Biejiajian Wan Regulates Polarization of Macrophages via HIF-1α/NF-κB Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the effect and mechanism of Biejiajian Wan on liver fibrosis by regulating the polarization of macrophages. MethodRaw264.7 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 （CoCl2）. The cells were randomly divided into blank group， CoCl2 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-1α were determined by cell immunofluorescence. ResultCompared 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-1β and IL-6 in the model group were increased （P<0.05）， the protein expression of HIF-1α and 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 kinase α/β （p-IKKα/β） and phosphorylated NF-κB inhibits protein α (p-IκBα） was elevated （P<0.05）， the nuclear expression of HIF-1α and 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-1β and IL-6 in macrophages were reduced （P<0.05）， the protein expression of HIF-1α， IL-6 and TNF-α was 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/β and p-IκBα was lowered （P<0.05）， the nuclear expression of HIF-1α and NF-κB p65 was inhibited. ConclusionBiejiajian 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.