The Effects And Mechanisms Of On Tongsaimai(TSM) Tablet On Diabetic Foot Rats And Mice Model

Diabetic foot(DF)is the common complication of diabetes mellitus patients that involves the exception of the local nerve and distal limb peripheral vascular lesions,leading to foot infection,ulcer and deep tissue destruction.DF is associated with multiple factors,including long-term glucolipid metabolic disorders,abnormal blood rheology,vascular endothelial injury and microcirculation dysfunction and some other elements.In this study,we evaluated the effect of Tongsaimai(TSM)in diabetic foot rats and mice model as well as explored the mechanism of TSM on diabetic foot by analyzing blood glucose,blood lipids,hemorheology,the function of vascular endothelial cell and VEGF signaling pathways.Methods(1)Diabetic foot rats model:The healthy male rats were employed and were randomly divided into two groups,one was normal control group,the other was diabetic model group.The diabetic rat model group was fed with high sugar and high fat for 8 weeks following intraperitoneal injection of STZ(35mg·kg-1).Then each animal received incisional wound on the left foot(2mmx5mm)for diabetic foot rats.The diabetic foot rats were randomly divided into 5 groups:model group,metformin treatment group,12.44 g·kg-1 dose TSM treatment group,6.22 g·kg-1 dose TSM treatment group,and 3.11 g·kg-1dose TSM treatment group.The foot ulcers were also made in normal control group.During the period of treatment for 18 days,the feed intake and water quantity were weighed every day.The ulcer wounds were took pictures on day 1,4,8,13,18 to determine the ulcer healing rate.The rats' body weight was respectively measured on day 8 and 18,as well as blood fasting blood glucose and random blood glucose.Eighteen days after administration,the food,blood,serum and plasma were acquired.Blood and plasma was draw to detect hemorheology.The contents of FINS,GSP,TC,TG,HDL,LDL,IVC and LN,TNF-a,IL-6,MDA,SOD,NO,NOS in serum,and the levels of ET,6-k-PGF1? and TXB2 in plasma were determined by biochemical method,radioimmunoassay and Elisa method.The contents of collagen ? and collagen ?on the ulcer of the rats'foot were also determined by Elisa method.The ulcer granulation tissue with hematoxylin and eosin(HE)and Masson staining was further examined at high magnification(200×).The pancreas morphology was observed by HE dyeing and electron microscopy(200×).The expression of VEGF,SDF1?,p-AKT and p-eNOS on the food ulcer tissue were observed by immunohistochemical method(200×).Western blot method was used to determine the release of VEGF,SDF1?,AKT,p-AKT,eNOS,p-eNOS in the ulcer area.The number of capillaries was counted in the granulation tissues section with immunohistochemical methods by Image-Proplus 6.0 image analysis software.(2)Diabetic lower extremity lesions mice model:The healthy male mice were employed and were randomly divided into two groups,one was normal control group,the other was diabetes model group.The diabetic mice model group was fed with high sugar and high fat for 8 weeks following the tail intravenous injection of alloxan(80mg·kg-1).Then each animal received incision wound on the bilateral foot(5 mm in diameter circle)for diabetic foot model.The diabetic foot mice were randomly divided into 5 groups:model group,metformin treatment group,17.42 g·kg-1 dose TSM treatment group,8.71 g·kg-1 dose TSM treatment group,and 4.35 g·kg-1dose TSM treatment group.The foot ulcers were also made in normal control group.During the period of treatment for 12 days,the feed intake and water quantity were weighed every day.The ulcer wounds were measured on day 1,4,8,12 to determine the ulcer healing rate.The mice body weight was respectively measured on day 4,8 and 12,as well as blood fasting blood glucose.Twelve days after administration,blood,serum were acquired.Blood was draw to detect glycosylated hemoglobin.The contents of FINS,GSP,TC,TG,HDL-C and LDL-C in serum were determined by biochemical method and radioimmunoassay.The ulcer granulation tissue with hematoxylin and eosin(HE)and Masson staining was further examined with light microscopy(200×).The pancreas morphology was observed by HE dyeing,light microscopy and electron microscopy(200×).The expression of VEGF,SDF1?,p-eNOS in the food ulcer tissue were observed by immunohistochemical method on the mice of day 4,8,12(100×).Western blot method was used to determine the release of VEGF,SDF1?,AKT,p-AKT,eNOS,p-eNOS in the ulcer area from legs of mice which were dissected and collected on day 4,8,12.Results1.The effect of TSM on the wound healing in DF