Effects Of Rhizoma Paridis Of Yunnan On Sepsis With Intestinal Dysfunction And Expression Of HMGB1

Part One.Effect of Rhizoma Paridis of yunnan total saponin on lipopolysaccharide induced intestinal epithelial barrier dysfunction of human colon carcinoma cellsObjective:To observe the effect of lipopolysaccharide.(LPS)induced intestinal epithelial barrier dysfunction of human colon carcinoma cells by Rhizoma Paridis of yunnan total saponin(RPTS).Methods:Caco-2 cells were plated on transwells at a density of 5×105/ml(500?l/well),and randomly divided into 5 groups(n=12):Control group,LPS group,RPTS group,RPTS+LPS group,Ethyl pyruvate(EP)+LPS group.When the transepithelial electrical resistance(TEER)>500 ?·cm2,intestinal epithelial cells monolayer model construct succeed.Cells were cultured in the medium containing 100 ?g/ml LPS in groups LPS,RPTS+LPS and EP+LPS,groups RPTS+LPS and RPTS was given 1 ?g/ml RPTS,and group EP+LPS was given 50 ?g/ml EP.TEER were measured at Oh,6h,12h,24h and permeability coefficient of FITC(PF)were measured at 24h.High mobility group protein B1(HMGB1)expression the level of mRNA and protein were measured by RT-PCR and Western blot at 6h,12h,24h.Results:?The inverted microscope showed that the cells in LPS group decreased significantly,with uneven distribution,irregular morphology,weakened refraction,and cloudy medium with floating cells.In RPTS+LPS group,the cells was more than LPS group,with more regular morphology,less floating cells,better refractive and clearer medium.There were no significant difference between RPTS group,EP+LPS group and Control group.?Compared with Control group,TEER were decreased in LPS group at 6h,12h and 24h(P<0.05);compared with LPS group,TEER in groups of RPTS,RPTS+LPS and EP+LPS were increased(P<0.05).?After 24h,compared with Control group,PF was increased significantly in LPS group(P<0.005);compared with LPS group,PF in groups of RPTS,RPTS+LPS and EP+LPS were decreased(P<0.05).?Compared with Control group,the expression of HMGB1 mRNA and protein were increased significantly in LPS group at 6h,12h and 24h(P<0.005).Compared with LPS group,the express of HMGB1 mRNA and protein were decreased significantly in groups RPTS+LPS,RPTS and EP+LPS at 6h,12h and 24h(P<0.005).? In LPS group,there were negative correlation between the expression of HMGB1 and TEER at 6h,12h and 24h(P<0.005).Conclusion:RPTS may improve the permeability and integrity of intestinal mucosal barrier by down-regulating the expression of HMGB1 in the model of human colon carcinoma cells,which led to inhibit the progression of intestinal barrier dysfunction.Therefore,RPTS plays an important role in the protection of intestinal mucosal.Part TWO.Effects of Rhizoma Paridis of yunnan down-regulation on the expression of HMGB1 and intestinal dysfunction in rats with sepsisObjectives:To explore the intestinal function protection of Rhizoma Paridis of yunnan and expression of HMGB1 in rats with sepsis induced by Lipopolysaccharide(LPS).Methods:150 SD-rats were randomly divided into 5 groups(n=30):sham group,model group,Low,Middle and High therapy groups(treated with 20mg/kg.po,40mg/kg.po,80mg/kg.po of Rhizoma Paridis of yunnan respectively).The sepsis model was established by LPS lmg/kg.iv,the sham group was given the same dosage of nomal saline.Continuous observation the behavioral changes in rats with sepsis.The concentration of WBC,ALT,CK-MB,Cre and Lac in blood of rats were detected by chemical laboratory method after 7d.The intestine propulsion of rats was assessed at 1d,3d,7d,14d.The alterations in intestinal were assessed by HE staining and histopathological score at Id,3d,7d,14d.The DAO of plasma and HMGB1 protein of intestinal were determined by ELISA at 1d,3d,7d,14d.Results:?Compared with control group,the mortality rates and intestinal propulsion rates were increased in model group,but the intestinal propulsion rates were decreased in the Low,Middle and High therapy groups were decreased(P<0.05).Compared model group,the mortality rates and intestinal propulsion rates in the Low,Middle and High therapy groups were decreased(P<0.05).?Compared with control group,the content of ALT,CK-MB and Cre were increased in model group,and WBC was decreased(P<0.05),but there was no significant difference in Lac(P>0.05).Compared with model group,the content of ALT,CK-MB and Cre were decreased in the Low,Middle and High therapy groups,and WBC was increased(P<0.05),but there was no significant difference in Lac(P>0.05).?According to HE staining of intestinal,the histopathological score who>? were evaluated.Compared with control group,the histopathological score who>? was increased significantly in model group(P<0.005).Compared with model group,the histopathological score who>? were decreased significantly in the Low,Middle and High therapy groups(P<0.005).? Compared with control group,the DAO of plasma and the expression of HMGB1 protein of intestinal were increased in model group(P<0.05).Compared with model group,the DAO of plasma and the expression of HMGB1 protein of intestinal were decreased in the Low,Middle and High therapy groups(P<0.05).? In model group,there was a positive correlation between the expression of HMGB1 protein of intestinal and DAO of plasma at 1d,3d,7d and 14d(P<0.005).Conclusions:Rhizoma Paridis of yunnan can down-regulating the expression of HMGB1 which have intestinal function protection.