基于PD-1/PD-L1轴探讨COPD痰浊壅肺型证候特点及麻杏蒌石汤干预的作用机制

【作者】 李莉；

【导师】 晏军；

【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通过研究PD-1、PD-L1等相关免疫指标在COPD痰浊壅肺证中的表达特点,探讨COPD痰浊壅肺型证候特点,旨在发现COPD痰浊壅肺型证候特点与PD-1、PD-L1等免疫指标的内在联系,为中医临床辨证提供客观依据。2通过观察一般实验室指标、CRP、IL-6、MCP-1、MIP-1α、TNF-α等炎症因子在COPD痰浊壅肺证中的表达特点,探讨COPD痰浊壅肺型证候特点,旨在发现COPD痰浊壅肺型证候特点与现代实验室指标、炎症指标的内在联系,为中医临床辨证提供客观依据。3通过建立COPD小鼠模型,基于PD-1/PD-L1轴探讨PD-1、PD-L1等相关免疫分子在COPD中的表达特点以及可能的调控机制,应用中药麻杏蒌石汤干预后探讨其对相关指标表达的影响及可能的作用机制,为进一步研究及治疗COPD提供新的客观依据。方法:1临床研究部分应用流行病学横断面研究方法。根据纳入及排除标准收集65例COPD 患者,进行 PD-1、PD-L1 等相关免疫指标和 CRP、IL-6、MCP-1、MIP-1α、TNF-α等炎症指标以及CAT评分、mMRC评分、FeNO检测、肺功能检查等一般实验室指标的分析,探讨观测指标在COPD痰浊壅肺证中的表达特点。2实验研究部分通过构建COPD动物模型,分为麻杏蒌石汤组(简称中药组)、正常组、PD-1抑制剂组(简称对照组)、COPD模型组(简称模型组),一共4组,分别给予不同的干预措施,采用ELISA法检测PD-1、PD-L1、IL-6、MCP-1、MIP-1α、TNF-α等指标的表达特点,通过Western Blot检测分析COPD模型小鼠肺组织NF-κB的表达,通过流式细胞术检测方法分析COPD模型小鼠脾脏、全血及肺泡灌洗液PD-1、PD-L1的表达特点,探讨PD-1、PD-L1等相关免疫分子在COPD中的表达特点以及可能的调控机制,并探讨中药麻杏蒌石汤干预后对检测指标表达的影响及可能的作用机制。结果:1 一般实验室指标在COPD痰浊壅肺型证候中的表达特点:痰浊壅肺证COPD患者血气分析PaCO2的表达低于肺肾气虚证患者(P<0.05)。痰浊壅肺证COPD患者WBC、PCT、PLR、TT4的表达高于肺肾气虚证患者(P<0.05)。痰浊壅肺证COPD患者肺功能指标FEV1、FEV1/FVC的表达高于肺肾气虚证COPD患者(P<0.05)。痰浊壅肺证COPD患者FeNO指标表达稍高于肺肾气虚证患者(P>0.05),CAT评分与mMRC评分及其他实验室指标在两个证候间差异无统计学意义(P>0.05)。2炎症指标在COPD痰浊壅肺型证候中的表达特点:痰浊壅肺证COPD患者MCP-1的表达高于肺肾气虚证患者(P<0.05),痰浊壅肺证COPD患者MIP-1α表达略低于肺肾气虚证(P>0.05)。痰浊壅肺证、肺肾气虚证COPD患者MCP-1、MIP-1α的表达均高于正常对照组(P<0.05)。痰浊壅肺证COPD患者ESR、IL-6、CRP的表达高于肺肾气虚证COPD患者(P<0.05)。3免疫指标在COPD痰浊壅肺型证候中的表达特点:痰浊壅肺证COPD患者PD-1的表达低于肺肾气虚证患者(P<0.05),PD-L1的表达高于肺肾气虚证患者(P<0.05)。痰浊壅肺证及肺肾气虚证COPD患者PD-1的表达均高于正常志愿者(P<0.05),痰浊壅肺证及肺肾气虚证COPD患者PD-L1的表达均低于正常志愿者(P<0.05)。痰浊壅肺证COPD患者IgE的表达高于肺肾气虚证患者(P<0.05),痰浊壅肺证COPD患者CD3+的表达高于肺肾气虚证患者(P<0.05);IgM、IgA、IgG、C3、C4、CD3+CD4+、CD3+CD8+、CD4+/CD8+等指标在两个证候间差异无统计学意义(P>0.05)。4实验研究:①肺组织HE染色:COPD模型组小鼠肺组织存在异常结构,可见肺泡蒌缩塌陷和肺泡壁增厚,还存在中性粒细胞浸润现象。而麻杏蒌石汤组及PD-1抑制剂组经用药干预后其肺组织病理表现较前改善。②ELISA检测血浆、肺泡灌洗液MCP-1、MIP-1α、IL-6、TNF-α表达:COPD模型组血浆、肺泡灌洗液MCP-1、MIP-1α的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),且麻杏蒌石汤组血浆MCP-1、MIP-1α的表达低于PD-1抑制剂组(P<0.05),麻杏蒌石汤组肺泡灌洗液MIP-1α的表达低于PD-1抑制剂组(P<0.05),麻杏蒌石汤组肺泡灌洗液MCP-1的表达略低于PD-1抑制剂组(P>0.05)。COPD模型组血浆、肺泡灌洗液IL-6、TNF-α的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),且麻杏蒌石汤组血浆、肺泡灌洗液IL-6、TNF-α的表达低于PD-1抑制剂组(P<0.05)。③Western Blot检测肺组织NF-κB表达:COPD模型组肺组织NF-κB的表达高于麻杏蒌石汤组、PD-1抑制剂组、正常组(P<0.05)。麻杏蒌石汤组肺组织NF-κB的表达低于PD-1抑制剂组(P<0.05)。④肺组织免疫组化IHC染色及IOD值:COPD模型组PD-1的表达高于正常组(P<0.05),COPD模型组PD-L1的表达低于正常组(P<0.05)。PD-1抑制剂组PD-1的表达低于COPD模型组、PD-L1的表达高于COPD模型组(P<0.05)。麻杏蒌石汤组PD-1的表达低于COPD模型组、PD-1抑制剂组(P<0.05),麻杏蒌石汤组PD-L1的表达高于COPD模型组、PD-1抑制剂组(P<0.05)。⑤ELISA检测血浆、肺泡灌洗液PD-1、PD-L1表达:COPD模型组血浆PD-1的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),COPD模型组血浆PD-L1的表达低于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),且麻杏蒌石汤组血浆PD-1的表达低于PD-1抑制剂组、血浆PD-L1的表达高于PD-1抑制剂组(P<0.05)。COPD模型组肺泡灌洗液PD-1的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),COPD模型组肺泡灌洗液PD-L1的表达低于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),且麻杏蒌石汤组肺泡灌洗液PD-1的表达低于PD-1抑制剂组、肺泡灌洗液PD-L1的表达高于PD-1抑制剂组(P<0.05)。⑥流式细胞术检测脾脏、全血、肺泡灌洗液PD-1、PD-L1表达:COPD模型组脾脏、全血PD-1+CD4细胞占T细胞比例的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05)。COPD模型组肺泡灌洗液PD-1+CD4细胞占T细胞比例的表达高于正常组、麻杏蒌石汤组(P<0.05)。