Cytokines Expression in Lymphocytes From Rats With Allergic Asthma Induced by Pleurotus sapidus besidiospores

Allergic asthma caused by mushroom spores (Pleurotus sapidus besidiospores) is a common health problem among mushroom-cultivating workers in China. An animal model of allergic asthma through the challenge of Pleurotus sapidus besidiospores in primed rats was developed for investigating the role of cytokines in the pathogenesis of the disease. In the study a series of related cytokines and their receptors, including their activity and mRNA levels of spleen lymphocytes isolated from asthmatic rats, were measured. Determined by 3H-TdR incorporation assay and NAG microcolorimetric assay, Con A-induced spleen lymphocyte proliferation and IL-2 activity in culture supernatants of spleen lymphocytes from 7-day challengedrats with allergic asthma increased significantly by 261% and 208%, respectively, as compared with those in the control. Cytokines and their receptor expression at mRNA levels were determined by RNA/cDNA hybridization, using (α-32P-dCTP radiolabeled cDNA probes for different cytokines and their receptors in vitro. The results showed that mRNA expression of IL-4, GM-CSF, IL-6, IL-2 and IL-2R, except IL-6R, in lymphocytes of 7-day-challenged asthma-suffering rats, increased significantly by 54%, 45%, 170%, 83% and 76%, respectively. In 2-day challenged-rats, mRNA levels of IL-4, IL-6 and IL-2 increased by 37%,58% and 125%, respectively, whereas mRNA leve1s of GM-CSF, IL-2R and IL-6R remained unchanged. Thus, the experimental results suggested a significant increase in TH2type cytokines in the pathogenesis of Pleurotus sapidus besidiospore-induced allergic asthma and IL-4 may play an essential role.

Effects of different concentrations of dexamethasone on pulmonary histology in rats with chronic asthma

Objective:To observe the intervention effects of different concentrations of dexamethasone on the general situation and pulmonary histology of chronic asthma rats,and to explore the suitable intragastric dose of dexamethasone in the rat model of chronic asthma.Methods:75 healthy male Sprague-Dawley(SD)rats were randomly divided into normal group,M group,dexamethasone low dose,middle dose and high dose group by random number table.The rat model of chronic asthma was established by sensitization and challenge of ovalbumin(OVA).From the 20th day of the experiment,dexamethasone groups were given intragastric administration of 0.0625mg/(kg·d),0.125mg/(kg·d)and 0.5mg/(kg·d),respectively.The general situation of rats was observed and recorded.The histological changes of rat lungs were observed by HE staining and Masson staining.Results:The degree of inflammatory infiltration of bronchus and surrounding tissue in the M group was significantly higher than that in the normal group(P<0.01).The thickness of airway wall and smooth muscle and the deposition of airway collagen in the M group were significantly higher than those in the normal group(P<0.01).After one week of intervention,the average daily food intake and body mass of rats in the high dose group were significantly lower than those in the M group(P<0.01),and the increase in body mass in the middle and low dose groups was significantly slower than that in the M group(P<0.01).After 2 weeks of intervention,the average daily food intake of rats in the middle and low dose groups was significantly lower than that in the M group(P<0.01).After 4 weeks of intervention,the average daily food intake in the middle and low dose groups was significantly lower than that in the low dose group(P<0.01).The inflammatory infiltration of lung tissue in the middle and low dose groups was significantly less than that in the model group,and the thickness of airway wall and smooth muscle and the deposition of airway collagen in the middle and low dose groups were significantly lower than those in the model group(P<0.01).There was no difference between the two groups(P>0.05).A large number of rats in the high dose group died after 5 times of intervention.Three rats died in the DEXM group at the 5th week of intervention,and there was no death in the other group.Conclusion:Both middle and low doses of dexamethasone can be used as the positive control dose in the experiment of chronic asthmatic rats,but the late side effects of low dose of dexamethasone are less,so the low dose of dexamethasone may be a safer and more suitable dose for the experiment of chronic asthmatic rats.

Effect of Yanghe Pingchuan granule on HMGB1-Related signalling pathway proteins and inflammatory factors in asthmatic rats

Objective To investigate the effect of Yanghe Pingchuan Granule(YHPCG)on the regulation of inflammatory factors and high-mobility histone(HMGB1),toll-like receptor 4(TLR4)and nuclear factor kappa B(NF-κB)signalling pathways in asthmatic rats.Methods Totally 60 SD male rats were divided into normal group,model group,dexamethasone group(0.25 g·kg^(-1)),low-dose YHPCGgroup(3.87g·kg^(-1)),medium-dose YHPCG group(7.74 g·kg^(-1)),and highdose YHPCG group(15.48 g·kg^(-1))according to random number method,10 rats in each group.

Detection of Circulating Autoantibodiestoβ_2-Adrenergic Receptors in Patients with Asthma

To investigate the roles of autoantibodies to β 2 adrenergic receptors in the pathogenesis of asthma, the positive chronotropic action of the β 2selective adrenergic agonist, clenbuterol, was investigated on cultured neonatal rat cardiomyocytes, firstly. Then we detected the autoantibodies to β 2adrenergic receptors through the sera of patients with asthma could inhibit the positive chronotropic action of clenbuterol. The results showed that all the sera of the patients with asthma (16 cases) had the autoantibodies to β 2 adrenergic receptors; in contrast, none of the control subjects (20 cases) had the autoantibodies to β 2 adrenergic receptors, and that the inhibitory autoantibodies were IgG type. This study suggests that the autoantibodies to β 2 adrenergic receptors may play an important role in the pathogenesis of asthma.

