基于TLR4/NF-κB/NLRP3通路探讨补阳还五汤调控巨噬细胞极化的作用机制

Mechanism of Buyang Huanwutang in Regulating Macrophage Cell Polarization Based on TLR4/NF-κB/NLRP3 Pathway

目的:基于Toll样受体4(TLR4)/核转录因子-κB(NF-κB)/核苷酸结合寡聚化结构域样受体3(NLRP3)通路探讨补阳还五汤调控巨噬细胞极化的作用机制。方法:本实验通过采用不同浓度(0、1.25、2.5、5、10、20、40、80 mg·L^(-1))脂多糖(LPS)对RAW264.7巨噬细胞干预24 h,细胞增殖与活性检测(CCK-8)法检测RAW264.7巨噬细胞存活率,筛选最适浓度建立体外LPS诱导RAW246.7巨噬细胞炎症模型。将实验分为空白组(20%空白血清),模型组(20%空白血清+10 mg·L^(-1)LPS),模型对照组(20%FBS+10mg·L^(-1)LPS),低、中、高(5%、10%、20%)补阳还五汤含药血清组及NLRP3抑制剂MCC950(50μmol·L^(-1))、活性氧(ROS)抑制剂NAC(10μmol·L^(-1))、NF-κB抑制剂PDTC(10μmol·L^(-1))联合补阳还五汤高剂量(20%)组。酶联免疫吸附测定法(ELISA)检测RAW264.7巨噬细胞白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)的表达情况;流式细胞术检测巨噬细胞ROS水平;蛋白免疫印迹法(Western blot)检测M1型巨噬细胞相关细胞因子诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子-α(TNF-α)、M2型巨噬细胞相关细胞因子精氨酸酶1(Arg-1)、白细胞介素-10(IL-10)蛋白表达,TLR4/NF-κB/NLRP3通路的相关蛋白表达。结果:CCK-8结果显示,在10 mg·L^(-1)LPS的刺激下,RAW264.7巨噬细胞的细胞活力值最高(P<0.01)。与空白组比较,模型组IL-1β、IL-18、TNF-α水平显著升高(P<0.01);ROS表达量显著上升(P<0.01);M1型巨噬细胞iNOS、TNF-α蛋白表达显著升高(P<0.01),M2型巨噬细胞Arg-1、IL-10蛋白表达显著降低(P<0.01);TLR4、髓样分化因子88(Myd88)、磷酸化核转录因子-κB抑制蛋白(p-IκB)/核转录因子-κB抑制蛋白(IκB)、磷酸化核转录因子-κB(p-NF-κB)p65/NF-κB p65、NLRP3、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1前体(pro-Caspase-1)蛋白表达水平明显升高(P<0.05,P<0.01)。与模型组比较,补阳还五汤各给药组及抑制剂组均可明显降低IL-1β、IL-18、TNF-α水平(P<0.05,P<0.01),抑制RWA264.7巨噬细胞炎症因子的表达;降低细胞ROS表达水平(P<0.01);下调M1型巨噬细胞iNOS、TNF-α蛋白(P<0.01),上调M2型巨噬细胞Arg-1、IL-10蛋白表达(P<0.05,P<0.01);降低TLR4、Myd88、p-IκB/IκB、p-NF-κB p65/NF-κB p65、NLRP3、ASC、pro-Caspase-1蛋白表达水平(P<0.05,P<0.01)。结论:补阳还五汤可改善巨噬细胞炎症反应,可能与降低巨噬细胞ROS水平,抑制RAW264.7巨噬细胞极化,下调TLR4/NF-κB/NLRP3通路蛋白表达水平有关。

Objective:To explore the mechanism of Buyang Huanwutang in regulating macrophage polarization based on the Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)/nucleotide-binding oligomerization domain-like receptor 3(NLRP3)pathway.Method:RAW264.7 macrophages were intervened with lipopolysaccharide(LPS)of different concentrations(0,1.25,2.5,5,10,20,40,and 80 mg·L^(-1))for 24 hours.Cell Counting Kit-8(CCK-8)assay was used to determine the cell viability of RAW264.7 macrophages.The optimal concentration was chosen to establish an in vitro inflammation model induced by LPS.Cells were divided into a blank group(20%blank serum),a model group(20%blank serum+10 mg·L^(-1) LPS),a model control group(20%FBS+10 mg·L^(-1) LPS),low-,medium-,and high-dose(5%,10%,and 20%)Buyang Huanwutang-containing serum groups,a high-dose(20%)Buyang Huanwutang combined with NLRP3 inhibitor MCC950(50μmol·L^(-1))group,a high-dose(20%)Buyang Huanwutang combined with reactive oxygen species(ROS)inhibitor NAC(10μmol·L^(-1))group,and a high-dose(20%)Buyang Huanwutang combined with NF-κB inhibitor PDTC(10μmol·L^(-1))group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of interleukin-1β(IL-1β),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α)in RAW264.7 macrophages.Flow cytometry was employed to measure ROS levels in macrophages.Western blot was used to determine the protein expression of M1-type macrophage-related factors inducible nitric oxide synthase(iNOS)and TNF-α,M2-type macrophage-related factors arginase-1(Arg-1)and interleukin-10(IL-10),as well as the proteins in the TLR4/NF-κB/NLRP3 pathway.Result:CCK-8 results indicated that under 10 mg·L^(-1) LPS stimulation,RAW264.7 macrophages exhibited the highest cell viability(P<0.01).Compared with the blank group,the model group showed significantly increased levels of IL-1β,IL-18,and TNF-α(P<0.05,P<0.01),increased ROS expression(P<0.05,P<0.01),increased protein expression of M1-type macrophage factors iNOS and TNF-α(P<0.01),decreased protein expression of M2-type macrophage factors Arg-1 and IL-10(P<0.05,P<0.01),and upregulated expression levels of TLR4,myeloid differentiation factor 88(MyD88),phosphorylated inhibitor of NF-κB(p-IκB)/NF-κB inhibitor(IκB),phosphorylated NF-κB(p-NF-κB)p65/NF-κB p65,NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),and pro-Caspase-1(P<0.05,P<0.01).Compared with the model group,all Buyang Huanwutang-treated groups and inhibitor groups significantly reduced levels of IL-1β,IL-18,and TNF-α(P<0.01),suppressed the expression of inflammatory factors in RAW264.7 macrophages,decreased cellular ROS expression levels(P<0.01),downregulated M1-type macrophages iNOS and TNF-αprotein expression(P<0.01),upregulated M2-type macrophages Arg-1 and IL-10 protein expression(P<0.01),and lowered protein expression levels of TLR4,MyD88,p-IκB/IκB,p-NF-κB p65/NF-κB p65,NLRP3,ASC,and pro-Caspase-1(P<0.05,P<0.01).Conclusion:Buyang Huanwutang can improve macrophage inflammation,potentially by reducing macrophage ROS levels,inhibiting RAW264.7 macrophage polarization,and downregulating the protein expression levels of the TLR4/NF-κB/NLRP3 pathway.