| Purpose:1.Explore the difficult healing mechanism of Diabetic ulcer（DU）based on the“lingering toxins”theory,which will provide a new insight for Chinese traditional medicine to promote healing of diabetic ulcers.2.To observe the healing effect and therapeutic mechanism of Badu shengji Powder on skin ulcer in diabetic rats.Material and method:1.Clinical research:The wound tissue of 20 Diabetic foot patients is collected and labeled as DU group.Normal skin tissues from the feet of 20 non-diabetic patients were collected and labeled as Normal control（NC）group.HE staining was used to observe the pathological manifestations.The protein contents of AGEs、RAGE、Wnt1 andβ-catenin were detected by ELISA.The protein expression levels of both Keap1 and Nrf2 were detected by Western blotting.Relatively amounts of RAGE、Keap1、Nrf2、Wnt1andβ-catenin were detected at the m RNA level by RT-PCR.Based on the theory of"lingering toxins",the thinking of"taking images and comparing types"in traditional Chinese medicine was used to differentiate AGEs.2.Experimental study:Forty-eight rats were injected intrabitoneally with streptozotocin to make a diabetes model.They were randomly divided into model group,Badu shengji Powder group,traditional Chinese medicine control group and Western medicine control group,and fed high fat and high sugar diet for 4 weeks.Twelve rats were selected as normal control group and fed ordinary diet for 4 weeks.After 4 weeks,the skin ulcer model was built.Start the medication once a day on the second day after modeling.Medication methods:After iodophor disinfection,normal control group and model group were washed with 0.9%sodium chloride solution;About 6mg powder was applied externally to Badu Shengji powder group;In the traditional Chinese medicine control group,a Yixiao ointment of approximately2mm thickness was applied;The western medicine control group was coated with Qingchuang glue about 2mm thick.After each group was coated,one layer of vaseline gauze and two layers of sterile gauze were applied externally,and the wounds were fixed with adhesive tape.On the 7th day after administration,5 rats were randomly selected from each group as normal control I group,model I group,Badu Shengji PowderⅠgroup,traditional Chinese medicine control I group and Western medicine control I group.The other rats were normal controlⅡgroup,modelⅡgroup,Badu Shengji PowderⅡgroup,traditional Chinese medicine controlⅡgroup and Western medicine controlⅡgroup,which were administered for 14 days.During administration,a normal control I group and a normal controlⅡgroup were fed a regular diet,and the remaining 8 groups were fed a high-sugar,high-fat diet.The samples were taken on the 7th and 14th days of drug administration,respectively.The modeling of diabetic rats was observed;Measuring body mass and blood sugar;Wound healing rates were measured within each group.HE staining was used to detect histopathology,Masson staining was used to detect collagen fibers in tissues,and immunohistochemistry was used to observe neovascularization.ELISA was used to determine the protein contents of the AGEs.The relative m RNA expression levels of RAGE、Keap1、Nrf2、Wnt、β-catenin and glycogen synthase exciter-3β（GSK-3β）were detected by RT-PCR.Results:1.The results of clinical studies showed that:（1）The results of HE staining showed that:In the NC group,the structure of the epidermis was complete,the squamous cells were normal and closely arranged,the dermis was rich in collagen fibers,and no obvious inflammation was observed.In DU group,a large number of epidermal and dermal cell necrosis,nuclear fragmentation or dissolution were observed,accompanied by connective tissue hyperplasia,the number of fibroblasts increased,more lymphocytes and neutrophils infiltrated,and the epidermal layer thickened around the injured area.