The Effect Mechanism Of Total Saponins Of Astragalus And Glycyrrhizic Acid On Liver Fibrosis

Objective The purpose of this study is to analyze the synergistic mechanism of anti-liver fibrosis with astragaloside and glycyrrhizic acid based on TGF-?/Smads signaling pathway and pro-inflammatory factor/NF-?B signaling pathway.Methods(1)Hepatic fibrosis/cirrhosis rat models were induced by 0.5%DMN for a total of 4 weeks.From the third week of modeling,AS,GA,AS/GA and HQD were used for intervention.The synergistic effects of AS and GA on anti-fibrosis were mainly analyzed by serum liver function,liver tissue hydroxyproline content(Hyp)and liver histopathological(H&E and sirius red stain).Immunohistochemical staining,immunofluorescence staining,qRT-PCR and Western Blot were used to reveal the expressionof inflammatory genes(NF?B1,NF?B2,NF?Bp65,IL-6,CCL2),fibrosis-related genes(?-SMA,TGF-?1,Col-I,Col-IV,MMP2,MMP9,TIMP1,TIMP2)and BMP And Activin Membrane Bound Inhibitor(BAMBI).(2)LX-2 and h HSCs were stimulated with 100 ng/ml LPS in the absence or presence of AS,GA,AS/GA or BAY11-117082(NF-?B inhibitor)for 1h or 4h.The expression of NF?Bp65,TNF-? and BAMBI were detected by Western blot or qRT-PCR.LX-2 and h HSCswere stimulated by 5ng/ml h TGF-?1 in the absence or presence of AS,GA,AS/GA or SB431542(T?RI inhibitor)for 1h or 24 h.Expression of ?-SMA,Col-I and Smad2/3 were determined by qRT-PCR and Western blot.Also LX-2 or h HSCs were co-stimulated with both LPS and h TGF-?1 in the absence or presence of AS,GA or AS/GA for 1h or 24 h,The expression of ?-SMA,Col-?,Smad2/3 and NF?Bp65 were assessed by Western blot analysis.Results(1)Animal experiments showed that AS,GA,AS/GA and HQD significantly restrained the progress of DMN-induced rat hepatic fibrosis and inflammation.Serum ALT,AST and liver hydroxyproline content in AS/GA group were lower than those in AS group and GA group,and statistically different from GA group(P<0.05).The m RNA expression of pro-fibrotic factors TGF-?1,?-SMA,Col-I,Col-IV,TIMP1/2,MMP2/9 and inflammatory factors NF?B1,NF?B2,CCL2 and IL-6 were decreased after each drug intervention,among which the expression of TGF-?1,Col-?,TIMP1/2 and MMP2/9 m RNA were lower in AS/GA group than in GA group(P<0.05 or P<0.01).The AS/GA group showed significantly decreased expression of ?-SMA m RNA and protein,which was statistically different from eitherthe AS group or the GA group(P<0.05).The expression of NF?B2,CCL2 and IL6 m RNA and phospho-NF?Bp65 protein in the AS/GA group were significantly lower than those in the AS and GA groups(P<0.05,P<0.01 or P<0.001).The expression of BAMBI was decreased in the model group compared with the normal group(P<0.01),and the expression of BAMBI was significantly increased after drug intervention(P<0.05,P<0.01 or P<0.001),among which the AS/GA group was more significant(P<0.001).(2)Compared with the control h HSCs,TNF-? m RNA levels were significantly increased while BAMBI m RNA and protein levels were significantly decreased in the LPS-treated h HSCs(P<0.05).The LPS-induced high expression of TNF-? and low expression of BAMBI were significantly reversed by the presence of AS,GA,AS/GA and BAY11-7082(P<0.05,P<0.01 or P<0.001);The effects of AS,GA,AS/GA on enhancing BAMBI expression were also demonstrated in LPS-treated LX-2 cells.In both h HSCs and LX-2 cells,AS and GA either alone or in combination significantly reduced TGF-?1-induced Smad2/3 phosphorylation,but the combined effect was more significant;AS,AS/GA and SB431542 significantly reduced TGF-?1-induced expression of ?-SMA and Col-I(P<0.05,P<0.01,P<0.001).Under the co-stimulation of h HSCs or LX-2 by LPS and TGF-?1,it was observed that the inhibition of Smad2/3 phosphorylation and NF?Bp65 phosphorylation by AS/GA combination was more obvious than the use of each alone.Conclusion Huangqi Decoction and its effective components AS and GA can reduce liver tissue inflammation and collagen deposition,through inhibiting TGF-?/Smads signaling pathway and NF-?B signaling pathway combined with up-regulating BAMBI expression in hepatic stellate cells,therefore attenuate liver fibrosis,with combination use of AS and GA having synergistic effect that is better than each alone.