AIM To investigate the role of Glycoprotein M6B (GPM6B) on the production of macrophage inflammatory cytokines, including IL-1β, TNF-α, IFN-γ and IL-6.
METHODS Western blot and qPCR were used to detect GPM6B expression in RAW264.7 macrophages classically activated M1 and alternatively activated M2. RAW264.7 macrophage lines were infected with lentivirus of the control group (sh-Scramble) and the knockdown GPM6B group (sh-GPM6B1 and sh-GPM6B2) respectively, and the infection efficiency of the virus was observed under fluorescence microscope and confirmed by Western blot and qPCR. The mRNA expression levels of pro-inflammatory factors IL-1β, TNF-α and IFN-γ in the control group and the GPM6B intervention group were detected by qPCR, and the contents of IL-1β and IL-6 in supernatant were detected by ELISA. The phosphorylation levels of inflammatory molecules p65 and Iκα were detected by Western blot after GPM6B interference.
RESULTS Compared with those in the control group, the protein and mRNA levels of GPM6B in RAW264.7 macrophages M1 type were significantly increased, while in M2 they were significantly decreased (P<0.05). Compared with those in the sh-Scramble group, the interference efficiency of GPM6B in the sh-GPM6B1 and sh-GPM6B2 groups was significantly decreased in protein and mRNA levels (P<0.05). The expression of pro-inflammatory factors mRNA, including IL-1β, TNF-α and IFN-γ, and the secretion levels of IL-1β and IL-4 in supernatant were significantly decreased after GPM6B knockdown (P<0.05). The phosphorylation levels of inflammatory molecules P65 and Iκα decreased after GPM6B intervention (P<0.05).
CONCLUSION Interference with GPM6B inhibits the expression of proinflammatory factors released by RAW264.7 macrophages.
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