| Objective:The therapeutic effect on sepsis rats treated by Huang Long Decoction and Meropenem were detected by routine blood test,pathological characteristic in rats’colon and the level of diamine oxidase(DAO),fatty acid-binding protein(FABP),endotoxin,procalcitonin(PCT).The effect on intestinal mucosal immuno-barrier of sepsis rats were detected by T lymphocyte subsets in ileal mucosa of rats.The effect on intestinal epithelial cell mechanical barrier were detected by the level of Claudin,Occludin mRNA and protein in ileal of rats.Method:Fifty-five Wistar rats were randomly divided into sham operation group(n=10),model group(n=15),western medicine treatment group(15 rats)and combined traditional Chinese and western medicine group(n=15).In sepsis model group,cecal ligation with puncture(CLP)was reproduced.In the sham operation group,the steps of operation performed were similar to those in the model group.but without CLP.Normal saline 2ml was given to sham operation group and model group by garage;meropenem 50 mg was given to the western medicine treatment group by intra—peritoneal injection;in the combined traditional Chinese and western medicine treatment group,besides meropenem 50 mg was injected intra—peritoneally,2 mL Huanglong decoction(containing raw herbal medicine 0.68 mL)was administered by gavage[ingredients of the decoction:rhubarb 15 g(put into water the latest),Glauber salt 10 g,fructus aurantii immaturus 15 g,mangnoliae officinalis 10 g,ginseng 10g,Angelica sinensis 20 g,Scutellaria baicalensis 15 g,Radix Glycyrrhizae 10 g,Jujube 12g,concentrated to a decoction 150 ml],in the four groups,the respective drugs or normal saline were given twice a day.After 4 days.Samples were collected.Diamine oxidase(DAO),fatty acid-binding protein(FABP),endotoxin,procalcitonin(PCT)in serum were detected by ELISA.Routine blood test were detected by five hematology analyzer for small animals.Pathological characteristic in rats’colon were detected HE staining.T lymphocyte subsets in ileal mucosa were detected flow cytometry.and mRNA expression levels of Claudin-1 and Occludin were detected by reverse transcription-polymerase chain reaction(RT-PCR),and protein expression levels of Claudin-1 and Occludin were detected by Western Blot.Results:1.The level of diamine oxidase(DAO)in serum of model group got a statistically significant higher than blank group(P<0.01).Meropenem group and Combination group got a statistically significant lower level than model group(P<0.01).Combination group got a statistically significant lower level than Meropenem group(P<0.01).2.The level of fatty acid-binding protein(FABP)in serum of model group got a statistically significant higher than blank group(P<0.01).Meropenem group and Combination group got a statistically significant lower level than model group(P<0.01).Combination group got a statistically significant lower level than Meropenem group(P<0.01).3.Pathology:The intestinal epithelium in blank group were intacted,no inflammatory cell infiltration in submucosa.There were focal epithelium degeneration,dropped and disorder of villus arrangement in model group,dense inflammatory cell infiltration in submucosa,vascular congestion,expansion,lymph tissue hyperplasia,inflammatory cell infiltration and even necrosis.Meropenem group got a less inflammatory cell infiltration in submucosa.There were orderly arrangement of villus in Combination group,occasional inflammatory cell infiltration in submucosa.4.Routine blood test:There were statistically significant higher level of WBC and neutrophils in all 3 oprated groups(model group,Meropenem group and combination group)than blank group(P<0.01).The 2 control groups(Meropenem group and combination group)got a lower level than model group and combination group also got a statistically significant lower level than Meropenem group(P<0.01).5.The model rat peripheral blood endotoxin were significantly increased compared withcontrol group(P<0.01).Meropenem group and combined Chinese medicine and western medicine group were significantly lower than the model group(P<0.01),the combined Chinese medicine and western medicine group was lower than the western medicine group(P<0.01).6.The level of procalcitonin(PCT)in serum of model group got a statistically significant higher than blank group(P<0.01).Meropenem group and combination group got a statistically significant lower level than model group(P<0.01).Combination group got a statistically significant lower level