环状RNA-胞裂蛋白2/miR-150-5p/聚腺苷二磷酸核糖聚合酶-1分子轴在重症急性胰腺炎中的作用及机制研究

The role and mechanism of circular RNA-septin 2/miR 150-5p/polyadenosine diphosphate ribose polymerase-1 molecular axis in severe acute pancreatitis

目的探讨环状RNA(circRNA)-胞裂蛋白2(SEPT2)/miR-150-5p/聚腺苷二磷酸核糖聚合酶-1(PARP-1)分子轴在重症急性胰腺炎(SAP)中的作用及机制。方法选取2019年12月至2021年12月赣南医学院第一附属医院收治的46例SAP患者作为重症组;并选取本院同期收治的41例轻症急性胰腺炎(MAP)患者作为轻症组;另选取本院同期35名健康体检者作为对照组。3组均采用逆转录-聚合酶链式反应(RT-PCR)法测定血清circRNA-SEPT2、miR-150-5p、PARP-1基因表达量,采用急性生理和慢性健康评分系统Ⅱ(APACHEⅡ)评估病情严重程度;采用重组慢病毒包装合成有效感染序列sh-SEPT2(干扰组)与过表达序列oe-SEPT2(过表达组)建立动物模型,采用酶联免疫吸附试验测定大鼠生化指标[白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)、D乳酸及二胺氧化酶(DAO)],采用RT-PCR及Western blot法检测大鼠circRNA-SEPT2、miR-150-5p、PARP-1及细胞凋亡相关基因半胱氨酸蛋白酶(Caspase)-3、Caspase-9 mRNA表达。比较3组circRNA-SEPT2、miR-150-5p、PARP-1基因表达量及APACHEⅡ评分,采用Pearson相关性分析SAP患者PARP-1与circRNA-SEPT2、miR-150-5p及APACHEⅡ评分的相关性;比较干扰组与过表达组大鼠苏木精-伊红染色法(HE)结果、肠黏膜损伤评分、炎症因子水平及cir-cRNA-SEPT2、miR-150-5p、PARP-1和Caspase-3、Caspase-9 mRNA表达量。结果重症组circRNA-SEPT2、miR-150-5p、PARP-1基因表达量及APACHEⅡ评分均高于轻症组和对照组,且轻症组高于对照组,差异有统计学意义(P<0.05)。Pearson相关性结果显示,SAP患者PARP-1与circRNA-SEPT2、miR-150-5p及APACHEⅡ评分均呈正相关(r>0,P<0.05)。干扰组HE染色下胰岛细胞界限清晰,胰岛丰富,胞浆丰富,形态完整;过表达组大鼠胰岛细胞界限不清,胰岛数量较少,细胞形态不规则,细胞体积较大,边缘不齐;干扰组胰腺损伤评分为(2.58±0.16)分,低于过表达组的(3.95±0.53)分,差异有统计学意义(P<0.05)。干扰组IL-1β、IL-18、TNF-α、D乳酸及DAO水平均低于过表达组,差异有统计学意义(P<0.05)。干扰组大鼠circRNA-SEPT2、miR-150-5p、PARP-1 mRNA表达及细胞凋亡相关基因Caspase-3、Caspase-9 mRNA表达量均低于过表达组,差异有统计学意义(P<0.05)。结论PARP-1通过靶向作用于circRNA-SEPT2/miR-150-5p参与SAP的发生与发展,抑制circRNA-SEPT2表达,可减轻SAP肠黏膜屏障损伤,能为SAP治疗提供新的靶点。

Objective To investigate the role and mechanism of circular RNA(circRNA)-septin 2(SEPT2)/miR-150-5p/polyadenosine diphos-phate ribose polymerase-1(PARP-1)molecular axis in severe acute pancreatitis(SAP).Methods 46 SAP patients admitted to the First Affiliated Hospital of Gannan Medical College from December 2019 to December 2021 were selected as the severe group,41 patients with mild acute pancre-atitis(MAP)admitted to our hospital during the same period were selected as the mild group,and another 35 healthy physical examination in our hos-pital during the same period were selected as the control group.Reverse transcription-polymerase chain reaction(RT-PCR)was used to determine the gene expressions of circRNA-SEPT2,miR-150-5p and PARP-1,acute physiology and chronic health evaluationⅡ(APACHEⅡ)scale was used to evaluate the symptoms;the effective infection sequence sh-SEPT2(interference group)and the overexpression sequence oe-SEPT2(overexpression group)were packaged by recombinant lentivirus to establish an animal mode,the biochemical indexes of rats(interleukin-1β[IL-1β],interleukin-18[IL-18],tumor necrosis factor-α[TNF-α],D-lactic acid and diamine oxidase[DAO])were detected by enzyme-linked immunosorbent assay,the mRNA expressions of circRNA-SEPT2,miR-150-5p,PARP-1 and apoptosis-related genes cysteine proteinase(Caspase)-3 and Caspase-9 were de-tected by RT-PCR and Western blot.The gene expression levels of circRNA-SEPT2,miR-150-5p,PARP-1 and APACHEⅡscore were compared among the three groups,and Pearson correlation analysis was used to analyze the correlation between PARP-1 and circRNA-SEPT2,miR-150-5p in SAP patients.Hematoxylin-eosin staining(HE)staining results,intestinal mucosal injury score,inflammatory factor levels,and expression of cir-cRNA-SEPT2,miR-150-5p,PARP-1,Caspase-3,Caspase-9 mRNA were compared between the interference group and the overexpression group.Results The gene expression levels of circRNA-SEPT2,miR-150-5p,PARP-1 and APACHEⅡscore in the severe group were higher than those in the mild group and the control group,and mild group was higher than control group,and the differences were statistically significant(P<0.05).Pearson correlation results showed that PARP-1 was positively correlated with circRNA-SEPT2,miR-150-5p and APACHEⅡscores in SAP pa-tients(r>0,P<0.05).In the interference group,HE staining showed the islet cells had clear boundaries,abundant islets,abundant cytoplasm,in-tact morphology;in the overexpression group,islet cells had unclear boundaries,fewer islets,irregular cell shape,larger cell volume,and irregular margins.The pancreatic injury score in the interference group was(2.58±0.16)scores,which was lower than(3.95±0.53)scores in the overexpres-sion group,and the difference was statistically significant(P<0.05).The levels of IL-1β,IL-18,TNF-α,D-lactic acid and DAO were lower than those in the overexpression group,and the differences were statistically significant(P<0.05);the mRNA expressions of circRNA-SEPT2,miR-150-5p,PARP-1 and apoptosis-related genes Caspase-3 and Caspase-9 in the interference group were lower than those in the overexpression group,and the differences were statistically significant(P<0.05).Conclusion PARP-1 is involved in the occurrence and development of SAP by targeting cir-cRNA-SEPT2/miR-150-5p,and inhibiting the expression of circRNA-SEPT2,can reduce the damage of the intestinal mucosal barrier in SAP,which can provide a new target for SAP treatment.