Mechanism of Panax notoginseng Saponins on macrophage M1 polarization via Notch pathway
Objective It is to observe the effect of Panax notoginseng Saponins(PNS)on hyperglycemia induced M1 polarization of RAW264.7 macrophages in mice,to observe the molecular mechanism of PNS in promoting early healing of diabetic foot ulcers(DFU).Methods The related pathways of PNS in the treatment of DFU were screened by network pharmacology.The NO(Nitric Oxide)kit was used to determine the high glucose concentration of mouse RAW264.7 mac-rophages polarized to M1 type,and the inflammatory model of mouse RAW264.7 macrophages polarized to M1 type induced by high glucose was established in vitro.CCK-8 experiment was used to detect the effects of PNS and Notch pathway activa-tor Jagged-1 on the proliferation of mouse RAW264.7 macrophages,and to screen the optimal concentrations of PNS and Jagged-1.According to the optimal concentrations of PNS and Jagged-1,the subsequent experiments were divided into con-trol group,high sugar group,high sugar+PNS group,high sugar+PNS+Jagged-1 group,and the macrophages in each group were cultured accordingly.The protein expression of cleaved Notch1,hairy division-associated enhancer 1(Hes1)in the cells were detected by Western blot,the mRNA expressions of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β)and Notch1 were detected by real-time fluorescence quantitative PCR,and the nuclear translo-cation of cleaved Notch1 in the cells was observed by immunofluorescence.Results Network pharmacology showed that PNS promoting the wound healing of DFU was most significantly correlated with Notch pathway.Glucose with concentration of 33.3 mmol/L was the best condition for inducing mouse RAW264.7 macrophages to transform into M1 type,150 μg/mL of PNS was the optimal concentration to protect mouse RAW264.7 macrophages from damage,and 10 μmol/L of Jagged-1 was the optimal concentration to inhibit the proliferation of mouse RAW264.7 macrophages.The relative protein expressions of cleaved Notch1 and Hes1 and the relative mRNA expressions of TNF-α,IL-6,IL-1β,and Notch1 mRNA in the high sugar group were significantly higher than those in the control group(all P<0.05),and cleaved Notch1 protein translocated from the cytoplasm to the nucleus of the cell;the relative protein expressions of cleaved Notch1 and Hes1 and the relative mRNA expressions of TNF-α,IL-6,IL-1β,and Notch1 in the high glucose+PNS group were significantly lower than those in the high glucose group and the high glucose+PNS+Jagged-1 group(all P<0.05),and the cleaved Notch1 protein was in the cytoplasm.Conclusion Panax notoginseng Saponins can inhibit the activation of Notch pathway and reduce the in-flammatory injury by inhibiting the M1-type polarization of macrophages and down-regulating the expression of cleaved Notch1 and Hes1 proteins as well as Notch1 mRNA in macrophages,thus to promote the repair of diabetic foot ulcer wound.
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