心阳片含药血清调控NLRP3介导细胞焦亡保护心肌细胞炎症损伤的作用及机制研究

Study on Effect and Mechanism of Xinyang Tablet(心阳片)-containing Serum Regulating NLRP3-Mediated Pyrocytosis and Protecting Myocardial Cell Inflammation

目的通过观察心阳片含药血清调控NLRP3介导的细胞焦亡对心肌细胞炎症损伤的保护作用,探讨心阳片防治心力衰竭的作用机制。方法随机将40只雄性SD大鼠分成空白组、心阳片低、中、高剂量组,分别给予临床等效剂量的1、2、4倍心阳片(0.24、0.48、0.96 g/kg)灌胃;空白组给予等量蒸馏水进行灌胃。各组大鼠灌胃2次/d, 2 mL/次,连续灌胃7 d。末次给药灌胃后,腹主动脉取血,分离血清。用LPS/ATP(1μg/mL LPS刺激细胞24 h,再用5 mmol/L ATP刺激细胞4 h)处理HL-1心肌细胞,并分为:空白组(NC)、模型组(LPS/ATP+空白血清)、心阳片低剂量组(LPS/ATP+低剂量心阳片血清)、心阳片中剂量组(LPS/ATP+中剂量心阳片血清)、心阳片高剂量组(LPS/ATP+高剂量心阳片血清)。CCK8检测细胞活性,免疫荧光检测各组细胞NLRP3的表达情况,RT-qPCR检测NLRP3、GSDMD、IL-1β、Caspase-1mRNA转录水平,Western Blot检测心肌细胞NLRP3、pro-IL-1β、IL-1β、IL-18、cleaved-GSDMD、pro-Caspase-1、Caspase-1、ASC蛋白表达情况。结果与空白组相比,LPS/ATP组HL-1细胞活力明显降低,NLRP3、GSDMD、IL-1β、Caspase-1mRNA水平升高(P<0.05),NLRP3、pro-IL-1β、IL-1β、IL-18、cleaved-GSDMD、pro-Caspase-1、Caspase-1、ASC蛋白表达明显升高(P<0.05);与LPS/ATP组相比,心阳片含药血清低、中、高剂量组细胞活力增加,NLRP3、GSDMD、IL-1β、Caspase-1mRNA水平降低(P<0.05),NLRP3、pro-IL-1β、IL-1β、IL-18、cleaved-GSDMD、pro-Caspase-1、Caspase-1、ASC蛋白表达明显降低(P<0.05)。结论心阳片含药血清能改善LPS/ATP诱导HL-1细胞炎症损伤,其机制可能是通过抑制NLRP3/IL-1β介导的细胞焦亡发挥作用。

Objective To observe the protective effect of Xinyang Tablet(心阳片)-containing serum regulating NLRP3-mediated pyrocytosis on myocardial cell inflammation and explore the mechanism of Xinyang Tablet preventing and treating heart failure. Methods Forty male SD rats were randomly into blank group, Xinyang Tablet low, medium and high dose groups(0.24 g/kg, 0.48 g/kg, 0.96 g/kg). The blank group was given the same amount of distilled water by gavage. Rats in each group were given gastric administration twice a day, 2 mL for each time, consecutively for 7 days. After the last administration, the blood was taken out from the abdominal aorta and the serum was separated. The HL-1 cardiomyocytes were treated with LPS/ATP(1 μg/mL LPS stimulated cells for 24 hours, and then 5 mmol/L ATP stimulated for 4 hours) and were divided into blank group(NC), model group(LPS/ATP+blank serum), Xinyang Tablet low dose group(LPS/ATP+low dose Xinyang Tablet), Xinyang Tablet medium dose group(LPS/ATP+medium dose Xinyang Tablet), Xinyang Tablet high dose group(LPS/ATP+high dose Xinyang Tablet). CCK8 detected the cell viability and the immunofluorescence detected the expression of NLRP3 of cells in each group. RT-qPCR detected the transcription levels of NLRP3, GSDMD, IL-1β and Caspase-1 mRNA. Western Blot detected the expressions of cardiomyocyte NLRP3, pro-IL-1β, IL-1β, IL-18, cleaved-GSDMD, pro-Caspase-1, Caspase-1 and ASC protein. Results Compared with those of the blank group, the viability of HL-1 cells in the LPS/ATP group was significantly reduced, and the levels of NLRP3, GSDMD, IL-1β and Caspase-1 mRNA were increased(P<0.05), and expressions of NLRP3, pro-IL-1β, IL-1β, IL-18, cleaved-GSDMD, pro-Caspase-1, Caspase-1 and ASC protein were significantly increased(P<0.05). Compared with those of the LPS/ATP group, the levels of NLRP3, GSDMD, IL-1β and Caspase-1 mRNA in Xinyang Tablets low, medium and high dose groups were decreased(P<0.05). The expressions of NLRP3, pro-IL-1β, IL-1β, IL-18, cleaved-GSDMD, pro-Caspase-1, Caspase-1 and ASC protein were significantly reduced(P<0.05). Conclusion Xinyang Tablet-containing serum can improve LPS/ATP-induced inflammatory injury of HL-1 cells. The mechanism may be through inhibiting NLRP3/IL-1β-mediated pyrocytosis.