基于Notch/eNOS信号通路探讨加味桃核承气汤改善肝窦毛细血管化的作用机制OA

Objective:To investigate whether modified Taohe Chengqitang(JTCT)treats liver fibrosis by inhibiting hepatic sinusoidal capillarization,and to verify whether its specific mechanism is related to the Homologous genes responsible for the notches along the edges of Drosophila wings(Notch)signaling pathway and endothelial nitric oxide synthase(eNOS)signaling pathway.Methods:Fifty C57BL/6 mice were randomized into 5 groups:Control,model(5 mL·kg-1·3 d-1),low-dose JTCT,medium-dose JTCT,and high-dose JTCT(JTCT-L,JTCT-M,JTCT-H).Except the control group,the other groups were intraperitoneally injected with 20%carbon tetrachloride(CCl4)olive oil solution every 3 days for 4 weeks to induce liver fibrosis.Meanwhile,JTCT-L,JTCT-M,and JTCT-H groups were administrated with JTCT by gavage at doses of 16.6,33.3,and 66.6 g·kg-1·d-1,respectively,for 4 consecutive weeks(the control and model groups received equal volumes of normal saline by gavage).Primary liver sinusoidal endothelial cells(LSECs)were isolated from rat livers through in situ perfusion of collagenase,Percoll density gradient centrifugation,and selective removal of Kupffer cells.After verification of cell phenotype,LSECs were used for experiments.For cells at the logarithmic growth phase,the small interfering RNA(siRNA)kit was used to knock down the expression of soluble guanylate cyclase(sGC)through transient transfection.Subsequently,the Notch agonist Jagged-1 was applied.Cell counting kit-8(CCK-8)was used to examine the cytotoxicity of JTCT on LSECs.Real-time PCR and Western blot were employed to measure the expression of molecules in the Notch and eNOS signaling pathways at mRNA and protein levels,respectively.Intracellular Ca2+was labeled with the fluorescent probe Fluo-4 acetoxymethyl ester(Fluo-4/AM).Results:The animal experiment showed that compared with the control group,the model group exhibited hepatic steatosis,inflammatory cell infiltration,hepatocyte degeneration and necrosis,lobular structural disorder,and fibrous tissue hyperplasia in the mouse liver tissue,significantly elevated serum levels of hydroxyproline(Hyp),alanine transaminase(ALT),and aspartate transaminase(AST)(P<0.01),significantly upregulated expression levels of Notch1,Hes family bHLH transcription factor 1(Hes1),Cluster of Differentiation 31(CD31),and von Willebrand factor(vWF)(P<0.01),and significantly downregulated phosphorylate(p)-eNOS/eNOS ratio and sGC expression(P<0.01).Compared with the model group,all the JTCT groups showed significantly reduced serum Hyp,ALT,and AST levels(P<0.01),downregulated expression of Notch1,Hes1,CD31,and vWF(P<0.05,P<0.01),and upregulated p-eNOS/eNOS ratio and sGC expression(P<0.05,P<0.01)in the liver tissue,with the JTCT-H group showing the most significant changes(P<0.01).The cell experiment showed that compared with the LSEC-1day group,the LSEC-5day group exhibited significantly increased expression of Notch1,Hes1,CD31,and vWF(P<0.01),significantly decreased p-eNOS/eNOS ratio and sGC expression(P<0.01),and elevated intracellular Ca2+concentration(P<0.01).Compared with the LSEC-5day group,the JTCT group showed decreased expression of Notch1,Hes1,CD31,and vWF(P<0.01),significantly increased p-eNOS/eNOS ratio(P<0.01),and significantly reduced intracellular Ca2+concentration(P<0.01).No significant differences were observed in the expression of Notch1,Hes1,eNOS,p-eNOS,CD31,vWF,sGC,or intracellular Ca2+concentration between the Jagged1 group and the Jagged1+JTCT group.Compared with the LSEC-5day group,the sGC knockout group(sGC-KO)showed significantly increased expression of CD31 and vWF(P<0.01).There was no significant difference in CD31 or vWF expression between the sGC-KO group and the sGC-KO+JTCT group.Conclusion:JTCT inhibits the activation of the Notch signaling pathway and restores the activity of the eNOS signaling pathway to suppress sinusoidal capillarization of LSECs,thereby treating liver fibrosis.