左氧氟沙星对肺结核大鼠肺组织自噬及磷脂酰肌醇3-激酶/蛋白激酶B通路的影响

Effect of levofloxacin on pulmonary autophagy and phosphatidylinositol 3-kinase/protein kinase B pathway in rats with pulmonary tuberculosis

目的探讨左氧氟沙星对肺结核大鼠肺组织自噬及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)通路的影响。方法按照随机数字表法将50只大鼠分为正常组、模型组及低、中、高剂量左氧氟沙星组,每组10只。正常组大鼠经尾静脉注射生理盐水,其余组大鼠经尾静脉注射结核杆菌悬液制备肺结核模型。造模后第2天开始,低、中、高剂量左氧氟沙星组大鼠每日分别灌胃6.5、13.0、26.0 mg·kg-1左氧氟沙星生理盐水溶液2 mL,正常组和模型组大鼠每日灌胃等体积的生理盐水,连续2个月。给药结束后处死大鼠取肺组织,采用苏木精-伊红染色观察各组大鼠肺组织病理学变化,免疫组织化学染色法检测各组大鼠肺组织中自噬标志分子微管相关蛋白1轻链3B(LC3B)的表达,实时荧光定量聚合酶链反应和Western blot分别检测各组大鼠肺组织中Beclin1、Atg5、PI3K、磷酸化Akt(p-Akt)mRNA和蛋白表达。结果正常组大鼠肺组织未见炎症反应;模型组大鼠肺组织结构破坏,出现大量结核结节,呼吸道周围症性细胞浸润明显,肺实质有肺泡管、肺泡腔不规则扩大,出现干酪样坏死;低、中、高剂量左氧氟沙星组大鼠肺组织病理损伤程度较模型组明显改善,并随着药物处理剂量增加,损伤程度逐渐减轻。模型组和低、中剂量左氧氟沙星组大鼠肺组织中LC3B阳性细胞数显著多于正常组(P<0.05);高剂量左氧氟沙星组大鼠肺组织中LC3B阳性细胞数与正常组比较差异无统计学意义(P>0.05);低、中、高剂量左氧氟沙星组大鼠肺组织中LC3B阳性细胞数显著少于模型组(P<0.05);中剂量左氧氟沙星组与低、高剂量左氧氟沙星组大鼠肺组织中LC3B阳性细胞数比较差异无统计学意义(P>0.05);高剂量左氧氟沙星组大鼠肺组织中LC3B阳性细胞数显著少于低剂量左氧氟沙星组(P<0.05)。模型组和低、中剂量左氧氟沙星组大鼠肺组织中Beclin1 mRNA表达水平显著高于正常组(P<0.05);高剂量左氧氟沙星组与正常组大鼠肺组织中Beclin1 mRNA表达水平比较差异无统计学意义(P>0.05)。与正常组比较,模型组和低、中、高剂量左氧氟沙星组大鼠肺组织中Atg5 mRNA表达水平显著升高,PI3K、p-Akt mRNA表达水平显著降低(P<0.05)。与模型组比较,低、中、高剂量左氧氟沙星组大鼠肺组织中Beclin1、Atg5 mRNA表达水平显著降低,PI3K、p-Akt mRNA表达水平显著升高(P<0.05)。低、中剂量左氧氟沙星组大鼠肺组织中Beclin1、Atg5、PI3K mRNA表达水平比较差异无统计学意义(P>0.05);中剂量左氧氟沙星组大鼠肺组织中p-Akt mRNA表达水平显著高于低剂量左氧氟沙星组(P<0.05)。与低剂量左氧氟沙星组比较,高剂量左氧氟沙星组大鼠肺组织中Beclin1、Atg5 mRNA表达水平显著降低,PI3K、p-Akt mRNA表达水平显著升高(P<0.05)。与中剂量左氧氟沙星组比较,高剂量左氧氟沙星组大鼠肺组织中Beclin1、Atg5 mRNA表达水平显著降低,PI3K、p-Akt mRNA表达水平显著升高(P<0.05)。与正常组比较,模型组和低、中剂量左氧氟沙星组大鼠肺组织中Beclin1蛋白表达水平显著升高,PI3K、p-Akt蛋白表达水平显著降低(P<0.05);高剂量左氧氟沙星组和正常组大鼠肺组织中Beclin1、PI3K、p-Akt蛋白表达水平比较差异无统计学意义(P>0.05);模型组和低、中、高剂量左氧氟沙星组大鼠肺组织中Atg5蛋白表达水平均显著高于正常组(P<0.05)。与模型组比较,低、中、高剂左氧氟沙星量组大鼠肺组织中Beclin1、Atg5蛋白表达水平显著降低,PI3K、p-Akt蛋白表达水平显著升高,且呈剂量依赖性(P<0.05)。结论左氧氟沙星可能通过激活肺组织中PI3K/Akt信号通路、抑制自噬相关蛋白表达,降低肺结核大鼠肺细胞自噬水平,减轻肺损伤。