Proteomics Analysis of Component in Serum with Anti-asthma Activity Derived from Rats Treated by Acupuncture

Objective： To identify the differential proteins in the serum with anti-asthma activity of acupuncture to pinpoint the substance basis of anti-asthma activity of acupuncture by proteomics method. Methods： Two-dimensional electrophoresis of protein was employed to analyze the differential proteins in the serum with anti-asthma activity of acupuncture, and identify the differential proteins with mass spectrum technique. Results： In adrenaleetomy （ADX）-asthma acupuncture group and asthma acupuncture group, two-dimensional eleetrophoresis showed that there was an obvious fuscous point （MW=44.1kD, PI=5.1）, which was 60S ribosomal protein L13, or hypothetical protein FLJ32191 analyzed by mass spectrum technique, different from other groups. In ADX-asthma acupuncture group, there were two obviously fuseous points （MW=39.8kD, PI=4.9 and MW=39.5kD, PI=5.0）, which were zinc finger protein 208, and thyroid hormone receptor-associated protein analyzed by mass spectrum technique, and two special proteins （MW=17.3kD, PI〈4.5 and 16.9kD, PI〈4.5）, which were Cyclophilin A and zinc finger protein 91 analyzed by mass spectrum technique, different from other groups. Conclusion： There are multi-target effects regulating the whole body in acupuncture treating asthma relating to immunoloregulation, gene expression, and protein svnthesization. The effects of acupuncture on the resnonse nroteins need further research.

High-throughput 16S rDNA sequencing of the pulmonary microbiome of rats with allergic asthma

A decrease in microbial infection in adolescents is implicated with an increase in the incidence of asthma and allergic diseases in adulthood,indicating that the microbiome plays a critical role in asthma.However,the microbial composition of the lower respiratory tract remains unclear,hindering the further exploration of the pathogenesis of asthma.This study aims to explore the microbial distribution and composition in the lungs of normal rats and rats with allergic asthma via 16S rDNA sequencing.The DNA of the pulmonary microbiome was extracted from the left lungs collected from normal control group(NC),saline control group(SC),and allergic asthma group(AA)under aseptic conditions.After the 16s rDNA V4eV5 region was amplified,the products were sequenced using Illumina high-throughput technology and subjected to operational taxonomic unit(OTU)cluster and taxonomy analysis.The OTU values of AA increased significantly compared with those of NC and SC.Microbiome structure analysis showed that the dominant phylum of the pulmonary microbiome changed from Proteobacteria in NC to Firmicutes in AA.Linear discriminant analysis indicated that the key microbiomes involved in the three groups varied.

SDS-PAGE Analysis of Components in Serum with Anti-asthma Activity Derived from Rats Treated by Acupuncture

Objective： To explore a method of analyzing the components of anti-asthma activity in serum derived from rats treated with acupuncture. Methods： SDS-PAGE was used to analyze the diversity of the components of serum derived from rats treated with or without acupuncture treatment. Results： SDS-PAGE images displayed that there are one deeply dyed band, whose molecular weight is 62.1 kDa, in the component I from asthma acupuncture group and ADX-asthma acupuncture group, two deeply dyed bands, whose molecular weight are 36. lkDa and 12.3 kDa, respectively, in the component Ⅲ of ADX-asthma acupuncture group, and one dyed band, whose molecular weight is 34.6 kDa, in the segment Ⅲ of ADX-asthma acupuncture group. Conclusion： Acupuncture treatment could produce different components in the serum of rats, and may have bidirectional regulation on protein components in serum.

Effect of moxibustion at Feishu(BL 13)on airway inflammation in asthma model rats

