle, and provided with unrestricted amount of rodent chow and drinkable water. All experiments were approved by the Institutional Animal Care & Use Committee of Tsinghua University and the Animal Welfare & Ethics Committee of Tsinghua University. 2.2 Plant extract Euphorbia kansui (E. KS) was identified by Prof. XIAO Xin-Yue. A voucher specimen was deposited in the herbarium (No. 080201) of Laboratory of Chinese Medicine, National Institutes for Food and Drug Control. This plant was dried in the shade and powdered for the extraction. Dried powder of E. kansui roots was refluxed with 30% ethanol three times, each time for 1 hour. The extract solution was collected and evaporated in vacuum at 40 ºC. The yield of E. kansui ethanol extract (EKEE) was 19.0% (W/W), and the content of euphol, the specific component of E.KS, was 1.48%, as determined by HPLC assay [25]. 2.3 Experimental 2.3.1 Experiment 1: Groups and dosages Mice were acclimated for one week and were fasted prior to dosing. EKEE, dissolved in saline solution, was administered to mice by gastric gavage in a volume of 20 mL·kg–1 body weight. Based on prior experiments, the animals were divided into four groups (n = 4), control group and three different dosage groups. Each group was consisting of six mice (n = 6). The three different dosages of EKEE were 1.2, 0.6 and 0.3 g·kg–1. Animals of vehicle groups (control group) received the normal saline solution alone in the same volume. 2.3.2 Experiment 2: Feces of mice The mice were separated to four groups randomly and the EKEE administered by intragastric (IG) oral administration at the different dosages (1.2, 0.6 and 0.3 g·kg–1). The mice in the normal control group were administered normal saline. The potentiation times were recorded from oral administration to the first appearance of feces with charcoal [26]. The feces in two hours were collected and dried at 56 ºC temperature for weighing. Mice were fasted for 12 hours before the experiment and the water was supplemented during the experiment. Each mouse was put into one clear cage during the experiment. 2.3.3 Experiment 3: Propulsion of small intestine ICR mice were separated into four groups randomly (control and three different dosages of EKEE, 1.2, 0.6 and 232 Chin J Nat Med May 2013 Vol. 11 No. 3
0.3 g·kg–1). Upper gastrointestinal transit was measured using a slightly modified Charcoal Meal Test [27-28]. Briefly, food was removed 12 hours prior to the experiment, but animals had free access to water until 20–30 min before the start of the experiment. In order to prevent coprophagy during fasting, each cage was refreshed at the time of food removal. On the day of the experiment, all animals received a charcoal (Fluka Chemika, Switzerland) meal by gavage (0.2 mL of charcoal suspension). Fifteen min after receiving the meal all mice were euthanized. The small intestine was excised, and the distance traveled by the black color marker along the gut from the pyloric sphincter was measured. Small bowel propulsion was evaluated by calculating the ratio between the distance traveled by the meal and the total length of the small bowel for each mouse [29]. Ratio of propulsion of gastrointestinal transit (= length of charcoal moving/length of small intestine × 100%). 2.3.4 Experiment 4: Contraction of smooth muscle of mouse small intestine in vitro The experiment was that described by Kirschstein [34] and Statoh [35], with some modification. ICR mice, fasting through the night before the experiment, were lightly anaesthetized with diethyl ether and then stunned by a blow on the head and bled via carotid arteries. Segments of the duodenum, jejunum and ileum were removed and placed in Krebs solution consisting of (mmol·L–1): NaCl 118.5, KCl 4.8, MgSO4 1.2, KH2PO4, NaHCO3 25, glucose 11.1, and CaCl2 2.5 at pH 7.4. The contents of the three excised segments were gently flushed out with Krebs solution. Whole segments of each intestinal region, except the ileum, were used. The segments, 2–3 cm in length, were excised from the central part of the ileum. Intestinal segments were suspended in an organ bath filled with Krebs solution aerated with 95% O2 and 5% CO2 and maintained at 37 ºC for 15–20 min until the stable contractile activity was spontaneous. The data of contraction before the extract of E. KS were recorded for 5 min. Normal saline was added for control and the extracts were added for the contraction observation. After the agents were added, the contraction of smooth muscle was recorded for 20 min. Lastly, 50 mmol·L–1 of KCl was added to check the integrity of the smooth muscle for use as the internal standard contraction. The extract was added to the organ bath in volumes of less than 1.0% of the bathing solution and the final concentration of 250 μg·mL–1 set. These volumes of normal saline did not affect either spontaneous contractile activity or muscle tone. For the contraction of the smooth muscle of the mouse small intestine in vivo, segments of the duodenum, jejunum and ileum of the mice were removed 6 h after oral administration of EKEE 1.2 g·kg–1, and the spontaneous contractile activity or muscle tone were observed as described previously. Data are shown as the percentage of the contraction by KCl (= Contraction of EKEE/Contraction of KCl × 100%). Isometric contractions and relaxations of the smooth muscle of mouse small intestine were measured in the perfu2013 年 5 月 第 11 卷 第 3 期