（2）The protein content of AGEs、RAGE、Wnt1 andβ-catenin in each group showed that:The protein content of AGEs in DU group was higher than that in NC group(1464.85±55.06pg·m L-1vs1033.51±50.99pg·m L-1,P<0.01),the RAGE protein content was higher than that in NC group(206.58±16.02pg·m L-1vs156.08±7.09 pg·m L-1,P<0.01),the protein content of Wnt1 was higher than that of NC group(32.58±2.89pg·m L-1vs25.22±1.29pg·m L-1,P<0.01),andβ-catenin protein content was less than that in the NC group(131.71±5.90pg·m L-1vs158.30±7.94pg·m L-1,P<0.01).（3）The protein expression levels of Keap1 and Nrf2 in each group showed that:The protein expression level of Keap1 in the DU group was greater than that of the NC group（1.19±0.20vs0.35±0.06,P<0.01）,and the protein expression level of Nrf2 was less than that of the NC group（0.47±0.06vs1.53±0.16,P<0.01）.（4）The relative expression levels of RAGE、Keap1、Nrf2、Wnt1 andβ-catenin m RNA in each group showed that:The m RNA expression quantity relatively of RAGE in DU group was higher than that in NC group（4.19±0.36vs1.00±0.09,P<0.01）,and The amount of relative Keap1 m RNA expression in the DU group was greater than that in the NC group（3.84±0.45vs1.00±0.13,P<0.01）.The amount of relative Wnt1 m RNA expression was greater than that of the NC group（2.86±0.34vs1.01±0.12,P<0.01）,The m RNA expression quantity relatively of Nrf2 was lower than that of NC group（0.43±0.10vs1.00±0.09,P<0.01）,The amount of relativeβ-catenin m RNA expression was lower than that in the NC groups（0.20±0.03vs1.01±0.13,P<0.01）.2.The results of experimental studies showed that:（1）The modeling of diabetic rats showed that:Diabetic rats showed increased water intake,food intake and urine volume.The body weight of the diabetic group was lower than that of normal control group（211.88±30.94gvs357.92±25.80g,P<0.01）,and blood glucose was elevated compared to normal control groups(26.80±2.33mmol·L-1vs6.20±0.80 mmol·L-1,P<0.01),and 16.7 mmol·L-1.（2）The appearance of healing of ulcerated surface showed that:In normal controlⅠgroup,the ulcer surface was significantly reduced,and the surface was covered by dry scab without obvious exudation.The ulcerative surface of modelⅠgroup had thick crusts and inflammatory exudation under the crusts.There was a significant reduction in ulcer surface area in Badu shengji Powder I group,traditional Chinese medicine control I group,and Western medicine control I group with mild red granulation,mild edema,and peripheral neophyte epithelium.The ulcer surface of normal controlⅡgroup was further reduced significantly,the dry scab was smaller,no exudation,and hair regeneration was observed.In modelⅡgroup,the middle of the ulcer scab was thin and inflammatory exudation was observed.After the scab was around,the epidermis of the wound margin was thickened and the new skin was less.There was a significant reduction in ulcer surface area in Badu shengji PowderⅡgroup,traditional Chinese medicine controlⅡgroup,and Western medicine controlⅡgroup,the scab was thin,and no obvious exudation was observed.（3）The results of ulcer surface healing rate showed that:The healing rate of modelⅠgroup was significantly lower than that of normal controlⅠgroup.（25.38±1.03%vs45.46±0.84%,P<0.01）.The healing rate of Badu shengji PowderⅠgroup was higher than that of modelⅠgroup（40.26±1.12%vs25.38±1.03%,P<0.01）.There was no significant difference with traditional Chinese medicine controlⅠgroup（40.26±1.12%vs40.29±1.06%,P>0.05）and western controlⅠgroup（40.26±1.12%vs38.47±1.51%,P>0.05）.The healing rate of the model II group was less than that of the normal control II group（49.86±1.10%vs80.41±0.75%,P<0.01）.The healing rate of Badu Shengji PowderⅡgroup was higher than that of modelⅡgroup（69.76±0.97vs49.86±1.10%,P<0.01）.There was no significant difference with traditional Chinese medicine controlⅡgroup（69.76±0.97%vs70.74±1.12%,P>0.05）and western controlⅡgroup（69.76±0.97%vs69.06±0.79%,P>0.05）.