基于Pink-1/Parkin信号通路补青颗粒含药血清对高糖诱导人晶状体上皮细胞线粒体自噬的影响

Effect of Buqing Granules Medicated Sera on Mitochondrial Autophagy in High Glucose-induced Human Lens Epithelial Cells Based on Pink-1/Parkin Signaling Pathway

目的:探讨补青颗粒含药血清对高糖诱导的人晶状体上皮(HLE)细胞线粒体自噬的影响。方法:体外培养HLE细胞,空白组将HLE细胞在正常培养液中培养48 h,模型组在空白组培养液中添加30 mmol·L^(-1)葡萄糖,实验组在模型组基础上加入不同含药血清刺激培养48 h。采用CCK-8法检测不同含药血清对高糖诱导的HLE细胞增殖活力的影响;利用免疫荧光技术及蛋白免疫印迹(Western blot)法检测Pink-1,Parkin,p62蛋白表达。结果:CCK-8法结果显示模型组较空白组HLE细胞增殖活力增强(P<0.01);与模型组比较,杞菊组与补青颗粒高、中、低剂量组细胞增殖活力均降低(P<0.01);与杞菊组比较,补青颗粒中剂量组细胞增殖活力明显降低(P<0.01);补青颗粒不同剂量组间比较,补青颗粒中剂量组细胞增殖活力显著降低(P<0.01)。细胞免疫荧光结果显示Pink-1,Parkin,p62蛋白主要在细胞胞浆表达,细胞核内亦有少量表达。Western blot结果显示与空白组比较,模型组Pink-1,Parkin表达增加(P<0.01),p62表达降低(P<0.01);与模型组比较,杞菊组与补青颗粒中剂量组Pink-1,Parkin表达增加(P<0.01),p62表达降低(P<0.01);与杞菊组比较,补青颗粒中剂量组Pink-1,Parkin表达增加(P<0.01,P<0.05),p62表达降低(P<0.01)。结论:补青颗粒对高糖诱导的HLE细胞线粒体自噬相关蛋白的表达有显著影响,线粒体自噬的改变可能是补青颗粒改善白内障发病的机制之一。

Objective： To discuss the effect of Buqing granules-medicated sera on the human lens epithelial cells induced by high glucose. Method： Human lens epithelial （HLE） cell lines were cultured in vitro. HLE cells of blank control group were cultured in the normal nutrient solution for 48 h. In addition to that, model group was also added with 30.0 mmol·L-1 glucose for 48 h. In addition to that, treatment group was also added with different medicated sera and cultured for 48 h. The cell＇s activity of proliferation was detected by CCK-8 assay. Protein expressions of Pink-l, Parkin, p62 were detected by Western blot. Result： According to CCK-8assay, model group showed a higher HLE cells proliferation activity than the blank group （ P 〈 0.01 ）. Compared with model group, the cell＇s activity of proliferation of Qiju group and high, middle and low-dose Buqing granules groups were lower （P 〈 0.01 ）. Compared with Qiju group, the cell＇s activity of proliferation of middle-dose Buqing granules group was obviously decreased （P 〈 0.01 ）. Among different Buqing granules groups, the cell＇s activity of proliferation of middle-dose Buqing Granules group was significantly decreased （P 〈 0.01 ）. According to immunofluorescence staining, Pink-l, Parkin, p62 proteins were expressed mainly in cell cytoplasm and rarely in nuclei. According to Western blot, compared with blank group, protein expression levels of Pink-l, Parkin of model group were increased （P 〈 0.01 ） , and p62 expression was decreased （ P 〈 0.01 ）. Compared with model group, protein expression levels of Pink-l, Parkin of Qiju group and middle-dose Buqing granules group were increased （P 〈 0.01 ）, and p62 expression was decreased （P 〈 0.01 ）. Compared with Qiju group, protein expression levels of Pink-1, Parkin of middle-dose Buqing granules group were increased （P 〈 0. 01 ）, and p62 expression was decreased （P 〈 0.01 ）. Conclusion： Buqing granules have a significant effect on protein expressions related to mitochondrial autophagy of HLE cells induced by high glucose. The change in mitochondrial autophagy may be one of the mechanisms of Buqing Granules in alleviating cataract.