当归对阴虚哮喘小鼠气道黏液高分泌及TNF-α/NF-κB信号通路的影响*

目的: 探讨当归对阴虚哮喘小鼠气道黏液高分泌及TNF-α/NF-κB信号通路的影响。方法: 取KM小鼠随机分为空白对照组、模型对照组、氨溴索组、当归低、中、高剂量(2、4、8 g/kg)组(n=12),采用卵蛋白与甲状腺片复制阴虚哮喘模型,观测当归对小鼠哮喘症状、IgE、TNF-α以及肺组织Muc5ac与NF-κB表达的影响。结果: 2、4、8 g/kg当归能明显缓解阴虚哮喘小鼠的哮喘症状,降低血清IgE与BALF中TNF-α水平,抑制肺组织Muc5ac与NF-κB的过度表达。结论: 当归具有明显的平喘作用,抑制TNF-α/NF-κB信号通路而缓解气道黏液高分泌是其平喘的作用机制之一。     

丹参对非酒精性脂肪肝Th17细胞及相关细胞因子的影响*

目的:研究丹参对非酒精性脂肪肝(NAFLD)大鼠Th17细胞及相关细胞因子的影响,为临床上丹参治疗NAFLD提供实验依据。方法:SPF级别SD大鼠32只,随机分为模型组、空白对照组、葛根对照组、丹参组,每组8只。模型组给予高脂饲料喂养4周以建立NAFLD模型;丹参治疗组及葛根对照组大鼠给予6.25g·kg^-1·d^-1的浓缩液每日1次灌胃;除了空白对照组外,其余各组大鼠均给予高脂饲料喂饲4周。实验完全结束(第4周),各组大鼠禁食12～14 h,禁水2 h后,采血,收集肝脏组织,检测各组大鼠血脂(TC、TG、LDL-C、HDL-C)水平及肝功能(AST、ALT)、肝脏指数,观察肝组织病理学改变。流式细胞仪检测外周血Th17、Treg细胞含量。通过ELISA法检测血清IL-6、IL-17、TNF-α水平;RT-PCR法检测肝脏组织RORγt基因表达。结果:连续4周喂养高脂饮食后,模型组大鼠肝脏发生脂肪变性,有大量炎症细胞浸润。与模型组相比,丹参治疗组、葛根对照组大鼠TC、TG、LDL-C、ALT、AST水平及肝脏指数明显低于模型组(P＜0.05),HDL-C水平明显高于模型组(P＜0.05)成功复制NAFLD大鼠模型。丹参治疗组大鼠外周血Th17细胞含量、IL-6、IL-17水平、大鼠RORγt基因表达量显著低于模型组及葛根对照组(P＜0.05);TNF-α水平低于模型组及葛根对照组;Treg细胞含量高于模型组(P＜0.05)及葛根对照组(P＞ 0.05);Treg/ Th17显著高于模型组及葛根对照组 (P＜0.05)。结论:丹参通过降低血清中IL-6、IL-17、TNF-α水平,抑制RORγt基因表达,降低外周血Th17细胞含量,升高Treg细胞含量,调整Th17/Treg平衡,从而抑制NAFLD发生发展。     

丁苯酞对哮喘小鼠气道黏液高分泌及IL-13、TNF-α的影响*

目的: 研究丁苯酞对哮喘小鼠气道黏液高分泌及白介素-13(IL-13)、肿瘤坏死因子-α(TNF-α)的影响。方法: 小鼠随机分为空白对照组、模型对照组、阳性对照组和丁苯酞高、中、低剂量(100、50、25 mg/kg)组(n=12)。实验第1日、8日、15日通过注射卵清白蛋白(OVA)致敏,第22日吸入OVA连续激发5周复制哮喘模型,同时在激发前给予丁苯酞20 mg/kg进行干预,观测哮喘行为学、气道杯状细胞及黏蛋白5ac(Muc5ac)的分泌,测定支气管肺泡灌洗液(BALF)粘度,采用ELISA法测定BALF中Muc5ac、IL-13及TNF-α的水平。结果: 与空白对照组比较,模型对照组小鼠打喷嚏、抓鼻及哮喘的程度显著加重(P＜0.01),小鼠气道上皮杯状细胞增生及Muc5ac的分泌显著增加(P＜0.01),BALF的粘度及其中的Muc5ac、IL-13和TNF-α的含量显著升高(P＜0.01);与模型对照组比较,25、50、100 mg/kg丁苯酞干预后哮喘行为学评分明显下降(P＜0.01);小鼠气道上皮杯状细胞增生、Muc5ac的分泌、BALF的粘度及其中的Muc5ac、IL-13和TNF-α的含量均明显降低(P＜0.05, 0.01)。结论: 丁苯酞具有抑制哮喘气道黏液高分泌而平喘的作用,缓解IL-13、TNF-α异常高表达是其抑制气道黏液高分泌的作用机制之一。     

