摘要
目的:探讨五味子乙素(Sch B)调控miR-22-3p/runt相关转录因子3 (RUNX3)分子轴对重症急性胰腺炎(SAP)腺泡细胞损伤的影响。方法:将大鼠胰腺腺泡细胞AR42J分为Con组、SAP组、SAP+Sch-L组、SAP+Sch-M组、SAP+Sch-H组、SAP+anti-miR-NC组、SAP+anti-miR-22-3p组、SAP+Sch+miR-NC组、SAP+Sch+miR-22-3p组。除Con组外,其余各组采用10 nmol/L雨蛙素联合10 mg/L脂多糖刺激AR42J细胞24 h构建SAP细胞模型。SAP+Sch-L组、SAP+Sch-M组、SAP+Sch-H组采用浓度为10、20、40μmol/L的Sch B处理细胞;SAP+anti-miR-NC组、SAP+anti-miR-22-3p组为分别转染antimiR-NC、anti-miR-22-3p;SAP+Sch+miR-NC组、SAP+Sch+miR-22-3p组为分别转染miR-NC、miR-22-3p mimics并用40μmol/L的Sch B干预。酶联免疫吸附法测定细胞培养液上清中白细胞介素-6 (IL-6)、肿瘤坏死因子-α(TNF-α)表达水平;流式细胞术检测细胞凋亡率;实时荧光定量PCR检测miR-22-3p和RUNX3 mRNA表达水平;BCA法检测RUNX3、B细胞淋巴瘤-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)表达水平;双荧光素酶报告基因实验检测miR-22-3p与RUNX3的靶向结合关系。结果:与Con组比较,SAP组AR42J细胞IL-6、TNF-α、miR-22-3p、Bax、cleaved-caspase3的表达及细胞凋亡率升高(P<0.05),RUNX3、Bcl-2的表达降低(P<0.05)。与SAP组比较,SAP+Sch-L组、SAP+Sch-M组、SAP+Sch-H组IL-L、TNF-α、miR-22-3P、Bcl-2表达降低(P<0.05),Bax、cleaved-caspase3、RUNX3表达及细胞凋亡率升高(P<0.05),且呈剂量依赖性(P<0.05)。与SAP+anti-miR-NC组比较,SAP+anti-miR-22-3p组细胞IL-6、TNF-α、Bcl-2的表达降低(P<0.05),细胞凋亡率及Bax、cleaved-caspase3、RUNX3的表达升高(P<0.05)。与SAP+Sch+miR-NC组比较,SAP+Sch+miR-22-3p组细胞IL-6、TNF-α、Bcl-2的表达升高(P<0.05),细胞凋亡率及Bax、cleavedcaspase3、RUNX3的表达降低(P<0.05)。结论:Sch B可通过下调miR-22-3p/RUNX3分子轴减轻SAP腺泡细胞损伤。
Objective: To discuss the effect of Schizandrin B(Sch B) regulating miR-22-3 p/runt-related transcription factor 3(RUNX3) molecular axis on injury of acinar cells with severe acute pancreatitis(SAP). Methods: The pancreatic acinar cells AR42 J of rats were divided into the Con group,the SAP group,the SAP+Sch-L group,the SAP+Sch-M group,the SAP+Sch-H group,the SAP+anti-miR-NC group,the SAP+anti-miR-22-3 p group,the SAP+Sch+miR-NC group,and the SAP+Sch+miR-22-3 p group. Except for the Con group,the other groups were given caerulein at a dose of 10 nmol/L and lipopolysaccharide at a dose of 10 mg/L to stimulate AR42 J cells for 24 hours to establish SAP cell model. In the SAP+Sch-L group,the SAP+Sch-M group,and the SAP+Sch-H group,the cells were treated with Sch B at concentrations of 10,20,and 40 μmol/L;in the SAP+anti-miR-NC group and the SAP+anti-miR-22-3 p group,the cells were transfected with anti-miR-NC and anti-miR-22-3 p respectively;in the SAP+Sch+miR-NC group and the SAP+Sch+miR-22-3 p group,the cells were transfected with miR-NC and miR-22-3 p mimics respectively and intervened with Sch B at a concentration of 40 μmol/L. The expression levels of interleukin-6(IL-6) and tumor necrosis factor α(TNF-α) in the culture supernatant of cells were detected by enzyme linked immunosorbent assay. The rate of cell apoptosis was detected by flow cytometry. The m RNA expression levels of miR-22-3 p and RUNX3 were detected by real-time fluorescence quantitative PCR. The expression levels of RUNX3,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cleaved-cysteinyl aspartate specific proteinase 3(cleaved-caspase3) were detected by BCA method. The targeted binding relationship between miR-22-3 p and RUNX3 was detected by dual-luciferase reporter assay. Results:Compared with those in the Con group,in the SAP group,the expression levels of IL-6,TNF-α,miR-22-3 p,Bax,and cleavedcaspase3 in AR42 J cells as well as the rate of cell apoptosis were increased(P<0.05),and the expression levels of RUNX3 and Bcl-2 were decreased(P<0.05). Compared with those in the SAP group, in the SAP+Sch-L group, SAP+Sch-M group and SAP+Sch-H group,the expression levels of IL-6,TNF-α,miR-22-3 p and Bcl-2 were decreased(P<0.05),and the expression levels of Bax,cleaved-caspase3,and RUNX3 as well as the rate of cell apoptosis were increased(P<0.05),and in a dose-dependent manner(P<0.05). Compared with those in the SAP+anti-miR-NC group,in the SAP+antimiR-22-3 p group,the expression levels of IL-6,TNF-α,and Bcl-2 were decreased(P<0.05);the expression levels of Bax,cleaved-caspase3,and RUNX3 as well as the rate of cell apoptosis were increased(P<0.05). Compared with those in the SAP+Sch+miR-NC group,in the SAP+Sch+miR-22-3 p group,the expression levels of IL-6,TNF-α,and Bcl-2 were increased(P<0.05);the expression levels of Bax,cleaved-caspase3,and RUNX3 as well as the rate of cell apoptosis were decreased(P<0.05). Conclusion:Sch B can relieve the injury of acinar cells with SAP by down-regulating miR-22-3 p/RUNX3 molecular axis.
作者
尹露西
丁文
王晓华
YIN Luxi;DING Wen;WANG Xiaohua
出处
《新中医》
CAS
2020年第21期13-18,共6页
New Chinese Medicine
