Anti-melanoma Effect Of Bitespiramycin And Its Immune-related Regulatory Mechanism

Background: Bitespiramycin(BT)is a broad-spectrum macrolide antibiotic developed through gene recombination technology.Cell experiments and clinical medications indicated that BT may inhibit the occurrence and development of tumors;and our previous studies had found that BT had an up-regulating effect on macrophages and neutrophils in normal mice.Therefore,this study aims to the antitumor effect of BT and to clarify the mechanism of BT inducing the polarization of macrophages.Discussing the anti-tumor and immunomodulatory effects of BT can provide a new medication basis for the treatment of tumor immunity and related inflammation.Methods:(1)The effect of BT on the immune sytem of normal mice by flow cytometry.(2)The anti-tumour effects of BT were evaluated with a murine subcutaneous tumour model.(3)Flow cytometry and CCK-8 method were used to detect the effect of BT on proliferation of macrophages.(4)The immune microbead phagocytosis experiment to detect the effect of BT on the phagocytosis of primary macrophages.(5)The effect of BT on macrophage differentiation was measured through inducing RAW cells with LPS,IFN-γ / IL-4.(6)Used Western Blot to detect the mechanism of BT on the polarization of M1 macrophages.Results:(1)BT significantly increased the proportion of monocytes(P<0.001)and neutrophils(P<0.01)in the peripheral blood of mice.It had no obvious effect on other immune cells.(2)In the tumour-bearing mouse model,the tumour weight of the BT group was significantly smaller than that of the control group(P<0.01),and the tumour inhibition rate was 41.6%;and the peripheral blood mononuclear cells and tumour tissues in the BT group.The proportion of macrophages increased significantly(P<0.05),and tended to M1 type(P<0.05).(3)BT could increase the proportion of M1 type macrophages in vivo and in vitro(P<0.05),and it also increased the proportion of macrophages phagocytosing fluorescent beads(P<0.05).(4)In the RAW264.7 cell polarization experiment,BT significantly up-regulated the expression level of i NOS in M1 macrophages(P<0.01),and at the same time inhibited Arg-1 in M1 macrophages Expression(P<0.05);there were significant differences in the up-regulation of TNF-α and i NOS expression and the down-regulation of Arg-1expression in M2 type(P<0.01,P<0.05,P<0.01).The results of the same trend were also obtained in the macrophage polarization experiment.(5)In the detection of protein expression level,BT up-regulated the expression of STAT1 protein in M1 type macrophages and enhanced the ratio of STAT1 and STAT3 protein phosphorylation.Conclusion: BT can promote the proliferation of some immune cells(macrophages and neutrophils)in mice.At the same time,BT has the effect of inhibiting the growth of melanoma,and this antitumor effect may be through promoting the proliferation of macrophages,enhancing the phagocytic function of macrophages,and inducing macrophages to M1 type by activating STAT1 and STAT3 Polarization and other immune-related regulatory mechanisms.