Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (21): 4315-4326.doi: 10.3864/j.issn.0578-1752.2022.21.017

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Effect of Matrine on NLRP3 Inflammasome Signaling Pathway in H9N2 AIV Infected Mice

WEI JingJie(),JIANG NingBo(),LIANG Yan,ZHANG Qian,SUN YingJian,HU Ge()   

  1. College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206
  • Received:2021-08-17 Accepted:2022-02-18 Online:2022-11-01 Published:2022-11-09
  • Contact: Ge HU E-mail:329974543@qq.com;1176693232@qq.com;bnhuge@126.com

RichHTML

40

PDF

58

Abstract:

【Objective】 Influenza virus infection causes inflammatory response and imbalance of immune homeostasis. The resulting cytokine storm (CS) is the main cause of death of infected hosts. The aim of this study was to explore the protective effect of Matrine on H9N2 AIV infected mice as well as the characteristics and preliminary mechanism of regulating NLRP3 inflammatory body signal pathway, so as to further improve the theoretical basis of antiviral of traditional Chinese medicine and lay a foundation for the development of new antiviral drugs.【Method】72 8-week-old BALB/c mice were randomly divided into blank control group (0.1 mL sterile chicken embryo allantoic fluid) and virus group (0.1 mL 4×105 PFU/mL H9N2 AIV), amantadine group (0.1 mL 4×105 PFU/mL H9N2 AIV + 100×10-6 99% amantadine), matrine high concentration treatment group (0.1 mL 4×105 PFU/mL H9N2 AIV + 40 mL·kg-1 matrine), medium concentration treatment group (0.1 mL 4×105PFU/mLH9N2 AIV + 20 mL·kg-1 matrine), and low concentration treatment group (0.1 mL 4×105PFU/mL H9N2 AIV + 10 mL·kg-1 matrine), and allantoic fluid nasal drops were used to construct the mouse model of viral pneumonia. The matrine was administered by gavage or drinking water for 5 consecutive days, and the test was conducted for 7 days. The weight changes of mice in different groups were observed. On the 1st, 3rd, 5th and 7th days, three mice were aseptically collected and killed, and the lung tissue was taken for histopathological observation, the expression of H9N2 and NLRP3 gene in mouse lung tissues was detected by RT-PCR, the expression of NLRP3 inflammatory body signal pathway related protein in lung tissue of H9N2 infected mice after matrine treatment was detected by Western blot, and the cytokine TNF in mouse serum was measured by ELISA- α and IL-1 β changes in the expression of IL-18 and IL-10.【Result】Compared with the virus group, the area of pulmonary edema and the number of inflammatory cells in the pathological section of H9N2 AIV infected mice in the high concentration matrine treatment group were significantly reduced, and the exudation of red blood cells decreased, while the effect was close to that in the amantadine group. At day 7, the alveolar wall of mice in the high concentration matrine treatment group was intact, the boundary between alveoli was clear, and the number of inflammatory cells and plasma cells in lung tissue decreased significantly, which was almost the same as that in the blank group; in the treatment group with medium concentration of matrine, there was a small amount of bleeding in the lung tissue, there was no swollen fluid in the alveoli, and the alveolar septum was intact; in the low concentration matrine treatment group, the red blood cells exuded from the alveoli, a large number of plasma cells were recruited, and there was fusion between the alveoli. The expression of H9N2 virus gene in lung tissue of mice treated with Matrine and amantadine decreased significantly on day 3, 5 and 7 (P<0.01). After 3 and 5 days of treatment, the expression of NLRP3 gene, protein and TNF- α, IL-1 β in matrine high concentration treatment group and amantadine group decreased significantly (P<0.01); on day 7, the expression of NLRP3 gene in lung tissue of mice in matrine high, medium and low treatment groups decreased significantly (P<0.01); the expression of NLRP3 protein, caspase-1 protein in low concentration group and caspase-1 protein in medium concentration group decreased significantly on day 5 (P<0.05); the expression of TNF- α and IL-1 β in matrine medium and low concentration groups on days 3 and 5 decreased significantly (P<0.01).The expression of IL-10 in Shegan medium concentration treatment group decreased significantly.【Conclusion】Matrine could inhibit the expression of H9N2 AIV in vivo, reduce the expression of TNF-α, IL-1β and IL-10 by down regulating NLRP3 inflammatory body signal pathway related proteins, and reduce the inflammatory response, which had good antiviral and anti-inflammatory effects.

