毛蕊异黄酮通过调控TREM-1表达对重症急性胰腺炎腺泡细胞损伤的影响

目的:探讨毛蕊异黄酮(Calycosin)对重症急性胰腺炎(SAP)腺泡细胞损伤的影响及其分子机制。方法:将大鼠胰腺腺泡细胞AR42J分为Con组(AR42J细胞)、SAP组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖)、SAP+Calycosin-L组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+10μmol/L毛蕊异黄酮)、SAP+Calycosin-M组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+50μmol/L毛蕊异黄酮)、SAP+Calycosin-H组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+100μmol/L毛蕊异黄酮)、SAP+si-NC组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+转染si-NC)、SAP+si-TREM-1组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+转染si-TREM-1)、SAP+Calycosin+pcDNA组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+100μmol/L毛蕊异黄酮+转染pcDNA)及SAP+Calycosin+pcDNA-TREM-1组(AR42J细胞+10 nmol/L雨蛙素+10 mg/L脂多糖+100μmol/L毛蕊异黄酮+转染pcDNA-TREM-1)。酶联免疫法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平;流式细胞术检测细胞凋亡;蛋白质印迹法检测B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved-caspase3)、髓样细胞表达触发受体-1(TREM-1)蛋白表达;实时荧光定量PCR(RT-qPCR)检测TREM-1 mRNA表达。结果:与Con组比较,SAP组细胞凋亡率,IL-6、TNF-α水平,Bax、cleaved-caspase3、TREM-1蛋白及TREM-1 mRNA表达增加(P<0.05),Bcl-2蛋白表达减少(P<0.05)。与SAP组比较,SAP+Calycosin-L组、SAP+Calycosin-M组及SAP+Calycosin-H组SAP腺泡细胞IL-6、TNF-α水平,Bcl-2、TREM-1蛋白及TREM-1 mRNA表达降低(P<0.05),细胞凋亡率、Bax、cleaved-caspase3蛋白表达增加,均呈浓度依赖性(P<0.05)。与SAP+si-NC组比较,SAP+si-TREM-1组SAP腺泡细胞TREM-1、Bcl-2蛋白表达及IL-6、TNF-α水平降低(P<0.05),细胞凋亡率、Bax、cleaved-caspase3蛋白表达增加(P<0.05)。与SAP+Calycosin+pcDNA组比较,SAP+Calycosin+pcDNA-TREM-1组腺泡细胞TREM-1、Bcl-2蛋白表达及IL-6、TNF-α水平升高(P<0.05),细胞凋亡率、Bax、cleaved-caspase 3蛋白表达减少(P<0.05)。结论:毛蕊异黄酮可通过调控TREM-1表达减轻SAP腺泡细胞炎症,并诱导细胞凋亡,保护SAP腺泡细胞免受损伤。

Effect of Calycosin on Acinar Cell Injury in Severe Acute Pancreatitis by Regulating TREM-1 Expression

Objective:To discuss the effect of calycosin on acinar cell injury in severe acute pancreatitis(SAP)and its molecular mechanism.Methods:Pancreatic acinar AR42J cells of rats were divided into the Con group(AR42J cells),the SAP group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide),the SAP+Calycosin-L group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+10μmol/L calycosin),the SAP+Calycosin-M group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+50μmol/L calycosin),the SAP+Calycosin-H group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+100μmol/L calycosin),the SAP+si-NC group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+transfected si-NC),the SAP+si-TREM-1 group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+transfected si-TREM-1),the SAP+Calycosin+pcDNA group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+100μmol/L calycosin+transfected pcDNA),the SAP+Calycosin+pcDNA-TREM-1 group(AR42J cells+10 nmol/L caerulein+10 mg/L lipopolysaccharide+100μmol/L calycosin+transfected pcDNA-TREM-1).The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured by enzyme-linked immunosorbnent assay.Cell apoptosis was detected by flow cytometry.Western blot method was used to detect the protein expression of B-cell lymphoma-2 gene(Bcl-2),Bcl-2-associated X protein(Bax),cleaved cysteinyl aspartate specific proteinase3(cleaved-caspase3),and triggering receptor expressed on myeloidcells-1 protein(TREM-1).The expression of mRNA of TREM-1 was detected by real time fluorescence quantitative PCR(RT-qPCR).Results:Compared with those in the Con group,in the SAP group,the cell apoptosis rate,and the levels of IL-6 and TNF-α,the protein expression of Bax,and cleaved-caspase3,as well as the expression of mRNA and protein of TREM-1 were increased(P<0.05);the protein expression of Bcl-2 was decreased(P<0.05).Compared with those in the SAP group,in the SAP+Calycosin-L group,the SAP+Calycosin-M group,and the SAP+Calycosin-H group,the levels of IL-6,TNF-α,the protein expression of Bcl-2,and the expression of mRNA and protein of TREM-1 were decreased in SAP acinar cells(P<0.05);the apoptosis rate,and the protein expression of Bax and cleavedcaspase3 were increased in a concentration-dependent manner(P<0.05).Compared with those in the SAP+si-NC group,in the SAP+si-TREM-1 group,the protein expression of TREM-1 and Bcl-2,and levels of IL-6 and TNF-αwere decreased in SAP acinar cells(P<0.05);the apoptosis rate,and the protein expression of Bax and cleaved-caspase3 were increased(P<0.05).Compared with those in the SAP+Calycosin+pcDNA group,in the SAP+Calycosin+pcDNA-TREM-1 group,the levels of IL-6 and TNF-α,and the protein expression of TREM-1 and Bcl-2 were increased in acinar cells(P<0.05);the cell apoptosis rate,and the protein expression of Bax and cleaved-caspase3 were decreased(P<0.05).Conclusion:Calycosin can relieve inflammation of SAP acinar cells by regulating the expression of TREM-1,induce cell apoptosis,and protect SAP acinar cells from injury.