21例侵袭性肺曲霉病患者Th以及Treg细胞的检测

目的：分析侵袭性肺曲霉病患者辅助性T细胞（Th）以及调节性T细胞（Treg）在外周血中单个核细胞中的表达情况及其临床相关性,探讨Th和Treg细胞介导的免疫反应在侵袭性肺曲霉病中的作用。方法分离21例侵袭性肺曲霉病患者及19例健康人外周血的单个核细胞,采用流式细胞术分析Th1、Th2、Th17、Treg细胞群的表达情况,Real-timePCR方法检测相关转录因子T-bet、GATA-3、RORγt以及Foxp3的表达,ELISA法检测血清中相关细胞因子IFN-γ、IL-4、IL-17以及TGF-β的表达。结果与健康人对照组相比,侵袭性肺曲霉病患者Th1细胞以及Treg细胞占CD4＋T细胞的比例较之对照组明显降低;Th1、Th17、Treg细胞相关转录因子T-bet、RORγt、Foxp3以及相关细胞因子IFN-γ、IL-17A、TGF-β与对照组相比表达明显降低。结论IPA患者的Th1、Th17以及Treg细胞所介导的免疫反应受抑制。     

IL-17A对慢性阻塞性肺疾病的干预作用及其机制*

目的:探讨白细胞介素-17A(IL-17A)对慢性阻塞性肺疾病(COPD)的干预作用及其机制。方法:C57BL/6小鼠随机分为野生型空白对照组、野生型COPD组和IL-7A敲除COPD组,每组20只。野生型空白对照组小鼠不做任何处理,其余两组小鼠暴露于香烟烟雾(1支/次,4次/日,每次45 min,每次间隔时间为1 h,总干预时间为90 d)制作COPD模型。干预结束24 h后,利用动物肺功能检测系统测定小鼠肺功能。收集小鼠支气管肺泡灌洗液(BALF),测定BALF细胞计数和分类。收集小鼠肺组织,采用流式细胞法测定气道上皮IL-17A表达水平,采用酶联免疫吸附法测定肺组织炎症因子水平。采用蛋白免疫印迹法测定小鼠肺组织JNK/AP1信号通路蛋白表达水平。结果:与野生型空白对照组小鼠比较,野生型COPD组小鼠气道上皮IL-17A表达水平明显升高,吸气峰流速(PIF)和呼气峰流速(PEF)明显降低,BALF中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞数明显升高,肺组织CXC类趋化因子1(CXCL1)、CXC类趋化因子2(CXCL2)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)表达水平明显升高,JNK、cJun和cFos磷酸化水平及AP1表达水平明显升高(P＜0.05);与野生型COPD组小鼠比较,IL-7A敲除COPD组小鼠气道上皮IL-17A表达水平明显降低,PIF和PEF明显升高,BALF中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞数明显降低,肺组织CXCL1、CXCL2、IL-1β和IL-6表达水平明显降低,JNK、cJun和cFos磷酸化水平及AP1表达水平明显降低(P＜0.05)。结论:香烟烟雾可诱导小鼠气道上皮产生IL-17A,降低(或抑制)IL-17A的产生(或表达或分泌),通过抑制JNK/AP1信号通路,减轻COPD气道炎症反应,改善COPD小鼠肺功能。     

过表达维甲酸相关孤核受体γt可导致自发性唾液腺炎

<正>舍格伦综合征(Sjgren's syndrome,SS)是一种累及外分泌腺(以唾液腺和泪腺为主)的慢性自身免疫系统疾病,临床上以口干和眼干为主要症状。已有文献报道,SS患者及小鼠模型体内唾液腺以及血清中IL-17和Th17相关细胞因子的表达量均显著升高。然而,作为调控Th17细胞分化及IL-17合成的必需核受体--维甲酸相关孤核受体γt(retinoic acid-related orphan receptorγt,RORγt)在SS发病中的作用还未见报道。因此,本研究在过表达RORγt的转基因(RORγt Tg)小鼠中探讨RORγt     

