Effects Of Ursolic Acid On Hedgehog Signaling Pathway In Rat Hepatic Stellate Cells

Background：All causes of chronic liver injury can cause of liver fibrosis and even developinto cirrhosis。The transition of quiescent hepatic cells （Q-HSCs）to Muscle intofibrous hepatic stellate cells（MF-HSCs）is pivotal in the pathogenesis of cirrhosis.Studies of injured adult human and rodent livers demonstrate that injury-relatedactivation of the Hedgehog pathway modulates several aspects of repair, including theactivation and proliferation of Muscle into fibrous hepatic stellate cells. The literaturesuggests that the expression of hedgehog signaling pathway related proteins wereup-regulated when hepatic stellate cells were activated and proliferated.Theseproteins including hedgehog ligand Sonic hedgehog, Desert hedgehog, Indianhedgehog, the receptor protein Patched, transmembrane protein Smoothened and zincfinger transcription factor Glioblastoma1/2.The expression of Hh ligand interactionprotein which is another hedgehog signal pathway related protein is down-regulated.Recent study found that NOX subunit Ras-related C3botulinum toxin substrate1active the hedgehog signal pathway, the result is the Q-HSC activation, proliferationand transformation。Previously, we performed the study that Ursolic Acid (UA) couldinhibit the expression of NOX subunit p22Phox and Rac1mRNA which wereinduced by leptin.This study will be based on the previous research results, furtherobservation the effect of UA on hedgehog signal pathway which induced by leptin.Objective：To explore the mechanism and effects of UA on hedgehog signal pathwayin HSCs(HSC-T6).Methods：HSC-T6in the exponential growth phase were devided: normal control group;leptin (100ng/ml) treated; UA（50μM）treated；DPI（20μM）treated；leptin treatedtogether with UA (50μM) and leptin treated together with NOX inhibitor DPI(20μM).HSC-T6were treated with medicine for12hours and mRNA expression ofShh，smo，Gli1/2were analyzed with RT-PCR. HSC-T6were treated with medicine for24hours and protein expression of Gli2and Rac1were analyzed with westernblotting. HSC-T6were treated with medicine for12hours,24hours,48hours andHSC-T6proliferation was analyzed with MTT.Results：1.The effect of UA on mRNA expression of Shh in HSC-T6HSC-T6were treated for12hours with Leptin, the mRNA expression of Shhwas increased compared with normal control group，but had no difference comparedwith normal control group (P>0.05)；Treatment with UA, the data were presentedreduce compared with leptin treatment and normal control group （bothP<0.05）;Treatment with DPI, the data were presented as relative reduce comparedwith leptin treatment and normal control group (both P<0.01). Here wedemonstrated that that UA and DPI both decrease the mRNA expression of Shhinduced by leptin. And DPI produced the inhibition is stronger than UA.2. The effect of UA on mRNA expression of Smo in HSC-T6HSC-T6were treated for12hours with Leptin, the mRNA expression of Smowas increased compared with normal control group（P<0.05）；Treatment with UA, thedata were presented as relative reduce compared with leptin treatment and normalcontrol group(P<0.01)；Treatment with DPI, the data were presented as relativereduce compared with leptin treatment and normal control group(P<0.01). Here wedemonstrated that UA and DPI both decreases the mRNA expression of Smo inducedby leptin.3.The effect of UA on mRNA expression of Gli1in HSC-T6HSC-T6were treated for12hours with Leptin, the mRNA expression ofSmo didn’t have statistical significance. Here we demonstrated that leptin、UAand DPI had no effect on the mRNA expression of Gli1.4.The effect of UA on mRNA expression of Gli2in HSC-T6HSC-T6were treated for12hours with Leptin, the mRNA expression ofGli2was increased compared with normal control group，but had no differencecompared with normal control group (P>0.05)；Treatment with UA and DPI, the data were presented as relative reduce compared with leptin treatment（均P<0.05）. Here we demonstrated that UA and DPI both decreases the mRNA ex pression of Gli2induced by leptin.5. The effect of UA on protein expression of NOX subunit Rac1induced byleptin. HSC-T6were treated for24hours with Leptin, the protein expression of Rac1was increased compared with normal control group(P<0.01); Treatment with UA, thedata were presented as relative reduce compared with leptin treatment and normalcontrol group（P<0.01）; Treatment with DPI, the data were presented as relativereduce compared with leptin treatment（P<0.01）. Here we demonstrated that UA andDPI both decreases the protein expression of Rac1induced by leptin.6. The effect of UA on protein expression of Gli2induced by leptin in HSC-T6.HSC-T6were treated for24hours with Leptin, the protein expression of Gli2was increased compared with normal control group(P<0.01); Treatment with UA, thedata were presented as relative reduce compared with leptin treatment (P<0.01)；Treatment with DPI, the data were presented as relative reduce compared with leptintreatment（P<0.01）. Here we demonstrated that UA and DPI both decreases theprotein expression of Gli2induced by leptin.7. The effect of UA on the HSC-T6proliferation induced by leptinHSC-T6were treated for12hours with Leptin, the growth inhibition rate of theHSC-T6were decreased compared with normal control group（P<0.01）; Treatmentwith UA, the data were increase compared with normal control group (P<0.01);Treatment with UA and DPI, the data were presented as relative reduce comparedwith leptin treatment (P<0.01). along with the treatment time,the growth inhibitionrate was decrease in the leptin group and were increased in the UA and DPI group，but the group treatment with UA and leptin didn’t have statistical significanceamong the time. Here we demonstrated that leptin promote the HSC-T6proliferation,and along with the treatment time the cell growth proliferation wasenhancemed.UA can inhibits the HSC-T6proliferation induced by leptin.Conclusions：UA can inhibit expression of Shh，Smo，Gli1/2mRNA and lower expression of Gli2protein in hedgehog signal pathway of HSC-T6induced by the leptin, and inhibit HSC-T6growth and proliferation. It may be related to decrea sing expression of NOX subunit Rac1protein by UA.