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sion chamber (Kent Science Corporation, USA) by force transducers (JZJ01, Chengdu Instruments, China), recorded with a bridge amplifier (SWF-2W, Chengdu Instruments, China) connected to an analog-to-digital converter (RM6240BD, Chengdu Instruments, China) and analyzed by the CAI software (Chengdu Instruments, China). 2.4 Morphology The mice were separated into four groups as previously described (control and three different dosages of EKEE, 1.2, 0.6 and 0.3 g·kg–1). Each group comprised six mice. After 6 h administration of EKEE, mice were sacrificed for morphological tests. All of the tissues were separated into two parts. One part of the intestinal tissues was placed at –80 ºC for mRNA and protein assays. The remainder of the intestinal tissue was fixed in 12% buffered formalin. Sections were prepared and evaluated by hematoxylin-eosin (HE) staining assay [30]. The histopathological diagnoses were made by two different pathologists of this laboratory. 2.5 Real-time PCR Each 50 mg sample of fresh intestinal tissue was mixed with 1 mL Trizol (Bio Basic Inc., Canada) for testing caspase 3 for apoptosis and TNFα and IL-1β for inflammation. Reverse transcription-polymerase chain reaction assay for mRNA expression of caspase 3, TNFα and IL-1β was that referenced in [31-33]. Total RNA was extracted with Trizol reagent (Bio Basic Inc., Canada) according to the manufacturer's recommended procedures. About 1 μg of total RNA was reverse-transcribed using an M-MuLV First Strand cDNA Synthesis Kit (Bio Basic Inc., Canada). PCR was performed using a real time PCR Kit, RealMater Mix (SYBR Green) (Tiangen Biotech Co., Ltd., China). PCR products were tested by Lightcycler 480II (Roche). All primer sequences used in these analyses are generally produced by Shanghai Shenggong Biotech Company. Cycling conditions were: 94 ºC for 3 min; 40 cycles of 94 ºC for 10 sec, 54 ºC for 10 sec, 95 ºC for 10 sec; 72 ºC for 10 min and cooled to 4 ºC. The data were dealt with by the software of Light Cycler 480 SW1.5. All PCR was performed in tandem with β-actin primers as an internal control. DNA sequences are as follows: caspase 3: sense:5’-CTGGACTGTGGCATTGAGACA-3’, antisense:5’-GCCTCCACC GGTATCTTCTG-3’. TNFα: sense: 5’-TCAGCCTCTCATTCCTGC-3’, antisense: 5’-TTGGTG GTTTGCTACGACGTG-3’. IL1β: sense: 5’-GCAA CTGTTCCTGAACTC-3’, antisense: 5’- C TCGGAGCCTGTAGTGCA-3’. β-actin sense: 5’-AGCCATGTACGTAGCCATCC-3’, antisense: 5’-CTCTCAGCTGTG GTGGTGAA-3’. 2.6 Western blotting Total protein was isolated from mice intestines with 3% SDS. Protein concentration was measured using a Bradford Protein Assay Kit (Sangon, China). Protein (10 μg) was loaded onto 5% to 12% SDS-PAGE gels, and transferred onto nitrocellulose membranes after electrophoresis. The membrane was blocked with 5% bovine serum albumin in 0.1% PBST for 2 h, and incubated with primary antibody in 2013 年 5 月
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0.1% PBST for 2 h in room temperature. The labeled membrane was washed three times (10 min each) with PBST and then incubated with secondary antibody in PBST for 1.5 h at room temperature. The membrane was again washed three times (10 min each) with PBST. The targeted proteins were visualized with the super signal ECL Western blot Substrate (Pierce, China) and the intensity of the visualized bands was measured using Quantity One software (Bio-Rad). The primary antibodies from rabbit for anti-mice (β-actin, Caspase3, IL-1β and TNFα) were purchased from Zhongshanqiao Biotech. Company (Beijing, China). The secondary antibody from goal for anti-rabbit was purchased also from Zhongshanqiao Biotech. Company (Beijing, China). The data are showed as the ratio to that of β-actin. 2.7 Data analysis All values were expressed as x ± s. Statistical analysis was carried out using Microsoft Excel Version 2007 (Microsoft, USA). Data were statistically analyzed by the one-way analysis of variance (ANOVA) with F value determination. Student t-test was performed to compare EKEE effects with the control group. P below 0.05 was considered statistically significant.
