蓝莓对大鼠肝星状细胞增殖、活化的影响及机制探讨

目的 观察蓝莓对大鼠肝星状细胞(HSC)增殖、活化的影响及其机制.方法 采用Ⅳ型胶原酶消化和Nycodenz密度梯度离心的方法,分离、培养大鼠HSC,加入10%蓝莓低剂量及10%蓝莓高剂量动物血清干预体外培养的HSC,并设10%丹芍化纤阳性对照血清组(阳性对照血清组)和10%生理盐水血清对照组.用MTT比色法测定各组HSC的增殖效应,ELISA方法测培养液上清Ⅰ型胶原(Col Ⅰ)的含量,免疫细胞染色法检测各组HSC内α-平滑肌肌动蛋白(α-SMA)的表达,采用Western印迹检测HSC、核转录相关因子(Nrf2)、Ⅰ型血红素氧化酶(HO-1)的表达.结果 与生理盐水血清组比较,蓝莓血清组均可显著抑制大鼠HSC的增殖(P＜0.05),其抑制率与阳性对照血清组接近;蓝莓低剂量、高剂量血清组培养液上清Col Ⅰ的含量明显减低(P＜0.05);HSC内α-SMA的表达强度降低(P＜0.05),与阳性对照血清组相当;Western印迹检测显示,与生理盐水血清组比较,蓝莓低剂量、高剂量血清组与阳性对照血清组HSC Nrf2和HO-1蛋白表达增多,以监莓低剂量、高剂量血清组增高更为明显(P＜0.05).结论 蓝莓可抑制体外大鼠HSC的增殖和活化,减少细胞外基质的合成,对大鼠肝纤维化有潜在的干预作用,其机制可能与激活HSC Nrf2增加HO-1的表达有关.

Effects of blueberry on the proliferation and activation of hepatic stellate cell and its mechanism

Objective To investigate the effects of blueberry on the proliferation and activation of hepatic stellate cell (HSC) and its mechanism. Methods Rat HSC were isolated by type Ⅳ collagenase digestion and Nycodenz density gradient centrifugation. The cultured HSC was incubated at different concentrations of 10% blueberry serum. The 10% DSHX serum was used as positive control and 10%normal rat serum group as control. MTT colorimetric assay was used to detect the HSC proliferation. Col Ⅰ of culture supernatant was detected by ELISA. The expression of α-SMA in HSC was measured by immunocytochemistry staining while the expressions of Nrf2 and HO-1 were determined by Western blot. Results Compared with controls, the low and high-dose blueberry serum groups significantly inhibited the HSC proliferation (P ＜0. 05 ). It had the same inhibitory effects as the positive control serum group( P ＞0. 05). Col Ⅰ of culture supernatant obviously decreased ( P ＜ 0. 05 ). And the expression levels of α-SMAin low and high-dose blueberry serum groups decreased significantly ( P ＜ 0. 05 ). And there were similareffects in low & high-dose blueberry serum and positive control serum groups. Western blot showed that theexpressions of Nrf2 and HO-1 in blueberry and positive control serum groups were higher than that in control group. And the increment was more significant in the low and high-dose blueberry serum groups( P ＜0.05 ).Conclusion Blueberry can significantly inhibit the proliferation and activation of HSC and reduce the synthesis of extra-cellular matrix. It may have potential preventive and protective effects on hepatic fibrosis.The mechanism may be related to an elevated expression of HO-1 through the Nrf2 pathway.