[1]黄健,于建,李明瑛,等.环鸟苷酸腺苷酸合成酶-环鸟苷酸腺苷酸-干扰素基因刺激分子信号通路在结核性胸膜炎大鼠中的作用[J].新乡医学院学报,2023,40(4):313-318.[doi:10.7683/xxyxyxb.2023.04.003]
　HUANG Jian,YU Jian,LI Mingying,et al.Role of cyclic guanosine adenylate synthetase-cyclic guanosine monophosphate adenosine monophosphate-stimulator of interferon gene signal pathway in rats with tuberculous pleurisy[J].Journal of Xinxiang Medical University,2023,40(4):313-318.[doi:10.7683/xxyxyxb.2023.04.003]
点击复制

环鸟苷酸腺苷酸合成酶-环鸟苷酸腺苷酸-干扰素基因刺激分子信号通路在结核性胸膜炎大鼠中的作用
分享到：

《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年4
页码:
313-318
栏目:
基础研究
出版日期:
2023-04-05

文章信息/Info

Title:
Role of cyclic guanosine adenylate synthetase-cyclic guanosine monophosphate adenosine monophosphate-stimulator of interferon gene signal pathway in rats with tuberculous pleurisy
作者:
黄健1; 2; 于建3; 李明瑛1; 2; 韩伟1; 2; 崔秀琴1; 2
（1.新乡医学院第一附属医院结核内科，河南　卫辉　453100；2.新乡市结核病学重点实验室，河南　卫辉　453100；3.新乡医学院第一附属医院病理科，河南　卫辉　453100）
Author(s):
HUANG Jian1; 2; YU Jian3; LI Mingying1; 2; HAN Wei1; 2; CUI Xiuqin1; 2
(1.Department of Tuberculosis,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China;2.Key Laboratory of Tuberculosis of Xinxiang City,Weihui 453100,Henan Province,China;3.Department of Pathology,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China)
关键词:
结核性胸膜炎; 环鸟苷酸腺苷酸; 环鸟苷酸腺苷酸合成酶; 干扰素基因刺激分子
Keywords:
tuberculous pleurisy; cyclic guanosine monophosphate-adenosine monophosphate; cyclic guanosine adenylate synthetase; stimulator of interferon gene
分类号:
R521.7
DOI:
10.7683/xxyxyxb.2023.04.003
文献标志码:
A
摘要:
目的探讨环鸟苷酸腺苷酸合成酶（cGAS）-环鸟苷酸腺苷酸（cGAMP）-干扰素基因刺激分子（STING）信号通路在结核性胸膜炎大鼠中的作用。方法30只6周龄无特定病原级Sprague Dawley雄性大鼠适应性饲养5 d后随机分为对照组、模型组和cGAS抑制剂组，每组10只。3组大鼠适应性饲养5 d后于右侧腹股沟内侧皮内注射100 μL卡介苗（BCG）悬液（0.06 mg）；接种5周后，模型组和cGAS抑制剂组大鼠于右侧肋弓角顶点注射1 mL结核分枝杆菌H37RV悬液（0.03 mg），建立结核性胸膜炎大鼠模型；对照组大鼠仅接种BCG，不注射结核分枝杆菌H37RV悬液。cGAS抑制剂组大鼠自造模第2天尾静脉注射cGAS抑制剂RU.521（600 μg·L-1） 200 μL，每日1次，连续7 d；对照组和模型组大鼠尾静脉注射等体积的生理盐水。第8天脱颈处死大鼠，立刻从大鼠剑突侧处沿胸骨剪开皮肤和胸骨，暴露胸腔和纵隔，观察大鼠胸腔积液的分布情况，收集、记录胸腔积液量；并观察胸膜粘连情况，采用胸腔积液中纤维蛋白原（FBG）水平和胸腔粘连带数量评分评估大鼠胸腔积液粘连性；取胸膜组织标本，以体积分数10%甲醛溶液固定。测量各组大鼠切片中平面状态下的胸膜厚度，苏木精-伊红（HE）染色观察各组大鼠胸膜组织病理学改变，采用Western blot法检测各组大鼠胸膜组织中cGAS和STING蛋白表达，采用酶联免疫吸附试验法检测各组大鼠胸膜组织中cGAMP蛋白和胸腔积液中基质金属蛋白酶（MMP）-1、MMP-9、白细胞介素（IL）-1、IL-6、可溶性细胞间黏附分子-1（sICAM-1）水平。结果模型组和cGAS抑制剂组大鼠胸腔积液显著多于对照组，胸膜厚度显著大于对照组（P<0.05）；cGAS抑制剂组大鼠胸腔积液显著少于模型组，胸膜厚度显著小于模型组（P<0.05）。cGAS抑制剂组大鼠胸腔积液中FBG水平、胸腔积液粘连性评分显著低于模型组（P<0.05）。HE染色显示，对照组大鼠肺间质和胸膜内血管排列、形态正常，胸膜无明显异常；模型组大鼠肺间质和胸膜内血管充血扩张，胸膜内可见大量中性粒细胞和淋巴细胞浸润、纤维组织增生、上皮样细胞团及凝固型坏死；与模型组相比，cGAS抑制剂组大鼠肺间质和胸膜内血管充血减轻，胸膜内中性粒细胞、淋巴细胞浸润减少，少量纤维组织增生，上皮样细胞团及凝固型坏死减少。模型组和cGAS抑制剂组大鼠胸膜组织中cGAS、cGAMP、STING蛋白表达显著高于对照组（P<0.05），cGAS抑制剂组大鼠胸膜组织中cGAS、cGAMP、STING蛋白表达显著低于模型组（P<0.05）。模型组和cGAS抑制剂组大鼠胸膜组织中MMP-1、MMP-9、IL-1、IL-6、sICAM-1水平显著高于对照组（P<0.05），cGAS抑制剂组大鼠胸膜组织中MMP-1、MMP-9、IL-1、IL-6、sICAM-1水平显著低于模型组（P<0.05）。结论结核性胸膜炎大鼠胸膜组织中cGAS、cGAMP、STING蛋白表达上调，抑制cGAS-cGAMP-STING信号通路活性可以改善大鼠的结核性胸膜炎，cGAS-cGAMP-STING信号通路可能参与了结核性胸膜炎的发生和发展。
Abstract:
ObjectiveTo investigate the role of cyclic guanosine adenylate synthetase(cGAS)-cyclic guanosine monophosphate adenosine monophosphate(cGAMP)-stimulator of interferon gene (STING) signal pathway in rats with tuberculous pleurisy.MethodsThirty-six-week-old Sprague Dawley male rats without specific pathogen free were randomly divided into control group,model group and cGAS inhibitor group after adaptive feeding for 5 days,with 10 rats in each group.After 5 days of adaptive feeding,100 μL(0.06 mg) bacillus calmette-guerin(BCG) suspension was injected intradermally into the right medial inguinal of rats in the three groups.Five weeks after inoculation,the rats in the model group and the cGAS inhibitor group were injected with 1 mL(0.03 mg) Mycobacterium tuberculosis H37RV suspension at the apex of the right angulus arcuum costarum to establish the models of tuberculous pleurisy,while the rats in the control group were only inoculated with BCG instead of Mycobacterium tuberculosis H37RV suspension.The rats in the