基于TLR4/NF-κB通路调补肺肾法干预细胞自噬对COPD肺血管重塑的影响和机制

The impact and mechanism of pulmonary and renal replenishing method intervention on cell autophagy in COPD-related pulmonary vascular remodeling via TLR4/NF-κB pathway

目的基于Toll样受体4(TLR4)/核转录因子-κB(NF-κB)通路研究调补肺肾法干预细胞自噬对慢性阻塞性肺疾病(COPD)肺血管重塑的影响和机制。方法取SD大鼠采用香烟烟雾暴露结合反复细菌感染的方案建立COPD模型,随机分为3组,模型组、补肺益肾方(3.7 g/kg)组、补肺益肾方(3.7 g/kg)+脂多糖(LPS)(TLR4激活剂,15 mg/kg)组,每组12只,另取12只大鼠正常呼吸并气管滴注等剂量生理盐水作为对照组,经补肺益肾方、LPS对大鼠分组干预后,检测各组大鼠肺功能指标:潮气量(TV)、呼气峰流速(PEF)、第0.3秒用力呼气容积(FEV0.3)/用力肺活量(FVC)。以HE染色检测各组大鼠肺组织病理形态和肺血管重塑,比较其管壁厚度(WT)占血管直径(VD)百分比WT/VD(%)、管腔面积(LA)占血管总面积(TA)百分比LA/TA(%)。以免疫荧光染色检测各组大鼠肺组织内肺血管内皮标记物CD34表达。以酶标仪检测各组大鼠肺泡灌洗液(BALF)及血清促炎因子:肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-17水平。以免疫印迹法检测各组大鼠肺组织自噬及TLR4/NF-κB信号通路相关蛋白表达。结果与对照组相比,模型组大鼠肺组织呈现明显病理损伤及肺血管重塑症状,TV、PEF、FEV0.3/FVC、LA/TA、肺组织Beclin-1蛋白表达与LC3Ⅱ/LC3Ⅰ显著降低(P<0.05),WT/VD、CD34相对阳性表达、BALF及血清促炎因子TNF-α与IL-17水平、肺组织TLR4蛋白表达与p-NF-κB p65/NF-κB p65显著升高(P<0.05)。与模型组相比,补肺益肾方组大鼠肺组织损伤及肺血管重塑症状减轻,TV、PEF、FEV0.3/FVC、LA/TA、肺组织Beclin-1蛋白表达与LC3Ⅱ/LC3Ⅰ升高(P<0.05),WT/VD、CD34相对阳性表达、BALF及血清促炎因子TNF-α与IL-17水平、肺组织TLR4蛋白表达与p-NF-κB p65/NF-κB p65降低(P<0.05)。与补肺益肾方组相比,补肺益肾方+LPS组大鼠肺组织损伤及肺血管重塑症状加重,TV、PEF、FEV0.3/FVC、LA/TA、肺组织Beclin-1蛋白表达与LC3Ⅱ/LC3Ⅰ降低(P<0.05),WT/VD、CD34相对阳性表达、BALF及血清促炎因子TNF-α与IL-17水平、肺组织TLR4蛋白表达与p-NF-κB p65/NF-κB p65升高(P<0.05)。结论补肺益肾方可通过抑制TLR4/NF-κB信号而抑制COPD体内炎症,并增强自噬,进而减轻大鼠肺组织病理损伤和肺血管重塑,改善其肺功能。

Objective To investigate the effects and mechanisms of the pulmonary and renal replenishing method(PRRM)intervention on cell autophagy in chronic obstructive pulmonary disease(COPD)-related pulmonary vascular remodeling,focusing on the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)pathway.Methods A COPD model was established in SD rats through a combination of cigarette smoke exposure and repeated bacterial infections.Rats were randomly divided into three groups:Model group,PRRM group(3.7 g/kg),and PRRM+Lipopolysaccharide(LPS)(TLR4 activator,15 mg/kg)group.Another 12 rats were maintained with normal breathing and received tracheal instillation of an equivalent volume of saline as the control group.After intervention with PRRM and LPS,lung function indicators[tidal volume(TV),peak expiratory flow(PEF),forced expiratory volume in 0.3 seconds(FEV0.3)/forced vital capacity(FVC)]were measured.Histopathological examination using Hematoxylin and Eosin(HE)staining assessed lung tissue morphology and pulmonary vascular remodeling,including wall thickness(WT)percentage of vessel diameter(VD)(WT/VD%)and lumen area(LA)percentage of total vessel area(TA)(LA/TA%).Immunofluorescence staining measured the expression of the pulmonary vascular endothelial marker CD34.Enzyme-linked immunosorbent assay(ELISA)measured levels of pro-inflammatory factors,tumor necrosis factor-α(TNF-α),and interleukin(IL)-17 in bronchoalveolar lavage fluid(BALF)and serum.Immunoblotting was employed to evaluate the expression of autophagy-related proteins and proteins in the TLR4/NF-κB signaling pathway in lung tissues.Results Compared to the control group,rats in the Model group exhibited significant pathological damage and symptoms of pulmonary vascular remodeling,decreased TV,PEF,FEV0.3/FVC,LA/TA,and reduced Beclin-1 protein expression,as well as a decreased LC3Ⅱ/LC3Ⅰratio(P<0.05).Furthermore,WT/VD%,CD34 positive expression,levels of TNF-αand IL-17 in BALF and serum,and the expression of TLR4 protein and p-NF-κB p65/NF-κB p65 in lung tissue were significantly elevated(P<0.05).In comparison to the Model group,rats in the PRRM group showed alleviated lung tissue damage and pulmonary vascular remodeling,increased TV,PEF,FEV0.3/FVC,LA/TA,and elevated Beclin-1 protein expression with an increased LC3Ⅱ/LC3Ⅰratio(P<0.05).Moreover,WT/VD%,CD34 positive expression,levels of TNF-αand IL-17 in BALF and serum,and the expression of TLR4 protein and p-NF-κB p65/NF-κB p65 in lung tissue were significantly reduced(P<0.05).Comparing the PRRM group to the PRRM+LPS group,rats in the PRRM+LPS group showed aggravated lung tissue damage and pulmonary vascular remodeling,decreased TV,PEF,FEV0.3/FVC,LA/TA,and decreased Beclin-1 protein expression with a reduced LC3Ⅱ/LC3Ⅰratio(P<0.05).Additionally,WT/VD%,CD34 positive expression,levels of TNF-αand IL-17 in BALF and serum,and the expression of TLR4 protein and p-NF-κB p65/NF-κB p65 in lung tissue were significantly increased(P<0.05).Conclusion PRRM can inhibit inflammation and enhance autophagy in the COPD rat model by suppressing the TLR4/NF-κB signaling pathway,thereby alleviating pathological damage and pulmonary vascular remodeling and improving lung function.