COPD模型组肺泡灌洗液中PD-1+CD8细胞占T细胞比例的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05)。COPD模型组脾脏、全血中PD-1+CD8细胞占T细胞比例的表达各组无显著差异(P>0.05)。COPD模型组脾脏、全血、肺泡灌洗液中PD-1+CD4细胞占总淋巴细胞比例的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05)。COPD模型组肺泡灌洗液中PD-1+CD8细胞占总淋巴细胞比例的表达高于正常组、麻杏蒌石汤组、PD-1抑制剂组(P<0.05),COPD模型组脾脏PD-1+CD8细胞占总淋巴细胞比例的表达高于正常组(P<0.05)且其他各组无显著差异(P>0.05)。COPD模型组全血PD-1+CD8细胞占总淋巴细胞比例的表达各组无显著差异(P>0.05)。COPD模型组脾脏、全血、肺泡灌洗液MHCⅡ+M1 Macrophage PD-L1+比例的表达高于正常组、麻杏蒌石汤组(P<0.05)。结论:1 WBC、PCT、PLR、PaCO2、TT4、肺功能等一般实验室指标、MCP-1、MIP-1α、IL-6、ESR、CRP等炎症指标以及PD-1、PD-L1、IgE、CD3+等免疫指标在COPD痰浊壅肺证患者中有其相应的表达特点。同时与肺肾气虚证患者进行对比发现COPD痰浊壅肺证患者的炎症反应及免疫反应更为明显,而免疫受损较肺肾气虚证轻。说明COPD痰浊壅肺型证候特点与现代实验室指标、炎症指标、免疫相关指标存在内在联系,提示了这些观察指标在COPD中医辨证上有一定的指导意义,为中医临床辨证分型提供依据,还可作为对COPD痰浊壅肺证与肺肾气虚证两个证候之间准确辨证分型的辅助参考指标。2 COPD模型小鼠肺组织存在明显的炎症反应。COPD可导致机体内炎症因子MCP-1、MIP-1α、IL-6、TNF-α的表达水平明显升高,也可使NF-κB p65蛋白的表达水平明显升高。而麻杏蒌石汤与PD-1抑制剂均可改善COPD机体的炎症反应,显著降低炎症因子MCP-1、MIP-1α、IL-6、TNF-α的表达,同时下调NF-κB p65蛋白的表达,并且麻杏蒌石汤的改善效果优于PD-1抑制剂,提示麻杏蒌石汤可以明显改善COPD模型小鼠的炎症反应。3 COPD模型小鼠肺组织存在明显的免疫反应,PD-1/PD-L1轴在其中起到重要作用。COPD可诱导小鼠肺组织、血浆及肺泡灌洗液中PD-1的高表达,抑制并下调PD-L1的表达,而麻杏蒌石汤及PD-1抑制剂可以显著降低了烟熏联合LPS诱导的PD-1高表达并提高PD-L1的表达,并且麻杏蒌石汤的效果优于PD-1抑制剂。提示麻杏蒌石汤可以通过调控PD-1/PD-L1轴改善COPD模型的免疫受损情况,调节PD-1、PD-L1免疫分子的表达,使PD-1下调,使PD-L1上调,从而改善免疫反应,提高机体免疫功能,起到免疫调节的作用,改善COPD的免疫失衡。4 PD-1、PD-L1在COPD中的表达特点揭示了 PD-1/PD-L1轴与COPD免疫机制之间存在相关性,可通过阻断PD-1/PD-L1轴作为COPD治疗的新靶点。麻杏蒌石汤与PD-1抑制剂均可以通过阻断PD-1/PD-L1轴调节COPD免疫功能,并且麻杏蒌石汤效果更优,为进一步应用中医药改善COPD免疫功能提供了新的治疗思路。更多还原

【Abstract】 Objective:1 By studying the expression characteristics of PD-1、PD-L1 and other related immune indicators in COPD phlegm-turbid obstructing lung syndrome,to explore the characteristics of COPD phlegm-turbid obstructing lung syndrome,in order to find the internal relationship between the characteristics of COPD phlegm-turbid obstructing lung syndrome and PD-1、PD-L1 and other immune indicators,to provide objective basis for TCM clinical syndrome differentiation.2 By observing the expression characteristics of general laboratory indicators,CRP,IL-6,McP-1,MIP-1α,TNF-α and other inflammatory factors in COPD phlegm-turbidity obstructing lung syndrome,to explore the characteristics of COPD phlegm-turbidity obstructing lung syndrome,in order to find the internal relationship between the characteristics of COPD phlegm-turbidity obstructing lung syndrome and modern laboratory indicators,inflammatory indicators.To provide objective basis for TCM clinical syndrome differentiation.3 By establishing a COPD mouse model,the expression characteristics and possible regulatory mechanisms of PD-1、PD-L1 and other related immune molecules in COPD were explored based on the PD-1/PD-L1 axis,and the influence on the expression of related indicators and possible mechanism of action were discussed after the intervention of Maxingloushi decoction,which has guiding significance for further clinical and research.To provide objective basis for clinical treatment of COPD.Methods:1 The clinical study was conducted in a cross-sectional epidemiological study 65 patients with COPD were collected according to the inclusion