（4）The results of HE staining showed that:In normal control group I,a large number of new epidermis,fibroblasts and neovascularization were observed,with a small amount of infiltration of fibrocytes,lymphocytes and neutrophils.In model I group,a large area of epidermal and dermal necrotic dissolution was observed,with a small amount of necrotic cell debris,a large number of fibroblasts and fibrocells,more new blood vessels,scattered lymphocyte and neutrophilic granulocyte infiltration,and slight local bleeding.A large number of new epidermal layer,a large number of fibroblasts and fibrocells,more new blood vessels,a small number of lymphocytes and neutrophils were observed in the Badu shengji PowderⅠgroup,traditional Chinese medicine controlⅠgroup and Western medicine controlⅠgroup.In normal control II group,a large number of new epidermal layer,a large number of fibroblasts and fibrocells,a large number of collagen fiber deposition,a large number of new blood vessels,and a small number of lymphocytes,neutrophils and macrophages were observed locally.In model II group,a large range of necrotic dissolution was observed in the epidermis and dermis,with necrotic cell debris.The dermis was repaired by proliferative connective tissue,with more fibroblasts and fibrocells,a large amount of collagen fiber deposition,more new blood vessels,scattered lymphocytes and neutrophil infiltration.In Badu shengji PowderⅡgroup,Chinese medicine controlⅡgroup and western medicine controlⅡgroup,necrotic dissolution of the large surface of the epidermis and dermis was observed,the dermis was repaired by proliferative connective tissue,a large number of fibroblasts and fibrocells,a large number of collagen fiber deposition,a large number of neovasculum,more lymphocytes and neutrophils infiltrated,and the new epidermis was visible at the edge of the damaged area.（5）The results of Masson staining showed that:The normal control II group showed a large amount of collagen fiber deposition and the arrangement was relatively neat and ordered.In the model II group,collagen fibers had less blue staining,a finer shape,a disordered arrangement,a sparse distribution,and a large void.More collagen fibres were deposited in the Badu shengji powder II groups,traditional Chinese medicine II control and western medicine controlⅡgroup,the shape was thicker,the gap was smaller,and the arrangement was relatively orderly.（6）The results of Immunohistochemistry showed that:In normal controlⅡgroup,there were more CD34 labeled neovascularization,and the diameter of neovascularization was larger.In modelⅡgroup,there were fewer CD34 labeled new vessels,and the new vessels were smaller in diameter.More CD34 labeled neovascularization and larger diameter of neovascularization were observed in Badu shengji PowderⅡgroup,traditional Chinese medicine controlⅡgroup and western medicine controlⅡgroup.（7）The results of AGEs and RAGE expression in ulcer granulation tissue of rats in each group showed that:（1）The protein content of AGEs in modelⅠgroup was higher than that in normal controlⅠgroup(968.41±18.87ng·L-1vs750.48±37.03ng·L-1,P<0.01).The protein content of AGEs in Badu shengji PowderⅠgroup was lower than that in modelⅠgroup(813.73±45.40ng·L-1vs968.41±18.87ng·L-1,P<0.01),and greater than that in the traditional Chinese medicine I control group(813.73±45.40ng·L-1vs746.58±30.19 ng·L-1,P<0.01),no significant difference compared with the western medicine control I group was observed(813.73±45.40ng·L-1vs869.19±35.67 ng·L-1,P>0.05).The protein content of AGEs in modelⅡgroup was greater than that of normal controlⅡgroup(880.89±64.78ng·L-1vs640.00±32.98ng·L-1,P<0.01).The protein content of AGEs in Badu shengji PowderⅡgroup was lower than that in modelⅡgroup(793.37±56.06ng·L-1vs880.89±64.78ng·L-1,P<0.05).No significant difference was found compared to the control group II of traditional Chinese medicine(793.37±56.06ng·L-1vs741.82±38.70ng·L-1,P>0.05)and western medicine controlⅡgroup(793.37±56.06ng·L-1vs852.73±25.95ng·L-1,P>0.05).