Effects Of Ursolic Acid On The NOX-Hh Signal Pathway Induced By TGF-β During The Activation Of Q-HSC

Background: Liver fibrosis is a wound-healing response to encapsulate injury, the progression of liver fibrosis often leads to cirrhosis and is associated with liver cancer. Liver fibrosis manifests as a progressive deposition of extracellular matrix(ECM), the activated HSCs are major producers of ECM and other fibrosis-associated proteins. Therefore the process of quiescent hepatic stellate cells transformate to MF-HSC is a very important step in liver fibrosis. The Hedgehog(Hh) pathway controls many aspects of cell differentiation and the development of tissues and organs during embryogenesis. Numerous studies have indicated that Hh signaling pathway plays a key role in transition of Q-HSC into MF-HSC and liver fibrosis. Moreover, activation of Rac1 promotes Hedgehog-mediated acquisition of the myofibroblastic phenotype in rat and human hepatic stellate cells. The previous researches of our group showed, the expression of NOX and the signal pathway in HSC-T6 induced by leptin could be inhibited by the intervention of Ursolic acid(UA). The inhibited effect of UA on Hh signaling pathway may be relative with the decreasing of Rac1. On the basis of previous research, this study will continue to explore the effect of UA on the activation of NOX and the related signaling pathway during the process of Q-HSC into MF-HSC, sought to clarify the target and anti-fibrotic mechanism intervened by UA in HSCs.Objective: To clarify the effect of UA on the activation of NOX and the downstream signaling pathway of NOX-Hh during Q-HSC into MF-HSC induced by TGF-beta.Methods: Primary HSC cultured to 5 day were randomly separated into the following groups: blank control group, UA control group(40μM), TGF-β group(5μg/L), UA intervention group(TGF-β treated along with UA 40 μM), DPI intervention group(TGF-β treated along with DPI 10μM). Primary HSC were treated with medicine for 12 hours and m RNA expression of rac1, α-SMA,Shh, smo, Gli2, type I collagen were analyzed with q RT-PCR. Primary HSC were treated with medicine for 24 hours and protein expression of rac1, Shh, smo, Gli2, α-SMA were analyzed with Western Blotting.Results: 1. The effect of UA on m RNA expression of type I collagen and α-SMA in primary HSC Primary HSC cultured to 5 day were treated with drugs for 12 hour, the expression of type I collagen and α-SMA m RNA of TGF-beta group was notably increased compared to blank control group(P<0.01); The expression of type I collagen and α-SMA m RNA of UA control group was lower than blank control group(P<0.05; P<0.01); UA intervention group was markedly lower than TGF-beta group(P<0.01); The expression of type I collagen and α-SMA m RNA of UA, DPI and UA intervention group had no significance of difference(P>0.05). The results show that the expression of type I collagen and α-SMA m RNA significantly increased during Q-HSC into MF-HSC, and UA could reverse it. 2. The effect of UA on m RNA expression of Rac1 in primary HSC Primary HSC cultured to 5 day were treated with drugs for 12 hour, the m RNA expression of Rac1 of TGF-beta group was notably increased compared to blank control group(P<0.01); The m RNA expression of Rac1 of UA control group was lower than blank control group(P<0.05); UA intervention group was markedly lower than TGF-beta group(P<0.05); The expression of Rac1 m RNA of UA, DPI and UA intervention group had no significance of difference(P>0.05). The results show that the expression of Rac1 m RNA significantly increased during Q-HSC into MF-HSC, and UA restrains the m RNA expression of Rac1 in primary HSCs. 3. The effect of UA on m RNA expression of Shh, Smo and Gli2 in primary HSC Primary HSC cultured to 5 day were treated with drugs for 12 hour, the m RNA expression of Shh, Smo and Gli2 of TGF-beta group was notably increased compared to blank control group(P<0.01; P<0.01; P<0.05); The m RNA expression of Shh,Smo and Gli2 of UA control group was lower than blank control group(P<0.01; P<0.01; P<0.05); UA intervention group was markedly lower than TGF-beta group(P<0.01); The expression of Shh, Smo and Gli2 m RNA of UA, DPI and UA intervention group had no significance of difference(P>0.05). The results show that the expression of Shh, Smo and Gli2 m RNA significantly increased during Q-HSC into MF-HSC, and UA restrains the m RNA expression of Shh, Smo and Gli2 in primary HSCs. 4. The effect of UA on protein expression of α-SMA in primary HSC Primary HSC cultured to 5 day were treated with drugs for 24 hour, the protein expression of α-SMA of TGF-beta group was notably increased compared to blank control group(P<0.01); The protein expression of α-SMA of UA control group was lower than blank control group(P<0.05); UA intervention group was markedly lower than TGF-beta group(P<0.01); The expression of α-SMA protein of UA, DPI and UA intervention group had no significance of difference(P>0.05). The results show that the expression of α-SMA protein significantly increased during Q-HSC into MF-HSC, and UA restrains the expression of α-SMA protein in primary HSCs. 5. The effect of UA on protein expression of Rac1 in primary HSC Primary HSC cultured to 5 day were treated with drugs for 24 hour, the protein expression of Rac1 of TGF-beta group was notably increased compared to blank control group(P<0.05); The protein expression of Rac1 of UA control group was lower than blank control group(P<0.01); UA intervention group was markedly lower than TGF-beta group(P<0.01); The expression of Rac1 protein of UA, DPI and UA intervention group had no significance of difference(P>0.05). The results show that the expression of Rac1 protein significantly increased during Q-HSC into MF-HSC, and UA restrains the expression of Rac1 protein in primary HSCs. 6. The effect of UA on protein expression of Shh, Smo, Gli2 in primary HSC Primary HSC cultured to 5 day were treated with drugs for 24 hour, the protein expression of Shh, Smo and Gli2 of TGF-beta group was notably increased compared to blank control group(P<0.05; P<0.05; P<0.01); The protein expression of Shh, Smo and Gli2 of UA control group was lower than blank control group(P<0.01); UA intervention group was markedly lower than TGF-beta group(P<0.05); Theexpression of Shh, Smo and Gli2 protein of UA, DPI and UA intervention group had no significance of difference(P>0.05). The results show that the expression of Shh, Smo and Gli2 protein significantly increased during Q-HSC into MF-HSC, NOX participated in the regulation of Shh, Smo and Gli2 protein, and UA restrains the expression of Shh, Smo and Gli2 protein in primary HSCs.Conclusions: 1. NOX could regulate the expression of Shh, Smo and Gli2 during Q-HSC into MF-HSC induced by TGF-beta. 2. UA could inhibit the activation of hedgehog signaling pathway and the expression of type I collagen and a-SMA, which may through decreasing the expression of Rac1.