丹皮酚对白蛋白诱导的肾小管上皮细胞自噬和NLRP3炎症小体影响的研究OA
Research of Effects of paeonol on autophagy and NLRP3 inflammasome in renal tubular epithelial cells induced by albumin
目的 观察丹皮酚对白蛋白(HSA)诱导的肾小管上皮细胞(HK-2)自噬和NOD样受体蛋白3(NLRP3)炎症小体的调控作用.方法 体外培养HK-2细胞并随机分为对照组、模型组和低、中、高剂量实验组.对照组细胞正常培养;模型组用20 mg·mL-1 HSA干预24 h;低、中、高剂量实验组在模型组的基础上分别加入25、50和100 μmol·L-1丹皮酚处理24 h.酶联免疫吸附实验法检测上清液肾损伤因子1(KIM-1)、单核细胞趋化蛋白-1(MCP-1)、白介素-1 β(IL-1β)和胱天蛋白酶-1(caspase-1)的含量,用实时荧光定量聚合酶链反应法检测KIM-1和MCP-1基因表达,用蛋白质印迹法检测含吡喃结构域的NLRP3、含CARD结构域的凋亡相关斑点样蛋白(ASC)、IL-1β、微管相关蛋白1轻链3(LC3)、泛素结合蛋白p62(p62)、自噬效应蛋白Beclin 1(Beclin-1)、PTEN诱导激酶1(PINK1)和Parkin蛋白的表达.结果 对照组、模型组和低、中、高剂量组细胞上清中KIM-1浓度分别为(8.65±0.53)、(17.33±2.04)、(10.76±1.41)、(8.48±0.47)和(8.47±0.24)pg·mL-1,MCP-1 含量为(531.38±15.64)、(2 198.55±148.21)、(1 966.29±53.52)、(1 605.49±234.68)和(937.33±36.96)pg·mL-1,IL-iβ 分泌水平为(71.66±2.29)、(109.92±4.41)、(91.68±4.75)、(80.66±4.00)和(62.38±3.24)pg·mL-1,caspase-1 含量为(66.57±10.52)、(210.50±17.50)、(201.88±7.22)、(195.42±13.95)和(129.81±13.20)pg·mL-1,KIM-1 mRNA基因相对表达水平分别为1.05±0.05、2.38±0.45、1.58±0.20、1.44±0.08 和 1.38±0.22,MCP-1 mRNA 相对表达水平分别为1.04±0.04、1.53±0.08、1.32±0.16、1.16±0.16 和0.62±0.13,NLRP3 蛋白相对表达水平为0.89±0.08、1.13±0.07、1.00±0.07、0.84±0.12和0.55±0.16,ASC蛋白相对表达水平分别为0.23±0.03、0.87±0.19、0.81±0.16、0.78±0.08和 0.46±0.05,IL-1β 蛋白相对表达水平分别为0.49±0.12、1.46±0.29、1.10±0.26、0.71±0.06 和0.53±0.13,微管相关蛋白1轻链3-2(LC3 Ⅱ)/LC3 Ⅰ蛋白相对表达水平分别为1.08±0.04、0.90±0.06、1.00±0.02和1.05±0.04,p62蛋白相对表达水平分别为0.38±0.06、1.23±0.11、1.05±0.20和 0.62±0.19,Beclin-1 蛋白相对表达水平分别为0.82±0.07、0.59±0.06、0.90±0.07 和 0.93±0.09,PINK1 蛋白相对表达水平分别为1.10±0.03、0.68±0.07、0.78±0.11 和 1.03±0.08,Parkin 蛋白相对表达水平为0.64±0.06、0.46±0.05、0.58±0.08 和 0.79±0.12.模型组的上述指标与对照组比较,在统计学上差异均有统计学意义(P＜0.05,P＜0.01,P＜0.001),高剂量实验组的上述指标与模型组比较,在统计学上差异均有统计学意义(P＜0.05,P＜0.01,P＜0.001).结论 丹皮酚可能通过促进线粒体自噬,抑制NLRP3炎症小体,缓解白蛋白导致的肾小管损伤.