Objective:To observe the effect of moxibustion at Feishu(BL 13)on related inflammatory cells and inflammatory factors in the bronchoalveolar lavage fluid of asthma model rats,and to explore the mechanism of moxibustion in treating asthma.Methods:A total of 48 Sprague-Dawley(SD)rats were randomly divided into a normal group,a model group,a moxibustion group and a medication group,with 12 rats in each group.Except for the normal group,the rats in the other three groups were subjected to ovalbumin sensitization to stimulate the asthma.At the same time,rats in the moxibustion group received moxibustion at bilateral Feishu(BL 13),and rats in the medication group received dexamethasone by intragastric administration.Rats in the normal and the model groups only received the same fixation and normal saline by intragastric administration.After the interventions,the inspiratory resistance,the expiratory resistance,and the pulmonary compliance were measured for rats in each group;the numbers of the inflammatory cells in the bronchoalveolar lavage fluid were counted;the levels of the involved inflammatory factors in bronchoalveolar lavage fluid were detected;the pathological morphologies of the lung tissues were observed under light microscope.Results:After modeling,compared with the normal group,the rats in the model group showed obvious asthma attack-like response,significantly increased inspiratory resistance and expiratory resistance(both P<0.01),and significantly reduced pulmonary compliance(P<0.01);thickened tracheal wall and the narrowed tracheal lumen observed under the light microscope;infiltration of inflammatory cells and increased eosinophils in and around the tracheal wall;significantly increased total number of inflammatory cells and proportion of eosinophils in the bronchoalveolar lavage fluid(all P<0.01);significantly reduced levels of interleukin(IL)-10,IL-12 and interferon(IFN)-γ(all P<0.01),and significantly increased levels of IL-4,IL-5 and tumor necrosis factor(TNF)-α(all P<0.01)in the bronchoalveolar lavage fluid.After intervention,compared with the model group,rats in the moxibustion and the medication groups showed significantly reduced asthma-like reaction,pathological morphological damage of lung tissue,inspiratory resistance and expiratory resistance(all P<0.01);significantly increased pulmonary compliance(P<0.01);significantly reduced total number of inflammatory cells,proportion of eosinophils,levels of IL-4,IL-5 and TNF-αin the bronchoalveolar lavage fluid(P<0.05 or P<0.01),while significantly increased IL-12 and IFN-γlevels(all P<0.01)in the bronchoalveolar lavage fluid;rats in the medication group also showed a significantly reduced IL-10 level in the bronchoalveolar lavage fluid(P<0.01);there was no statistically significant difference between the moxibustion and the medication groups(all P>0.05).Conclusion:Both moxibustion at Feishu(BL 13)and intragastric administration of dexamethasone can improve the asthma attack-like symptoms of ovalbumin-sensitized rats;regulating the inflammatory cell numbers and the inflammatory factor contents in the lung may be one mechanism of moxibustion in treating asthma.

Effect of adrenalectomy on rat epididymidis

AIM: To investigate the effect of adrenalectomy (ADX) on the epididymidis of Sprague-Dawley rats. METHODS: The histological, biochemical (cholesterol protein, zinc, copper, alkaline and acid phosphatase aryl sulphatase, lactic dehydrogenase and leucine amino peptidase) and hormonal (FSH, LH and testosterone) changes of caput and cauda epididymis in ADX rats were observed. RESULTS: Organ wet weight, histological studies and morphometric measurements indicated a cellular degeneration in caput and cauda epididymis of ADX rats. Serum testosterone level was significantly lower in ADX than in sham-operated rats, while the serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham-operated rats. Epididymal zinc level increased whereas copper level decreased in ADX rats compared to the sham-operated. CONCLUSION: Adrenalectomy leads to degeneration of caput and cauda epididymidis epithelial cells as a result of decreased supply of testosterone.

基于MAPK/NF-κB信号通路探讨调气止咳方对咳嗽变异性哮喘大鼠气道炎症的影响及机制

目的基于MAPK/NF-κB信号通路探讨调气止咳方(麻黄、苦杏仁、浙贝母、黄芩、瓜蒌子等)对咳嗽变异性哮喘大鼠气道炎症的影响及机制。方法采用卵清蛋白致敏复制咳嗽变异性哮喘大鼠模型。将SD大鼠随机分组为空白组、模型组、地塞米松组(0.5 mg·kg^(-1))、孟鲁司特组(1.0 mg·kg^(-1))及调气止咳方低、中、高剂量组(9.6、19.2、38.4 g·kg^(-1)),每组10只;灌胃给药,每日1次,连续14 d。观察大鼠一般状况并记录雾化后2 min内大鼠的咳嗽次数;采用HE染色、Masson染色法观察大鼠肺组织病理变化;ELISA法检测血清白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)含量;Western Blot法检测肺组织p38 MAPK、p-p38、NF-κB p65、p-p65蛋白表达水平;RT-qPCR法检测肺组织p38 MAPK、NF-κB p65 mRNA表达水平。结果与空白组比较,模型组大鼠的咳嗽次数显著增加(P<0.01),肺损伤病理评分及胶原纤维面积比显著升高(P<0.01);血清IL-1β、IL-6、TNF-α水平均显著升高(P<0.01);肺组织中p-p38、p-p65、p-p38/p38、p-p65/p65蛋白表达均显著上调(P<0.01),p38 MAPK、NF-κB p65 mRNA表达显著上调(P<0.01)。与模型组比较,各给药组大鼠的咳嗽次数显著减少(P<0.01),肺损伤病理评分及胶原纤维面积比显著降低(P<0.01),血清IL-1β、IL-6、TNF-α水平均显著降低(P<0.01),肺组织中p-p38、p-p65蛋白表达及p38 MAPK、NF-κB p65 mRNA表达均明显下调(P<0.05,P<0.01);调气止咳方高剂量组大鼠肺组织中p-p38/p38、p-p65/p65蛋白表达比值均显著下调(P<0.01)。与地塞米松组和孟鲁司特组比较,调气止咳方高剂量组大鼠的咳嗽次数明显减少(P<0.05);与地塞米松组比较,调气止咳方高剂量组大鼠的血清IL-1β、IL-6、TNF-α水平明显降低(P<0.01);与孟鲁司特组比较,调气止咳方高剂量组大鼠的血清IL-1β、TNF-α水平明显降低(P<0.05,P<0.01)。结论调气止咳方能够改善卵清蛋白诱导的咳嗽变异性哮喘大鼠的咳嗽症状,减轻气道炎症细胞浸润及气道重塑,降低炎症细胞因子IL-1β、IL-6、TNF-α水平,其机制可能与调控MAPK/NF-κB信号通路有关。

