The Role Of STING In The Diabetic Skin Wound Healing

ObjectivesDiabetic patients have a slow rate of wound healing,which can lead to diabetic foot problems and amputations.As the most common complication of diabetes,it is associated with a high disability rate.Therefore,it is significant to find the key molecular mechanisms and intervention targets of diabetic skin healing delay.Wound healing involves the cooperation of multiple cells and is a complex,highly regulated process in maintaining normal skin barrier function.Uncovering the complex pathophysiological mechanism of diabetic wound healing difficulties,especially the key molecular mechanisms underlying inflammatory responses,has important guiding significance for the treatment of the disease and the development of related clinical drugs.Stimulator of interferon genes(STING)is a key effector molecule that mediates inflammatory response,oxidative stress and ECM remodeling.STING is a multifunctional adaptor protein encoded by the TMEM173 gene.When STING is activated,it can further promote the phosphorylation of downstream transcription factor IRF-3 by TBK1 to induce a type I IFN response,thereby producing a variety of downstream biological effects.Therefore,STING is a key gene for initiating and maintaining inflammatory responses,yet its role in wound healing remains unclear.In this study,we focus on the role of STING in diabetic skin wound healing and demonstrated the critical role of STING from multiple perspectives such as expression,functional effects,regulatory mechanisms,and novel intervention strategies.Contents1.To explore the expression pattern of stimulator of interferon genes(STING)in the process of skin damage repair in diabetic mice.2.To verify the role of STING in the skin damage of diabetic mice.3.To clarify the regulation mechanism of STING expression,and to further verify the effects of related drugs(autophagy agonists Rapamycin and acetylshikonin)on damage repair.MethodsAnimal models,several cell lines,molecular biology experiments,gene silencing,and drug intervention were used to clarify the role of STING in the repair of diabetic skin damage and its related regulatory mechanisms.The main research methods are as follows:1.db/m control mice and db/db diabetic mice were used to establish a skin wound model and calculate the healing time.2.Real-time RT-PCR and WB were used to detect the changes of STING expression in the wound area of control mice and db/db mice.3.Real-time RT-PCR and WB were used to detect the changes of STING expression in the wound area of wild-type mice and diabetic model mice.4.HaCaT and RAW264.7 cells were cultured in vitro with high glucose.Real-time RT-PCR and WB were used to detect the expression changes of STING in cultured cells.5.siRNA was used to interfere with STING expression in HaCaT cells.RT-PCR and WB were used to test the efficiency of siRNA interference.6.FITC-Annexin V and PI staining were used to detect the effect of STING knockout on HaCaT by flow cytometry.7.Real-time RT-PCR was used to detect the effect of STING inhibitor on the expression of inflammatory factors in wound healing area.8.Immunofluorescence staining was used to detect the autophagy levels in db/db mouse skin wound healing tissue sections.9.LC3-GFP-RFP autophagy tandem sensor was used to detect changes in autophagy levels in HaCaT cells treated with high glucose10.Rapamycin was used to treat HaCaT cells cultured with high glucose.WB and RT-PCR were used to detect the effect of Rapamycin on the expression level of STING in high glucose cultured cells.11.RT-PCR and WB were used to detect the effect of acetylshikonin treatment on the expression level of STING in HaCaT cells cultured with high glucose.ResultsPart Ⅰ The upregulation of STING in diabetic mouse skin lesions and the role of STING in diabetic skin wound healing1.The rate of skin wound healing in db/db mice and STZ-induced diabetic mice was significantly decreased.2.The expression of STING in the skin wound area of db/db mice and STZ-induced diabetic mice was increased.3.The expression of STING was significantly increased in cultured HaCaT cells under high glucose conditions.4.siRNA interference can significantly reduce STING expression.5.Flow cytometry results showed that STING gene silencing significantly reduced cell apoptosis.6.In vivo treatment with STING inhibitor C176 has a significant protective effect on wound healing in mice.7.STING inhibitors significantly improved the level of inflammatory factors in the wound area,indicating that STING inhibitors could improve the diabetic wound healing by inhibiting the inflammatory response in the wound area.Part Ⅱ The effect of autophagy level on STING expression1.Immunofluorescence results showed that the level of autophagy in db/db mouse skin wound healing tissue sections was significantly reduced.2.HG treatment significantly decreased authopahgy level in HaCaT cells treated with high glucose.3.Rapamycin significantly inhibited the expression of STING in HaCaT cells.4.Rapamycin could significantly reduce the mRNA levels of inflammatory factors under high glucose culture conditions.Part Ⅲ Study on the effect of acetylshikonin on STING in vitro1.Acetylshikonin treatment increased the expression level of LC3B in HaCaT cells cultured with high glucose,suggesting that it can increase the level of autophagy.2.Acetylshikonin treatment could reduce the expression of STING and related pro-inflammatory cytokines in HaCaT cells cultured with high glucose,while 3-MA treatment abolished the effect of acetylshikonin,indicating that acetylshikonin could reduce the expression of STING by regulating the level of autophagy.Conclusions and innovationTo our knowledge,it is the first time to demonstrate that the expression of STING was significantly up-regulated during the healing of diabetic skin wounds.Furthermore,we found that STING inhibitors could improve the healing process of diabetic skin wounds.These results may provide an important experimental basis for the development of STING-targeted drugs for diabetic skin wound healing.In addition,we found that autophagy can regulate the expression of STING protein.In vitro studies further found that acetylshikonin regulates the expression of STING via affecting autophagy processes.