长链非编码RNA np-5318对大鼠肠道缺血再灌注损伤作用研究

Effect of long non-coding RNA np-5318 on intestinal ischemia-reperfusion injury in rats

目的 探讨长链非编码RNA(lncRNA)np-5318在肠道缺血/再灌注(I/R)损伤中的表达及作用,并分析np-5318与表皮生长因子受体(EGFR)信号通路的相互作用。方法 选取20只雄性SD大鼠随机分为假手术组(n=10)与I/R组(n=10)。I/R组麻醉后沿腹部中线做1.0~1.5 cm切口,显露肠系膜上动脉(SMA),使用微动脉夹夹住SMA以阻断血流,45 min后经原切口入腹,取出动脉夹,恢复血供,构建大鼠肠道I/R损伤模型。假手术组仅分离SMA,但不夹闭SMA。I/R组再灌注后6 h处死大鼠,同时,处死假手术组大鼠,并立即取出肠道组织。计算假手术组与I/R组肠道干湿比(W/D)。采用实时定量聚合酶链式反应(qRT-PCR)检测np-5318在假手术组与I/R组肠道组织中的表达。采用酶联免疫吸附试验(ELISA)检测肠道转化生长因子-β(TGF-β)、γ干扰素(INF-γ)、白细胞介素4(IL-4)、白细胞介素6(IL-6)、白细胞介素17A(IL-17A)和环氧化酶-2(Cox-2)水平。取大鼠肠道上皮细胞分为对照组、I/R模型组与转染si-np-5318组。转染si-np-5318组与I/R模型组的大鼠肠道上皮细胞构建体外I/R模型,并分别转染si-np-5318抑制lncRNA np-5318表达和相应的对照(si-NC)。采用MTT比色法检测细胞活性,并通过qRT-PCR检测np-5318以及EGFR通路相关基因的表达量。结果 I/R组肠道组织W/D及炎症因子TGF-β、INF-γ、IL-4、IL-6、IL-17A、Cox-2表达水平均高于假手术组,差异有统计学意义(P<0.05)。qRT-PCR结果显示,I/R组np-5318表达水平为(2.07±0.14),高于假手术组的(1.02±0.11),差异有统计学意义(P<0.05)。MTT比色测定结果显示,I/R模型组细胞活性低于对照组,而转染si-np-5318组细胞活性高于I/R模型组,差异有统计学意义(P<0.05)。对照组与转染si-np-5318组细胞活性比较,差异无统计学意义(P>0.05)。I/R模型组EGFR信号通路基因HBEGF、CSK、HGS表达量较对照组显著下降,而转染si-np-5318组EGFR信号通路基因HBEGF、CSK、HGS表达量较I/R模型组明显升高,差异有统计学意义(P<0.05)。结论 在肠道I/R中,lncRNA np-5318可抑制EGFR信号通路,可能是lncRNA np-5318加重肠道I/R损伤的潜在机制。

Objective To investigate the expression and role of long non-coding RNA(lncRNA)np-5318 in intestinal ischemiareperfusion(I/R)injury,and to analyze interaction between np-5318 and epidermal growth factor receptor(EGFR)signaling pathway.Methods Twenty male SD rats were randomly divided into sham operation group(n=10)and I/R group(n=10).After the anesthesia,1.0-1.5 cm incision was made along the midline of the abdomen in the I/R group to expose the superior mesenteric artery(SMA),and the SMA was clamp with arteriolar clamp to block the blood flow.Forty-five minutes later,the artery clamp was removed through the incision to restore the blood supply,and the rat intestinal I/R injury model was established.In the sham operation group,the SMA was isolated but not clamped.The rats in the I/R group were killed 6 hours after the reperfusion,meanwhile the rats in the sham operation group were killed,and the intestinal tissues were removed immediately.The dry-wet ratio(W/D)of intestinal tract in sham operation group and I/R group was measured.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of np-5318 in the intestinal tissues of the sham operation group and the I/R group.Enzyme linked immunosorbent assay(ELISA)was used to detect intestinal transforming growth factor-β(TGF-β),interferon-γ(INF-γ),interleukin-4(IL-4),interleukin-6(IL-6),interleukin-17A(IL-17A)and cyclooxidase-2(Cox-2)levels.The intestinal epithelial cells of rats were divided into control group,I/R model group and transfected si-np-5318 group.In-vitro I/R model was constructed in the intestinal epithelial cells of transfecting si-np-5318 group and I/R model group,and si-np-5318 was transfected to inhibit the expression of lncRNA np-5318 in transtecting si-np-5318 group and corresponding control(si-NC)was transfecting in I/R model group,respectively.The cell activity was detected by MTT colorimetry,and the expression levels of np-5318 and EGFR signaling pathway related genes were detected by qRT-PCR.Results The W/D and expression levels of inflammatory factors TGF-β,INF-γ,IL-4,IL-6,IL-17A and Cox-2 in intestinal tissue of the I/R group were higher than those of the sham operation group,and the differences were statistically significant(P<0.05).The results of qRT-PCR showed that the expression level of np-5318 in the I/R group was(2.07±0.14),which was higher than that in the sham operation group(1.02±0.11),and the difference was statistically significant(P<0.05).MTT colorimetric assay results showed that the cell activity in the I/R model group was lower than that in the control group,while the cell activity in the transfected si-np-5318 group was higher than that in the I/R model group,with statistical significance(P<0.05).There was no significant difference in cell activity between the control group and the transfected si-np-5318 group(P>0.05).The expression levels of EGFR signaling pathway genes HBEGF,CSK and HGS in the I/R model group were significantly lower than those in the control group,while the expression levels of EGFR signaling pathway genes HBEGF,CSK and HGS in the transfected si-np-5318 group were significantly higher than those in the I/R model group,and the differences were statistically significant(P<0.05).Conclusion In intestinal I/R,lncRNA np-5318 can inhibit EGFR signaling pathway,that may be the potential mechanism of lncRNA np-5318 aggravating intestinal I/R injury.