DMKG INHIBITS THE ACTIVATION OF HEPATIC STELLATE CELLS AND HEPATIC FIBROSIS THROUGH ACETYL COENZYME A-EP300 PATHWAY

Hongxin Yang¹, Xiaoling Liu², Xiaobin Guo¹, Hao Guo¹, Minxiang Dong³, Hongxin Yang^{4, *}

¹Department of Pharmacy, Inner Mongolia Autonomous Region People’s Hospital, Hohhot, PR China - ²Department of Scientific Research, Inner Mongolia Autonomous Region People’s Hospital, Hohhot, PR China - ³Inner Mongolia Institute of Traditional Chinese Medicine, Hohhot, PR China - ⁴Inner Mongolia Autonomous Region People’s Hospital, Hohhot, PR China

Objective: To analyse the mechanism of DMKG inhibiting the activation of hepatic stellate cells and hepatic fibrosis. 

Methods: Hepatic stellate cell lines were cultured in vitro and treated with different concentrations of DMKG. The MTT method was used to detect the effect of DMKG on the survival rate of hepatic stellate cells with the real-time quantitative PCR method and Western blot applied to determine the effect of DMKG on α - SMA and collagen I mRNA and protein of hepatic stellate cells. The liver fibrosis model of rats was established and divided into a control group, DMKG group, model group and DMKG intervention model group. The Masson-staining technique was employed to establish the impact of DMKG on hepatic stellate cell fibrosis; a Western blot method was further utilized to determine the effect of DMKG on the LC3B-Ⅱ/Ⅰratio and Beclin-1 protein expression levels of hepatic stellate cell. 

Results: The MTT assay showed that 1 ~ 16 mM DMKG had no significant difference in cell survival rate compared with the control group (P>0.05), and 20 ~ 32 mM DMKG could significantly inhibit the cell survival rate compared with the control group, which was concentration dependent (P<0.05). Compared with the control group, the mRNA levels of α - SMA and collagen I in hepatic stellate cells were significantly reduced by 8 mM DMKG, and the mRNA levels of collagen I in hepatic stellate cells were significantly reduced by 1 mM DMKG (P<0.05). The results of the Western blot indicated that 1 and 8 mM DMKG could significantly reduce the expression of α-SMA and collagen I in hepatic stellate cells compared with the control group (P<0.05). The Masson-staining results showed that there was no obvious deposition of liver tissue fibres in the control group or DMKG group, and that liver tissue morphology was normal, while a large number of collagen fibres were deposited in the model group, and the area of collagen fibres in the DMKG intervention model group was significantly reduced compared with the model group. The expression of LC3B-Ⅱ/I and Beclin-1 in hepatic stellate cells decreased significantly when DMKG was added. After hepatic stellate cells were cultured with C464 (specific inhibitors of acetyl CoA and EP300), there was no significant change in LC3B-Ⅱ/I and Beclin-1 compared with the control group (P>0.05). 

Conclusion: A high concentration of DMKG can significantly inhibit the activation of hepatic stellate cells and reduce the degree of liver fibrosis. The mechanism may be achieved by the inhibition of autophagy of hepatic stellate cells by the acetyl coenzyme A-EP300 pathway.