Effects Of Ursolic Acid On The Expression And Fuction Of NADPH Oxidase Subunits During The Activation Of Quiescent HSC

Background:Liver fibrosis is caused by excessive wound healing response due to varies of chronic liver injury,which characterized by extracellular matrix(ECM),mainly type I collagen,excessive deposition in the liver.The actived hepatic stellate cells(HSCs) are the most important cellular source of ECM,so the activation and transformation of the quiescent HSCs is crucial for the pathogenesis of Hepatic fibrosis.NADPH oxidase(NOX) and it derived reactive oxygen species(ROS) induce oxidative stress,and the oxidative stress is proved playing very important role in the activation of HSCs and both in the occurrence and development of liver fibrosis.Our previous researches showed that Ursolic acid(UA) inhibited the proliferation of actived HSCs and induced them to apoptosis efficiently by decreasing the effects of leptin,Ang II and PDGF,which can all induce the up-regulation of the NOX subunits expression,the NOX activity,the ROS generated by NOX,the activation of NOX downstream signal pathways PI3K/Akt and p38 MAPK in the active HSCs.But whether UA can inhibit the activation of the quiescent HSC by down-regulating the NOX subunits expression during the activation of the quiescent HSC to prevent the liver fibrosis is still unclear. On the basis of our previous researches,this study will use rat primary quiescent HSCs as interesting cells,TGF-β as pro-activation factor,investigate the effects of UA on the expression of NOX subunits during the activation of quiescent HSC and the relationship between the effects and the HSC activation.This study will clarify the anti-fibrotic mechanism of UA on a deeper level. Objective:Objective: To observe the effect of ursolic acid on the expression of NOX subunits during the activation of rat quiescent HSC,and explore the relationship between the effects and the quiescent HSC activationMethods: The quiescent HSCs in our experiment were isolated from healthy SD rats by using liver perfusion in situ and density gradient centrifugation.The primary HSCs which were cultured naturally for five days were randomly divided into five groups and treated with different medicine:normal control group(cultured naturally); UA group(40μM); TGF-β group(5μg/L);UA intervention group(treated with UA 40μM previous TGF-β 5μg/L); DPI intervention group(treated with DPI 10μM previous TGF-β 5μg/L).After treated with medicine for 24 hours,the total protein of the five group cells were extracted,then the protein expression levels of the NOX subunits gp91 phox,p22phox,p67 phox and α-SMA which was the specific mark of active HSCs were detected by Western Blot.In the meantime,we observe the impact of UA on m RNA expression of NOX subunits Rac1,p22 phox and collagen I by RT-q PCR.Results: 1 The impact of UA on the ptrotein expression of NOX subunits during the activation of quiescent HSC1.1 The impact of UA on the ptrotein expression of gp91 phox during the activation of quiescent HSCAfter treating the primary HSCs TGF-β for 24 hours, the gp91 phox protein expression was significantly higher than blank control group(P<0.01).The UA group had obviously lower gp91 phox protein expression compared to blank control group(P<0.05).The primary HSCs which was intervented with UA before TGF-βstimulation had remarkablely lower gp91 phox protein expression level than pure TGF-β treatment group(P<0.01),as the same time,there was no statistic difference between UA intervention group and the NOX inhibitor DPI intervention group(P>0.05).These above data suggested that the protein expression of gp91 phox significantly increased during the activation of quiescent HSC,UA intervention could inhibit the protein expression of gp91 phox during the activation of quiescent HSC.1.2 The impact of UA on the ptrotein expression of p67 phox during the activation of quiescent HSCThe p67 phox protein expression of primary HSCs,which was treatmented with TGF-β for 24 hours, was remarkablely higher than blank control group(P<0.01).The UA group had obviously lower p67 phox protein expression compared to blank control group(P<0.05).Pretreating the primary HSCs with UA before TGF-βstimulation,the protein expression level of p67 phox was significantly lower than pure TGF-β treatment group(P<0.01),and there was no statistic difference between UA intervention group and DPI intervention group(P>0.05).The upper data suggested that the protein expression of p67 phox increased during the activation of quiescent HSC,UA intervention could inhibit the protein expression of p67 phox during the activation of quiescent HSCs.1.3 The impact of UA on the ptrotein expression of p22 phox during the activation of quiescent HSCThe primary