清热消癥方对高糖刺激下HK-2细胞自噬相关蛋白表达的影响  

Effect of decoction for clearing heat and dispersing mass on autophagy-related protein expression in HK-2 cells stimulated by high glucose

作　　者：王健 王玉洁 周静威 WANG Jian;WANG Yujie;ZHOU Jingwei(The First Clinical Medical College of Beijing University of Chinese Medicine,Beijing 100700,China;Hospital of Traditional Chinese Medicine Affiliated to Guangzhou Medical University,Guangzhou 510140,Guangdong,China)

机构地区：[1]北京中医药大学第一临床医学院,北京100700 [2]广州医科大学附属中医医院,广东广州510140

出　　处：《现代中西医结合杂志》2023年第22期3067-3073,3143,共8页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：国家自然科学基金资助项目(81874401)。

摘　　要：目的探讨清热消癥方对高糖刺激下HK-2细胞自噬相关蛋白表达的影响。方法以人近端肾小管上皮细胞系HK-2细胞为干预对象,采用CCK-8法检测细胞活力对高糖模型造模条件和清热消癥方最佳干预浓度进行筛选;采用Western blot法对海藻糖干预浓度进行筛选。将HK-2细胞分为对照组、模型组、清热消癥方组、海藻糖组、清热消癥方+海藻糖组,分别予含有24.4 mmol/L甘露醇的完全培养基、30 mmol/L高糖培养基、30 mmol/L高糖+200μg/mL清热消癥方培养基、30 mmol/L高糖+50 mmol/L海藻糖培养基、30 mmol/L高糖+50 mmol/L海藻糖+200μg/mL清热消癥方培养基培养48 h。采用Western blot法和Immunofluorescence法检测HK-2细胞自噬相关蛋白表达情况。结果与对照组比较,模型组p62蛋白相对表达量和阳性表达平均荧光强度均明显升高(P均<0.05),溶酶体相关膜蛋白1(LAMP1)、B细胞淋巴瘤2(Bcl-2)蛋白相对表达量均明显降低(P均<0.05)。与模型组比较,清热消癥方组自噬相关蛋白7(Atg7)、LAMP1、Bcl-2相互作用蛋白1(Beclin 1)、Bcl-2蛋白相对表达量均明显升高(P均<0.05),p62蛋白相对表达量和p62阳性表达平均荧光强度均明显降低(P均<0.05);海藻糖组Atg7、LC3Ⅱ/LC3Ⅰ、LAMP1、Beclin 1、Bcl-2蛋白相对表达量和LC3B阳性表达平均荧光强度均明显升高(P均<0.05),p62蛋白相对表达量和清热消癥方组p62阳性表达平均荧光强度均明显降低(P均<0.05);清热消癥方+海藻糖组LC3Ⅱ/LC3Ⅰ、LAMP1、Bcl-2蛋白相对表达量和LC3B阳性表达平均荧光强度均明显升高(P均<0.05),p62蛋白相对表达量明显降低(P<0.05)。结论清热消癥方可上调Atg7、LAMP1、Beclin 1、Bcl-2表达,下调p62表达,改善溶酶体功能,增强自噬货物的吞噬消化,减少细胞凋亡,保护高糖刺激的HK-2细胞。Objective It is to investigate the effect of decoction for clearing heat and dispersing mass(DCHDM)on the expression of autophagy-related protein in HK-2 cells stimulated by high glucose.Methods The human proximal renal tubular epithelial cell line HK-2 cells were used as the intervention object,and the cell viability was detected by CCK-8 assay to screen the modeling conditions of the high glucose model and the optimal intervention concentration of DCHDM.The intervention concentration of trehalose was screened by Western blot method.HK-2 cells were divided into control group,model group,DCHDM group,trehalose group,DCHDM+trehalose group,and were respectively cultured for 48h with complete medium containing 24.4 mmol/L mannitol,30 mmol/L high glucose medium,30 mmol/L high glucose+200μg/mL DCHDM medium,30 mmol/L high glucose+50 mmol/L trehalose medium,30 mmol/L high glucose+50 mmol/L trehalose+200μg/mL DCHDM medium.The expression of autophagy-related proteins in HK-2 cells were detected by Western blot method and immunofluorescence method.Results Compared with the control group,the relative expression and the average fluorescence intensity of positive expression of p62 protein were significantly increased,and the relative expressions of lysosome-associated membrane protein 1(LAMP1)and B-cell lymphoma 2(Bcl-2)were significantly decreased in the model group(all P<0.05).Compared with the model group,the relative expressions of autophagy-related protein 7(Atg7),LAMP1,Bcl-2 interacting protein 1(Beclin 1),and Bcl-2 protein were significantly increased,and the relative expression and average fluorescence intensity of positive expression of p62 were significantly decreased in the DCHDM group(all P<0.05);the relative expressions of Atg7,LC3Ⅱ/LC3Ⅰ,LAMP1,Beclin 1,Bcl-2 proteins and the average fluorescence intensity of LC3B positive expression were significantly increased in the trehalose group(all P<0.05),the relative expression of p62 protein in the trehalose group and the average fluorescence intensity of positive e

关 键 词：糖尿病肾病 清热消癥法 HK-2细胞 自噬 

分 类 号：R-33[医药卫生]