鳖甲煎丸通过HIF-1α/NF-κB信号通路调控巨噬细胞极化的机制探讨

Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway

目的:探讨鳖甲煎丸通过调控巨噬细胞极化防治肝纤维化的作用和机制。方法:运用血清药理学方法,体外培养小鼠巨噬细胞RAW264.7,通过氯化钴(CoCl_(2))刺激RAW264.7细胞建立体外缺氧模型,将细胞随机分为空白组、CoCl2模型组(200 mmol·L^(-1))、鳖甲煎丸低(0.55 g·kg^(-1))、中(1.1 g·kg^(-1))、高(2.2 g·kg^(-1))和扶正祛瘀胶囊组(0.56 g·kg^(-1)扶正祛瘀胶囊)。采用细胞增殖与活性检测(CCK-8)法检测各组细胞增殖水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测巨噬细胞缺氧诱导因子-1α(HIF-1α)、白细胞介素-1β(IL-1β)、IL-6 mRNA表达水平;蛋白免疫印迹法(Western blot)检测巨噬细胞极化相关蛋白与HIF-1α/核转录因子-κB(NF-κB)信号通路相关蛋白表达水平;细胞免疫荧光检测NF-κB信号通路相关蛋白、HIF-1α的分布及表达。结果:与空白组比较,CoCl2刺激24 h后,RAW264.7细胞增殖受抑制明显(P<0.05);与模型组比较,用相应含药血清处理24 h后,各用药组能促进RAW264.7细胞增殖(P<0.05)。与空白组比较,模型组HIF-1α、IL-1β、IL-6 mRNA表达均有明显升高(P<0.05);与模型组比较,鳖甲煎丸与扶正祛瘀胶囊处理后能不同程度的降低巨噬细胞中HIF-1α、IL-1β、IL-6 mRNA的表达水平(P<0.05)。与空白组比较,模型组HIF-1α及M1巨噬细胞表型蛋白IL-6、肿瘤坏死因子-α(TNF-α)表达明显升高(P<0.05),M2巨噬细胞表型蛋白IL-10表达明显降低(P<0.05)。与模型组比较,鳖甲煎丸与扶正祛瘀胶囊组HIF-1α、IL-6、TNF-α蛋白表达降低(P<0.05),CD163、IL-10蛋白表达升高(P<0.05)。与空白组比较,模型组NF-κB p65、磷酸化(p)-NF-κB p65、磷酸化NF-κB抑制蛋白激酶α/β(p-IKKα/β)、磷酸化NF-κB抑制蛋白α(p-IκBα)蛋白表达明显升高(P<0.05);与模型组比较,鳖甲煎丸与扶正祛瘀胶囊组NF-κB p65、p-NF-κB p65、p-IKKα/β、p-IκBα蛋白表达明显降低(P<0.05)。与空白组比较,模型组促进HIF-1α和NF-κB p65的入核表达;与模型组比较,鳖甲煎丸和扶正祛瘀胶囊组能抑制HIF-1α和NF-κB p65的入核表达。结论:鳖甲煎丸可调控巨噬细胞极化,减轻缺氧环境下巨噬细胞相关炎症反应损伤,从而延缓肝纤维化进展,其机制可能与其调控HIF-1α/NF-κB信号通路有关。

Objective:To investigate the effect and mechanism of Biejiajian Wan on liver fibrosis by regulating the polarization of macrophages.Method:Raw264.7 cells were cultured in vitro by serum pharmacological method,and the hypoxia model of RAW264.7 cells was established by stimulating RAW264.7 cells with cobalt chloride(CoCl_(2)).The cells were randomly divided into blank group,CoCl_(2) hypoxia model group(200 mmol·L^(-1)),Biejiajian Wan low-dose group(200 mmol·L^(-1)+0.55 g·kg^(-1)Fuzheng Quyu capsules),medium-dose group(200 mmol·L^(-1)+1.1 g·kg^(-1)Biejiajian Wan),and high-dose group(200 mmol·L^(-1)+2.2 g·kg^(-1)Biejiajian Wan)and Fuzheng Quyu capsule group(200 mmol·L^(-1)+0.56 g·kg^(-1)Biejiajian Wan).Cell proliferation was detected by cell counting kit-8(CCK-8),and the gene expression of hypoxia inducible factor-1α(HIF-1α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in macrophages was detected by real time fluorescence quantitative polymerase chain reaction(Real-time PCR).The expression of macrophage polarization-related protein and HIF-1α/nuclear factor-kappa B(NF-κB)signaling pathway-related protein was tested by Western blot,and the distribution and expression of NF-κB signaling pathway-related protein and HIF-1αwere determined by cell immunofluorescence.Result:Compared with the conditions in the blank group,the proliferation of RAW264.7 cells was inhibited after CoCl2 stimulation for 24 hours(P<0.05),the mRNA expression of HIF-1α,IL-1βand IL-6 in the model group were increased(P<0.05),the protein expression of HIF-1αand M1 macrophage phenotypic proteins IL-6 and tumor necrosis factor-α(TNF-α)was boosted while that of M2 macrophage phenotypic protein interleukin-10(IL-10)was reduced(P<0.05),the protein expression of NF-κB p65,phosphorylation(p)-NF-κB p65,phosphorylated NF-κB inhibits protein kinaseα/β(p-IKKα/β)and phosphorylated NF-κB inhibits proteinα(p-IκBα)was elevated(P<0.05),the nuclear expression of HIF-1αand NF-κB p65 was promoted.Compared with the conditions in the model group,after 24 hours of treatment with corresponding drug-containing serum,each treatment group promoted the proliferation of RAW264.7 cells(P<0.05),the mRNA expression levels of HIF-1α,IL-1βand IL-6 in macrophages were reduced(P<0.05),the protein expression of HIF-1α,IL-6 and TNF-αwas decreased,while that of CD163 and IL-10 was increased(P<0.05),the protein expression of NF-κB p65,p-NF-κB p65,p-IKKα/βand p-IκBαwas lowered(P<0.05),the nuclear expression of HIF-1αand NF-κB p65 was inhibited.Conclusion:Biejiajian Wan could modulate the polarization of macrophages,attenuate the injury of macrophage-associated inflammatory response under hypoxia,and thus delay the progression of liver fibrosis,which might be related to its regulation of HIF-1α/NF-κB signaling pathway.