（2）The amount of relative RAGE m RNA expression in the model I group was greater than that in the normal control I group（3.44±0.24vs1.00±0.05,P<0.01）.The m RNA expression quantity relatively of RAGE in Badu shengji PowderⅠgroup was lower than that in modelⅠgroup（2.03±0.17vs3.44±0.24,P<0.01）,greater than that of traditional Chinese medicine controlⅠgroup（2.03±0.17vs1.56±0.14,P<0.01）,and lower than that in western medicine controlⅠgroup（2.03±0.17vs2.36±0.16,P<0.01）.The amount of relative RAGE m RNA expression in the model II group was higher than that of the normal control II group（3.30±0.2vs1.00±0.07,P<0.01）.The m RNA expression quantity relatively of RAGE in Badu shengji PowderⅡgroup was lower than that in modelⅡgroup（1.96±0.12vs3.30±0.20,P<0.01）,and greater than that in the control group II of traditional Chinese medicine（1.96±0.12vs1.68±0.06,P<0.05）.It was lower than that of western medicine controlⅡgroup（1.96±0.12vs2.83±0.16,P<0.01）.（8）The expression of Keap1 and Nrf2 in ulcer granulation tissue of rats in each group showed that:（1）The m RNA expression quantity relatively of Keap1 in modelⅠgroup was greater than that of normal controlⅠgroup（2.85±0.17vs1.00±0.07,P<0.01）.The m RNA expression quantity relatively of Keap1 in Badu shengji PowderⅠgroup was lower than that of modelⅠgroup（1.67±0.13vs2.85±0.17,P<0.01）,and lower than that of traditional Chinese medicine controlⅠgroup（1.67±0.13vs1.95±0.15,P<0.05）.It was lower than western control groupⅠ（1.67±0.13vs2.34±0.15,P<0.01）.The m RNA expression quantity relatively of Keap1 in model groupⅡwas greater than that of normal controlⅡgroup（3.11±0.19vs1.00±0.07,P<0.01）.The m RNA expression quantity relatively of Keap1 in Badu shengji PowderⅡgroup was lower than that in modelⅡgroup（1.62±0.07vs3.11±0.19,P<0.01）,but did not differ significantly from that in the control group II of traditional Chinese medicine（1.62±0.07vs1.80±0.14,P>0.05）.It was lower than that in western medicine controlⅡgroup（1.62±0.07vs2.10±0.19,P<0.01）.（2）The amount of relative Nrf2 m RNA expression in the model I group was less than that in the normal control I group（0.32±0.04vs1.00±0.06,P<0.01）.The m RNA expression quantity relatively of Nrf2 in Badu shengji PowderⅠgroup was higher than that in modelⅠgroup（0.93±0.06vs0.32±0.04,P<0.01）,greater than that of traditional Chinese medicine controlⅠgroup（0.93±0.06vs0.76±0.05,P<0.01）,and higher than that in western medicine controlⅠgroup（0.93±0.06vs0.68±0.03,P<0.01）.The m RNA expression quantity relatively of Nrf2 in model groupⅡwas lower than that in normal controlⅡgroup（0.37±0.04vs1.00±0.09,P<0.01）.The m RNA expression quantity relatively of Nrf2 in Badu shengji PowderⅡgroup was higher than that in modelⅡgroup（0.90±0.07vs0.37±0.04,P<0.01）,but had no significant difference from that in traditional Chinese medicine controlⅡgroup（0.90±0.07vs0.84±0.05,P>0.05）.It was higher than that of western medicine controlⅡgroup（0.90±0.07vs0.58±0.03,P<0.01）.