Key words: matrine, H9N2 AIV, NLRP3 inflammasome signaling pathway

Table 1

Sequence and fragment size of primers for NA, NLR and reference GAPDH gene"

基因 Gene 引物序列 Primer sequence (5′ to 3′) 片段大小 Fragment size (bp)
NA Forward primer：TGTGGGGCATAAATCATCA
Forward primer：ATGAGGCGACAGTCGAATTAA		 224
GAPDH Forward primer：TTGGCTACAGCAACAGGGTG
Forward primer：GAGGAGATGCTCGGTGTGTT		 236
NLR Forward primer：TGAAGTGGATTGAAGTGAAAGCC
Forward primer：TGTGAAAAAAACCCAGGGAAAGC		 256

Fig. 1

HE staining results of mice in the experimental group after two drug treatments on the third day of virus infection A. Matrine high dose; B. Matrine medium dose; C. Matrine low dose; D. Amantadine; E. Virus positive; F. Blank control; ★ : Inflammatory cell recruitment; ▲ : Pulmonary edema"

Fig. 2

H.E staining results of mice in the experimental group after four times of drug treatment on the fifth day of virus infection A. Matrine high dose; B. Matrine medium dose; C. Matrine low dose; D. Amantadine; E. Virus positive; F. Blank control; ★ : Inflammatory cell recruitment"

Fig. 3

H.E staining results of experimental group mice after five times of drug treatment on the seventh day of virus infection A. Matrine high dose; B. Matrine medium dose; C. Matrine low dose; D. Amantadine; E. Virus positive; F. Blank control; ★: Inflammatory cell recruitment"

Fig. 4

Expression of mRNA in lung tissues of mice infected with H9N2 AIV Compared with virus group *P<0.05; **P<0.01"

Fig. 5

Expression of NLRP3mRNA in lung tissues of mice infected with H9N2 AIV Compared with virus group *P<0.05; **P<0.01"

Fig. 6

Relative expression of NF-κB, NLRP3, Caspase-1 protein after five drug treatments on day 7 of viral infection"

Fig. 7

Gray value analysis of NF-κB, NLRP3, Caspase-1 Compared with virus group *P<0.05; **P<0.01; Compared with control group #P<0.05; ##P<0.01"

Fig. 8

Expression of TNF-α in lung tissues of mice infected with H9N2 AIV Compared with virus group *P<0.05; **P<0.01; Compared with the control group #P<0.05; ##P<0.01. The same as below"

Fig. 9

Expression of IL-1β in H9N2 AIV infected mice treated with drugs Compared with virus group *P<0.05; **P<0.01; Compared with the control group #P<0.05; ##P<0.01"

Fig. 10

Expression of IL-10 in mice infected with H9N2 AIV after drug treatment Compared with virus group *P<0.05; **P<0.01; Compared with the control group #P<0.05; ##P<0.01"

