滋金补水膏对慢性阻塞性肺疾病模型大鼠的治疗作用及对lncRNAPVT1、miR-30b-5p表达的影响

Therapeutic Effect of Zijin Bushui Soft Extract on a Rat Model of Chronic Obstructive Pulmonary Disease and Its Effect on the Expressions of lncRNA PVT1,miR-30b-5p

【目的】探讨滋金补水膏对慢性阻塞性肺疾病(COPD)模型大鼠的治疗作用及机制。【方法】将96只大鼠随机分为正常组,模型组,滋金补水膏低、中、高剂量组,盐酸氨溴索组、滋金补水膏高剂量+pcDNA组,滋金补水膏高剂量+pcDNA-人浆细胞瘤转化迁移基因1(PVT1)组,每组12只。除正常组外,其他各组大鼠采用气道滴注脂多糖(LPS)联合烟雾暴露法构建COPD模型。建模成功后,进行给药干预。给药结束后,采用酶联免疫吸附分析(ELISA)检测大鼠血清中白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平;流式细胞术检测大鼠外周血中辅助性T细胞17/调节性T细胞(Th17/Treg)水平,苏木素-伊红(HE)染色法观察肺组织病理变化,实时定量聚合酶链反应(qRT-PCR)法检测血清和肺组织中长链非编码RNA(lncRNA)PVT1、miR-30b-5p表达,Western Blot法检测肺组织中维甲酸相关孤核受体γt(RORγt)、叉头框蛋白P3(Foxp3)蛋白表达,双荧光素酶报告基因实验验证lncRNA PVT1与miR-30b-5p的关系。【结果】与正常组比较,模型组肺泡间隔变大,支气管周围有大量炎症细胞浸润,Treg细胞比例、miR-30b-5p表达水平、Foxp3蛋白表达水平降低,IL-6、TNF-α水平,Th17细胞比例、Th17/Treg比值,lncRNA PVT1表达水平,RORγt蛋白表达水平升高(均P<0.05);与模型组比较,滋金补水膏低、中、高剂量组及盐酸氨溴索组大鼠肺组织病理损伤减轻、Treg细胞比例、miR-30b-5p表达水平、Foxp3蛋白表达水平升高,IL-6、TNF-α水平,Th17细胞比例,Th17/Treg比值,lncRNA PVT1表达水平,RORγt蛋白表达水平降低(均P<0.05)。lncRNA PVT1靶向调控miR-30b-5p表达。【结论】滋金补水膏可能通过调节lncRNA PVT1/miR-30b-5p轴促进Th17/Treg细胞平衡来抑制COPD大鼠炎症反应。

Objective To investigate the therapeutic effect and mechanism of Zijin Bushui Soft Extract on chronic obstructive pulmonary disease(COPD)model rats.Methods Ninety-six rats were randomly divided into the normal group,the model group,the low-,medium-and high-dose groups of Zijin Bushui Soft Extract,the Ambroxol Hydrochloride group,the high-dose of Zijin Bushui Soft Extract+pcDNA group,and the high-dose of Zijin Bushui Soft Extract+pcDNA-PVT1 group,with 12 rats in each group.Except for the normal group,the COPD model was constructed by using the airway drip lipopolysaccharide(LPS)combined with smoke exposure method in all other groups of rats.After successful modelling,drug administration intervention was carried out.At the end of drug administration,the serum levels of interleukin 6(IL-6)and tumor necrosis factorα(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA);the level of Th17/Treg was detected by flow cytometry in the peripheral blood of rats,and pathological changes in the lung tissues were observed by hematoxylin-eosin(HE)staining,and the expressions of lncRNA PVT1 and miR-30b-5p in serum and lung tissues were detected by quantitative real-time polymerase chain reaction(qRT-PCR),the expressions of nuclear hormone receptor retinoic acid receptor-related orphan receptorγt(RORγt)and forkhead box P3(Foxp3)in lung tissues were detected by Western Blot,and the double luciferase reporter gene assay was performed to verify the relationship between lncRNA PVT1 and miR-30b-5p.Results Compared with the normal group,the alveolar septa of the model group were enlarged,with a large number of inflammatory cells infiltrating around the bronchioles,the proportion of Treg cells,the expression level of miR-30b-5p,and the protein expression level of Foxp3 were reduced,and the levels of IL-6 and TNF-α,the proportion of Th17 cells,and the ratio of Th17/Treg,the expression level of lncRNA PVT1 and the protein expression level of RORγt were elevated(all P<0.05);compared with the model group,the lung histopathological injury of rats in the Zijin Bushui Soft Extract low-,medium-and high-dose groups and the Ambroxol Hydrochloride group was reduced,the proportion of Treg cells,the expression level of miR-30b-5p,the protein expression level of Foxp3 was elevated,and the levels of IL-6 and TNF-α,the proportion of Th17 cells,the ratio of Th17/Treg,the expression level of lncRNA PVT1,and RORγt protein expression level were decreased(all P<0.05).LncRNA PVT1 targeted miR-30b-5p expression.Conclusion Zijin Bushui Soft Extract may inhibit the inflammatory response in COPD rats by regulating the lncRNA PVT1/miR-30b-5p axis to promote Th17/Treg cell balance.