3
Results
3.1
Feces of mice and propulsion of intestines After oral administration of EKEE, the mice showed different responses in both feces and anus. For EKEE with large dosage 1.2 g·kg–1, the mice responded with a loose stool and an unclean anus. While the normal reaction with the fecal mass and clean anus was observed in the normal control. The potential time for feces of EKEE with three different dosages (1.2, 0.6 and 0.3 g·kg–1) were shorter, showing the stool passed through the intestines quickly (Fig. 1A). The quantity of feces of EKEE was not larger than that of normal mice (Fig. 1B), except for the feces produced with 0.3 g·kg–1 of EKEE. EKEE at a large dosage (1.2 g·kg–1) promoted the propulsion of mice small intestine significantly, but had no significant effect on the propulsion of the small intestine of mice using the medium and low doses (0.6 and 0.3 g·kg–1) (Fig. 1C). When the mice were euthanized for intestines for the pathological test, most of the animals with EKEE treatment showed severe gastro-intestinal distension with a lot of fluid accumulation, especially at the higher dosage 1.2 g·kg–1 (Fig. 2). There was no distension observed in the normal mice. The mice receivning 0.6 and 0.3 g·kg–1 of EKEE showed nearly the same appearance as normal mice. To detect the function of such distension of the intestines, the small intestinal segment was taken out and placed in an organ bath filled with Krebs solution for its physiological movement. In the group of EKEE with large dose (1.2 g·kg–1) in vivo, the segment lost the spontaneous contraction itself (Fig. 3A) in comparison with the contraction of the small intestine of normal mouse (Fig. 3B). The extract of E.KS inhibited the contraction of normal small intestinal segment nearly completely with a final concentration of 250 μg·mL–1 (Fig. 3C) in vitro. Chin J Nat Med May 2013
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Fig. 1 Alteration of mice intestines after oral administration of Euphorbia kansui ethanol extract (EKEE). (A) the potentiation time for feces of mice after oral administration of EKEE. F (3, 44) = 72.809, **P < 0.01 vs the control. (B) the feces of mice after oral administration of EKEE. F (3, 44) = 10.05, **P < 0.01 vs the control. (C) the rate of propulsion of small intestine of mice after oral administration of EKEE. F (3, 44) = 4.927, **P < 0.01 vs the control. Normal saline was used as the vehicle control. EKEE was dosed of 1.2, 0.6 and 0.3 g·kg–1. x ± s, n = 12
Fig. 2 Observation of the gastro-intestinal tract of mice after oral administration of Euphorbia kansui ethanol extract (EKEE). (A) Normal mouse. (B) Severe appearance of distension observed after the administration of EKEE (1.2 g·kg–1). The obvious distension of the stomach and intestine can be seen. (C) Mild distension appearance after the extract with dosage of 0.6 g·kg–1. (D) Nearly normal appearance after the extract with dosage of 0.3 g·kg–1
Fig. 3 Contraction of the smooth muscle of mouse small intestines after oral administration of Euphorbia kansui ethanol extract (EKEE). (A) Normal mice as control without any attenuated contraction of small intestines. (B) The contraction of smooth muscle of small intestine at 6 h after oral administration of EKEE (1.2 g·kg–1). The concentration of KCl was 50 mmol·L–1; (C) The contraction of smooth muscle in vitro. Arrow of “EKEE” showed that with final concentration of EKEE 250 μg·mL–1, immediately the contraction of small intestines was inhibited; (D) The statistical results between normal control (A) and EKEE of 1.2 g·kg–1 administered in vivo (B). The data showed the ratio to the contraction of KCl (50 mmol·L–1). F (1, 10) = 6.285, *P < 0.05 vs the control (E). The statistical results between normal control (A) and the extract 250 μg·mL–1 in vitro (C). F (1, 10) = 109.51, **P < 0.01 vs the control. The data showed the ratio to the contraction of NaCl. x ± s, n = 6
