Study Of Kangxian Ruangan Granule Regulate Hedgehog Pathway's Nuclear Factor Gli1 For Anti-activation Of HSC

Objective: Kangxian Ruangan granule is a kind of preparation of Hubei Provincial Traditional Chinese Medical Hospital, and long term clinical applications show that it has significant effect on reducing and delaying the progression of hepatic fibrosis. In this study, we make use of Kangxian Ruangan granule to interfere in activated Hepatic Stellate Cells(HSC) which induced by Platelet Derived Growth Factor(PDGF), and analyze its effect on the expression of nuclear transcription factor Gli1 and Other key molecules of Hedgehog(Hh) signal pathway, furthermore, investigate its mechanism of anti-hepatic fibrosis through regulating the Hh signaling pathway and inhibiting the activation of HSC.Methods: To introduce Human Hepatic Stellate Cell line HSC-LX2, and culture and passage them in conventional methods. We set up blank control group, model group and Kangxian Ruangan granule medium, low does group. Conventional cultured cells as the blank control group. We induce HSCs with PDGF-BB whose final concentration is 10ng/ml and set up the model group. And then, we treat the activated HSCs with Kangxian Ruangan granule by concentration of 5mg/ml, 1.25mg/ml( Kangxian Ruangan granule medium, low does group) for 24 hours. Followed, the cells were collected. We extract total RNA using TRIzol method, and synthesize the first-strand c DNA by reverse transcription. Then, we determine the expression of Hh signaling pathway's related signal molecules such as Gli1, Shh, Ptc and Smo by Real Time Fluorescence Quantitative PCR, and analyze the effect of Kangxian Ruangan granule on HSC activation and synthesis of transforming growth factor-beta 1(TGF-1) and PDGF-B.Results: After induced by PDGF-BB, HSCs were activated and proliferate. Real time fluorescence quantitative PCR results show that, in HSC cells of the model group, the expression of Hh signaling pathway's related signal molecules include Shh, Ptc, Smo, Gli1 m RNA were increased, and compared with the blank control group, the difference was significant(P < 0.05). In Kangxian Ruangan granule medium, low does group, the expression of Gli1, Ptc, Smo m RNA were lower than that in the model group, the expression of Shh m RNA was higher than it in the model group, and the difference was significant(P < 0.05), and there was no significant difference among different does groups. After HSCs activated by PDGF-BB, in the model group, the synthesis of TGF-?1 and PDGF-B increased, and compared with the blank control group, the difference was significant(P < 0.05). In Kangxian Ruangan granule medium, low does group, the synthesis of TGF-?1 and PDGF-B decreased, and compared with the model group, the difference was significant(P < 0.05), and there was no significant difference among different does groups.Conclusion: Kangxian Ruangan granule can regulate the expression of nuclear transcription factor Gli1 and its related signal molecules include Shh, Ptc and Smo of Hedgehog(Hh) signal pathway, and then inhibit the activation of HSCs and reduce the synthetise of TGF-?1, PDGF-B. It may be one of the mechanisms of Kangxian Ruangan granule to reduce the severity of hepatic fibrosis and delay the progression of the disease.