Effects Of Huiyang Shengji Decoction On Skin Wound Healing And Phenotypic Transformation Of Macrophages In Db/db Mice

Chronic skin ulcer(CSU)is a clinically refractory skin disease.Hyperglycemia and other factors cause vascular and neuropathy around the wound.A large number of neutrophils,monocytes/macrophages,lymphocytes and other immune cells gather to release inflammation factors and inflammatory mediators keeping the wound in a chronic inflammatory state for a long time.It is difficult to regenerate and remodel the extracellular matrix,and to transition from the inflammatory phase to the proliferative phase,leading to prolonged healing time.Regulating anti-inflammatory macrophage(M2 type)directed differentiation may be a therapeutic approach for chronic skin ulcers.In the wound surface,pro-inflammatory monocytes(M1 type)could not polarize to M2 type,releasing a large number of inflammatory factors and mediators,making it difficult for M2 type to promote repair.Therefore,promoting the polarization of M1 macrophages to M2 type can accelerate the healing of chronic wounds.At the same time,increased mobilization and recruitment of pro-inflammatory monocytes(Ly6Chi/+)and decreased mobilization and recruitment of anti-inflammatory monocytes(Ly6Clow/-)can also lead to the increase of monocyte-derived M1 type macrophages on the wound surface,and insufficient recruitment of M2 type macrophages.Huiyang Shengji Decoction(HYSJD)is an effective prescription for treating "Yin syndrome" of CSU.The development of CSU to the later stage for the sore selection of "Yin syndrome" category.Due to deficiency of qi and blood,deficiency of spleen and kidney Yang and deficiency of kidney essence,the source and function of macrophages are impaired,and the transformation to M2 type cannot be timely and effective.Moreover,the deficiency of monocytes derived M2 type makes the wound repair cannot be carried out on time.Therefore,HYSJD is often used in clinical treatment.In this study,the application of HYSJD for tonifying the kidney and strengthening the spleen was investigated to explore whether it could promote the proliferation of repair cells through the mobilization and recruitment of bone marrow monocytes and the differentiation of monocytes/macrophages,so as to achieve the effect of tonifying the torminal muscle.In the animal experiment of this study,db/db mouse full-thickness excision model was used to verify the influence of HYSJD on the healing of skin wounds with slow healing,and the number of macrophages with different phenotypes on the wounds was observed.The expression of inflammation-related proteins in wound tissue was detected,and the proportion of total monocytes,Ly6chi/+type and Ly6clow/-type monocytes in bone marrow and blood was counted to explore the effect of HYSJD on the directed differentiation of monocytes from bone marrow.By cultured mouse mononuclear macrophage leukemia cells(RAW264.7)in vitro,the expression of mannose receptor(CD206)and CD86 molecules were detected.M1 and M2 macrophages related cytokines secretion;Nitric oxide(NO)release;the expression levels of M1-type symbolic protein nitric oxide synthase(iNOS)and M2-type symbolic protein arginase-1(Arg-1)were investigated to investigate the effect of HYSJD on directional polarization of M2-type macrophages.Experimental StudyExperiment 1:Effect of Huiyang Shengji Decoction on wound healing and mononuclear macrophage phenotype transformation in db/db diabetic miceAIM:To explore the effect of HYSJD on wound healing in db/db mice,and to clarify the regulation effect of HYSJD on phenotypic transformation of monocyte macrophages,so as to provide scientific basis for the treatment of chronic skin ulcer.METHODS:10 db/m mice were the blank group,and 30 db/db mice were randomly divided into model group(Model),HYSJD group,and basic fibroblast growth factor group(bFGF);The mice in each group were weighed and blood glucose was tested before modeling.The full-thickness skin on the back was excised with a 6mm diameter wound.Topical medication was applied,and the dressing was changed every other day.the wound was photographed and the wound area was measured.The skin tissues of the back of mice were taken 7 days after trauma,and hematoxylin-eosin(HE)staining was used to observe histopathological changes;multiple immunofluorescence staining was used to detect the number of M1 and M2 macrophages in wound tissue;protein chip technology was used to detect M1 and M2 related protein expression;Flow cytometry to detect the proportion of total monocytes,Ly6clow/-and Ly6ch1/+type monocytes in mouse bone marrow and blood.RESULTS:The weight and blood glucose of the model group were significantly higher than those of the blank group(P<0.01);the wound healing rate of the model group was significantly lower than that of the blank group(P<0.01),and the wound healing rate of the HYSJD group was significantly higher than that of the model group(P<0.01);Compared with the model group,granulation tissues were newly formed,fibroblasts and blood vessels were abundant,and the fibers were arranged neatly in the HYSJD group.Compared with the blank group,the number of M1 types in the model group increased and the number of M2 types decreased on the 7th day(P<0.01);Compared with the model group,the number of M1 types in the HYSJD group decreased and the number of M2 increased(P<0.01).The number of M1 types decreased,and the number of M2 types increased in the bFGF group(P<0.01).Model the expression level of group interleukin 1?