Anti-Prolifelation And Inducing Apoptosis Effects Of Boschniakia Rossica On Hepatic Stellate Cell-T6and Molecular Mechenisems

Objective:Hepatic fibrosis usually results liver cirrhosis. Hepatic stellate cells (HSCs) are the major cells in hepatic fibrosis, and are responsible for the development of fibrosis. It is well known that activation and proliferation of HSCs is the key to fibrogenesis, while apoptosis of HSCs is associated with the resolution of fibrosis. Therefore, inhibiting the proliferation and inducing the apoptosis of HSCs is potential anti-fibrosis strategies. However, there is no any approved drug available for anti-fibrosis of liver.Boschniakia rossica Fedtsch. et Flerov (Orobanchaceae) is a parasite growing on the root of plants of the genus Alnus (Betulaceae). Boschniakia rossica (BR) is widely used as a substitute for Cistanchis Herba, a well known staminal tonic agent in oriental medicine. BR have been reported a variety of pharmacological activities, including hepatoprotective activity, antioxidative, anti-inflammatory and anti-tumor activities. Our previous study has shown that BR can prevent pig serum-induced liver fibrosis in rat in vivo. However, the potential role of BR on HSCs and the molecular pathways have not been investigated.To observe the effects and mechanism of anti-proliferation and inducing apoptosis on the Hepatic stellate cell-T6induced by Ethanol Extract from Boschniakia rossica (EEBR), and provide the experimental evidence for Boschniakia rossica as a new anti-hepatic fibrosis drug.Methods:Hepatic stellate cell-T6(HSC-T6) was cultured in vitro. The inhibition on the proliferation of HSC-T6by the EEBR was assessed by MTT assay.The apoptosis of HSC-T6was detected by inverted microscope, light microscope.fluorescent microscope,TUNEL, and the ultrastructure of HSC-T6was observed by electronic microscope. The cell cycle distribution and the apoptotic rate of HSC-T6was analyzed by flow cytometry. RE-PCR and Western blotting was utilized to measure the activation or inhibition of signal molecules and expression of type I collagen.Results:①MTT assay revealed that the EEBR could inhibit HSC-T6proliferation in a dose-and time-dependent manner.②The typical morphological changes of the apoptosis HSC-T6was detected by light microscope, fluorescent microscope, TUNEL and electronic microscope: cell surface microvilli disappeared, cell size became smaller, cytoplasm concentrated, nuclear chromatin marginationed, nucleus pyknosis, karyorrhexis, blebbing.③FFlow cytometry analysis showed that the HSC-T6treated with the EEBR was arrested in G0/G1phases. The apoptotic rate increased significantly in drug treated HSC-T6group compared with the control group and positively correlated with the dose increasement of EEBR.④EBR significantly incrased the activated caspase-3and caspase-9, and decreased the ratio of Bcl-2to Bax.⑤EBR remarkably suppressed the level of both phosphor-Akt and phosphor-p70S6K without changes the level of total p70S6K and Akt levels.⑥EEBR inhibited mRNA level of collagen al (Ⅰ) and the protein expression of type Ⅰ collagen.Conclusions:①The EEBR had a significant anti-proliferation effect on HSC-T6in vitro.②The EEBR could induce HSC-T6apoptosis and arrest HSC-T6in G0/G1phases.③EEBR significantly incrased the activated caspase-3and caspase-9, and decreased the ratio of Bcl-2to Bax.④EEBR can down-regulated of Akt/p70S6k pathway.⑤EEBR inhibited mRNA level of collagen al (Ⅰ) and the protein expression of type I collagen.