伏虎平咳饮对咳嗽变异性哮喘大鼠血清 IL-4、IL-10、TGF-β1影响的实验研究

目的:探讨伏虎平咳饮对咳嗽变异性哮喘大鼠的作用机制。方法:选取60只幼龄SD大鼠,根据体质量随机分为6组,正常对照组,模型对照组,伏虎平咳饮高、中、低剂量组及阳性对照组(布地奈德),给药2周后,ELISA测定各组大鼠血清白细胞介素-4(IL-4)、白细胞介素-10(IL-10)、转化生长因子-β1(TGF-β1)的含量。结果:与正常对照组比较,模型对照组大鼠血清IL-4水平升高,IL-10、TGF-β的含量降低,均有显著性差异(P<0.01);与模型对照组比较,伏虎平咳饮高剂量组大鼠血清IL-4含量明显降低(P<0.01),IL-10含量明显升高(P<0.01),TGF-β含量升高(P<0.05)。结论:伏虎平咳饮能够通过升高咳嗽变异性哮喘大鼠血清IL-10、TGF-β1水平,降低血清IL-4水平,减轻气道炎症反应。

基于上皮细胞-间充质转化的搜风愈喘方调控哮喘大鼠气道重塑的机制研究

目的探讨搜风愈喘方通过调控上皮细胞-间充质转化(EMT)抑制哮喘气道重塑的作用机制。方法将60只大鼠随机分为正常对照组、模型对照组、地塞米松组及搜风愈喘方高、中、低剂量组,每组10只。除正常对照组外,其余各组均采用卵清白蛋白(OVA)注射和雾化复制哮喘大鼠模型。各组大鼠给予相应药物灌胃21 d后处死大鼠,苏木素-伊红(HE)染色,观察肺组织病理形态学变化;透射电镜观察大鼠肺组织中上皮细胞超微结构情况;酶联免疫吸附测定法(ELISA)检测血清中的转化生长因子β1(TGF-β1)、白细胞介素17(IL-17)的含量;实时荧光定量聚合酶链式反应(RT-PCR)法检测肺组织匀浆中TGF-β1、α-平滑肌肌动蛋白(α-SMA)、E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)的mRNA表达。蛋白免疫印迹法(Western Blot)检测肺组织中TGF-β1、α-SMA、E-cadherin、N-cadherin蛋白表达水平。结果与正常对照组比较,模型对照组大鼠血清中TGF-β1、IL-17含量明显升高(P<0.01),肺组织中TGF-β1、α-SMA和N-cadherin mRNA和蛋白表达明显上调(P<0.01),E-cadherin mRNA和蛋白表达明显下调(P<0.01);HE染色显示气道壁及肺泡壁增厚,视野内可见大量炎症细胞浸润,炎症评分明显升高(P<0.01);透射电镜显示肺组织中上皮细胞水肿程度严重,胞内基质局部溶解、变淡,细胞器肿胀明显。与模型对照组比较,地塞米松组和搜风愈喘方各剂量组大鼠血清中TGF-β1、IL-17含量明显降低(P<0.05,P<0.01),TGF-β1、α-SMA和N-cadherin mRNA和蛋白表达明显下调(P<0.05,P<0.01),E-cadherin mRNA和蛋白表达明显上调(P<0.05,P<0.01);肺组织炎症细胞浸润减轻,支气管壁及肺泡壁增厚程度减轻,地塞米松组和搜风愈喘方高剂量组炎症评分降低(P<0.05);超微结构显示上皮细胞水肿程度明显减轻,细胞膜完整,胞内基质均匀,细胞器大多轻微肿胀。结论搜风愈喘方可降低TGF-β1水平,从而改善肺组织EMT,达到防治哮喘气道重塑的目的。

