The mechanism of Jiuyi powder and its effective components in inhibiting the M1 phenotype polarization of macrophages on chronic and refractory wounds infected by MRSA

Objective:To observe the effect of Jiuyi powder and its active components on the bacterial culture and macrophage phenotypic factors of the chronic refractory wound rat model,and to explore its mechanism of removing decay and promoting muscle growth.Methods:SD rats were divided into control group,Jiuyi powder group,Shengdan group,and calcined gypsum group,with 8 rats in each group.MRSA-infected skin lesions and wounds were used to build a model of chronic and difficult-to-heal wounds in rats.After the model was formed,the control group was treated with daily routine nitrofural disinfection and replaced with sterile gauze.On the basis of the control group,quantitative Jiuyi powder,Shengdan powder,and calcined gypsum powder were used for dressing change,once a day for 7 consecutive days.Before and after the last administration,collect rat wound secretions for bacterial culture,inducible monoxide nitrogen synthase content.At the same time,after the last administration,the rat wound tissue was excised for histopathology and immunofluorescence double staining to label macrophages and their M1 phenotype.Results:After the last dressing change,the wound healing of Jiuyi powder group was better than the other groups,and the wound healing rate of each group had significant difference(P<0.05).The histomology showed that the inflammation of Jiuyi powder group was controlled and had a healing trend.After the last drug change,the contents of TNF-α,IL-6 and iNOS in serum of all groups decreased,and the contents of IL-6,TNF-αand iNOS in serum of Jiuyi powder group decreased significantly before and after medication(P<0.05).There was statistical significance in serum IL-6 content between calcined gypsum group and Shengdan group before and after medication(P<0.05).In addition,the results showed that the contents of IL-6 and iNOS in serum of Jiuyi powder group were statistically different from those of the control group(P<0.05).Tissue immunofluorescence double staining showed that the positive rate of M1 macrophages in Jiuyi powder group and Shengdan group was significantly lower than that in control group(P<0.05).The MRSA negative conversion rate of Jiuyi powder group and Shengdan group was better than that of the control group and calcined gypsum group(P<0.05).Conclusion:Jiuyi powder can improve the inflammation of chronic refractory wounds,and has antibacterial,anti-corrosion and myogenic effects.Its mechanism may be related to the inhibition of macrophage M1 phenotype polarization.

C-X3-C motif chemokine ligand 1/receptor 1 regulates the M1 polarization and chemotaxis of macrophages after hypoxia/reoxygenation injury

Background:Macrophages play an important role in renal ischemia reperfusion injury,but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further clarified.Methods:The effects of hypoxia/reoxygenation on functional characteristics of RAW264.7 macrophages were analyzed through the protein expression detection of pro-inflammatory factors TNF-αand CD80,anti-inflammatory factors ARG-1 and CD206.The functional implications of C-X3-C motif chemokine receptor 1(CX3CR1)down-regulation in hypoxic macrophages were explored using small interfering RNA technology.Significance was assessed by the parametrict-test or nonparametric Mann-Whitney test for two group comparisons,and a one-way ANOVA or the Kruskal-Wallis test for multiple group comparisons.Results:Hypoxia/reoxygenation significantly increased the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and chemokine C-X3-C motif chemokine ligand 1(CX3CL1)/CX3CR1 and inhibited the protein expression of M2-related anti-inflammatory factors ARG-1 and CD206 in a time-dependent manner in RAW264.7 cells.However,the silencing of CX3CR1 in RAW264.7 cells using specific CX3CR1-siRNA,significantly attenuated the increase in protein expression of TNF-α(P<0.05)and CD80(P<0.01)and the inhibition of ARG-1(P<0.01)and CD206(P<0.01)induced by hypoxia/reoxygenation.In addition,we also found that hypoxia/reoxygenation could significantly enhance the migration(2.2-fold,P<0.01)and adhesion capacity(1.5-fold,P<0.01)of RAW264.7 macrophages compared with the control group,and CX3CR1-siRNA had an inhibitory role(40%and 20%reduction,respectively).For elucidating the mechanism,we showed that the phosphorylation levels of ERK(P<0.01)and the p65 subunit of NF-κB(P<0.01)of the RAW264.7 cells in the hypoxic/reoxygenation group were significantly increased,which could be attenuated by down-regulation of CX3CR1 expression(P<0.01,both).ERK inhibitors also significantly blocked the effects of hypoxic/reoxygenation on the protein expression of M1-related pro-inflammatory factors TNF-α,CD80 and M2-related anti-inflammatory factors ARG-1 and CD206.Moreover,we found that conditioned medium from polarized M1 macrophages induced by hypoxia/reoxygenation,notably increased the degree of apoptosis of hypoxia/reoxygenation-induced TCMK-1 cells,and promoted the protein expression of pro-apoptotic proteins bax(P<0.01)and cleaved-caspase 3(P<0.01)and inhibited the expression of anti-apoptotic protein bcl-2(P<0.01),but silencing CX3CR1 in macrophages had a protective role.Finally,we also found that the secretion of soluble CX3CL1 in RAW264.7 macrophages under hypoxia/reoxygenation was significantly increased.Conclusions:The findings suggest that hypoxia/reoxygenation could promote M1 polarization,cell migration,and adhesion of macrophages,and that polarized macrophages induce further apoptosis of hypoxic renal tubular epithelial cells by regulating of CX3CL1/CX3CR1 signaling pathway.