五味子乙素调控miR-22-3p/RUNX3分子轴对重症急性胰腺炎腺泡细胞损伤影响的研究
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Study on Effect of Schizandrin B Regulating MiR- 22- 3p/RUNX3 Molecular Axis on Injury of Acinar Cells with Severe Acute Pancreatitis
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    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、40 μmol/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40 μmol/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。

    Abstract:

    Abstract: Objective: To discuss the effect of Schizandrin B(Sch B) regulating miR- 22- 3p/runt- related transcription factor 3(RUNX3) molecular axis on injury of acinar cells with severe acute pancreatitis(SAP). Methods: The pancreatic acinar cells AR42J 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-22-3p group,the SAP+Sch+miR-NC group,and the SAP+Sch+miR-22-3p 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 AR42J 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-22-3p group,the cells were transfected with anti-miR-NC and anti-miR-22-3p respectively; in the SAP+Sch+miR-NC group and the SAP+ Sch+miR-22-3p group,the cells were transfected with miR-NC and miR-22-3p 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 mRNA expression levels of miR- 22- 3p 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- 3p 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-22-3p,Bax,and cleavedcaspase3 in AR42J 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-22-3p 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- 22-3p 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-22-3p 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-22-3p/RUNX3 molecular axis.

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尹露西,丁文,王晓华.五味子乙素调控miR-22-3p/RUNX3分子轴对重症急性胰腺炎腺泡细胞损伤影响的研究[J].新中医,2020,52(21):13-18

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  • 在线发布日期: 2020-11-05
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