α-倒捻子素通过NF-κB途径抑制LPS/ATP诱导的小胶质细胞中NLRP3炎症小体的激活

α-mangostin inhibits LPS/ATP-induced NLRP3 inflammasome activation in microglia via the NF-κB pathway

目的探究α-倒捻子素(α-mangostin)在脊髓损伤后小胶质细胞炎症模型中作用及相关机制。方法体外培养小鼠小胶质细胞系BV-2细胞,利用脂多糖和三磷酸腺苷(LPS/ATP)联合诱导的方式建立BV-2炎症模型。CCK-8法检测不同浓度(0、10、20、40、80μmol/L)的α-mangostin对LPS/ATP刺激下的细胞增殖活力影响以筛选适宜的α-mangostin浓度范围;将BV-2细胞分为Ctrl组、LPS/ATP组、40μmol/Lα-mangostin组和不同浓度(10、20、40μmol/L)的α-mangostin干预组(分别记为LPS/ATP+10μmol/Lα-mangostin组、LPS/ATP+20μmol/Lα-mangostin组与LPS/ATP+40μmol/Lα-mangostin组)。ELISA实验检测各组BV-2细胞上清液中促炎因子白介素-6/1β/18(IL-6、IL-1β、IL-18)和肿瘤坏死因子(TNF-α)含量,Western blot检测各组细胞中NOD样受体蛋白3(NLRP3)炎症小体相关蛋白NLRP3、凋亡相关斑点样蛋白(ASC)、裂解型半胱氨酸蛋白酶1(cleaved caspase-1)和白介素1β(IL-1β)表达及核因子κB(NF-κB)途径中p65的磷酸化水平(p-p65/p65)和BV-2细胞核中p65的表达。结果与Ctrl组相比,LPS/ATP组细胞增殖活力明显降低(P<0.05),但低浓度(10、20、40μmol/L)的α-mangostin可显著改善LPS/ATP对小胶质细胞增殖活力的抑制作用(P<0.05),但高浓度(80μmol/L)α-mangostin可促进LPS/ATP对小胶质细胞的损伤(P<0.05)。与Ctrl组相比,40μmol/Lα-mangostin组小胶质细胞上清液中炎症因子IL-6、IL-1β、IL-18、TNF-α含量和细胞中NLRP3、ASC、cleaved caspase-1、IL-1β和p-p65/p65比值及细胞核中p65蛋白均无明显改变(P>0.05),而LPS/ATP组均显著增加(P<0.05);与LPS/ATP组相比,不同浓度α-mangostin干预组中IL-6、IL-1β、IL-18、TNF-α含量和BV-2细胞中NLRP3、ASC、cleaved caspase-1、IL-1β和p-p65/p65比值及细胞核中p65蛋白表达随α-mangostin浓度的增加而依次降低,其中以LPS/ATP+40μmol/Lα-mangostin组的降低程度最为明显(P<0.01)。结论α-mangostin可通过NF-κB途径抑制BV-2细胞中NLRP3炎性小体活化所介导的神经炎症反应。

Objective To investigate the effects and underlying mechanisms ofα-mangostin in a spinal cord injury model of microglial cell inflammation.Methods Mouse microglial cell line BV-2 was cultured in vitro,and an inflammation model was established by co-treatment with lipopolysaccharide and adenosine triphosphate(LPS/ATP).The CCK-8 assay was used to test the influence of different concentrations(0,10,20,40,80μmol/L)ofα-mangostin on cell proliferation vitality under LPS/ATP stimulation to select an appropriate concentration range ofα-mangostin;BV-2 cells were divided into Ctrl group,LPS/ATP group,40μmol/Lα-mangostin group,and intervention groups with different concentrations(10,20,40μmol/L)ofα-mangostin(designated as LPS/ATP+10μmol/Lα-mangostin group,LPS/ATP+20μmol/Lα-mangostin group,and LPS/ATP+40μmol/Lα-mangostin group,respectively).ELISA experiments were conducted to detect the levels of pro-inflammatory cytokines interleukin-6/1β/18(IL-6,IL-1β,IL-18)and tumor necrosis factor(TNF-α)in the supernatants of each group,and Western blot was used to detect the expression of NLRP3,ASC,cleaved caspase-1,IL-1β,and the phosphorylation levels of p65(p-p65/p65)in the NF-κB pathway,as well as the expression of p65 in the nuclei of BV-2 cells.Results Compared with the Ctrl group,cell proliferation vitality in the LPS/ATP group was significantly reduced(P<0.05),but low concentrations(10,20,40μmol/L)ofα-mangostin significantly improved the inhibitory effect of LPS/ATP on microglial cell proliferation vitality(P<0.05),while a high concentration(80μmol/L)ofα-mangostin exacerbated the damage to microglial cells caused by LPS/ATP(P<0.05).Compared with the Ctrl group,the levels of inflammatory factors IL-6,IL-1β,IL-18,TNF-α,and the expression of NLRP3,ASC,cleaved caspase-1,IL-1β,and the p-p65/p65 ratio in the 40μmol/Lα-mangostin group,as well as the expression of p65 protein in the nuclei,showed no significant changes(P>0.05),whereas these significantly increased in the LPS/ATP group(P<0.05).Compared with the LPS/ATP group,the levels of IL-6,IL-1β,IL-18,TNF-α,and the expression of NLRP3,ASC,cleaved caspase-1,IL-1β,and the p-p65/p65 ratio in the intervention groups,as well as the expression of p65 protein in the nuclei,decreased in a concentration-dependent manner with increasingα-mangostin concentration,with the most significant reduction observed in the LPS/ATP+40μmol/Lα-mangostin group(P<0.01).Conclusionα-mangostin can inhibit the neuroinflammatory response mediated by NLRP3 inflammasome activation in BV-2 cells through the NF-κB pathway.