Study On Mechanism Of Chinese Medicine Extract Of Maggot To Promote Diabetic Wound Healing By Regulating Inflammatory Response

Background:As one of the most common complications of diabetes,diabetic ulcer(DU)is a chronic and difficult wound that often occurs in the foot or lower limb.The pathogenesis of DU is complex and the treatment period is long,which brings great burden to patients’ life.Research shows that the overactivated inflammatory response mediated by the NLRP3 inflammasome plays an important role in the pathogenesis of diabetic ulcer wounds.At present,the traditional Chinese medicine Wu Gu Chong(maggot)is widely used in the clinical treatment of chronic refractory wounds,with antibacterial and healing promoting effects,which may be achieved by regulating inflammatory response,but the research on its related molecular mechanism is still not comprehensive and in-depth.Objective:To investigate the effect of the maggot extract on diabetic wound by regulating Nod-like receptor protein 3(NLRP3)inflammasome and NLRP3/ cysteinyl aspasrtate specific proteinase-1(Caspase-1)/gasdermin D(GSDMD)signaling pathway and related mechanism..Methods:1.Maggot extract was prepared by the water extraction method.2.Animal experiments:(1)Preparation of animal model: The diabetes model of C57BL/6J mice was constructed by high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin(STZ)solution(30mg/kg).Full-layer skin wounds with a diameter of 6mm were constructed on the back skin of mice under full anesthesia.The experiment was divided into three groups,namely Normal Control group(NC group),Diabetes Mellitus model group(DM group)and Maggot extract group(WGC group).(2)The body weight and blood glucose of mice were recorded weekly.The hyaline membrane was used to map the wound profile of mice and calculate the healing rate.(3)Hematoxylin-Eosin(HE)staining was performed to observe the morphological changes of wound tissue in mice.(4)Western Blot and RT-qPCR were used to detect the protein and mRNA expression levels of NLRP3,Caspase-1,apoptosis-associated speck-like protein(ASC),GSDMD,interleukin-1 beta(IL-1β)and interleukin-18(IL-18)in skin wound tissue of diabetic mice in each group.3.Cell experiments(1)By Lipopolysaccharide(LPS)combined with Adenosine 5 ’-triphosphate(ATP)to construct the inflammation model of mouse mononuclear macrophage leukemia cells(RAW264.7 cells).The experimental cells were divided into four groups: Control group,Model group(LPS+ATP group),Maggot group(Maggot group)and MCC950 group(MCC950 group).(2)CCK-8 assay was used to detect the toxicity and efficacy of maggot extract on RAW264.7 cells.Cell membrane integrity levels were measured by lactate dehydrogenase(LDH)release test.(3)Western Blot and RT-qPCR were used to detect NLRP3 inflammassome and NLRP3/Caspase-1/GSDMD pathway related proteins and mRNA expression levels in RAW264.7 cells of each group.Results:1.Results of animal experiments(1)The fasting blood glucose(FBG)of the mice remained above 16.7mmol/L after modeling.The wound healing of mice in NC group was the fastest,and the wound healing of mice in WGC group was significantly faster than that in DM group,indicating that maggot extract can effectively promote the wound healing of diabetic mice.(2)The results of wound histomorphology showed that the inflammatory infiltration of the DM group was more obvious than that of the NC group,which was alleviated after treatment with maggot extract.(3)The results of WB and RT-qPCR showed that the expression levels of NLRP3,Caspase-1,ASC,GSDMD,IL-1β,IL-18 protein and mRNA in wound tissue of DM group were significantly higher than those of NC group.However,the expression levels of related proteins and mRNA were significantly decreased after the treatment with maggot extract compared with the DM group,suggesting that maggot extract may promote diabetic wound healing by regulating the activation of NLRP3 inflammasome and NLRP3/Caspase-1/GSDMD signaling pathway.2.Cell experiment results(1)The results of CCK-8 showed that the extract could promote the activity of RAW264.7 cells in a certain range of concentration,and the cell activity reached the highest when the extract concentration was 50μg/ml.(2)After cell model construction,the integrity level of RAW264.7 cell membrane in LPS+ATP group was decreased,and the damage of cell membrane was improved after pretreatment with maggot extract and MCC950,a specific inhibitor of NLRP3 inflammasome.(3)The results of WB and RT-qPCR showed that NLRP3,Caspase-1,ASC,GSDMD,IL-1β and IL-18 protein and mRNA expression levels in LPS+ATP group were significantly increased compared with those in Control group,suggesting the existence of excessive inflammatory reaction.After pretreatment with maggot extract and MCC950,the expression levels of related proteins and mRNA were lower than those in the Model group.These results suggested that maggot extract may have the same action trend as MCC950,which can reduce inflammatory response and accelerate diabetic wound healing by regulating the level of NLRP3 inflammasome and the expression of NLRP3/Caspase-1/GSDMD signaling pathway.Conclusion:The maggot extract may accelerate wound healing in diabetic mice by reducing the activation of NLRP3 inflammasome and down-regulate the expression of NLRP3/Caspase-1/GSDMD signaling pathway.It can reduce excessive inflammatory response and cell pyroptosis,protect cell function,reduce the maturation and release of inflammatory cytokines IL-1β and IL-18,and improve the microenvironment of diabetic mice wound skin tissue.