基于肺脾核心两本多脏虚慢阻肺稳定期模型构建及相关机制研究

【作者】 李磊；

【导师】 李友林；

【作者基本信息】 北京中医药大学 ， 中医内科学（专业学位）， 2022， 博士

【摘要】 目的1从呼吸功能的物质基础、脏腑的功能之源等角度探究慢性阻塞性肺疾病(COPD)稳定期的脏腑功能特征,为后续临床和基础研究提供理论依据。2分析COPD稳定期患者的临床症状体征分布情况,深化对本病脏腑功能特征的认识。3以动物为载体,探索并建立反映COPD稳定期的中医脏腑功能特征的动物模型,观察模型宏观和微观特征,初步确立模型评价方法。4依托COPD稳定期小鼠模型,评价理肺汤(RLD)对COPD稳定期的疗效,并揭示RLD调控本病的可能机制。方法1分析传统中医对COPD稳定期的相关理论与标志性成果及其对八纲辨证和施治的影响,探索可能更加吻合本病临床实际的脏腑功能特征;2采用观察性研究方法,通过分析COPD稳定期患者的主要症状、兼症及舌脉等情况,以及构成比分析、聚类分析和因子分析本病临床特征,阐释其脏腑功能特征;3探索研究中比较番泻叶水煎剂、隔天禁食、噪音、氢化可的松中单个、两个和多个干预因素影响的差异;其次,结合烟雾暴露(CS)和脂多糖(LPS)干预;再者,综合临床对肺脾核心两本多脏虚的认识及造模后多重检测指标结果,最终初步确立肺脾核心两本多脏虚COPD稳定期的造模方法。4正式实验中按探索研究最终确立的方法建立肺脾核心两本多脏虚COPD稳定期模型,检测模型小鼠一般情况、肺组织病理等生物学表征及微观表征。5将正式实验中小鼠随机分为模型组、阳性药组、RLD低剂量组、中剂量组、高剂量组和空白对照组。给药28d后,评估小鼠一般状况,肺功能和BALF炎症细胞计数,采用酶联免疫吸附分析(ELISA)法检测肺组织中炎症因子和氧化应激指标,包括IL-6、IL-8、IL-10、TNF-α、IL-1β和巨噬细胞炎症蛋白2(MIP-2)、8羟基脱氧鸟苷(8-OHdG)和总抗氧化能力(T-AOC);再利用WB和Rt-PCR技术检测TLR4-PI3K-Akt-mTOR蛋白和mRNA表达水平,探索RLD对COPD稳定期模型小鼠的疗效及可能作用机制。结果1肺脾为核心两本多脏虚反映了 COPD稳定期复杂的脏腑功能关系,此脏腑功能定位具有吻合COPD稳定期临床实际的先进性。2共纳入231例COPD稳定期患者,构成比分析显示,主症出现频次由多到少依次为咳嗽、喘息、气短和胸闷;兼症频次较多的前十位依次为起夜频数、畏寒肢冷、口干、入睡困难、眠浅易醒、自汗、耳鸣、乏力、食欲减退、腹胀等;舌象以暗红舌、薄白苔为主,脉象以细脉、沉细脉、弦细脉为主。因子分析显示,35个症状变量旋转后得到14个公因子,R型层次聚类将COPD稳定期临床症状体征分为三大类,较为客观地反映出COPD稳定期以肺脾为核心两本多脏虚的脏腑功能特征和本虚标实、寒热错杂的病理特征。3本研究成功建立了肺脾核心两本多脏虚COPD稳定期(以下简称两本多脏虚COPD)小鼠模型。3.1探索研究中综合各组小鼠的一般情况,如体重、食水量、肛温和症状总分,肺功能,病因符合程度和造模死亡率等生物学表征发现,联合应用氢化可的松、隔天禁食、噪音和番泻叶及CS和LPS是两本多脏虚COPD模型的最佳建立方法。3.2正式实验发现,两本多脏虚COPD组与单纯COPD模型组较空白对照组体重、食物量、肛温降低,饮水量和症状总分较空白对照组升高,两本多脏虚COPD组较单纯COPD模型组肛温明显下降(P<0.01),症状总分升高(P<0.05)。肺组织病理显示,两本多脏虚COPD组和单纯COPD模型组均出现肺泡壁变薄或破裂,肺泡腔扩大,气道周围有炎性细胞浸润等病理特征,且两本多脏虚COPD组在炎性浸润、基底膜水肿、管腔内渗出方面程度较重。通过ELISA探索模型的微观表征发现,两本多脏虚COPD组小鼠生长抑素较空白对照组升高(P<0.01),D-木糖、胃泌素、雌二醇、皮质醇较空白对照组降低(P<0.01或P<0.05),反三碘甲状原氨酸、睾酮较空白对照组降低(P>0.05)。4 RLD对两本多脏虚COPD小鼠具有良好疗效,可改善其呼吸功能。4.1 一般状态:两本多脏虚COPD模型组较空白对照组的体重、食物量、肛温降低(P<0.05或P<0.01),饮水量降低(P>0.05),症状总分显著升高(P<0.01)。阳性药组和RLD中、高剂量组在给药治疗后一般状态得到不同程度改善。其中体重、食物量、饮水量方面虽有增加,但未见统计学差异(P>0.05);各给药组肛温较模型组得到显著改善(P<0.01);RLD低剂量组和阳性药组症状总分虽较模型组下降,但未见差异(P>0.05),RLD中、高剂量组症状总分较模型组明显下降(P<0.01)。4.2肺功能:两本多脏虚COPD模型组小鼠动态弹性(Ers)、最大呼气流速(PEF)、FEV0.1/FVC、FEV0.05/FVC、FEV0.2/FVC较空白对照组降低,两本多脏虚COPD模型组小鼠动态阻力(Rrs)、动态顺应性(Crs)、深吸气量(IC)、用力肺活量(FVC)、FEV0.1、FEV0.05、FEV0.2较空白对照组升高。各给药组在给药治疗后肺功能得到改善,改善程度以阳性药组和RLD高剂量组更为明显,中剂量组次之,但优于模型组。4.3肺组织病理:与空白对照组相比,两本多脏虚COPD模型组小鼠气道呈高分泌状态,肺泡内有炎症细胞浸润,肺泡腔增大或破裂融合,肺泡毛细血管扩张增粗,小气道上皮增生或水肿。各给药组较模型组小鼠的以上病理情况情况得到不同程度减轻,以RLD中、高剂量组和阳性药组明显。4.4 BALF炎症细胞计数:两本多脏虚COPD模型组小鼠BALF中中性粒细胞、淋巴细胞、巨噬细胞数量较空白对照组均升高(P<0.05或P<0.01),给药后RLD中、高剂量组和阳性药组淋巴细胞数量较模型组小鼠降低(P<0.05或P<0.01),RLD中、高剂量组中性粒细胞、噬细胞数量较模型组小鼠降低,但未见差异(P>0.05),下降幅度均以RLD高剂量组为优。4.5抗炎症损伤和氧化应激:两本多脏虚COPD模型组小鼠IL-6、IL-8、IL-10、IL-1β、TNF-α、MIP-2水平较空白对照组显著升高(P<0.01)。给药干预后,阳性药组和RLD高剂量组治疗后的IL-6、IL-8、IL-10、IL-1β、TNF-α、MIP-2水平降低程度更为明显(P<0.05或P<0.01),中剂量组次之,但优于模型组(P<0.05或P<0.01),其中RLD高剂量组IL-8、TNF-α水平的下降幅度较阳性药组更大。两本多脏虚COPD模型组小鼠8-OHdG水平较空白对照组显著升高(P<0.01),给药干预后,各给药组治疗后的8-OHdG水平均降低(P<0.05或P<0.01),以RLD高剂量组效优,RLD中剂量组次之;两本多脏虚COPD模型组小鼠T-AOC水平较空白对照组降低,各给药组在给药干预后均有不同程度升高,但未见差异。5 RLD通过TLR4-PI3K-Akt-mTOR途径可能是调控COPD稳定期实现抑制炎症反应的途径。两本多脏虚COPD模型组小鼠TLR4、PI3K、Akt、mTOR mRNA和TLR4/GAPDH、p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR 蛋白表达水平较空白对照组显著升高(P<0.01),给药干预后,各给药组治疗后均降低,以阳性组和RLD中、高剂量组效优。说明RLD可能通过降低模型小鼠肺组织中TLR4蛋白的表达水平,从而抑制PI3K-Akt-mTOR信号通路的激活。结论1肺脾为核心两本多脏虚反映了 COPD稳定期复杂的脏腑功能关系,肺脾为核心旨在强调中医肺主气呼吸功能的物质基础,两本提示要重视脏腑功能之源。2临床COPD稳定期患者表现为虚实并存,寒热错杂征象,呈现出肺脾核心两本多脏虚的脏腑功能特征。3联合应用番泻叶、隔天禁食、噪音和氢化可的松及CS和LPS复合方式能够建立两本多脏虚COPD稳定期模型。模型评价须通过一般情况、肺组织病理、肺功能以及证候微观指标等综合判定。4 RLD治疗两本多脏虚COPD稳定期具有良好疗效,可能通过下调TLR4蛋白的表达水平,使得下游PI3K-Akt-mTOR信号通路得到抑制,改善呼吸功能,但仍需深入研究完善其疗效机制。更多还原

