目的 探讨黄芩苷对缺氧缺糖（oxygen-glucose deprivation，OGD）诱导的神经元细胞增殖、凋亡、炎症、氧化应激的影响及其对miR-190的调控作用。方法 采用OGD处理小鼠海马神经元HT22细胞建立细胞损伤模型，使用不同剂量（10、30 mg/L）的黄芩苷处理HT22细胞，分别将miR-NC、miR-190 mimics转染至HT22细胞后进行OGD处理（OGD+miR-NC组、OGD+miR-190组）；分别将anti-miR-NC、anti-miR-190转染至HT22细胞后使用黄芩苷处理24 h，随后进行OGD处理（OGD+黄芩苷+anti-miR-NC组、OGD+黄芩苷+anti-miR-190组）；采用MTT法检测细胞增殖情况；采用流式细胞术检测细胞凋亡率；采用ELISA法检测白细胞介素-1β（interleukin-1β，IL-1β）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的水平；采用2，4-二硝基苯肼显色法检测乳酸脱氢酶（lactate dehydrogenase，LDH）的活性；采用黄嘌呤氧化酶法检测超氧化物歧化酶（superoxide dismutase，SOD）的含量；采用qRT-PCR法检测miR-190的表达量；Western blotting法检测细胞周期蛋白1（Cyclin D1）、未剪切的半胱天冬酶-3（pro-cysteinyl aspartate-specific protease-3，pro-Caspase-3）、p21、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved-cysteinyl aspartate-specific protease-3，cleaved-Caspase-3）蛋白表达量。结果 OGD可明显降低HT22细胞活力及CyclinD1、pro-Caspase-3蛋白水平（P<0.05），升高细胞凋亡率及p21、cleaved-Caspase-3蛋白水平（P<0.05），并可提高IL-1β、TNF-α的水平及LDH的活性（P<0.05），降低SOD的含量（P<0.05）；黄芩苷可明显提高OGD诱导的HT22细胞活力及CyclinD1、pro-Caspase-3蛋白水平（P<0.05），降低细胞凋亡率及p21、cleaved-Caspase-3蛋白水平（P<0.05），并可降低IL-1β、TNF-α的水平及LDH的活性（P<0.05），升高SOD的含量（P<0.05）；OGD可明显降低HT22细胞中miR-190的表达水平（P<0.05），而黄芩苷可明显升高miR-190的表达水平（P<0.05）；miR-190过表达对OGD诱导的HT22细胞增殖、凋亡、炎症及氧化应激的作用与黄芩苷的作用相似；抑制miR-190表达可明显逆转黄芩苷对OGD诱导的HT22细胞增殖、凋亡、炎症及氧化应激的作用。结论 黄芩苷可通过上调miR-190的表达而促进细胞增殖及抑制炎症反应、氧化应激、细胞凋亡从而减轻OGD诱导的神经元细胞损伤。

Objective To explore the effects of baicalin on the proliferation, apoptosis, inflammation, and oxidative stress of neuronal cells induced by oxygen-glucose deprivation (OGD) and its regulation on miR-190. Methods OGD was used to treat mouse hippocampal neuronal cells HT22 to establish a cell injury model, and different doses (10, 30 mg/L) of baicalin was used to treat the cells. HT22 cells were transfected with miR-NC or miR-190 mimics and then treated with OGD (OGD + miR-NC group, OGD + miR-190 group). HT22 cells were transfected with anti-miR-NC and anti-miR-190 and treated with baicalin for 24 h, followed by OGD treatment (OGD + baicalin high-dose group + anti-miR-NC group, OGD + baicalin high-dose group + anti-miR-190 group). The MTT method and flow cytometry were used to detect cell proliferation and apoptosis rate, respectively. The levels of IL-1β and TNF-α were detected by ELISA. The 2,4-dinitrophenylhydrazine color method was performed to assess the activity of lactate dehydrogenase (LDH). Xanthine oxidase method was used to detect the content of SOD. The expression of miR-190 was determined by qRT-PCR. And the protein expression of CyclinD1, pro-Caspase-3, p21, and cleaved-Caspase-3 was detected by Western blot. Results OGD significantly reduced HT22 cell viability and the protein levels of CyclinD1 and pro-Caspase-3 (P<0.05), increased cell apoptosis rate and the protein levels of p21 and cleaved-Caspase-3 (P<0.05), as well as increased the levels of IL-1β, TNF-α and the activity of LDH (P<0.05), reduced the content of SOD (P<0.05). Baicalin could significantly increase OGD-induced HT22 cell viability and the protein levels of CyclinD1 and pro-Caspase-3 (P<0.05), reduce cell apoptosis rate and the protein levels of p21 and Cleaved-caspase3 (P<0.05), and reduce the levels of IL-1β, TNF-α and the activity of LDH (P<0.05), and increase the content of SOD (P<0.05). OGD significantly reduced the expression level of miR-190 in HT22 cells (P<0.05), while baicalin significantly increased the expression level of miR-190 (P<0.05). The effects of miR-190 overexpression on the proliferation, apoptosis, inflammation and oxidative stress of HT22 cells induced by OGD were similar to those of baicalin. Inhibition of miR-190 expression reversed the effects of baicalin on the proliferation, apoptosis, inflammation and oxidative stress of OGD-induced HT22 cells. Conclusion Baicalin could promote cell proliferation and inhibit inflammation, oxidative stress, and apoptosis by up-regulating the expression of miR-190, thereby alleviating OGD-induced neuronal cell damage.

R285.5

国家中医药管理局国家中医临床研究基地业务建设科研专项（ZDZX20120660）；河南省中医药科学研究专项（2018JDZX109）