(IL-1?),interleukin 1?(IL-1)?,interferon ?(IFN-y),tumor necrosis factor ?(TNF-?),tumor necrosis factor receptor ?(TNF-RII)was higher than that of the blank group(P<0.05 or P<0.01);the HYSJD group the expression of IL-1?,IL-1?,IFN-y,TNF-? and TNF-RII was lower than the model group(P<0.05or P<0.01);the expression levels of IL-1? and TNF-RII in the bFGF group were lower than the model group(P<0.01),but the expression levels of IFN-y and TNF-? was higher than the model group Group(P<0.05 or P<0.01),IL-1? expression level was not statistically different(P>0.05).Compared with the blank group,the expression of monocyte chemokine L2(CCL2;MCP-1)in the model group increased,and the expression of macrophage colony stimulating factor(M-CSF)decreased;compared with the model group,Huiyang Shengji Decoction can inhibit the expression of MCP-1 and promote the expression of M-CSF.There was no statistical difference in GM-CSF among the groups.Compared with the blank group,the ratio of total monocytes and Ly6clow/-monocytes decreased in the model group;the ratio of Ly6chi/+ monocytes increased(P<0.05orP<0.01);the ratio of total monocytes and Ly6chi/+monocytes in the blood increased,and the ratio of Ly6clow/-/-monocytes decrease(P<0.05orP<0.01).Compared with the model group,the ratio of total bone marrow mononuclear cells and Ly6clow/-in the HYSJD group increased,the ratio of Ly6chi/+decreased(P<0.05orP<0.01),and the total monocytes in the blood.The ratio of Ly6chi/+decreased,and the ratio of Ly6clow/-increased(P<0.01);the ratio of bone marrow,blood total monocytes,Ly6clow/-and Ly6chi/+monocytes in the bFGF group was not significantly different from that of the model group(P>0.05).CONCLUSION:Huiyang Shengji Decoction can promote wound healing in db/db mice,and its mechanism may be through down-regulating the expression of MCP-1,inhibiting the local mobilization and recruitment of Ly6chi/+monocytes to the wound;at the same time up-regulating the expression of M-CSF,promote the mobilization and recruitment of Ly6clow/-monocytes,thereby reducing the number of monocyte-derived M1 types,and increasing the number of monocyte-derived M2 type in the wound.Experiment 2:Effect of Huiyang Shengji Decoction on the phenotypic polarization of RAW264.7 macrophagesAIM:To investigate the polarization effect of HYSJD on macrophages and its possible mechanism.METHODS:In this experiment,RAW264.7 macrophages were cultured in vitro,and lipopolysaccharide(LPS,100ng/mL)and interferon ?(IFN-?,20ng/mL)were used to induce macrophages into M1 type,interleukin 4(IL-4,20ng/mL)was used to induce M2 type.The CCK-8 method was used to detect the proliferation effect of the extract of HYSJD on RAW264.7 macrophages.The extract of HYSJD was pre-acted for 24h,and after 12h stimulation with LPS+IFN-?,the level of nitric oxide(NO)in the cell supernatant was detected by colorimetry;the interleukin 1?(IL-1?)interleukin 6(IL-6)?interleukin 12(IL-12)?tumor necrosis factor ?(TNF-?)?interleukin 10(IL-10)?vascular endothelial growth factor(VEGF)in the cell supernatant was detected by luminex high-throughput detection technology;The expression of mannose receptor(CD206)protein was detected by flow cytometry;The expression of nitric oxide synthase(iNOS),arginase-1(Arg-1)and signal transduction and activation of transcription protein 6(STAT6)was detected by Western blotting.RESULTS:The extract of HYSJD at 130?520 mg·L-1 has a proliferation effect on macrophages;it could significantly reduce there lease of NO of macrophages stimulated by LPS+IFN,reduce the secretion of IL-1?,IL-6,and IL-12;simultaneously promote the secretion of IL-10 and the expression of CD206(p<0.05),and there was no significant difference between the expression of CD206 in macrophages induced by IL-4(p>0.05);and reduced iNOS protein expression,but increased Arg-1 and p-STAT6 protein expression(p<0.05).CONCLUSION:The extract of HYSJD can inhibit LPS+IFN-y induced macrophages to polarize to M1 type,and promote the polarization to M2 type,which may play a role by activating the STAT6 signaling pathway.EPILOGUEIn summary,the wound healing of db/db mice is slow,and HYSJD can promote the healing.HYSJD improves the micro-environment of wounds by invigorating qi and blood,invigorating the spleen and kidney,down-regulating the expression of MCP-1,inhibiting the mobilization and recruitment of Ly6chi/+monocytes to the wound,and up-regulating the expression of M-CSF to promote Ly6clow/-monocyte mobilization and recruitment,increase the monocyte-derived M2 type.HYSJD can promote the directional polarization of macrophages into M2 type,reduce the expression of IL-1?,IL-1?,IFN-?,TNF-?,TNF-RII and other inflammatory proteins,and promote granulation regeneration and epidermis crawling to achieve the role of nourishment and muscle growth.