【Abstract】 Objective1 To explore the functional characteristics of the viscera in the stable stage of chronic obstructive pulmonary disease(COPD)from the perspectives of the material basis of respiratory function,the source of viscera function and the relationship between cold and heat may provide theoretical basis for subsequent clinical and experimental research.2 To analyze the distribution of clinical symptoms and signs and the essence of pathogenesis in patients with stable COPD and to deepen the understanding of the functional characteristics of the viscera.3 We explore and establish a TCM disease-symptom combination animal model that reflects the pathogenesis of stable COPD,observe the macroscopic and microscopic features of the model,and initially establish the model evaluation method by the carrier of animals.4 This study observes the curative effect of Recuperating lung decoction(RLD)on stable COPD mouse model by measuring multiple indicators and illustrates that RLD may regulate the stable phase of COPD through TLR4-PI3K-Akt-mTOR pathway,which is a key way to achieve the inhibition of inflammatory response.Methods1 By analyzing the related theories and landmark achievements of traditional Chinese medicine on the stable COPD stage and its influence on the eight principles of syndrome differentiation and treatment,we will explore the pathogenesis and zang-fu function characteristics that may be more in line with the clinical reality of the disease.2 The observational research method was used to uniformly input the basic data,main symptoms,concurrent symptoms,tongue and pulse,etc.of the included patients with stable COPD.The distribution of symptoms and signs in stable COPD was mined through component ratio analysis,cluster analysis and factor analysis to explain the zang-fu function characteristics.3 In the exploratory study,firstly,according to the possible influencing factors of deficiency of the lung and spleen core two organs,choose senna decoction,fasting on alternate days,noise stimulation and hydrocortisone.Then compare single,two and multiple intervention factors to determine the optimal modeling method of the lung and spleen core two multi-organ deficiency model.Secondly,combined with smoke exposure(CS)and lipopolysaccharide(LPS)intervention.Thirdly,Combining the clinical understanding of the multiple viscera deficiency in the core of the lung and spleen,as well as the general condition of the mice after modeling,mortality,lung function and the degree of compliance with the etiology and other factors,the modeling method of stable COPD of multiple viscera deficiency in the core of the lung and spleen was initially determined.4 In the formal experiment,90 BALB/c mice were divided into 20 blank control group,10 simple COPD stable stage group,and 60 COPD stable stage group with multiple viscera deficiency in the core of the lung and spleen.According to the method finally established in the preliminary experiment to establish a stable COPD model with lung and spleen core two multiple viscera deficiency.Randomly select 10 mice in each group to test the general condition,lung tissue pathology and lung function to determine the stability and repeatability of the experiment.And then explore their possible microscopic characteristics of D-xylase,somatostatin,cortisol,trans-triiodothyronine,testosterone and estradiol expression levels.5 The 50 mice in the stable COPD group with multiple viscera deficiency in the core of the lung and spleen were randomly divided into model group,positive drug group,RLD low-dose group,medium-dose group and the high-dose group,the other 10 were blank control groups.After 28 days of drug administration and intervention,the general conditions of the mice were evaluated,such as body weight,rectal temperature,water volume,lung function,etc..Experimental specimens such as lung tissue,serum and BALF were prepared,and the enzymelinked immunosorbent assay(ELISA)method