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Effect of polysaccharides from Sijunzi decoction on Ca2+ related regulators during intestinal mucosal restitution
Phytomedicine 58 (2019) 152880
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Original Article
Effect of polysaccharides from Sijunzi decoction on Ca2+ related regulators during intestinal mucosal restitution
T
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Shi Yuxia1, Zhu Huibin1, Li Ruliu , Wang Dongxu, Zhu Yiping, Hu Ling, Chen Weiwen Pi-wei Institute, Guangzhou University of Chinese Medicine, 12 Jichang road, Guangzhou 510405, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sijunzi Decoction polysaccharides intestinal epithelial cells cell migration polyamine mucosal restitution
Background: Sijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been widely used for treating patients with gastrointestinal diseases and spleen-deficiency syndrome, however, the exact effects and mechanisms are remained unclear. Hypothesis/purpose: The migration of intestinal epithelial (IEC-6) cells has been demonstrated to be one of the major repair modalities during the healing of mucosal wounds. Ca2+ is the key factor in regulating cell migration. Thus, this study aimed to elucidate the potential effects and mechanisms of polysaccharides (SJZDP) extracted from Sijunzi decoction in intestinal mucosal restitution. Method: Cell migration was detected by scratch method with micropipette tip. Western blotting was adopted to evaluate the expression of STIM1 and STIM2 proteins. Immunofluorescence was carried out to assess the translocation of STIM1 protein. Immunoprecipitation was used to determine the levels of STIM1/TRPC1 and STIM1/STIM2 complexes. A indomethacin-induced intestinal mucosal injury rat model was applied. The content of polyamines in intestinal mucosa was detected by high-performance liquid chromatography. The morphological changes in the intestinal mucosa were observed at the end of animal experiment. Results: The results showed that treatment with SJZDP significantly promoted cell migration, enhanced the level of STIM1 protein and STIM1/TRPC1 complexes, reduced the level of STIM2 protein and STIM1/STIM2 complexes. Further more, SJZDP exposure promoted the translocation of STIM1 to the plasma membrane. In vivo experiment results, the administration of SJZDP effectively reduced intestinal mucosal injury. Conclusion: These results revealed that SJZDP promotes intestinal epithelial restitution after wounding, presumably by regulating cellular levels of STIM1 and STIM2 differentially, stimulating the translocation of STIM1, and inducing STIM1/TRPC1 association as well as decreasing the level of STIM1/STIM2.
Introduction Gastrointestinal mucosal injury is one of the most common histopathological features in gastrointestinal disease and spleen-deficiency syndrome which is both the organic disorders and functional changes in the theory of traditional Chinese medicine (Xu et al., 1987). After injury, gastrointestinal mucosa undergoes a rapid restitution which reseals the superficial wounds by epithelial cell migration. This early mucosal restitution is one of the major repair modalities in the gastrointestinal tract and is controlled by numerous intracellular and extracellular factors, including polyamines and cytosolic free Ca2+ ([Ca2+]cyt) (Ridley et al., 2003).
Sijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been extensively applied to the treatment of gastrointestinal diseases and spleen-deficiency syndrome. It is composed of four Chinese herbs: Panax ginseng (Renshen); Atractylodes macrocephala (Baizhu); Poria cocos (Fulin); Glycyrrhiza uralensis (Gancao), of which polysaccharides are considered the major active ingredient (Liu et al., 2005). In clinical practice, Sijunzi decoction effectively ameliorates gastrointestinal mucosal lesion, and promote the healing of peptic ulcer (Xie, 2012). Our previous studies found that Sijunzi decoction had prophylactic effect on gastric mucosal injury in rats with stress ulcer presumably by increasing polyamines content in the mucosa of stomach and small intestine (Nian, 2013). Then, we
Abbreviations: [Ca2+]cyt, cytosolic free Ca2+; CCE, capacitative Ca2+ entry; DFMO, DL-α-difluoromethylornithine; IEC-6, intestinal epithelial cells; SJZDP, Sijunzi decoction polysaccharides; SOCs, store-operated Ca2+channel; STIM1, stromal interaction molecule 1; STIM2, stromal interaction molecule 2; TRPC1, canonical transient receptor potential 1 ⁎ Corresponding author. E-mail address: [email protected] (R. Li). 1 These authors contributed to the work equally and should be regarded as first joint authors. https://doi.org/10.1016/j.phymed.2019.152880 Received 12 October 2018; Accepted 27 February 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
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Fig. 1. The HPGPC analysis of SJZDP3.
investigated the effect and mechanism of polysaccharides extracted from Sijunzi decoction (SJZDP) on promoting cell migration via regulating polyamine and [Ca2+]cyt. We found that administration of SJZDP promoted IEC-6 migration. SJZDP enhanced the contents of polyamines, then up-regulated the expression of potassium channel protein Kv1.1, associated with cell membrane hyperpolarization which could enhance the driving force for Ca2+ influx (Tu et al., 2016a). [Ca2+]cyt is the key factor in regulating epithelial cell migration. Increasing [Ca2+]cyt accelerates intestinal epithelial restitution after wounding both in vivo and in vitro (Putney, 2005). [Ca2+]cyt is regulated by extracellular Ca2+ influx via Ca2+-permeable channels and intracellular Ca2+ release from reticulum (Song et al., 2017). Canonical transient receptor potential 1 (TRPC1) acts as store-operated Ca2+channels, regulates the balance of [Ca2+]cyt, and then has influence upon epithelial restitution (Rao et al., 2006). Stromal interaction molecule 1 (STIM1) and stromal interaction molecule 2 (STIM2), the sensors of Ca2+ within the store, act essential parts in regulating TRPC1-mediated Ca2+ influx after store depletion (Pani et al., 2012). Since, Ca2+ related regulators have a great impact on cell migration, we presumed that SJZDP may promote intestinal mucosal restitution through modulating the Ca2+ related regulators. Thus, the purpose of this research was to evaluate the effects of SJZDP on intestinal mucosal restitution, and to further elucidate the potential mechanism of action in IEC-6 migration through Ca2+ related regulators pathway.
(MO, U.S.A). Rabbit antibodies against STIM2 and STIM1 were provided by Cell Signaling Technology (MA, U.S.A). Rabbit antibodies against TRPC1 and GAPDH, and Goat secondary antibody to rabbit IgG (HRP) were obtained from Abcam (MA, U.S.A)
Materials and methods
Properties of SJZDP
Herbs and reagents
Determination of the total carbohydrate and proteins Phenol-sulfuric acid assay, which was described by Dubois (Dubois et al., 1951), was conducted to determine the total carbohydrate of SJZDP with D-glucose as a standard. The protein content of SJZDP2 was detected in according with Lowry's method (Lowry et al., 1951), with bovine serum albumin used as a standard.
Preparation of SJZDP Panax ginseng, Atractylodes macrocephala, Poria cocos, and Glycyrrhiza uralensis in the ratio of 1:1:1:1 were minced and mixed, soaked in 12-times volume of double distilled water for 2 h, and then decocted for 2 h, and filtered. Subsequently, the herbs were decocted and filtered again in the same way, and then the decoction were pooled together and concentrated. The extracts were added with 4-times volume of 95% ethanol to precipitate crude polysaccharides at 4℃ overnight. Crude polysaccharides were dissolved and precipitated which was repeated in three times. Then the precipitates were dissolved and centrifugated (8000 rpm/min,15 min), solution were collected and freeze-dried to produce SJZDP1. SJZDP1 were deproteinized by the Sevag method (Sevag et al., 1938) to produce SJZDP2 which were used in vivo. 2.0164 g SJZDP2 were dissolved in 15 ml ultrapure deionized water and were loaded onto DEAE-52 cellulose anion exchange chromatography column which was washed with ultrapure water at a flow rate of 1.7 ml/min. Then, the elution from SJZDP2 were collected and freeze-dried to produce SJZDP3 which was used in vitro.
