迷走神经电刺激对急性呼吸窘迫综合征中Th17和Treg 蛋白变化及炎性因子的影响

目的 探讨迷走神经电刺激（Vagus nerve stimulation, VNS）调控脂多糖（LPS）诱导的急性呼吸窘迫征（acute respiratory distress syndrome, ARDS）体内Th17和Treg 蛋白变化控制炎症的机制。方法 以2mg/kg LPS 鼻管滴注SD大鼠,6h后给予左侧颈部迷走神经以10min,5V,5Hz,2ms频率刺激, 2h后取双肺及脾组织进行检测。大鼠随机分为Control组、LPS组和LPS+VNS组,每组10只。检测肺泡灌洗液总蛋白含量,测定肺部湿干比,H-E染色检测肺部炎症程度,蛋白免疫印迹法及免疫组化检测肺及脾组织Foxp3及Rorγt蛋白表达,ELISA检测肺部灌洗液中TNF-α、IL-1β、IL-6、IL-17、IL-10水平。结果 成功构建大鼠ARDS模型。与Control组相比,LPS组肺泡灌洗液总蛋白含量增高（P<0.001）,湿干比增高（P<0.001）,IL-1β、IL-6、IL-17、IL-10表达升高（均P <0.01）和TNF-α表达升高,肺和脾组织中Foxp3和Rorγt蛋白表达上升;与LPS组相比,LPS+VNS组肺泡灌洗液总蛋白含量降低（P<0.05）,湿干比下降（P<0.05）,IL-6、IL-17降低（P<0.05或P<0.01）,IL-10升高（P<0.01）,肺及脾组织中Foxp3蛋白表达上调（P<0.05）和Rorγt蛋白表达下降。结论 VNS可通过调控肺和脾组织Th17和Treg蛋白进而调控炎性因子,减轻LPS诱导的ARDS病理改变,保护组织。     

慢性乙型肝炎患者外周血细胞因子IL-17A、IL-2、IL-21、IL-4表达水平及意义

目的 探索初治慢性乙型肝炎（CHB）患者外周血中细胞因子表达水平及其与病毒载量和肝脏炎症程度的关系,以期为临床动态评估病情和预后方面提供新思路。方法 选择2018年10月至2019年11月就诊于海军军医大学第一附属医院感染科的初治慢性乙型肝炎病毒（HBV）感染者68例,健康对照者12名,通过ELISA检测外周血中细胞因子IL-17A、IL-2、IL-21和IL-4表达水平,同时检测HBV DNA及转氨酶水平。统计学处理采用Mann-Whitney U检验、Kruskal-Wallis H检验及Spearman相关性分析。结果 相较于健康对照者,初治CHB患者外周血IL-17A高 [17.50（11.99,25.36）vs14.14（9.01,23.68）pg/ml,Z=-2.001,P=0.045],IL-2低[57.19（31.10,79.92）vs73.06（62.41,105.84）pg/ml,Z=-2.509,P=0.012],IL-21高[37.12（23.85,77.66）vs（20.95±5.72）pg/ml,Z=-3.485,P<0.01],IL-4表达水平无差异。不同免疫状态的IL-17A表达有明显差异（H=8.870,P=0.031）。炎症活动状态CHB患者的IL-17A和IL-21更低、IL-2更高（P＜0.05）,IL-4无差异（P＞0.05）。HBeAg阳性CHB患者、HBeAg阴性CHB患者与健康对照者外周血细胞因子IL-17A、IL-2、IL-21比较,差异有统计学意义（IL-17A：H=10.061,P=0.007;IL-2：H=6.576,P=0.037;IL-21：H=12.444,P=0.002）。初治CHB患者外周血IL-17A、IL-21、IL-4与HBV DNA无相关性（r=0.02、0.23、0.07,均P>0.05）,IL-2与HBV DNA存在弱相关（r=0.32,P=0.01）。初治HBV患者外周血IL-17A、IL-21与ALT存在相关性（IL-17A：r=0.59,P<0.01;IL-21：r=0.49,P<0.01）,与AST存在相关性（IL-17A：r=0.47,P<0.01;IL-21：r=0.36,P<0.01）,而IL-2、IL-4与ALT和AST均无明显相关。ALT≥300U/L的初治CHB组、ALT＜300U/L的初治CHB组与健康对照者外周血细胞因子IL-17A、IL-2、IL-21比较,差异有统计学意义（IL-17A：H=27.557,P<0.01;IL-2：H=8.581,P=0.014;IL-21：H=21.438,P<0.01）。ROC曲线分析结果显示,IL-17A判断肝脏炎症程度的AUC值为0.8933（95%CI：0.7930-0.9936）,IL-21判断肝脏炎症程度的AUC值为0.7600（95%CI：0.6227-0.8973）。结论 IL-17A、IL-2和IL-21参与慢性HBV感染进程：初治CHB患者无论HBeAg阳性与否或炎症程度高低,外周血IL-17A和IL-21升高,IL-2下降;IL-2与HBV DNA有一定相关性;IL-17A和IL-21与ALT及AST均存在正相关;检测IL-17A和IL-2有助于病情评估与预后判断。     