and exclusion criteria,and analyzed related immune indexes such as PD-1 and PD-L1,inflammatory indexes such as CRP,IL-6,MCP-1,MIP-1α and TNF-α,as well as general laboratory indexes such as CAT score,mMRC score,FeNO test and lung function test.To explore the expression characteristics of observation indexes in COPD phlegm-turbidities obstructing lung syndrome.2 In the experimental study,the animal model of COPD was constructed and divided into four groups,namely,Maxingloushi decoction group(TCM group),normal group,PD-1 inhibitor group(control group)and COPD model group(model group).Different intervention measures were given respectively.The expression characteristics of PD-1,PD-L1,IL-6,MCP-1,MIP-1α and TNF-α were detected by ELISA.The expression of NF-κB in lung tissues of COPD model mice was analyzed by Western Blot.Flow cytometry was used to analyze the expression characteristics of PD-1 and PD-L1 in spleen,whole blood and alveolar lavage fluid of COPD model mice,to explore the expression characteristics of PD-1,PD-L1 and other related immune molecules in COPD and the possible regulatory mechanism,and to explore the influence of Chinese traditional medicine Maxingloushi decoction on the expression of detection indicators and the possible mechanism of action.Results:1 Expression characteristics of laboratory indicators in COPD phlegm-turbidities obstructing lung syndrome:The expression of PaCO2 in blood gas analysis of COPD patients with phlegm-turbidities obstructing lung was lower than that of patients with lung-kidney qi deficiency syndrome(P<0.05).The expressions of WBC,PCT,PLR and TT4 in COPD patients with phlegm-turbidity obstructing lung were higher than those in patients with lung-kidney qi deficiency(P<0.05).The expression of FEV1 and FEV1/FVC in COPD patients with phlegm-turbidity obstructing lung was higher than that in COPD patients with lung-kidney qi deficiency(P<0.05).The expression of FeNO index in COPD patients with phlegm-turbidness obstructing lung was slightly higher than that in patients with lung-kidney qi deficiency(P>0.05),there was no significant difference in CAT score,mMRC score and other laboratory indicators between the two syndromes(P>0.05).2 The expression characteristics of inflammatory indicators in COPD patients with phlegm-turbidities obstructing lung syndrome were as follows:the expression of MCP-1 in COPD patients with phlegm-turbidities obstructing lung was higher than that in patients with lung-kidney qi deficiency syndrome(P<0.05),MIP-1α was slightly lower in COPD patients with phlegm-turbidities obstructing lung than in lung-kidney qi deficiency syndrome(P>0.05).The expression of MCP-1 and MIP-1α in COPD patients with phlegm-turbidity obstruction of lung and deficiency of lung-kidney Qi was higher than that in normal control group(P<0.05).The expression of ESR,IL-6 and CRP in COPD patients with phlegm-turbidity obstructing lung was higher than that in COPD patients with pneumon-kidney qi deficiency(P<0.05).3 The expression characteristics of immune indexes in COPD patients with