Study On The Effect Of Chinese Herbal Formulation On Wound Healing In Normal Model And Type Ⅱ Diabetes Mellitus Model Rats

Diabetes mellitus(DM)is a very complicated metabolic disease characterized mostly by the occurrence of chronic hyperglycemia due to a deficiency of insulin secretion or insulin resistance.DM is identified as one of the leading causes that can most commonly affect the wound healing process.Furthermore,impaired wound healing is a major source of morbidity and mortality in diabetic patients.With the increasing prevalence of diabetes worldwide,the incidence of diabetic foot ulcers(DFUS)have increased,along with the increase in morbidity,mortality and medical costs.Impairment of wound healing is a major complication of diabetes,and despite the associated risks,treatment strategies for diabetic wounds remain limited.Chinese herbal medicine is an important pharmaceutical resource with a long history and has great potential in the development of pharmacology.In recent years,traditional Chinese medicine(TCM)has been widely used as an alternative to mainstream treatments for different diseases.Due to the difficulty of single-drug treatment for diabetic wounds,this study used a combination of Coptis chinensis,Folium Isatidis and Radix Isatidis as the main components to prepare Chinese medicine formulation(CMF)and studied its promoting effects on wound healing in normal and DM model rats.Objective:This study aims to prepare CMF ointment and investigate its effect on the wound healing of normal model rats and type Ⅱ diabetic(T2DM)model rats.Methods:1.The formulation is mainly composed of Coptis chinensis,Folium Isatidis,Radix Isatidis,and other traditional Chinese medicines.The dried material was powdered and processed separately.The distilled water was used to extract the medicine,then evaporated the solvent by rotary evaporator,after that dried by freeze dryer to obtain medicine extract.CMF ointment was prepared by mixing the medicine extract with petroleum jelly(1: 10),which was used as a topical treatment.2.The animals grouping design and wounding :(1)Normal model rat experiment,72 SD rats were divided into 3 groups(n = 24): normal control group(NC),normal positive drug treatment group(Iodine,NPC),normal CMF treatment group(NCMF).(2)Diabetes model experiment,96 SD rats were divided into 4 groups(n = 24): diabetes control group(DC),diabetes negative control group(DPJ),diabetes positive control group(Iodine,DPC),diabetes CMF treatment group(DCMF).Wounds(3 × 3 cm)were made on the dorsal surface of all animals in a sterile condition on the day before medicine administration.3.Induction of T2 DM rat model: after 5 weeks of feeding the rats on the high-fat diet,rats were intraperitoneally injected with streptozotocin(STZ,30mg/kg),and the rats with fasting(12 hours)blood glucose ≥ 13.6 mmol/L were established as T2 DM model animals.4.On days 3,7,14 and 21 days,body weight was measured,wound area was recorded,fasting blood glucose was measured,and samples were collected.1)Serum concentrations of C-peptide,insulin,IL-6 and NF-κB were determined by ELISA kit.2)H&E staining was used to detect the histological changes of the wound tissue,and the expressions of CD31,PCNA and e NOS were analyzed by immunohistochemistry.3)The m RNA expressions of VEGF,EGF,TGF-β1,SDF-1α,IL-10,IL-1B,TNF-α,NF-κB and MMP-9 in wound tissues were detected by RT-q PCR.4)The protein levels of VEGF,TGF-β1,PDGF,EGF,MMP-9 and Collagen IV were detected by Western blotting.Results:1.In the normal model experiment:1)Compared with NC and NPC groups,the wound area of the NCMF treatment group significantly decreased on days 3,7,14 and 21(p < 0.05),and the wound contraction was significantly increased(p <0.05).2)Compared with the NC group and NPC group,the histological changes of NCMF group were significantly improved.Neovascularization,collagen deposition,and fibroblast expression in the NCMF group were higher than those in the NC and NPC groups.Moreover,CD31,e NOS and PCNA levels in the NCMF group were significantly higher than those in the NC and NPC groups(p< 0.05).3)Compared with NC and NPC,CMF alleviated inflammation by significantly reducing the expression of NF-κB,TNF-α,IL-1B and MMP-9 on days 7,14,and21 postwounded.4)CMF induced angiogenesis and neovascularization by significantly increasing the levels of VEGF,TGF-β1,EGF,PDGF and SDF-1α on days 3,7 and 14 compared with the NC and NPC groups(p < 0.05),but there was no significant difference on day 21(p > 0.05).2.In the diabetes model experiment:1)After successful induction of T2 DM,body weight and blood glucose did not change significantly on days 3,7 and 14.But on day 21,the DCMF group significantly decreased them compared with DC,DPJ and DPC groups.On days14 and 21,there were significant differences in insulin and C-peptide between the DCMF group and the DC group.Compared with DC,DPJ and DPC groups,the blood plasma levels of NF-κB and IL-6 in DCMF group were significantly decreased on days 7,14 and 21 days.Compared with DC,DPJ and DPC groups,the wound area of the DMCF group was significantly decreased(p < 0.05)and wound contraction was significantly increased(p < 0.05)on days 3,7,14 and 21 in the DMCF group.2)Compared with DC,DPJ and DPC groups,the histological changes of wound tissue in DCMF group were improved.Neovascularization,collagen deposition and fibroblast expression in the DCMF group were higher than those in DC,DPJ and DPC groups.In addition,compared with DC,DPJ and DPC groups,CMF significantly up-regulated the expression of CD31,e NOS and PCNA(p < 0.05).3)Compared with DC,DPJ and DPC groups,CMF significantly reduced the expression of NF-κB,TNF-α,IL-1B and MMP-9 on days 7,14 and 21 postwounded.4)CMF induced angiogenesis and neovascularization by significantly increasing the levels of VEGF,TGF-β1,EGF,PDGF and SDF-1α compared with DC,DPJ and DPC groups on days 3,7 and 14(p < 0.05).Conclusion:This study revealed that CMF for refractory ulcers has a positive effect on diabetes-impaired wounds.The improved wound healing is associated with reduced inflammation,increased angiogenesis,and collagen deposition after CMF treatment.So the topical application of CMF could provide an alternative and effective approach for diabetic wound therapy.