髓系抑制细胞、Th17细胞在哮喘患儿外周血中的表达

目的 分析髓系抑制细胞(MDSCs)及Th17细胞在哮喘患儿外周血中的表达情况和临床意义.方法 选取自贡市第一人民医院儿科2013年6月至2015年8月收治的120例哮喘患儿作为观察组,另外选择87例健康儿童作为对照组.应用流式细胞术检测MDSCs、Th17细胞在哮喘患儿外周血中的表达情况.结果 和对照组相比,观察组MDSCs占有核细胞的百分比和Th17细胞占单个核细胞的百分比都显著升高(Hc值分别为56.14、45.98,均P<0.05),差异具有统计学意义.结论 从MDSCs、Th17细胞在哮喘患儿外周血中的表达情况来看,它们可能参与了婴幼儿哮喘的发生与发展过程.     

IL-17A与自发性高血压大鼠肾上皮间质转化密切相关

目的探究IL-17A在原发性高血压大鼠肾上皮间质转化（EMT）中的作用。方法4周龄的SPF级自发性高血压大鼠（SHR）（实验组）、京都维斯塔尔大鼠（WKY）（对照组）各20只，分别随机分为4组，每组5只，在4、6、10、30周龄进行实验观察。采用无创尾动脉血压测量仪动态检测大鼠的血压情况；流式细胞技术分析大鼠脾淋巴细胞中Th17细胞的频数；RT-PCR法及免疫组化技术检测大鼠肾脏IL-17A、iNOS、Arg-1、E-cadherin、α-SMA mRNA和蛋白的表达水平；ELISA法检测大鼠血浆中IL-17A的水平。结果从6周龄始SHR大鼠血压显著高于同期的WKY组（P < 0.05或P < 0.01），SHR大鼠Th17细胞频数以及肾脏和血浆中IL-17A的表达亦显著高于同期WKY组（P < 0.05或P < 0.01）；在30周龄时SHR大鼠肾脏中E-cadherin mRNA及蛋白的表达显著低于WKY组（P < 0.01），而Arg-1 mRNA及蛋白的表达显著高于WKY组（P < 0.01）；iNOS mRNA及蛋白的表达在6、10周龄时显著高于WKY组（P < 0.05或P < 0.01），α-SMA mRNA及蛋白从10周龄始显著高于WKY组（P < 0.05或P < 0.01）。SHR大鼠肾脏中IL-17A mRNA及蛋白表达水平从10周龄始与E-cadherin mRNA及蛋白表达水平呈显著负相关（r=-0.731，P < 0.05；r=-0.827，P < 0.01），而与α-SMA mRNA及蛋白表达水平呈显著正相关（r=0.658，P < 0.05；r=0.968，P < 0.01）。结论IL-17A与SHR大鼠肾EMT的过程密切相关，可能是通过介导肾脏浸润的巨噬细胞M1/M2极化发挥作用。     

