活血解毒化瘀方通过MR/NLRP3通路抑制UUO 6个月大鼠对侧肾脏血管平滑肌细胞焦亡的机制研究  

Mechanism of Huoxue Jiedu Huayu Recipe Ameliorates Kidney Vascular Smooth Muscle Cell Pyroptosis in the Contralateral Kidneys of 6-Month UUO Rats via MR/NLRP3 Pathway

作　　者：羡云倩 韩雨彤 强盼盼 郝娟 杨帆 许庆友 Xian Yunqian;Han Yutong;Qiang Panpan;Hao Juan;Yang Fan;Xu Qingyou(Graduate School,Hebei University of Chinese Medicine,Shijiazhuang 050091,China;Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns,Shijiazhuang 050091,China;Department of Internal Medicine,Hebei University of Chinese Medicine,Shijiazhuang 050200,China;Institute of Integrative Medicine,College of Integrative Medicine,Hebei University of Chinese Medicine,Shijiazhuang 050200,China)

机构地区：[1]河北中医药大学研究生学院,石家庄050091 [2]河北省中西医结合肝肾病证研究重点实验室,石家庄050091 [3]河北中医药大学内科教研室,石家庄050200 [4]河北中医药大学中西医结合研究所,石家庄050200

出　　处：《世界科学技术-中医药现代化》2023年第10期3336-3346,共11页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会面上项目(82174317):醛固酮刺激MR活化诱导UUO大鼠对侧肾脏淋巴管生成及益气解毒化瘀中药的保护作用,负责人:许庆友

摘　　要：目的观察活血解毒化瘀方对单侧输尿管梗阻(Unilateral ureteral obstruction,UUO)6个月大鼠模型对侧肾脏血管平滑肌细胞(Vascular smooth muscle cell,VSMC)焦亡的干预作用,探讨活血解毒化瘀方对慢性肾脏病(Chronic kidney disease,CKD)的部分治疗机制。方法40只雄性Wistar大鼠随机分为假手术(Sham)组、单侧输尿管梗阻(UUO)组、依普利酮治疗(EPL)组和活血解毒化瘀方干预(HJHR)组(n=10)。术后EPL组给予依普利酮,HJHR组给予中药。6个月后摘取右侧肾脏。通过HE染色和Masson染色评估对侧肾脏病理改变。免疫组化检测含半胱氨酸的天冬氨酸蛋白水解酶1(Cysteinyl aspartate specific proteinase1,Caspase-1)、白细胞介素1β(Interleukin-1β,IL-1β),核因子κappa-B,p65(Nuclear Factorκappa-B,NF-κB,p65)和血清和糖皮质激素诱导的激酶1(Serum and glucocorticoid-induced kinase 1,SGK1)的表达。通过TUNEL染色观察肾脏血管平滑肌细胞的DNA损伤情况。体外实验将大鼠血管平滑肌细胞随机分为4组:空白对照(CON)组、醛固酮刺激(ALD)组、醛固酮加依普利酮治疗(EPL)组和醛固酮加活血解毒化瘀方干预(HJHR)组。ALD组给予醛固酮刺激24 h,EPL组和HJHR组在醛固酮刺激前分别给予依普利酮和10%大鼠含中药血清预处理。通过流式细胞术检测细胞凋亡率。通过免疫荧光观察细胞膜中GSDMD蛋白(Gasdermin D,GSDMD)的穿孔和核受体亚家族3C组成员2(Nuclear Receptor subfamily 3 group C member 2,NR3C2)、编码盐皮质激素受体(Mineralocorticoid receptor,MR)的核转位。通过蛋白免疫印迹法检测炎症和细胞焦亡相关信号分子的蛋白质表达。通过实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)检测细胞焦亡信号通路相关分子的信使RNA(Messenger RNA,mRNA)表达。结果与Sham组比较,UUO组肾脏血管及血管平滑肌细胞的DNA损伤加重,Caspase-1、IL-1β、SGK1、NF-κB的表达增高;与UUO组比较,EPL组和HJHR组肾脏血管及血管平Objective The intervention effect of Huoxue Jiedu Huayu Recipe on the pyroptosis of vascular smooth muscle cells(VSMC)on the contralateral renal vessel of 6-month unilateral ureteral obstruction(UUO)rat model was observed,and part of the treatment mechanism of Huoxue Jiedu Huayu Recipe on chronic kidney disease(CKD)was investigated.Methods Forty male wistar rats were randomly divided into 4 groups:the sham operation(Sham)group,the unilateral ureteral obstruction(UUO)group,the eplerenone treatment(EPL)group and the Huoxue Jiedu Huayu Recipe intervention(HJHR)group,10 rats in each group.The EPL group was given eplerenone and the HJHR group was given traditional Chinese medicine.The right kidney was collected after 6 months.Pathological changes were assessed by HE staining and Masson staining.The expressions of Cysteinyl aspartate specific proteinase1(Caspase-1),Interleukin-1β(IL-1β),Serum and glucocorticoid-induced kinase 1(SGK1)and nuclear factorκappa-B(NF-κB)were detected by immunohistochemistry.The DNA damage in renal VSMC was examined by TUNEL staining.Rat VSMCs were used for in vitro experiments.The cells were divided into 4 groups using the random number method:the control(CON)group,the aldosterone(ALD)group,the aldosterone plus eplerenone treatment(EPL)group and the aldosterone plus Huoxue Jiedu Huayu Recipe treatment(HJHR)group.The ALD group was given aldosterone stimulation for 24 h.The EPL group and the HJHR group were given eplerenone and rat serum containing 10%Huoxue Jiedu Huayu Recipe pretreatment before aldosterone stimulation,respectively.The apoptosis rate was detected by flow cytometry.Perforation of gasdermin D(GSDMD)and nuclear translocation of NR3C2(Encode MR)in cell membranes were examined by immunofluorescence.The protein expression of inflammation and pyroptosis-related signaling molecules were examined by western blotting.The mRNA expression of pyroptosis signaling pathway were detected by qRT-PCR.Results Compared with the Sham group,DNA damage of renal blood vessels and VSMCs was incre

关 键 词：活血解毒化瘀方 细胞焦亡 单侧输尿管梗阻 血管平滑肌细胞 依普利酮 

分 类 号：R285.5[医药卫生—中药学]