Effects Of Maggot Extract On Skin Wound Healing In Diabetic Rats And Its Molecular Mechanism

Background and objectives: Diabetes mellitus is a metabolic disease which seriously threatens human health.Its incidence rate is increasing,and its complications are numerous and harmful.Diabetic foot is one of its complications.It often causes amputation due to severe ulcers,infection and wound healing.It brings great pain and financial burden to patients and families.At present,the difficulty in healing of diabetic skin wounds is a common problem in the clinical treatment of diabetes,and there is no effective treatment.In the face of this dilemma,it is of great clinical value and social significance to explore effective preparations from natural products to promote the healing of diabetic skin wounds,and to clarify their biological effects and internal mechanisms by using modern cell and molecular biological techniques.In our previous study,we found that maggot extract can effectively promote skin wound healing in rats.The activation of STAT3 signaling pathway is closely related to the process of skin wound healing.Therefore,in this study,we want to establish a diabetic rat skin wound model,and systematically study and analyze the curative effect and molecular mechanism of maggot extract on wound healing.Methods: 50 SPF SD rats(male,weight 200-220g)were selected as the research objects,which were provided by SPF experimental animal center of Dalian Medical University.The usage and related research contents were approved by the medical ethics committee of Dalian Medical University,which was in line with the welfare and protection principle.40 SD rats were injected intraperitoneally with streptozotocin to build diabetic animal model.The full-thickness skin defect model was established on the back of SD rats under aseptic conditions.Subsequently,the rats were divided into 5groups: diabetic blank group(diabetic control group,10 rats),normal group(non-diabetic control group,10 rats),vaseline group(diabetic vaseline treatment group,10 rats),Maggot group(diabetic maggot extract treatment group,10 rats,250μg/g),and rh EGF group(diabetic recombinant human epidermal growth factor treatment group,10 rats,200μg/g).The wound healing of rats in each group was observed,photographed and recorded daily,and the healing rate of each group at different time points was calculated.On the 14 th day,the wound tissues of rats in each group were taken,fixed,paraffin embedded,sectioned and HE staining(Hematoxylin-eosin staining,HE)for histopathological observation.The expression of STAT3,p-STAT3 and its downstream target genes CyclinD1,VEGF and Bcl-2 in tissues of rats were detected by immunohistochemical staining,immunofluorescence staining and Western blot to investigate its molecular mechanism.Results:(1)72h after intraperitoneal injection of streptozotocin,the fasting tail venous blood glucose concentration of SD rats was detected by blood glucose meter,and the blood glucose value was ≥ 16.7mmol/L,which determined the success of diabetic modeling.(2)After 3 days of wound modeling,the back wounds of rats in each group healed to varying degrees.On the 14 th day,the wound healing rate of rats was calculated.The results showed that the healing rate of normal group(91.7 ± 3.7 %)>Maggot group(79.6 ± 4.2 %)> rh EGF group(71.0 ± 4.0 %)> vaseline group(55.1± 5.0 %)> diabetic blank group(43.3 ± 2.3 %)(P < 0.05).(3)HE staining results showed that compared with vaseline group and diabetes blank group,the granulation tissue formation and the number of inflammatory cells decreased in the Maggot group.(4)In order to explore the molecular mechanism of maggot extract promoting skin healing,immunohistochemical staining,immunofluorescence double staining and Western blot were used to detect p-STAT3,Bcl-2,CyclinD1 and VEGF.(1)Compared with the normal group,p-STAT3,Bcl-2,CyclinD1 and VEGF showed a low expression pattern in the Diabetic blank group(p<0.05).(2)Compared with the vaseline group,the expression levels of p-STAT3,Bcl-2,CyclinD1 and VEGF in the Maggot group and rh EGF group were increased(p<0.05),and the degree of increase in the Maggot group was more obvious.Conclusion:(1)Maggot extract can effectively promote skin wound healing in diabetic rats.(2)Maggot extract can promote the activation of STAT3 signal transduction pathway and up regulate the expression of its downstream target genes: Bcl-2,cyclin D1 and VEGF,thereby promote the wound healing of skin with diabetic.(3)The protein levels of p-STAT3,Bcl-2,CyclinD1 and VEGF in the skin wound tissue of rats in the diabetic blank group are lower than those in the skin wound tissue of healthy rats,which may be one of the reasons why the wound of diabetic rats was not easy to heal.