黄芪丹参配伍调控ASIC1a/CaMKⅡδ信号抑制酸性微环境中心肌细胞焦亡

Huangqi-Danshen in the Regulation of ASIC1a/CaMKⅡδ Signaling to Inhibit Cardiomyocytes from Pyroptosis in Acidic Microenvironment

    摘要
  • 摘要:
      目的  探讨黄芪丹参配伍对乳酸诱导的酸性微环境中心肌细胞焦亡损伤的保护作用及基于酸敏感离子通道1a(ASIC1a)/钙离子-钙调蛋白依赖性激酶Ⅱδ(CaMKⅡδ)信号通路的调控机制。
      方法  采用Lactate刺激H9C2心肌细胞建立pH 6.5酸性微环境细胞损伤模型。分为正常对照组、模型组、黄芪丹参提取物(HQ)组、HQ+ASIC激动剂Nocistatin组和ASIC抑制剂NS383阳性药对照组。采用CCK-8法测定心肌细胞活力, 流式细胞术检测心肌细胞凋亡, 免疫荧光检测ASIC1a表达, qPCR分析NLRP3、Caspase-1、IL-1β及GSDMD mRNA表达, Western blot检测ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白的表达。
      结果  在pH 6.5酸性环境中, 心肌细胞活力显著降低(P < 0.01), 细胞凋亡明显增加(P < 0.01), NLRP3、Caspase-1、IL-1β及GSDMD mRNA表达明显增加(P < 0.01), ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白表达明显增加(P < 0.01)。60 μg · mL-1HQ可显著促进pH6.5酸性微环境中心肌细胞活力(P < 0.01), 抑制心肌细胞凋亡(P < 0.01), 下调NLRP3、Caspase-1、IL-1β、GSDMD mRNA和ASIC1a、CaMKⅡδ、NLRP3、Caspase-1、Cleaved Caspase-1、GSDMD及GSDME-N蛋白表达(P < 0.01)。并且HQ的以上效应可被ASIC激动剂Nocistatin逆转。
      结论  黄芪丹参配伍可下调ASIC1a/CaMKⅡδ信号抑制酸性微环境下NLRP3炎症小体介导的心肌细胞焦亡。

     

    Abstract:
      OBJECTIVE  To observe the protective effect of Huangqi-Danshen on cardiomyocytes pyroptosis or injury in acidic microenvironment induced by lactic acid and the regulatory mechanism based on the acid-sensitive ion channel 1a (ASIC1a) or calcium/calmodulin-dependent protein kinase Ⅱδ (Ca MKⅡδ) signaling pathway.
      METHODS  Lactate was used to stimulate H9C2 cardiomyocytes to establish the cell injury model in the acidic environment of pH 6.5. The rats were divided into the normal control group, model group, Huangqi-Danshen extract (HQ) group, HQ + ASIC agonist Nocistatin group, as well as ASIC inhibitor NS383 positive drug control group. The viability of cardiomyocytes was measured by CCK-8 method. The apoptosis of cardiomyocytes was detected by flow cytometry. Besides, the expression of ASIC1a was detected by immunofluorescence. The mRNA expression of NLRP3, Caspase-1, IL-1β, and GSDMD was analyzed by qPCR. In addition, the protein expression of ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, and GSDME-N was detected by Western blot.
      RESULTS  In the acidic environment of pH 6.5, the viability of cardiomyocytes was significantly reduced (P < 0.01), but the apoptosis was significantly increased (P < 0.01). The mRNA expressions of NLRP3, Caspase-1, IL-1β and GSDMD were significantly increased (P < 0.01), and the protein expressions of ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD and GSDME-N were also significantly increased (P < 0.01). In addition, we found that HQ (60 μg·mL-1) could significantly promote the viability of cardiomyocytes in acidic microenvironment (P < 0.01), inhibit cardiomyocyte apoptosis (P < 0.01), and down-regulate the mRNA expressions (NLRP3, Caspase-1, IL-1β, GSDMD) combined with protein expressions (ASIC1a, CaMKⅡδ, NLRP3, Caspase-1, Cleaved Caspase-1, GSDMD, GSDME-N) (P < 0.01). Besides, the above effects of HQ can be reversed by ASIC agonist Nocistatin.
      CONCLUSION  Huangqi-Danshen can down-regulate ASIC1a/CaMKⅡδ signaling to inhibit NLRP3 inflammasome -mediated cardiomyocyte pyroptosis in acidic microenvironment.

     

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