当归与激素合用对哮喘小鼠的治疗作用及其机制

目的：探讨当归与激素合用对哮喘小鼠的治疗作用及其机制。方法：取BALB/c小鼠随机分为对照组、血瘀模型组、哮喘模型组、氢化可的松组、当归组、氢化可的松与当归合用组（n=12）。采用卵白蛋白致敏、激发,复制哮喘模型;使用氢化可的松复制血瘀模型。观测当归、氢化可的松及其合用对小鼠血液流变学（全血黏度与血清TXB2含量）和哮喘（哮喘行为学、肺功能、肺指数与肺组织含水量）的影响,采用ELISA法、免疫组化法测定相关细胞因子和HMGB1/TLR4/NF-κB蛋白质的表达水平。结果：8 g/kg当归、0.05 g/kg氢化可的松及其合用可明显缓解哮喘行为学指标,提高肺功能,降低肺指数及肺组织含水量,降低BALF中TNF-α、IL-1β及IL-6水平,抑制肺组织HMGB1、TLR4及NF-κB蛋白质的高表达;当归与氢化可的松合用对提高哮喘小鼠肺功能、降低相关细胞因子（TNF-α、IL-1β及IL-6）水平明显优于单独使用当归组,抑制肺组织中TLR4和NF-κB蛋白质表达的作用优于单独使用当归或氢化可的松。二者合用后可缓解氢化可的松所致的小鼠血清TXB2含量升高、血液粘稠度增加。结论：当归与激素及其合用具有明显的平喘作用,抑制HMGB1/TLR4/NF-κB蛋白质合成是其平喘的机制之一。当归与激素合用改善肺功能的作用更强,可能与加强抑制TLR4/NF-κB合成有关。合用当归可缓解激素在治疗哮喘时引起的血液流变学异常变化。     

木犀草素调节SIRT3/AMPK/mTOR信号通路对溃疡性结肠炎小鼠Th17/Treg免疫平衡的影响CSCD

探究木犀草素对溃疡性结肠炎(ulcerative colitis,UC)小鼠模型中辅助性T细胞17(Th17)/调节性T细胞(Treg)免疫平衡的影响,并分析其潜在机制。60只BALB/c雄性小鼠随机分为正常组、模型组、阳性对照组、木犀草素组、木犀草素+3-TYP组,每组12只。采用葡聚糖硫酸钠(DSS)诱导建立UC模型,观察并记录小鼠体重变化、大便稠度和大便潜血情况,计算疾病活动指数(DAI);HE染色观察结肠组织病理学变化;流式细胞术检测脾脏Th17、Treg细胞比例,计算Th17/Treg比值;ELISA检测结肠组织中IL-6、IL-10、IL-17和IL-23含量;RT-qPCR检测结肠组织RORγt和Foxp3的mRNA表达;Western blot检测结肠组织SIRT3、AMPK、p-AMPK、mTOR、p-mTOR蛋白表达。与正常组相比,模型组小鼠DAI评分、组织病理学评分、脾脏Th17/Treg比值、结肠组织IL-6、IL-17、IL-23、RORγt mRNA水平和p-mTOR/mTOR比值升高,IL-10、Foxp3 mRNA和SIRT3蛋白水平以及p-AMPK/AMPK比值降低(P<0.05)。经药物干预后,小鼠DAI评分、组织病理学评分、脾脏Th17/Treg比值、结肠组织IL-6、IL-17、IL-23、RORγt mRNA水平和p-mTOR/mTOR比值降低,IL-10、Foxp3 mRNA和SIRT3蛋白水平以及p-AMPK/AMPK比值升高(P<0.05);3-TYP可减弱木犀草素对UC小鼠Th17/Treg细胞分化平衡的影响(P<0.05)。木犀草素可改善DSS诱导的UC小鼠模型中Th17/Treg失衡,其机制可能与激活SIRT3/AMPK/mTOR通路有关。     