Panax ginseng (Renshen), Atractylodes macrocephala (Baizhu), Poria cocos (Fulin), and Glycyrrhiza uralensis (Gancao) were purchased from Guangzhou Tong Kang Pharmaceutical Co. Ltd and were identified by associate professor Tong JY from the Department of Chinese medicine identification in Guangzhou University of Chinese Medicine. Spermidine, spermine, putrescine, indomethacin, and DL-α-difluoromethylornithine (DFMO) were purchased from Sigma-Aldrich
High-performance gel permeation chromatograph HPGPC equipped with a Waters G 4000 PWXL column (MA, U.S.A) 2
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Fig. 2. Effect of SJZDP on cell migration in vitro. Images of IEC-6 cell migration in plates with different treatment containing DFMO (B) or not (A). (C) Quantitative analysis of migration were described by the ratio of the covered wound area to the initial scratched area. Values were presented as means ± SD (n = 15). ## p < 0.01, compared with DFMO group. **p < 0.01, compared with Control. Original magnification, ×100.
previous study (Ray et al., 2007). 5 parallel lines were drawn on the bottom of 6-well plates before seeding cells. The scratch-wound was carried out perpendicular to the marked lines using a 200 μl micropipette tip. Damaged cells in plates were washed away using PBS. Subsequently, the respective medium were treated with drugs as described. Photomicrographs of the area of cell migration were taken using an Olympus inverted phase contrast microscope (Tokyo, Japan) at each intersection of the wounded edge and the marked lines at 0 h (S0) and at 12 h (S12h) after administration of drugs. Cell migration was worked out as wound area (%) covered at 12 h in according with this formula (S12h-S0) /S¯ 0 by the Image-Pro Plus software.
was used to detected the purity of SJZDP3. Briefly, after being cleared through a 0.45 μm filter, 20 μl SJZDP3 (1 mg/ml, dissolved in ultrapure water) was applied to the column. The column and refractive index detector were maintained at a temperature of 30 ℃. The mobile phase was ultrapure water at a flow rate of 0.6 ml/min. Cell culture IEC-6 cells were provided by the American Type Culture Collection (VA, U.S.A, NO. 63139935). Cells were seeded in T-75 flasks containing of DMEM supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 5% fetal bovine serum. Flasks were maintained in a humidified atmosphere of 95% air-5% CO2 at 37 ℃. When IEC-6 cells grew to about 90% of the flask's area, cells were digested with trypsinEDTA, transferred into 6-well plates, and cultured for 24 h in the above conditions. Subsequently, cells were scratched and treated with control, spermidine (5 μmol/l, SPD), and SJZDP3 (20, 40, 80 and 160 mg/l) respectively, containing DFMO (2.5 mmol/l) or not for appropriate time.
Western-blot analysis Western-blot of protein expression STIM1, STIM2 and GAPDH in IEC-6 cells after different treatment were performed and quantified by Image Lab software. The semi-quantitative of STIM1 and STIM2 immunoblots were normalized by corresponding GAPDH bands. Immunofluorescence staining
Cell migration assays Immunofluorescence was detected according to the previous research (Rao et al., 2010) to determine the location of STIM1 in IEC-6
Measurement of IEC-6 migration were detected according to the 3
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Fig. 3. Effect of SJZDP on the expression of STIM1 and STIM2 proteins in IEC-6 cells after wounding. Representative bands of STIM1 and STIM2 in IEC-6 cells with different treatment containing DFMO (B) or not (A). (C) Semi-quantitative analysis of STIM1 and STIM2 immunoblots were normalized by corresponding GAPDH bands. Values were presented as means ± SD from three separate experiments. ##p < 0.01,compared with DFMO group. *p < 0.05 and **p < 0.01, compared with control.
group (n = 12). After 3 days acclimation period, rats in model and SJZDP groups were treated with indomethacin (6 mg/kg, i.p., dissolved in NaHCO3 solution) once daily for 6 days to induce intestinal mucosal injury, while rats in normal group received the same volume of saline. One hour before model inducing, each rats in SJZDP groups was treated with SZJDP2 (0.358 g/kg or 1.074 g/kg, dissolved in double distilled water) by intragastric administration, while rats in other groups received the same volume of double distilled water, once daily for 6 days. 24 h after the last administration of drugs, the rats were sacrificed, and the tissue samples of small intestine were collected.
cells. Immunoprecipitation assays Equal amounts of proteins from IEC-6 cells were incubated overnight with the rabbit anti-STIM1 (4 μg) at 4℃. Then, protein A + G Agarose was added and incubated for 3 h at 4 ℃. The supernatant was removed by centrifugation, while the agarose was resuspended in 50 μl 1×SDS-PAGE buffer. For immunoblotting, samples were detected by western-blot analysis, and incubated either with rabbit anti-STIM1 (1:1000), rabbit anti-STIM2 (1:000), or rabbit anti-TRPC1 (1:5000).
Gross appearance of intestinal mucosal injury Experimental protocol in vivo The gross appearance of intestinal mucosal injury was scored according to the degree of ulcer and adhesion (Zhu et al., 1998). Grading scale of ulcer: 0 point (no mucosal hyperemia, edema or ulcer); 1 point (localized hyperemia and edema; no ulcer); 2 points (mild ulcer, without obvious mucosal hyperemia or edema); 3 points (one ulcer with mucosal hyperemia and edema); 4 points (multiple ulcers with inflammation, ulcer diameter<1 cm); 5 points (multiple ulcers with inflammation, at least one of the ulcer diameters>1 cm). Grading scale of adhesion: 0 point (no adhesion); 1 point (the adhesion is light, the small intestine and other tissues can be separated by a little strength); 2 points (the adhesion is heavy).
Male Sprague-Dawley rats (SPF, approximately 180–220 g) were provided by the Animal Laboratory Center, Guangzhou University of Chinese Medicine (NO.44005800005951). Animal were housed in separated cages in the SPF laboratory animal room provided by the Animal Laboratory Center, Guangzhou University of Chinese Medicine (NO.00180626). Animal were given water and food ad libitum. All animal experiments were approved by the committee of animal ethics in Guangzhou University of Chinese Medicine. Rats were randomized into 4 groups: normal group (n = 8), model group (n = 16), SJZDP2 0.358 g/kg group (n = 12), SJZDP2 1.074 g/kg 4
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Fig. 4. Effect of SJZDP on the translocation of STIM1 protein in IEC-6 cells after wounding. Subcellular localization of STIM1 (Green) in IEC-6 cells with different treatment containing DFMO (B) or not (A). Nuclei were stained with DAPI (blue). Image (original magnification, ×630) were taken by laser confocal microscopy.
Fig. 5. Effect of SJZDP on the changes of STIM1/TRPC1 and STIM1/STIM2 complexes. Representative bands of STIM1, STIM2 and TRPC1 proteins in the complexes which were immunoprecipitated by antibody against STIM1 from IEC-6 cells with different treatment containing DFMO (B) or not (A) after wounding.
by capillary congestion; Grade 2: extension of subepithelial space with moderate edema in lamina propria, and dilated central lacteal; Grade 3: severe edema in lamina propria, epithelial cell degeneration and necrosis in intestinal mucosa, a few villis may be denuded; Grade 4: denuded villis with lamina propria and dilated capillaries exposed; Grade 5: disintegration and digestion of lamina propria, bleeding or ulceration.
Histopathological examination of intestinal mucosal injury After scoring the gross appearance of intestinal mucosal injury, the tissue samples were fixed in 10% paraformaldehyde, dehydrated in ethanol, embedded in paraffin, and sectioned. The sections were stained by H&E, observed by optical microscope. According to the previous research (Chiu et al., 1970), pathological changes in intestinal mucosa were dividing into 6 grades with some modifies: Grade 0: normal mucosal villis; Grade 1: development of subepithelial Gruenhagen'space at the apex of the villis, accompanied 5
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Fig. 6. Gross evaluation of intestinal mucosal injury in different groups. (A) Representative photographs of the intestinal mucosa in different groups. (B) Bar graphs showing the score of gross evaluation for intestinal mucosal injury. #p < 0.05 and ##p < 0.01, compared with Indomethacin group. ⁎⁎p < 0.01, compared with control.
Polyamines assay in small intestinal mucosa
Results
Pre-column derivatization reversed phase high performance liquid chromatography with Hypersil ODS2 column (Thermo Fisher Scientific, MA, U.S.A) was adopted with the polyamines (putrescine, spermidine and spermine) as indices in according with our previous works (Sui et al., 2011). The polyamines contents (μmol/mg) from mucosal samples were described by the ratio of the detected value of polyamines divided by the corresponding weight of mucosal, to the amounts of proteins.
Extraction yield and properties of SJZDP The extraction yields of SJZDP1, SJZDP2, and SJZDP3 were 8.77%, 7.16% and 4.52% respectively. And the total carbohydrate content in SJZDP1, SJZDP2, and SJZDP3 were 91.34%, 81.58%, and 91.54% respectively. The content of protein in SJZDP2 was 0.049964 mg/ml, which meant the protein was almost removed by the Sevag method. The HPGPC of SJZDP3 was determined in Fig. 1. SJZPD3 had 3 absorption peaks, suggesting that SJZDP3 was not homogeneous polysaccharides. The retention time of the max absorption peak was 20.30 min, accounted for 82.99% of the polysaccharides, indicating that the SJZDP3 was extracted with high purity.
Statistical analysis All values were expressed as means ± SD, and SPSS 21.0 software was used for analyzing. Differences in mean values was determined by one-way ANOVA or Kruskal-Wallis H test. Then, multiple comparisons between the groups were determined using Dunnett's multiple range test or Nemenyi test. p < 0.05 was accepted as statistical significance.
