Based On The Effect Of LSEC On HSC, The Mechanism Of The Anti-hepatic Fibrosis Effect Of The Chemical Component F-03 Of Fuzheng Huayu Recipe Was Analyzed

Background and objective:Previous studies found that Fuzheng Huayu Recipe(FZHY)can effectively inhibit liver fibrosis and reduce the generation of pathological angiogenesis.Based on the screening of the main chemical constituents of four-stage dr?gs,F-01,F-02 and F-03 were found to be the most exposed in extracts,portal vein,liver and whole blood.Based on the above results,the effects of F-01,F-02 and F-03 in inhibiting the dedifferentiation of liver sinusoidal endothelial cell(LSEC)and activation of hepatic stellate cell(HSC)were evaluated.The chemical component F-03 which inhibits the dedifferentiation of LSEC was screened out.The mechanism of F-03 anti-hepatic fibrosis in inhibiting the dedifferentiation of LSEC,and the mechanism of inhibiting the activation of HSC through inhibiting the dedifferentiation of LSEC was further explored.Method:(1)To study the effects of chemical constituents F-01,F-02 and F-03 of FZHY Recipe on the dedifferentiation of LSEC:After intervention with FZHY recipe and its chemical constituents F-01,F-02 and F-03 respectively for 24 hours,the proliferation of human hepatic sinusoidal endothelial cell(HHSEC),AML12 and L02 cells was detected by CCK8 method;HHSEC was induced by 100 ?M COCl2,and FZHY recipe and its chemical constituents F-01,F-02 and F-03 were given respectively.RT-PCR and Western blot were used to detect the expression of CD31 and CD44 in HHSEC and mouse primary liver sinusoidal endothelial cell(MP-LSEC),and immunofluorescence was used to detect the fluorescence expression of CD44 and chemokine receptor 4(CXCR4).(2)To study the effects of chemical constituents F-01,F-02 and F-03 of FZHY Recipe on HSC activation:LX-2 and JS1 cells were activated by 5ng/ml TGF-?1.After 24 hours of intervention with FZHY recipe and its chemical constituents F-01,F-02 and F-03 respectively,RT-PCR and Western blot were used to detect the expression levels of LX-2 and JS1 cell activation indicators(?-SMA and COL-?).The fluorescence expression of ?-SMA and COL-? was detected by immunofluorescence method.(3)In HHSEC-LX-2 co-culture system,the pharmacodynamic differences of F-02 and F-03 in inhibiting HHSEC dedifferentiation and affecting LX-2 activation by inhibiting HHSEC dedifferentiation were observed.The expression levels of CD31,CD44,?-SMA and COL-? were detected by RT-PCR,and the fluorescence expressions of ?-SMA and COL-? were detected by immunofluorescence.(4)HHSEC-LX-2 co-culture system was used to observe the effect of F-03 on HHSEC dedifferentiation and its effect on HSC activation by inhibiting HHSEC dedifferentiation.HHSEC was dedifferentiated by 100 ?M CoCl2 and intervened by F-03,YC-1,AMD3100,sh-CXCR4 gene silencing and STI 571,respectively.CD31,CD44 and HIF-1a/CXCR4/PDGF/CXCR7 signal pathway indices(HIF-1a,SDF-1,CXCR4,PDGF,PDGF?R,CXCR7)were detected by RT-PCR and Western blot.The expression of PDGF?R,?-SMA and COL-? in LX-2 cells were detected by by RT-PCR and Western blot,The fluorescence expression of a-SMA and COL-? in LX-2 cells were detected by immunofluorescence.(5)Observation of the anti-hepatic fibrosis effect of F-03 in experimental animal model:?The rat hepatic fibrosis model was prepared by subcutaneous injection of 50%CCL4-olive oil solution 1 ml/kg body weight twice a week;from the seventh week,F-03 was given to intervene with FZHY and Sorafenib as positive control.At the end of the ninth week,the rats were sacrificed.Normal group and model group were given 0.3%CMC-Na by gavage.?The liver fibrosis model of BDL rats was randomly divided into three groups:sham-operated group,model group and F-03 drug group.The rats were given F-03 intervention at the second week of modeling,and the rats were sacrificed at the end of 4 weeks.The general conditions of rats were observed and recorded,including body weight,liver weight,spleen weight,liver-body ratio and spleen-body ratio changes;?Serum ALT,AST and GGT were detected;hepatic inflammation was detected by hematoxylin-eosin(H&E)staining;collagen fibers in liver tissue were detected by Sirius red(SR)staining,and collagen fibers area ratio was semi-quantitatively analyzed;hydroxyproline(Hyp)in liver tissue was determined by alkali hydrolysis method.The expression of ?