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Volume 39 Issue 2
Mar.  2021
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HUA Rong, CHEN Yao. Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(2): 143-147. doi: 10.12206/j.issn.1006-0111.202101003
Citation: HUA Rong, CHEN Yao. Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(2): 143-147. doi: 10.12206/j.issn.1006-0111.202101003

Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome

doi: 10.12206/j.issn.1006-0111.202101003
  • Received Date: 2021-01-03
  • Rev Recd Date: 2021-03-07
  • Available Online: 2021-03-31
  • Publish Date: 2021-03-25
  •   Objective  To find the effect of leonurine on LPS-induced macrophages activation and its potential mechanism.  Methods  Mouse primary peritoneal macrophages were isolated and pretreated for 24 h with LPS and leonurine. MTT assay was used to detect the cell viability of macrophages. The production of IL-1β, IL-6, TNF-α and IL-18 in culture medium were tested by ELISA, and the production of NO was detected by Griess reagent. The mRNA expression of NLRP3, ASC, caspase-1, TNF-α, iNOS, Arg-1 and CD206 were detected by RT-PCR, and the protein expression of NLRP3, ASC and caspase-1 were detected by Western blotting.  Results  LPS can significantly increase the releases of NO、IL-1β、IL-6、TNF-α and IL-18 from macrophages. Leonurine can suppress the expression of pro-inflammatory factor levels, such as IL-1β (P<0.05), IL-18 (P<0.05), NO(P<0.05), IL-6(P<0.05) and TNF-α (P<0.05). Leonurine can decrease the activation of macrophage as well as the expression of NLRP3 Inflammasome.Protein expressions of NLRP3、ASC、caspase-1 were mitigated.   Conclution   Leonurine exerts beneficial effects through M1/M2 phenotypic differentiation of peritoneal macrophage via inhibiting overactivation of NLRP3 inflammasome. These findings suggest that leonurine might have a therapeutic potential for pelvic inflammatory disease.
  • [1] ZAPOROZHAN V, MARICHEREDA V, SITNIK P. Inflammation biomarkers in pelvic inflammatory disease[J]. Eur J Obstet Gynecol Reproductive Biol,2019,234:e43.
    [2] CHYLIKOVA J, DVORACKOVA J, TAUBER Z, et al. M1/M2 macrophage polarization in human obese adipose tissue[J]. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub,2018,162(2):79-82. doi:  10.5507/bp.2018.015
    [3] LANDIS R C, QUIMBY K R, GREENIDGE A R. M1/M2 macrophages in diabetic nephropathy: Nrf2/HO-1 as therapeutic targets[J]. Curr Pharm Des,2018,24(20):2241-2249. doi:  10.2174/1381612824666180716163845
    [4] COLIN S, CHINETTI-GBAGUIDI G, STAELS B. Macrophage phenotypes in atherosclerosis[J]. Immunol Rev,2014,262(1):153-166. doi:  10.1111/imr.12218
    [5] 乔晶晶, 吴啟南, 薛敏, 等. 益母草化学成分与药理作用研究进展[J]. 中草药, 2018, 49(23):5691-5704.
    [6] LIU H, ZHANG X, DU Y, et al. Leonurine protects brain injury by increased activities of UCP4, SOD, CAT and Bcl-2, decreased levels of MDA and Bax, and ameliorated ultrastructure of mitochondria in experimental stroke[J]. Brain Res,2012,1474:73-81. doi:  10.1016/j.brainres.2012.07.028
    [7] CAO H, SETHUMADHAVAN K, LI K, et al. Cinnamon polyphenol extract and insulin regulate diacylglycerol acyltransferase gene expression in mouse adipocytes and macrophages[J]. Plant Foods Hum Nutr,2019,74(1):115-121. doi:  10.1007/s11130-018-0709-7
    [8] 谢怡. 蒿芩清胆汤联合克林霉素磷酸酯治疗盆腔炎性疾病后遗症的效果及对血清辅助性T细胞1/辅助性T细胞2和粒细胞-巨噬细胞集落刺激因子水平的影响[J]. 中国医药, 2018, 13(7):1083-1086.
    [9] TRACEY K J. The inflammatory reflex[J]. Nature,2002,420(6917):853-859. doi:  10.1038/nature01321
    [10] 丘甜美, 何援利, 蔡慧华. 子宫内膜炎性反应与宫腔粘连的相关性[J]. 现代妇产科进展, 2019, 28(4):317-318, 320.
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Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome

doi: 10.12206/j.issn.1006-0111.202101003

Abstract:   Objective  To find the effect of leonurine on LPS-induced macrophages activation and its potential mechanism.  Methods  Mouse primary peritoneal macrophages were isolated and pretreated for 24 h with LPS and leonurine. MTT assay was used to detect the cell viability of macrophages. The production of IL-1β, IL-6, TNF-α and IL-18 in culture medium were tested by ELISA, and the production of NO was detected by Griess reagent. The mRNA expression of NLRP3, ASC, caspase-1, TNF-α, iNOS, Arg-1 and CD206 were detected by RT-PCR, and the protein expression of NLRP3, ASC and caspase-1 were detected by Western blotting.  Results  LPS can significantly increase the releases of NO、IL-1β、IL-6、TNF-α and IL-18 from macrophages. Leonurine can suppress the expression of pro-inflammatory factor levels, such as IL-1β (P<0.05), IL-18 (P<0.05), NO(P<0.05), IL-6(P<0.05) and TNF-α (P<0.05). Leonurine can decrease the activation of macrophage as well as the expression of NLRP3 Inflammasome.Protein expressions of NLRP3、ASC、caspase-1 were mitigated.   Conclution   Leonurine exerts beneficial effects through M1/M2 phenotypic differentiation of peritoneal macrophage via inhibiting overactivation of NLRP3 inflammasome. These findings suggest that leonurine might have a therapeutic potential for pelvic inflammatory disease.

HUA Rong, CHEN Yao. Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(2): 143-147. doi: 10.12206/j.issn.1006-0111.202101003
Citation: HUA Rong, CHEN Yao. Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(2): 143-147. doi: 10.12206/j.issn.1006-0111.202101003
  • 盆腔炎是一种常发病于年轻女性上生殖道感染的妇科疾病。临床研究发现,盆腔炎患者上生殖道内存在大量激活的巨噬细胞[1]。在炎症和病原体的刺激下,巨噬细胞过度激活可以释放出大量炎症因子,包括肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)。研究发现,NLRP3炎性小体激活可以促使caspase-1活化并切割IL-18、IL-1β前体,促进IL-18、IL-1β的成熟与释放,而抑制NLRP3炎性小体过度激活以减轻盆腔炎临床症状,并且与降低炎症因子、趋化因子释放有关。巨噬细胞在不同刺激下可以活化为不同表型:经典活化的M1型和替代活化的M2型。在含有脂多糖(LPS)和IFN-γ微环境中,巨噬细胞活化为变形虫样的M1型,参与炎症的发生。巨噬细胞在含有IL-4、IL-10、IL-13等抗炎因子微环境中被活化为M2型,表达精氨酸酶 1(Arg-1)和甘露糖受体1(CD206) 等特异性标志分子,参与炎症消退和组织重塑。研究发现,M1 /M2比例失衡是多种炎症性疾病的病理标志,如肥胖[2]、糖尿病[3]、动脉粥样硬化[4]等。

    益母草碱(LEO)是一种具有抗炎、抗氧化和抗肿瘤作用的天然黄酮类化合物[5],研究报道显示益母草碱可抑制Bax/Bcl-2信号通路激活抑制炎症因子的表达[6]。但鲜有益母草碱对巨噬细胞中NLRP3炎症小体影响的报道。本实验以益母草碱为研究对象,探讨其对巨噬细胞中NLRP3炎症小体激活的影响,以及对巨噬细胞M1/M2表型的调节作用。

  • 益母草碱(纯度>98%,西格玛奥德里奇贸易有限公司,上海);脂多糖(L6143)、DMEM高糖培养基、胎牛血清、NuPAGE 10% Bis-Tris Gel、10×MOPS SDS 运行缓冲液、10×传输缓冲液、预制蛋白Marker、荧光定量PCR、反转录试剂盒(美国赛默飞世尔科技公司);青-链霉素混合液、胰蛋白酶(美国Hyclone公司);Griess试剂盒(江苏碧云天生物试剂公司);IL-1β、IL-18、IL-6、TNF-α等ELISA试剂盒(武汉伊莱瑞特生物公司);PVDF膜(美国Millipore公司);caspase-1兔抗单克隆抗体、β-actin兔抗单克隆抗体、羊抗兔/羊抗鼠单克隆二抗(武汉三鹰生物技术有限公司);NLRP3兔抗单克隆抗体(英国Biorbyt生物试剂公司);四甲基偶氮唑蓝溶液(MTT,美国Bio-Rad公司);TRIzol Regent试剂盒(日本TaKaRa公司)。

  • C57BL/6小鼠,6周龄,雌性,体重(20±2) g,购于江苏集萃药康生物科技股份有限公司。小鼠饲养于实验室SPF级动物房,温度(22 ± 1)°C和湿度(60 ± 2)%,动物自由饮食。动物实验操作均通过实验动物伦理委员会批准。