than Meropenem group(P<0.01).7.The mortality in model group was significantly higher than that in sham operation group(P<0.05).Meropenem group and combination group were significantly lower than the model group((P<0.05).8.T lymphocyte subsets:The percentage of CD4+T、CD8+T and the ratio of CD4+T/CD8+T in ileal mucosa of model group got a statistically significant lower than blank group(P<0.01).Meropenem group and combination group got a statistically significant higher level than model group(P<0.01 or 0.05).9.The level of Claudin-1 mRNA in ileal of all 3 oprated groups(model group,Meropenem group and combination group)got a statistically significant lower than blank group(P<0.01).The 2 control groups(Meropenem group and combination group)got a higher level than model group and combination group got a statistically significant lower level(P<0.01 or0.05).Combination group also got a statistically significant lower level than Meropenem group(P<0.05).10.The level of Occludin mRNA in ileal of all 3 oprated groups(model group,Meropenem group and combination group)got a statistically significant lower than blank group(P<0.01).The 2 control groups(Meropenem group and combination group)got a higher level than model group and combination group got a statistically significant higher level(P<0.01 or0.05).Combination group also got a statistically significant higher level than Meropenem group(P<0.05).11.The level of Claudin-1 proteins in ileal of all 3 oprated groups(model group,Meropenem group and combination group)got a statistically significant lower than blank group(P<0.01).The 2 control groups(Meropenem group and combination group)got a higher level than model group and combination group got a statistically significant higher level(P<0.01 or0.05).Combination group also got a statistically significant highr level than Meropenem group(P<0.05).12.The level of Occludin mRNA in ileal of all 3 oprated groups(model group,Meropenem group and combination group)got a statistically significant lower than blank group(P<0.01).The 2 control groups(Meropenem group and combination group)got a higher level than model group and combination group got a statistically significant higher level(P<0.O1 or0.05).Combination group also got a statistically significant higher level than Meropenem group(P<0.05).Conclusion:1.The content of serum diamine oxidase and intestinal fatty acid binding protein in septic rats increased significantly,which may be due to the destruction of intestinal mucosa epithelial cells release a large amount of diamine oxidase and intestinal fatty acid binding protein into blood.2.The combination of Huang Long Decoction and Meropenem got a significant lower level of diamine oxidase(DAO)and fatty acid-binding protein(FABP)in serum of rats in sepsis.The combination of Huang Long Decoction and Meropenem also relieved the injury of intestinal mucosal inflammation and protected the Intestinal villus epithelium and mucosal barrier through the analyzing of pathology.3.There were higher levels of WBC,neutrophils,endotoxin and procalcitonin(PCT)in model group and the mortality rate was significantly higher,which indecated the successfully rebuilding of sepsis and severe infection.4.The combination of Huang Long Decoction and Meropenem got a significant effect on infection of sepsis in experiment,which got a lower level of WBC,neutrophils,endotoxin and procalcitonin(PCT)in serum of rats in sepsis.The combined treatment group reduce the mortality rate of rats,The combination got a better effect than Meropenem alone.5.There were disorder of T lymphocyte subsets in sepsis rats which caused the decreased of percentage of CD4~+T、CD8~+T and the ratio of CD4~+T/CD8~+T,which may be one of the mechanisms of the hypofunction of intestinal mucosal immunity.6.The combination of Huang Long Decoction and Meropenem got a regulation effect on the intestinal mucosal immunity by increasing the percentage of CD4~+T、CD8~+T and the ratio of CD4~+T/CD8~+T.7.The level o of Claudin-1 and Occludin mRNA and protein expression in ileal tissue of sepsis rats decreased significantly,which may be one of the mechanisms of the hypofunction of intestinal mucosal barrier in sepsis.8.Huang Long Decoction increased the expression of Claudin、Occludin mRNA and proteins in ileal tissue of septic rats,which may be one of the mechanisms of Huang longtang on the protective effect of intestinal mucosal barrier in septic rats. |
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