黄芩清热除痹胶囊含药血清对强直性脊柱炎患者外周血单核细胞氧化应激的影响

Effects of Huangqin Qingre Chubi Capsule Containing Serum on Oxidative Stress in PBMCs of Patients with Ankylosing Spondylitis

目的探讨黄芩清热除痹胶囊(HQC)含药血清对强直性脊柱炎(AS)患者外周血单核细胞(PBMCs)氧化应激的影响。方法将40只大鼠按随机数字表法分为HQC低、中、高剂量组,空白组、阴性组(生理盐水),其中HQC低、中、高3组大鼠连续给药7天处死取血清;采用四甲基偶氮唑盐(MTT)法检测HQC含药血清对PBMCs增殖的影响;将PBMCs分为正常组、模型组、HQC组、腺苷酸活化蛋白激酶(AMPK)抑制剂(Compound C)组、HQC含药血清+Compound C组;采用酶联免疫吸附法(ELISA)检测超氧化物歧化酶(SOD)、丙二醇(MDA)、脂质过氧化产物(LPO)、总抗氧化能力(TAOC)的表达;采用实时荧光定量聚合酶链反应(RT-PCR)及蛋白免疫印迹(Western Blot)检测AMPK/叉头转录因子O3a(FOXO3a)/锰超氧化物歧化酶(MnSOD)信号通路相关基因及蛋白的表达。结果中剂量HQC含药血清具有显著抑制PBMCs增殖的作用,确定其最佳作用时间为24 h。与正常组比较,模型组MDA、LPO明显升高,SOD、TAOC明显降低(P<0.01);与模型组比较,HQC、HQC+Compound C组MDA、LPO明显降低,SOD、TAOC明显升高(P<0.01),Compound C组MDA、LPO明显升高,SOD、TAOC明显降低(P<0.01);与Compound C组比较,HQC+Compound C组MDA、LPO明显降低,SOD、TAOC明显升高(P<0.01);与正常组比较,模型组AMPK、FOXO3a、MnSOD mRNA及AMPK、p-AMPK、FOXO3a、p-FOXO3a、MnSOD蛋白表达水平明显降低(P<0.01);与模型组比较,HQC组AMPK、FOXO3a、MnSOD mRNA及AMPK、p-AMPK、FOXO3a、p-FOXO3a、MnSOD蛋白表达水平明显升高(P<0.01),Compound C组AMPK、FOXO3a、MnSOD mRNA及AMPK、p-AMPK、FOXO3a、p-FOXO3a、MnSOD蛋白表达水平明显降低(P<0.05,P<0.01);与Compound C组比较,HQC+Compound C组AMPK、FOXO3a、MnSOD mRNA及AMPK、p-AMPK、FOXO3a、p-FOXO3a、MnSOD蛋白表达水平明显升高(P<0.01)。结论HQC含药血清能通过活化AMPK、FOXO3a、MnSOD信号通路,改善AS患者PBMCs氧化应激。

Objective To observe the effects of Huangqin Qingre Chubi Capsule(HQC)containing serum on oxidative stress in peripheral blood mononuclear cells(PBMCs)of patients with ankylosing spondylitis(AS).Methods Forty rats were divided into low,middle,and high dose HQC groups,blank control group,and normal saline group.After 7 successive days of intragastric administration,serum was taken from mice in low,middle,and high HQC dose groups.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the effects of HQC containing serums on the proliferation of PBMCs.The cultured PBMCs were divided into the normal group,model group,HQC group,Compound C group,Compound C+HQC containing serum group.Contents of superoxide dismutase(SOD),total antioxidant capacity(TAOC),malondialdehyde(MDA),lipid peroxide(LPO)were detected using ELISA.mRNA and protein expressions of AMPK-FOXO3a-MnSOD were detected using realtime fluorescence quantitative polymerase chain reaction(RT-PCR)and Western Blot.Results The proliferation of PBMCs was significantly inhibited by middle dose HQC containing serum,and the optimal effect was achieved at 24 h treatment.Compared with the normal group,contents of MDA and LPO significantly increased,activities of SOD and TAOC significantly decreased in the model group(P<0.01).Compared with the model group,contents of MDA and LPO significantly decreased,activities of SOD and TAOC significantly increased in the HQC group and Compound C+HQC containing serum group(P<0.01).Contents of MDA and LPO significantly increased,activities of SOD and TAOC significantly decreased in Compound C group(P<0.01).Compared with Compound C group,contents of MDA and LPO significantly decreased,activities of SOD and TAOC significantly increased in Compound C+HQC containing serum group(P<0.01).Compared with the normal group,mRNA expressions of AMPK,FOXO3a,MnSOD,and protein expressions of AMPK、p-AMPK,FOXO3a,p-FOXO3a,MnSOD significantly decreased in model group(P<0.01).Compared with the model group,mRNA expressions of AMPK,FOXO3a,MnSOD,and protein expressions of AMPK,p-AMPK,FOXO3a,p-FOXO3a,MnSOD significantly increased in HQC group(P<0.01);mRNA expressions of AMPK,FOXO3a,MnSOD,and protein expressions of AMPK,p-AMPK,FOXO3a,p-FOXO3a,MnSOD significantly decreased in Compound C group(P<0.05,P<0.01).Compared with Compound C group,mRNA expressions of AMPK,FOXO3a,MnSOD,and protein expressions of AMPK,p-AMPK,FOXO3a,p-FOXO3a,MnSOD significantly increased in Compound C+HQC containing serum group(P<0.01).Conclusion HQC containing serum improved oxidative stress in PBMCs of AS patients possibly by activating AMPK-FOXO3a-MnSOD signaling pathways.