Effect of SJZDP on cell migration in vitro The process of cell migration after wounding was shown in Fig. 2. Treatment with SJZDP (40, 80, 160 mg/l) remarkably promoted cell migration as compared to control group (p < 0.01, Fig. 2A and C). Polyamine depletion by DFMO significantly inhibited cell migration, and the migration rate in DFMO group had decreased to 78% of control 6
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10 min after wounding without any treatments. STIM1 still located in the plasma membrane from 30 min to 3 h, and then declined at 6 h after wounding. In comparison with control group, treatment with SJZDP (80 mg/l) stimulated the translocation of STIM1 whose immunostaining in the plasma membrane was still obvious at 6 h after wounding (Fig. 4A), indicating that SJZDP could maintain the translocation of STIM1 over a longer period of time. Polyamine depletion by DFMO slowed the translocation of STIM1, whose immunostaining in the plasma membrane was also weaker than those in control groups after wounding (Fig. 4B). However, treatment with SJZDP (80 mg/l) as well as SPD (5 μmol/l) prevented the effects of DFMO on STIM1 translocation (Fig. 4B). Effect of SJZDP on the changes of STIM1/TRPC1 and STIM1/STIM2 complexes We further determine whether the level of STIM1/TRPC1 and STIM1/STIM2 complexes were affected by the treatment of SJZDP. As shown in Fig. 5, SJZDP (80 mg/l) significantly enhanced the levels of STIM1/TRPC1 association, but reduced the levels of STIM1/STIM2 complex. In contrast, polyamine depletion by DFMO decreased STIM1/ TRPC1 complex and increased STIM1/STIM2 association, and these effects of DFMO were prevented by the administration of SJZDP (80 mg/l) and SPD (5 μmol/l). Effect of SJZDP on experimental intestinal mucosal injury in rats Gross evaluation of intestinal mucosal injury The results of gross evaluation were presented in Fig. 6. There were not obvious lesions in control group, while the indomethacin group showed intense intestinal mucosal injury appeared as heavy adhesion, obvious mucosal hyperemia, and multiple ulcers (Fig. 6A). The administration of SJZDP considerably reduced intestinal mucosal damage and decreased the ulcer formation in comparison with indomethacin group (p < 0.05, Fig. 6A and B).
Fig. 7. Effect of SJZDP on histopathological lesions in indomethacin-induced intestinal injury. (A) Histopathological observation of intestinal mucosa stained with H&E. (B) The scoring of histopathological evaluation of intestinal mucosal injury. #p < 0.05 and ##p < 0.01, compared with indomethacin group. ⁎⁎p < 0.01, compared with control original magnification,×100.
Histopathological evaluation of intestinal mucosal injury As displayed in Fig. 7, there was no disruption of mucosa in control group, while the indomethacin group exhibited obvious damage to the intestinal mucosa appeared as degenerative and necrotic epithelial cells, denuded villis, disintegrative lamina propria, and ulceration. SJZDP 0.358 g/kg group presented with moderate disruption of intestinal mucosa, a much fewer denuded villis and lamina propria as compared to indomethacin group (p < 0.01, Fig. 7A and B). SJZDP 1.074 g/kg group exhibited much better histopathological appearance with almost intact epithelial cells, and thus showed protection from indomethacin-induced mucosal damage (Fig. 7A and B).
at 12 h (Fig. 2B). The inhibition of cell migration by DFMO could be almost reversed and restored to normal level by concomitant treatment with SJZDP (Fig. 2B and C), suggesting that the effect of SJZDP on cell migration is related to polyamines. Effect of SJZDP on the expression of STIM1 and STIM2 protein To investigate the underlying mechanism of SJZDP on cell migration, STIM1 and STIM2 were detected by western blot. The results showed that SJZDP (20, 40, 80 and 100 mg/l) enhanced the expression of STIM1 protein, while reduced the level of STIM2 protein, in comparison with control group (p < 0.05, Fig. 3A and C), suggesting that the SJZDP may promote cell migration by changing the expression of stromal interaction molecule. Polyamine depletion by DFMO decreased the expression of STIM1 protein, and increased the level of STIM2 (p < 0.05, Fig. 3B and C). However, the effects of DFMO on STIM1 and STIM2 protein expression were reversed by the administration of SJZDP (40, 80 and 160 mg/l) as well as SPD (5 μmol/l) (p < 0.05, Fig. 3B and C).
Contents of polyamines in small intestinal mucosa There were not significant differences of putrescine or spermine content among each group (p > 0.05, Fig. 8). However, the spermidine content in indomethacin group was significantly decreased in comparison with control group (p < 0.05), and reduced to ∼47.7% of control group. Treatment with SJZDP significantly increased the level of spermidine as compared to indomethacin group (p < 0.05, Fig. 8B). Discussion The gastrointestinal tract is lined by a monolayer of differentiated epithelial cells which constitute a key element of mucosal barriers to separate the luminal pathogens and the host (Jankowski et al., 1994). The impairment of mucosal barriers is a common pathological event which is observed in the pathogenesis of multiple gut diseases, such as NSAID-induced enteropathy, inflammatory bowel disease, and peptic ulcer (Catalioto et al., 2011). In response to injury, intestinal mucosa
Effect of SJZDP on the translocation of STIM1 protein To determine the location of STIM1 in IEC-6 cells after wounding, immunofluorescence of STIM1 was carried out. The result in Fig. 4A showed that STIM1 obviously retained in IEC-6 cytoplasm at 0 min. The translocation of STIM1 to the plasma membrane was increased within 7
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Fig. 8. Polyamines contents in small intestinal mucosa. (A) HPLC chromatogram of polyamines content from different group. (B) Quantitative analysis of spermidine were presented as bar graphs. #p < 0.05 and ##p < 0.01, compared with Indomethacin group. *p < 0.05, compared with control.
increasing [Ca2+]cyt is a critical factor (Tu et al., 2016b). Therefore, we further elucidate the potential mechanism of SJZDP in promoting IEC-6 cell migration through Ca2+ related signal pathway, in order to investigate the regulation of extracellular calcium influx by SJZDP during cell migration. Moreover, we used indomethacin to induce the rat model of intestinal mucosal injury, and further evaluate the effect of SJZDP on accelerating the restitution of mucosal damage in vivo. An increasing of [Ca2+]cyt is a important trigger for intestinal epithelial restitution. It depends on the release of intracellular calcium from endoplasmic reticulum, or the influx of extracellular Ca2+ which is the critical mechanism involved in maintaining suitable [Ca2+]cyt (Berridge et al., 2000). Ca2+ influx owing to store depletion is also known as capacitative Ca2+ entry (CCE) which is mediated through Ca2+-permeable channels termed store-operated Ca2+channel (SOCs). TRPC1 functions as a component of native SOCs. It acts an essential role in controlling CCE, in maintaining a sustained intracellular [Ca2+]cyt, and in regulating intestinal epithelial restitution. Induced TRPC1 expression enhances CCE as well as cells migration after wounding,
goes through a rapid restitution which appears as a result of remaining viable epithelial cell migration to seal the wounds and re-built the barriers (Rathor et al., 2014). What's more, the healing of damaged mucosa is highly controlled by cellular polyamines pathway (Gao et al., 2013). Thus, promoting the mucosal healing via polyamines pathway may be a possible therapeutical intervention in gut diseases. Using herbal medicine to adjust the homeostasis of the body is common practice among the East Asians. Sijunzi decoction, a classical Chinese herbal formula, is commonly used for the treatment of disorders of gastrointestinal tract with the following symptoms: indigestion, diarrhea, and loss of appetite. Our previous studies showed that Sijunzi decoction could protect against experimental stress ulcer in rats, partly through improving the contents of polyamines in gastrointestinal mucosa (Nian, 2013). Polysaccharides, the most abundant constituent and the major active ingredient in Sijunzi decoction, have been proved to have protective effects on the epithelial barriers (Gao et al., 2018). Our previous research has further revealed that SJZDP promoted cell migration after injury via the polyamine signaling pathway, in which the 8
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spermidine content in vivo.
whereas inhibition of TRPC1 expression reduced both CCE and cells migration (Rao et al., 2006). Our previous works have demonstrated that SJZDP could significantly increase the level of TRPC1 protein and reserve the inhibition of DFMO on TRPC1 protein expression during restitution, indicating that SJZDP enhances IEC-6 cells migration partly through its regulation of TRPC1 (Tu et al., 2016b). Increasing evidences have noted that STIM1 acts a vital role in the regulating SOCs activation (Huang et al., 2006). As a intracellular sensor of Ca2+, STIM1 is able to sense the decline of store [Ca2+]cyt. Then, it activates the SOCs, contributing to an increase in Ca2+ influx. In response to the damage of epithelial cells, STIM1 swiftly transfers to the plasma membrane from endoplasmic reticulum, interacts with TRPC1 channels, and then forms the complex of STIM1/TRPC1 which enhances TRPC1-mediated Ca2+ influx as well as promoting cell migration (Rao et al., 2010). The data from the current research presented that the expression of STIM1 protein was enhanced by the administration of SJZPD. Then, we further detected the immunostaining of STIM1 during restitution. The results indicated that not only did SJZDP promote the translocation of STIM1, but it also maintained the redistribution of STIM1 at the plasma membrane over a longer period of time. Moreover, treatment with SJZDP significantly increased the level of STIM1/TRPC1 complex which has been demonstrated to up-regulate TRPC1-mediated CCE. In contrast to the regulation of STIM1 on TRPC1, STIM2 is a negative regulator of SOCs, it can form heteromers with STIM1 and block STIMI1-activate store-operated Ca2+ entry (Rao et al., 2010; Soboloff et al., 2006). Our study found that SJZDP reduced the level of both STIM2 protein and STIM1/STIM2 complex. What's more, depletion of polyamines by DFMO inhibited the expressions of STIM1 protein and STIM1/TRPC1 complex, but enhanced the levels of STIM2 protein and STIM1/STIM2 complex. However, treatment with SJZDP significantly reversed the effects of DFMO on the expression of STIM1, STIM2, STIM1/TRPC1, and STIM1/STIM2 complexes. These findings suggest that SJZDP could effectively promote cell migration, via regulating the expression of Ca2+ receptor protein (STIM1, STIM2) and protein complexes (STIM1/TRPC1, STIM1/STIM2). Cell migration is a vital process throughout intestinal mucosal healing which is the critical event in early defense against undesirable luminal antigens in ulcerative mucosal injuries (Choi et al., 2016). Therefore, we further investigate the effects of SJZDP on intestinal mucosal damage in vivo. In our current study, administration of SJZDP considerably reduced the evaluation of both gross appearance and histopathological examination, suggesting that SJZDP could effectively promote the healing of intestinal mucosal injury. Other studies have demonstrated that the delay of gastrointestinal ulcer healing induced by NSAID is connected with the inhibition of epithelial cell migration, but not the cell proliferation in vivo (Penney et al., 1995). Moreover, the inhibition of indomethacin on epithelial cell migration has also been examined and confirmed in cultured IEC-6 cells in vitro (Silver et al., 2015). Thus, combined with our current work in vitro, SJZDP promotes the healing of intestinal mucosal injury partly through enhancing cell migration. Since polyamines act an important role in mucosal recovery, we further detected the contents of polyamines in small intestinal mucosa, results showed that treatment with SJZDP significantly increased the level of spermidine. These results indicate that SJZDP accelerates mucosal healing, possibly by increasing spermidine content which is an essential regulator in cell migration.
Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This work was supported by the National Natural Science Foundation of China (NO. 81173254, 81673940), Guangzhou Science Technology and Innovation Commission (NO. 201607010335), and First-class discipline construction major project of Guangzhou University of Chinese Medicine, Guangzhou University of Chinese Medicine Planning (2018) No.6. We thank “Lingnan medicial research center of Guangzhou university of Chinese medicine” for providing us with some experimental equipments during our research. References Berridge, M.J., Lipp, P., Bootman, M.D., 2000. Signal transduction. The calcium entry pas de deux. Science 287, 1604–1605. Catalioto, R.M., Maggi, C.A., Giuliani, S., 2011. Intestinal epithelial barrier dysfunction in disease and possible therapeutical interventions. Curr. Med. Chem. 18 (3), 398–426. Chiu, C.J., McArdle, A.H., Brown, R., Scott, H.J., Gurd, F.N., 1970. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch. Surg. 101, 478–483. Choi, H.J., Do, K.H., Park, J.H., Kim, J., Yu, M., Park, S.H., Moon, Y., 2016. Early epithelial restitution by nonsteroidal anti-inflammatory drug–activated gene 1 counteracts intestinal ulcerative injuries. J. Immunol. 197 (4), 1415–1424. Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., Smith, F., 1951. A colorimetric method for the determination of sugars. Nature 168, 167. Gao, B., Wang, R., Peng, Y., Li, X., 2018. Effects of a homogeneous polysaccharide from Sijunzi decoction on human intestinal microbes and short chain fatty acids in vitro. J. Ethnopharmacol. 224, 465–473. Gao, J.H., Guo, L.J., Huang, Z.Y., Rao, J.N., Tang, C.W., 2013. Roles of cellular polyamines in mucosal healing in the gastrointestinal tract. J. Physiol. Pharmacol. 64, 681–693. Huang, G.N., Zeng, W., Kim, J.Y., Yuan, J.P., Han, L., Muallem, S., Worley, P.F., 2006. STIM1 carboxyl-terminus activates native SOC, Icrac and TRPC1 channels. Nat. Cell Biol. 8 (9), 1003–1010. Jankowski, J.A., Goodlad, R.A., Wright, N.A., 1994. Maintenance of normal intestinal mucosa: function, structure, and adaptation. GUT 35 (1 Suppl), S1–S4. Liu, L., Han, L., Wong, D.Y., Yue, P.Y., Ha, W.Y., Hu, Y.H., Wang, P.X., Wong, R.N., 2005. Effects of Si-Jun-Zi decoction polysaccharides on cell migration and gene expression in wounded rat intestinal epithelial cells. Br. J. Nutr. 93, 21–29. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Nian, L.Q., The study of the effect of Yiqi Jianpi herbs on stress ulcer in rats and polyamines. Unpublised results., p. 73. 2013. Pani, B., Bollimuntha, S., Singh, B.B., 2012. The TR (i)P to Ca²⁺ signaling just got STIMy: an update on STIM1 activated TRPC channels. Front. Biosci. (Landmark edition) 17, 805. Penney, A.G., Malcontenti-Wilson, C., O'Brien, P.E., Andrews, F.J., 1995. NSAID-induced delay in gastric ulcer healing is not associated with decreased epithelial cell proliferation in rats. Digestive Dis. Sci. 40 (12), 2684–2693. Putney, J.W., 2005. Capacitative calcium entry sensing the calcium stores. J. Cell Biol. 169, 381–382. Rao, J.N., Platoshyn, O., Golovina, V.A., Liu, L., Zou, T., Marasa, B.S., Turner, D.J., Yuan, J.X.J., Wang, J.Y., 2006. TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding. Am. J. Physiol.-Gastr. L 290 (4), 782–792. Rao, J.N., Rathor, N., Zou, T., Liu, L., Xiao, L., Yu, T., Cui, Y., Wang, J.Y., 2010. STIM1 translocation to the plasma membrane enhances intestinal epithelial restitution by inducing TRPC1-mediated Ca2+ signaling after wounding. Am. J. Physiol.-Cell PH 299 (3), 579–588. Rathor, N., Chung, H.K., Wang, S.R., Wang, J.Y., Turner, D.J., Rao, J.N., 2014. Caveolin-1 enhances rapid mucosal restitution by activating TRPC1-mediated Ca2+ signaling. Physiol. Rep. 2 (11), e12193. Ray, R.M., Guo, H., Patel, M., Jin, S., Bhattacharya, S., Johnson, L.R., 2007. Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells. Am. J. Physiol.-Gastr. L 292 (4), 983–995. Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T., Horwitz, A.R., 2003. Cell migration: integrating signals from front to back. Science 302, 1704–1709. Sevag, M.G., Lackman, D.B., Smolens, J., 1938. The isolation of the components of streptococcal nucleoproteins in serologically active form. J. Biol. Chem. 124, 42–49. Soboloff, J., Spassova, M.A., Hewavitharana, T., He, L.P., Xu, W., Johnstone, L.S.,
Conclusion This study provides evidences indicating that SJZDP differentially regulates cellular levels of STIM1 and STIM2, stimulates the translocation of STIM1, and then induces STIM1/TRPC1 association as well as decreases the level of STIM1/STIM2, subsequently up-regulates TRPC1mediated Ca2+ signaling pathway and promotes cell migration after wounding. What's more, SJZDP is effective in accelerating indomethacin-induced intestinal mucosal healing, and enhancing 9
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potential. J. Chin. Med. Mater. 39, 856–862. Tu, X.H., Li, R.L., Deng, J., Zeng, D., Cai, J.Z., Chen, W.W., 2016b. Effects of Sijunzi decoction polysaccharide on polyamine mediated calcium signaling pathway during intestinal epithelial cell migration. China J. Traditional Chin. Med. Pharm. 31, 1665–1673. Xie, P.F., 2012. Clinical observation on treating gastric ulcer with the Sijunzi decoction. Clin. J. Chin. Med. 81–82. Xu, C.Z., Zhang, Y.Y., Liu, L.L., Zhu, Q.D., Tian, Q.P., Sha, J.H., 1987. A pathomorphological and histochemical study in the duodenum of spleen deficiency patients. Zhongguo Zhong Xi Yi Jie He Za Zhi 7, 722–725. Zhu, F., Qian, J.M., Pang, G.Z., 1998. The establishment of TNBS-induced experimental colitis. ACTA Academiae Medicinae Sinicae 20, 271–278.