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Fig. 4 Histogram of mice small and large intestine after oral administration of Euphorbia kansui ethanol extract (EKEE). Control: normal control in A and B. C and D represent the injury in small and large intestinal mucosa after oral administration of EKEE (1.2 g·kg–1) for 6 h (magnification 100 ×). E and F represent small and large intestines after EKEE (0.6 g·kg–1) administration (magnification 100 ×). G and H represent small and large intestines after EKEE (0.3 g·kg–1) administration without any pathological appearance in mucosa (magnification 100 ×)
3.2
Morphology of small and large intestines The abnormal appearance was found by light microscopic observation. In normal mice, the mucosa of the small and large intestines appeared normal without any pathological changes (Figs. 4A & B). After 6 h of the oral administration of EKEE, the mucosa of both small and large intestines showed necrosis with acute inflammatory injury. In the small intestine, there appeared mucosal hyperemia, intestinal villus scaling, parietal relaxing, cavity enlargement with much secretion (Fig. 4C). In the large intestine, relaxation, secretion and hyperemia were noted the same as in small intestine, except for the intestinal glands with cellular swelling (Fig. 4D). In the medium dosage of 0.6 g·kg–1 of EKEE, the necrosis and inflammation were not apparent, but secretion, relaxation and mucosal stimulation in both small and large intestines were observed (Figs. 4E&F). There were no distinct pathologic changes in the intestines in the group dosed with 0.3 g·kg–1 of EKEE, except a little mucosa stimulation in the large intestine (Figs. 4G & H). 3.3 mRNA express of Caspase 3, TNFα and IL-1β in mice intestines Using real time PCR (qPCR), the mRNA expression of 2013 年 5 月
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the factors of inflammation, TNFα and IL-1β, and the factor of apoptosis, caspase 3, were expressed to detect the promotion of feces and the distention of gastrointestines. As shown in Fig. 5, the mRNA expression of TNFα and IL-1β in the group of 1.2 g·kg–1 of EKEE were up-expressed markedly in both the small and large intestines, implying that an inflammatory reaction was occurring (Fig. 5). However, there were negative differences between the control and EKEE of mRNA express of caspase 3 in both small and large intestines, indicating there is no apoptosis reaction with the administration of 1.2 g·kg–1 EKEE. There were no positive response of TNFα and IL-1β with the 0.6 and 0.3 g·kg–1 doses of EKEE. 3.4 Protein expression of Caspase 3, TNFα and IL-1β in mice intestines In Fig. 6, the protein of TNFα and IL-1β expressed up in the small and large intestines. However, IL-1β was up regulated apparently in the small intestines with the different dosages of 1.2 and 0.6 g·kg–1. And the expression of TNFα was increased distinctly in the large intestines with EKEE at the three different dosages (1.2, 0.6 and 0.3 g·kg–1). In the small intestines, only TNFα with the EKEE medium dosage (0.6 g·kg–1) was up regulated. There was no positive reaction of Caspase 3 in the small and large intestines (Figs. 6A & B), the same as that that observed for mRNA expression. The differential expression of TNFα and IL-1β in the small and large intestines implied that there were somewhat different pathologic changes produced in the intestines, inflammation and necrosis.