was used to detect inflammatory factors,including IL-6,IL-8,IL-10,TNF-α,IL-1β and macrophage inflammatory protein 2(MIP-2)and oxidative stress indicator,such as 8-hydroxydeoxyguanosine(8-OHdG)and total antioxidant capacity(T-AOC).Then the expression levels of TLR4-PI3K-Akt-mTOR protein and mRNA were detected by Rt-PCR and WB technology to explore the curative effect and possible mechanism of RLD on stable COPD model mice.Results1 Multiple viscera deficiency in the core of the lung and spleen reflect the complex functional relationship of the viscera in COPD,which requires a correct understanding of COPD stable specimens and the relationship between cold and heat.The lung and spleen as the core aims to emphasize the material basis of the respiratory function of the lungs in TCM and the latter are intended to focus on the source of the functions of the zang-fu organs.Therefore,the functional positioning of the multiple viscera deficiency in the core of the lung and spleen has the advanced nature of matching the clinical reality of COPD in stable stage.2 A total of 231 patients with stable COPD were included.The composition ratio analysis showed that the main symptoms of the included patients were cough,wheezing,shortness of breath,and chest tightness in descending order.The other symptoms are night frequency,chills and cold limbs,dry mouth,difficulty falling asleep,light sleep and easy awakening,spontaneous perspiration,tinnitus,fatigue,loss of appetite,abdominal distension,etc..The tongue color is mainly dark red and the tongue coating is mainly thin white coating,followed by white greasy coating and thin yellow moss.The pulse condition is mainly thin veins.Factor analysis obtained 14 common factors through factor rotation of 35 symptom variables.The symptom variables included were divided the clinical symptoms and signs of stable COPD into three categories by R-type hierarchical clustering,which more objectively reflected the zangfu functional characteristics of stable COPD and the pathological characteristics of the deficiency and excess and the mixed cold and heat.3 In this study,a mouse model of COPD with multiple viscera deficiency in the core of the lung and spleen was successfully established in the stable period.3.1 We comprehensively detect body weight,water intake,rectal temperature,symptom total score,lung function and the degree of compliance with the cause of each group of mice after the end of the syndrome and 2,4,8,and 12 weeks of smoke exposure(CS).It was found that the combined application of hydrocortisone,hunger and satiety,noise stimulation,senna,CS and LPS was the best method for establishing the COPD model with multiple viscera deficiency in the core of the lung and spleen.3.2 In the formal experiment,it was found that the body weight,food intake and rectal temperature of the COPD group with multiple viscera deficiency in the core of the lung and spleen and the simple COPD model group were lower than those of the blank control group,and the water intake and total symptom score were higher than those of the blank control group.Compared with the simple COPD model group,the rectal temperature in the group decreased significantly(P<0.01),and the total symptom score increased(P<0.05).This indicated that the mice in the COPD group of multiple viscera deficiency in the core of the lung and spleen had more obvious visceral dysfunction.Lung histopathology showed that compared with the normal control group,the alveolar wall became thinner or ruptured,the alveolar cavity was enlarged and there were inflammatory cell infiltration around the airway in the COPD group with multiple viscera deficiency in the core of the lung and spleen and the