槐杞黄颗粒通过调控AMPK/ACC通路影响IgA血管炎肾炎小鼠Th17细胞的分化

Huaiqihuang Granules Affect Differentiation of Th17 Cells in IgA Vasculitis Nephritis Mice by Regulating AMPK/ACC Pathway

目的:观察槐杞黄颗粒对免疫球蛋白(Ig)A血管炎肾炎(IgAVN)小鼠的干预作用并探讨该方药的疗效机制。方法:SPF级昆明种雄性小鼠50只,随机分为正常组、IgAVN模型组、地塞米松组(2.5 mg·kg^(-1)·d^(-1))、槐杞黄低、高剂量组(4、8 g·kg^(-1)·d^(-1))。采用口服麦胶蛋白结合尾静脉注射印度墨水法建立小鼠模型。判断造模成功后依据分组灌胃给药4周麻醉处死小鼠。检测各组小鼠24 h尿蛋白定量、尿β2-微球蛋白、血清总蛋白、白蛋白、IgA等;流式测定脾脏单细胞悬液辅助性T细胞17(Th17)比例;蛋白免疫印迹法(Western blot)检测Th17细胞腺苷酸激活蛋白激酶α(AMPKα)、磷酸化腺苷酸激活蛋白激酶α(p-AMPKα)、乙酰辅酶A羧化酶1(ACC1)、磷酸化乙酰辅酶A羧化酶1(p-ACC1)蛋白表达;观察脾脏与肾脏病理变化。结果:与正常组比较,模型组小鼠24 h尿蛋白定量、尿β2-微球蛋白、总胆固醇(P<0.05)、血清白细胞介素-17(IL-17)、IgA及脾单细胞悬液Th17比例、脾组织IL-17表达显著升高(P<0.01);血清总蛋白、白蛋白、Th17细胞p-AMPKα/AMPKα与p-ACC1/ACC1表达显著下降(P<0.01)。与IgAVN模型组比较,第4周各治疗组24 h尿蛋白定量、尿β2-微球蛋白与血清IL-17、IgA水平及肾脏IgA沉积均显著降低(P<0.01),脾脏组织中Th17比例与IL-17表达明显降低(P<0.05,P<0.01);血清白蛋白水平明显上升(P<0.05)。与IgAVN模型组比较,地塞米松组与槐杞黄高剂量组血清总蛋白显著上升(P<0.01)、Th17细胞p-AMPKα/AMPKα与p-ACC1/ACC1表达明显升高(P<0.05,P<0.01);槐杞黄高剂量组总胆固醇水平明显下降(P<0.05)。各治疗组脾脏与肾脏病理变化均有不同程度改善。结论:槐杞黄颗粒可能通过调控AMPK/ACC通路影响Th17细胞分化,纠正免疫炎症紊乱从而发挥治疗IgAVN的效应。

Objective:To observe the intervention effect of Huaiqihuang granules(HQH)on immunoglobulin A vasculitis nephritis(IgAVN)mice and explore the underlying therapeutic mechanism.Method:Fifty SPF-grade male Kunming mice were randomly divided into a normal group,an IgAVN model group,a dexamethasone group(2.5 mg·kg^(-1)·d^(-1)),a low-dose HQH group(4 g·kg^(-1)·d^(-1)),and a high-dose HQH group(8 g·kg^(-1)·d^(-1)).The mouse model was established using oral administration of gliadin combined with intravenous injection of India ink.After successful modeling,the mice were euthanized after 4 weeks of gastric gavage according to groups.The 24 h urinary total protein(24 h UTP),urineβ_(2)-microglobulin(β_(2)-MG),serum total protein,albumin,IgA,etc.were detected in each group.Flow cytometry was used to determine the proportion of T helper 17(Th17)cells in spleen cell suspension.Western blot was employed to detect the expression of adenosine 5'-monophosphate-activated protein kinaseα(AMPKα),phosphorylated AMPKα(p-AMPKα),acetyl-CoA carboxylase 1(ACC1),and phosphorylated ACC1(p-ACC1)in Th17 cells.Pathological changes in the spleen and kidneys were observed.Result:Compared with the normal group,the IgAVN model group showed significant increases in 24 h UTP,urineβ_(2)-MG,total cholesterol(P<0.05),serum interleukin-17(IL-17),IgA,Th17 proportion in the spleen cell suspension,and IL-17 expression in the spleen tissue(P<0.01),and significantly decreased serum total protein,albumin,p-AMPKα/AMPKα,and p-ACC1/ACC1 expression of Th17 cells(P<0.01).Compared with the IgAVN model group,in the 4th week,the 24 h UTP,urineβ_(2)-MG,serum IL-17,IgA levels,and renal IgA deposition were significantly reduced in each treatment group(P<0.01),and the Th17 proportion and IL-17 expression in spleen tissue were significantly decreased(P<0.05,P<0.01).Serum albumin levels significantly increased(P<0.05).Compared with the IgAVN model group,the dexamethasone group and the high-dose HQH group showed increases in serum total protein(P<0.01),p-AMPKα/AMPKα,and p-ACC1/ACC1 expression of Th17 cells(P<0.05,P<0.01).The high-dose HQH group showed a significant decrease in total cholesterol level(P<0.05).Various treatment groups showed different degrees of improvement in spleen and kidney pathological changes.Conclusion:HQH may affect Th17 cell differentiation by regulating the AMPK/ACC pathway,correcting immune inflammatory disorders,and exerting therapeutic effects on IgAVN.