4
Discussion
Based on the literature of Euphorbia kansui (E. KS) in clinical practice, the research was conducted in order to evaluate the toxicity and pharmacological effect of E.KS, respectively. It was noted that EKEE could promote the feces, but in the form of diarrhea and loose stool. Morphologically, the mucosa of the intestines showed injury with liquid secretion. The inflammatory factors, IL-1β in the small intestines and TNFα in large intestines, were up-regulated. It was noted that after the extract the mice showed diarrhea and loose stool in the anus in the groups dosed with 1.2 and 0.6 g·kg–1 of EKEE. Although the potentiation time of feces was shorter than that of normal mice, the quantity of fecal mass of EKEE was no more than that of normal mice. The propulsion of small intestine of mice after oral administration showed that EKEE at a dosage of 1.2 g·kg–1 promoted the movement of mice small intestines. It was suggested that the short stool time of EKEE was caused probably by a heavier secretion from the intestines. To prove this hypothesis, the experiment on the smooth muscle contraction in vivo and in vitro was conducted. When EKEE was used at the concentration of 250 μg·mL–1, the contraction of smooth muscle of mouse small intestine was completely inhibited in vitro. Meanwhile, the spontaneous contraction of the smooth muscle of mouse small intestines nearly disappeared after 6 h Chin J Nat Med May 2013
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of extract administration (1.2 g·kg–1) in vivo. It was suggested that what the fecal stool did rapidly after the administration of EKEE might not be caused by the propulsion of smooth muscle of intestines, but by secretion of mucosa by the intestines. To confirm the pathological injury further, a morphological study was conducted to answer the question of what caused the secretion of the intestines by EKEE. In the experiment, the small and large intestines were studied histologically. Under the microscope, injury to the intestines was found. In Fig. 4, the specimen of E.KS extract at a dosage of 1.2 g·kg–1 showed the injury of hyperemia, intestinal villus scaling, parietal relaxation, and cavity enlarging with much secretion, indicating the appearance of an acute inflammatory injury and exudation. Although the injury from the extract doses of 0.6 and 0.3 g·kg–1 was not as marked as that of the dose of 1.2 g·kg–1, the pathological irritation was also observed in both small and large intestines. Therefore, it was concluded that EKEE caused an acute toxic injury with inflammatory reaction in the intestinal mucosa after its oral administration in a dose-dependent
manner. TNFα and IL-1β reflect the inflammatory reaction , while caspase 3 reflects the apoptotic reaction in the intestines. In both the small and large intestines at the dose of 1.2 g·kg–1, TNFα and IL-1β, in both mRNA and proteins, was up-expressed significantly, which showed that this reaction of the intestines to the administration of EKEE might correlate with necrosis of inflammation. In both mRNA and protein expression, caspase 3 was not up-regulated apparently in the intestines, suggesting there is no occurrence of apoptosis. Combined with inflammatory factor expressions, the damage in the mucus of mouse small and large intestines after oral administration of EKEE was elucidated with inflammatory hyperemia, intestinal villus scaling, and enlarged cavity with heavier exudation. The results suggested that the morphological appearance was a toxic reaction. Shu’s report proved partly this conclusion regarding the toxic inflammatory reaction induced by E. KS [40] in models of inflammation using exoteric mice splenic lymphocytes (SPL) and rat peritoneal macrophages (PMphi) in vitro. Thus, we may draw the conclusion that the diarrhea produced by EKEE was caused by the inflammatory exuda[36-38]
Fig. 5 Expression of mRNA of caspase 3, TNFα and IL-1β in mice small intestines after oral administration of Euphorbia kansui ethanol extract (EKEE) with the assay of qPCR. E.KS extract was used at three dosages of 1.2, 0.6 and 0.3 g·kg–1 by oral administration. TNFα of small intestines, F (3, 20) = 6.89, *P < 0.05 vs control. TNFα of large intestines, F (3, 20) = 8.997, *P < 0.05 vs control. IL-1β of small intestines, F (3, 20) = 5.812, *P < 0.05 vs control. IL1β of large intestines, F (3, 20) = 4.851, *P < 0.05 vs control. x ± s, n = 6