simple COPD model group.The inflammatory infiltration,basement membrane edema and intraluminal exudation were more severe in the COPD group with multiple viscera deficiency in the core of the lung and spleen.Through ELISA to explore the microscopic characteristics of the model,it was found that the somatostatin in the COPD group with multiple viscera deficiency in the core of the lung and spleen was higher than that in the blank control group(P<0.01),and the levels of D-xylose,gastrin,estradiol and cortisol were lower than those in the blank control group(P<0.01 or P<0.05).The levels of transtriiodothyronine and testosterone were lower than those in the blank control group,but there was no difference(P>0.05).4 RLD can partially relieve the general state,lung function,lung tissue pathology,inflammatory injury and oxidative stress state of COPD mice with multiple viscera deficiency in the core of the lung and spleen and improve respiratory function.4.1 General status:The body weight,food amount,and rectal temperature of the two COPD model of multiple viscera deficiency in the core of the lung and spleen group were significantly lower than the blank control group(P<0.05 or P<0.01),while the total symptom score was significantly increased(P<0.01).The general state of the positive drug group and the RLD middle and high dose groups were improved to varying degrees after administration and treatment.Among them,the body weight,food amount and water intake increased,but there was no statistical difference(P>0.05).The rectal temperature of each administration group was significantly improved compared with the model group(P<0.01).The RLD lowdose group and the positive drug group were significantly improved(P<0.01).Although the total symptom score of the RLD group was lower than that of the model group,there was no statistical difference(P>0.05).It is suggested that the RLD middle and high dose groups have a good effect on improving the macroscopic performance of mice,which indicates that the RLD middle and high dose groups have certain advantages in regulating the state of viscera function deficiency.4.2 Pulmonary function:Ers,PEF,FEV0.1/FVC,FEV0.05/FVC,FEV0.2/FVC of the mice in the COPD model group of multiple viscera deficiency in the core of the lung and spleen were lower than those in the blank control group,while the Rrs,Crs,IC,FVC,FEV0.1,FEV0.05,and FEV0.2 of mice were higher than those in the blank control group.It indicates that the model mice have increased airway resistance and the possibility of small airway obstruction.The pulmonary function of each administration group was improved after administration.The improvement was more obvious in the positive drug group and the RLD high-dose group,followed by the middle-dose group.Therefore RLD can improve the airway elasticity to a certain extent and reduce the airway resistance and the degree of obstruction in the model mice.4.3 Pathology of lung tissue:Compared with the blank control group,the airways of the mice in the COPD model group were in a state of hypersecretion,inflammatory cells infiltrated in the alveoli,enlarged alveolar cavity and small airway epithelial hyperplasia or edema,etc..Compared with the model group,the above pathological conditions of the mice in each administration group were relieved to varying degrees.The RLD middle and high dose groups and the positive drug group were more obvious.4.4 BALF inflammatory cell count:The numbers of neutrophils,lymphocytes,and macrophages in the B ALF of the mice in the COPD model group of multiple viscera deficiency in the core of the lung and spleen were higher than those in the blank control group(P<0.05 or P<0.01).The number of lymphocytes in the