青光安Ⅱ号方对诱导损伤的RGC-5细胞中NF-κB/HIF-1α通路相关细胞因子的影响

Effects of QingguanganⅡFormula on NF-κB/HIF-1αPathway Related Cytokines in Injury-induced RGC-5 Cells

目的观察青光安Ⅱ号方对谷氨酸诱导损伤的RGC-5细胞中核因子κB(nuclear factor kappa-B,NF-κB)、低氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)、BCL2/腺病毒E1B相互作用蛋白3(BCL2/adenovirus E1B 19kDa interacting protein 3,BNIP3)、超氧化物歧化酶(superoxide dismutase,SOD)及丙二醛(malondialdehyde,MDA)的影响。方法体外培养RGC-5细胞,分为4组:空白组、模型组、含药血清组和阻断剂组。模型组、含药血清组和阻断剂组予以谷氨酸诱导细胞损伤,模拟青光眼对神经节细胞的损伤,含药血清组加入青光安Ⅱ号方含药血清,阻断剂组加入KC7F2进行HIF-1α通路的阻断。以CCK-8法摸索含药血浆以及谷氨酸的最佳干预浓度;采用Hoechst法检测各组细胞的凋亡情况;Western blot检测NF-κB、HIF-1α、BNIP3蛋白表达情况;比色法检测SOD、MDA的表达情况。结果CCK8法确定青光安Ⅱ号方5倍组含药血清为实验量,确定谷氨酸的最佳干预浓度为200μM。与空白组比较,模型组NF-κB、HIF-1α、BNIP3、SOD、MDA表达显著升高(P<0.05);与模型组比较,含药血清组、阻断剂组NF-κB、HIF-1α、BNIP3和SOD的表达显著降低(P<0.05);含药血清组与阻断剂组的NF-κB、HIF-1α、BNIP3、SOD及MDA差异均无统计学意义(P>0.05)。结论青光安Ⅱ号方对NF-κB具有抑制作用,进而抑制了HIF-1α相关通路的激活,减弱了由于氧化应激所致RGC-5细胞的凋亡,对RGC具有保护作用。

Objective To observe the effects of QingguanganⅡFormula on nuclear factor kappa-B(NF-κB),hypoxia inducible factor-1(HIF-1α),BCL2/adenovirus E1B 19kDa interacting protein 3(BNIP3),superoxide dismutase(SOD)and malondialdehyde(MDA)in RGC-5 cells injury induced by glutamate.Methods The RGC-5 cells cultured in vitro were divided into 4 groups blank group,model group,drug-containing serum group and blocker group.Glutamic acid was added to model group,drug-containing serum group and blocker group to simulate retinal ganglion cell damage caused by glaucoma,and QingguanganⅡFormula serum was added to drug-containing serum group,and the blocker group was added with KC7F2 to block the HIF-1αpathway.The CCK-8 method was used to find optimal intervention concentration of drug-containing plasma and glutamate;Hoechst method was used to detect the apoptosis of cells in each group;Western blot was used to detect the expression of NF-κB,HIF-1α,BNIP3;colorimetry was used to detect the expression of SOD and MDA.Results CCK8 method determined 5 times serum content as the QingguanganⅡFormula experimental amount,and the optimal intervention concentration of glutamate was 200μM.Compared with blank group,the expression of NF-κB,HIF-1α,BNIP3,SOD and MDA increased significantly in the model group(P<0.05);compared with the model group,the expression of NF-κB,HIF-1α,BNIP3 and SOD in the drug-containing serum group and blocker group significantly decreased(P<0.05);there was no statistically significant difference between NF-κB,HIF-1α,BNIP3,SOD and MDA in drug-containing serum group and blocker group(P>0.05).Conclusion Qingguangan II Formula has an inhibitory effect on NF-κB,thereby inhibiting the activation of HIF-1αrelated pathways,reducing the apoptosis of RGC-5 cells caused by oxidative stress,it also has a protective effect on RGC.