Study On The Mechanism Of Action And Clinical Effect Of Modified Sanmiao Pigmentum On Diabetic Foot Ulcer

Objective: The efficacy of Modified Sanmiao pigmentum(SM)on patients with diabetic foot ulcer was proved through clinical data.To predict the therapeutic target and related mechanism of SM on diabetic foot ulcer by network pharma-cology,and then use diabetic ulcer rat model for in vivo verification,and further evaluate the therapeutic effect of SM on diabetic footulcer patients by clinical study.Methods: 48 patients treated in the ward and outpatient department of peripheral vascular disease in the First Affiliated Hospital of Heilongjiang University of Chinese Medicine were randomly divided into Kangfuxin liquid group(KF)and modified Sanmiao pigmentum group,with 24 cases in each group.Based on the basic treatment,patients in the Kangfuxin liquid group were externally applied to Kangfuxin Liquid every day,and patients in the modified Sanmiao pigmentum group were externally applied to Sanmiao pigmentum every day,and the dressing was changed once a day until they were cured.The target information of compounds and diabetic foot ulcer was collected from database.The common targets of TCM and disease were obtained form Venn Diagrame to present the interaction between proteins in the form of network diagram.The diabetic rat model was bulit by intrapitoneal injection of streptozotocin,and diabetic ulcer rat model was prepared after successful construction of diabetic model.Then,30 diabetic ulcer model rats were divided into model group(Model),Kangfuxin liquild solution group(KF),Modified Sanmiao pigmentum group(SM),and blank control group(NC,n=10).The rats in the KF group and the modified SM group were wet applied on the wound with gauze containing the medicinal solution,and the dressing was changed once a day.To weigh the body weight of each group on days 0,7,14 and 21 after administration.Fasting blood glucose(FBG)was measured before,after,and on days 0,7,14 and 21 after medication,and the ulcer area was measured,and the wound healing rate was calculated.Serum levels of CRP,IL-6 and p-NF-κB were determined by ELISA.The expression of AKT,PI3 K and m TOR genes in rat wound tissue was detected by RT-PCR.The index of ulcer area,healing rate,healing days,contents of IL-1β,IL-6,TNF-α,WBC,NE% and CRP in serum or whole blood of the two groups were compared.HE staining was used to observe the pathological tissue of the wound.The expression of p-AKT and p-PI3 K protein in wound tissue was detected by immunohistochemistry.The protein contents of AKT,p-AKT,PI3 K,p-PI3 K,m TOR and p-m TOR in serum were determined by WB method.Results: At day 0,between the two groups,there was no significant difference in ulcer area(P>0.05).However,at day 7 and 14,in modified Sanmiao pigment-um group,the ulcer area was significantly lower than in Kangfuxin liquid group(P<0.01),and the ulcer healing rate of the modified Sanmiao pigmentum group was significantly higher than that of the Kangfuxin liquid group(P<0.01).In addition,the healing days of patients in the modified Sanmiao pigmentum group were(14.8±2.54)days,significantly lower than those in the Kangfuxin solution group(19.6±3.2)days(P<0.01).Before treatment and 14 days after administration,the ulcer depth and exudation score of the modified Sanmiao pigmentum group were significantly lower than those of the Kangfuxin solution group(P<0.01).Network pharmacological analysis showed that 123 active compounds in the modified Sanmiao pigmentum had the potential to treat diabetic foot ulcer,and 237 potential therapeutic targets were involved.The treatment of diabetic foot ulcer with modified Sanmiao pigmentum mainly involved Pathways in cancer,Metabolic pathways and PI3K-Akt signaling pathway.There was no significant difference in ulcer area among all groups on day 0 of administration.At the 7th,14 th and 21 st day of administration,compared with blank control group,the ulcer area of model group was signifi-cantly larger and the ulcer healing rate was significantly lower.The ulcer area and healing rate of rats in the modified Sanmiao pigmentum group were significantly smaller than in model group.ELISA results showed that the contents of CRP,IL-6 and p-NF-κB in model group were significantly more than in blank control group(P<0.01).Compared with model group,modified Sanmiao pigmentum could significantly reduce the levels of the above proinflammatory factors(P<0.01).RT-PCR results showed that compared with the blank control group,the relative expression levels of AKT,PI3 K and m TOR genes in the model group were significantly decreased,but the expression levels of the above genes were significantly increased after the treatment with the modified Sanmiao pigmentum(P<0.01).The levels of inflammatory factors IL-6,IL-1β,TNF-α,NE%,WBC and CRP of the patients with diabetic foot ulcer in the Kangfuxin solution group and the modified Sanmiao pigmentum group were detected on day 0 before treatment and day 14 after treatment.It was found that the levels of the above inflammatory factors were significantly decreased in the two groups after 14 days of treatment(P<0.01).HE staining showed inflammatory cells were infiltrated and fibroblasts were less in the patient wound who were treated brfore.Although a small number of inflammatory cells were observed in the new wound tissue after the treatment of Kangfuxin solution and the modified Sanmiao pigmentum group,there were also a large number of new capillaries and fibrocytes.WB results showed that,the protein expression levels of P-Akt /AKT,P-PI3 K /PI3 K and P-m TOR /m TOR in modified Sanmiao pigmentum group were significantly increased before the patient received treatment(P<0.01).Immunohistoc-hemical results also showed that the expression of p-AKT and p-PI3 K protein was lower in the model group and higher in the wound tissue of the modified Sanmiao group(P<0.01).Conclusions: The modified Sanmiao pigmentum can significantly promote the healing of ulcer area and inhibit inflammation in diabetic ulcer model rats,which may be related to the activation of PI3K/AKT signaling pathway.Moreover,the clinical data further indicated that the modified Sanmiao pigmentum had significant curative effect on diabetic foot ulcer.The modified Sanmiao pigmentum has potential development and research value for the treatment of diabetic foot ulcer.