丹参酮ⅡA对过敏性紫癜肾炎小鼠肾组织中Cosmc和AOPP表达的影响及其治疗作用

doi:10.13481/j.1671-587x.20190421

摘要/Abstract

摘要： 目的：探讨丹参酮ⅡA对过敏性紫癜肾炎小鼠肾组织中半乳糖基转移酶伴侣蛋白（Cosmc）、晚期氧化蛋白终末产物（AOPP）表达及胞外信号调节激酶/丝裂原活化蛋白激酶（ERK/MAPK）信号通路蛋白水平的影响，阐明丹参酮ⅡA对过敏性紫癜肾炎的治疗机制。方法：38只小鼠随机分为对照组、模型组（建立过敏性紫癜肾炎模型）和丹参酮ⅡA组（建立过敏性紫癜肾炎模型+丹参酮ⅡA治疗），其中2只小鼠用于验证造模是否成功，其余每组12只。测定各组小鼠尿红细胞计数和尿蛋白水平，采用过碘酸希夫反应（PAS）染色观察各组小鼠肾组织病理形态表现，采用酶联免疫吸附法（ELISA）测定小鼠肾组织中AOPP蛋白水平，采用Western blotting法测定小鼠肾组织中ERK和磷酸化ERK（p-ERK）蛋白表达水平。采用逆转录-聚合酶链反应（RT-PCR）法测定各组小鼠肾组织中ERK、p-ERK和Cosmc mRNA表达水平。结果：对照组小鼠肾小球基底膜无增生，无肾小球硬化；模型组小鼠肾小球基底膜增生明显，可见炎症细胞浸润，出现肾小球硬化；丹参酮ⅡA组小鼠肾小球基底膜增生和炎症浸润不明显，少量炎症细胞浸润。与对照组比较，模型组和丹参酮ⅡA组小鼠尿红细胞计数、24 h尿蛋白水平及肾组织中AOPP蛋白水平和p-ERK mRNA表达水平升高（P<0.05或P<0.01），Cosmc mRNA表达水平降低（P<0.01）；与模型组比较，丹参酮ⅡA组小鼠尿红细胞计数、24 h尿蛋白水平及肾组织中AOPP蛋白水平和p-ERK蛋白表达水平降低（P<0.05或P<0.01），Cosmc mRNA表达水平升高（P<0.01）。模型组小鼠肾组织中Cosmc mRNA表达水平与AOPP蛋白水平及p-ERK蛋白表达水平呈负相关关系（r=-0.573，P<0.01；r=-0.602，P<0.01），AOPP蛋白水平与p-ERK蛋白表达水平呈正相关关系（r=0.614，P<0.01）。结论：丹参酮ⅡA可能通过降低肾组织Cosmc水平、升高肾组织中AOPP水平以及激活ERK/MAPK信号通路对过敏性紫癜肾炎小鼠发挥治疗作用。

关键词: 丹参酮ⅡA, 过敏性紫癜肾炎, 小鼠, 肾组织, 半乳糖基转移酶伴侣蛋白, 晚期氧化蛋白终末产物, 胞外信号调节激酶, 丝裂原活化蛋白激酶

Abstract: Objective:To explore the influence of tanshinone ⅡA on the expressions of molecular chaperone Cosmc, advanced oxidation protein products (AOPP) in the kidney tissue of the allergic purpura nephritis mice and the levels of extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) signaling pathway proteins,and to clarify mechanism of tanshinone Ⅱ A in the treatment of allergic purpura nephritis. Methods:A total of 38 mice were divided into control group, model group (allergic purpura nephritis model) and tanshinone Ⅱ A group (allergic purpura nephritis model + tanshinone Ⅱ A treatment), with 12 in each group; the other 2 mice were used to verify the success of modeling. The urine red blood cell counts and urine protein levels of the mice in various groups were measured, and the pathomorphology of kidney tissue of the mice in various groups were observed by periodate schiff-reaction (PAS) staining. The AOPP protein levels in the kidney tissue of the mice were determined by enzyme linked immunosorbent assay (ELISA), and the ERK and phosphorylated ERK (p-ERk) protein expression levels were determined by Western blotting method. The expression levels of ERK, p-ERK and Cosmc mRNA in kidney tissue of the mice were determined by RT-PCR. Results:In control group, there was no hyperplasia of glomerular basement membrane and no glomerular sclerosis;in model group, the proliferation of glomerular basement membrane was obvious, the inflammatory cells were infiltrated, and glomerular sclerosis appeared;in tanshinone ⅡA group, glomerular basement membrane hyperplasia and inflammatory infiltration were not obvious in the mice, there was only small amount of infiltrated inflammatory cells. Compared with control group, the urine red blood cell count,the 24 h urine protein level,the p-ERK mRNA and AOPP protein levels of the mice in model group and tanshinone Ⅱ A group were increased(P<0.05 or P<0.01); the Cosmc mRNA expression levels were decreased (P<0.01). Compared with model group, the urine red blood cell count, 24 h urine protein level, the p-ERK and AOPP protein levels in the kidney tissue of the mice in tanshinone Ⅱ A group were decreased (P<0.05 or P<0.01), and the Cosmc mRNA expression level was increased (P<0.01). In model group, the Cosmc mRNA expression level in the kidney tissue was negatively correlated with the expression levels of AOPP protein and the expression level of p-ERK protein (r=-0.573, P<0.01; r=-0.602, P<0.01), and the level of AOPP protein was positively correlated with the expression level of p-ERK protein (r=0.614, P<0.01). Conclusion:Tanshinone Ⅱ A may play a role in the treatment of allergic purpura nephritis in the mice by lowering the levels of Cosmc,increasing the AOPP in kidney tissue and activating the ERK/MAPK signaling pathways.