  • 以颈椎脱臼的方式处死小鼠,置于75%的乙醇溶液中浸泡10 min,将15 ml PBS缓冲液注入小鼠腹中,仰卧平放,揉捏小鼠腹部5 min,吸出腹液,离心分离巨噬细胞,用DMEM培养液调整细胞浓度,在细胞培养箱中以5%CO2、37 ℃恒温孵育24 h后,换液,去除未贴壁细胞,即得到纯化的小鼠腹腔巨噬细胞[7]。以1.5×105个/ml密度接种于24孔(或96孔)板中培养24 h后,随机分为空白组、益母草碱(10 μmol/L)组、脂多糖(1 μg/ml)组、脂多糖+益母草碱(10 μmol/L)组,益母草碱预处理1 h之后加入脂多糖,放回孵箱中培养,24 h后,提取上清液和细胞蛋白,检测相关指标。

  • 脂多糖预处理巨噬细胞24 h后,在96孔板中每孔加入10 μl(5 mg/ml)MTT溶液,于37 ℃孵箱中培养,4 h后取出,移除细胞上清液,每孔加入200 μl二甲基亚砜,放置于恒温摇床震摇10 min,于562 nm处测定OA值,检测相关指标。

  • Griess试剂盒取出,恢复至室温。将细胞上清液和标准品和加入到96孔板中,将试剂I 和试剂II 混匀加入96孔板中,避光,放置于恒温摇床震摇30 min,于470 nm处测定OA值,检测NO含量。

  • 提前将ELISA试剂盒取出,恢复至室温。稀释细胞上清液,配置标准品工作液,按照ELISA试剂盒说明书要求依次加入反应液,最后加入终止液,于450 nm处检测OD值,计算IL-1β、IL-18、IL-6、TNF-α含量。

  • 按照Trizol法提取巨噬细胞中总RNA,根据逆转录试剂盒说明书,进行RT-PCR反应。设定反应条件为:5 ℃预变性30 s,接着95 ℃变性6 s,最后60 ℃退火,延伸37 s,重复反应40个循环。RT-PCR引物设计见(表1)。以GAPDH为内参,利用2−∆∆Ct方法分析结果。

    基因引物序列(5′→3′)
    NLRP3F: AGAAGAGACCACGGCAGAAG
    R: CCTTGGACCAGGTTCAGTGT
    ASCF: TGGATGCTCTGTACGGGAAG
    R: CCAGGCTGGTGTGAAACTGAA
    caspase-1F: CTTGGAAATAGCTCCCAGAA
    R: CATTTGGGAACTTCTCATCC
    TNF-αF: CCAATGGCAGAGTGGGTATG
    R: TGAAGAGGACCTGGGAGTAG
    iNOSF: GGGAATCTTGGAGCGAGTTG
    R: GTGAGGGCTTGGCTGAGTGA
    CD206F: CAGGTGTGGGCTCAGGTAGT
    R: TGGTGAGCTGAAAGGTGA
    Arg-1F: TTGCTGTGCTCCATAGTTTCCA
    R: CCATGCAAGTTTCCACTTGT
    GAPDHF: GGAGAAACCTGCCAAGTATG
    R: TTACTCCTTGGAGGCCATGTAG
  • 脂多糖处理巨噬细胞24 h后,弃去细胞上层培养基,置于冰上,PBS洗涤3次,加入RIPA裂解液(含1%PMSF)反应30 min,离心,收集上清液。BCA蛋白定量试剂盒检测蛋白含量,配置缓冲液,变性。每孔10 μl加入到10%预制胶中,设置电压200 V电泳30 min,设置电压25 V电转30 min,TBST洗涤,5%脱脂牛奶封闭2 h,TBST洗涤,4 ℃一抗孵育过夜,TBST洗涤,二抗孵育30 min,TBST洗涤,加入曝光剂,曝光。

  • 采用SPSS18.0分析实验中所涉及的数据,组间比较方差齐,用LSD检验,方差不齐采用 Dunnett’s T3检验,以P < 0.05为统计学差异,数据结果用均数 ± 标准误($\bar x \pm s$)表示。

  • 首先,观察益母草碱和脂多糖对巨噬细胞活力的影响(图1)。结果显示,脂多糖和益母草碱均能提高巨噬细胞的活力(P<0.05),当益母草碱与脂多糖共同刺激巨噬细胞时,巨噬细胞活力得到了进一步的增强(P<0.05)。