Effects of Interleukin-17A on the Early Stages of Arterial Thrombosis in Mice

PurposeInterleukin (IL)-17A has been suggested to play a role in the growth and organization of thrombi. We examined whether IL-17A plays a role in the early stages of thrombosis and whether there are sex differences in the effects of IL-17A.Materials and MethodsWe performed a blinded, randomized, placebo-controlled study to compare time to thrombotic occlusion and sex differences therein between mice treated with IL-17A and those treated with saline using a ferric chloride-induced model. We also assessed thrombus histology, blood coagulation, and plasma levels of coagulation factors.ResultsTime to occlusion values did not differ between the IL-17A group and the control group (94.6±86.9 sec vs. 121.0±84.4 sec, p=0.238). However, it was significantly shorter in the IL-17A group of female mice (74.6±57.2 sec vs. 130.0±76.2 sec, p=0.032). In rotational thromboelastometry, the IL-17A group exhibited increased maximum clot firmness (71.3±4.5 mm vs. 66.7±4.7 mm, p=0.038) and greater amplitude at 30 min (69.7±5.2 mm vs. 64.5±5.3 mm, p=0.040) than the control group. In Western blotting, the IL-17A group showed higher levels of coagulation factor XIII (2.2±1.5 vs. 1.0±0.9, p=0.008), monocyte chemoattractant protein-1 (1.6±0.6 vs. 1.0±0.4, p=0.023), and tissue factor (1.5±0.6 vs. 1.0±0.5, p=0.003).ConclusionIL-17A plays a role in the initial st ages of arterial thrombosis in mice. Coagulation factors and monocyte chemoattractant protein-1 may be associated with IL-17A-mediated thrombosis.     

Role of miR‐21‐5p/FilGAP axis in estradiol alleviating the progression of monocrotaline‐induced pulmonary hypertension

BackgroundAberrant expression of microRNAs (miRNAs) has been associated with the pathogenesis of pulmonary hypertension (PH). It is, however, not clear whether miRNAs are involved in estrogen rescue of PH.MethodsFresh plasma samples were prepared from 12 idiopathic pulmonary arterial hypertension (IPAH) patients and 12 healthy controls undergoing right heart catheterization in Shanghai Pulmonary Hospital. From each sample, 5 μg of total RNA was tagged and hybridized on microRNA microarray chips. Monocrotaline‐induced PH (MCT‐PH) male rats were treated with 17β‐estradiol (E₂) or vehicle. Subgroups were cotreated with estrogen receptor (ER) antagonist or with antagonist of miRNA.ResultsMany circulating miRNAs, including miR‐21‐5p and miR‐574‐5p, were markedly expressed in patients and of interest in predicting mean pulmonary arterial pressure elevation in patients. The expression of miR‐21‐5p in the lungs was significantly upregulated in MCT‐PH rats compared with the controls. However, miR‐574‐5p showed no difference in the lungs of MCT‐PH rats and controls. miR‐21‐5p was selected for further analysis in rats as E₂ strongly regulated it. E₂ decreased miR‐21‐5p expression in the lungs of MCT‐PH rats by ERβ. E₂ reversed miR‐21‐5p target gene FilGAP downregulation in the lungs of MCT‐PH rats. The abnormal expression of RhoA, ROCK2, Rac1 and c‐Jun in the lungs of MCT‐PH rats was inhibited by E₂ and miR‐21‐5p antagonist.ConclusionsmiR‐21‐5p level was remarkably associated with PH severity in patients. Moreover, the miR‐21‐5p/FilGAP signaling pathway modulated the protective effect of E₂ on MCT‐PH through ERβ.     

Th17 cell‐derived miR‐155‐5p modulates interleukin‐17 and suppressor of cytokines signaling 1 expression during the progression of systemic sclerosis

Background miR‐155‐5p is associated with autoimmune diseases. T helper 17 (Th17) cells, interleukin (IL)‐17, and suppressor of cytokines signaling 1 (SOCS1) have important roles in the pathogenesis of systemic sclerosis (SSc). The purpose of this study was to explore the role of miR‐155‐5p in the regulation of IL‐17 and SOCS1 expression in Th17 cells and the subsequent effect on SSc disease progression.MethodsTh17 cells were isolated from peripheral blood mononuclear cells of SSc patients and healthy controls (HCs). RT‐qPCR and western blotting were used to examine the expression patterns of miR‐155‐5p, IL‐17, and SOCS1. Luciferase reporter assays were performed to confirm SOCS1 as a target of miR‐155‐5p. RNA pull‐down assays were performed to detect the interaction of IL‐17 and SOCS1 with miR‐155‐5p. In situ hybridization was performed to analyze the co‐expression pattern of miR‐155‐5p and IL17A in Th17 cells.ResultsThe levels of Th17 cell‐derived miR‐155‐5p were significantly up‐regulated in SSc patients compared with HCs, and its levels were negatively correlated with SOCS1 levels. Meanwhile, miR‐155‐5p positively regulated IL‐17 expression levels in Th17 cells isolated from SSc patients as the disease progressed. Using pmirGLO vectors, SOCS1 was confirmed as a target of miR‐155‐5p. The binding status of IL‐17 and SOCS1 to miR‐155‐5p was related to SSc progression. An increase in the co‐localization of miR‐155‐5p and IL‐17 was associated with greater SSc progression.ConclusionsIL‐17 and SOCS1 expression modulated by Th17 cell‐derived miR‐155‐5p are critical for SSc progression, which may provide novel insights into the pathogenesis of SSc.     

