Objective

2

To explore the mechanism by which Yiguanjian in inhibiting the proliferation of hepatic stellate cells （HSCs） based on the Wnt/β-catenin signaling pathway.

Methods

2

The MTT assay was used to determine the optimal concentration of carbon tetrachloride （CCl4） to induce activation of rat HSCs-T6 and the optimal drug intervention concentration of Yiguanjian. The cells were divided into the normal control group， model group， and Yiguanjian low， medium， and high-dose groups. Activated HSCs-T6 were treated with different concentrations of Yiguanjian freeze-dried powder. The MTT assay was used to determine the proliferation inhibition rate of HSCs-T6 in each group， and cell morphology was observed under an inverted microscope. Reverse transcription PCR （RT-PCR） was used to detect the mRNA expression levels of activation-related proteins Col-1 and α-SMA in HSCs-T6 in each group. Western blot was used to assess the protein expression of Col-1， α-SMA， and key proteins in the Wnt/β-catenin signaling pathway， including Wnt1， β-catenin， and GSK-3β.

Results

2

After 24 hours of stimulation with 6 mmol/L CCl4， the highest proliferation rate of HSCs-T6 was observed， so 6 mmol/L was selected as the optimal experimental concentration. Compared with the negative control group， cell viability was significantly lower after intervention with 2 000 and 4 000 µg/mL Yiguanjian （

P

<

0.05）， so 500， 1 000， and 1 500 µg/mL were chosen as the experimental concentrations of Yiguanjian. Compared with the normal control group， the model group showed a lower proliferation inhibition rate after 24 hours （

P

<

0.05）. Compared with the model group， the Yiguanjian low， medium， and high-dose groups exhibited higher proliferation inhibition rates after 24 hours （

P

<

0.05）. In the Yiguanjian low， medium， and high-dose groups， cells showed varying degrees of decreased density， nuclear shrinkage， and rounding compared with the normal control and model groups. Compared with the normal control group， the model group showed higher mRNA expression levels of Col-1 and α-SMA （

P

<

0.05）. Compared with the model group， the Yiguanjian low， medium， and high-dose groups showed significantly reduced mRNA expression

levels of Col-1 and α-SMA （

P

<

0.05， with the high-dose group showing lower mRNA expression levels of Col-1 and α-SMA than the low and medium-dose groups （

P

<

0.05）. Compared with the normal control group， the model group showed higher protein expression levels of Col-1 and α-SMA （

P

<

0.05）. Compared with the model group， the Yiguanjian low and medium-dose groups showed lower protein expression levels of Col-1， and the Yiguanjian low， medium， and high-dose groups showed lower protein expression levels of α-SMA （

P

<

0.05）. In particular， the Yiguanjian high-dose group showed higher expression of Col-1 protein and lower expression of α-SMA protein compared with the low and medium-dose groups （

P

<

0.05）. Compared with the normal control group， the model group showed higher protein expression levels of Wnt1 and lower expression levels of GSK-3β and β-catenin （

P

<

0.05）. Compared with the model group， the Yiguanjian low， medium， and high-dose groups showed lower protein expression levels of Wnt1 and β-catenin， and higher protein expression levels of GSK-3β （

P

<

0.05）， with the Yiguanjian high-dose group showing lower expression levels of Wnt1 and β-catenin and higher expression of GSK-3β compared with the low and medium-dose groups （

P

<

0.05）.

Conclusion

2

Yiguanjian effectively inhibits the proliferation of HSCs， and its mechanism of action may be related to the inhibition of Wnt/β-catenin signaling pathway activation.