[1] 陈超, 池晓娟, 白庆玲, 陈吉龙. 甲型流感病毒感染过程中干扰素介导的天然免疫应答机制. 生物工程学报, 2015, 31(12): 1671-1681. doi:10.13345/j.cjb.150296.
CHEN C, CHI X J, BAI Q L, CHEN J L. Mechanisms underlying interferon-mediated host innate immunity during influenza A virus infection. Chinese Journal of Biotechnology, 2015, 31(12): 1671-1681. doi:10.13345/j.cjb.150296. (in Chinese)
[2] 张雨婷, 韩旭. 流感病毒性肺炎与细菌性肺炎的临床特点分析. 中国基层医药, 2020, 27(8): 932-935. doi:10.3760/cma.j.issn.1008-6706.2020.08.009.
ZHANG Y T, HAN X. Analysis of the different clinical characteristics between influenza virus pneumonia and bacterial pneumonia. Chinese Journal of Primary Medicine and Pharmacy, 2020, 27(8): 932-935. doi:10.3760/cma.j.issn.1008-6706.2020.08.009. (in Chinese)
[3] 秦颖, 赵伟. NLRP3炎症小体的负向调控机制. 中国科学: 生命科学, 2018, 48(11): 1187-1196.
QIN Y, ZHAO W. Negative regulatory mechanisms of NLRP3 inflammasome. Scientia Sinica (Vitae), 2018, 48(11): 1187-1196. (in Chinese)
[4] TATE M D, ONG J D H, DOWLING J K, MCAULEY J L, ROBERTSON A B, LATZ E, DRUMMOND G R, COOPER M A, HERTZOG P J, MANSELL A. Reassessing the role of the NLRP3 inflammasome during pathogenic influenza A virus infection via temporal inhibition. Scientific Reports, 2016, 6: 27912. doi:10.1038/srep27912.
[5] 李卫东, 颜源均, 赵世桥, 冯尧, 蒲志强, 冯飞. NLRP3对病毒感染心肌细胞凋亡及炎症反应的作用. 西部医学, 2021, 33(7): 963-969. doi:10.3969/j.issn.1672-3511.2021.07.006.
LI W D, YAN Y J, ZHAO S Q, FENG Y, PU Z Q, FENG F. Effect of NLRP3 on inflammatory response and apoptosis of cardiomyocytes with virus infection in vitro. Northwest Pharmaceutical Journal, 2021, 33(7): 963-969. doi:10.3969/j.issn.1672-3511.2021.07.006. (in Chinese)
[6] 宋祯彦, 夏小芳, 王玉珂, 郑雨珊, 陈佩莹, 罗德勇, 贺春香, 于文静, 李平, 成绍武. 当归芍药散通过调控NLRP3/Caspase-1信号通路抑制AD大鼠神经炎症的作用. 中国实验方剂学杂志, 2021, 27(19): 1-8. doi:10.13422/j.cnki.syfjx.20211802.
SONG Z Y, XIA X F, WANG Y K, ZHENG Y S, CHEN P Y, LUO D Y, HE C X, YU W J, LI P, CHENG S W. Danggui Shaoyaosan inhibits neuroinflammation in AD rats by regulating NLRP3/caspase-1 pathway. Chinese Journal of Experimental Traditional Medical Formulae, 2021, 27(19): 1-8. doi:10.13422/j.cnki.syfjx.20211802. (in Chinese)
[7] 董婉茹, 李寒, 李云凤, 王宁, 马伯艳, 陆阁玲, 辛相如, 陈冰冰. 基于骨骼肌NLRP3/caspase-1/IL-1β、IL-18通路的黄连温胆汤改善IGT机制研究. 中国中药杂志, 2021, 46(17): 4480-4487. doi:10. 19540/j.cnki.cjcmm.20210621.401.
DONG W R, LI H, LI Y F, WANG N, MA B Y, LU G L, XIN X R, CHEN B B. Mechanism of Huanglian Wendan Decoction in improving impaired glucose tolerance based on skeletal muscle NLRP3/caspase-1/IL-1β, IL-18 pathway. China Journal of Chinese Materia Medica, 2021, 46(17): 4480-4487. doi:10.19540/j.cnki.cjcmm.20210621.401. (in Chinese)
[8] 魏小林, 谢敏. NALP3炎性复合体与MAPK、NF-κB及ROS信号通路之间的关系. 国际呼吸杂志, 2015, 35(6): 476-480. doi:10.3760/cma.j.issn.1673-436X.2015.06.017.
WEI X L, XIE M. Relationship between NALP3 inflammasome and MAPK, NF-κB, ROS signal pathway. International Journal of Respiration, 2015, 35(6): 476-480. doi:10.3760/cma.j.issn.1673-436X.2015.06.017. (in Chinese)
[9] SUN P P, SUN N, YIN W, SUN Y G, FAN K H, GUO J H, KHAN A, HE Y M, LI H Q. Matrine inhibits IL-1β secretion in primary porcine alveolar macrophages through the MyD88/NF-κB pathway and NLRP3 inflammasome. Veterinary Research, 2019, 50(1): 53. doi:10.1186/s13567-019-0671-x.
