细胞质核苷酸结合寡聚化域样受体蛋白3炎症小体通路激活对硫酸吲哚酚诱导的心肌细胞肥大蛋白表达的影响

Effects of indoxyl sulphate-induced nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes activation on protein expression in hypertrophic cardiomyocytes

目的探讨硫酸吲哚酚(IS)诱导细胞质核苷酸结合寡聚化域样受体蛋白3(NLRP3)炎症小体通路激活与心肌细胞肥大蛋白表达的关系及可能机制。方法提取原代心肌细胞,分为对照组和IS(10 mmol/L)组,IS干预后检测两组脑钠肽蛋白表达情况。细胞计数套盒8(CCK8)法检测IS刺激小鼠单核巨噬细胞白血病细胞(RAW264.7)的最佳刺激浓度和时间。将RAW264.7分为对照组和IS组,Western blot检测NLRP3、含半胱氨酸的天冬氨酸蛋白水解酶-1前体(Pro-caspase 1)、白细胞介素-1β(IL-1β)、IL-18蛋白表达情况。酶联免疫吸附试验(ELISA)法检测对照组和IS组细胞上清液中炎症因子IL-18和IL-1β水平。将巨噬细胞培养基上清液与心肌细胞直接共培养,分为空白对照组、巨噬细胞培养基组、巨噬细胞培养基+IS组,检测各组NLRP3、Pro-caspase 1、IL-1β、IL-18、脑钠肽蛋白表达情况。结果IS刺激原代心肌细胞后,心房钠尿肽(ANP)和脑钠肽蛋白表达显著上调。由CCK8法检测发现IS刺激RAW264.7的最佳浓度是15 mmol/L,最佳干预时间为3 h。IS刺激RAW264.7后,与对照组相比炎症相关蛋白NLRP3、Pro-caspase 1、IL-1β、IL-18表达明显增加(均P<0.05)。通过ELISA法检测发现IS组巨噬细胞培养基上清液中炎症因子IL-1β和IL-18水平较对照组显著增加。在直接共培养实验中发现IS+巨噬细胞培养基组与空白对照组和巨噬细胞培养基组相比,NLRP3、Pro-caspase 1、IL-1β、IL-18、ANP和脑钠肽蛋白表达显著上调(均P<0.05)。结论IS可诱导NLRP3炎症小体活化,促进心肌细胞肥大蛋白表达。

Objective To investigate the relationship and possible mechanism between IS(indoxyl sulphate)-induced nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3)inflammasomes activation and protein expression in hypertrophic cardiomyocytes.Methods Primary cardiomyocytes were extracted and one part were divided into control group and IS(10 mmol/L)group.And the expression of natriuretic peptide was detected for both groups after the intervention.Cell counting kit 8(CCK8)was used to identify the optimal stimulus duration and concentration of IS stimulating mice leukemic monocyte/macrophage(RAW264.7).RAW264.7 were then divided into control and IS groups.After the intervention,their expression of NLRP3,Pro-caspase 1,interlenkin 1β(IL-1β)and IL-18 were measured by Western blot.And their cell supernatant was determined by enzyme-linked immunosorbent assay(ELISA)to assess the levels of inflammatory factors,including IL-18 and IL-1β.Subsequently,the other primary cardiomyocytes were cultured in the supernatant of macrophages and divided into control group,supernatant group and supernatant+IS group.Their expression of NLRP3,Pro-caspase 1,IL-1β,IL-18 and natriuretic peptide were detected after the intervention of each group.Results The expression of atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)in primary cardiomyocytes was significantly up-regulated after IS stimulation.CCK8 assays showed that the optimal stimulus concentration of IS stimulating RAW264.7 was 15 mmol/L,and the optimal stimulus duration was 3 hours.Compared with the control group,the expression of inflammation-related proteins,including NLRP3,Pro-caspase 1,IL-1β,IL-18,and p-P65,were significantly increased after IS stimulation in RAW264.7(all P<0.05).ELISA revealed that the levels of inflammatory factors,including IL-1βand IL-18,in the supernatant of the IS group were significantly increased.In the experiment of culturing cardiomyocytes in the supernatant of macrophages,it was found that the expression of NLRP3,Pro-caspase 1,IL-1β,IL-18,ANP and BNP was significantly up-regulated in the supernatant+IS group compared with the control group(all P<0.05).Conclusion IS can induce the activation of NLRP3 inflammasomes and promote protein expression in hypertrophic cardiomyocytes.