基于微小RNA-30b-5p/B细胞淋巴瘤2样11轴分析滋金补水膏对慢性阻塞性肺疾病大鼠肺功能的影响及机制

Effect of Zijin Bushui Ointment on rats with chronic obstructive pulmonary disease based on the miR-30b-5p/BCL2L11 axis and the underlying mechanism

目的基于微小RNA-30b-5p/B细胞淋巴瘤2样11轴(miR-30b-5p/BCL2L11轴)分析滋金补水膏对慢性阻塞性肺疾病(COPD)大鼠肺功能的影响及机制。方法选用84只SPF雄性Wistar大鼠,按照随机数字表法分为7组,即对照组、模型组、滋金补水膏低剂量组、滋金补水膏中剂量组、滋金补水膏高剂量组、antagomir NC组、miR-30b-5p antagomir组,每组12只组。除对照组外,其余6组采用气道滴注脂多糖(LPS)联合烟雾暴露构建COPD大鼠模型,建模成功后,各组灌胃、尾静脉注射相应药物,每日1次,连续12周。小动物肺功能检测仪检测各组大鼠肺功能;实时荧光定量聚合酶链反应(RT-qPCR)法检测各组大鼠肺组织miR-30b-5p mRNA表达;苏木精-伊红(HE)染色法观察各组大鼠肺组织病理损伤情况,并测量肺组织小气道管壁厚度;原位缺口末端标记法(TUNEL)染色观察各组大鼠肺上皮细胞凋亡情况;酶联免疫吸附(ELISA)法检测各组大鼠血清白细胞介素(IL)-8、IL-1β水平;Western blot法检测各组大鼠B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)及BCL2L11蛋白表达。双荧光素酶报告实验验证miR-30b-5p和BCL2L11之间的靶向关系。结果与对照组比较,模型组大鼠静息呼吸频率、RI、肺组织小气道管壁厚度、肺上皮细胞凋亡指数、血清IL-8和IL-1β水平及肺组织Bax、BCL2L11蛋白表达水平显著升高(P<0.05),肺组织病理损伤严重,PIF、C_(dyn)值、肺组织miR-30b-5 p mRNA表达水平和Bcl-2蛋白表达下降(P<0.05);与模型组比较,滋金补水膏低、中、高剂量组大鼠静息呼吸频率、RI、肺组织小气道管壁厚度、肺上皮细胞凋亡指数、血清IL-8和IL-1β水平及肺组织Bax、BCL2L11蛋白表达水平显著降低(P<0.05),肺组织病理损伤减轻,PIF、C_(dyn)值、肺组织miR-30b-5p mRNA表达水平和Bcl-2蛋白表达显著升高(P<0.05),且除滋金补水膏中剂量组静息呼吸频率与滋金补水膏低剂量组比较差异无统计学意义(P>0.05)外,其余指标滋金补水膏低、中、高剂量组两两比较差异均有统计学意义(P<0.05);与滋金补水膏高剂量组、antagomir NC组比较,miR-30b-5p antagomir组大鼠静息呼吸频率、RI、肺组织小气道管壁厚度、肺上皮细胞凋亡指数、血清IL-8和IL-1β水平及肺组织Bax、BCL2L11蛋白表达水平显著升高(P<0.05),肺组织病理损伤加重,PIF、C_(dyn)值、肺组织miR-30b-5p mRNA表达水平和Bcl-2蛋白表达下降(P<0.05),miR-30b-5p antagomir减弱了高剂量的滋金补水膏对COPD大鼠的改善作用。miR-30b-5p靶向负调控BCL2L11表达。结论滋金补水膏可抑制COPD大鼠肺上皮细胞凋亡和炎症反应,改善肺功能,缓解相关症状,其机制可能与促进miR-30b-5p表达、降低BCL2L11表达相关。

Objective To explore the effect of Zijin Bushui Ointment on rats with chronic obstructive pulmonary disease(COPD)rats and its potential mechanism.Methods Eighty-four male Wistar rats in specific pathogen free(SPF)level were selected and randomly assigned into 7 groups(12 rats/group)according to the random number table method:control group,model group,low-dose,medium-dose and high-dose Zijin Bushui Ointment groups,antagomir NC group(high-dose Zijin Bushui Ointment+transfection of antagomir NC),and miR-30b-5p antagomir group(high-dose Zijin Bushui Ointment+transfection of miR-30b-5p antagomir).Except for rats in the control group,the COPD rat model was constructed by airway instillation of lipopolysaccharide(LPS)combined with smoke exposure.After successful modeling,rats in each group were given corresponding drugs by oral gavage and tail vein injection,once a day for 12 weeks.Small animal lung function testing instrument was used to detect the lung function of rats in each group.The mRNA expression of miR-30b-5p in lung tissue was detected by real-time fluorescence quantitative PCR(RT-qPCR).Hematoxylin and eosin(H&E)staining was performed to visualize pathological damage of lung tissue and measure the thickness of small airway wall in lung tissue.Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining was applied to observe the apoptosis of rat lung epithelial cells.Serum interleukin(IL)-8 and IL-βlevels were detected by enzyme linked immunosorbent assay(ELISA).Western blot was applied to detect the protein expressions of anti-apoptotic factor B cell lymphocyte tumor 2(Bcl-2),Bcl-2 associated X protein(Bax)and BCL2L11.Dual luciferase reporter assay was applied to verify the targeting relationship between miR-30b-5p and BCL2L11.Results Compared with those of the control group,rats in the model group had significantly higher resting respiratory rate,resistance index(RI),thickness of small airway wall in lung tissue,apoptosis index of lung epithelial cells,serum levels of IL-8 and IL-β,and protein expressions of Bax and BCL2L11,severer pathological damages in the lung,and significantly lower peak inspiratory flow(PIF),compliance dynamic(C_(dyn)),mRNA level of miR-30b-5p,and the protein expression of Bcl-2(P<0.05).Compared with those of Model group,rats in the low-dose,medium-dose and high-dose Zijin Bushui Ointment groups had significantly lower resting respiratory rate,RI,thickness of small airway wall in lung tissue,apoptosis index of lung epithelial cells,serum levels of IL-8 and IL-β,and protein expressions of Bax and BCL2L11,milder pathological damages in the lung,and significantly higher PIF,C_(dyn),mRNA level of miR-30b-5p,and the protein expression of Bcl-2(P<0.05).There was no significant difference in resting respiratory rate between the medium-dose and low-dose Zijin Bushui Ointment groups(P>0.05),and significant differences in the other indicators were detected in the pairwise comparison among the low-dose,medium-dose and high-dose Zijin Bushui Ointment groups(P<0.05).Compared with those of high-dose Zijin Bushui Ointment group and antagomir NC group,rats in the miR-30b-5p antagomir group had significantly higher resting respiratory rate,RI,thickness of small airway wall in lung tissue,apoptosis index of lung epithelial cells,serum levels of IL-8 and IL-β,and protein expressions of Bax and BCL2L11,severer pathological damages in the lung,and significantly lower PIF,C_(dyn),mRNA level of miR-30b-5p,and the protein expression of Bcl-2(P<0.05).Transfection of miR-30b-5p antagomir weakened the protective effect of high-dose Zijin Bushui Ointment on COPD.MiR-30b-5p negatively regulated the expression of BCL2L11.Conclusion Zijin Bushui Ointment down-regulates BCL2L11 by regulating miR-30b-5p,thereby inhibiting inflammatory response,reducing apoptosis of lung epithelial cells,improving lung function,and alleviating symptoms of COPD rats.