腰突颗粒通过调控miR-221表达干预髓核细胞的增殖与凋亡

Yaotu Granule deals with the proliferation and apoptosis of nucleus pulposus cells via regulating the expression of microRNA-221

背景:microRNA与椎间盘退变密切相关,腰突颗粒能显著延缓椎间盘退变进展。目的:通过研究miR-221对髓核细胞增殖与凋亡功能的影响及腰突颗粒对miR-221的调控作用探讨腰突颗粒延缓椎间盘退变的机制。方法:应用miR-221模拟物(模拟物组)及抑制剂(抑制剂组)转染大鼠髓核细胞,肿瘤坏死因子α诱导髓核细胞退变模型(模型组),并分别应用100,200 mg/L腰突颗粒冻干粉处理退变的髓核细胞(腰突颗粒低、高浓度组),模拟物+腰突颗粒组为同时加入miR-221模拟物和200 mg/L腰突颗粒冻干粉处理的髓核细胞。采用CCK-8法检测细胞增殖,TUNEL法检测凋亡情况,RT-qPCR检测miR-221的mRNA表达,Western blot检测凋亡相关蛋白Bax和Bcl-2的蛋白表达。结果与结论:①与模拟物阴性对照组比较,模拟物组在24,48,72 h细胞增殖减缓(P <0.05),凋亡增多(P <0.05);与抑制剂阴性对照组比较,抑制剂组在24,48,72 h细胞增殖明显(P <0.05),凋亡减少(P <0.05);与正常组比较,模型组细胞增殖抑制(P <0.05),凋亡增加(P <0.05),miR-221表达上调(P <0.05);②与模型组比较,腰突颗粒低、高浓度组细胞明显增殖(P <0.05),细胞凋亡率下降(P <0.05),miR-221表达下调(P <0.05);与模拟物组比较,模拟物+腰突颗粒组细胞凋亡降低(P <0.05),细胞增殖改善(P <0.05),Bax蛋白表达下降(P <0.05),Bcl-2蛋白表达增高(P <0.05)。③提示:miR-221影响髓核细胞增殖与凋亡,腰突颗粒通过下调miR-221的表达发挥延缓椎间盘退变的作用。

BACKGROUND:MicroRNA is involved in intervertebral disc degeneration,and Yaotu Granule can significantly delay the progress of intervertebral disc degeneration.OBJECTIVE:To investigate the effect of Yaotu Granule in the proliferation and apoptosis of nucleus pulposus cells by regulating the expression of microRNA-221(miR-221).METHODS:Mi R-221 mimics and inhibitors were transfected into rat nucleus pulposus cells to establish miR-221 overexpression and inhibition models.Tumor necrosis factor-αwas used to induce nucleus pulposus cell degeneration model.The degenerated nucleus pulposus cells were treated with 100 and 200 mg/L Yaotu Granule freeze-dried powder(low and high dose groups).In the miR-221 mimics+Yaotu Granule group,miR-221 mimic and 200 mg/L Yaotu Granule freeze-dried powder were added.Cell counting kit-8 and TUNEL assay were used to detect the cell proliferation and apoptosis,RT-qPCR was used to detect mRNA expression of miR-221,and western blot was used to detect the expression of Bax and Bcl-2.RESULTS AND CONCLUSION:MiR-221 mimics reduced cell prolifecation at 24,48,72 hours(P<0.05)and promoted cell apoptosis at 48 hours(P<0.05);miR-221 inhibitors had the opposite results(P<0.05).Compared with the normal group,tumor necrosis factor-αinhibited cell proliferation,increased cell apoptosis,and upregulated miR-221 expression(all P<0.05).Compared with the model group,low-and high-dose Yaotu Granules increased cell proliferation,reduced cell apoptosis,and downregulated miR-221 expression(all P<0.05).Compared with the mimics group,cell proliferation improved,apoptosis rate decreased,the protein expression of Bax reduced,and the Bcl-2 protein expression up-regulated in the miR-221 mimics+Yaotu Granule group(all P<0.05).To conclude,miR-221 has an effect on the proliferation and apoptosis of nucleus pulposus cells,and Yaotu Granule can delay the progression of intervertebral disc degeneration by downregulating the expression of miR-221.