modelCompared with control and treatment group,the wound healing was at a longer profile in the diabetic foot ulcer model.A trend of greater reduction of the ulcer area from days 4 to 18(days 4 to 12)was observed in the TSM groups,which showed that TSM could prevent the development of diabetic foot through reducing the ulcer area of DF rats and mice.Three drugs all could improve the ulcer healing,but the effect of high dose TSM treatment group was better than middle and low dose TSM treatment group,which showed that TSM presented a certain dose dependent.The epithelialization and scar formation did not well develope under the model group,whereas they developed well in high TSM treated groups.The local skin squamous epithelial cells in DF model were degenerated,epidermal and dermal inflammatory cells were infiltrated moderately.Furthermore,the blood capillary and fibrous connective tissue had hyperplasia and the skin attachment was significantly reduced.TSM could significantly reduce these pathological injury,especially in dose TSM treatment group(P<0.05~0.01).After treating for 18 days,we found that collagen synthesis was reduced in the treatmen groups of control and TSM by Masson staining,but model group was obviously more,and the average IOD value was higher than the control and high dose TSM treatment group.It implied that the collagen synthesis was greater than the decomposition in wound repair phase which leading to wound healing slowly in the model group.At the same time,in the control and TSM treatment groups collagen ?was higher,type ? collagen was less and the ratio of collagen type ?/? significantly was lower than the model group.It showed that the wound healing has better elasticity and less scarring comparing with the model group wound.It implied that TSM might have improved wound healing by accelerating the collagen synthesis,facilitating tissue regeneration and reducing tissue lesions.2.The effect of TSM on the general cases in DF model(1)Glucose and Insulin:The levels of fasting and postprandial blood sugar in model group was increased significantly in model group,glycated albumin(GSP),glycosylated hemoglobin(HbAlc)and fasting insulin level was elevated compared with control group(P<0.05~0.01)which resulted in the decrease of insulin sensitivity(ISI)and the aggravation of insulin resistance(IR).We observed that the pancreas islet structure was not clear,the number of cells was decreased that revealed degeneration and necrosis moderately,while part of the catheter vacuoles were degenerated.Under the treatment of TSM,the levels of fasting postprandial blood sugar,GSP and HbAlc were significantly decreased.TSM could also significantly reduce the IR,improve the ISI and reduce the pancreas degeneration and necrosis(P<0.05-0.01).It indicated that TSM could promote wound healing by lowering blood sugar,reducing the content of the GSP and HbAlc,regulating IR and improving insulin sensitivity and islet structure and form.(3)Lipid Level:Compared with control group,the levels of total cholesterol(TC),triglyceride(TG)and low-density lipoprotein cholesterol(LDL-C)in serum in model group increased significantly,and HDL-C levels were reduced(P<0.01).These data were improved significantly after given with TSM,which showed that TSM could prevent the development of DF through reducing the blood lipid levels of DF model and increasing the levels of HDL-C.Three dose drugs all could reduce the levels of TC,TG and LDL-C in serum,increase the level of HDL-C,especially the high dose TSM treatment group(P<0.01).The regulation of TSM on the level of blood lipid contributed to improving wound healing.3.The effect of TSM on the oxidative stress and proinflammatory factor in DF rats(1)Oxidative Stress:The level of MDA in DF rats was significantly escalated,and the SOD level was decreased,compared with control group(P<0.01).TSM could regulate the balance of SOD and MDA against the oxidation stress injury in endothelial cell.The regulated balance between SOD and MDA with TSM might improve the ability to removal of oxygen free radical,enhance its antioxidant capacity,and thus reduce peroxide direct injury of vascular endothelial cells.