Study On The Function Of PD-1/PD-L1 Axis In Diabetic Ulcer Repairation And The Intervention Of Sheng-ji Hua-yu Formula

Object:Diabetic ulcer(DU)is one of the most common complications of diabetes,which is the primary reason for the hospitalization of refractory ulcer in China.Based on the academic view-point-“removing blood stasis is good for generating muscle,and generating muscle does not cause blemish”-we selected Sheng-ji Hua-yu 3(SJHY-3)as the most effective formula for DU treatment during the early repairation.Based on previous studies,the purpose of this research is to clarify the role SJHY-3 formula on regulating re-epithelization and effecting via PD-1/PD-L1 axis,immune cells,inflammatory factors and growth factor in DU pathological process.Results can enrich the immunologic mechanism and internal law of SJHY therapy in DU treatment.Methods:Part I: The expression and distribution of PD-1 protein in patients’ wounds among different ulcer types were detected by immunohistochemical staining.The expression and distribution of PD-1 protein in DU rat wounds were verified by immunohistochemistry and flow cytometry analysis.Part II: Immunohistochemistry,flow cytometry,western blot and q PCR analysis were used to detect the expression of PD-1,PD-L1,immune cells,inflammatory factors and growth factor during DU re-epithelization.Cell cloning,scratch test,and q PCR analysis were used to detect cellular proliferation,migration,and inflammation with or without PD-1 stimulation.Part III: Immunohistochemistry,flow cytometry,western blot and q PCR analysis were used to detect the expression of PD-1,PD-L1,immune cells,inflammatory factors and growth factor in SJHY-3 treated DU mice,as well as clarifying the role of PD-1/PD-L1 axis in wound reconstruction.Results:The signals of PD-1/PD-L1 axis were inhibited in DU patients and DU animals.It was showed that PD-1 application promotes keratinocytes proliferation,improves the immune status of wound regions,and regulates the expression of inflammation and growth factor in vivo and in vitro.SJHY-3 application can increase the expression of PD-1 and PD-L1 in the early stage of DU.SJHY-3 accelerates the process of DU re-epithelization via promoting keratinocytes proliferation,improving the immune status of wound regions,and regulating the secretion of inflammatory factors and growth factor.Conclusion:SJHY-3 application accelerates DU renovation through up-regulating the expression of PD-1/PD-L1 axis,promoting keratinocytes proliferation,improving the immune status of wound regions,reducing the expression of inflammatory factors and growth factor.Results of this study enriched the connotation of Traditional Chinese Medicine in DU treatment.