cGAS inhibitor group were injected with 200 μL cGAS inhibitor RU.521 (600 μg·L-1) by the vena caudalis from the second day of modeling,once a day for 7 days.The rats in the control group and model group were injected with the same volume of normal saline through the vena caudalis.On the eighth day,the rats were decapitated and killed,then the skin and sternum were immediately cut along the sternum from the side of the xiphoid process of the rats,and the thoracic cavity and mediastinum were exposed.The distribution of pleural effusion was observed,and the amount of pleural effusion was collected and recorded;the pleural adhesions were observed.The level of fibrinogen (FBG) in pleural effusion and the number of pleural adhesions were used to evaluate the adhesiveness of pleural effusion in rats.The distribution of pleural effusion of rats was observed,and the volume of pleural effusion was collected and recorded.The pleural adhesions were observed,and the adhesiveness of pleural effusion was evaluated by the level of FBG in pleural effusion and the number of pleural adhesions bands.The pleural thickness in plane state was measured in the sections of rats in each group.The histopathological changes of pleural tissues of rats in each group were observed by hematoxylin-eosin (HE) staining,and the expression of cGAS and STING protein in pleural tissues of rats in each group was detected by Western blot.The levels of cGAMP protein in pleural tissues and matrix metalloproteinase (MMP)-1,MMP-9,interleukin (IL)-1,IL-6,soluble intercellular adhesion molecule-1 (sICAM-1) in pleural effusion were detected by enzyme-linked immunosorbent assay.ResultsThe pleural effusion and pleural thickness of rats in the model group and cGAS inhibitor group was significantly greater than that in the control group (P<0.05),and the pleural effusion and pleural thickness of rats in the cGAS inhibitor group were significantly less than those in the model group (P<0.05).The FBG level in pleural effusion and the adhesion score of pleural effusion of rats in the cGAS inhibitor group was significantly lower than that in the model group (P<0.05).The HE staining showed that the arrangement and morphology of blood vessels in the lung interstitium and pleura of rats in the control group were normal,and there was no obvious abnormality in the pleura.In the model group,the blood vessels in the lung interstitium and pleura were congested and dilated,and a large number of neutrophils and lymphocytes,fibrous tissue hyperplasia,epithelioid cell mass and coagulative necrosis could be seen in the pleura.Compared with the model group,the vasocongestion in the lung interstitium and pleura,neutrophils and lymphocytes in the pleura,fibrous tissue hyperplasia,epithelioid cell mass and coagulative necrosis of rats in the cGAS inhibitor group were reduced.The expressions of cGAS,cGAMP and STING protein in pleural tissues of rats in the model group and cGAS inhibitor group were significantly higher than those in the control group (P<0.05).The expressions of cGAS,cGAMP and STING protein in the pleural tissues of rats in the cGAS inhibitor group were significantly lower than those in the model group (P<0.05).The levels of MMP-1,MMP-9,IL-1,IL-6 and sICAM-1 in pleural tissues of rats in the model group and cGAS inhibitor group were significantly higher than those in the control group (P<0.05).The levels of MMP-1,MMP-9,IL-1,IL-6 and sICAM-1 in the pleural tissues of rats in the cGAS inhibitor group were significantly lower than those in the model group (P<0.05).ConclusionThe expressions of cGAS,cGAMP and STING protein in pleural tissues of rats with tuberculous pleurisy are up-regulated,inhibiting the activity of cGAS-cGAMP-STING signal pathway can improve the tuberculous pleurisy of rats,and the cGAS-cGAMP-STING signal pathway may be involved in the occurrence and development of tuberculous pleurisy.