黄芩总黄酮对支气管哮喘大鼠的治疗作用及miR-133a-3p/NOX4/NLRP3信号轴表达的影响

目的黄芩总黄酮对支气管哮喘大鼠的治疗作用及miR-133a-3p/NOX4/NLRP3信号轴表达的影响。方法清洁级SD大鼠100只分为正常对照组、模型组、地塞米松组(200 mg/kg)、黄芩总黄酮低剂量组(100 mg/kg)、黄芩总黄酮高剂量组(200 mg/kg),每组20只。除正常对照组外,模型组、地塞米松组、黄芩总黄酮低高剂量组通过卵清蛋白(OVA)建立支气管哮喘模型,并予以相应药物干预。实验结束后,检测肺泡灌洗液淋巴细胞百分比、中性粒细胞百分比、白细胞计数,以及肺/体比值、肺损伤评分、动脉血氧分压(PaO_(2))水平;反逆转录聚合酶链反应(RT-PCR)及蛋白印迹法检测肺支气管组织微小RNA-133a-3p(miR-133a-3p)、NADPH氧化酶4(NOX4)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)水平。结果与正常对照组比较,模型组肺泡灌洗液淋巴细胞百分比、中性粒细胞百分比、白细胞计数、肺/体比值、肺损伤评分,以及支气管组织中NOX4、NLRP3蛋白和mRNA表达水平升高,PaO_(2)水平、支气管组织中miR-133a-3p表达水平降低,差异有统计学意义(P<0.05);与模型组比较,地塞米松组、黄芩总黄酮低剂量组和黄芩总黄酮高剂量组肺泡灌洗液淋巴细胞百分比、中性粒细胞百分比、白细胞计数、肺/体比值、肺损伤评分,以及支气管组织中NOX4、NLRP3蛋白和mRNA表达水平降低,PaO_(2)水平、支气管组织中miR-133a-3p表达水平升高,差异有统计学意义(P<0.05)。与地塞米松组比较,黄芩总黄酮低剂量组、黄芩总黄酮高剂量组肺泡灌洗液淋巴细胞百分比、中性粒细胞百分比、白细胞计数、肺/体比值、肺损伤评分,以及支气管组织中NOX4、NLRP3蛋白和mRNA表达水平升高,PaO_(2)水平、支气管组织中miR-133a-3p表达水平降低,差异有统计学意义(P<0.05)。与黄芩总黄酮低剂量组比较,黄芩总黄酮高剂量组肺泡灌洗液淋巴细胞百分比、中性粒细胞百分比、白细胞计数、肺/体比值、肺损伤评分,支气管组织中NOX4、NLRP3蛋白和mRNA表达水平升高,PaO_(2)水平、支气管组织中miR-133a-3p表达水平降低,差异有统计学意义(P<0.05)。结论黄芩总黄酮对大鼠支气管哮喘具有明显治疗作用,其机制可能与黄芩总黄酮促进miR-133a-3p的表达进而抑制NOX4/NLRP3信号轴的激活相关。

祛风止痉胶囊对支气管哮喘大鼠气道炎症及TRPV1通路的影响

目的:观察祛风止痉胶囊对支气管哮喘大鼠气道平滑肌瞬时受体电位香草酸亚型1(TRPV1)表达以及外周血白介素(IL)-4、IL-13、IL-15、IL-17水平的影响。方法:将60只健康雄性大鼠随机分为正常组、阳性对照组、模型组与低剂量组、中剂量组、高剂量组,每组10只。除正常组外,其余各组大鼠于实验的第1、15天给予10%OVA腹腔注射1 ml以致敏,第15天将致敏的大鼠给予1%OVA溶液进行超声雾化建立哮喘模型。连续干预10 d。采用ELISA法测定大鼠外周血IL-4、IL-13、IL-5、IL-17水平,采用荧光定量PCR及Western blot测定大鼠肺组织TRPV1 mRNA和蛋白的表达。结果:低剂量、中剂量组、高剂量组IL-4、IL-13、IL-5、IL-17水平低于模型组(均P<0.05),高剂量组IL-4、IL-13、IL-5、IL-17水平低于阳性对照组(均P<0.05)。模型组及阳性对照组的TRPV1 mRNA和蛋白表达高于正常组(均P<0.05),模型组的TRPV1 mRNA和蛋白表达高于阳性对照组、低剂量组、中剂量组、高剂量组(均P<0.05)。结论:祛风止痉胶囊可降低哮喘大鼠气道炎症因子水平,抑制哮喘大鼠气道平滑肌TRPV1表达。

基于IL-6/STAT3和IL-2/STAT5信号通路探讨伏九贴敷药物调控Th17/Treg免疫平衡的抗哮喘作用机制

目的考察伏九贴敷药物(白芥子、甘遂、延胡索、细辛组成)通过IL-6/信号转导器和转录激活因子3(STAT3)、IL-2/信号转导器和转录激活因子5(STAT5)信号通路对哮喘大鼠CD4+T辅助细胞17(Th17)/CD4+CD25+调节性T细胞(Treg)平衡的调节作用,揭示其抗哮喘的作用机制。方法采用卵白蛋白(OVA)和氢氧化铝混合液致敏激发造模,然后于大鼠大椎穴、肺俞穴和肾俞穴敷上伏九贴敷药物,每次贴敷4 h,隔日1次,共给药7次。采用免疫组化检测肺组织Th17特异细胞因子IL-17、Treg转录因子Foxp3的阳性表达;流式细胞术检测外周血中Th17、Treg细胞比例;免疫蛋白印迹法检测肺组织IL-6/STAT3和IL-2/STAT5通路相关蛋白的表达。结果与模型组比较,伏九贴敷组大鼠肺中IL-17的阳性表达量明显减少(P<0.01),Foxp3的阳性表达明显增加(P<0.05);同时IL-6蛋白和STAT3磷酸化蛋白表达水平明显降低(P<0.01),而IL-2蛋白和STAT5磷酸化蛋白表达水平明显升高(P<0.01),但总STAT3和STAT5蛋白表达均没有明显变化(P>0.05)。此外,伏九贴敷组大鼠外周血中Th17细胞比例低于模型组,而Treg细胞比例高于模型组,差异均具有统计学意义(P<0.01)。结论哮喘大鼠存在Th17/Treg免疫失衡,伏九贴敷药物可通过抑制哮喘大鼠IL-6表达,下调磷酸化STAT3的表达,降低产生IL-17的Th17细胞水平;同时增加IL-2表达,介导STAT5磷酸化,升高表达Foxp3的Treg细胞水平,促进Th17/Treg趋于平衡,抑制大鼠哮喘免疫应答,从而发挥抗哮喘效应。

