Effect of Huazhuojiedu medicated serum on the proliferation and activation of hepatic stellate cells and the expression of PI3K and p-Akt in rats

摘要

To observe the effect of Huazhuojiedu medicated serum on the proliferation and activation of hepatic stellate cells, as well as the expression of PI3K and p-Akt in rats, and to explore the underlying mechanism of Huazhuojiedu prescription against hepatic fibrosis. Hepatic stellate cells harvested from rats were resuscitated and subcultured, followed by the intervention of Huazhuojiedu equivalent dose, Huazhuojiedu double dose, and positive drug (Compound Biejiaruangan Troche) medicated serum of rats. After in vitro culture, hepatic stellate cells were stimulated with 5 ng/mL transforming growth factor-β1. At 24, 48, 72 hours, the proliferation of hepatic stellate cells was detected with MTT assay; at 48 hours, α-SMA mRNA and protein expression in hepatic stellate cells were determined with RT-PCR assay and western blot analysis, respectively, to evaluate the activation of hepatic stellate cells; in addition, PI3K and p-Akt protein expression levels were also assayed with western blot analysis at 48 hours. The results showed that, 24-hour transforming growth factor-β1 stimulation significantly promoted the proliferation of hepatic stellate cells (P < 0.01). Each medicated serum inhibited the proliferation of hepatic stellate cells (P < 0.01). Huazhuojiedu equivalent dose had the similar inhibition effect with positive drug (P > 0.05), and Huazhuojiedu double dose achieved more apparent inhibition effect (P < 0.01). After 48 and 72 hours of transforming growth factor-β1 stimulation, hepatic stellate cells still proliferated significantly (P < 0.01), which was inhibited by each medicated serum (P < 0.01). Huazhuojiedu equivalent dose showed a weaker inhibition effect than positive drug (P < 0.05), and Huazhuojiedu double dose exerted a strong inhibition effect (P < 0.05). After hepatic stellate cells were stimulated with transforming growth factor-β1 for 48 hours, the expression of α-SMA mRNA and protein in hepatic stellate cells was significantly increased (P < 0.01); the medicated serums significantly down-regulated α-SMA mRNA and protein expression, and inhibited the activation of hepatic stellate cells (P < 0.01). Huazhuojiedu equivalent dose showed the similar inhibition effect with positive drug (P > 0.05), and Huazhuojiedu double dose exerted a significant inhibition effect (P < 0.05), which was stronger than Huazhuojiedu equivalent dose (P < 0.05). After hepatic stellate cells were stimulated with transforming growth factor-β1 for 48 hours, PI3K and p-Akt protein expression levels were increased (P < 0.05); each medicated serum down-regulated the elevated expression levels of PI3K and p-Akt (P < 0.05). Huazhuojiedu equivalent dose had the similar down-regulation effect with positive drug (P > 0.05), and Huazhuojiedu double dose achieved more apparent inhibition effect on PI3K expression (P < 0.05). Huazhuojiedu double dose significantly decreased the PI3K and p-Akt protein expression compared with Huazhuojiedu equivalent dose (P < 0.05). Huazhuojiedu medicated serum inhibits the proliferation and activation of hepatic stellate cells induced by transforming growth factor-β1 in vitro, reduces the expression of PI3K and p-Akt protein, and the mechanisms of preventing hepatic fibrosis is mediated by the intervention on PI3K/Akt pathway.