The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

Dendritic cells (DCs) play a critical role in controlling T helper 2 (Th2) cell-dependent diseases, but the signaling mechanism that triggers this function is not fully understood. We showed that p38α activity in DCs was decreased upon HDM stimulation and dynamically regulated by both extrinsic signals and Th2-instructive cytokines. p38α-specific deletion in cDC1s but not in cDC2s or macrophages promoted Th2 responses under HDM stimulation. Further study showed that p38α in cDC1s regulated Th2-cell differentiation by modulating the MK2−c-FOS−IL-12 axis. Importantly, crosstalk between p38α-dependent DCs and Th2 cells occurred during the sensitization phase, not the effector phase, and was conserved between mice and humans. Our results identify p38α signaling as a central pathway in DCs that integrates allergic and parasitic instructive signals with Th2-instructive cytokines from the microenvironment to regulate Th2-cell differentiation and function, and this finding may offer a novel strategy for the treatment of allergic diseases and parasitic infection.     

儿童原发免疫性血小板减少症miR-106b-5p的表达及其与T细胞的相关性研究

目的探讨血浆miR-106b-5p在原发免疫性血小板减少症（primary immune thrombocytopenia，ITP）中的表达及其与辅助性T细胞17（T helper cells 17，Th17）、调节性T细胞（T regulatory cell，Treg）、Th17/Treg的相关性。方法选取79例ITP患儿（ITP组）和40例健康儿童（对照组）为研究对象。79例ITP患儿根据治疗效果分为完全有效组（40例）、有效组（18例）、无效组（21例）。采用实时荧光定量PCR技术检测miR-106b-5p表达水平，流式细胞技术检测Th17和Treg，计算Th17/Treg，分析血浆miR-106b-5p表达水平与Th17、Treg及Th17/Treg的相关性。结果ITP组miR-106b-5p、Th17及Th17/Treg水平高于对照组（P<0.05），Treg水平低于对照组（P<0.05）。ITP组患儿治疗后miR-106b-5p、Th17、Th17/Treg水平低于治疗前（P<0.05），Treg水平高于治疗前（P<0.05）。完全有效组miR-106b-5p、Th17、Th17/Treg水平低于有效组和无效组（P<0.05），Treg水平高于有效组和无效组（P<0.05）。相关分析显示，ITP患儿血浆miR-106b-5p表达水平与Th17、Th17/Treg均呈正相关（分别r=0.730、0.816，均P<0.001），与Treg呈负相关（r=-0.774，P<0.001）。结论ITP患儿存在miR-106b-5p高表达和Th17/Treg比例失衡，在ITP患儿治疗过程中检测miR-106b-5p、Th17、Treg及Th17/Treg水平，对ITP患儿的疗效判断具有较好的指导作用。     

Early and late diagnoses of 17β-Hydroxysteroid dehydrogenase type-3 deficiency in two unrelated patients

17β-hydroxysteroid dehydrogenase type 3 deficiency is a rare cause of 46 XY disorders of sexual development. Mutations in the HSD17B3 gene result in reduced activity of the 17β-HSD3 enzyme, decreasing the conversion of androstenedione to testosterone. In this report, two cases, admitted with different clinical findings in the neonatal and adolescent periods and were decided to be raised in different genders are presented. The first case who had complete female external genitalia presented on the third postnatal day with the complaint of swelling in the groin. He was decided to be raised as a male and was treated successfully with parenteral testosterone in order to increase phallus size before surgical correction of the external genitalia. The second case was an adolescent girl who presented due to pubertal virilisation and primary amenorrhoea and chose female gender. Molecular genetic analyses of the HSD17B3 gene revealed two different previously reported homozygous variants. We emphasise that patients with 17β-hydroxysteroid dehydrogenase type 3 deficiency can present with heterogeneous clinical findings in different age groups. Early diagnosis is important to prevent future gender confusion and related problems.