-SMA,COL-?,CD31,CD44,CXCR4,CXCR7 and PDGF was detected by RT-PCR or Western blot,and the expression of a-SMA,COL-? and CD31 was detected by immunohistochemistry.Result:(1)FZHY recipe and its chemical constituents F-01,F-02 and F-03 can significantly inhibit the dedifferentiation of HHSEC cells and MP-LSEC cells.Among them,FZHY recipe has the best effect at 25 ?g/ml,F-01 at 50 ?M,F-02 at 10 ?M and F-03 at 2 ?M.(2)FZHY recipe and its chemical constituents F-01,F-02 and F-03 can significantly inhibit the activation of LX-2 cells and JS1 cells.Among them,the best concentration is 25 ?g/ml in FZHY recipe,5 ?M in F-01,10?M in F-02 and 2 u?M in F-03.(3)In HHSEC-LX-2 co-culture system,compared with the normal control group,the mRNA expression of CD31 was significantly increased,and the mRNA expression of CD44 was significantly decreased in HHSEC cells in Transwell inferior chamber of model group,while the expression of ?-SMA and COL-? in LX-2 cells in Transwell superior chamber was significantly increased.Compared with the model group,both 10 ?M F-02 and 2 ?M F-03 could significantly reduce the mRNA expression of CD31 and increase the expression of CD44 in dedifferentiated HHSEC cells,Compared with 10 ?M F-02,2 ?M F-03 could significantly reduce the mRNA expression of CD31.Both of them can significantly reduce the mRNA expression of COL-? in activated LX-2 cells,and the expression effect of 2 ?M F-03 is better,the difference is statistically significant;only 2 ?M F-03 can significantly reduce the mRNA expression of ?-SMA.(4)In the HHSEC-LX-2 co-culture system,compared with the normal control group,the mRNA expression of CD31,HIF-1a,CXCR4 and PDGF of HHSEC cells in Transwell inferior chamber was significantly increased in model group,while the expression of CD44 and CXCR7 was significantly decreased.The expression of a-SMA,COL-? and PDGF?R of LX-2 cells in Transwell superior chamber was significantly increased.Compared with the model group,the mRNA expression of CD31,HIF-1?,CXCR4,and PDGF were significantly decreased,and the mRNA expression of CD44,CXCR7 were significantly increased in HHSEC cells in Transwell inferior chamber,while the expression of ?-SMA,COL-? and PDG?R in LX-2 cells in Transwell superior chamber was significantly decreased,and the effect was not significantly different from that of YC-1,AMD3100 and sh-CXCR4 gene silencing and STI 571 intervention.(5)In CC14 and BDL rat models,compared with the normal control group,the activity of serum ALT,AST and GGT increased significantly in the model group;pathological staining showed necrosis of hepatocytes,infiltration of inflammatory cells,proliferation of collagen fibers,formation of fibrous septum;semi-quantitative increase of Hyp content and Sirius red staining,increased expression of hepatic fibrosis-related indicators ?-SMA and COL-?;hepatic angiogenesis indicators CD31,CXCR4 increased significantly.The expressions of PDGF and CD44 and CXCR7 were significantly increased,while the expressions of CD44 and CXCR7 were significantly decreased.Compared with the model group,F-03 group can significantly reduce serum ALT and AST activity;F-03 group can significantly reduce the expression of ?-SMA,COL-I Hyp content and collagen deposition;significantly reduce the expression of CD31,CXCR4,PDGF,and significantly increase the expression of CD44,CXCR7.Conclusion:(1)Upregulation of HIF-1? expression in LSEC promotes the expression of CXCR4.Overexpression of CXCR4 promotes the expression and secretion of PDGF.On the one hand,PDGF binds with PDGF?R on HHSEC to inhibit the expression of CXCR7 and promote the dedifferentiation of HHSEC;on the other hand,PDGF secreted by HHSEC binds with PDGF?R on HSC to promote the activation of HSC.It is s?ggested that the HIF-1?/CXCR4/PDGF/CXCR7 axis plays an important role in the dedifferentiation of LSEC and the relationship between LSEC and HSC,providing a new target pathway for anti-hepatic fibrosis.(2)F-03 has good anti-hepatic fibrosis effect.Its mechanism is related to alleviating inflammation,reducing collagen deposition and LSEC dedifferentiation.(3)F-03 inhibits the dedifferentiation of LSEC by inhibiting the activity of HIF-1?/SDF-1/CXCR4 signal,and promotes the expression of CXCR7.Furthermore,F-03 inhibits the activation of HSC by inhibiting the secretion of PDGF by LSEC,and further reduces the binding of PDGF to PDGF?R of HSC.These findings provide some scientific basis for the clinical application and development of FZHY recipe and the further development of new anti-fibrosis drugs.