Dziadek, M.A., Gill, D.L., 2006. STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ entry. Curr. Biol. 16 (14), 1465–1470. Silver, K., Littlejohn, A., Thomas, L., Marsh, E., Lilich, J.D., 2015. Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling. Biochem. Pharmacol. 98 (4), 614–628. Song, H.P., Hou, X.Q., Li, R.Y., Yu, R., Li, X., Zhou, S.N., Huang, H.Y., Cai, X., Zhou, C., 2017. Atractylenolide I stimulates intestinal epithelial repair through polyaminemediated Ca2+ signaling pathway. Phytomedicine 28, 27–35. Sui, J.J., Lu, W.B., Li, R.L., Wen, P., 2011. Determination of polyamines in rat intestinal epithelial cell line IEC-6 by RP-HPLC. Chin. Pharmacologyical Bull. 1309–1312. Tu, X.H., Li, R.L., Deng, J., Cai, J.Z., Wu, T.T., Chen, W.W., 2016a. Effect of Sijunzi decoction polysaccharide on IEC-6 cell migration, potassium channel and membrane
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Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Original Article
Effect of polysaccharides from Sijunzi decoction on Ca2+ related regulators during intestinal mucosal restitution
T
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Shi Yuxia1, Zhu Huibin1, Li Ruliu , Wang Dongxu, Zhu Yiping, Hu Ling, Chen Weiwen Pi-wei Institute, Guangzhou University of Chinese Medicine, 12 Jichang road, Guangzhou 510405, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sijunzi Decoction polysaccharides intestinal epithelial cells cell migration polyamine mucosal restitution
Background: Sijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been widely used for treating patients with gastrointestinal diseases and spleen-deficiency syndrome, however, the exact effects and mechanisms are remained unclear. Hypothesis/purpose: The migration of intestinal epithelial (IEC-6) cells has been demonstrated to be one of the major repair modalities during the healing of mucosal wounds. Ca2+ is the key factor in regulating cell migration. Thus, this study aimed to elucidate the potential effects and mechanisms of polysaccharides (SJZDP) extracted from Sijunzi decoction in intestinal mucosal restitution. Method: Cell migration was detected by scratch method with micropipette tip. Western blotting was adopted to evaluate the expression of STIM1 and STIM2 proteins. Immunofluorescence was carried out to assess the translocation of STIM1 protein. Immunoprecipitation was used to determine the levels of STIM1/TRPC1 and STIM1/STIM2 complexes. A indomethacin-induced intestinal mucosal injury rat model was applied. The content of polyamines in intestinal mucosa was detected by high-performance liquid chromatography. The morphological changes in the intestinal mucosa were observed at the end of animal experiment. Results: The results showed that treatment with SJZDP significantly promoted cell migration, enhanced the level of STIM1 protein and STIM1/TRPC1 complexes, reduced the level of STIM2 protein and STIM1/STIM2 complexes. Further more, SJZDP exposure promoted the translocation of STIM1 to the plasma membrane. In vivo experiment results, the administration of SJZDP effectively reduced intestinal mucosal injury. Conclusion: These results revealed that SJZDP promotes intestinal epithelial restitution after wounding, presumably by regulating cellular levels of STIM1 and STIM2 differentially, stimulating the translocation of STIM1, and inducing STIM1/TRPC1 association as well as decreasing the level of STIM1/STIM2.
Introduction Gastrointestinal mucosal injury is one of the most common histopathological features in gastrointestinal disease and spleen-deficiency syndrome which is both the organic disorders and functional changes in the theory of traditional Chinese medicine (Xu et al., 1987). After injury, gastrointestinal mucosa undergoes a rapid restitution which reseals the superficial wounds by epithelial cell migration. This early mucosal restitution is one of the major repair modalities in the gastrointestinal tract and is controlled by numerous intracellular and extracellular factors, including polyamines and cytosolic free Ca2+ ([Ca2+]cyt) (Ridley et al., 2003).
Sijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been extensively applied to the treatment of gastrointestinal diseases and spleen-deficiency syndrome. It is composed of four Chinese herbs: Panax ginseng (Renshen); Atractylodes macrocephala (Baizhu); Poria cocos (Fulin); Glycyrrhiza uralensis (Gancao), of which polysaccharides are considered the major active ingredient (Liu et al., 2005). In clinical practice, Sijunzi decoction effectively ameliorates gastrointestinal mucosal lesion, and promote the healing of peptic ulcer (Xie, 2012). Our previous studies found that Sijunzi decoction had prophylactic effect on gastric mucosal injury in rats with stress ulcer presumably by increasing polyamines content in the mucosa of stomach and small intestine (Nian, 2013). Then, we
Abbreviations: [Ca2+]cyt, cytosolic free Ca2+; CCE, capacitative Ca2+ entry; DFMO, DL-α-difluoromethylornithine; IEC-6, intestinal epithelial cells; SJZDP, Sijunzi decoction polysaccharides; SOCs, store-operated Ca2+channel; STIM1, stromal interaction molecule 1; STIM2, stromal interaction molecule 2; TRPC1, canonical transient receptor potential 1 ⁎ Corresponding author. E-mail address: [email protected] (R. Li). 1 These authors contributed to the work equally and should be regarded as first joint authors. https://doi.org/10.1016/j.phymed.2019.152880 Received 12 October 2018; Accepted 27 February 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
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Fig. 1. The HPGPC analysis of SJZDP3.
investigated the effect and mechanism of polysaccharides extracted from Sijunzi decoction (SJZDP) on promoting cell migration via regulating polyamine and [Ca2+]cyt. We found that administration of SJZDP promoted IEC-6 migration. SJZDP enhanced the contents of polyamines, then up-regulated the expression of potassium channel protein Kv1.1, associated with cell membrane hyperpolarization which could enhance the driving force for Ca2+ influx (Tu et al., 2016a). [Ca2+]cyt is the key factor in regulating epithelial cell migration. Increasing [Ca2+]cyt accelerates intestinal epithelial restitution after wounding both in vivo and in vitro (Putney, 2005). [Ca2+]cyt is regulated by extracellular Ca2+ influx via Ca2+-permeable channels and intracellular Ca2+ release from reticulum (Song et al., 2017). Canonical transient receptor potential 1 (TRPC1) acts as store-operated Ca2+channels, regulates the balance of [Ca2+]cyt, and then has influence upon epithelial restitution (Rao et al., 2006). Stromal interaction molecule 1 (STIM1) and stromal interaction molecule 2 (STIM2), the sensors of Ca2+ within the store, act essential parts in regulating TRPC1-mediated Ca2+ influx after store depletion (Pani et al., 2012). Since, Ca2+ related regulators have a great impact on cell migration, we presumed that SJZDP may promote intestinal mucosal restitution through modulating the Ca2+ related regulators. Thus, the purpose of this research was to evaluate the effects of SJZDP on intestinal mucosal restitution, and to further elucidate the potential mechanism of action in IEC-6 migration through Ca2+ related regulators pathway.
(MO, U.S.A). Rabbit antibodies against STIM2 and STIM1 were provided by Cell Signaling Technology (MA, U.S.A). Rabbit antibodies against TRPC1 and GAPDH, and Goat secondary antibody to rabbit IgG (HRP) were obtained from Abcam (MA, U.S.A)
Materials and methods
Properties of SJZDP
Herbs and reagents
Determination of the total carbohydrate and proteins Phenol-sulfuric acid assay, which was described by Dubois (Dubois et al., 1951), was conducted to determine the total carbohydrate of SJZDP with D-glucose as a standard. The protein content of SJZDP2 was detected in according with Lowry's method (Lowry et al., 1951), with bovine serum albumin used as a standard.
Preparation of SJZDP Panax ginseng, Atractylodes macrocephala, Poria cocos, and Glycyrrhiza uralensis in the ratio of 1:1:1:1 were minced and mixed, soaked in 12-times volume of double distilled water for 2 h, and then decocted for 2 h, and filtered. Subsequently, the herbs were decocted and filtered again in the same way, and then the decoction were pooled together and concentrated. The extracts were added with 4-times volume of 95% ethanol to precipitate crude polysaccharides at 4℃ overnight. Crude polysaccharides were dissolved and precipitated which was repeated in three times. Then the precipitates were dissolved and centrifugated (8000 rpm/min,15 min), solution were collected and freeze-dried to produce SJZDP1. SJZDP1 were deproteinized by the Sevag method (Sevag et al., 1938) to produce SJZDP2 which were used in vivo. 2.0164 g SJZDP2 were dissolved in 15 ml ultrapure deionized water and were loaded onto DEAE-52 cellulose anion exchange chromatography column which was washed with ultrapure water at a flow rate of 1.7 ml/min. Then, the elution from SJZDP2 were collected and freeze-dried to produce SJZDP3 which was used in vitro.