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with the first reaction of abdominal pain, and then loose stool . In these experiments, the same observation was made as that in the clinic. The loose stool of the mice appeared ahead of the charcoal feces observation after oral administration of EKEE. Although the potentiation time of the charcoal feces was shorter than that of the control, the quantity of fecal mass of EKEE was not increased compared with that of the control. Meanwhile, the small intestinal contraction was inhibited by the extract both in vivo and in vitro, and the intestines showed relaxation in morphology. It was suggested that this kind of loose stool in clinical practice was positively related with heavier exudation in the intestines due to the irritation caused of EKEE. This study showed that the promotion of feces by the extract of E. KS occurred because of inflammatory irritation and caused exudation, which is the typical characteristic of E. KS on gastrointetines. This partly explains the usage of E. KS for the inner water expulsion in traditional Chinese medicine. However, several questions still remain. Why the inner water (water retention) is dispelled just by the inflammatory exudation and how is the retained water retention dispelled? What is the molecular mechanism in pharmacology? To answer these questions further research is required. This recent work showed that EKEE could reduce inner water of water-loaded mice, accompanied with IL-1β and TNFα increasing in the kidney [43]. That means there is a complex mechanism operating to be evaluated for this kind of toxic inflammation of E. KS. In conclusion, this is the first study to evaluate the extract of Euphorbia kansui (E. KS) on mice gastrointestines concerning inflammatory injury in vivo. Comprehensive and interesting results showed that EKEE could promote a loose stool in mice, inhibit the contraction of smooth muscle of mouse small intestine, and irritate the acute inflammatory injury on mucosa of intestines with heavier exudation. It was demonstrated that the effect of E. KS on the gastrointestines is correlated with irritation of inflammatory exudation. Care must be taken in the use of E. KS because of its acute toxicity in the intestinal mucosa by direct irritation. It was proposed that the function of E. KS maybe for the fecal mass, and for the body liquid by the exudation and the indication of E. KS, relieving constipation and for other pathological changes in the body, such as pancreatitis, ascites due to cirrhosis, etc. [41-42]
Fig. 6 Expression of protein of Caspase 3, TNFα and IL1β in mice small (A) and large (B) intestines after oral administration of Euphorbia kansui ethanol extract (EKEE) using Western blotting. The extract of E.KS was used at the three dosages of 1.2, 0.6 and 0.3 g·kg–1 by oral administration. IL-1β of small intestines, F (3, 20) = 8.07, *P < 0.05 vs the control. TNFα of large intestines, F (3, 20) = 51.04, **P < 0.01 vs the control. x ± s, n = 6
tion. E. KS contains diterpenoids such as euphol, kansuinin A, kansuinin B, etc., which are thought of as toxic components [20] , and which maybe the inducer for the inflammatory injury. Yu reported that they used the inflammatory irritant action of the ethanol extract of E. KS on the peritoneal macrophage cells harvested from SD rats and evaluated the fruit of Ziziphus jujuba for a protecting effect from the inflammatory irritation of the ethanol extract of E. KS [39]. That also suggests that the components of the ethanol extract of E. KS could irritate the intestines with the inflammatory reaction and exudation. As an original indication from the Chinese medical literature, the plant was classed in the category of “Jun-Xia-Zhu-Shui” (dispelling the inner water by promoting feces vigorously). So, the Chinese doctors often use E.KS not only for the feces, but also for water retention in the body, such as ascites due to cirrhosis. This is an interesting practical experience. During the treatment with E. KS in modern Chinese clinical practice, the E. KS reaction was recorded 2013 年 5 月
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甘遂致小鼠稀便与炎性分泌相关 柴玉爽 1, 胡 珺 1, 王秀坤 1, 王玉刚 1, 肖新月 2, 程宪隆 2, 花 雷 1, 雷 帆 1, 邢东明 1, 杜力军 1* 1
清华大学教育部蛋白质科学重点实验室, 生命科学学院医学院 分子药物药理研究室, 北京 100084;
2
国家食品药品检定研究院, 北京 100050
【摘 要】 目的：甘遂在我国有着几千年的应用历史, 主要用于促进肠运动。通过实验观察甘遂通便促进肠推进同时是否 伴有病理变化。方法：应用 30%甘遂提取物给小鼠灌胃后排便及其大小肠的影响, 进行组织学检查。同时观察大小肠 IL-1β, TNFα 和 Caspase 3 的 mRNA 和蛋白表达的变化。结果：甘遂能够明显促进小鼠排稀便, 抑制肠平滑肌收缩, 肠粘膜出现明显炎性损伤, 并伴有 IL-1β 和 TNFα 表达的上调。结论：甘遂促进排便与刺激肠粘膜分泌和炎性反应有关。 【关键词】 甘遂; 肠; 炎症; 小鼠
【基金项目】 国家自然科学基金资助项目(Nos. 30801523, 30973896, 81073092), 国家“重大新药创制”科技重大专项“十二五” 计划资助项目(No. 2011ZX09101-002-11, 2012ZX09103-201-041, 2012ZX09102-201-008), “十一五” 国家科技支撑项目(No. 2006BAI1 4B01)
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