The Function And Mechanism Of Zisheng Decoction On Slow Transit Constipation

Objective:(1)To investigate the multi-component,multi-target and multi-pathway mechanism of Zisheng Decoction in the treatment of STC by TCM network pharmacology.(2)To determine the effect of Zisheng Decoction on fecal water content and small intestinal propulsion rate of loperamide induced STC mice;3)To investigate the effect of Zisheng Decoction on colon cajal cell apoptosis induced by loperamide in STC mice.The key targets and pathways of network pharmacology were verified.(4)To investigate the effect of Zisheng Decoction serum on primary cajal cell apoptosis induced by TNF α and verify the pathway.(5)To explore the mechanism of Zisheng Decoction in treating slow transit constipation by improving intestinal flora.Research methods:(1)Collect active ingredients and action targets of Shengtang by using TCMSP and BATMAN databases,and explore constipation related targets by using DisGenet,TTD,OMIM and other databases.The intersection targets were analyzed by GO and KEGG enrichment analysis,and cytoscape was used to construct the drugdisease-target-pathway network.(2)Male ICR mice were randomly divided into:Blank(Ctrl)group,model(Lop)group,Low dose ZSD-L group,medium dose ZSD-M group,high dose ZSD-H group,Prucapride(Pru)group,linalotide(Lina)group,STC mouse model was prepared by subcutaneous injection of loperamide suspension.The laxative effect of Zisheng Decoction was evaluated by general observation,fecal moisture content,small intestine propulsion rate and colonic electromyography.The colonic neurotransmitters SP,Ach,5-HT,NO and VIP were detected by ELISA.(3)The colonic physiological structure of mice was observed by HE staining;The expression of cajal cells in mouse colon was evaluated by IHC.The apoptosis of cajal cells was evaluated by transmission electron microscopy and WB.The expression of AKT/NFkB p65/IKBKB pathway was verified by Western Blot.(4)Primary cajal cells were extracted from the colon of neonatal mice and identified by immunofluorescence method.The apoptosis of cajal cells was induced by TNF α,and the cells were divided into blank(Ctrl)group,model(TNF α)group,ZSD-L group,ZSD-M group and ZSD-H group.After treatment,CCK-8 method was used to detect cell viability.The expressions of BAX,BCL2,caspase 3 and AKT/NFkB p65/IKBKB pathways in ICC cells were detected by WB after TNF-αinduction.(5)Colon contents of mice were sequenced by 16sRNA.Results:(1)The network pharmacological results showed that there were 71 intersection targets between the active ingredients of Zisheng Decoction and constipation.Advanced functional analysis showed that the mechanism of Zisheng Decoction in treating constipation was related to viral interaction,cell apoptosis,ion transport,biological rhythm,etc.The key targets of TNF,TGFB,CASP,IKBKB,NFkB and p53 were obtained by PPI network analysis.(2)①The results of in vivo experiments showed that mice in Lop group were listless and tended to curl up,and fecal water content and small intestine propulsion rate were significantly lower than those in Ctrl group(p<0.05).Compared with Lop group,fecal water content in groups ZSD-M,ZSD-H and Lina was significantly increased(P<0.05);The small intestinal propulsion rate in ZSD-H and Pru groups was increased(P<0.05).(2)The colonic electromyography showed that compared with Ctrl group,the colonic slow wave frequency and amplitude of mice in Lop group slowed down.Compared with Lop,ZSD-M group has higher amplitude and more regular frequency.The colonic emg of ZSD-H group tended to Ctrl group.(3)Western Blot showed that the protein expressions of AKT,p-AKT,IKBKB and p-IKBKB in colon tissue of mice Lop group were decreased.The expression of p65 and p p65 protein was increased.Compared with Lop group,the protein expressions of AKT,P-Akt,IKBKB and P-ikbKB in colon tissue of ZSD-M and ZSD-H groups were increased,while the protein expressions of p65 and p p65 were decreased(P<0.05).(3)The results of HE staining showed that the colonic muscle layer was thinner and goblet cells were decreased in Lop group compared with Ctrl group.After ZSD-H intervention,the colonic structural integrity was restored as before ②IHC results showed that the expressions of SCF and c-Kit protein in colonic tissues of mice in Lop group were decreased compared with those in Ctrl group(P<0.05).Compared with Lop group,expressions of SCF and cKit protein in colon tissues of mice in ZSD-M and ZSD-H groups were increased(P<0.05).(3)Transmission electron microscopy(TEM)showed that ICC cells in Ctrl group showed long spindle or star type,large nuclei and mature Golgi bodies.Ultrastructure of ICC in colon of mice in Lop group was damaged,with abnormal nuclear morphology and partial dissolution of cytoplasm.Occasional apoptotic bodies were observed in group ZSD-H.ICC mitochondrial swelling was not obvious,apoptotic body structure was not obvious and protrusion was normal.④The results of WB showed that c-kit and SCF were decreased and the expressions of BCL2,BAX and caspase 3 were increased in the colon tissue of mice in Lop group(P<0.05).Compared with Lop group,c-kit and SCF of colon tissue in ZSD-and ZSD-H groups were significantly increased,while BCL2 and BAX were decreased(P<0.05).(4)In vivo experiments showed that apoptosis of ICC cells was observed after TNF-α intervention.WB results showed that compared with TNF-α group,the expressions of BCL2,BAX and caspase 3 in ZSD-L,ZSD-M and ZSDH groups were decreased(P<0.05).(5)The αand β diversity analysis of sequencing results showed that the intestinal microbial community diversity of STC mice was significantly lower than that of NC group,and these changes were restored after ZSD intervention.Meanwhile,LEFSe analysis also showed that ZSD had an up-regulation effect on Bacteroidetes,Firmicutes,Lactobacillus and other beneficial bacteria.Conclusion:(1)ZSD can improve STC by promoting intestinal motility,increasing fecal water content and affecting colonic neurotransmitters.(2)Both Zisheng Decoction and Zisheng Decoction medicated serum can inhibit ICC apoptosis,and the mechanism may be related to the fact that Zisheng decoction and Zisheng Decoction medicated serum can inhibit cajal apoptosis by regulating AKT/P65/IKBKB pathway,and thus play a role in the treatment of STC.(3)ZSD can improve STC by up-regulating bacteroidetes,Firmicutes,Lactobacillus and other beneficial bacteria.