phlegm-turbidity obstructing lung syndrome:the expression of PD-1 in COPD patients with phlegm-turbidity obstructing lung syndrome was lower than that in patients with lung-kidney qi deficiency syndrome(P<0.05),the expression of PD-L1 was higher than that of patients with lung-kidney qi deficiency syndrome(P<0.05).The expression of PD-1 in COPD patients with phlegm-turbidities and lung-kidney qi deficiency was higher than that in healthy volunteers(P<0.05),PD-L1 expression in COPD patients with phlegm-turbidities obstructing lung and pneumon-kidney qi deficiency was lower than that in healthy volunteers(P<0.05).The IgE expression in COPD patients with phlegm-turbidity obstructing lung was higher than that in patients with lung-kidney qi deficiency(P<0.05),CD3+expression in COPD patients with phlegm-turbidity obstructing lung was higher than that in patients with lung-kidney qi deficiency(P<0.05);IgM,IgA,IgG,C3,C4,CD3+CD4+,CD3+CD8+,CD4+/CD8+showed no significant difference between the two syndroms(P>0.05).4 Experimental research:① Lung tissue HE staining:There were abnormal structures in the lung tissues of COPD model mice,including alveolar atrophy and collapse,alveolar wall thickening,and neutrophil infiltration.The pathological manifestations of lung tissue in Maxingloushi decoction group and the PD-1 inhibitor group were improved after drug intervention.②ELISA detected MCP-1,MIP-1α,IL-6 and TNF-α:The levels of MCP-1 and MIP-1αin plasma and alveolar lavage fluid of model group were higher than those of normal group,Maxingloushi decoction group and control group(P<0.05),and the levels of plasma MCP-1 and MIP-1α in Maxingloushi decoction group were lower than those in control group(P<0.05),the expression of MIP-1α in Chinese herbal group was lower than that in control group(P<0.05),the expression of MCP-1 in alveolar lavage fluid in Maxingloushi decoction group was slightly lower than that in control group(P>0.05).The levels of IL-6 and TNF-α in plasma and alveolar lavage fluid of model group were higher than those of normal group,Maxingloushi decoction group and control group(P<0.05),and the levels of IL-6 and TNF-αin plasma and alveolar lavage fluid in Maxingloushi decoction group were lower than those in control group(P<0.05).③Lung tissue NF-κB was detected by Western Blot:the expression of NF-κB in model group was higher than that in Maxingloushi decoction group,control group and normal group(P<0.05),and the expression of NF-κB in lung tissues of Maxingloushi decoction group was lower than that of control group(P<0.05).④IHC staining and IOD value in lung tissue:The expression of PD-1 in model group was higher than that in normal group(P<0.05),PD-L1 expression in model group was lower than that in normal group(P<0.05).The expression of PD-1 in control group was lower than that in model group,and the expression of PD-L1 was higher than that in model group(P<0.05).The expression of PD-1 in Maxingloushi decoction group was lower than that in model group and control group(P<0.05),the expression of PD-L1 in Maxingloushi decoction group was higher than that in model group and control group(P<0.05).