补阳还五汤调节铁代谢蛋白改善脑缺血神经元损伤的研究

Buyang Huanwu Decoction improved cerebral ischemia injury by regulating iron transporter

目的探讨补阳还五汤通过调节铁代谢转运蛋白改善脑缺血神经元损伤的作用机制。方法选择大鼠肾上腺嗜铬细胞瘤细胞系PC12细胞,采用氧糖剥夺(oxygen-glucose deprivation,OGD)造模建立体外脑缺血模型,制备补阳还五汤含药血浆(极低、低、中、高剂量组),其中极低、低、中、高剂量浓度比为0.5∶1∶2∶4。将细胞依次分为正常组、模型组、空白血浆组以及补阳还五汤含药血浆组(极低、低、中、高剂量组)。除正常组外,其余各组造模6 h后分别给予等体积不同浓度含药血浆干预24 h,正常组和模型组为普通培养基培养,其余组分别使用含10%各自浓度含药血浆的基础培养基培养。CCK-8筛选补阳还五汤含药血浆的最佳干预浓度。制备去铁酮含药血浆为阳性对照药。细胞分为正常组、模型组、补阳还五汤最佳血浆浓度组(低剂量组)、去铁酮组。正常组和模型组使用含有10%空白血浆的基础培养基培养,低剂量组和去铁酮组使用含10%各自浓度含药血浆的基础培养基处理24 h。分别进行细胞形态观察、细胞活力检测、活性氧(reactive oxygen species,ROS)测定比较各组间细胞的损伤情况;超氧化物歧化酶活力(superoxide dismutase,SOD)测定比较各组间清除氧自由基能力;丙二醛含量(malondialdehyde,MDA)测定比较各组间细胞过氧化损伤程度;Western blot检测铁代谢相关蛋白铁调素(hepcidin,HEP)、转铁蛋白受体(transferrin receptor,TFR)和膜铁转运蛋白(ferroportin,FPN)的表达水平。结果与模型组比较,补阳还五汤组细胞损伤明显改善,其中低剂量组CCK-8细胞活力明显提高(P<0.01),ROS含量显著下降(P<0.01),SOD活力明显上升(P<0.01),MDA含量下降(P<0.01),TFR和HEP蛋白表达降低(P<0.05),FPN蛋白表达增加(P<0.01),差异有统计学意义。结论补阳还五汤可能通过抑制TFR、HEP表达,促进FPN表达,纠正脑缺血后铁稳态失衡,发挥改善神经元损伤的作用。

Objective To investigate the mechanism of Buyang Huanwu Decoction(BYHWD)in improving cerebral ischemic neuronal injury by regulating iron metabolism transporter.Methods Rat adrenal pheochromocytoma cell line PC12 cells were selected,and an in vitro cerebral ischemia model was established by oxygen-glucose deprivation(OGD).The plasma containing BYHWD(very low,low,medium and high dose)was prepared.The concentration ratio of very low,low,medium and high doses was 0.5∶1∶2∶4.The cells were divided into normal group,model group,blank plasma group and BYHWD medicated plasma groups(very low,low,medium and high dose groups).Except for the normal group,the other groups were given the same volume and different concentrations of medicated plasma for 24 h after modeling for 6 h.The normal group and the model group were cultured in ordinary medium,and the other groups were cultured in basal medium containing 10%medicated plasma with different concentrations respectively.CCK-8 was used to screen the optimal intervention concentration of plasma containing BYHWD.The deferiprone medicated plasma was prepared as a positive control drug.The cells were divided into normal group,model group,BYHWD group with the optimal concentration(low dose group)and deferiprone group.Normal group and model group were cultured with basal medium containing 10%blank plasma.The low dose group and deferiprone group were treated with basal medium containing 10%medicated plasma of their respective concentrations for 24 h.Cell morphology observation,CCK-8 cell viability detection,and reactive oxygen species(ROS)measurement were performed to compare the damage of cells among the groups.The superoxide dismutase(SOD)activity was measured to compare the oxygen free radical scavenging ability among the groups.The level of malondialdehyde(MDA)was measured to compare the degree of cell peroxidative damage among the groups.The expression levels of iron metabolism-related proteins hepcidin(HEP),transferrin receptor(TFR)and ferroportin(FPN)were detected by Western blot.Results The viability of CCK-8 cells in the low-dose BYHWD group significantly increased(P<0.01),the level of ROS was markedly reduced(P<0.01),the activity of SOD was obviously increased(P<0.01),the level of MDA was lower(P<0.01),and the protein expression levels of TFR and HEP decreased(P<0.05).The expression of FPN protein increased(P<0.01).The difference was statistically significant.Conclusion BYHWD may play a role in improving neuronal damage by inhibiting the expression of TFR and Hepcidin,promoting the expression of FPN,and correcting the imbalance of iron homeostasis after cerebral ischemia.