RLD middle and high dose groups and the positive drug group was significantly lower than that in the model group(P<0.05 or P<0.01).The numbers of neutrophils and phages in the RLD middle and high dose groups were lower than those in the model group,but there was no difference(P>0.05).4.5 Anti-inflammatory injury and oxidative stress:The levels of IL-6,IL-8,IL-10,IL-1β,TNF-α and MIP-2 in the COPD model group of multiple viscera deficiency in the core of the lung and spleen were significantly higher than those in the blank control group(P<0.01).The levels of these indicators in the positive drug group and the RLD high-dose group decreased more significantly after treatment(P<0.05 or P<0.01),followed by the middle-dose group which always better than the model group(P<0.05 or P<0.01).Compared with the blank control group,the 8-OHdG level in the COPD model group of multiple viscera deficiency in the core of the lung and spleen was significantly higher(P<0.01).The levels of 8-OHdG in each administration group decreased after treatment(P<0.05 or P<0.01).And the RLD highdose group had the best effect,followed by the RLD medium-dose group.The T-AOC level of the mice in the COPD model group of multiple viscera deficiency in the core of the lung and spleen was lower than that in the blank control group.There were different degrees of elevation,but no difference was found,which suggests that RLD may reduce oxidative stress injury mainly by inhibiting the level of oxidative stress.5 RLD through the TLR4-PI3K-Akt-mTOR pathway may be the key way to regulate the stable phase of COPD to suppress the inflammatory response.Compared with the blank control group,the expression levels of TLR4,PI3K,Akt,mTOR mRNA and TLR4/GAPDH、pPI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR protein in the COPD model group with multiple viscera deficiency in the core of the lung and spleen were significantly higher(P<0.01).After intervention,all administration groups decreased after treatment,especially the positive group and RLD middle and high dose groups.It shows that RLD may inhibit the stimulation of PI3KAkt-mTOR signaling pathway by reducing the expression level of TLR4 protein in the lung tissue of model mice to reduce lung inflammation and improving respiratory function.Conclusion1 Multiple viscera deficiency in the core of the lung and spleen reflects the complex functional relationship of the viscera during the stable COPD period.The lung and spleen as the core aims to emphasize the material basis of the TCM lung governing qi and respiratory function and the two viscera pay attention to the source of the functions of the zang-fu organs.2 Patients in the stable stage of clinical COPD are characterized by coexistence of deficiency and excess,mixed with cold and heat,which shows the functional characteristics of the multiple viscera deficiency in the core of the lung and spleen.3 Combined application of senna,hunger and satiety,noise stimulation,hydrocortisone,CS and LPS compound methods can establish stable COPD models with multiple viscera deficiency in the core of the lung and spleen that reflect the clinical practice of traditional Chinese medicine and reflect the overall functional relationship of the viscera.Model evaluation must be comprehensively judged by general conditions,lung tissue pathology,lung function and microscopic indicators of syndromes.4 RLD has good curative effect in the treatment of COPD with multiple viscera deficiency in stable stage.RLD may reduce the pulmonary inflammatory response and improve respiratory function by downregulating the expression level of TLR4 protein and inhibiting the activation of the downstream PI3K-Akt-mTOR signaling pathway.更多还原