(1)Proinflammatory Factor:The contents of TNF-a and IL-6 in DF rats were higher than that in control group(P<0.01).TSM of 12.44 g·kg-1 dose could notably reduce the levels of TNF-a and IL-6(P<0.05),indicating that TSM could delay the development of the DF by inhibiting inflammatory factors production and reducing inflammatory response.4.The effect of TSM on the vascular endothelial function in DF model(1)Blood Rheology:The PT,TT and APTT in DF rats was significantly shortened compared with control group,while whole blood viscosity,plasma viscosity,platelet aggregation rate increased significantly(P<0.01).TSM could extended PT,TT and APTT of plasma obviously,at the same time,whole blood viscosity,plasma viscosity,platelet aggregation rate significantly decreased(P<0.05-0.01).It indicated that TSM had the comprehensive effects of anticoagulant,reducing blood viscosity,improve microcirculation and endothelial cell function,and promote the DF wound healing.(2)Vascular Endothelial Active Factors:Compared with control group,the levels of NO,NOS and 6-k-PGF1? were decreased,while the levels of ET,TXB2 and the ratios of ET/NO and TXB2/6-K-PGF1? were increased in DF rats(P<0.01).TSM could significantly increase the contents of NO,NOS and decrease ET/NO,TXB2/6-K-PGF1? ratio.In addition,12.44 g·kg-1 dose TSM might significantly increase the levels of 6-K-PGF1?(P<0.05~0.01).It could prevent the development of type 2 DF by improving their balance,protecting endothelial cells,and inhibiting the microcirculation disturbance of blood vessels.(3)Vascular Basement Membrane:The contents of LN and IVCL were markedly increased in DF rats.TSM could significantly reduce the contents of LN and IVCL in blood serum(P<0.05~0.01).As for the decrease of extracellular matrix components,TSM showed the ability of reducing vascular basement membrane,strengthening the protection of vascular endothelium,and thus reducing DF lesions to accelerate the restoration of ulcer.5.The effects of TSM on the VEGF-PI3K/AKT-eNOS of wounds in DF modelAfter treatment for 18 days,the expression of VEGF,SDF1?,p-AKT,p-eNOS and the number of capillary in DF rats was higher than the control group(P<0.050.01).The expression of VEGF,SDF1?,p-AKT and p-eNOS in 12.44 g·kg-1 dose TSM treatment groups was significantly lower than the model rats group(P<0.01),and the number of capillary was lower than the model rats group(P<0.05-0.01).On the day 4 and 8,the expression of VEGF,SDF1?,eNOS in the type 2 DF mice was lower than the control group(P<0.05~0.01),the expression of VEGF,SDF1?,eNOS in the type 2DF mice was higher than the control group(P<0.05-0.01)on the day 12;Instead,TSM could change these expression.It implied that the expression of VEGF-PI2K/AKT-eNOS is not consistent in the different stages of wound healing.In the early stage,the expression of VEGF-PI3K/AKT-eNOS is more to help wound repair while in the late stage of wound repair the expression of VEGF-PI3K/AKT-eNOS showed a trend of decrease suggesting the wound had been largely repaird.These results showed that TSM could promote early wound ulcer healing to speed up the repair by adjusting the expression of VEGF-PI3K/AKT-eNOS signaling pathways.Conclusions1.TSM has a positive therapeutic effect on DF model.In the three dose groups of TSM,and the three dose groups present a dose dependent.2.TSM can reduce blood glucose of DF model animals,increase insulin sensitivity,improve insulin resistance and lipid metabolism disorder,regulate the balance of SOD and MDA.Moreover,TSM can reduce inflammation to promote wound healing in DF,shorten the course of the disease,improve the morphology of wound epidermis and the dermis and accelerate the formation of collagen fibers and the fibers.3.TSM can adjust the vascular endothelial active factors of DF animal model,reduce contents of NO,NOS of serum and 6-k-PGF1? of plasma,increase the contents of ET and TXB2,improve ET and NO,6-k-PGF1?and TXB2 balance,reduce blood viscosity and platelet aggregation rate,regulate the blood rheology,which leading to protect vascular endothelial cells,reduce the synthesis of extracellular matrix components and vascular basement membrane and inhibit blood microcirculation obstacle.4.The effects of TSM in DF are associated with VEGF-PI3K/AKT-eNOS signaling pathways.TSM can regulate the protein contents of VEGF,SDF1?,AKT and eNOS,which prompts that TSM can promote the formation of new capillaries of the wound site by providing good blood supply to the wound site to accelerate wound healing.In summary,TSM plays a pivotal role in treating DF.TSM can influence the foot ulcer healing by reducing blood sugar,blood fat and insulin resistance,inhibiting peroxidation and excessive inflammation,improving the blood rheology and vascular endothelial active factors and excessive deposition of extracellular matrix components in blood vessels and reducing repair late VEGF-PI3K/AKT-eNOS phosphorylation.It can prevent further deterioration of wound,and it is of great significance on reducing the occurrence of DF.