Key words: tanshinone ⅡA, allergic purpura nephritis, mice, kidney, Cosmc, advanced oxidation protein, extracellular signal-regulated kinase, mitogen-activated protein kinase

中图分类号:

R285.5

杨云, 马喜兴, 王大虎, 张环环, 马耀辉, 任翠敏, 刘强. 丹参酮ⅡA对过敏性紫癜肾炎小鼠肾组织中Cosmc和AOPP表达的影响及其治疗作用[J]. 吉林大学学报(医学版), 2019, 45(04): 867-871.

YANG Yun, MA Xixing, WANG Dahu, ZHANG Huanhuan, MA Yaohui, REN Cuimin, LIU Qiang. Influence of tanshinone Ⅱ A on expressins of Cosmc and AOPP in kidney tissue of allergic purpura nephritis mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2019, 45(04): 867-871.

参考文献

[1] CHEN JY, MAO JH.Henoch-Schnleinpurpuranephritisin children:incidence, pathogenesis and management[J].World J Pediatr,2015,11(1):29-34.
[2] 李向新,闫纪平,姜威,等. 护肾除癜方辅助西医药物治疗过敏性紫癜肾炎疗效及对凝血指标的影响[J]. 中国中医急症, 2018, 27(5):877-880.
[3] XIE YX, HE LY, CHEN X,et al.Potential diagnostic biomarkers for IgA nephropathy:a comparative study pre-and post-tonsillectomy[J].Int Urol Nephrol,2016,48(11):1855-1861.
[4] 胥会英,童晓,李成万. 丹参酮ⅡA提高小儿过敏性紫癜血浆组织因子途径抑制物水平及干预肾损害的研究[J]. 中医儿科杂志, 2018,14(4):26-28.
[5] 李灵. 芪元颗粒及有效部位对HSP模型大鼠治疗作用的实验研究[D].太原:山西中医药大学,2017.
[6] 杨扬,杨洁.丹参酮ⅡA对EAE小鼠临床表现及外周Treg细胞的影响[J].天津医科大学学报,2017,23(1):17-20.
[7] 王恒东,顾教伟,王勇,等. 复方甘草酸苷注射液联合他克莫司治疗过敏性紫癜肾炎的临床研究[J]. 现代药物与临床, 2018,33(11):3021-3025.
[8] 邢丽娜,任金海,王颖,等. 孟鲁司特钠对ITP模型小鼠T淋巴细胞亚群及晚期氧化蛋白产物的影响[J]. 中国免疫学杂志, 2018,34(6):826-829.
[9] 白晓峰,冯梅,李凯,等.晚期氧化蛋白产物抑制CD4⁺CD25⁺调节性T细胞的增殖[J].中华实验外科杂志,2018,35(1):123-126.
[10] 季伟星,金慧英. 过敏性紫癜患儿血清骨桥蛋白、核转移因子含量检测及其与氧化应激、细胞免疫功能的相关关系[J]. 中国卫生检验杂志, 2018,28(19):2356-2358.
[11] HU S, BAO H, XU XD,et al.Increased miR-374b promotes cell proliferation and the production of aberrant glycosylated IgA1 in B cells of IgA nephropathy[J].FEBS Lett,2015,589(24 Pt B):4019-4025.
[12] 霍建丽. 晚期氧化蛋白产物(AOPPs)在过敏性紫癜的血清水平及其与异常糖基化IgA1及Cosmc mRNA表达的关系[D]. 石家庄:河北医科大学, 2017.
[13] GAO J, WU LL, WANG Y,et al.Knockdown of Cxcl10 inhibits mesangial cell proliferation in murine habu nephritis via ERK signaling[J].Cell Physiol Biochem,2017,42(5):2118-2129.
[14] WU H, ZHOU SS, KONG LL,et al.Metallothionein deletion exacerbates intermittent hypoxia-induced renal injury in mice[J].Toxicol Lett,2015,232(2):340-348.
[15] 曾辉. 儿童过敏性紫癜血浆miRNAs差异表达谱研究[D]. 衡阳:南华大学,2016.
[16] REN B, ZHANG Y X, ZHOU H X,et al.Tanshinone ⅡA prevents the loss of nigrostriatal dopaminergic neurons by inhibiting NADPH oxidase and iNOS in the MPTP model of Parkinson's disease[J].J Neurol Sci,2015,348(1/2):142-152.
[17] 范茂虓,何飞,丁岚,等. 丹参酮ⅡA磺酸钠注射液对过敏性紫癜患者紫癜消退及肾受累的影响[J]. 中国临床研究, 2016,29(2):262-265.
[18] 王乐琪,谢志茹,李莎莎,等. 丹参及其复方制剂用于与微循环障碍有关疾病的研究进展[J]. 中国药房, 2018,29(23):3297-3302.
[19] LIU X, GUO C Y, MA X J,et al.Anti-inflammatory effects of tanshinone ⅡA on atherosclerostic vessels of ovariectomized ApoE mice are mediated by estrogen receptor activation and through the ERK signaling pathway[J].Cell Physiol Biochem,2015,35(5):1744-1755.
[20] 叶丽娟,李锦开,萧庆文. 孟鲁司特联合丹参酮ⅡA磺酸钠治疗儿童单纯型过敏性紫癜的临床效果分析[J]. 广西医科大学学报, 2018,35(10):1452-1455.