Objective To investigate the regulatory effects of paeonol on autophagy and the NOD-like receptor protein 3(NLRP3)inflammasome in human renal proximal tubular epithelial cells(HK-2)induced by human serum albumin(HSA).Methods HK-2 cells were cultured in vitro and randomly assigned to control group,model group,as well as low-,medium-and high-dose experimental groups.Cells in the control group were cultured under normal conditions;those in the model group were treated with 20 mg·mL-1 HSA for 24 h;and cells in the low-,medium-and high-dose experimental groups were administered 25,50 and 100 μmol·L-1 paeonol,respectively,for 24 h on the basis of the model group intervention.Enzyme-linked immunosorbent assay(ELISA)was performed to determine the concentrations of kidney injury molecule 1(KIM-1),monocyte chemoattractant protein 1(MCP-1),interleukin 1 β(IL-1β)and caspase-1 in the cell supernatant.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression levels of KIM-1 and MCP-1.Western blotting was applied to measure the protein expression levels of NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),interleukin-1 β(IL-1 β),microtubule-associated protein 1 light chain 3(LC3),sequestosome 1(p62),Beclin-1,PTEN-induced kinase 1(PINK1)and Parkin.Results The concentrations of KIM-1 in the cell supernatant of the control group,model group,and low-,medium-and high-dose experimental groups were(8.65±0.53),(17.33±2.04),(10.76±1.41),(8.48±0.47)and(8.47±0.24)pg·mL-1,respectively;the contents of MCP-1 were(531.38±15.64),(2 198.55±148.21),(1 966.29±53.52),(1 605.49±234.68)and(937.33±36.96)pg·mL-1,respectively;the secretion levels of IL-1 β were(71.66±2.29),(109.92±4.41),(91.68±4.75),(80.66±4.00)and(62.38±3.24)pg·mL-1,respectively;the contents of caspase-1 were(66.57±10.52),(210.50±17.50),(201.88±7.22),(195.42±13.95)and(129.81±13.20)pg·mL-1,respectively;the relative expression levels of KIM-1 mRNA in each group were 1.05±0.05,2.38±0.45,1.58±0.20,1.44±0.08 and 1.38±0.22,respectively;while those of MCP-1 were 1.04±0.04,1.53±0.08,1.32±0.16,1.16±0.16 and 0.62±0.13,respectively;the relative expression levels of NLRP3 protein were 0.89±0.08,1.13±0.07,1.00±0.07,0.84±0.12 and 0.55±0.16,respectively;those of ASC were 0.23±0.03,0.87±0.19,0.81±0.16,0.78±0.08 and 0.46±0.05,respectively;those of IL-1 β were 0.49±0.12,1.46±0.29,1.10±0.26,0.71±0.06 and 0.53±0.13,respectively;the LC3 Ⅱ/Ⅰ ratios were 1.08±0.04,0.90±0.06,1.00±0.02 and 1.05±0.04,respectively;the expression levels of p62 protein were 0.38±0.06,1.23±0.11,1.05±0.20 and 0.62±0.19,respectively;those of Beclin-1 were 0.82±0.07,0.59±0.06,0.90±0.07 and 0.93±0.09,respectively;those of PINK1 were 1.10±0.03,0.68±0.07,0.78±0.11 and 1.03±0.08,respectively;those of Parkin were 0.64±0.06,0.46±0.05,0.58±0.08 and 0.79±0.12,respectively.Compared with the control group,all the above indicators in the model group exhibited statistically significant differences(P＜0.05,P＜0.01,P＜0.001).Moreover,the indicators in the high-dose experimental group showed statistically significant differences compared with the model group(P＜0.05,P＜0.01,P＜0.001).Conclusion Paeonol may alleviate albumin-induced renal tubular injury by promoting mitophagy and inhibiting the activation of the NLRP3 inflammasome.
郝永熙;梁卫;时乐;秦子怡;梁涛
南京中医药大学药学院,江苏南京 210023中国人民解放军东部战区空军医院中医科,江苏南京 210002南京中医药大学药学院,江苏南京 210023南京中医药大学药学院,江苏南京 210023南京中医药大学药学院,江苏南京 210023
医药卫生
丹皮酚白蛋白自噬炎症小体肾小管上皮细胞
paeonolalbuminautophagyinflammasomerenal tubular epithelial cell
《中国临床药理学杂志》 2026 (1)
53-58,6
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