葛根素通过circ-TLK1/miR-367-3p/TLR4抑制SK-N-SH细胞氧糖剥夺损伤

Puerarin inhibits oxygen-glucose deprivation injury of SK-N-SH cells through circ-TLK1/miR-367-3p/TLR4

目的探讨葛根素对缺血性脑卒中(ischemicstroke,IS)神经细胞损伤的保护作用及机制。方法建立氧糖剥夺(oxygen-glucose deprivation,OGD)诱导的人神经母细胞瘤细胞SK-N-SH损伤模型,以葛根素进行干预,采用q RT-PCR法检测细胞circ-丝氨酸/苏氨酸蛋白扰动样激酶1(tousled-like kinase1,TLK1)和miR-367-3p mRNA表达情况;采用MTT法检测细胞存活率;采用流式细胞术检测细胞凋亡情况;采用ELISA法检测细胞上清液中白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;采用Western blotting检测细胞Toll样受体4(Toll-like receptor 4,TLR4)蛋白表达情况。采用生物信息学在线软件预测miR-367-3p与circ-TLK1、TLR4之间的靶向关系;采用双荧光素酶标记实验和RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation,RIP)实验验证靶向结合关系。结果与模型组比较,葛根素能够明显提高OGD诱导的SK-N-SH细胞存活率(P<0.05、0.01、0.001),显著降低细胞凋亡率(P<0.01、0.001),显著降低IL-6和TNF-α水平(P<0.01、0.001)。模型组circ-TLK1 mRNA表达水平显著升高(P<0.001),葛根素抑制circ-TLK1 mRNA表达水平(P<0.001),过表达circ-TLK1能够逆转葛根素对OGD诱导的SK-N-SH细胞损伤保护作用(P<0.01、0.001)。circ-TLK1能够靶向负调控miR-367-3p表达,模型组miR-367-3p mRNA表达水平显著降低(P<0.001),葛根素显著上调miR-367-3p m RNA表达水平,抑制miR-367-3p能够逆转葛根素对OGD诱导的SK-N-SH细胞损伤保护作用(P<0.05、0.001)。miR-367-3p能够靶向负调控TLR4蛋白表达,模型组TLR4蛋白表达水平显著升高(P<0.001),葛根素显著下调TLR4蛋白表达水平(P<0.001),过表达TLR4能够逆转葛根素对OGD诱导的SK-N-SH细胞损伤保护作用(P<0.05、0.001)。circ-TLK1能够通过结合miR-367-3p调控TLR4表达。结论葛根素能够通过作用于circ-TLK1/miR-367-3p/TLR4对IS神经细胞损伤起到保护作用。

Objective To explore the protective effect and mechanism of puerarin on neuronal damage in ischemic stroke(IS). Methods Oxygen glucose deprivation(OGD) induced SK-N-SH cells injury model was established. Puerarin was used to intervene SK-N-SH cells induced by OGD. Expression of circ-TLK1 and miR-367-3 p mRNA was detected by qRT-PCR. Cell viability was measured by MTT. Apoptosis was detected by flow cytometry. Levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were analyzed by ELISA. Expression of Toll-like receptor 4(TLR4) was detected by Western blotting. Bioinformatics online software was used to predict the targeting relationship between miR-367-3 p and circ-TLK1 or TLR4. Double luciferase labeling assay and RNA binding protein immunoprecipitation(RIP) assay were used to verify the targeted binding relationship. Results Compared with model group,puerarin significantly increased survival rate of SK-N-SH cells induced by OGD(P < 0.05,0.01,0.001),reduced rate of apoptosis(P < 0.01,0.001),reduced levels of IL-6 and TNF-α(P < 0.01,0.001). circ-TLK1 mRNA level of SK-N-SH cells in model group was significantly increased(P < 0.001),puerarin inhibited circ-TLK1 m RNA level(P < 0.001),overexpression of circ-TLK1 reversed the protective effect of puerarin on SK-N-SH cells induced by OGD(P < 0.01,0.001). circ-TLK1 targeted and negatively regulated miR-367-3 p expression. miR-367-3 p mRNA level of SK-N-SH cells in model group was significantly reduced(P < 0.001),puerarin significantly up-regulated miR-367-3 p mRNA level,inhibited miR-367-3 p reversed the protective effect of puerarin on SK-N-SH cells induced by OGD(P < 0.05,0.001). miR-367-3 p targeted and negatively regulated TLR4 expression. TLR4 protein expression of SK-N-SH cells in model group was significantly increased(P < 0.001),puerarin significantly down-regulated TLR4 protein expression(P < 0.001),overexpression of TLR4 reversed protective effect of puerarin on SK-N-SH cells induced by OGD(P < 0.05,0.001). circ-TLK1 regulated TLR4 expression by binding miR-367-3 p. Conclusion Puerarin can protect IS nerve cells from damage via circ-TLK1/miR-367-3 p/TLR4.