基于NLRP3/Caspase-1信号通路探讨苁蓉舒痉颗粒调控小胶质细胞极化抑制神经炎症的作用及机制OA北大核心CSTPCD
Exploring the role and mechanism of Congrong Shujing Granules in regulating microglial polarization and inhibiting neuroinflammation based on the NLRP3/Caspase-1 signaling pathway
目的 探讨苁蓉舒痉颗粒通过核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(NLRP3)/半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)信号通路促进小胶质细胞极化进而抑制神经炎症的作用及其机制.方法 20 只SD大鼠按随机数字表法分为空白血清组和苁蓉舒痉颗粒含药血清组,每组10 只.苁蓉舒痉颗粒含药血清组大鼠灌胃苁蓉舒痉颗粒(2.57 g/kg),空白血清组灌胃等量生理盐水.制备空白血清和苁蓉舒痉颗粒含药血清.体外培养小鼠小胶质细胞BV-2,利用CCK-8 法和免疫荧光法筛选出1-甲基-4-苯基-吡啶离子(MPP+)最佳造模浓度和苁蓉舒痉颗粒含药血清最优体积分数,并以 NLRP3 抑制剂 MCC950 为阳性对照.将细胞分为空白血清组(10%空白血清)、模型组(10%空白血清+500 μmol/L MPP+)、苁蓉舒痉颗粒含药血清组(10%苁蓉舒痉颗粒含药血清+500 μmol/L MPP+)、MCC950 组(10%空白血清+10 μmol/L MCC950+500 μmol/L MPP+),分别进行干预.干预 14 h后,观察BV-2 细胞形态学变化;采用酶联免疫吸附测定检测白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、IL-6 和IL-4 含量;实时荧光定量PCR法检测分化簇 86(CD86)、诱导型一氧化氮合酶(iNOS)、分化簇 206(CD206)、精氨酸酶 1(Arg1)mRNA表达;免疫荧光法检测CD86、Arg1、离子钙结合衔接分子 1(Iba1)、NLRP3 平均荧光强度;蛋白质印迹法检测 iNOS、Arg1、TNF-α、IL-6、IL-4、NLRP3、半胱氨酸天冬氨酸蛋白酶-1 前体(pro-Caspase-1)、Caspase-1 蛋白表达.结果 MPP+(12 h、500 μmol/L)处理下Iba1 激活表达增强(P<0.05),细胞活力未受影响;10%苁蓉舒痉颗粒含药血清单独或联合MPP+处理对细胞活力影响均减少(P>0.05).与空白血清组比较,模型组BV-2 细胞多分枝突起,呈阿米巴状;IL-1β、TNF-α、IL-6含量增加,IL-4 含量降低;CD86、iNOS mRNA表达升高,CD206、Arg1 mRNA表达降低;CD86、Iba1、NLRP3 平均荧光强度增强,Arg1 平均荧光强度减弱;iNOS、TNF-α、IL-6、NLRP3、pro-Caspase-1、Caspase-1 蛋白表达升高,Arg1、IL-4 蛋白表达下降;均P<0.05.与模型组比较,苁蓉舒痉颗粒含药血清组和MCC950 组细胞突起分枝减少,细胞激活减少;IL-1β、TNF-α、IL-6 的含量减少,IL-4 的含量增多;CD86、iNOS mRNA表达降低,CD206 mRNA表达升高;CD86、Iba1、NLRP3 平均荧光强度降低,Arg1 平均荧光强度增强;iNOS、TNF-α、IL-6、NLRP3、pro-Caspase-1、Caspase-1 蛋白表达下降,Arg1、IL-4 蛋白表达升高;均 P<0.05.结论 苁蓉舒痉颗粒含药血清可能通过抑制 NLRP3/Caspase-1 信号通路调控小胶质细胞极化,缓解MPP+诱导的神经炎症反应.