  • 观察益母草碱对巨噬细胞炎症因子释放的影响,在脂多糖刺激下,巨噬细胞上清液中NO的释放增加,而益母草碱可以抑制巨噬细胞NO释放(图2A)。检测脂多糖对巨噬细胞上清液中IL-1β、IL-18和IL-6释放的影响,结果发现,益母草碱可以降低巨噬细胞IL-1β、IL-18和IL-6释放(图2B-2D)。结果显示,益母草碱可以减少脂多糖引起的巨噬细胞炎症因子的释放。

  • 细胞内IL-1β、IL-18等炎症因子的释放需要经过NLRP3炎症小体的激活,为此,观察了益母草碱对NLRP3炎症小体激活的影响。RT-PCR结果显示(图3),脂多糖刺激后,NLRP3、ASC、caspase-1的mRNA表达增加,益母草碱可以降低mRNA表达。Western blot结果也证实益母草碱可以抑制脂多糖引起的巨噬细胞中NLRP3、ASC、caspase-1蛋白表达(图4)。

  • 应用RT-PCR检测益母草碱对巨噬细胞M1/M2表型的影响。正常情况下,巨噬细胞M1型标志物TNF-α和iNOS表达量较低,经脂多糖诱导刺激后TNF-α和iNOS的表达水平显著升高,益母草碱可以降低脂多糖引起的TNF-α和iNOS的mRNA表达(图5A5B)。另一方面,脂多糖诱导刺激后,巨噬细胞M2型标志物Arg-1和CD206的表达水平降低,益母草碱预处理可以增加脂多糖引起的Arg-1和CD206表达(图5C5D)。表明益母草碱可以调控脂多糖引起的巨噬细胞由M1向M2型转化。

  • 研究发现在盆腔炎患者的上生殖道内存在大量激活的巨噬细胞[8]。巨噬细胞是参与炎症反应的天然免疫细胞,当病原体入侵或者组织发生病变时,巨噬细胞分泌多种炎症因子,诱导更多的巨噬细胞活化、募集,加强局部抗炎作用。正常生理情况下,炎症因子的含量极少,具有维持机体免疫和调节心脑血管等功能[9]。但当机体长期受到病原微生物、致炎因子刺激时,会导致一系列病理改变,如长期慢性子宫内膜炎刺激可以增加子宫纤维化的发病率[10]。基于此,本实验利用革兰阴性菌来源的脂多糖刺激巨噬细胞,观察益母草碱对脂多糖诱导的巨噬细胞激活和炎症因子表达的影响。结果显示,脂多糖刺激可以引起巨噬细胞过度激活,相关炎症因子表达增加,益母草碱预处理可以减少炎症因子的表达和分泌,提示益母草碱的抗炎作用与抑制巨噬细胞中炎症因子的产生有关。

    为了进一步阐明益母草碱的抗炎作用,本实验对NLRP3炎症小体进行了研究。大量研究发现脂多糖可以激活NLRP3炎症小体,引起细胞因子释放增加。鉴于盆腔炎是炎性刺激引起的病理变化,且抑制NLRP3炎症小体可以降低炎症,推测抑制NLRP3炎症小体过度激活可能对盆腔炎起到一定的治疗效果。本实验中发现,益母草碱可以通过抑制脂多糖引起的巨噬细胞中NLRP3炎症小体相关蛋白表达,从而减少炎症因子释放,证实益母草碱可以抑制脂多糖诱导的巨噬细胞内NLRP3炎症小体的过度激活发挥抗炎作用。

    巨噬细胞的表型转化在盆腔炎的病理进程中发挥着重要作用,M1型巨噬细胞主要发挥促炎、吞噬病原体的作用,M2型巨噬细胞主要发挥促进组织重塑、损伤修复等。因此,在盆腔炎疾病中,M1型巨噬细胞能够加重上生殖道炎症进展,而M2型巨噬细胞能抑制疾病进展。为了明确脂多糖对巨噬细胞分化的影响,使用RT-PCR实验验证不同处理方式对巨噬细胞分型的影响。结果显示,脂多糖能够促进M1型标志物TNF-α和iNOS的mRNA表达,而益母草碱能明显抑制 TNF-α和iNOS的mRNA表达,同时促进M2型标志物Arg-1和CD206的mRNA表达。上述结果提示,益母草碱能抑制脂多糖诱导的巨噬细胞向M1型分化以及IL-18、IL-1β、TNF-α表达,促进巨噬细胞向M2型分化。

    在炎症反应过程中,脂多糖可以引起巨噬细胞中IL-1β、IL-18、IL-6、TNF-α等炎症因子表达增加,益母草碱可以通过抑制NLRP3炎症小体激活发挥其抗炎作用,提示抑制NLRP3炎症小体过度激活可能成为盆腔炎治疗的新策略,同时,本研究也为进一步开发益母草碱作为妇科用药提供理论基础。

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