Panax ginseng (Renshen), Atractylodes macrocephala (Baizhu), Poria cocos (Fulin), and Glycyrrhiza uralensis (Gancao) were purchased from Guangzhou Tong Kang Pharmaceutical Co. Ltd and were identified by associate professor Tong JY from the Department of Chinese medicine identification in Guangzhou University of Chinese Medicine. Spermidine, spermine, putrescine, indomethacin, and DL-α-difluoromethylornithine (DFMO) were purchased from Sigma-Aldrich
High-performance gel permeation chromatograph HPGPC equipped with a Waters G 4000 PWXL column (MA, U.S.A) 2
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Fig. 2. Effect of SJZDP on cell migration in vitro. Images of IEC-6 cell migration in plates with different treatment containing DFMO (B) or not (A). (C) Quantitative analysis of migration were described by the ratio of the covered wound area to the initial scratched area. Values were presented as means ± SD (n = 15). ## p < 0.01, compared with DFMO group. **p < 0.01, compared with Control. Original magnification, ×100.
previous study (Ray et al., 2007). 5 parallel lines were drawn on the bottom of 6-well plates before seeding cells. The scratch-wound was carried out perpendicular to the marked lines using a 200 μl micropipette tip. Damaged cells in plates were washed away using PBS. Subsequently, the respective medium were treated with drugs as described. Photomicrographs of the area of cell migration were taken using an Olympus inverted phase contrast microscope (Tokyo, Japan) at each intersection of the wounded edge and the marked lines at 0 h (S0) and at 12 h (S12h) after administration of drugs. Cell migration was worked out as wound area (%) covered at 12 h in according with this formula (S12h-S0) /S¯ 0 by the Image-Pro Plus software.
was used to detected the purity of SJZDP3. Briefly, after being cleared through a 0.45 μm filter, 20 μl SJZDP3 (1 mg/ml, dissolved in ultrapure water) was applied to the column. The column and refractive index detector were maintained at a temperature of 30 ℃. The mobile phase was ultrapure water at a flow rate of 0.6 ml/min. Cell culture IEC-6 cells were provided by the American Type Culture Collection (VA, U.S.A, NO. 63139935). Cells were seeded in T-75 flasks containing of DMEM supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 5% fetal bovine serum. Flasks were maintained in a humidified atmosphere of 95% air-5% CO2 at 37 ℃. When IEC-6 cells grew to about 90% of the flask's area, cells were digested with trypsinEDTA, transferred into 6-well plates, and cultured for 24 h in the above conditions. Subsequently, cells were scratched and treated with control, spermidine (5 μmol/l, SPD), and SJZDP3 (20, 40, 80 and 160 mg/l) respectively, containing DFMO (2.5 mmol/l) or not for appropriate time.
Western-blot analysis Western-blot of protein expression STIM1, STIM2 and GAPDH in IEC-6 cells after different treatment were performed and quantified by Image Lab software. The semi-quantitative of STIM1 and STIM2 immunoblots were normalized by corresponding GAPDH bands. Immunofluorescence staining
Cell migration assays Immunofluorescence was detected according to the previous research (Rao et al., 2010) to determine the location of STIM1 in IEC-6
Measurement of IEC-6 migration were detected according to the 3
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Fig. 3. Effect of SJZDP on the expression of STIM1 and STIM2 proteins in IEC-6 cells after wounding. Representative bands of STIM1 and STIM2 in IEC-6 cells with different treatment containing DFMO (B) or not (A). (C) Semi-quantitative analysis of STIM1 and STIM2 immunoblots were normalized by corresponding GAPDH bands. Values were presented as means ± SD from three separate experiments. ##p < 0.01,compared with DFMO group. *p < 0.05 and **p < 0.01, compared with control.
group (n = 12). After 3 days acclimation period, rats in model and SJZDP groups were treated with indomethacin (6 mg/kg, i.p., dissolved in NaHCO3 solution) once daily for 6 days to induce intestinal mucosal injury, while rats in normal group received the same volume of saline. One hour before model inducing, each rats in SJZDP groups was treated with SZJDP2 (0.358 g/kg or 1.074 g/kg, dissolved in double distilled water) by intragastric administration, while rats in other groups received the same volume of double distilled water, once daily for 6 days. 24 h after the last administration of drugs, the rats were sacrificed, and the tissue samples of small intestine were collected.
cells. Immunoprecipitation assays Equal amounts of proteins from IEC-6 cells were incubated overnight with the rabbit anti-STIM1 (4 μg) at 4℃. Then, protein A + G Agarose was added and incubated for 3 h at 4 ℃. The supernatant was removed by centrifugation, while the agarose was resuspended in 50 μl 1×SDS-PAGE buffer. For immunoblotting, samples were detected by western-blot analysis, and incubated either with rabbit anti-STIM1 (1:1000), rabbit anti-STIM2 (1:000), or rabbit anti-TRPC1 (1:5000).
Gross appearance of intestinal mucosal injury Experimental protocol in vivo The gross appearance of intestinal mucosal injury was scored according to the degree of ulcer and adhesion (Zhu et al., 1998). Grading scale of ulcer: 0 point (no mucosal hyperemia, edema or ulcer); 1 point (localized hyperemia and edema; no ulcer); 2 points (mild ulcer, without obvious mucosal hyperemia or edema); 3 points (one ulcer with mucosal hyperemia and edema); 4 points (multiple ulcers with inflammation, ulcer diameter<1 cm); 5 points (multiple ulcers with inflammation, at least one of the ulcer diameters>1 cm). Grading scale of adhesion: 0 point (no adhesion); 1 point (the adhesion is light, the small intestine and other tissues can be separated by a little strength); 2 points (the adhesion is heavy).
Male Sprague-Dawley rats (SPF, approximately 180–220 g) were provided by the Animal Laboratory Center, Guangzhou University of Chinese Medicine (NO.44005800005951). Animal were housed in separated cages in the SPF laboratory animal room provided by the Animal Laboratory Center, Guangzhou University of Chinese Medicine (NO.00180626). Animal were given water and food ad libitum. All animal experiments were approved by the committee of animal ethics in Guangzhou University of Chinese Medicine. Rats were randomized into 4 groups: normal group (n = 8), model group (n = 16), SJZDP2 0.358 g/kg group (n = 12), SJZDP2 1.074 g/kg 4
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Fig. 4. Effect of SJZDP on the translocation of STIM1 protein in IEC-6 cells after wounding. Subcellular localization of STIM1 (Green) in IEC-6 cells with different treatment containing DFMO (B) or not (A). Nuclei were stained with DAPI (blue). Image (original magnification, ×630) were taken by laser confocal microscopy.
Fig. 5. Effect of SJZDP on the changes of STIM1/TRPC1 and STIM1/STIM2 complexes. Representative bands of STIM1, STIM2 and TRPC1 proteins in the complexes which were immunoprecipitated by antibody against STIM1 from IEC-6 cells with different treatment containing DFMO (B) or not (A) after wounding.
by capillary congestion; Grade 2: extension of subepithelial space with moderate edema in lamina propria, and dilated central lacteal; Grade 3: severe edema in lamina propria, epithelial cell degeneration and necrosis in intestinal mucosa, a few villis may be denuded; Grade 4: denuded villis with lamina propria and dilated capillaries exposed; Grade 5: disintegration and digestion of lamina propria, bleeding or ulceration.
Histopathological examination of intestinal mucosal injury After scoring the gross appearance of intestinal mucosal injury, the tissue samples were fixed in 10% paraformaldehyde, dehydrated in ethanol, embedded in paraffin, and sectioned. The sections were stained by H&E, observed by optical microscope. According to the previous research (Chiu et al., 1970), pathological changes in intestinal mucosa were dividing into 6 grades with some modifies: Grade 0: normal mucosal villis; Grade 1: development of subepithelial Gruenhagen'space at the apex of the villis, accompanied 5
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Fig. 6. Gross evaluation of intestinal mucosal injury in different groups. (A) Representative photographs of the intestinal mucosa in different groups. (B) Bar graphs showing the score of gross evaluation for intestinal mucosal injury. #p < 0.05 and ##p < 0.01, compared with Indomethacin group. ⁎⁎p < 0.01, compared with control.
Polyamines assay in small intestinal mucosa
Results
Pre-column derivatization reversed phase high performance liquid chromatography with Hypersil ODS2 column (Thermo Fisher Scientific, MA, U.S.A) was adopted with the polyamines (putrescine, spermidine and spermine) as indices in according with our previous works (Sui et al., 2011). The polyamines contents (μmol/mg) from mucosal samples were described by the ratio of the detected value of polyamines divided by the corresponding weight of mucosal, to the amounts of proteins.
Extraction yield and properties of SJZDP The extraction yields of SJZDP1, SJZDP2, and SJZDP3 were 8.77%, 7.16% and 4.52% respectively. And the total carbohydrate content in SJZDP1, SJZDP2, and SJZDP3 were 91.34%, 81.58%, and 91.54% respectively. The content of protein in SJZDP2 was 0.049964 mg/ml, which meant the protein was almost removed by the Sevag method. The HPGPC of SJZDP3 was determined in Fig. 1. SJZPD3 had 3 absorption peaks, suggesting that SJZDP3 was not homogeneous polysaccharides. The retention time of the max absorption peak was 20.30 min, accounted for 82.99% of the polysaccharides, indicating that the SJZDP3 was extracted with high purity.
Statistical analysis All values were expressed as means ± SD, and SPSS 21.0 software was used for analyzing. Differences in mean values was determined by one-way ANOVA or Kruskal-Wallis H test. Then, multiple comparisons between the groups were determined using Dunnett's multiple range test or Nemenyi test. p < 0.05 was accepted as statistical significance.
