Regulatory Effect Of Flavonoids From Spatholobus Suberectus Dunn On Escherichia Coli Sepsis

Objective:Sepsis,one of the most serious infectious disease in the clinic,is threating human life and animal health.The aim of this study was to investigate the regulatory effect of Total Flavonoids of Spatholobus suberctus Dunn(TFSD)and its main component,Formononetin(FMN),on Escherichia coli(E.coli)sepsis,and to analysize its molecular mechanism.Methods:90 healthy mice were randomly divided into 9 groups:Control group,E.coli group,Colistin group(positive control),TFSD 25 group,TFSD 50group,TFSD 100 group,FMN 25 group,FMN 50 group and FMN 100 group,with5 male and 5 female mice in each group.After adapting cultured for 3 days,mice were intragastrically administered with 0.2 m L/g.bw of normal saline,TFSD,FMN or intraperitoneal injected with colistin according to experimental design for 7consecutive days.At the evening of the 7th day,mice in Control group were intraperitoneally injected with normal saline of 0.01 m L/g,and mice in other groups were intraperitoneally injected with 0.01 m L/g.bw E.coli at a concentration of 1.0×10~7 CFU/m L.12 hours later,the clinical manifestations such as spirit,hair,breathing and eyelid secretion were observed,and the pathological changes of liver,spleen,thymus,kidney and lung were observed from pathological sections.The changes of blood routine indices were detected,and the levels of serum biochemical indices,inflammatory factors and immunoglobulins were analyzed.The expression levels of TLR2,TLR4,My D88,CD14,IL-6,i NOS and other related genes in liver tissues were also analyzed.Results:(1)12 h after E.coli injection,the mice in E.coli group were depressed,with rough hair,red eyes,increased eye secretions and shortness of breath.Some mice developed convulsions and shock.TFSD and FMN pretreatment relieved the above clinical manifestations.(2)Comparing to Control group,the liver index of mice in E.coli group was significantly increased,while the spleen and thymus indices drametically decreased(P<0.05).In general observation,liver swelling,thymus and spleen atrophy were observed in mice with E.coli sepsis.Pathological sections with HE staining showed that the microstructures of liver,spleen,kidney,lung and thymus of mice with E.coli infection were greatly altered,while TFSD and FMN pretreatment could alleviate the above changes in E.coli infected mice.(3)The number of lymphocytes(LMN),total white blood cells(WBC),red blood cell count(RBC),platelet count(PLT),platelet crit(PCT)and hemoglobin(HGB)in the blood of mice with E.coli sepsis decreased sharply(P<0.05)comparing to those of mice in Control group.The levels of WBC,LMN,Mon,Gran,RBC,PLT and PCT in TFSD and FMN pretreated mice were significantly higher than those in mice with E.coli sepsis(P<0.05).(4)The AST and ALT levels of E.coli group were significantly higher than those of the Control group(P<0.05),while those in the colistin group,TFSD and FMN pretreated groups were decreased.(5)After E.coli infection,the levels of IL-6 and TNF-αin the serum were significantly increased(P<0.05),and the levels of IFN-γ,IL-2 and IL-10 were greatly decreased(P<0.05).TFSD and FMN treatment inhibited E.coli induced changes in cytokine secretions.(6)The levels of Ig A,Ig M and Ig G in the serum of E.coli group were significantly lower than those in the control group(P<0.05),and these changes were inhibited by TFSD and FMN pretreatment.(7)Compared with Control group,the expression levels of TLR4,i NOS,IL-6 and CD14 m RNA in the liver tissue of E.coli group were significantly increased(P<0.05).Both TFSD and FMN pretreatments were able to inhibited the overexpression of TLR4,i NOS,My D88,IL-6 and CD14.Conclusion:The flavonoids of Spatholobus suberectus Dunn was able to alleviate the clinical pathological manifestations and tissue damages in mice with E.coli sepsis,regulate the secretion of inflammatory factors and immunoglobulin,and inhibit the sepsis induced expression of TLR4,i NOS,IL-6,CD14 and My D88genes in liver tissue.These results suggested that the flavonoids of Spatholobus suberectus Dunn might protect the host body against E.coli sepsis induced inflammation and immunosuppression by inhibiting the activation of TLR4/My D88/NF-κB signaling pathway.Formononetin may be the main active ingredient in the regulation of E.coli sepsis in the flavonoids of Spatholobus suberectus Dunn.