Effect of oxaliplatin on the activation of hepatic stellate cells and its mechanism / 临床肝胆病杂志

ABSTRACT

ObjectiveTo investigate the effect of oxaliplatin on the activation of hepatic stellate cells （HSCs）， as well as the association of oxaliplatin with microRNA-30a-5p and autophagy. MethodsHSC-LX2 cells were cultured and divided into groups according to the following three protocols： control group， PDGF treatment group， oxaliplatin treatment group， oxaliplatin+PDGF treatment group； control group， microRNA-30a-5p transfection group， PDGF treatment group， microRNA-30a-5p transfection+PDGF treatment group； control group， 3-MA group， microRNA-30a-5p inhibitor group， microRNA-30a-5p inhibitor+3-MA group. Western Blot was used to measure the expression of HSC activation-related proteins （Collagen-I and alpha-smooth muscle actin ［α- SMA］） and HSC autophagy-related proteins （Beclin-1， P62， and LC3B）； LysoTracker staining and immunofluorescence assay were used to measure the expression of LC3B autophagosomes； RT-PCR was used to measure the expression level of microRNA-30a-5p； bioinformatics techniques were used to predict the potential targets of microRNA-30a-5p in HSCs. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsAfter the cells were treated with oxaliplatin， RT-PCR results showed that the oxaliplatin treatment group had a significantly higher expression level of microRNA-30a-5p than the control group （P<0.01）； Western Blot showed that the oxaliplatin treatment group had significant reductions in the expression levels of the HSC activation-related proteins α-SMA and Collagen-‍Ⅰ and the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ （all P<0.001）； immunofluorescence assay showed that the oxaliplatin treatment group had a significantly lower number of autophagosomes than the control group （P<0.05）. After HSC-LX2 cells were transfected with microRNA-30a-5p mimic， compared with the control group， the microRNA-30a-5p mimic group had significant reductions in the expression levels of the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ （P<0.05） and the HSC activation-related protein Collagen-‍‍Ⅰ （P<0.001）； after HSC-LX2 cells were transfected with microRNA-30a-5p inhibitor， Western Blot showed that compared with the control group， the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC activation-related proteins Collagen-‍Ⅰ and α-SMA and the autophagy-related protein Beclin 1 （t=2.41， 2.32， and 4.57， all P<0.05）. Western Blot showed that compared with the control group， the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC autophagy-related protein Beclin 1 and the HSC activation-related protein α-SMA （both P<0.05）， and after the treatment with the autophagy inhibitor 3-MA， there were no significant differences in the expression of these proteins between the two groups （P>0.05）. The bioinformatics analysis using TargetScan， PicTar， and miRanda databases showed that the autophagy-related protein Beclin-1 might be a potential target of miRNA-30a-5p. ConclusionOxaliplatin can inhibit the activation of HSCs by upregulating the expression of microRNA-30a-5p， which provides new ideas and a new target for the treatment of liver fibrosis.