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目的探讨莱菔硫烷(SFN)对体外培养皮质神经元氧糖剥夺(OGD)/复氧(R)损伤的保护作用。方法出生0～1 d的SD乳大鼠皮质神经元原代培养5～6 d后,随机分为正常对照组、OGD组及SFN组。建立皮质神经元的OGD模型,1 h后复氧,模拟再灌注模型,OGD/R期间给予0.1、0.5、1、2.5μmol/L SFN,复氧24 h后采用四甲基偶氮唑盐(MTT)微量酶反应比色法测定细胞存活率,测定乳酸脱氢酶(LDH)的释放量来衡量细胞损伤程度,免疫荧光和Westernblot观察神经元OGD/R后8-羟基脱氧鸟苷(8-OHDG)和NADPH醌氧化还原酶(NQO1)的表达。结果 MTT检测示神经元的细胞存活率1μmol/L SFN组显著高于OGD组[(54.56±8.75)%vs(37.51±9.39)%,P<0.01]。SFN组LDH的释放量显著低于OGD组[(330.91±152.33)vs(769.87±275.63)mmol/L,P<0.01]。免疫荧光检测示SFN组8-OHDG的阳性表达率显著低于OGD组[(42.10±8.53)%vs(72.50±10.85)%,P<0.01],而SFN组NQO1的阳性表达率显著高于OGD组[(90.47±5.46)%vs(70.90±7.92)%,P<0.01]。Western blot检测示SFN组NQO1的相对灰度值显著高于OGD组[(0.760±0.063)vs(0.497±0.056),P<0.01]。结论 SFN可减轻OGD/R引起的皮质神经元的损伤。降低8-OHDG的表达和增加NQO1的表达是其可能机制之一。
Objective To investigate the protective effect of sulforaphane (SFN) on oxygen glucose deprivation (OGD) / reoxygenation (R) injury in cultured cortical neurons. Methods Primary cultured cortical neurons from SD rats aged 0 to 1 day were cultured for 5-6 d. They were randomly divided into normal control group, OGD group and SFN group. OGD models of cortical neurons were established. After 1 h of reoxygenation and reperfusion, 0.1, 0.5, 1 and 2.5 μmol / L SFN were given during OGD / R, MTT assay was used to measure the cell viability. The level of lactate dehydrogenase (LDH) release was measured to measure the degree of cell injury. Immunofluorescence and Western blotting were used to observe the neuronal OGD / R after 8-hydroxydeoxyguanosine (8- OHDG) and NADPH quinone oxidoreductase (NQO1). Results MTT assay showed that the survival rate of neurons in 1μmol / L SFN group was significantly higher than that in OGD group [(54.56 ± 8.75)% vs (37.51 ± 9.39)%, P <0.01]. The release of LDH in SFN group was significantly lower than that in OGD group [(330.91 ± 152.33) vs (769.87 ± 275.63) mmol / L, P <0.01]. Immunofluorescence showed that the positive expression rate of 8-OHDG in SFN group was significantly lower than that in OGD group (42.10 ± 8.53)% vs (72.50 ± 10.85)%, P <0.01], while the positive expression rate of NQO1 in SFN group was significantly higher than that in OGD group Group [(90.47 ± 5.46)% vs (70.90 ± 7.92)%, P <0.01]. Western blot showed that the relative gray value of NQO1 in SFN group was significantly higher than that in OGD group [(0.760 ± 0.063) vs (0.497 ± 0.056), P <0.01]. Conclusion SFN can reduce the damage of cortical neurons caused by OGD / R. Reducing the expression of 8-OHDG and increasing the expression of NQO1 are one of its possible mechanisms.