HSCs which was treatmented with TGF-β for 24 hours had obviously higher p22 phox protein expressin than blank control group(P<0.01).The p22 phox protein expression of UA group was obviously lower than blank control group(P<0.05).Interventing the primary HSCs with UA before TGF-βstimulation,the p22 phox protein expression was remarkablely lower than pure TGF-β stimulation group(P<0.01),and there was no statistic difference between the UA intervention group and DPI intervention group(P>0.05).These data suggested that the protein expression of p22 phox increased during the activation of quiescent HSC,and UA intervention could efficiently inhibit the protein expression of p22 phox during the activation of quiescent HSC.2. The impact of UA on the m RNA expression of NOX subunits during the activation of quiescent HSC2.1 The impact of UA on the m RNA expression of Rac1 during the activation of quiescent HSCThe Rac1 m RNA expression of primary HSCs which were stimulated with TGF-β for 12 hours was significantly higher than blank control group(P<0.01).The UA treatment group had lower Rac1 m RNA expression than the blank control group(P<0.05).The primary HSCs of UA intervention group and DPI intervention group,which were pretreated with UA and DPI individually before TGF- βstimulation,had both remarkablely lower Rac1 m RNA expression than the TGF-β group(both P<0.05),and there was no statistic difference between the two intervention groups(P>0.05).These data suggested that the Rac1 m RNA expression increased during the activation of quiescent HSC,and UA intervention could efficiently inhibit the m RNA expression of Rac1 during the activation of quiescent HSC.2.2 The impact of UA on the m RNA expression of p22 phox during the activation of quiescent HSCThe primary HSCs which were stimulated with TGF-β had apparently higher p22 phox m RNA expression than blank control group(P<0.01).The UA treatment group had lower p22 phox m RNA expression than the blank control group(P<0.01). The p22 phox m RNA expression of primary HSCs which were pretreated with UA before TGF- β stimulation was remarkablely lower than the pure TGF-β stimulation group(P<0.01),and there was no statistic difference between the UA intervention group and DPI intervention group(P>0.05).These upper data suggested that the p22 phox m RNA expression increased during the activation of quiescent HSC,and UA intervention could efficiently inhibit the m RNA expression of p22 phox during the activation of quiescent HSC.3. The impact of UA on the ptrotein expression of α-SMA during the activation of quiescent HSCThe primary HSCs which were stimulated by TGF-β for 24 hours had obviously higher α-SMA protein expressin than blank control group(P<0.05).The cultured naturally primary HSCs which were treatmented with UA had lower α-SMA protein expression than blank control group(P<0.05).Interventing the primary HSCs with UA and DPI individually before TGF-βstimulation,the α-SMA protein expression were both remarkablely lower than pure TGF-β stimulation group(both P<0.01),and were both lower than blank control group(both P<0.05),there was no statistic difference between the UA intervention group and DPI intervention group(P>0.05).These above data suggested that NOX induced the HSC activation,and UA intervention could efficiently decrease the protein expression of α-SMA during HSC activation to inhibit the activation of quiescent HSC.4. The impact of UA on m RNA expression of collagenⅠduring the activation of quiescent HSCThe m RNA expression of collagen I of primary HSCs which were stimulated with TGF-β for 12 hours was significantly higher than blank control group(P<0.05).The UA treatment group had lower collagen I m RNA expression than the blank control group(P<0.05).The primary HSCs of UA internvention group and DPI intervention group,which were pretreated with UA and DPI individually before TGF-βstimulation,had both remarkablely lower collagen I m RNA expression than the TGF-β group(both P<0.01),and there was no statistic difference between the two intervention groups(P>0.05).These upper data suggested that NOX mediated the synthesis of collagen I,and UA intervention could inhibit the m RNA expression of collagen I during the activation of quiescent HSC.Conclusions:1. During the activation of primary quiescent HSC,the protein and m RNA expression of NOX subunits gp91 phox,p67phox,p22 phox,Rac1 can both increase,and the UA internvention can inhibit the up-regulation of the NOX expression.2. NOX mediates the HSC activation and the collagen synthesis I during the activation of primary quiescent HSC,UA intervention can inhibit the HSC activation and collagen synthesis,and it’s mechanism may be related to the UA’s inhibiting effect on NOX subnits expression.