参考文献/References:

［1］　MASCOLO L,BALD D.Cytochrome bd in Mycobacterium tuberculosis:a respiratory chain protein involved in the defense against antibacterials［J］.Prog Biophys Mol Biol,2020,152(1):55-63.
［2］　KIM S B,SHIN B,LEE J H,et al.Pleural fluid ADA activity in tuberculous pleurisy can be low in elderly,critically ill patients with multi-organ failure［J］.BMC Pulm Med,2020,20(1):1779-1787.
［3］　HAMEED H A,TAN Y,ISLAM M M,et al.Detection of novel gene mutations associated with pyrazinamide resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates in Southern China［J］.Infect Drug Resist,2020,13(1):217-227.
［4］　THOMSEN M K,SKOUBOE M K,BOULARAN C,et al.The cGAS-STING pathway is a therapeutic target in a preclinical model of hepatocellular carcinoma［J］.Oncogene,2020,39(2):1652-1664.
［5］　LI T,CHEN Z J.The cGAS-cGAMP-STING pathway connects DNA damage to inflammation,senescence,and cancer［J］.J Exp Med,2018,215(5):1287-1299.
［6］　COLLINS A C,CAI H,LI T,et al.Cyclic GMP-AMP synthase is an innate immune DNA sensor for Mycobacterium tuberculosis［J］.Cell Host Microbe,2015,17(6):820-828.
［7］　黄超文，冯起校，于斌，等.胸腔内注入尿激酶治疗大鼠结核性胸膜炎的实验研究［J］.广东医学，2011，32（14）：1814-1816.
HUANG C W,FENG Q X,YU B,et al.Experimental study on intrapleural injection of urokinase to treat tuberculous pleurisy in rats ［J］.Guangdong Med J,2011,32 (14):1814-1816.
［8］　VINCENT J,ADURA C,GAO P,et al.Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice［J］.Nat Commun,2017,8(1):750-762.
［9］　ZHANG M,NIU Y R,LIU J Y,et al.Interleukin-26 upregulates interleukin-22 production by human CD4+ T cells in tuberculous pleurisy［J］.J Mol Med,2019,97(5):619-631.
［10］　COLL F,PHELAN J,HILLCAWTHORNE G A,et al.Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis［J］.Nate Genet,2018,50(2):307-316.［11］　唐先梅，赵英仁，江自成，等.新方法初治结核性胸膜炎的疗效观察［J］.中国感染控制杂志，2018，17（1）：52-55.
TANG X M,ZHAO Y R,JIANG Z C,et al.Efficacy of a new therapeutic approach as initial treatment for tuberculous pleuritis［J］.Chin J Infect Control,2018,17(1):52-55.
［12］　SAEED A F,RUAN X,GUAN H,et al.Regulation of cGAS-mediated immune responses and immunotherapy［J］.Adv Sci,2020,7(6):1902599.
［13］　BAI J,LIU F.The cGAS-cGAMP-STING pathway:a molecular link between immunity and metabolism［J］.Diabetes,2019,68(6):1099-1108.
［14］　MARINHO F V,BENMERZOUG S,ROSE S,et al.The cGAS/STING pathway is important for dendritic cell activation but is not essential to induce protective immunity against Mycobacterium tuberculosis infection［J］.J Innate Immun,2018,10(3):239-252.
