Protective Effect And Mechanisms Of Kushenlu On Lipopolysaccharide-induced Small Intestinal Inflammation In Rats

The herb Kushen is the dried root of leguminous plant Sophora flavescens Ait.In Traditional Chinese medicine(TCM),its property is classified as cold,with the effect of heat clearing and damp eliminating.Kushen is commonly used in TCM to treat enteritis,dysentery and other diseases.Distillation product from the herb(known as Yaolu in TCM)has been widely used in TCM clinics since the Qing Dynasty.In this study,we distilled Kushen to make Kushenlu(KSL)which is one kind of Yaolu.The purpose of this study was to evaluate the protective effect of KSL on lipopolysaccharide(LPS)-induced small intestinal inflammation.The tests were conducted on animal model and cells model.Method:Animal test:24 Sprague-Dawley rats were randomly divided into 4 groups(n=6 per group):control,LPS,LPS+H2O,LPS+KSL.Rats were placed into the fog box for 30 minutes,daily for five days.The control and LPS group received no ultrasonic spray inhalation,whereas the LPS+H2O and LPS+KSL group received spray inhalation with 80mL distilled water and 80mL KSL respectively,while in the fog box.On the fifth day,the control group rats received an intraperitoneal injection of normal saline;the other group received an intraperitoneal injection of 10mg/kg LPS solution.Seven hours after injection,all rats were sacrificed by decapitation,blood and proximal jejunum samples were collected from each rat.The protective effect of KSL on LPS-induced small intestinal inflammation were evaluated by assessing its body wight change rate,concentrations of inflammatory factors in serum,blood cell counts and expression of inflammation related proteins in small intestine tissue.Cell test:Rat small intestinal epithelial cells(IEC-6)and rat small intestine microvascular endothelial cells(RIMVEC)were used as model cells,after different concentrations of KSL and LPS administration,the concentrations of inflammatory factors in supernatant and the expression of inflammatation related proteins in cells were determined by ELISA and Western Blot.Results:(1)Rat models of small intestinal infalmmation can be successfully obtain through intraperitoneal injection of 10mg/kg LPS solution;(2)Ultrasonic spray inhalation of KSL can significantly relieved diarrhea,decreased concentration on important blood parameters(RBC,HGB,WBC,PLT),increased concentration in inflammatory factors in serum(IL-1??IL-6?TNF-?),increased expression of TLR4,degradation of i?B? and increased phosphorylation of p65 in small intestine tissues;(3)Pretreatment of KSL inhibited the activation and transduction of NF-?B and MAPKs signaling pathway in IEC-6 and RIMVEC cells by down-regulating of TLR4,MyD88 and TRIF;directly inhibited the activation of NF-?B by reduction of p65 phosphorylation and I?B? degradation;directly inhibited the activation of MAPKs signaling pathway by reduction of ERK,JNK,p38 phosphorylation of.Through inhibition of TLR4-NF-?B-MAPKs pathways activation,KSL inhibits production of downstream proinflammatory cytokines(IL-1??IL-6?TNF-?)in both cells which is caused by LPS.Conclusion:KSL can effectively relieve LPS-induced small intestinal inflammation in rats.This effect was in part achieved by inhibition of TLR4,NF-?B and MAPKs signaling pathway activation in epithelium and capillary endothelium,which inhibits production of downstream proinflammatory cytokines.