The Effect Of Ursolic Acid On The Activation Of NADPH Oxidase And Its Regulation On The Signaling Pathway Of PI3K/Akt、P38MAPK In Rat Hepatic Stellate Cells Induced By Angiotensin Ⅱ

Background：Hepatic fibrosis is the chronic liver injury caused by any etiology(such ashepatitis, schistosomiasis, alcohol and fat, etc), characterized by the extracellularmatrix deposition in the liver. The stage of liver fibrosis is a inevitabily experience inthe process of chronic liver disease to cirrhosis. Although this process can bereversed, there is no effective hepatic antifibrotic therapies available.The hepatic stellate cells(HSCs) are a major fibrogenic cell type in the liver,HSC activation, conversion is considered to be central event in the pathogenesis ofhepatic fibrosis. There are many pro-fibrotic factors, such as angiotensin Ⅱ (AngⅡ),transforming growth factor-β (TGF-β), leptin involved in HSC activation andproliferation. Angiotensin Ⅱ (AngⅡ) is considered to be one of the importantpro-fibrotic factors that can promote the liver fibrosis, It can induce the liver fibrosisthrough the promotion of HSC activation, proliferation. Some studies have shownthat the NADPH oxidase(NOX) mediates the signal transduction of AngⅡ, TGF-β,leptin and other pro-fibrotic factors. Thus, the NOX is considered an important newtarget of antifibrotic therapy.In our previous study which use the leptin as a pro-fibrotic factor found that UAcan inhibit the expression of NOX subunits, thereby block the the activation of NOXand signal pathways which induce the liver fibrosis in the HSC-T6. But whetherursolic acid can block the activation of signaling pathways induced by AngⅡ, TGF-βand other pro-fibrotic factor is unclear. we use the AngⅡ as a pro-fibrotic factor toobserve whether UA has a inhibitory effect on NOX subunit p47Phoxand itsdownstream signaling pathway of PI3K/Akt, P38MAPK, the mRNA expression ofcollagenⅠand proliferation of HSC or not, which can provide the theoretical basisand experimental datas for UA be used in clinical treatment of liver fibrosis.Objective：To clarify the effect of Ursolic acid on the activation of NADPH oxidase and thedownstream signaling pathway of PI3K/Akt, P38MAPK in rat hepatic stellate cells induced by AngⅡMethods：1.To observe the impact of UA on aversion of NOX subunit p47Phoxinduced byAngⅡin the HSC-T6with western blotting2.To observe the impact of UA on signaling pathway of P38MAPK,PI3K/Aktinduced by AngⅡin the HSC-T6with western blotting.3. To observe the impact of UA on mRNA expression of collagenⅠinduced byAngⅡin the HSC-T6by RT-PCR, the proliferation of HSC-T6by using the CCK8kit.Results：1. The impact of UA on moving of NOX subunit p47Phoxinduced by AngⅡHSC-T6were treated for15minutes with AngⅡ, the protein expression ofp47phoxin membrane was higher than normal control group (P<0.05); Beingintervened by DPI and UA, The data were significantly decreased than AngⅡtreatment (both P<0.05). so we consider that UA can stop the p47Phoxfrom moving tothe membrane.2. The impact of UA on PI3K、p-Akt and p-P38MAPK expression inducedby AngⅡ in HSC-T62.1The impact of UA on the expression of PI3K induced by AngⅡinHSC-T6HSC-T6were treated for30minutes with AngⅡ, the expression of PI3K washigher than normal control group (P<0.05); while being intervened by UA, DPI andLY294002, The data were showed as distinctly decrease compared with AngⅡtreatment (all P<0.05). so we consider that the UA can reduce the expression of PI3Kinduced by AngⅡ.2.2The impact of UA on p-Akt expression induced by AngⅡin HSC-T6HSC-T6were treated for30minutes with AngⅡ, the expression of p-Akt wasincreased (P<0.05); Intervene with DPI, UA and LY294002, The data were reducecompared with AngⅡ treatment (all P<0.05). Here we consider that the UA inhibitsthe expression of p-Akt induced by AngⅡ.2.3The impact of UA on expression of p-P38MAPK induced by AngⅡin HSC-T6HSC-T6were treated for30minutes with AngⅡ, the p-P38MAPK levels werehigher than normal control group (P<0.05); Intervene with SB203580, UA and DPI,the data were showed as reduce compared with AngⅡ treatment (all P<0.05); Herewe consider that the UA inhibits the expression of p-P38MAPK induced by AngⅡ.3. The impact of UA on expression of collagenⅠinduced by AngⅡHSC-T6were treated for12hours with AngⅡ, the expression of collagenⅠwas higher than normal control group (P<0.05); Intervene with SB203580, LY294002,UA and DPI, the data were reduce compared with AngⅡtreatment (all P<0.05), Herewe consider that the UA can decrease the expression of collagenⅠinduced by AngⅡ.4. The impact of UA on the the proliferation of HSC-T6induced by AngⅡHSC-T6were treated for12hours,24hours,48hours with AngⅡ, the HSC-T6proliferation was rised compared with normal control group; Intervene withLY294002, SB203580, UA and DPI, the data were reduce compared with AngⅡtreatment (all P<0.05). Here we conside that the UA can inhibit the proliferation ofHSC-T6induced by AngⅡ.Conclusions：1.AngⅡcan induce aversion of NOX subunit p47phoxin HSC-T6and activateNOX, upregulatethe activity of PI3K/Akt, P38MAPK, thus promote the HSC-T6proliferation and the mRNA expresstion of collagenⅠ, which induce the liverfibrosis.2.UA can inhibit aversion of NOX subunit p47phoxin HSC-T6, thusdownregulate the activity of PI3K/Akt, P38MAPK.3.UA can inhibit mRNA expression of collagenⅠand the proliferation ofHSC-T6. The mechanism may be related to inhibting activation of PI3K/Akt andP38MAPK signal net which regulated by NOX.