解毒活血汤调控YAP/ACSL4通路抑制铁死亡治疗急性肾损伤的作用与机制研究  

Effect and mechanism of Jiedu Huoxue Decoction in regulating YAP/ACSL4 pathway to inhibit ferroptosis in treatment of acute kidney injury

作　　者：程冉 田瑞敏 黄丽华[1,2,3] 王晓婉 郭珊[1,2] 吴爱君 黎创 陈静 李淑菊[5] 陈明 毛炜[1,2,3] 徐鹏 CHENG Ran;TIAN Rui-min;HUANG Li-hua;WANG Xiao-wan;CUO Shan;WU Ai-jun;LI Chuag;CHEN Jing;LI Shu-ju;CHEN Ming;MAO Wei;XU Peng(State Key Laboratory of Dampness Syndrome of Chinese Medicine,the Second Afiliated Hospital of Guangzhou University of Chinese Medicine&Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,China;the Second Clinical Medical College,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Guangdong Provincial Academy of Chinese Medical Sciences,Guangzhou 510006,China;Faculty of Chinese Medicine,Macao University of Science and Technology,Macao 999078,China;Heilongjiang Academy of Chinese Medicine Sciences,Harbin 150036,China)

机构地区：[1]广州中医药大学第二附属医院&广东省中医院省部共建中医湿证国家重点实验室,广东广州510120 [2]广州中医药大学第二临床医学院,广东广州510405 [3]广东省中医药科学院,广东广州510006 [4]澳门科技大学中医药学院,中国澳门999078 [5]黑龙江省中医药科学院,黑龙江哈尔滨150036

出　　处：《中国中药杂志》2024年第1期151-161,共11页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82374356,82004157);广东省中医院朝阳人才科研专项(ZY2022KY13);广东省中医药局科研项目(20225002);省部共建中医湿证国家重点实验室专项(SZ2021ZZ10)。

摘　　要：解毒活血汤出自王清任《医林改错》,是国医大师张琪教授根据急性肾损伤(acute kidney injury,AKI)热毒瘀滞病机从古方中筛选出应用临床数十年的有效方剂,该文拟从调控铁死亡(ferroptosis)角度阐释解毒活血汤对AKI的治疗作用及其潜在机制。将32只雄性C57BL/6小鼠随机分为4组:正常组、模型组及解毒活血汤低、高剂量组,每组8只。由于临床AKI的治疗普遍以支持或替代疗法为主,尚无特效药,故该实验未设置阳性药物组。解毒活血汤低剂量组对应0.5倍的临床等效剂量,高剂量组则对应临床等效剂量,予以每日晨间灌胃,连续干预7 d;正常组及模型组小鼠分别灌以相应体积的纯水。给药第5天时,除正常组外其余各组小鼠均按20 mg·kg^(-1)的剂量腹腔注射顺铂溶液进行造模,正常组给予生理盐水注射。造模72 h后取材,留取小鼠血清及肾脏组织,检测血清肌酐(serum creatinine,Scr)、尿素氮(blood urea nitrogen,BUN)水平以评价小鼠肾脏功能变化;酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测血清肾损伤分子1(kidney injury molecule 1,KIM-1)的表达水平;苏木素-伊红(hematoxylin-eosin,HE)染色法观察小鼠肾组织病理学改变;过碘酸雪夫(periodic acid-Schiff,PAS)染色法观察肾脏糖原沉积变化;普鲁士蓝染色观察肾组织铁沉积水平;生化比色法检测肾组织中还原型谷胱甘肽(glutathione,GSH)含量、超氧化物歧化酶(superoxide dismutase,SOD)以及过氧化氢酶(catalase,CAT)活力。采用Wes-tern blot法检测小鼠肾组织中酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)、溶血磷脂酰胆碱酰基转移酶3(lysophosphatidylcholine acyltransferase 3,LPCAT3)及Hippo通路关键分子Yes相关蛋白(Yes-associated protein,YAP)蛋白表达;免疫组化法检测YAP在肾组织中的定位与相对表达;再采用实时荧光定量PCR法检测ACSL4及谷胱甘肽过氧化物酶4(Jiedu Huoxue Decoction(JDHX), first recorded in the Correction on Errors in Medical Works by WANG Qing-ren, is an effective formula screened out from ancient formulas by the traditional Chinese medicine(TCM) master ZHANG Qi to treat acute kidney injury(AKI) caused by heat, toxicity, stasis, and stagnation. This paper elucidated the therapeutic effect of JDHX on AKI and probed into the potential mechanism from ferroptosis. Thirty-two male C57BL/6 mice were randomized into four groups(n=8): normal, model, and low-and high-dose JDHX. Since the clinical treatment of AKI depends on supportive or alternative therapies and there is no specific drug, this study did not include a positive drug group. The low dose of JDHX corresponded to half of clinically equivalent dose, while the high dose corresponded to the clinically equivalent dose. Mice were administrated with JDHX by gavage daily for 7 consecutive days, while those in the normal group and the model group were administered with the corresponding volume of distilled water. On day 5 of drug administration, mice in other groups except the normal group were injected intraperitoneally with cisplatin solution at a dose of 20 mg·kg^(-1) to induce AKI, and the normal group was injected with saline. All of the mice were sacrificed 72 h after modeling, blood and kidney samples were collected for subsequent analysis. The levels of serum creatine(Scr) and blood urea nitrogen(BUN) were measured by the commercial kits. The expression level of kidney injury molecule 1(KIM-1) in the serum was measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin(HE) staining, periodic acid-Schiff(PAS) staining, and Prussian blue staining were employed to observe the pathological changes, glycogen deposition, and iron deposition, respectively, in the renal tissue. In addition, the levels of glutathione(GSH), superoxide dismutase(SOD), and catalase(CAT) in the renal tissue were examined by biochemical colorimetry. Western blot was performed to determine the protein levels of acyl-CoA syn

关 键 词：解毒活血汤 急性肾损伤(AKI) 铁死亡 Yes相关蛋白(YAP) 酰基辅酶A合成酶长链家族成员4(ACSL4) 

分 类 号：R285.5[医药卫生—中药学]