麻杏石甘汤对咳嗽变异型哮喘大鼠IL-6/STAT3信号通路及TRPV1感受器的影响

【目的】探讨麻杏石甘汤对咳嗽变异型哮喘(CVA)大鼠的治疗作用及机制。【方法】将60只大鼠随机分为正常组,模型组,麻杏石甘汤低、高剂量组,麻杏石甘汤高剂量+信号转导和转录激活因子3(STAT3)激活剂Colivelin(Col)组,每组12只。除正常组外,其他各组大鼠采用腹腔注射卵清蛋白结合艾条熏蒸法构建CVA模型。对应治疗后,观察大鼠体征和咳嗽次数,肺功能仪检测气道阻力(RE),Diff-Quik染色计数嗜酸性粒细胞(EOS),苏木素-伊红(HE)染色法观察肺、支气管组织病理学特征,酶联免疫吸附分析(ELISA)检测肺组织单核细胞趋化蛋白1(MCP-1)、肿瘤坏死因子α(TNF-α)含量,Western Blot法检测肺组织白细胞介素6(IL-6)、STAT3、瞬时受体电位香草酸亚型1(TRPV1)蛋白表达水平。【结果】与正常组比较,模型组大鼠出现明显哮喘症状,肺组织可见炎性细胞浸润严重,支气管上皮细胞坏死、纤毛粘连、黏液多,RE,EOS数目,MCP-1、TNF-α含量,以及IL-6、STAT3、TRPV1蛋白表达水平均升高(P<0.05);与模型组比较,麻杏石甘汤低、高剂量组大鼠哮喘症状明显改善,肺及支气管损伤减轻,RE,EOS数目,MCP-1、TNF-α含量以及IL-6、STAT3、TRPV1蛋白表达水平呈剂量依赖性降低(P<0.05);与麻杏石甘汤高剂量组比较,麻杏石甘汤高剂量+Col组大鼠哮喘加重,肺及支气管损伤加重,RE,EOS数目,MCP-1、TNF-α含量以及IL-6、STAT3、TRPV1蛋白表达水平升高(P<0.05)。【结论】麻杏石甘汤可有效改善CVA大鼠症状,其机制与抑制IL-6/STAT3信号通路及TRPV1高表达有关。

金合欢素对哮喘幼年大鼠的改善作用及机制

目的基于沉默信息调节因子1(SIRT1)/AMP活化蛋白激酶(AMPK)信号通路探讨金合欢素对哮喘幼年大鼠的改善作用及机制。方法将幼年SD大鼠随机分为对照组、哮喘组、金合欢素组(13.33 mg/kg,灌胃)、SIRT抑制剂EX-527组(1 mg/kg,腹腔注射)、金合欢素+EX-527组(13.33 mg/kg金合欢素,灌胃+1 mg/kg EX-527,腹腔注射),每组12只(雌雄各半)。除对照组外,其余各组大鼠以腹腔注射卵清蛋白致敏+雾化吸入卵清蛋白增敏的方式复制哮喘模型。造模后,各药物组大鼠灌胃或(和)腹腔注射相应药液,每天1次,持续2周。末次给药后,检测其支气管肺泡灌洗液(BALF)中细胞总数、嗜酸性粒细胞数占比和白细胞介素5(IL-5)、IL-4、肿瘤坏死因子α(TNF-α)水平,观察肺组织病理形态学变化和杯状细胞异常增生情况,检测肺组织中丙二醛(MDA)、超氧化物歧化酶(SOD)水平,以及SIRT1、AMPK和过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1α)蛋白的表达情况。结果与对照组比较,哮喘组大鼠肺组织中有大量的炎症细胞浸润和明显的杯状细胞异常增生;BALF中细胞总数、嗜酸性粒细胞数占比和IL-5、IL-4、TNF-α水平,肺组织过碘酸希夫染色(PAS)评分,以及肺组织中MDA水平均显著升高(P<0.05);肺组织中SOD水平、SIRT1、PGC-1α蛋白的表达水平和AMPK蛋白的磷酸化水平均显著降低(P<0.05)。与哮喘组比较,金合欢素组大鼠肺组织的病理改变和杯状细胞异常增生均有所减轻,各定量指标均显著改善(P<0.05),而EX-527组大鼠肺组织的病理改变和杯状细胞异常增生均有所加重,各定量指标均显著恶化(P<0.05);联用EX-527可显著逆转金合欢素对哮喘幼年大鼠氧化应激及气道炎症的改善作用。结论金合欢素可抑制哮喘幼年大鼠的氧化应激和气道炎症,上述作用可能与激活SIRT1/AMPK信号通路有关。