⑤PD-1 and PD-L1 in plasma and alveolar lavage fluid were detected by ELISA:The expression of PD-1 in plasma in model group was higher than that in normal group,Maxingloushi decoction group and control group(P<0.05),the plasma PD-L1 expression in model group was lower than that in normal group,Maxingloushi decoction group and control group(P<0.05),and plasma PD-1 expression in Maxingloushi decoction group was lower than that in control group,and plasma PD-L1 expression was higher than that in control group(P<0.05).The expression of PD-1 in alveolar lavage fluid in model group was higher than that in normal group,Maxingloushi decoction group and control group(P<0.05),the expression of PD-L1 in alveolar lavage fluid in model group was lower than that in normal group,Maxingloushi decoction group and control group(P<0.05),and the expression of PD-1 in alveolar lavage fluid in Maxingloushi decoction group was lower than that in control group,and the expression of PD-L1 in alveolar lavage fluid in Maxingloushi decoction group was higher than that in control group(P<0.05).⑥Flow cytometry detected PD-1 and PD-L1 in spleen,blood and alveolar lavage fluid:The expression of PD-1+CD4 cells in spleen and whole blood in the model group was higher than that in normal group,Maxingloushi decoction group and control group(P<0.05),the proportion of PD-1+CD4 cells in T cells in alveolar lavage fluid in model group was higher than that in normal group and Maxingloushi decoction group(P<0.05).The expression of PD-1+CD8 cells to T cells in alveolar lavage fluid in model group was higher than that in normal group,Maxingloushi decoction group and control group(P<0.05),there was no significant difference in the ratio of PD-1+CD8 cells to T cells in spleen and whole blood of model group(P>0.05).The expression of PD-1+CD4 cells in spleen,whole blood and alveolar lavage fluid in model group was higher than that in normal group,Maxingloushi decoction group and control group(P<0.05).The expression of PD-1+CD8 cells in alveolar lavage fluid in model group was higher than that in normal group,Maxingloushi decoction group and control group(P<0.05),the expression of PD-1+CD8 cells in spleen in the proportion of total lymphocytes in model group was higher than that in normal group(P<0.05)and there was no significant difference in other groups(P>0.05),there was no significant difference in the expression of PD-1+CD8 cells in the proportion of total lymphocytes in model group(P<0.05).The expression of MHCII+M1 Macrophage PD-L1+in spleen,whole blood and alveolar lavage fluid of model group was higher than that of normal group and Maxingloushi decoction group(P<0.05).Conclusion:1 General laboratory indicators,such as WBC,PCT,PLR,PaCO2,TT4,lung function;inflammatory indicators,such as MCP-1,MIP-1α,IL-6,ESR,CRP;and immune indicators,such as PD-1,PD-L1,IgE,CD3+,have corresponding expression characteristics in COPD patients with phlegm-turbide-obstructed lung syndrome.At the same time,compared with