小檗碱通过抑制细胞焦亡减轻糖尿病肾小球纤维化的实验研究  

Effect of berberine on renal fibrosis by inhibiting pyroptosis in glomerular endothelial cells in diabetic kidney disease in rats

作　　者：马泽军[1] 张曼[1] 王珊珊 郭欣 孙蓓[1] 陈莉明[1] Ma Zejun;Zhang Man;Wang Shanshan;Guo Xin;Sun Bei;Chen Liming(Blood Purification Center,National Health Council of Key Laboratory of Hormones and Development,Tianjin Key Laboratory of Metabolic Diseases,Chu Hsien-I Memorial Hospital and Tianjin Institute of Endocrinology,Tianjin Medical University,Tianjin 300314,China)

机构地区：[1]天津医科大学朱宪彝纪念医院血液净化中心、天津市内分泌研究所、国家卫生健康委员会激素与发育重点实验室、天津市代谢性疾病重点实验室,天津300314

出　　处：《中华糖尿病杂志》2024年第7期799-807,共9页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(81470187)。

摘　　要：目的探讨小檗碱(BBR)对糖尿病肾小球内皮细胞焦亡的影响及机制研究。方法 30只SD大鼠按照随机数字表法分为3组:正常对照组(NC组)、糖尿病肾脏病组(DKD组)、BBR干预组(DKD+BBR组),每组各10只。高糖高脂喂养联合小量链脲佐菌素注射的方法构建DKD模型,DKD+BBR组每天给予BBR 150 mg/kg治疗12周。检测空腹血糖、糖化血红蛋白、甘油三酯、总胆固醇、血肌酐及尿素氮、肾脏指数(KWI)等指标。病理染色观察肾脏病理学变化。免疫组化和免疫荧光染色观察肾小球内皮细胞焦孔素D(GSDMD)的表达变化,Western blotting和实时定量聚合酶链反应(RT-PCR)检测肾组织胶原-Ⅳ、纤维连接蛋白(FN)、NOD样受体蛋白3(NLRP3)、剪切的半胱天冬氨酸蛋白酶-1(cleaved caspase-1)、白细胞介素-1β(IL-1β)的表达变化。体外培养大鼠肾小球内皮细胞,分为正常糖组(5.6 mmol/L葡萄糖)、高渗组(5.6 mmol/L葡萄糖+24.4 mmol/L甘露醇)、高糖组(30.0 mmol/L葡萄糖)、BBR低剂量组(30.0 mmol/L葡萄糖+1 μmol/L BBR)、BBR中剂量组(30.0 mmol/L葡萄糖+10 μmol/L BBR)、BBR高剂量组(30.0 mmol/L葡萄糖+100 μmol/L BBR)。药物作用72 h后检测肾小球内皮细胞GSDMD、NLRP3、cleaved caspase-1、IL-1β的表达变化,酶联免疫吸附测定法(ELISA)检测细胞上清液IL-1β、白细胞介素-18(IL-18)含量。多组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果 DKD组大鼠血糖、24 h尿蛋白、KWI较NC组显著升高(均P<0.05)。DKD+BBR组大鼠24 h尿蛋白、KWI较DKD组显著减少(均P<0.05)。肾组织病理检查结果显示,DKD大鼠肾小球肥大,基底膜增厚,肾小球系膜增生,BBR干预后上述病理改变明显减轻。免疫组织化学染色和免疫荧光染色显示,与NC组大鼠相比,DKD组大鼠肾小球内皮细胞中GSDMD-N蛋白表达增加,BBR治疗后GSDMD-N蛋白表达明显减少。Western blotting和RT-PCR结果显示,与NC组比较,DKD组大鼠肾小球内皮细胞GObjective To assess whether berberine could alleviate pyroptosis in glomerular endothelial cells and the possible molecular mechanism.Methods Thirty Sprague-Dawley rats were randomly divided into 3 groups:normal control rats(NC group),diabetic kidney disease without drug treatment(DKD group),diabetic rats treated with berberine(BBR group)for 12 weeks.The DKD model was established using a high-sugar and high-fat diet and a small amount of streptozotocin injection.At the end of the study,blood glucose,glycated hemoglobin A1c,total cholesterol,triglycerides,serum creatinine,24-hour urine protein and kidney weight index(KWI)were determined,and pathological morphology and kidney fibrosis were quantified by hematoxylin and eosin(HE)and Masson′s staining.The expression of pyroporin D(GSDMD)in glomerular endothelial cells was observed by immunohistochemistry and immunofluorescence staining.The expressions of CollagenⅣ,fibronectin(FN),cleaved caspase-1,NOD-like receptor protein 3(NLRP3),caspase-1,interleukin-1β(IL-1β)were detected by real-time polymerase chain reaction(RT-PCR)and Western blotting analysis.Glomerular endothelial cells were divided into six groups:normal control group(NG group,5.6 mmol/L glucose);osmotic pressure control group(OP group,5.6 mmol/L glucose+24.4 mmol/L mannitol);high glucose group(HG group,30 mmol/L glucose);different concentrations of BBR(1,10 or 100μmol/L)+high glucose group(BBR+HG group).The expressions of GSDMD,NLRP3,cleaved caspase-1,IL-1βwere detected by RT-PCR and Western blotting analysis.The concentration of IL-1βand interleukin-18(IL-18)in the cell supernatant was determined by enzyme-linked immunosorbent assay.Statistical analysis was performed using analysis of variance(ANOVA)to compare differences among three and more groups and unpaired Student′s t-tests to compare differences between two groups.Results The blood glucose,24-hour urine protein,and renal weight index of rats in the DKD group were significantly higher than those in the NC group(all P<0.05).The 24-hour uri

关 键 词：糖尿病 糖尿病肾脏病 小檗碱 细胞焦亡 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]