穗花杉双黄酮调节cGAS-STING信号通路对哮喘幼年大鼠气道炎症的影响

目的探讨穗花杉双黄酮(AF)对哮喘幼年大鼠气道炎症的影响并初步探讨其机制。方法采用腹腔注射联合雾化吸入卵白蛋白(OVA)法诱导幼年SD大鼠建立哮喘模型,将大鼠随机分为哮喘模型(M)组、地塞米松(DXMS)组、AF低(AF L)、中(AF M)、高(AF H)剂量组和正常对照(CT)组。给药结束后,无创肺功能仪检测气道反应性,通过吉姆萨(Giemsa)染色分析支气管肺泡灌洗液(BALF)中炎性细胞类型并计数,使用苏木素-伊红(HE)染色评价肺组织和支气管组织病理特征,酶联免疫吸附剂实验(ELISA)检测血清炎症因子的含量,Western blot检测肺组织GMP-AMP合酶(cGAS)、干扰素基因刺激物(STING)、磷酸化-干扰素调节因子3(p-IRF3)和干扰素调节因子3(IRF3)蛋白表达。结果与CT组相比,M组幼年大鼠肺组织和支气管组织可见明显病理损伤,气道反应性、肺组织、支气管组织病理评分、BALF中炎性细胞总数及单核细胞、嗜酸性粒细胞、中性粒细胞和淋巴细胞的数量显著增加、血清炎症因子以及肺组织c GAS、STING、p-IRF3/IRF3蛋白表达显著增加(P<0.05);与M组相比,DXMS和高剂量AF治疗哮喘大鼠后肺、支气管组织病理损伤缓解,气道反应性、肺组织、支气管组织病理评分、BALF中炎性细胞总数显著降低、血清炎症因子以及肺组织c GAS、STING、p-IRF3/IRF3蛋白表达明显降低(P<0.05)。结论AF可缓解哮喘幼年大鼠的气道炎症,其可能是通过抑制cGAS-STING信号通路实现的。

平喘宁调节IRE-1α-XBP-1s信号轴干预哮喘大鼠气道炎症的机制研究

目的:探究平喘宁对哮喘大鼠气道炎症的防治作用及机制。方法:将105只雄性SD大鼠按随机数字表法分为正常组、模型组、地塞米松组、桂龙咳喘宁组、平喘宁高剂量组、平喘宁中剂量组、平喘宁低剂量组,每组15只。以卵清蛋白联合氢氧化铝复制大鼠哮喘模型,造模21 d后,各给药组分别灌胃给予相应药物,正常组、模型组灌胃给予等体积的生理盐水,1次/d,连续4周。4周后,在进行哮喘激发试验后2 h内解剖大鼠并取出肺组织,收集支气管肺泡灌洗液(BALF)。HE染色观察肺组织中平滑肌厚度、炎症细胞浸润程度等相应病理的改变;ELISA法检测BALF中IL-5、IL-17水平;RT-qPCR法检测肺组织IRE-1αmRNA、XBP-1s mRNA、NF-κB p65 mRNA、Hgsnat mRNA、Pdgfrb mRNA、Scara3 mRNA相对表达量;Western blotting法检测肺组织中IRE-1α、XBP-1s、NF-κB p65蛋白相对表达量。结果:HE染色结果显示,与模型组比较,各给药组(平喘宁低剂量组、平喘宁中剂量组、平喘宁高剂量组、地塞米松组、桂龙咳喘宁组)大鼠肺组织病理情况都有不同程度的缓解。ELISA结果显示,与正常组比较,模型组大鼠BALF中IL-5、IL-17水平均明显升高(P<0.01);与模型组比较,各给药组大鼠BALF中IL-5、IL-17水平均明显降低(P<0.01),且平喘宁具有剂量依赖性;平喘宁低、中剂量组大鼠BALF中IL-5、IL-17水平均高于地塞米松组和桂龙咳喘宁组(P<0.01);平喘宁高剂量组大鼠BALF中IL-5水平明显高于地塞米松组和桂龙咳喘宁组(P<0.01),而IL-17水平与地塞米松组和桂龙咳喘宁组比较,差异无统计学意义(P>0.05)。RT-qPCR结果显示,与正常组比较,模型组大鼠肺组织IRE-1αmRNA、XBP-1s mRNA、NF-κB p65 mRNA相对表达量均明显升高(P<0.01),Hgsnat mRNA、Pdgfrb mRNA、Scara3 mRNA相对表达量均明显降低(P<0.01);与模型组比较,各给药组大鼠肺组织IRE-1αmRNA、XBP-1s mRNA、NF-κB p65 mRNA相对表达量均明显降低(P<0.01),Hgsnat mRNA、Pdgfrb mRNA、Scara3 mRNA相对表达量均明显升高(P<0.05或P<0.01);平喘宁低剂量组大鼠肺组织IRE-1αmRNA、XBP-1s mRNA、NF-κB p65 mRNA相对表达量均明显高于地塞米松组和桂龙咳喘宁组(P<0.01),Hgsnat mRNA、Pdgfrb mRNA、Scara3 mRNA相对表达量均明显低于地塞米松组和桂龙咳喘宁组(P<0.01);平喘宁中剂量组大鼠肺组织XBP-1s mRNA、NF-κB p65 mRNA相对表达量均明显高于地塞米松组和桂龙咳喘宁组(P<0.01),Hgsnat mRNA、Pdgfrb mRNA、Scara3 mRNA相对表达量均明显低于地塞米松组和桂龙咳喘宁组(P<0.05或P<0.01),而IRE-1αmRNA相对表达量与地塞米松组和桂龙咳喘宁组比较,差异无统计学意义(P>0.05);平喘宁高剂量组大鼠肺组织IRE-1αmRNA相对表达量均明显低于地塞米松组和桂龙咳喘宁组(P<0.01),而Hgsnat mRNA、Pdgfrb mRNA相对表达量均明显高于地塞米松组(P<0.01);平喘宁高剂量组Hgsnat mRNA、Pdgfrb mRNA相对表达量与桂龙咳喘宁组比较,差异无统计学意义(P>0.05),XBP-1s mRNA、NF-κB p65 mRNA、Scara3 mRNA与地塞米松组和桂龙咳喘宁组比较,差异无统计学意义(P>0.05)。Western blotting结果显示,与正常组比较,模型组大鼠肺组织IRE-1α、XBP-1s、NF-κB p65蛋白相对表达量均明显升高(P<0.01);与模型组比较,各给药组大鼠肺组织IRE-1α、XBP-1s、NF-κB p65蛋白相对表达量均明显降低(P<0.01);平喘宁低剂量组大鼠肺组织IRE-1α、XBP-1s、NF-κB p65蛋白相对表达量均明显高于地塞米松组和桂龙咳喘宁组(P<0.01);平喘宁中剂量组大鼠肺组织XBP-1s、NF-κB p65蛋白相对表达量均明显高于地塞米松组和桂龙咳喘宁组(P<0.05或P<0.01),IRE-1α蛋白相对表达量明显高于桂龙咳喘宁组(P<0.05),而IRE-1α蛋白相对表达量与地塞米松组比较,差异无统计学意义(P>0.05);平喘宁高剂量组大鼠肺组织IRE-1α、XBP-1s、NF-κB p65蛋白相对表达量与地塞米松组和桂龙咳喘宁组比较,差异无统计学意义(P>0.05)。结论:平喘宁可通过调节IRE-1α-XBP-1s信号轴改善OVA诱导的哮喘大鼠气道炎症性损伤。