（9）The expression of Wnt1、β-catenin and GSK-3βin the granulation tissue of rats in each group showed that（1）The amount of relative Wnt1 m RNA expression in the model I group was less than that in the normal control I group（0.37±0.06vs1.00±0.07,P<0.01）.The m RNA expression quantity relatively of Wnt1 in Badu shengji PowderⅠgroup was higher than that in modelⅠgroup（0.94±0.08vs0.37±0.06,P<0.01）,and higher than that in traditional Chinese medicine controlⅠgroup（0.94±0.08vs0.68±0.07,P<0.01）.It was greater than that in western medicine controlⅠgroup（0.94±0.08vs0.81±0.07,P<0.05）.The amount of relative Wnt1 m RNA expression in the model II group was less than that in the normal control II group（0.35±0.04vs1.00±0.10,P<0.01）.The m RNA expression quantity relatively of Wnt1 in Badu shengji PowderⅡgroup was higher than that in modelⅡgroup（0.82±0.08vs0.35±0.04,P<0.01）,but did not differ significantly from that in the control group II of traditional Chinese medicine（0.82±0.08vs0.72±0.06,P>0.05）and western medicine controlⅡgroup（0.82±0.08vs0.78±0.09,P>0.05）.（2）The amount of relativeβ-catenin m RNA expression in the model I group was less than that in the normal control I group（0.25±0.02vs1.00±0.08,P<0.01）.The m RNA expression quantity relatively ofβ-catenin in Badu shengji PowderⅠgroup was higher than that in modelⅠgroup（0.70±0.07vs0.25±0.02,P<0.01）,and less than that of the control group（0.70±0.07vs0.90±0.09,P<0.05）in traditional Chinese medicine.There was no significant difference compared to the control group of western medicine I（0.70±0.07vs0.80±0.05,P>0.05）.The amount of relativeβ-catenin m RNA expression in the model II group was less than that in the normal control II group（0.28±0.02vs1.00±0.09,P<0.01）.Relatively high amount ofβ-catenin m RNA expression in Badu shengji PowderⅡgroup was higher than that in modelⅡgroup（0.72±0.05vs0.28±0.02,P<0.01）,and lower than that of traditional Chinese medicine controlⅡgroup（0.72±0.05vs0.92±0.07,P<0.01）.It was lower than that in western medicine controlⅡgroup（0.72±0.05vs0.86±0.10,P<0.05）.（3）The amount of relative GSK-3βm RNA expression in the model I group was greater than that in the normal control I group（3.90±0.11vs1.01±0.11,P<0.01）.The m RNA expression quantity relatively of GSK-3βin Badu shengji PowderⅠgroup was lower than that in modelⅠgroup（2.05±0.13vs3.90±0.11,P<0.01）,but had no significant difference from that in traditional Chinese medicine controlⅠgroup（2.05±0.13vs1.86±0.11,P>0.05）.It was lower than that of western medicine controlⅠgroup（2.05±0.13vs2.67±0.22,P<0.01）.The amount of relative GSK-3βm RNA expression in the modelⅡgroup was greater than that in the normal controlⅡgroup（3.37±0.23vs1.00±0.08,P<0.01）.The m RNA expression of GSK-3βwas significantly lower in Badu shengji PowdeⅡgroup than in GSK-3βgroup I group（2.06±0.08vs3.37±0.23,P<0.01）,and greater than that of traditional Chinese medicine controlⅡgroup（2.06±0.08vs1.59±0.10,P<0.01）.It was lower than that of western medicine controlⅡgroup（2.06±0.08vs2.40±0.19,P<0.01）.Conclusion:1.Pathological manifestations of cell necrosis,connective tissue hyperplasia,lymphocyte and neutrophil infiltration,high expression of AGEs、RAGE、Keap1 protein and m RNA,low expression of Nrf2、β-catenin protein and m RNA were observed in diabetic ulcer wound tissue.2.The pathogenicity of AGEs is consistent with the characteristics of"Lingering toxins";AGEs are the pathological material basis of"Lingering toxins".The deposition of"Lingering toxins"in the wound tissue of diabetic ulcers leads to the reduction of antioxidant and cell proliferation ability,which is One of the mechanism of diabetic ulcer refractory.3.Badu Shengji Powder can promote skin ulcer healing in diabetic rats.4The multi-target mechanism includes removing the"Lingering toxins"deposited in the skin ulcer wound of diabetic rats by targeting AGEs,up-regulating the expression of Nrf2、Wnt1andβ-catenin in the granulation tissue,down-regulating the expression of AGEs、RAGE、Keap1 and GSK-3β,and increasing the deposition of collagen fibers、angiogenesis and antioxidant capacity,Inhibiting ulcerative surface inflammation.By this mechanism,Badu shengji Powder promotes skin ulcer healing in diabetic rats. |
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