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

  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.