| Research Purposes1 To observe the clinical efficacy and safety of the external application of Fufang Xuejie powder in the treatment of diabetic foot ulcer(DFU)through a clinical trial study.2 Based on the animal experimental study,we observed the effect of Dracorhodin perchlorate(Dp),the active ingredient of the main component of Fufang Xuejie powder,on promoting the wound healing of DFU rats,and explored its potential mechanism of action through network pharmacology combined with metabolomics and molecular docking,protein blotting(WB)as well as real-time quantitative polymerase chain reaction(Q-PCR)techniques.Research Methods1 Clinical ResearchThe 68 included patients with DFU were randomly divided into treatment and control groups,with 34 patients in each group.The treatment group received Fufang Xuejie powder as a topical treatment,and the control group was given topical treatment with Estaacridine Lactate Solution.The treatment period is 28 days.Laboratory indexes,traumatic area,VAS score,trauma central temperature infrared calorimetric value,and Traditional Chinese Medicine(TCM)Syndrome Score were recorded for both groups.2 Animal ExperimentsAfter 7 days of acclimatization,40 SPF SD rats were randomly divided into 2 groups,i.e.blank control group(n=10)and diabetic group(n=30).The blank control group was fed with normal diet,and the diabetes group was fed with high sugar and high fat diet After 1 month,the diabetic group was constructed by disposable intraperitoneal injection of Streptozotocin solution.After that,the rats in the diabetic group were successfully modeled and the rats in the blank control group were wounded by surgical excision of the skin,and the diabetic group was coated with glacial acetic acid to construct the DFU model.The rats with successful diabetic foot ulcer modeling were randomly divided into 3 groups:The first group was the model group(Mod),the second group was the western medicine control group(ELS group),and the third group was the treatment group(Dp group).The blank control group was used as the normal trauma group(Con),with 6 rats in each group.Normal trauma group and model group:no intervention.Topical application of Ethacridine lactate solution to the trauma of ELS group and topical application of Dp dressing to the trauma of Dp group.The general condition,wound area and wound healing rate of each group were recorded before,before and after the intervention,and the pathological changes of skin tissue of ulcerated wounds in each group were viewed by HE staining and Masson’s trichrome staining.The levels of TNF-α,IL-1,IL-6,VEGF,bFGF and MMP-9 were tested by enzyme-linked immunosorbent assay(ELISA)in the serum of each group of rats.Network pharmacology was used to predict the targets of Dp for the treatment of DFU,metabolomics was used to detect the metabolites in the wound skin tissues group of rats in the normal wound group,model group,and Dp group.And through the integration of metabolomics and network pharmacology,a "compound-reaction-enzyme-gene" network map was constructed to screen key targets,differential metabolites,and metabolic pathways for Dp treatment of DFU.The core target was screened through molecular docking,and the expression of the core target in the traumatic skin tissues of rats in each group was verified by WB and Q-PCR.Research Results1 Clinical Research(1)General data:The two groups were comparable regarding general information like gender,age,height,weight,disease duration and Wagner classification at baseline(P>0.05).Before treatment,there were no statistical differences between the two groups in laboratory test indicators,trauma area,VAS score,trauma central temperature infrared caloric value and TCM syndrome score(P>0.05).(2)Laboratory indicators:Fasting blood glucose,white blood cell count,neutrophil ratio,sedimentation,and C-reactive protein were lower in the treatment and control groups after treatment than before treatment,a difference that was statistically significant was observed(P<0.05).The treatment group had lower white blood cell count,neutrophil ratio,blood sedimentation and C-reactive protein than the control group at the end of treatment,and there was a statistically significant variance(P<0.05).(3)Trauma area:Both the treatment and control groups had smaller trauma areas at the end of treatment than before treatment,and there was a statistically significant variance(P<0.05).The trauma area of the treatment group was smaller than that of the Con at the end of treatment,and there was a statistically significant variance(P<0.05).(4)VAS pain score:VAS scores at the end of treatment and at the follow-up after three months were lower than before treatment in both the treatment and control groups,and there was a statistically significant variance(P<0.05).VAS scores were lower in the treatment group than in the control group at the end of treatment and at the follow-up at three months,and there was a statistically significant variance(P<0.05).(5)Infrared thermal value of the central temperature of the trauma surface:Both the treatment group and the control group had higher infrared thermal values of the central temperature of the trauma at the end of treatment than before treatment,there was a statistically significant variance(P<0.05).The treatment group had higher infrared thermal values of the central temperature of the trauma at the end of treatment than the control group and there was a statistically significant variance(P<0.05).(6)Comparison of the main symptom score:Both the treatment group and the control group had lower scores of main symptoms at the end of treatment than before treatment,and there was a statistically significant variance(P<0.05).The treatment group had a lower score of main symptoms at the end of treatment than the control group.The difference between the two groups was statistically significant(P<0.05).(7)Comparison of secondary symptom scores:The treatment group had lower secondary symptom symptom scores at the end of treatment than before treatment,and the difference was statistically significant(P>0.05).No significant difference was found between the secondary symptom symptom scores of the treatment group and the control group at the end of treatment(P>0.05).