Effect of SJZDP on cell migration in vitro The process of cell migration after wounding was shown in Fig. 2. Treatment with SJZDP (40, 80, 160 mg/l) remarkably promoted cell migration as compared to control group (p < 0.01, Fig. 2A and C). Polyamine depletion by DFMO significantly inhibited cell migration, and the migration rate in DFMO group had decreased to 78% of control 6
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10 min after wounding without any treatments. STIM1 still located in the plasma membrane from 30 min to 3 h, and then declined at 6 h after wounding. In comparison with control group, treatment with SJZDP (80 mg/l) stimulated the translocation of STIM1 whose immunostaining in the plasma membrane was still obvious at 6 h after wounding (Fig. 4A), indicating that SJZDP could maintain the translocation of STIM1 over a longer period of time. Polyamine depletion by DFMO slowed the translocation of STIM1, whose immunostaining in the plasma membrane was also weaker than those in control groups after wounding (Fig. 4B). However, treatment with SJZDP (80 mg/l) as well as SPD (5 μmol/l) prevented the effects of DFMO on STIM1 translocation (Fig. 4B). Effect of SJZDP on the changes of STIM1/TRPC1 and STIM1/STIM2 complexes We further determine whether the level of STIM1/TRPC1 and STIM1/STIM2 complexes were affected by the treatment of SJZDP. As shown in Fig. 5, SJZDP (80 mg/l) significantly enhanced the levels of STIM1/TRPC1 association, but reduced the levels of STIM1/STIM2 complex. In contrast, polyamine depletion by DFMO decreased STIM1/ TRPC1 complex and increased STIM1/STIM2 association, and these effects of DFMO were prevented by the administration of SJZDP (80 mg/l) and SPD (5 μmol/l). Effect of SJZDP on experimental intestinal mucosal injury in rats Gross evaluation of intestinal mucosal injury The results of gross evaluation were presented in Fig. 6. There were not obvious lesions in control group, while the indomethacin group showed intense intestinal mucosal injury appeared as heavy adhesion, obvious mucosal hyperemia, and multiple ulcers (Fig. 6A). The administration of SJZDP considerably reduced intestinal mucosal damage and decreased the ulcer formation in comparison with indomethacin group (p < 0.05, Fig. 6A and B).
Fig. 7. Effect of SJZDP on histopathological lesions in indomethacin-induced intestinal injury. (A) Histopathological observation of intestinal mucosa stained with H&E. (B) The scoring of histopathological evaluation of intestinal mucosal injury. #p < 0.05 and ##p < 0.01, compared with indomethacin group. ⁎⁎p < 0.01, compared with control original magnification,×100.
Histopathological evaluation of intestinal mucosal injury As displayed in Fig. 7, there was no disruption of mucosa in control group, while the indomethacin group exhibited obvious damage to the intestinal mucosa appeared as degenerative and necrotic epithelial cells, denuded villis, disintegrative lamina propria, and ulceration. SJZDP 0.358 g/kg group presented with moderate disruption of intestinal mucosa, a much fewer denuded villis and lamina propria as compared to indomethacin group (p < 0.01, Fig. 7A and B). SJZDP 1.074 g/kg group exhibited much better histopathological appearance with almost intact epithelial cells, and thus showed protection from indomethacin-induced mucosal damage (Fig. 7A and B).
at 12 h (Fig. 2B). The inhibition of cell migration by DFMO could be almost reversed and restored to normal level by concomitant treatment with SJZDP (Fig. 2B and C), suggesting that the effect of SJZDP on cell migration is related to polyamines. Effect of SJZDP on the expression of STIM1 and STIM2 protein To investigate the underlying mechanism of SJZDP on cell migration, STIM1 and STIM2 were detected by western blot. The results showed that SJZDP (20, 40, 80 and 100 mg/l) enhanced the expression of STIM1 protein, while reduced the level of STIM2 protein, in comparison with control group (p < 0.05, Fig. 3A and C), suggesting that the SJZDP may promote cell migration by changing the expression of stromal interaction molecule. Polyamine depletion by DFMO decreased the expression of STIM1 protein, and increased the level of STIM2 (p < 0.05, Fig. 3B and C). However, the effects of DFMO on STIM1 and STIM2 protein expression were reversed by the administration of SJZDP (40, 80 and 160 mg/l) as well as SPD (5 μmol/l) (p < 0.05, Fig. 3B and C).
Contents of polyamines in small intestinal mucosa There were not significant differences of putrescine or spermine content among each group (p > 0.05, Fig. 8). However, the spermidine content in indomethacin group was significantly decreased in comparison with control group (p < 0.05), and reduced to ∼47.7% of control group. Treatment with SJZDP significantly increased the level of spermidine as compared to indomethacin group (p < 0.05, Fig. 8B). Discussion The gastrointestinal tract is lined by a monolayer of differentiated epithelial cells which constitute a key element of mucosal barriers to separate the luminal pathogens and the host (Jankowski et al., 1994). The impairment of mucosal barriers is a common pathological event which is observed in the pathogenesis of multiple gut diseases, such as NSAID-induced enteropathy, inflammatory bowel disease, and peptic ulcer (Catalioto et al., 2011). In response to injury, intestinal mucosa
Effect of SJZDP on the translocation of STIM1 protein To determine the location of STIM1 in IEC-6 cells after wounding, immunofluorescence of STIM1 was carried out. The result in Fig. 4A showed that STIM1 obviously retained in IEC-6 cytoplasm at 0 min. The translocation of STIM1 to the plasma membrane was increased within 7
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Fig. 8. Polyamines contents in small intestinal mucosa. (A) HPLC chromatogram of polyamines content from different group. (B) Quantitative analysis of spermidine were presented as bar graphs. #p < 0.05 and ##p < 0.01, compared with Indomethacin group. *p < 0.05, compared with control.
increasing [Ca2+]cyt is a critical factor (Tu et al., 2016b). Therefore, we further elucidate the potential mechanism of SJZDP in promoting IEC-6 cell migration through Ca2+ related signal pathway, in order to investigate the regulation of extracellular calcium influx by SJZDP during cell migration. Moreover, we used indomethacin to induce the rat model of intestinal mucosal injury, and further evaluate the effect of SJZDP on accelerating the restitution of mucosal damage in vivo. An increasing of [Ca2+]cyt is a important trigger for intestinal epithelial restitution. It depends on the release of intracellular calcium from endoplasmic reticulum, or the influx of extracellular Ca2+ which is the critical mechanism involved in maintaining suitable [Ca2+]cyt (Berridge et al., 2000). Ca2+ influx owing to store depletion is also known as capacitative Ca2+ entry (CCE) which is mediated through Ca2+-permeable channels termed store-operated Ca2+channel (SOCs). TRPC1 functions as a component of native SOCs. It acts an essential role in controlling CCE, in maintaining a sustained intracellular [Ca2+]cyt, and in regulating intestinal epithelial restitution. Induced TRPC1 expression enhances CCE as well as cells migration after wounding,
goes through a rapid restitution which appears as a result of remaining viable epithelial cell migration to seal the wounds and re-built the barriers (Rathor et al., 2014). What's more, the healing of damaged mucosa is highly controlled by cellular polyamines pathway (Gao et al., 2013). Thus, promoting the mucosal healing via polyamines pathway may be a possible therapeutical intervention in gut diseases. Using herbal medicine to adjust the homeostasis of the body is common practice among the East Asians. Sijunzi decoction, a classical Chinese herbal formula, is commonly used for the treatment of disorders of gastrointestinal tract with the following symptoms: indigestion, diarrhea, and loss of appetite. Our previous studies showed that Sijunzi decoction could protect against experimental stress ulcer in rats, partly through improving the contents of polyamines in gastrointestinal mucosa (Nian, 2013). Polysaccharides, the most abundant constituent and the major active ingredient in Sijunzi decoction, have been proved to have protective effects on the epithelial barriers (Gao et al., 2018). Our previous research has further revealed that SJZDP promoted cell migration after injury via the polyamine signaling pathway, in which the 8
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spermidine content in vivo.
whereas inhibition of TRPC1 expression reduced both CCE and cells migration (Rao et al., 2006). Our previous works have demonstrated that SJZDP could significantly increase the level of TRPC1 protein and reserve the inhibition of DFMO on TRPC1 protein expression during restitution, indicating that SJZDP enhances IEC-6 cells migration partly through its regulation of TRPC1 (Tu et al., 2016b). Increasing evidences have noted that STIM1 acts a vital role in the regulating SOCs activation (Huang et al., 2006). As a intracellular sensor of Ca2+, STIM1 is able to sense the decline of store [Ca2+]cyt. Then, it activates the SOCs, contributing to an increase in Ca2+ influx. In response to the damage of epithelial cells, STIM1 swiftly transfers to the plasma membrane from endoplasmic reticulum, interacts with TRPC1 channels, and then forms the complex of STIM1/TRPC1 which enhances TRPC1-mediated Ca2+ influx as well as promoting cell migration (Rao et al., 2010). The data from the current research presented that the expression of STIM1 protein was enhanced by the administration of SJZPD. Then, we further detected the immunostaining of STIM1 during restitution. The results indicated that not only did SJZDP promote the translocation of STIM1, but it also maintained the redistribution of STIM1 at the plasma membrane over a longer period of time. Moreover, treatment with SJZDP significantly increased the level of STIM1/TRPC1 complex which has been demonstrated to up-regulate TRPC1-mediated CCE. In contrast to the regulation of STIM1 on TRPC1, STIM2 is a negative regulator of SOCs, it can form heteromers with STIM1 and block STIMI1-activate store-operated Ca2+ entry (Rao et al., 2010; Soboloff et al., 2006). Our study found that SJZDP reduced the level of both STIM2 protein and STIM1/STIM2 complex. What's more, depletion of polyamines by DFMO inhibited the expressions of STIM1 protein and STIM1/TRPC1 complex, but enhanced the levels of STIM2 protein and STIM1/STIM2 complex. However, treatment with SJZDP significantly reversed the effects of DFMO on the expression of STIM1, STIM2, STIM1/TRPC1, and STIM1/STIM2 complexes. These findings suggest that SJZDP could effectively promote cell migration, via regulating the expression of Ca2+ receptor protein (STIM1, STIM2) and protein complexes (STIM1/TRPC1, STIM1/STIM2). Cell migration is a vital process throughout intestinal mucosal healing which is the critical event in early defense against undesirable luminal antigens in ulcerative mucosal injuries (Choi et al., 2016). Therefore, we further investigate the effects of SJZDP on intestinal mucosal damage in vivo. In our current study, administration of SJZDP considerably reduced the evaluation of both gross appearance and histopathological examination, suggesting that SJZDP could effectively promote the healing of intestinal mucosal injury. Other studies have demonstrated that the delay of gastrointestinal ulcer healing induced by NSAID is connected with the inhibition of epithelial cell migration, but not the cell proliferation in vivo (Penney et al., 1995). Moreover, the inhibition of indomethacin on epithelial cell migration has also been examined and confirmed in cultured IEC-6 cells in vitro (Silver et al., 2015). Thus, combined with our current work in vitro, SJZDP promotes the healing of intestinal mucosal injury partly through enhancing cell migration. Since polyamines act an important role in mucosal recovery, we further detected the contents of polyamines in small intestinal mucosa, results showed that treatment with SJZDP significantly increased the level of spermidine. These results indicate that SJZDP accelerates mucosal healing, possibly by increasing spermidine content which is an essential regulator in cell migration.
Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgments This work was supported by the National Natural Science Foundation of China (NO. 81173254, 81673940), Guangzhou Science Technology and Innovation Commission (NO. 201607010335), and First-class discipline construction major project of Guangzhou University of Chinese Medicine, Guangzhou University of Chinese Medicine Planning (2018) No.6. We thank “Lingnan medicial research center of Guangzhou university of Chinese medicine” for providing us with some experimental equipments during our research. References Berridge, M.J., Lipp, P., Bootman, M.D., 2000. Signal transduction. The calcium entry pas de deux. Science 287, 1604–1605. Catalioto, R.M., Maggi, C.A., Giuliani, S., 2011. Intestinal epithelial barrier dysfunction in disease and possible therapeutical interventions. Curr. Med. Chem. 18 (3), 398–426. Chiu, C.J., McArdle, A.H., Brown, R., Scott, H.J., Gurd, F.N., 1970. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch. Surg. 101, 478–483. Choi, H.J., Do, K.H., Park, J.H., Kim, J., Yu, M., Park, S.H., Moon, Y., 2016. Early epithelial restitution by nonsteroidal anti-inflammatory drug–activated gene 1 counteracts intestinal ulcerative injuries. J. Immunol. 197 (4), 1415–1424. Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., Smith, F., 1951. A colorimetric method for the determination of sugars. Nature 168, 167. Gao, B., Wang, R., Peng, Y., Li, X., 2018. Effects of a homogeneous polysaccharide from Sijunzi decoction on human intestinal microbes and short chain fatty acids in vitro. J. Ethnopharmacol. 224, 465–473. Gao, J.H., Guo, L.J., Huang, Z.Y., Rao, J.N., Tang, C.W., 2013. Roles of cellular polyamines in mucosal healing in the gastrointestinal tract. J. Physiol. Pharmacol. 64, 681–693. Huang, G.N., Zeng, W., Kim, J.Y., Yuan, J.P., Han, L., Muallem, S., Worley, P.F., 2006. STIM1 carboxyl-terminus activates native SOC, Icrac and TRPC1 channels. Nat. Cell Biol. 8 (9), 1003–1010. Jankowski, J.A., Goodlad, R.A., Wright, N.A., 1994. Maintenance of normal intestinal mucosa: function, structure, and adaptation. GUT 35 (1 Suppl), S1–S4. Liu, L., Han, L., Wong, D.Y., Yue, P.Y., Ha, W.Y., Hu, Y.H., Wang, P.X., Wong, R.N., 2005. Effects of Si-Jun-Zi decoction polysaccharides on cell migration and gene expression in wounded rat intestinal epithelial cells. Br. J. Nutr. 93, 21–29. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Nian, L.Q., The study of the effect of Yiqi Jianpi herbs on stress ulcer in rats and polyamines. Unpublised results., p. 73. 2013. Pani, B., Bollimuntha, S., Singh, B.B., 2012. The TR (i)P to Ca²⁺ signaling just got STIMy: an update on STIM1 activated TRPC channels. Front. Biosci. (Landmark edition) 17, 805. Penney, A.G., Malcontenti-Wilson, C., O'Brien, P.E., Andrews, F.J., 1995. NSAID-induced delay in gastric ulcer healing is not associated with decreased epithelial cell proliferation in rats. Digestive Dis. Sci. 40 (12), 2684–2693. Putney, J.W., 2005. Capacitative calcium entry sensing the calcium stores. J. Cell Biol. 169, 381–382. Rao, J.N., Platoshyn, O., Golovina, V.A., Liu, L., Zou, T., Marasa, B.S., Turner, D.J., Yuan, J.X.J., Wang, J.Y., 2006. TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding. Am. J. Physiol.-Gastr. L 290 (4), 782–792. Rao, J.N., Rathor, N., Zou, T., Liu, L., Xiao, L., Yu, T., Cui, Y., Wang, J.Y., 2010. STIM1 translocation to the plasma membrane enhances intestinal epithelial restitution by inducing TRPC1-mediated Ca2+ signaling after wounding. Am. J. Physiol.-Cell PH 299 (3), 579–588. Rathor, N., Chung, H.K., Wang, S.R., Wang, J.Y., Turner, D.J., Rao, J.N., 2014. Caveolin-1 enhances rapid mucosal restitution by activating TRPC1-mediated Ca2+ signaling. Physiol. Rep. 2 (11), e12193. Ray, R.M., Guo, H., Patel, M., Jin, S., Bhattacharya, S., Johnson, L.R., 2007. Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells. Am. J. Physiol.-Gastr. L 292 (4), 983–995. Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T., Horwitz, A.R., 2003. Cell migration: integrating signals from front to back. Science 302, 1704–1709. Sevag, M.G., Lackman, D.B., Smolens, J., 1938. The isolation of the components of streptococcal nucleoproteins in serologically active form. J. Biol. Chem. 124, 42–49. Soboloff, J., Spassova, M.A., Hewavitharana, T., He, L.P., Xu, W., Johnstone, L.S.,
Conclusion This study provides evidences indicating that SJZDP differentially regulates cellular levels of STIM1 and STIM2, stimulates the translocation of STIM1, and then induces STIM1/TRPC1 association as well as decreases the level of STIM1/STIM2, subsequently up-regulates TRPC1mediated Ca2+ signaling pathway and promotes cell migration after wounding. What's more, SJZDP is effective in accelerating indomethacin-induced intestinal mucosal healing, and enhancing 9
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potential. J. Chin. Med. Mater. 39, 856–862. Tu, X.H., Li, R.L., Deng, J., Zeng, D., Cai, J.Z., Chen, W.W., 2016b. Effects of Sijunzi decoction polysaccharide on polyamine mediated calcium signaling pathway during intestinal epithelial cell migration. China J. Traditional Chin. Med. Pharm. 31, 1665–1673. Xie, P.F., 2012. Clinical observation on treating gastric ulcer with the Sijunzi decoction. Clin. J. Chin. Med. 81–82. Xu, C.Z., Zhang, Y.Y., Liu, L.L., Zhu, Q.D., Tian, Q.P., Sha, J.H., 1987. A pathomorphological and histochemical study in the duodenum of spleen deficiency patients. Zhongguo Zhong Xi Yi Jie He Za Zhi 7, 722–725. Zhu, F., Qian, J.M., Pang, G.Z., 1998. The establishment of TNBS-induced experimental colitis. ACTA Academiae Medicinae Sinicae 20, 271–278.
Dziadek, M.A., Gill, D.L., 2006. STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ entry. Curr. Biol. 16 (14), 1465–1470. Silver, K., Littlejohn, A., Thomas, L., Marsh, E., Lilich, J.D., 2015. Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling. Biochem. Pharmacol. 98 (4), 614–628. Song, H.P., Hou, X.Q., Li, R.Y., Yu, R., Li, X., Zhou, S.N., Huang, H.Y., Cai, X., Zhou, C., 2017. Atractylenolide I stimulates intestinal epithelial repair through polyaminemediated Ca2+ signaling pathway. Phytomedicine 28, 27–35. Sui, J.J., Lu, W.B., Li, R.L., Wen, P., 2011. Determination of polyamines in rat intestinal epithelial cell line IEC-6 by RP-HPLC. Chin. Pharmacologyical Bull. 1309–1312. Tu, X.H., Li, R.L., Deng, J., Cai, J.Z., Wu, T.T., Chen, W.W., 2016a. Effect of Sijunzi decoction polysaccharide on IEC-6 cell migration, potassium channel and membrane
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