颗粒蛋白前体通过抑制IL-6表达减轻哮喘气道炎症

目的: 探究颗粒蛋白前体(PGRN)在过敏性哮喘中的作用及机制。方法: 分别在野生鼠和IL-6 缺陷鼠中设置对照组和哮喘模型组,每组8只。模型组中,在第0日和第7日致敏小鼠(腹腔注射OVA 100 μg),从第14日起连续激发8 d(5％OVA雾化吸入,30 min/d,每日1次),末次激发24 h后取标本;对照组用PBS代替OVA做相同处理。采集支气管肺泡灌洗液(BALF)进行白细胞计数和分类计数;HE染色观察肺组织病理情况;Q-PCR及ELISA检测小鼠肺匀浆、血清和BALF中细胞因子水平。用IL-13刺激A549或BEAS-2B细胞建立体外哮喘炎症模型,每组3个复孔,共4组:PBS处理组、IL-13处理组、IL-13与重组人PGRN蛋白(rhPGRN)共同处理组及p38磷酸化抑制剂(SB203508)处理组。0 min~48 h后收集细胞及上清,用Q-PCR及ELISA检测PGRN和IL-6的表达;Western blot检测p38的磷酸化。结果: 与对照组相比,哮喘组小鼠肺匀浆和BALF中PGRN均显著降低(P＜ 0.01),血清PGRN有降低的趋势,然而哮喘小鼠BALF中IL-6显著升高(P＜0.05)。与野生鼠哮喘组相比,IL-6缺陷鼠哮喘组BALF中白细胞总数降低(P＜0.05),中性粒细胞数降低(P＜0.05),PGRN显著升高(P＜0.05),肺部病理损伤也减轻。体外实验中,IL-13处理组与PBS处理组相比,PGRN显著降低(P＜0.05),IL-6显著增高(P＜ 0.05),p38的磷酸化增加;p38抑制剂处理组比未处理组中IL-6水平降低(P＜0.05)。IL-13与rhPGRN共同处理组的IL-6显著低于IL-13处理组(P＜0.05),p38的磷酸化降低(P＜0.05)。结论: PGRN通过抑制p38磷酸化降低IL-6水平从而减轻哮喘小鼠气道炎症。     

乳杆菌肽聚糖对β-乳球蛋白致敏小鼠脾细胞Th1/Th2及Treg/Th17失衡的体外影响

【目的】观察嗜酸乳杆菌完整肽聚糖(Whole peptidoglycan,WPG)对致敏脾淋巴细胞Th1/Th2及Treg/Th17平衡的体外调节作用。【方法】通过腹腔注射β-乳球蛋白(β-Lg)建立BALB/c小鼠牛乳过敏模型。造模成功后,分离致敏小鼠的脾淋巴细胞并分别与不同剂量的WPG共同孵育,酶联免疫法检测细胞上清液中抗体(总IgE和特异性IgE),Th1/Th2及Treg/Th17相关细胞因子(IFN-γ,IL-4,TGF-β,IL-17)水平,流式细胞术检测脾淋巴细胞中CD3+、CD4+和CD8+的百分含量,荧光定量PCR法检测过敏小鼠脾细胞中Th1/Th2及Treg/Th17相关转录因子T-bet、GATA-3、Foxp3和RORγt mRNA的表达量。【结果】WPG体外刺激致敏脾细胞后可显著抑制IgE的产生,上调CD3+、CD4+细胞数及CD4+/CD8+比值,下调Th2型因子(IL-4,GATA-3mRNA)和Th17型因子(IL-17,RORγt mRNA)的表达,且与过敏组相比,中、高剂量WPG的作用效果显著(P0.05);另外,WPG体外作用还上调了Th1型因子(IFN-γ,T-bet mRNA)及Treg型因子(TGF-β,Foxp3 mRNA)的表达,且具有剂量依赖性。【结论】嗜酸乳杆菌WPG体外刺激可有效纠正致敏脾淋巴细胞的Th1/Th2及Treg/Th17失衡。     

A novel inhibitory role of microRNA-224 in particulate matter 2.5-induced asthmatic mice by inhibiting TLR2

Epidemiological studies have shown that elevated concentrations of particulate matter 2.5 (PM2.5) correlate with increased incidence of asthma. Studies have highlighted the implication of microRNAs (miRNAs) in asthmatic response. Here, the objective of this study is to explore the effect of miR-224 on PM2.5-induced asthmatic mice. Ovalbumin (OVA) was utilized to establish asthmatic mouse models, which were then exposed to PM2.5, followed by miR-224 expression detection. Next, lesions and collagen deposition area in lung tissue, ratio Treg/Th17, the expression of TLR4 and MYD88, inflammation, eosinophils (EOS) and airway remodelling were evaluated in OVA mice after injection with miR-224 agomir. Following isolation of mouse primary bronchial epithelial cells, miR-224 mimic and TLR2/TLR4 inhibitor were introduced to assess inflammation and the expression of TGF-β, MMP9, TIMP-1, Foxp3, RORγt, TLR2, TLR4 and MYD88. After exposure to PM2.5, lesions and collagen deposition were promoted in lung tissues, inflammation and EOS were increased in bronchoalveolar lavage fluid (BALF), and airway remodelling was enhanced in OVA mice. miR-224 was down-regulated, whereas TLR2/TLR4/MYD88 was up-regulated in OVA mice after treatment with PM2.5, accompanied by Treg/Th17 immune imbalance. Of note, bioinformatic prediction and dual luciferase reporter gene assay confirmed that TLR2 was a target gene of miR-224. Overexpressed miR-224 reduced expression of TGF-β, MMP9, TIMP-1 and RORγt and inflammation but increased Foxp3 expression in bronchial epithelial cells through down-regulating TLR2. In summary, overexpressed miR-224 suppressed airway epithelial cell inflammation and airway remodelling in PM2.5-induced asthmatic mice through decreasing TLR2 expression.     