[10] 庞博, 董军杰, 庞国勋. 清热解毒类中药的药理作用及临床应用探讨. 临床合理用药杂志, 2013, 6(31): 180-181. doi:10.15887/j.cnki.13-1389/r.2013.31.017.
PANG B, DONG J J, PANG G X. Pharmacological action and clinical application of traditional Chinese medicine for clearing heat and detoxification. Chinese Journal of Clinical Rational Drug Use, 2013, 6(31): 180-181. doi:10.15887/j.cnki.13-1389/r.2013.31.017. (in Chinese)
[11] COOK D N, BECK M A, COFFMAN T M, KIRBY S L, SHERIDAN J F, PRAGNELL I B, SMITHIES O. Requirement of MIP-1 alpha for an inflammatory response to viral infection. Journal of Medicinal Chemistry, 1995, 269(5230): 1583-1585. doi:10.1126/science.7667639.
[12] GUZMAN J R, KOO J S, GOLDSMITH J R ", MÜHLBAUER M, NARULA A, JOBIN C. Oxymatrine prevents NF-κB nuclear translocation and ameliorates acute intestinal inflammation. Scientific Reports, 2013, 3: 1629. doi:10.1038/srep01629.
[13] LIANG J, CHANG B Y, HUANG M C, HUANG W C, MA W K, LIU Y, TAI W, LONG Y, LU Y. Oxymatrine prevents synovial inflammation and migration via blocking NF-κB activation in rheumatoid fibroblast- like synoviocytes. International Immunopharmacology, 2018, 55: 105-111. doi:10.1016/j.intimp.2017.12.006.
[14] ZHANG Y, SUN S G, CHEN J, REN P C, HU Y S, CAO Z, SUN H H, DING Y. Oxymatrine induces mitochondria dependent apoptosis in human osteosarcoma MNNG/HOS cells through inhibition of PI3K/Akt pathway. Tumor Biology, 2014, 35(2): 1619-1625. doi:10.1007/s13277-013-1223-z.
[15] JIANG G J, LIU X H, WANG M, CHEN H, CHEN Z Y, QIU T. Oxymatrine ameliorates renal ischemia-reperfusion injury from oxidative stress through Nrf2/HO-1 pathway. Acta Cirurgica Brasileira, 2015, 30(6): 422-429. doi:10.1590/S0102-865020150060000008.
[16] YAO N, WANG X. In vitro immunomodulatory activity of oxymatrine on Toll-like receptor 9 signal pathway in chronic hepatitis B. The American Journal of Chinese Medicine, 2014, 42(6): 1399-1410. doi:10.1142/s0192415x14500888.
[17] SHI L J, SHI L, SONG G Y, ZHANG H F, HU Z J, WANG C, ZHANG D H. Oxymatrine attenuates hepatic steatosis in non- alcoholic fatty liver disease rats fed with high fructose diet through inhibition of sterol regulatory element binding transcription factor 1 (Srebf1) and activation of peroxisome proliferator activated receptor alpha (Pparα). European Journal of Pharmacology, 2013, 714(1/2/3): 89-95. doi:10.1016/j.ejphar.2013.06.013.
[18] LIU Y, XU Y, JI W D, LI X Y, SUN B, GAO Q G, SU C Q. Anti-tumor activities of matrine and oxymatrine: Literature review. Tumor Biology, 2014, 35(6): 5111-5119. doi:10.1007/s13277-014-1680-z.
[19] FEI Z W, QIU M K, QI X Q, DAI Y X, WANG S Q, QUAN Z W, LIU Y B, OU J M. Oxymatrine suppresses proliferation and induces apoptosis of hemangioma cells through inhibition of HIF-1a signaling. International Journal of Immunopathology and Pharmacology, 2015, 28(2): 201-208. doi:10.1177/0394632015578342.
[20] WEN J B, ZHU F Q, CHEN W G, JIANG L P, CHEN J, HU Z P, HUANG Y J, ZHOU Z W, WANG G L, LIN H, ZHOU S F. Oxymatrine improves intestinal epithelial barrier function involving NF-κB-mediated signaling pathway in CCl4-induced cirrhotic rats. PLoS ONE, 2014, 9(8): e106082. doi:10.1371/journal.pone.0106082.
[21] 孙娜. 苦参碱抗PRRSV/PCV2共感染及其作用机制[D]. 太谷: 山西农业大学, 2016.
SUN N. Matrine inhibited co-infection of PRRSV/PCV2 and its mechanism[D]. Taigu: Shanxi Agricultural University, 2016. (in Chinese)
