Sennoside A Inhibits The Clostridia To Increase The Melanosis Coli Risk

Research Background and PurposeAnthraquinones and Chinese herbal preparations containing anthraquinones are widely used clinically and is under a high frequency of use.Especially in obese and refractory constipation patients,long-term use of anthraquinone laxatives is very common.But long-term use of anthraquinone laxatives leads to colonic injury and colon melanosis,and increases even carcinogenic risk.The exact mechanism of anthraquinone laxatives inducing colon melanosis is elusive and needs to be further studied.Gut microbiota as a direct contact with the intestinal microenvironment is closely involved in tumor and metabolic diseases.Therefore,we choose the representative anthraquinone compound sennoside A to study its effect and the underlying mechanisms on colon melanosis and carcinogenesis from the perspective of intestinal flora.This study provides experimental evidence and reference for effective clinical use avoiding the side effects of anthracene Quinone laxatives.Research Contents and ResultsIn this study,C57BL/6 mice were divided into control group and administration group.The administration group was given 100 mg/kg sennoside A for 12 weeks.By assessing the body weight of the mice,we found that long-term administration of sennoside A significantly inhibited weight gain in mice.At the same time,through the analysis of H&E staining,lipofusin staining and melanin staining of the administration group and control group colon tissues,we found that the senoside A caused colonic tissue damage and caused the deposition of lipid and melanin in the colon tissue.The profile of IL-1β,IL-6,IL-10 and TNF-α mRNA was measured by extraction of colonic tissue mRNA.The results showed that long-term administration of sennoside A caused mild inflammation in colon.The apoptosis level of colonic tissue was detected by TUNEL method.The results showed that the apoptosis rate of colon tissue was increased after long-term administration of sennoside A.At the same time,the proliferation in colonic tissue was observed by Ki67 immunofluorescence.The results showed that the concentration of sennoside A increased the proliferation of colonic fossa.These results suggest that long-term administration of sennoside A can increase the cell apoptosis and produce pigment deposition,at the same time increase potential carcinogenic risk.Considering the phenomenon of colonic cell apoptosis induced by sennoside A,the effect of sennoside A on normal colonic epithelial cells was analyzed by MTT assay in vitro.The results showed that sennoside A had no effect on normal colonic epithelial cells in vitro significant inhibitory effect.As the active form of sennoside A in vivo is rhein anthraquinone produced by intestinal microflora from theβ-glucosidase metabolism,we co-incubated β-glucosidase and sennoside A with normal human colon epithelial cells and determined cell viability by MTT assay.MTT assay showed that sennoside A metabolites on normal human colonic epithelial cells also did not significantly inhibit the cells.This proves that the sennoside A-induced colon melanosis coli requires the participation of other biological substances in the body.Considering that the gut microbiota is a microenvironment having direct contact with colonic tissue and the action of sennoside A depends on the intestinal flora,we further examined the effect of sennoside A on the gut microbiota of mice.The results showed that sennoside A could inhibit the production of intestinal bacteria by high-throughput sequencing analysis.The butyric acid bacteria mainly synthesize the short-chain fatty acids necessary for the growth of colon cells,so we further detected changes in intestinal short-chain fatty acids.Using GC/MS to detect the concentration of short chain fatty acids in mice,it demonstrated that sennoside A had a significant inhibitory effect on butyric acid.The results showed that exogenous administration of butyric acid could significantly inhibit the apoptosis of epithelial cells induced by sennoside A by Ki67 immunohistochemistry staining and TUNEL assay.The above results showed that sennoside A inhibits colonic epithelial cell apoptosis and colonic melanosis by inhibiting the intestinal bacteria producing butyric acid in mice.Further by fecal transplantation method,we verified the intestinal flora played an important role in this process.The results showed that fecal transplantation could effectively inhibit the injury of colon tissue.It was found that fecal transplantation significantly up-regulated the relative abundance of butyric acid bacteria in the intestine of the mice,and elevated the concentration of butyric acid in the intestinal of the mice.Conclusion and SignificanceBased on the above results,we found that sennoside A through the inhibition of intestinal production of butyric acid and intestinal butyric acid concentration led to colonic epithelial cell apoptosis and melanosis coli,while causing increased proliferation of colonic fossa site potential carcinogenesis risk.Using enema to give butyric acid or fecal transplantation surgery can effectively inhibit damage on colon tissue caused by sennoside A.This research from the intestinal flora perspective clarifies the long-term use of sennoside A caused colonic injury,the potential risk of cancer-causing mechanism,and found that fecal transplantation can effectively inhibit the adverse reactions of sennoside A,so as to provide a reference for effectively avoid adverse reactions from long-term use of sennoside A or anthraquinone laxatives.