Objective To investigate the effect of levofloxacin on pulmonary autophagy and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)pathway in rats with pulmonary tuberculosis.Methods Fifty rats were divided into the normal group,model group,low-,medium-and high-dose levofloxacin group,according to the random number method,with 10 rats in each group.The rats in the normal group were injected with normal saline via the tail vein;the rats in the other groups were made the pulmonary tuberculosis model by tail vein injection of Mycobacterium tuberculosis suspension.From the second day after modeling,the rats in the low-dose levofloxacin group,medium-dose levofloxacin group and high-dose levofloxacin group were given 6.5,13.0 and 26.0 mg·kg-1 levofloxacin saline solution 2 mL by gavage every day for 2 months;the rats in the normal group and the model group were intragastrically administered with equal volume of normal saline every day for 2 months.After administration,all rats were killed and the lurg tissues were taken.The pathological changes of lung tissue of all rats were detected by haematoxylin eosin staining;the expression levels of autophagy marker microtubule associated protein 1 light chain 3 B(LC3B)in lung tissue of all rats were detected by immunohistochemical staining;and the expression levels of Beclin1,Atg5,PI3K,phosphorylated Akt(p-Akt)mRNA and protein in lung tissues of all rats were detected by real-time fluorescence quantitative polymerase chain reaction and Western blot,respectively.Results There were no inflammatory reaction in lung tissue of the rats in the normal group;the lung tissue structure of the rats in the model group was destroyed,a large number of tuberculosis nodules appeared in the lung tissue,the inflammatory cells around the airway were infiltrated obviously,and the lung parenchyma had alveolar ducts and irregularly enlarged alveolar cavities,with cheese-like necrosis.Compared with the model group,the pathological damage of the lung tissue of rats in the low-,medium-and high-dose of levofloxacin groups was improved,and the damage was gradually reduced with the increase of the dose of drug.The number of LC3B positive cells in the lung tissue of rats in the model group,low-and medium-dose levofloxacin groups was significantly higher than that in the normal group(P<0.05);there was no statistical difference in the number of LC3B positive cells in the lung tissue of rats between the high-dose levofloxacin group and the normal group(P>0.05).The number of LC3B positive cells in the lung tissue of rats in the low-,medium-and high-dose levofloxacin groups was significantly less than that in the model group(P<0.05);there were no statistical difference in the number of LC3B positive cells in the lung tissue of rats between the medium-dose levofloxacin group and the low-dose levofloxacin group,high-dose levofloxacin group(P>0.05);the number of LC3B positive cells in the lung tissue of rats in the high-dose levofloxacin group was significantly less than that in the low-dose levofloxacin group(P<0.05).The expression levels of Beclin1 mRNA in lung tissues of rats in model group,low-and medium-dose levofloxacin groups were significantly higher than that in the normal group(P<0.05);there was no statistical difference in the expression of Beclin1 mRNA in lung tissues of rats between the high-dose levofloxacin group and the normal group(P>0.05).Compared with the normal group,the expression of Atg5 mRNA in lung tissues of rats in the model group,low-,medium-and high-dose levofloxacin groups increased,and the expression of PI3K,p-Akt mRNA decreased(P<0.05).Compared with the model group,the expression of Beclin1,Atg5 mRNA in lung tissues of rats in low-,medium-and high-dose levofloxacin groups decreased and the expression levels of PI3K,p-Akt mRNA increased(P<0.05).There was no statistical difference in the expression level of Beclin1,Atg5,PI3K mRNA between the low-dose levofloxacin group and the medium-dose levofloxacin group(P>0.05);the expression of p-Akt mRNA in lung tissues of rats in medium-dose levofloxacin group was significantly higher than that in the low-dose levofloxacin group(P<0.05).Compared with the low-dose levofloxacin group,the expression levels of Beclin1,Atg5 mRNA in lung tissues of rats in the high-dose levofloxacin group decreased and the expression levels of PI3K,p-Akt mRNA increased(P<0.05).Compared with the normal group,the expression levels of Beclin1 protein in lung tissues of rats in the model group,low-dose levofloxacin group and medium-dose levofloxacin group increased,and the expression levels of PI3K,p-Akt protein decreased(P<0.05);there was no statistical difference in the expression of Beclin1,PI3K,p-Akt protein between the high-dose levofloxacin group and the normal group(P>0.05);the expression levels of Atg5 protein in lung tissues of rats in the model group,low-,medium-and high-dose levofloxacin groups were significantly higher than that in the normal group(P<0.05).Compared with the model group,the expression levels of Beclin1,Atg5 protein in lung tissues of rats in the low-,medium-and high-dose levofloxacin group decreased and the PI3K,p-Akt protein increased in a dose-dependent manner(P<0.05).Conclusion Levofloxacin can reduce the pulmonary autophagy level of pulmonary tuberculosis rats and alleviate lung injury by inhibiting the expression of autophagy-related protein and activating PI3K/Akt signaling pathway in lung tissue.