Role of Enteric Glial Cells in Gastric Motility in Diabetic Rats at Different Stages

Summary： Diabetes patients tend to have the gastrointestinal motility disorder. Although the relationship between the motility disorder and both the neurons and Cajal cells in the enteric nervous system （ENS） is well established, little is known about the role of enteric glial cells （EGCs） in gastric motility in diabetes. This study aimed to examine the expression of the glial marker S100B and morphology of EGCs in gastric tissues and the relationship between activated EGCs and the damage of gastric emptying in diabetic models. The diabetic model of rat was induced with 1% streptozotocin （STZ）. The model rats at 7-14 days and at 56-63 days were defined as early diabetic rats and advanced diabetic rats, re- spectively, and normal rats at the two time periods served as their corresponding controls. The gastric emptying rate of the rats was tested by using the phenol red solution. The ultrastructure of EGCs in the gastric antrum was observed by the transmission electron microscopy, and the expression of S100B in the myenteric plexus was immunohistochemically detected. The results showed that the gastric emptying rate was significantly increased in the early diabetic rats and decreased in the advanced diabetic rats when compared with their corresponding control rats （P〈0.01 for both）. The ultrastructure of EGCs was mostly normal in both the early diabetic and control groups. Vacuolization of mitochondria and expan- sion of endoplasmic reticulum occurred in both the advanced diabetic group and its control group, and even the structure of smooth muscle cells and intestinal neurons was destroyed in the advanced diabetic group. The expression level of S100B in the advanced diabetic group was significantly decreased com- pared with its control group （P〈0.05）. It was obviously increased in the early diabetic control group when compared with the advanced diabetic control group （P〈0.05）. However, there was no significant difference in the S 100B expression between the early diabetic group and its control group （P〉0.05）. The findings suggested that the gastric motility dysfunction in diabetes may be associated with the changes of morphology and number of EGCs in the myenteric plexus.

Effect of modified Wendan Decoction on enteric glia cells in depression model rats

Background:To explore the effect of modified Wendan Decoction on expression of enteric glial cells in depression model rats.Methods:Eighteen rats were randomly divided into the Blank group,Model group,and modified Wendan Decoction group(Decoction group).Depression was induced by isolation combined with chronic unpredictable mild stress in rats of all groups except for the Blank group.Changes of protein and mRNA expressions of the specific markers of enteric glial cells,glial fibrillary acidic protein,and S100 calcium-binding proteinβsubunit(S100β)in the stomach and colon tissues of rats in each group were detected respectively using immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR).Results:Compared with the Blank group,the percentage of positive cells and the mRNA expression of GFAP and S100βwere increased in the Model group,and the difference was statistically significant(P<0.05);compared with the Model group,the percentage of positive cells and the mRNA expression of GFAP and S100βin gastrointestinal tissues of rats were decreased in the Decoction group,and the difference was statistically significant(P<0.05).Conclusion:The depression model rats developed hyperplasia,and enteric glial cells(EGCs)was activated.The mechanism of action of the Wendan Decoction on improving depression and promoting gastrointestinal motility may be achieved by inhibiting the hyperplasia and activation of EGCs in rats with depression.

Ji-Chuan decoction ameliorates slow transit constipation via regulation of intestinal glial cell apoptosis

BACKGROUND Slow transit constipation(STC)is a common intestinal disease with increasing incidence.STC results from various factors,such as the enteric nervous system and metabolic changes.As a classical formula of traditional Chinese medicine,Ji-Chuan decoction(JCD)has been extensively and effectively used in STC treatment,yet its pharmacological mechanism remains unclear.AIM To explore the integrated regulatory pattern of JCD against STC through hyphenated techniques from metabolism,network pharmacology and molecular methods.METHODS STC model mice were generated by intragastric administration of compound diphenoxylate(10 mg/kg/d)for 14 d.The STC mice in the low dose of JCD(3.04 g/kg),middle dose of JCD(6.08 g/kg)and high dose of JCD(12.16 g/kg)groups were orally administered JCD solution once a day for 2 wk.The acetylcholine(ACH)level was examined by enzyme-linked immunosorbent assay.The pathological features of colon tissue were observed by hematoxylin and eosin staining.The differentially expressed metabolites and metabolic pathways were tested by nontargeted metabolomics.The main targets and core ingredients of JCD were identified by network pharmacology,and the expression of AKT was confirmed by immunohistochemistry.Finally,the pathways involved in JCD treatment were predicted using a combination of differentially expressed metabolites and targets,and intestinal glial cell apoptosis was demonstrated by immunofluorescence.RESULTS JCD significantly promoted intestinal motility,increased the levels of the excitatory neurotransmitter ACH and reduced intestinal inflammation in STC mice.Untargeted metabolomics results showed that JCD significantly restored metabolic dysfunction and significantly affected taurine and hypotaurine metabolism.Network pharmacology and molecular experiments showed that JCD regulates AKT protein expression,and the core component is quercetin.Combined analysis demonstrated that apoptosis may be an important mechanism by which JCD relieves constipation.Further experiments showed that JCD reduced enteric glial cell(EGC)apoptosis.CONCLUSION This work demonstrated that reducing EGC apoptosis may be the critical mechanism by which JCD treats STC.These findings call for further molecular research to facilitate the clinical application of JCD.

Electroacupuncture Alleviates Functional Constipation in Mice by Activating Enteric Glial Cell Autophagy via PI3K/AKT/mTOR Signaling

Objective: To investigate autophagy-related mechanisms of electroacupuncture(EA) action in improving gastrointestinal motility in mice with functional constipation(FC). Methods: According to a random number table, the Kunming mice were divided into the normal control, FC and EA groups in Experiment Ⅰ. The autophagy inhibitor 3-methyladenine(3-MA) was used to observe whether it antagonized the effects of EA in Experiment Ⅱ. An FC model was established by diphenoxylate gavage. Then the mice were treated with EA stimulation at Tianshu(ST 25) and Shangjuxu(ST 37) acupoints. The first black stool defecation time, the number, weight, and water content of 8-h feces, and intestinal transit rate were used to assess intestinal transit. Colonic tissues underwent histopathological assessment, and the expressions of autophagy markers microtubuleassociated protein 1 light chain 3(LC3) and Beclin-1 were detected by immunohistochemical staining. The expressions of phosphoinositide 3-kinases(PI3K)-protein kinase B(AKT)-mammalian target of rapamycin(m TOR) signaling pathway members were investigated by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. The relationship between enteric glial cells(EGCs) and autophagy was observed by confocal immunofluorescence microscopy, localization analysis, and electron microscopy. Results: EA treatment shortened the first black stool defecation time, increased the number, weight, and water content of 8-h feces, and improved the intestinal transit rate in FC mice(P<0.01). In terms of a putative autophagy mechanism, EA treatment promoted the expressions of LC3 and Beclin-1 proteins in the colonic tissue of FC mice(P<0.05), with glial fibrillary acidic protein(GFAP) and LC3 significantly colocalized. Furthermore, EA promoted colonic autophagy in FC mice by inhibiting PI3K/AKT/m TOR signaling(P<0.05 or P<0.01). The positive effect of EA on intestinal motility in FC mice was blocked by 3-MA. Conclusion: EA treatment can inhibit PI3K/AKT/m TOR signaling in the colonic tissues of FC mice, thereby promoting EGCs autophagy to improve intestinal motility.