[22] 王伟, 丁萌楠, 张敏, 张艺凡, 孟凡飞, 徐欢, 张冲冲, 卢春凤, 宋伦. 热应激通过激活NLRP3/IL-1β/IL-6途径介导肝脏炎症损伤效应. 军事医学, 2021, 45(7): 481-484. doi:10.7644/j.issn.1674-9960.2021.07.001.
WANG W, DING M N, ZHANG M, ZHANG Y F, MENG F F, XU H, ZHANG C C, LU C F, SONG L. Heat stress mediates hepatic inflammatory injury by activating the NLRP3/IL-1β/IL-6 pathway. Military Medical Sciences, 2021, 45(7): 481-484. doi:10.7644/j.issn.1674-9960.2021.07.001. (in Chinese)
[23] YANG Y, WANG H N, KOUADIR M, SONG H H, SHI F S. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death & Disease, 2019, 10(2): 128. doi:10.1038/s41419-019-1413-8.
[24] LUO M, YAN D, SUN Q, TAO J, XU L, SUN H, ZHAO H. Ginsenoside Rg1 attenuates cardiomyocyte apoptosis and inflammation via the TLR4/NF-kB/NLRP3 pathway. Journal of Cellular Biochemistry, 2020, 121(4): 2994-3004. doi:10.1002/jcb.29556.
[25] 顾晓凌, 宋勇. NALP3炎性体在急性肺损伤中的研究进展. 中国呼吸与危重监护杂志, 2012, 11(6): 607-609.
GU X L, SONG Y. Research progress of nalp3 inflammatory body in acute lung injury. Chinese Journal of Respiratory and Critical Care Medicine, 2012, 11(6): 607-609. (in Chinese)
[26] ZHANG R H, AI X, DUAN Y J, XUE M, HE W X, WANG C L, XU T, XU M J, LIU B J, LI C H, WANG Z J, ZHANG R H, WANG G H, TIAN S F, LIU H F. Kaempferol ameliorates H9N2 swine influenza virus-induced acute lung injury by inactivation of TLR4/MyD88- mediated NF-κB and MAPK signaling pathways. Biomedicine & Pharmacotherapy, 2017, 89: 660-672. doi:10.1016/j.biopha.2017.02.081.
[27] WANG Q, LIN B F, LI Z F, SU J, FENG Y L. Cichoric acid ameliorates monosodium urate-induced inflammatory response by reducing NLRP3 inflammasome activation via inhibition of NF-kB signaling pathway. Evidence-Based Complementary and Alternative Medicine, 2021, 2021: 8868527. doi:10.1155/2021/8868527.
[28] 陈娇, 钱晓明, 聂时南. 急性肺损伤动物模型的研究现状. 医学研究生学报, 2013, 26(8): 851-854. doi:10.16571/j.cnki.1008-8199.2013.08.008.
CHEN J, QIAN X M, NIE S N. Current situation of the research about the animal models of acute lung injury. Journal of Medical Postgraduates, 2013, 26(8): 851-854. doi:10.16571/j.cnki.1008-8199.2013.08.008. (in Chinese)
[29] 李俏琦, 杨茜, 高玲, 易成, 黄英. 细胞因子风暴与病毒性肺炎. 中国呼吸与危重监护杂志, 2021, 20(1): 70-75.
LI Q Q, YANG X, GAO L, YI C, HUANG Y. Cytokine storm and viral pneumonia: A review. Chinese Journal of Respiratory and Critical Care Medicine, 2021, 20(1): 70-75. (in Chinese)
[30] 魏东, 刘英, 贾宁, 徐彤, 刘贵河. H9N2亚型猪流感病毒诱导小鼠急性肺损伤中TNF-α、IL-1β、IL-6和IL-10的变化和作用. 中国实验动物学报, 2013, 21(2): 72-74. doi:10.3969/j.issn.1005-4847.2013.02.015.
WEI D, LIU Y, JIA N, XU T, LIU G H. Changes and roles of TNF-α, IL-1β, IL-6 and IL-10 in acute lung injury induced by H9N2 subtype swine influenza virus in mice. Acta Laboratorium Animalis Scientia Sinica, 2013, 21(2): 72-74. doi:10.3969/j.issn.1005-4847.2013.02.015. (in Chinese)
[1] YU Yong-Tao, HE Sheng-Hu, ZHAO Qing-Mei. Isolation and Identification of Matrine-Producing Fungal Endophytes from Sophora alopecuroides in Ningxia [J]. Scientia Agricultura Sinica, 2013, 46(13): 2643-2654.
[2] ,,,,. Bacteriostatic Action and Mechanism of Sophora flavescens Ait on Escherichia coli 01 C84010 [J]. Scientia Agricultura Sinica, 2006, 39(05): 1018-1024 .
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd