| 张威,王帅,王喜梅,等.葛根素调控miR-7抗小鼠肝细胞缺氧-复氧存活和细胞凋亡的作用机制[J].安徽医药,2020,24(9):1718-1724. |
| 葛根素调控miR-7抗小鼠肝细胞缺氧-复氧存活和细胞凋亡的作用机制 |
| Mechanism of puerarin regulating miR-7 against hypoxia- reoxygenation survival and apoptosis in mouse hepatocytes |
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| DOI：10.3969/j.issn.1009-6469.2020.09.006 |
| 中文关键词: 葛根素 再灌注损伤 细胞低氧 小鼠肝细胞 细胞凋亡 miR-7 核因子 -κB |
| 英文关键词: Puerarin Reperfusion injury Cell hypoxia Mouse hepatocytes Apoptosis MicroRNA-7 Nuclear factor-κB |
| 基金项目:洛阳市科技计划资助项目( 1603003A-6)；洛阳市科技计划项目(1812002A) |
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| 中文摘要: |
| 目的探讨葛根素对缺氧 -复氧( H-R)诱导小鼠肝细胞损伤的影响及其分子机制。方法将小鼠肝细胞采用随机数字表法分为对照组、 H-R组、不同浓度( 70 μmol/L、140 μmol/L、280 μmol/L、560 μmol/L)葛根素组、 H-R+葛根素组、 H-R+miR-7组、 H-R+miR-7阴性对照( miR-con)组、 H-R+葛根素 +anti-miR-7组、 H-R+葛根素 +ant-miR-7阴性对照( anti-miR-con)组、 H-R+葛根素 +佛波酯( PMA)组。实时荧光定量 PCR(qPCR)检测 miR-7水平，四甲基偶氮唑盐微量酶反应比色法( MTT法)检测细胞活性，流式细胞仪检测细胞凋亡，蛋白质印迹法( Western Blot)检测细胞中细胞核相关抗原 Ki-67(Ki-67)、 B细胞淋巴瘤 /白血病 -2(Bcl-2)、 Bcl-2相关 X蛋白( Bax)、核因子 -κB(NF-κB)p65蛋白表达。结果葛根素预处理后， H-R小鼠肝细胞活性明显增强(P＜0.05)Ki-67蛋白表达量、 miR-7表达量显著增加( P＜0.05)。与 H-R组比较， 280 μmol/L葛根素预处理后， H-R小鼠肝细胞凋亡率［(14.7，2±1.20)%比( 25.85±1.58)%］、 Bax、NF-κB p65表达量降低， Bcl-2蛋白表达量增加(均 P＜0.05)。与 H-R+miR-con组相比，过表达 miR-7显著提高 H-R小鼠肝细胞 miR-7水平、细胞活性［(81.29±7.56)比( 0.28±0.04)］、 Ki-67、Bcl-2蛋白水平，降低细胞凋亡率［(15.65±1.56)%比( 26.65±1.25)%］、 Bax蛋白水平(均 P＜0.05)。干扰 miR-7表达部分逆转葛根素对小鼠肝细胞 H-R后细胞凋亡、 Bax和 NF-κB p65表达的抑制作用，以及部分逆转葛根素对小鼠肝细胞 H-R后细胞活性、 miR-7、Ki-67、Bcl-2表达的促进作用。结论葛根素显著促进 H-R诱导的小鼠肝细胞增殖并抑制细胞凋亡，可能是通过调控 miR-7表达，抑制 NF-κB信号通路活性发挥作用。 |
| 英文摘要: |
| Objective To investigate the effect and molecular mechanism of puerarin on hepatocytes injury induced by hypoxia and reoxygenation(H-R)inmice.Methods mouse hepatocytes were randomly divided into control group H-R group，puerarin group with different concentrations(70 μmol/L，140 μmol/L，280 μmol/L，560 μmol/L)，H-R+puerarin group，H-R+microRNA-7(miR-7) group，H-R+miR-7 negative control miR-con group，H-R+puerarin+anti-miR-7 group，H-R+puerarin+anti-miR -7 negative control anti-miR-con group，H-R + puerarin + phorbol ester(PMA)group by random number table method.miR-7 level was determined by real-time quantitative PCR(qPCR)，cell viability was detected by methyl thiazolyl tetrazolium(MTT)assay，cellapoptosis was de-tected by flow cytometry，and expressions of Nuclear associated antigen Ki67(Ki-67)、 B cell lymphoma/lewkmia-2(Bcl-2)，Bcl-2 associated X protein(Bax)and nuclear factor-κB(NF-κB)p65 were analyzed by Western Blot.Results After pretreatment with puerarin，the cell viability of mouse hepatocytes in H-R was obviously increased(P＜0.05)，and the expression of Ki-67 protein、miR-7 notably increased(P＜0.05).Compared with H-R group，after pretreatment of 280 μmol/L puerarin，the apoptosis rate［( 14.72±1.20)% vs.(25.85±1.58)%］，expressions of Bax and NF-κB p65 decreased and Ki-67、Bcl-2 protein expressions in- creased in H-R mouse hepatocytes，and all the differences reached significant(all P＜0.05).Compared with H-R+miR-con group， overexpression of miR-7 greatly increased the level of miR-7，cell viability［(81.29±7.56)vs.(0.28±0.04)］and Bcl-2 protein in H-R mouse hepatocytes，and decreased the apoptosis rate［(15.65±1.56)% vs.(26.65±1.25)%］and Bax protein level，and all the dif- ferences reached significant(all P＜0.05).Interfering with miR-7 expression partially reversed the inhibitory effect of puerarin on apoptosis，Bax and NF-κB p65 expression after H-R of mouse hepatocytes，and partially reversed the promoting effect of puerarin on cell activity，miR-7，Ki-67 and Bcl-2 expression after H-R of mouse hepatocytes.Conclusion Puerarin significantly promotes the proliferation and inhibits the apoptosis of mouse hepatocytes induced by H-R，possibly by regulating the expression of miR-7 and inhibiting the the activity ofNF-κB signaling pathway. |
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