ABSTRACT
Objective To investigate the effect of miRNA-200b-specific inhibitor on hepatic stellate cells(HSCs) activation,proliferation,and extracellular matrix production.Methods The miRNA-200b-specific inhibitors were designed,synthesized,and transfected into HSCs with lipofectamine 2000.The supernatant and HSCs were collected after incubation for 48 h.The expression of miR-200b was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).The expression ofα-smooth muscle actin (oα-SMA) protein in HSCs was detected by Western blotting.The cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) method.Contents of type Ⅲ procollagen and hyaluronic acid in supernatant were determined by radioimmunoassay.Results Compared to the control group,miRNA-200b expression was decreased in the miRNA-200b inhibitor group by 82％ (P ＜ 0.01),α-SMA protein expression was reduced in the miRNA-200b inhibitor group by (19 ± 3) ％ (P ＜ 0.05),and the activity of HSCs proliferation was reduced by(33 ± 5)％ (P ＜0.01),and the contents of type Ⅲ procollagen and hyaluronic acid in supernatant were reduced in miRNA-200b inhibitor group by (35 ± 4)％ and (31 ± 2)％,respectively(P ＜0.01).Conclusions The miRNA-200b-specific inhibitor could significantly reduce the expression of miRNA-200b,and inhibit HSC proliferation,activation,and extracellular matrix production.
ABSTRACT
Objective To observe the effects of adiponectin on mRNA and protein expressions of connective tissue growth factor (CTGF) in hepatic stellate cells (HSCs) and the levels of procollagen type Ⅲ (PC Ⅲ) and hyaluronic acid (HA).Methods Cultured rat HSCs were treated with different concentrations of adiponectin.CTGF mRNA and protein expressions were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot.The levels of PCⅢ and HA were detected by enzymelinked immunosorbent assay (ELISA).Results Compared with the control group,the result of RT-PCR showed that the four groups had different degrees of inhibitory effect,of which group D exhibited the strongest inhibitory effect.The absorbance ratio was 1.54 ±0.18,1.21 ±0.14,0.96 ±0.10,and 0.79 ± 0.08,respectively (t =2.42,P ＜0.05;t =2.73,P ＜0.05;t =3.28,P ＜0.01;t =4.67,P ＜0.01).Western Blot also indicated that four groups had different degrees of inhibitory effect,of which D group exhibited the strongest inhibitory effect.The ratio of integral absorption was 1.54 ± 0.18,1.21 ±0.14,0.96±0.10,and 0.79 ±0.08,respectively (t =2.84,P ＜0.01;t =4.05,P ＜0.01;t =6.25,P ＜0.01;t =9.72,P ＜0.01).The levels of PCⅢ and HA secreted in culture media were also decreased.It was significantly decreased with the concentration of adiponectin increased.Conclusion Adiponectin can inhibit the levels of PC Ⅲ and HA,which may be achieved through reducing CTGF mRNA and protein expressions.