Restricted microbiota and absence of cognate TCR antigen leads to an unbalanced generation of Th17 cells

Retinoic acid-related orphan receptor (ROR)γt(+) TCRαβ(+) cells expressing IL-17, termed Th17 cells, are most abundant in the intestinal lamina propria. Symbiotic microbiota are required for the generation of Th17 cells, but the requirement for microbiota-derived Ag is not documented. In this study, we show that normal numbers of Th17 cells develop in the intestine of mice that express a single TCR in the absence of cognate Ag, whereas the microbiota remains essential for their development. However, such mice, or mice monocolonized with the Th17-inducing segmented filamentous bacteria, fail to induce normal numbers of Foxp3(+) RORγt(+) T cells, the regulatory counterpart of IL-17(+)RORγt(+) T cells. These results demonstrate that a complex microbiota and cognate Ag are required to generate a properly regulated set of RORγt(+) T cells and Th17 cells.     

Bulleyaconitine A inhibits the lung inflammation and airway remodeling through restoring Th1/Th2 balance in asthmatic model mice

ABSTRACT

The current study aimed to study the effects of Bulleyaconitine A (BLA) on asthma. Asthmatic mice model was established by ovalbumin (OVA) stimulation, and the model mice were treated by BLA. After BLA treatment, the changes in lung and airway resistances, total and differential leukocytes in the bronchoalveolar lavage fluid (BALF) were detected, and the changes in lung inflammation and airway remodeling were observed. Moreover, the secretion of IgE, Th1/Th2-type and IL-17A cytokines in BALF and serum of the asthmatic mice were determined. The resuts showed that BLA attenuated OVA-induced lung and airway resistances, inhibited the inflammatory cell recruitment in BALF and the inflammation and airway remodeling of the asthmatic mice. In addition, BLA suppressed the secretion of IgE, Th2-type cytokines, and IL-17A, but enhanced secretions of Th1-type cytokines in BALF and serum. The current study discovered that BLA inhibited the lung inflammation and airway remodeling via restoring the Th1/Th2 balance in asthmatic mice.     

CXCR3 antagonist AMG487 suppresses rheumatoid arthritis pathogenesis and progression by shifting the Th17/Treg cell balance

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by uncontrolled joint inflammation and damage to bone and cartilage. Previous studies have shown that chemokine receptors have important roles in RA development, and that blocking these receptors effectively inhibits RA progression. Our study was undertaken to investigate the role of AMG487, a selective CXCR3 antagonist, in DBA/1J mice bearing collagen-induced arthritis (CIA). Following induction of CIA, animals were treated with 5 mg/kg AMG487 intraperitoneally every 48 h, starting from day 21 until day 41 and evaluated for clinical score, and histological hallmarks of arthritic inflammation. We further investigated the effect of AMG487 on Th1 (T-bet), Th17 (IL-17A, RORγt, STAT3), Th22 (IL-22), and T regulatory (Treg; Foxp3 and IL-10) cells in splenic CXCR3⁺ and CD4⁺ T cells using flow cytometry. We also assessed the effect of AMG487 on T-bet, RORγt, IL-17A, IL-22, Foxp3, and IL-10 at both mRNA and protein levels using RT-PCR and Western blot analyses of knee samples. The severity of clinical scores, and histological inflammatory damage decreased significantly in AMG487-treated compared with CIA control mice. Moreover, the percentage of Th1, Th17, and Th22 cells decreased significantly and that of Treg cells increased in AMG487-treated mice. We further observed that AMG487-treatment downregulated T-bet, IL-17A, RORγt, and IL-22, whereas it upregulated Foxp3 and IL-10 mRNA and protein levels. This study demonstrates the antiarthritic effects of AMG487 in CIA animal model and supports the development of CXCR3 antagonists as a novel strategy for the treatment of inflammatory and arthritic conditions.