［15］　李强，刘春法，何小丽，等.cGAS介导牛分枝杆菌诱导小鼠髓源树突状细胞成熟［J］.畜牧兽医学报，2017，48（5）：914-921.
LI Q,LIU C F,HE X L,et al.Cyclic GMP-AMP synthase-mediated maturation of murine bone marrow derived dendritic cells after infection with Mycobacterium bovis［J］.Acta Vet Et Zootech Sin,2017,48(5):914-921.
［16］　CHAROENPAK R,SANTIMALEEWORAGUN W,SUWANPIMOLKUL G,et al.Association between the phenotype and genotype of isoniazid resistance among Mycobacterium tuberculosis isolates in Thailand［J］.Infect Drug Resist,2020,13(1):627-634.
［17］　陈刚，徐旭东，陈达，等.结核性胸膜炎大鼠胸水中基质金属蛋白酶-1和基质金属蛋白酶-9浓度的变化［J］.中华实验外科杂志，2017，34（10）：1710.
CHEN G,XU X D,CHEN D,et al.Dynamic variation of matrix metalloproteinase-1 and matrix metalloproteinase-9 concentrations in tuberculous pleurisy pleural effusion of rats ［J］.Chin J Exp Surg,2017,34 (10):1710.
［18］　周安，徐巧玲，李明强，等.结核患者血清TNF-α、IL-1、IL-10和HMGB-1动态变化及临床意义［J］.实用医学杂志，2017，33（2）：124-127.
ZHOU A,XU Q L,LI M Q,et al.The dynamic changes of serum TNF-α，IL-1，IL-10 and HMGB-1 during tuberculosis disease progression ［J］.J Pract Med,2017,33 (2):124-127.
［19］　陈英，陈兴年，叶志坚.结核性胸膜炎患者sIL-2R和IL-6的临床意义［J］.当代医学，2019，25（4）：104-106.
CHEN Y,CHEN X N,YE Z J.Clinical significance of sIL-2R and IL-6 levels in patients with tuberculous pleurisy ［J］.Contemp Med,2019,25 (4):104-106.
［20］　PANTOVIC＇-STEFANOVIC＇ M,PETRONIJEVIC＇ N,DUNJIC＇-KOSTIC＇ B,et al.sVCAM-1,sICAM-1,TNF-α and IL-6 levels in bipolar disorder typeⅠ:acute,longitudinal and therapeutic implications［J］.World J Biol Psychiatry,2018,19(Suppl 2):S41-S51.

相似文献/References:

[1]张志强,郭磊.结核性包裹性胸膜炎形成原因探讨[J].新乡医学院学报,2005,22(01):057.
[2]高景利,崔俊伟,石卓林,等.胸腔积液中微RNA-29a和腺苷脱氨酶联合检测对结核性胸膜炎的临床诊断效能[J].新乡医学院学报,2021,38(8):746.[doi:10.7683/xxyxyxb.2021.08.010]
　GAO Jingli,CUI Junwei,SHI Zhuolin,et al.Clinical efficacy of combined detection of microRNA-29a and adenosine deaminase in pleural effusion for the diagnosis of tuberculous pleuritis[J].Journal of Xinxiang Medical University,2021,38(4):746.[doi:10.7683/xxyxyxb.2021.08.010]
[3]崔俊伟,王永亮,蔡瑞艳,等.中心静脉导管胸腔闭式引流联合超声引导靶位给药治疗结核性胸膜炎疗效观察[J].新乡医学院学报,2018,35(5):400.[doi:10.7683/xxyxyxb.2018.05.011]
　CUI Jun-wei,WANG Yong-liang,CAI Rui-yan,et al.Clinical effect of central venous catheter thoracic drainage combined with ultrasound guided target therapy in treating patients with tuberculous pleurisy[J].Journal of Xinxiang Medical University,2018,35(4):400.[doi:10.7683/xxyxyxb.2018.05.011]

更新日期/Last Update: 2023-04-05