Objective To investigate the effect and mechanism of Congrong Shujing Granules on promoting microglial polarization and inhibiting neuroinflammation through the nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)/Caspase-1 signaling pathway.Methods Twenty Sprague-Dawley rats were assigned to the blank serum and Congrong Shujing Granules containing serum groups using random number table method,with 10 rats in each group.Rats in the Congrong Shujing Granules containing serum group received intragastric administration of Congrong Shujing Granules(2.57 g/kg)and the rats in the blank serum group received intragastric administration of physiological saline of equal volume.Blank serum and Congrong Shujing Granules containing serum were prepared separately.Mouse microglia cells BV-2 were cultured in vitro,and the optimal concentration of 1-methyl-4-phenylpyridine(MPP+)and optimal volume fraction of Congrong Shujing Granules containing serum were selected by the CCK-8 assay and immunofluorescence staining.And the NLRP3 inhibitor MCC950 was used as a postive control.Cells were divided into the blank serum group(10%blank serum),model group(10%blank serum+500 μmol/L MPP+),Congrong Shujing Granules containing serum group(10%Congrong Shujing Granules containing serum+500 μmol/L MPP+),and MCC950 group(10%blank serum+10 μmol/L MCC950+500 μmol/L MPP+),and intervened separately.After 14 h of intervention,morphological changes in BV-2 cells were observed.The contents of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6,and IL-4 were detected by an enzyme-linked immunosorbent assay.The mRNA expressions of differentiation cluster 86(CD86),inducible nitric oxide synthase(iNOS),CD206,and arginase 1(Arg1)were detected by real-time fluorescence PCR.The expressions of CD86,Arg1,Ionized calcium binding adaptor molecule 1(Iba1),and NLRP3 were detected by immunofluorescence staining.The expression of iNOS,Arg1,TNF-α,IL-6,IL-4,NLRP3,pro-cysteinyl aspartate specific proteinase 1(pro-Caspase-1),and Caspase-1 proteins was detected by Western blotting.Results Iba1 activation and expression increased under the MPP+(12 h,500 μmol/L)intervention(P<0.05),and cell viability was not affected.There was no statistically significant effect on cell viability after treatment with 10%Congrong Shujing Granules containing serum alone or in combination with MPP+(P>0.05).Compared to the blank serum group,BV-2 cells in the model group showed multiple branches and protruded in the shape of an amoeba.The contents of IL-1β,TNF-α,and IL-6 increased,while the contents of IL-4 decreased.The mRNA expressions of CD86 and iNOS increased,while mRNA expressions of CD206 and Arg1 decreased.The mean fluorescence intensity of CD86,Iba1,and NLRP3 increased,while the mean fluorescence intensity of Arg1 decreased.The protein expressions of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 increased,while the protein expressions of Arg1,IL-4 decreased,P<0.05.Compared to the model group,the Congrong Shujing Granules containing serum group and MCC950 group showed a decrease in the branch of cell protrusions,reduced cell activation,decreased levels of IL-1β,TNF-α,and IL-6,increased levels of IL-4,decreased expression of CD86 and iNOS mRNA,increased expression of CD206 mRNA,the decreased mean fluorescence intensity of CD86,Iba1,and NLRP3,the increased mean fluorescence intensity of Arg1,decreased expression of iNOS,TNF-α,IL-6,NLRP3,pro-Caspase-1,and Caspase-1 proteins,and increased expression of Arg1 and IL-4 proteins,P<0.05.Conclusion Congrong Shujing Granules containing serum may alleviate the MPP+-induced neuroinflammatory response by inhibiting the NLRP3/Caspase-1 signaling pathway to regulate M1/M2 phenotype polarization of microglia.
周美;林瑶;许茜;高正涛;林萍燕;周炳灿;陈明恒;张楚天;成慧灵;刘海鑫;蔡晶
福建中医药大学中西医结合学院 中西医结合研究院 福州 350122
中医学
苁蓉舒痉颗粒神经炎症小胶质细胞极化核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3/半胱氨酸天冬氨酸蛋白酶-1信号通路大鼠
Congrong Shujing Granulesneuroinflammationmicroglia polarizationnucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3/Caspase-1 signaling pathwayrats
《北京中医药大学学报》 2024 (012)
1710-1723 / 14
国家自然科学基金青年项目(No.81904263),福建省自然科学基金面上项目(No.2024J01123,No.2024J01745),福建省中医药科技项目(No.2021zyjc02),福建中医药大学中医健康管理学联合省级临床重点专科建设项目(老年病科)(No.XJG2023018),福建中医药大学校管课题青年项目(No.X2023010),福建省"大学生创新创业训练计划"项目 National Natural Science Foundation of China(No.81904263)
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