人重组骨形态发生蛋白7通过下调半胱氨酸蛋白酶-1/焦孔素D信号通路抑制高糖诱导的近曲小管上皮细胞的焦亡

    Recombinant human bone morphogenetic protein 7 suppressed high glucose-induced pyroptosis of proximal tubular epithelial cells through down-regulating caspase-1/GSDMD signaling pathway

      摘要
    • 摘要:
      目的  观察人重组骨形态发生蛋白-7（recombinant human bone morphogenetic protein-7，rhBMP-7）对高糖诱导的人肾皮质近曲小管上皮细胞（human kidney-2，HK-2）的焦亡和炎症反应的作用。
      方法  体外培养高糖（high glucose，HG）刺激的HK-2细胞模拟糖尿病肾病模型。分为正常浓度葡萄糖对照组（Control）、高浓度葡萄糖组（HG）、Control+rhBMP-7组、HG+rhBMP-7组。采用免疫荧光方法检测HK-2细胞中E-钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-smooth muscle actin，α-SAM）、焦孔素D（gasdermin D，GSDMD）蛋白和焦孔素E（gasdermin E，GSDME）蛋白；TUNEL染色检测细胞死亡情况；蛋白质印迹法检测各组HK-2细胞中焦亡相关蛋白半胱氨酸蛋白酶-1（caspase-1）、半胱氨酸蛋白酶-4（caspase-4）、半胱氨酸蛋白酶-5（caspase-5）、N-GSDMD和GAPDH的表达；酶联免疫吸附试验（enzyme linked immunosorbent assay，ELISA）检测各组HK-2细胞培养上清液中炎症因子白细胞介素-18（interleukin-18，IL-18）和白细胞介素-1β（interleukin-1β，IL-1β）的表达。
      结果  与Control组相比，高糖组HK-2细胞Tunel染色的阳性率明显增加，差异具有统计学意义（P＜0.0001），α-SMA蛋白和α-SMA mRNA表达量也显著增加（P＜0.0001），E-cadherin蛋白和E-cadherin mRNA表达量显著下降（P = 0.0006，P＜0.0001）；HG组添加rhBMP-7后α-SMA蛋白和mRNA水平显著下降（P = 0.0117，P = 0.002），E-cadherin蛋白和mRNA水平明显升高 （P = 0.0015，P＜0.0001）。与对照组比较，HG组HK-2细胞GSDME/GSDMD蛋白的荧光信号明显增强。进而，HG组添加rhBMP-7后GSDME/GSDMD蛋白荧光信号显著减弱。同样，HG组HK-2细胞中焦亡相关蛋白caspase-1、caspase-4、caspase-5以及GSDMD的表达量显著高于对照组，差异明显（均P＜0.001）。HG组添加rhBMP-7后焦亡相关蛋白的表达明显下降（分别P＜0.001，P = 0.002，P＜0.001）。与对照组比较，HG组HK-2细胞培养上清液中IL-1β和IL-18蛋白浓度显著增加（均P＜0.001）。进而导致rhBMP-7干预后HG组细胞上清液中IL-10和IL-1β蛋白浓度明显减少（P＜0.001）。
      结论  rhBMP-7具有抑制HK-2细胞焦亡的作用，其通过调控caspase-1/GSDMD信号通路减缓高糖诱导的HK-2细胞焦亡。

       

      Abstract:
      Objective  To observe the effect of recombinant human bone morphogenetic protein-7 （rhBMP-7） on pyroptosis and inflammatory response of human kidney-2 （HK-2） cell after a stimulation of high glucose （HG）.
      Methods  HK-2 cell stimulated by high glucose （HG） was cultured in vitro for diabetic nephropathy modeling. There were four groups of normal glucose control （control）, HG, control + rhBMP-7 and HG + rhBMP-7. The levels of E-cadherin, a-smooth muscle actin （a-SAM）, Gasdermin D （GSDMD） and Gasdermin E （GSDME） were detected by immunofluorescence. Cell death was detected by TdT-mediated dUTP nick end labeling （TUNEL） stain. Western blot was utilized for detecting the expressions of pyroptosis-related proteins caspase-1/4/5, N-GSDMD and GAPDH. And the expressions of inflammatory factors of interleukin-18 （IL-18） and interleukin-1β （IL-1β） in cell supernatant were detected by enzyme-linked immunosorbent assay （ELISA）.
      Results  Compared with control group, positive rate of TUNEL stain spiked sharply in HG group and the difference was statistically significant （P＜0.0001）. The expressions of α-SMA protein/mRNA spiked significantly （P＜0.0001） while the expression of E-cadherin protein/RNA dropped markedly （P = 0.0006, P＜0.0001）; Furthermore, the level of α-SMA protein/mRNA declined significantly in HG group after adding rhBMP-7 （P = 0.0117, P = 0.002） while the level of E-cadherin protein/mRNA jumped significantly （P = 0.0015, P＜0.0001）. Compared with control group, fluorescent signal of GSDME/GSDMD protein became significantly strengthened and then weakened in HG group after an addition of rhBMP-7. Similarly, the expression levels of caspase-1/4/5 and GSDMD of HG group were significantly higher than those of control group （P＜0.001）. The expressions of pyroptosis-related proteins were significantly down-regulated in HG group after adding rhBMP-7 （P＜0.001, P = 0.002, P＜0.001）. Compared with control group, protein concentrations of IL-1β and IL-18 in supernatant of HG group jumped significantly （both P＜0.001）. Furthermore, after adding rhBMP-7, the protein concentrations of IL-10 and IL-1β in supernatant declined markedly in HG group （P＜0.001）.
      Conclusions  rhBMP-7 can alleviate the pyroptosis of HK-2 cell induced by HG through regulating caspase-1/GSDMD signaling pathway.

       

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