the patients with lung-kidney qi deficiency syndrome,it was found that the inflammatory response and immune response of COPD patients with phlegm-turbidities obstructing lung were more obvious,while the immune damage was less severe than that of the patients with lung-kidney qi deficiency syndrome.It indicates that the characteristics of the syndrome of COPD phlegm-turbidium-obstructing lung have internal relationship with modern laboratory indicators,inflammatory indicators and immune-related indicators,suggesting that these observation indicators have certain guiding significance in COPD TCM syndrome differentiation,and provide a basis for TCM clinical syndrome differentiation.It can also be used as an auxiliary reference index for accurate syndrome differentiation and classification between the two syndromes of COPD:phlegm-turbidity obstructing lung and deficiency of lung-kidney qi.2 There were obvious inflammatory reactions in lung tissues of COPD model mice.COPD can significantly increase the expression of inflammatory factors MCP-1,MOP-1α,IL-6 and TNF-α,as well as NF-κB P65 protein.Both Maxingloushi decoction and PD-1 inhibitor can improve the inflammatory response of COPD,significantly reduce the expression of inflammatory factors MCP-1,MIP-1α,IL-6 and TNF-α,and down-regulate the expression of NF-κB P65 protein,and the improvement effect of Maxingloushi decoction is better than that of PD-1 inhibitor.It is suggested that Maxingloushi decoction can significantly improve the inflammatory response of COPD model mice.3 The PD-1/PD-L1 axis plays an important role in the obvious immune response in the lung tissues of COPD model mice.COPD can induce the high expression of PD-1 in lung tissue,plasma and alveolar lavage fluid of mice,and inhibit and down-regulate the expression of PD-L1.However,Maxingloushi decoction and PD-1 inhibitor can significantly reduce the high expression of PD-1 induced by smoking combined with LPS,and improve the expression of PD-L1,and the effect of Maxingloushi decoction is better than that of PD-1 inhibitor.It is suggested that Maxingloushi decoction can improve the immune impairment of COPD model by regulating The PD-1/PD-L1 axis,regulating the expression of PD-1 and PD-L1 immune molecules,down-regulating PD-1 and up-regulating PD-L1,thus improving the immune response,improving the immune function of the body,playing the role of immune regulation,and improving the immune imbalance of COPD.4 Through clinical and experimental studies,the expression characteristics of PD-1 and PD-L1 in COPD were found,revealing the correlation between the PD-1/PD-L1 axis and the immune mechanism of COPD,and blocking the PD-1/PD-L1 axis can be used as a new target for the treatment of COPD.In this study,it was found that both Maxingloushi decoction and PD-1 inhibitor could regulate the immune function of COPD by blocking the PD-1/PD-L1 axis,and Maxingloushi decoction had a better effect,providing a new treatment idea for further application of Traditional Chinese medicine to improve the immune function of COPD.更多还原