Autophagy Of Human Hepatoma G2 Cell Line Could Be Induced By The Effect Of Matrine Via MTOR Signaling Pathway

AimsTo explore the molecular mechanism of autophagy induced by the effect of Matrine in human hepatoma G2 cell line.MethodsUsing human hepatoma G2 cell line as the experiment objects, cell morphology was observed under reverse microscope, the changes of ultrastructure of cells were observed by transmission electron microscope, the expression of Benlin 1 mRNA which was associated with autophagy were determined by Real-time quantitative RT-PCR, the expression of LC3-â…¡protein which was related to autophagy, mTOR/p-mTOR and p70s6k/p-p70s6k proteins were determined by Western blotting method.ResultsAutophagy of human hepatoma G2 cell could be induced by the effect of Matrine at 0.25, 0.5,1.0,2.0 mg/ml for 12h,24h,48h in time and does dependent manners. Cells growed well, maintained normal grows morphology and anchored well in the control group (Matrine at 0 mg/ml), on the contrary, cells bubbled unequal in size, vacuoles increased and became larger in the Matrine group under inverted microscope. After cells were treated with Matrine at 1.0,2.0 mg/ml for 12h,24h, some suspension cells appeared, became smaller and round. After cells were treated with Matrine at 2.0 mg/ml for 48h, the whole culture medium showed a scene of desolation under inverted microscope.Furthermore it showed that compared with the control group (Matrine at 0 mg/ml), swelling organelles, cytoplasm encapsulated by a double membrane and obvious autophagic vacuoles appeared in Matrine treated groups when human hepatoma G2 cells were treated with Matrine at 2.0 mg/ml for 48h under transmission electron microscope.Real-time quantitative RT-PCR and Western blotting results showed Matrine could up-regulated the mRNA expression of Beclin 1 which is considered indispensable for autophagy and the protein expression of LC3-â…¡which is the unique validated marker of autophagosome formation. Western blotting results also showed that the effect of autophagy induced by Matrine in human hepatoma G2 cell line may via mTOR signaling pathway. The expression of mTOR and p-70s6k proteins had no obvious statistical differece (p>0.05). The expression of p-mTOR and p-p70s6k proteins down-regulated after cells were treated with Matrine at 0,0.25,1.0,2.0 mg/ml for 24h in a does dependent manner.ConclusionMatrine could induce the autophagy in human hepatoma G2 cell line in time and does dependent manners. The molecular mechanism of autophagy induced by the effect of Matrine in human hepatoma G2 cell line via down-regulatd mTOR signaling pathway.