盐酸纳洛酮调控TRL4/NF-κB通路对缺氧缺糖诱导的BV2细胞炎症反应的影响

Effect of Naloxone Hydrochloride on the Inflammatory Response of BV2 Cells Induced by Hypoxia and Glucose-deficiency by Regulating TRL4/NF-κB Pathway

目的:观察盐酸纳洛酮通过调控Toll样受体4(TRL4)/核转录因子κB(NF-κB)通路对缺氧缺糖诱导的BV2细胞炎症反应的影响。方法:培养BV2细胞,采用细胞计数试剂盒(CCK)-8测定不同浓度(0、0.01、0.10、1.00、5.00、10.00、20.00、40.00μmol/L)盐酸纳洛酮对BV2细胞活性的影响。将BV2细胞分为对照组、缺氧缺糖诱导组、盐酸纳洛酮低浓度组(0.01μmol/L)、盐酸纳洛酮高浓度组(5.00μmol/L)、盐酸纳洛酮高浓度+TLR4激活剂组(5.00μmol/L盐酸纳洛酮+1 mg/L脂多糖)。采用乳酸脱氢酶(LDH)试剂盒检测盐酸纳洛酮对BV2细胞的毒性;CCK-8法检测盐酸纳洛酮对BV2细胞增殖率的影响;倒置显微镜观察BV2细胞形态;检测BV2细胞培养上清液一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β水平;逆转录定量聚合酶链式反应(qRT-PCR)检测BV2细胞TNF-α mRNA、IL-6 mRNA、IL-1β mRNA水平;蛋白免疫印迹法(Western Blot)检测BV2细胞TRL4/NF-κB通路相关蛋白表达。结果:浓度为0.01~5.00μmol/L的盐酸纳洛酮对BV2细胞活性无明显影响。与对照组比较,缺氧缺糖诱导组BV2细胞增殖率及LDH、NO、IL-1β、TNF-α、IL-6、IL-1β mRNA、TNF-α mRNA、IL-6 mRNA、TRL4、细胞核NF-κB p65均升高(P<0.05),细胞质NF-κB p65水平降低(P<0.05);与缺氧缺糖诱导组比较,盐酸纳洛酮低浓度组、盐酸纳洛酮高浓度组BV2细胞增殖率及LDH、NO、IL-1β、TNF-α、IL-6、 IL-1βmRNA、TNF-α mRNA、IL-6 mRNA、TRL4、细胞核NF-κB p65降低(P<0.05),细胞质NF-κB p65水平升高(P<0.05);与盐酸纳洛酮高浓度组比较,盐酸纳洛酮高浓度+TLR4激活剂组BV2细胞增殖率及LDH、NO、IL-1β、TNF-α、IL-6、IL-1β mRNA、TNF-α mRNA、IL-6 mRNA、TRL4、细胞核NF-κB p65升高(P<0.05),细胞质NF-κB p65水平降低(P<0.05)。结论:盐酸纳洛酮可能通过抑制TRL4/NF-κB通路,降低缺氧缺糖诱导的BV2细胞炎症反应。

Objective:To investigate the effect of naloxone hydrochloride on the Inflammatory response of BV2 cells induced by hypoxia and glucose-deficiency by regulating Toll-like receptor 4(TRL4)/nuclear transcription factor κB(NF-κB) pathway.Methods:The effects of naloxone hydrochloride at different concentrations(0,0.01,0.10,1.00,5.00,10.00,20.00,40.00 μmol/L) on BV2 cell activity were determined by Cell Counting Kit(CCK)-8 method.BV2 cells were divided into the control group,hypoxia and glucose-deficiency induced group,naloxone hydrochloride low-dose group(0.01 μmol/L),naloxone hydrochloride high-dose group(5.00 μmol/L),and naloxone hydrochloride high-dose+TLR4 activator group(5.00 μmol/L naloxone hydrochloride+1 mg/L lipopolysaccharide).Lactate dehydrogenase(LDH) kit was used to detect the toxicity of naloxone hydrochloride on BV2 cells.Cell counting kit(CCK8) assay was used to detect the effect of naloxone hydrochloride on the proliferation rate of BV2 cells.Inverted microscope was used to observe the morphology of BV2 cells.The levels of nitric oxide(NO),tumor necrosis factor α(TNF-α),interleukin(IL)-6,and IL-1β in the supernatant of BV2 cell culture were detected.The levels of TNF-α mRNA,IL-6 mRNA,and IL-1β mRNA in BV2 cells were detected by reverse transcription quantitative polymerase chain reaction(qRT-PCR).The expressions of TRL4/NF-κB pathway-related proteins in BV2 cells were detected by protein immunoblotting(Western Blot).Results:Naloxone hydrochloride at the concentrations from 0.01 to 5.00 μmol/L showed no significant effect on the activity of BV2 cells.Compared with the control group,the proliferation rate of BV2 cells,LDH,NO,IL-1β,TNF-α,IL-6,IL-1β mRNA,TNF-α mRNA,IL-6 mRNA,TRL4,and cytosolic NF-κB p65 were increased in the hypoxia and glucose-deficiency induced group,and level of cytoplasmic NF-κB p65 was decreased(P0.05).Compared with the hypoxia and glucose-deficiency induced group,the proliferation rate of BV2 cells,LDH,NO,IL-1β,TNF-α,IL-6,IL-1β mRNA,TNF-α mRNA,IL-6 mRNA,TRL4,and cytosolic NF-κB p65 were decreased in the naloxone hydrochloride low-dose group and the naloxone hydrochloride high-dose group,and level of cytoplasmic NF-κB p65 was increased(P0.05).Compared with the naloxone hydrochloride high-dose group,the proliferation rate of BV2 cells,LDH,NO,IL-1β,TNF-α,IL-6,IL-1β mRNA,TNF-α mRNA,IL-6 mRNA,TRL4,and cytosolic NF-κB p65 were increased in the naloxone hydrochloride high-dose+TLR4 activator group,and level of cytoplasmic NF-κB p65 was decreased(P0.05).Conclusion:Naloxone hydrochloride alleviated inflammatory response of BV2 cells induced by hypoxia and glucose-deficiency by inhibiting the TRL4/NF-κB pathway.