慢阻肺气虚证大鼠模型的建立及评价

Establishment and Evaluation of Chronic Obstructive Pulmonary Qi Deficiency Syndrome Model

目的为进一步研究慢阻肺相关机制及中医证候,建立病证结合的慢阻肺气虚证大鼠模型,探究慢阻肺气虚证大鼠模型评价指标。方法采用肺部滴入LPS合并烟熏和单纯肺部滴入LPS两种方法建立的慢性阻塞性肺疾病大鼠模型为研究对象,采用病理、肺功能等进行慢阻肺判定;通过宏观表征采集、体重观察、抓力测试、旷场实验、ATP含量和线粒体动力学蛋白印迹检测进行大鼠证候判定。结果从第3周开始,LPS联合烟熏组体重显著小于空白组和LPS组(P<0.05),持续至第12周;第8周时,LPS联合烟熏组抓力最大值显著小于空白组及LPS组(P<0.05),三组之间抓力平均值无统计学差异;LPS联合烟熏组旷场总距离和平均速率均显著小于空白组和LPS组(P<0.05);第12周结束时,LPS联合烟熏组抓力最大值和平均值均显著小于空白组和LPS组(P<0.05);LPS联合烟熏组旷场总距离和平均速率均显著小于空白组和LPS组(P<0.05);第12周结束时,ATP含量检测结果显示,LPS联合烟熏组肺组织和骨骼肌中ATP含量较空白组有明显的下降(P<0.05);LPS联合烟熏组骨骼肌ATP含量较空白组和LPS组均有明显的下降(P<0.05);Western Blot结果显示,与空白组和LPS组相比,LPS联合烟熏组大鼠肺和骨骼肌组织均表现出Drp1蛋白表达量升高(P<0.05)、Mfn1蛋白表达量降低(P<0.05);肺功能结果显示LPS联合烟熏组肺顺应性低于空白组和LPS组,而肺呼吸阻力高于空白组和LPS组;病理结果显示,LPS联合烟熏组和LPS组均表现出慢阻肺特征性病理变化,LPS联合烟熏组比LPS组病理改变更加显著。结论LPS滴入结合烟熏的方法可以成功的建立慢阻肺大鼠气虚证模型,这种模型较为符合临床上慢阻肺患者气虚证阶段的症状表现,且慢阻肺气虚证大鼠模型出现的各种证候表现与线粒体融合/分裂蛋白之间的平衡及能量代谢有着非常密切的关系,Drp1、Mfn1蛋白表达和ATP含量变化可以作为慢阻肺气虚证大鼠模型的评价指标。

Objective To further study the related mechanism of COPD and TCM syndromes,establishing the rat model of COPD qi deficiency syndrome combining diseases and syndromes,and exploring the evaluation indexes of rat model of copd qi deficiency syndrome.Methods the COPD rat model was established by LPS combined with smoking and LPS alone.The syndromes of rats were determined by macroscopic characterization collection,weight observation,scratch test,open-field experiment,ATP content and mitochondrial dynamics western blot test.Results From week 3,the weight of LPS combined smoking group was significantly lower than that of the blank group and LPS group(P<0.05),which lasted until week 12.At week 8,the maximum grasping force of the LPS combined smoking group was significantly lower than that of the blank group and LPS group(P<0.05),and there was no statistical difference in the mean grasping force between the three groups.The total distance and average speed of the open field in the LPS combined smoking group were significantly lower than those in the blank group and LPS group(P<0.05).At the end of week 12,the maximum and average grasping power of LPS combined with smoking group was significantly lower than that of blank group and LPS group(P<0.05).The total distance and average speed of the open field in the LPS combined smoking group were significantly lower than those in the blank group and LPS group(P<0.05).At the end of week 12,ATP content in lung tissues and skeletal muscles of the LPS combined smoking group was significantly lower than that of the blank group(P<0.05).Compared with the blank group and LPS group,the ATP content of skeletal muscle in LPS combined smoking group was significantly decreased(P<0.05).Western Blot results showed that compared with the blank group and LPS group,lung and skeletal muscle tissues of the LPS combined smoking group showed increased Drp1 protein expression(P<0.05)and decreased Mfn1 protein expression(P<0.05).Lung function results showed that lung compliance in LPS combined with smoking group was lower than that in blank group and LPS group,and lung respiratory resistance was higher than that in blank group and LPS group.Pathological results showed that the LPS combined smoking group and LPS group showed characteristic pathological changes of COPD,and the LPS combined smoking group showed more significant pathological changes than the LPS group.Conclusion LPS combined with smoking can successfully establish the model of qi deficiency syndrome in rats with COPD,which is in line with the clinical symptoms of qi deficiency syndrome in patients with COPD.And,through further research,we found that the qi deficiency COPD rat model of syndrome performance has a very close relationship with the balance of mitochondria fusion protein/division and energy metabolism,the changes of Drp1,Mfn1 protein expression and ATP content can be used as evaluation index of COPD qi deficiency syndrome in rats model.