基于JAK/STAT通路探讨益气温阳护卫法干预支气管哮喘大鼠防御素水平的机制

目的:基于Janus激酶(Janus kinase,JAK)/信号转导子和转录激活子(signal transducer and activator of transcription,STAT)通路探讨益气温阳护卫法干预支气管哮喘大鼠防御素水平的机制。方法:SPF级雄性SD大鼠50只,随机分为正常组、模型组及益气温阳护卫汤低剂量组(5 g·kg^(-1))、中剂量组(10 g·kg^(-1))、高剂量组(20 g·kg^(-1)),每组10只。除正常组外,其余各组均采用腹腔注射抗原液(卵清蛋白+氢氧化铝)结合雾化(卵清蛋白)激发的方式建立支气管哮喘大鼠模型。造模第8天,各药物组开始灌胃相应剂量的益气温阳护卫汤,正常组、模型组灌胃等体积生理盐水,每天2次,持续4周。RT-PCR检测肺组织β-防御素基因表达水平;ELISA检测血清干扰素-γ(interferon gamma,IFN-γ)、白细胞介素-4(interleukin-4,IL-4)水平;RT-PCR检测肺组织JAK mRNA、STAT6 mRNA表达水平;瑞氏染色法检测肺泡灌洗液中免疫细胞水平。结果:(1)与正常组比较,模型组和各药物组大鼠肺组织β-防御素基因表达水平升高(P<0.05);与模型组比较,各药物组β-防御素基因表达水平降低(P<0.05);各药物组β-防御素基因表达水平比较,差异无统计学意义(P>0.05)。(2)与正常组比较,模型组和各药物组大鼠血清IFN-γ水平降低(P<0.05);与模型组比较,各药物组血清IFN-γ水平升高(P<0.05);与正常组比较,模型组和各药物组大鼠血清IL-4水平升高(P<0.05);与模型组比较,各药物组血清IL-4水平降低(P<0.05);各药物组血清IFN-γ、IL-4水平比较,差异无统计学意义(P>0.05)。(3)与正常组比较,模型组和各药物组大鼠肺组织JAK mRNA、STAT6 mRNA表达水平升高(P<0.05);与模型组比较,各药物组肺组织JAK mRNA、STAT6 mRNA表达水平降低(P<0.05);各药物组肺组织JAK mRNA、STAT6 mRNA表达水平比较,差异无统计学意义(P>0.05)。(4)与正常组比较,模型组和各药物组大鼠肺泡灌洗液中,中性粒细胞、巨噬细胞、嗜酸性粒细胞和淋巴细胞水平升高(P<0.05);与模型组比较,各药物组中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞水平降低(P<0.05);各药物组中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞水平比较,差异无统计学意义(P>0.05)。结论:益气温阳护卫法可减轻支气管哮喘大鼠气道炎症反应,降低β-防御素基因表达水平,其机制可能与调节JAK/STAT信号通路有关。