| Liver fibrosis is a dynamic and progressive process of excessive deposition of extracellular matrix,which is the result of a combination of multiple chronic liver diseases.Hepatic stellate cells activate and proliferate,leading to unbalanced synthesis and degradation of extracellular matrix and ultimately to liver fibrosis HBYYRJ is composed of nine herbs,such as Artemisia annua lucidum,Anemarrhena,rhubarb and so on.It has good curative effect on the treatment of chronic liver disease and hepatic fibrosis.Objective: To investigate the effect of HBYYRJ extracted by butanol on liver fibrosis and to reavel the mechanism of anti-fibrosis effect through Nrf2/HO-1 signaling pathway,providing the theoretical basis for the clinical application of HBRJYY in the treatment of liver fibrosis.Method:(1)The proliferation of LX-2 cells was determined by MTT assay to select the best best inhibitory effect on LX-2 cells.To detected the activity of lactate LDH colorimetry.To detected the content of hydroxyproline in the supernatant by enzyme digestion.To measure the level of α-SMA and collagen I protein.(2)LX-2 cells were cultured in vitro and divided into control group,model group, middle dose group and high dose group of HBRJYY,ATRA group and ATRA+ HBRJYY high dose group.To measure the level of Nrf2,HO-1,α-SMA and collagen I proteins by western blot.To measure the level of Nrf2 in nuclear by western blot.Results:(1)Compared with the control group,TGF-β1 stimulation group significantly stimulated the proliferation of LX-2 cells,P<0.05.The MTT results showed that after pretreatment of cells with HBYYRJ,the proliferation of LX-2 cells was significantly inhibited compared with TGF-β1 stimulation group,and the concentration of 1mg/mL of HBYYRJ extracted by butanol has the most significant inhibitory effect on LX-2 proliferation.(2)Compared with the control group,TGF-β1 significantly stimulated Hypsecretion in LX-2 cells,P<0.05.After pretreatment with different dose of HBRJYY extracted by butabol,the Hyp content was significantly decreased, and with the increase of HBYYRJ concentration,the content of Hyp decreased more significantly.Compared with the control group,there was no significant change in LDH activity in the HBYYRJ group of each concentration,P>0.05, indicating that HBYYRJ are not toxic to LX-2 cells.(3)Compared with the control group,the level of collagen I and α-SMA in TGF-β1 stimulation group increased significantly,P<0.05.After pretreatment with different dose of HBRJYY extracted by butabol can significantly down-regulated the level of collagen I and α-SMA protein,indicating that HBYYRJ extracted by butanol can inhibit fibrogenesis by inhibiting the production of extracellular matrix and activation of hepatic stellate cells.(4)Compared with TGF-β1 stimulation group,the expression of Nrf2 and HO-1 were significantly up-regulated in LX-2 total protein and nuclear protein in the middle and high doses of HBYYRJ group,P<0.05.Compared with high dose group of HBYYRJ,inhibition of Nrf2/HO-1 signaling pathway can reverse the decrease of α-SMA and collagen I protein level.Conclusions:(1)HBYYRJ extracted by butanol can inhibit the proliferation of LX-2 cells stimulated by TGF-β1,and it could effectively inhibit the secretion of collagen by LX-2,reduce the activation of LX-2,and inhibit the activation of liver fibrogenesis.(2)HBYYRJ extracted by butanol promotes Nrf2 translocation into the nucleus, and up-regulates the Nrf2/HO-1 signaling pathway,produces anti-oxidative stress,and subsequently reduces LX-2 proliferation and activation,and reduces collagen secretion eventually inhibits liver fibrogenesis. |
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