(8)Comparison of the total symptom syndrome score:Both the treatment and control groups had lower total symptom scores at the end of treatment than before treatment,and there was a statistically significant variance(P<0.05).The treatment group had a lower total symptom score than the control group at the end of the treatment,and there was a statistically significant variance(P<0.05).(9)Clinical efficacy:<1>Comparison of the efficacy of the main symptoms of trauma:Compared with the pre-treatment period,and there was a statistically significant variance in the improvement of the ulcer wound area,wound depth,granulation,pus,and Peritraumatic redness and swelling between the therapy group and the Con after treatment(P<0.01).The improvement of the trabecular area,granulation,and Peritraumatic redness and swelling in the treatment group was superior than that in the Con at the end of therapy,and there was a statistically significant variation.(P<0.05).<2>Comparison of the overall clinical efficacy:the total effective rate of the treatment group(93.33%)was greater than that of the Con(76.67%),and there was a statistically significant variance(P<0.05).No adverse reactions were observed in both groups.2 Animal Experiments(1)General data:<1>In terms of fasting blood glucose:compared to rats in the normal trauma group,rats in the Mod,ELS group,and Dp group had significantly higher blood glucose from day 3 to day 28(P<0.05).Compared to the Mod,rats in the ELS group and Dp group had no significant difference in blood glucose from day 3 to day 28(P>0.05).It is suggested that the blood glucose values of rats were more than 16.7 mmol/L during the whole experiment,which reached the standard of diabetes model.<2>Trauma area:Compared with the pre-intervention period,the trauma areas of the normal trauma group,the model group,the ELS group and the Dp group were not significantly different on day 3(P>0.05),and the trauma areas of the ulcers in all groups decreased from day 7 to day 28,with statistically significant differences(P<0.05).The wound area of Dp group and ELS group was significantly smaller than that of the Mod(P<0.05).<3>Wound healing rate:compared to the Mod,the wound healing rates of the Dp and ELS groups from day 7 to day 28 of the intervention were better than those of the Mod,and there was a significant statistical difference observed(P<0.05).(2)Pathology:<1>HE staining-Quantitative statistics of regenerated epidermal thickness:The thickness of regenerated epidermis in both Dp and ELS groups was better than that in the Mod,and there was a statistically significant variance(P<0.05).The thickness of regenerated epidermis in the Dp group was better than that in the ELS,and there was a statistically significant variance(P<0.05).<2>Masson Staining-Collagen Synthesis Quantitative Statistics:Masson staining showed that the collagen volume fraction in both the Dp and ELS groups was better than that in the Mod,and there was a statistically significant variance(P<0.05).There was a statistically significant difference observed between the collagen volume fraction in the Dp group and the ELS group,with the former group exhibiting better values(P<0.05).(3)The expression of TNF-α,IL-1,hs-CRP and growth factors VEGF,bFGF and MMP9 in serum was detected by ELISA:the expression levels of TNF-α,IL-1 and hs-CRP in the Dp and ELS groups were below those in the Mod,and the differences were statistically significant(P<0.05).The levels of TNF-α,IL-1 and hs-CRP in the Dp group were all below those in the ELS group,and there was a statistically significant variance(P<0.05).The levels of VEGF and bFGF in the ELS and Dp groups were higher than those in the Mod,while the expression of MMP-9 was lower than that in the Mod,and there was a statistically significant variance(P<0.05).The levels of VEGF and bFGF in the Dp group were all higher than those in the ELS group,while the expression of MMP-9 was below those in the ELS group,and there was a statistically significant variance(P<0.05).(4)Metabolomic detection of metabolic profiles of rat skin tissues in normal trauma,model and Dp groups:The main metabolites regulated by Dp treatment of DFU are uridine,thymidine,thymine,etc.The key metabolic pathways are mainly related to leukotriene metabolism,urea cycle and arginine,proline,glutamate and tyrosine metabolism,etc.Integrating network pharmacology and metabolomics with molecular docking to obtain core targets for Dp treatment of DFU,including CAT,GSTM1,DHFR,and PAH,which are closely related to oxidative stress,inflammation,and energy supply.(5)Western Blot detected CAT,GSTM1,DHFR and PAH expression in wound skin tissue of rats in each group:CAT and GSTM1 expression were significantly increased in the Dp group than in the Mod,while DHFR and PAH expression were decreased than in the Mod,and the difference was statistically significant(P<0.05).(6)Detection of mRNA expression of CAT,GSTM1,DHFR,and PAH in wound skin tissue of rats in each group by Q-PCR:The mRNA expression of CAT and GSTM1 in the Dp group were both higher than that in the model group,while the mRNA expression of DHFR and PAH were both lower than that in the model group,and the differences were statistically significant(P<0.05).Research Conclusions1 The Fufang Xuejie Powder can safely and effectively reduce the area of DFU wounds with damp-heat toxicity,promote the growth of granulation tissue,reduce periwound redness and swelling,relieve pain and improve its physiological indicators,thus accelerating wound healing.2 Dp can promote the re-epithelialization of skin tissue and collagen deposition on the wounds of DFU rats,inhibit the serum levels of inflammatory factors TNF-α,IL-1 and hsCRP,increase the levels of growth factors VEGF and bFGF,reduce the expression of MMP9,relieve inflammation,improve local blood circulation,promote the proliferation of fibroblasts,and then improve the healing rate of wounds and accelerate the repair of ulcerated wounds.3 Dp is involved in the treatment of DFU by regulating metabolites such as uridine,thymidine,thymine,etc,and metabolic pathways such as leukotriene metabolism,urea cycle,arginine,proline,glutamic acid,tyrosine metabolism,etc,and reversing the abnormal expression of the core targets CAT,DHFR,GSTM1,and PAH in wound skin tissue caused by DFU,to inhibit the inflammatory response,antioxidant,regulate energy supply and immune function,etc.It is a potential mechanism of action of Dp to promote wound healing in DFU rats. |
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