Protective Effect Of Ursolic Acid On Hepatocytes In The Development Of Liver Fibrosis In Rat And Its Potential Mechanism

Background:Recent studies indicate that NADPH oxidase(NOX)plays a key role in the development of various tissue and organ fibrosis.It's a new important target to develop anti-fibrosis drugs.Once NOX in hepatic stellate cells(HSCs)was activated,a large number of reactive oxygen species(ROS)can be produced.ROS can mediate the transduction of pro-fibrotic signals in HSCs and gradually lead to liver fibrosis.It's worthy of attention that ROS derived from NOX promotes HSCs proliferation,transformation and survival.However,it induces apoptosis in hepatocytes and aggravates liver damage.Our previous studies showed that Ursolic Acid(UA)resisted liver fibrosis in an unique way.It could selectively induce apoptosis of activated HSCs(HSC-T6)and inhibit their proliferation in vitro,but did not cause apoptosis of hepatocytes.In contrast,UA promoted hepatocyte proliferation.We also observed that UA could block lipid peroxidation,decrease fibrous tissue,and protect the damaged hepatocytes against apoptosis and necrosis in vivo,which significantly improved the tissue structure of the fibrotic liver in rats.However,the molecular mechanism of how UA protects hepatocytes in the development of liver fibrosis is so far unclear.We propose the hypothesis that UA would inhibit hepatocyte apoptosis and promote hepatocyte regeneration by inhibiting NOX activity and reducing ROS generation in hepatocytes,so as to protect hepatocytes.In this research,we'll use TGF-?1 as the pro-fibrotic factor to study the influence of UA intervention on NOX activity and ROS generation in primary hepatocytes from SD rats,and its relationship with proliferation and apoptosis of hepatocytes.Oobjective:To observe the influence of UA intervention on hepatocyte proliferation and apoptosis,and the influence of UA intervention on NOX activation and ROS generation induced by TGF-?1 in primary hepatocytes,finally clarify the possible mechanism that UA ptotects hepatocytes in the development of liver fibrosis.Methods Extract primary hepatocytes by situ perfusion from male healthy SD rats,culture 12-24 hours until the cells have grown as adherent cords,islands,more than80% of the total area,randomly divide the cells into the following groups: blank control group,UA control group(25?M/L),TGF-?1 group(2.5ng/ml),UA intervention group(TGF-?1 2.5ng/ml treated along with UA 25 ?M/L),DPI intervention group(TGF-?1 2.5ng/ml treated along with DPI 0.5?M/L),Rosup positive control group(10u M/L).Primary hepatocytes were treated with drugs for30 min and expression of protein p47 phox was analyzed with Western Blotting.Primary hepatocytes were treated with drugs for 1h and ROS generation was detected with reactive oxygen detection kits.Primary hepatocytes were treated with drugs for 6h and CD95 m RNA expression was analyzed with q RT-PCR.Primary hepatocytes were treated with drugs for 12 h and hepatocyte proliferation and apoptosis were analyzed with Flow Cytometry,expression of protein CD95 was analyzed with Western Blotting.Results:1.The influence of UA intervention on proliferation and apoptosis of primary hepatocytes treated by TGF-?11.1 The influence of UA intervention on proliferation of primary hepatocytes treated by TGF-?1Primary hepatocytes were treated with TGF-?1 alone for 12 hours,and the proliferation index was obviously lower compared to blank control group(P<0.01);The data of UA control group was markedly increased than blank control group(P<0.01);the data of UA intervention group was notably higher than TGF-?1 group(P<0.01),and had no significant difference with DPI intervention group(P>0.05).The results showed that UA intervention weakened the inhibition of TGF-?1 on hepatocyte proliferation,in contrast,UA intervention promoted hepatocyte regeneration.1.2 The influence of UA intervention on apoptosis of primary hepatocytes induced by TGF-?1Primary hepatocytes were treated with TGF-?1 alone for 12 hours,and the apoptosis rate was obviously increased compared to blank control group(P<0.01);The data of UA control group was markedly lower than blank control group(P<0.01);the data of UA intervention group was obviously lower than TGF-?1 group(P<0.01),and had no significant difference with DPI intervention group(P>0.05).The results showed that UA intervention reduced the apoptosis of primary hepatocytes induced by TGF-?1.1.3 The influence of UA intervention on CD95 m RNA expression induced by TGF-?1 in primary hepatocytes Primary hepatocytes were treated with TGF-?1 alone for 6 hours,and the expression of CD95 m RNA was markedly increased compared to blank control group(P<0.01);The data of UA control group was notably lower than blank control group(P<0.01);the data of UA intervention group was markedly lower than TGF-?1 group(P<0.01),and had no significant difference with DPI intervention group(P>0.05).The results showed that UA intervention restrained the CD95 m RNA expression induced by TGF-?1 in primary hepatocytes.1.4 The influence of UA intervention on expression of protein CD95 induced by TGF-?1 in primary hepatocytes Primary hepatocytes were treated with TGF-?1 alone for 12 hours,and the expression of protein CD95 was markedly increased compared to blank control group(P<0.01);The data of UA control group was lower than blank control group(P<0.05);the data of UA intervention group was lower than TGF-?1 group(P<0.05),and had no significant difference with DPI intervention group(P>0.05).The results showed that UA intervention restrained the expression of protein CD95 induced by TGF-?1 in primary hepatocytes.2.The influence of UA intervention on NOX subunit p47 phox membrane translocation induced by TGF-?1 in primary hepatocytes Primary hepatocytes were treated with TGF-?1 alone for 30 minutes,and p47 phox subunits that have translocated to cell membrane were notably increased than blank control group(P<0.01);The data of UA control group was less than blank control group(P<0.05);the data of UA intervention group was markedly lower than TGF-?1group(P<0.01),and had no significant difference with DPI intervention group(P>0.05).The results showed that UA intervention restrained the membrane translocation of subunit p47 phox induced by TGF-?1 so as to inhibit NOX in primary hepatocytes.3.The influence of UA intervention on ROS generation induced by TGF-?1 in primary hepatocytes Primary hepatocytes were treated with TGF-?1 alone for 1 hour,and the DCF fluorescence intensity was notably higher than blank control group(P<0.01),and had no significant difference compared to Rosup positive group(P>0.05);The DCF fluorescence intensity of UA control group was lower than blank control group(P<0.05);the data of UA intervention group was markedly lower than TGF-?1 group(P<0.01),and had no significant difference compared to DPI intervention group(P>0.05).The results showed that UA intervention restrained the generation of ROS in primary hepatocytes induced by TGF-?1.Conclusion:1.TGF-?1 might induce hepatocyte apoptosis by up-regulating the expression of CD95(Fas)after it activated NOX in hepatocytes and caused ROS generation.2.The mechanism that UA protected hepatocytes was likely to be that UA intervention reduced hepatocyte apoptosis and promoted hepatocyte proliferation by inhibiting generation of ROS from NOX and expression of CD95 in primary hepatocytes induced by TGF-?1.