细胞焦亡相关分子在急性胰腺炎模型小鼠胰腺中的表达

Expression of pyroptosis-related molecules in pancreas of acute pancreatitis model mice

目的探究3种不同造模方式制备的急性胰腺炎(AP)模型小鼠胰腺中细胞焦亡相关分子的表达水平和意义。方法选取20只SPF级雄性C57BL/6小鼠,采用随机数表法分为对照组、雨蛙肽组(CAE组)、CAE+脂多糖(LPS)组、L-精氨酸(L-Arg)组,每组5只,后3组分别制备AP模型。观察4组的胰腺组织病理变化并予以评分,检测血清淀粉酶、脂肪酶、TNF-α、IL-1β、IL-18水平,应用实时荧光定量PCR法检测胰腺组织中凋亡相关斑点样蛋白(ASC)、天冬氨酸特异性半胱氨酸蛋白酶-1(Caspase-1)、Gasdermin D(GSDMD)、IL-1β的mRNA表达水平,应用蛋白质印迹法和免疫荧光染色法检测胰腺组织中NLRP3、Caspase-1及IL-1β的蛋白表达水平。结果3组AP模型小鼠的血清淀粉酶、脂肪酶、TNF-α、IL-1β、IL-18及病理评分均较对照组升高,除CAE组的血清TNF-α和IL-18水平与对照组的差异无统计学意义(P>0.05)外,各组间其余指标的差异均有统计学意义(P均<0.05)。3组AP模型小鼠的胰腺组织中ASC、Caspase-1、GSDMD及IL-1β的mRNA表达水平均较对照组明显升高,除CAE组ASC和GSDMD的mRNA表达水平与对照组的差异无统计学意义(P>0.05)外,各组间其余指标的差异均有统计学意义(P均<0.05)。对照组胰腺组织中NLRP3、活化的Caspase-1(Cleaved-Caspase-1)、IL-1β蛋白呈低表达,Caspase-1前体(Pro-Caspase-1)和IL-1β前体(Pro-IL-1β)蛋白呈高表达;3组AP模型小鼠的胰腺组织中NLRP3、Cleaved-Caspase-1、IL-1β蛋白表达水平较对照组升高,除L-Arg组与对照组的NLRP3表达水平差异无统计学意义(P>0.05)外,其余差异均有统计学意义(P均<0.05);而3组AP模型小鼠的胰腺组织中Pro-Caspase-1和Pro-IL-1β表达水平则较对照组降低,其中Pro-Caspase-1与对照组的差异均有统计学意义(P均<0.05)。结论AP模型小鼠的胰腺组织中细胞焦亡相关分子NLRP3、Caspase-1、ASC、GSDMD、IL-1β的表达均上调,提示细胞焦亡可能在AP发生和发展过程中发挥了重要作用。

Objective This paper intends to explore the expression levels and significance of pyroptosis-related molecules in the pancreas of acute pancreatitis(AP)model mice prepared by three different modeling methods.Methods Twenty SPF grade male C57BL/6 mice were selected and randomly assigned to the control group,the cerulein group(CAE group),the CAE+lipopolysaccharide(LPS)group,and the L-arginine(L-Arg)group according to the number table method.Each group had 5 mice,and AP models were prepared in the last 3 groups.The pathological changes of the pancreatic tissue in the 4 groups were observed and scored.The serum amylase,lipase,TNF-α,IL-1β,and IL-18 levels were detected,and real-time quantitative PCR was used to detect apoptosis-related spotting proteins(ASC),cysteinyl aspartate specific proteinase-1(Caspase-1),Gasdermin D(GSDMD),and IL-1βmRNA expression levels in the pancreatic tissue.Western blotting and immunofluorescence staining were used to detect expression levels of NLRP3,Caspase-1,and IL-1βprotein in pancreatic tissue.Results The serum amylase,lipase,TNF-α,IL-1β,IL-18,and pathological scores of the AP model mice in the three groups are higher than those in the control group.Except for the levels of serum TNF-αand IL-18 in the CAE group and the control group,there are no significant differences(P>0.05),and the differences in other indicators between the groups are statistically significant(P<0.05).The mRNA expression levels of ASC,Caspase-1,GSDMD,and IL-1βin the pancreatic tissue of the three AP model mice groups are significantly higher than those in the control group.Except for the mRNA expression levels of ASC and GSDMD between the CAE group and the control group,there are no significant differences(P>0.05),and the differences of other indicators between the groups are statistically significant(P<0.05).The expression of NLRP3,activated Caspase-1(Cleaved-Caspase-1),and IL-1βprotein in the pancreatic tissue of the control group is low,and Caspase-1 precursor(Pro-Caspase-1)and IL-1βprecursor(Pro-IL-1β))protein was highly expressed.The expression levels of NLRP3,Cleaved-Caspase-1 and IL-1βin the pancreatic tissue of AP model mice in the three groups are higher than those in the control group,except that there is no significant difference in the expression levels of NLRP3 between the L-Arg group and the control group(P>0.05),the rest of the differences are statistically significant(P<0.05).However,the expression levels of Pro-Caspase-1 and Pro-IL-1βin the pancreatic tissue of AP model mice in the three groups are lower than those in the control group,and the difference between Pro-Caspase-1 and the control group is statistically significant(P<0.05).Conclusion The expression levels of pyroptosis-related molecules,including NLRP3,Caspase-1,ASC,GSDMD,and IL-1β,are all upregulated in the pancreatic tissue of AP model mice,suggesting that pyroptosis may play an important role in the occurrence and development of AP.