健脾清化方改善局灶节段性肾小球硬化的效应机制研究  

Study on the effect mechanism of spleen⁃strengthening and purifying formula in improving focal segmental glomerulosclerosis

作　　者：刘珊 钟逸斐[1] 李屹 LIU Shan;ZHONG Yifei;LI Yi(Department of Nephrology A,Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院肾病一科,上海徐汇200032

出　　处：《海南医学院学报》2024年第20期1535-1546,共12页Journal of Hainan Medical University

基　　金：国家自然科学基金面上项目(82374589);上海市科委“科技创新行动计划”临床医学领域项目(18401970603);上海中医药大学产业发展中心医养结合科创项目(602059D)。

摘　　要：目的:探讨健脾清化方对局灶节段性肾小球硬化(FSGS)小鼠及阿霉素诱导足细胞损伤的保护作用,并观察其对炎症反应、氧化应激及细胞凋亡的影响。方法:(1)体内实验:实验组balb/c小鼠,采用单次尾静脉注射阿霉素(10.5 mg/kg)的方法建立FSGS动物模型,造模后连续灌胃给药6周。测定24 h尿蛋白(定量)、血肌酐,及肾组织内超氧化物歧酶、丙二醛、过氧化氢酶的水平。并采用H&E、Masson、PAS染色,观察肾组织病理损伤程度。透射电镜观察肾脏超微结构。免疫荧光nephrine/p-JAK2双染观察足细胞上p-JAK2的表达。免疫印迹法检测(western blotting,WB)肾组织中p-JAK2/JAK2、p-STAT3/STAT3、TGF-β1蛋白表达水平。(2)体外实验:实验1:采用CCK-8法检测阿霉素及健脾清化方对细胞活力的影响,并筛选出造模和健脾清化方干预浓度。DCFH-DA荧光探针检测各组足细胞ROS的含量。流式细胞术测定足细胞凋亡情况。WB检测p-JAK2/JAK2、p-STAT3/STAT3、TGF-β1的蛋白表达水平以及RT-qPCR检测STAT3下游凋亡因子BAX、BAD、Caspase3、p21、Blc-2、和炎症因子IL-6、IL-1β、TNF-α的mRNA表达情况。实验2:WB检测JAK2激动剂C-A1对p-JAK2/JAK2,p-STAT3/STAT3蛋白表达的影响。结果:(1)体内实验:与对照组相比,模型组小鼠肾小球硬化,足细胞足突广泛融合,肾组织p-JAK2荧光表达明显增加。24 h尿蛋白定量、血肌酐、MDA升高,SOD、CAT降低(P<0.05)。p-JAK2/JAK2、p-STAT3/STAT3、TGF-β1表达升高。与模型组相比健脾清化方干预后足细胞损伤缓解,肾组织P-JAK2荧光表达降低,24 h尿蛋白定量、血肌酐、MDA降低,SOD、CAT升高。p-JAK2/JAK2、p-STAT3/STAT3、TGF-β1表达降低(P<0.05)。(2)体外实验:实验1:CCK-8法确定阿霉素造模浓度为0.4μg/mL,健脾清化方干预低中高浓度分别为1 mg/mL,2 mg/mL,4 mg/mL。与对照组比较,模型组凋亡细胞数量、ROS含量增多,p-JAK2/JAK2、p-STAT3/STAT3、TGF-β1蛋白表达升高(P<0.05),Objective:To observe the effects of strengthening spleen and cleansing formula on adriamycin-induced balb/c mice and mouse renal podocytes(MPC-5)to explore the potential effect mechanism of its anti-oxidative stress against focal segmental glomerulosclerosis(FSGS)to ameliorate podocyte injury.Methods:(1)In vivo experiments:balb/c mice were randomly divided into five groups:the blank group(CON),the model group(MOD),the low-dose group(L:1.13 g/kg),the medium-dose group(Z:2.26 g/kg)and the high-dose group(H:4.52 g/kg)of the strengthening spleen and clearing harmonizing formula.In the ex-perimental group of balb/c mice,FSGS animal model was established by single tail vein injection of adriamycin(10.5 mg/kg),and the drug was administered by continuous gavage for 6 weeks after modeling.The 24-h urine protein(quantitative),blood cre-atinine,and the levels of superoxide dismutase(SOD),malonaldehyde(MDA),catalase(CAT)in renal tissues were measured.H&E,Masson and PAS staining were used to observe the degree of renal histopathological damage.Transmission electron micros-copy(TEM)was used to observe the morphological changes of podocytes.Immunofluorescence nephrine/p-JAK2 double staining was used to observe the expression of p-JAK2 on podocytes.The protein expression levels of p-JAK2/JAK2,p-STAT3/STAT3,and TGF-β1 in renal tissues were detected by immunoblotting.(2)In vitro experiments:Experiment 1:The podocytes were divided into 5 groups:the control group(CON),the model group(MOD),the low-dose group(L),the medium-dose group(M),and the high-dose group(H)of JianSheng QingHua Fang.Experiment 2:Foot cells were divided into the CON group(CON),the model group(ADR),the high dose group of strengthening the spleen and cleansing and harmonizing formula(ADR+H),and the C-A1 group and the ADR+H+C-A1 group.Experiment 1:The effects of adriamycin and spleen-health clearing for-mula on cell viability were detected by CCK-8 assay,and the intervention concentrations of modeling and spleen-health clearing formula were screened.DCFH-DA fluoresc

关 键 词：局灶节段性肾小球硬化 足细胞损伤 健脾清化方 JAK2/STAT3 

分 类 号：R285[医药卫生—中药学]