| Background:Liver fibrosis is a chronic liver injury caused by various factors, and therewound healing response exist in the development of fibrosis. As the main source ofthe extracellular matrix (ECM), the activation, proliferation, and transformation ofhepatic stellate cells (HSCs) become the key pathogenesis of liver fibrosis. In recentyears, studies have shown that the signal transduction in HSCs is mainly regulated byreactive oxygen species (ROS) produced by NADPH oxidase (NOX). Therefore, byinhibiting the activity of NOX and blocking the signal transmission of the fibrosisfactors in HSCs, NOX is expected to become an important target for resisting liverfibrosis. The previous researches of our group showed, the expression of NOX andthe signal pathway in HSCs induced by leptin could be inhibited by the interventionof Ursolic acid(UA). Many factors which could promote fibrosis, such as PDGF andTGF-β, are participating in HSCs activation and proliferation, whether UA can blockthe signal pathway activation mediated by factors which can promote fibrosis, besidesleptin, will be a matter for further research. On the basis of previous research, thisstudy will continue to explore the effect of UA on the activation of NOX and therelated signaling pathway evoked by PDGF in activated HSCs, sought to clarify thetarget and anti-fibrotic mechanism intervened by UA in activated HSCs.Objective:To observe the effect of UA on the activation of NOX and the downstreamsignaling pathways evoked by PDGF in activated hepatic stellate cells (HSC-T6) inrats.Methods:Exponential phase of HSC-T6cells were randomly separated into the followinggroups: blank control group, Rosup positive control group (5μg/ml), UA group (50μM), PDGF-BB group (10ng/ml), UA intervention group (PDGF-BB treated along with UA50μM), DPI intervention group (PDGF-BB treated along with DPI20μM),SB203580intervention group (PDGF-BB treated along with SB20358010μM),LY294002intervention group (PDGF-BB treated along with LY29400210μM). Toobserve the influence of UA on type I collagen HSC-T6were treated with drugs for12h,24h, using RT-PCR method to detect the expression of type I collagen; Toobserve the influence of UA on the membrane shift of NOX subunit p47phoxprotein,HSC-T6were treated with drugs for15min,30min,60min, then using Westernblotting to test the expression of membrane protein p47phox. To observe the influenceof UA on ROS level in HSCs, HSC-T6were treated with drugs for10min,30min,1h,2h, using reactive oxygen detection kits, living cells workstation and enzymestandard instrument to test DCF model. To observe the influence of UA on thePI3K-AKT, P38MAPK signaling pathways NOX controlled, HSC-T6were treatedwith drugs for15min,30min,60min, using Western blotting to test the expressionof PI3K, P-AKT and P-P38MAPK protein.Results:1. The influence of UA on type I collagen induced by PDGF in HSC-T6HSCs were treated with drugs for12hour, the expression of type I collagenmRNA of PDGF group was notably boosted compared to blank control group(P<0.01); UA intervention group was markedly lower than PDGF group (P<0.01) andhad no difference compared to blank control group (P>0.05); The expression of type Icollagen mRNA of UA group was lower than blank control group (P<0.01); The datain the DPI, SB203580and LY294002intervention group were reduced compared toPDGF group (P<0.01); The expression of type I collagen mRNA of UA, DPI,SB203580and LY294002intervention group had no notable difference (P>0.05).The results show that UA restrains the expression of type I collagen mRNA in HSCsevoked by PDGF-BB.2. The influence of UA on p47phoxprotein membrane displacement inducedby PDGFHSCs were treated with drugs for15min, the expression of membrance protein p47phoxof PDGF group was notably raised compared to blank control group (P<0.05);UA intervention group was markedly less than PDGF group (P<0.05), and there wasno difference compared to the blank control group (P>0.05); The expression ofp47phoxof UA group was lower than blank control group (P<0.01); In the DPI,LY294002and SB203580intervention group, the data were less than PDGF group(P<0.05). The data of UA intervention group had no notable difference compared toDPI intervention group, SB203580intervention group and LY294002interventiongroup in statistics (P>0.05). The results show that UA restrains the membranetranslocation of the p47phoxevoked by PDGF.3. The influence of UA on ROS level induced by PDGF in HSC-T6HSCs were treated with drugs for1hour, the DCF model fluorescence intensityof PDGF group was notably higher than blank control group (P<0.01), and had nonotable difference compared to Rosup positive group (P>0.05); The DCF modelfluorescence intensity of UA intervention group and DPI intervention group weremarkedly less than PDGF group and Rosup positive group (P<0.01), and had nodifference compared to blank control group (P>0.05); The DCF model fluorescenceintensity of UA group was lower than blank control group (P<0.01); The data of UAintervention group had no notable difference compared to DPI intervention group(P>0.05). The results show that UA restrains the generation of ROS in HSCs evokedby PDGF.4. The influence of UA on PI3K-Akt, P38MAPK signaling pathway inducedby PDGF in HSC-T64.1The influence of UA on the protein expression of PI3K evoked by PDGFHSCs were treated with drugs for30min, the expression of PI3K protein ofPDGF group was notably higher than blank control group (P<0.01); The data in UAintervention group was markedly lower than PDGF group and blank control group(P<0.01, P<0.05); The expression of PI3K of UA group was lower than blank controlgroup (P<0.01); The data in DPI and LY294002intervention group were less thanPDGF group (P<0.01); The data of UA intervention group had no notable difference compared to DPI and LY294002intervention group (P>0.05). The results show thatUA restrains the protein expression of PI3K evoked by PDGF.4.2The influence of UA on protein expression of p-Akt evoked by PDGFHSCs were treated with drugs for30min, the expression of p-Akt protein ofPDGF group was markedly higher than blank control group (P<0.01); The data in UAintervention group was notably less than PDGF group (P<0.01), and had nodifference compared to blank control group (P>0.05); The expression of p-Akt of UAgroup was lower than blank control group (P<0.01); The data in DPI and LY294002intervention group were reduced compared to PDGF group (P<0.05); The expressionof p-Akt in UA intervention group had no notable difference compared to DPI andLY294002intervention group (P>0.05). The results show that UA restrains theprotein expression of p-Akt evoked by PDGF.4.3The influence of UA on protein expression of P-P38MAPK evoked byPDGF in HSC-T6HSCs were treated with drugs for30min, the protein expression of P-P38MAPKof PDGF group was markedly higher than blank control group (P<0.01); The data inUA intervention group was notably less than PDGF group (P<0.01), and had nodifference compared to blank control group (P>0.05); The expression ofP-P38MAPK of UA group was lower than blank control group (P<0.01); The data inDPI and SB203580intervention group were lower than PDGF group (P<0.01); Theexpression of P-P38MAPK of UA intervention group had no notable differencecompared to DPI and SB203580intervention group (P>0.05). The results show thatUA restrains the protein expression of P-P38MAPK evoked by PDGF.Conclusions:1. PDGF could induce the membrane displacement of NOX subunit p47phox,activate NOX, and then initiate the activation of PI3K-Akt, P38MAPK signalpathways, promote the expression of type I collagen mRNA in HSC-T6.2. UA could inhibit the expression of type I collagen mRNA induced by PDGFin HSC-T6, the mechanism is probably related to restraining the membrane displacement of NOX subunit p47phox, blocking NOX activation, thus cutting theactivation of PI3K-Akt, P38MAPK signal pathways. |
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