RND3对雨蛙素诱导的大鼠AR42J细胞损伤的影响

Effect of RND3 on injury of AR42J cell in rats induced by caerulein

目的:观察干预RND3对雨蛙素诱导的大鼠胰腺腺泡细胞AR42J损伤的影响,并探究RND3在急性胰腺炎(AP)中的调控机制。方法:分别构建RND3沉默和过表达的雨蛙素诱导的AR42J细胞AP模型,共6组(NC对照组、雨蛙素处理组、si-con阴性对照组、pcDNA阴性对照组、si-RND3沉默组和pcDNA-RND3过表达组)。qRT-PCR和Western blot分别在雨蛙素给药后的0 h、4 h、8 h、12 h和24 h检测AR42J细胞中RND3 mRNA、蛋白表达水平;CCK-8法检测细胞的存活情况和LDH漏出量,流式细胞术检测AR42J细胞的凋亡率,qRT-PCR检测相关炎症因子(TNF-α、IL-6、IL-1β和IL-18)的mRNA表达水平,Western blot检测AR42J细胞中凋亡相关蛋白(GSDMD、NLRP3、Cleaved caspase-1和caspase-1)表达。结果:雨蛙素诱导的AR42J细胞能抑制RND3 mRNA和蛋白表达,且随着雨蛙素处理时间延长,其对RND3的抑制作用越显著;相比NC对照组,siRND3组、雨蛙素处理组大鼠AR42J细胞中的相关炎症因子TNF-α、IL-6、IL-1β和IL-18表达上调(P<0.05),同时,凋亡相关蛋白GSDMD、NLRP3、Cleaved caspase-1和caspase-1表达升高(P<0.05);相反,pcDNA-RND3过表达组细胞炎症和凋亡水平显著降低(P<0.05)。结论:RND3可减轻大鼠胰腺腺泡细胞AR42J的炎症反应和细胞凋亡,其作用机制与NLRP3/caspase-1/GSDMD通路的活性有关。

Objective:To observe the effect of RND3 intervention on injury of pancreatic acinar cells AR42J induced by caerulein in rats,and explore regulatory mechanism of RND3 in acute pancreatitis(AP).Methods:Lentiviral AP models with RND3 gene silencing and overexpression,and AR42J cell AP models induced by caerulein were constructed in 6 groups(NC control group,caerulein treatment group,si-con negative control group,pcDNA negative control group,si-RND3 silencing group,and pcDNA-RND3 overexpression group).After the administration of caerulein,mRNA and protein expression level of RND3 in AR42J cells were quantified at time points of 0 h,4 h,8 h,12 h and 24 h by qRT-PCR and Western blot,respectively.Cell survival and LDH leakage were detected by CCK-8 method.Apoptosis rate of AR42J cells was detected by flow cytometry.mRNA expression levels of related inflammatory factors(TNF-α,IL-6,IL-1βand IL-18)were detected by qRT-PCR.Western blot was used to detect expressions of apoptosisrelated proteins(GSDMD,NLRP3,Cleaved caspase-1 and caspase-1)in AR42J cells.Results:Expression of RND3 mRNA and protein in AR42J cells induced by caerulein was inhibited,and expression of RND3 was inhibited more significantly with the increase of caerulein treatment time.Compared with NC control group,expressions of TNF-α,IL-6,IL-1βand IL-18 in AR42J cells of rats in si-RND3 group and caerulein treatment group were up-regulated(P<0.05),at the same time,expressions of apoptosis-related proteins GSDMD,NLRP3,Cleaved caspase-1 and caspase-1 were increased.On the contrary,levels of cell inflammation and apoptosis were significantly reduced in pcDNA-RND3 overexpression group(P<0.05).Conclusion:RND3 can reduce the inflammatory reaction and apoptosis of rats pancreatic acinar cells AR42J,which mechanism is related to the activity of NLRP3/caspase-1/GSDMD pathway.