Effect And Mechanism Of Berberine On Diabetic Wound Healing

Objective:The abnormal repair of diabetic wound is mainly manifested by decreased neovascularization and collagen deposition,prolonged inflammatory reaction and increased oxidative stress.In recent years,it has been proved by clinical and animal experiments that Berberine hydrochloride(BBR)has the effects of lowering blood sugar,lowering blood lipid,reducing insulin resistance,anti-inflammatory and anti-oxidation,and has certain curative effect on diabetes and its complications.This study is to explore the effects and mechanisms of BBR on diabetic wound healing through animal and cell experiments.Method:1.Diabetic rats were induced by intraperitoneal injection of SD rats(220g-260g)with STZ.A full-thickness skin defect model of diabetic rats was prepared by full skin resection of the back.Rats were randomly divided into normal control group(group NC),blank control group(group DM),drug solvent group(BBR Solvent),low dose drug group(LBBR)and high dose drug group(HBBR).The general situation of wound healing and the change of wound closure area were observed.The wound tissues were taken for analysis on the 3rd,8th,12th and 16th day of wound healing.HE and Masson staining were used to observe the changes of wound histomorphology.The formation and accumulation of advanced glycation end products(AGEs)were detected by ELISA.Western blotting was performed to detect the expression of RAGE receptor and type I collagen(Collagen I).The proliferation and apoptosis of fibroblasts in diabetic wound were detected by immunofluorescence.2.Human dermal fibroblasts(HDFs)were isolated and identified by the expression of vimentin.Glycosylated albumin(AGEs-HSA)was prepared in vitro.Cell experiment was divided into control group,AGEs-HSA groups,and HSA group.The appropriate concentration and effects of AGEs-HSA were explored on HDFS.The protein expression of RAGE was detected.The apoptosis-related indicators and caspase family proteins were further detected to identify the signaling pathway of apoptosis induced by AGEs-HSA.The changes of related indexes in mitochondrial apoptosis pathway were detected,including Bax/Bcl-2,mitochondrial membrane potential,Cytochrome c,and the levels of intracellular ROS.3.To explore whether BBR can regulate the effect of AGEs-HSA on HDFs,the cells were divided into control group,AGEs-HSA treatment group,HSA treatment group,BBR solvent group,and AGEs-HSA combined with low-dose or high-dose BBR groups.Firstly,the appropriate concentration of BBR was determined by detecting cell proliferation and toxicity.Then the protein level of RAGE was detected,and the apoptosis level was detected by TUNEL and flow cytometry.The level of ROS in fibroblasts was further detected,and whether ROS is the target of BBR was explored by using ROS activator instead of BBR.The changes of mitochondrial apoptosis signal pathway were detected,including mitochondrial membrane potential,Cytochrome c,Caspase-9 and Caspase-3 protein expression.Results:1.After injecting SD rats intraperitoneally with STZ,the blood glucose of diabetic rats was significantly higher than that of normal rats.Compared with NC group,the wound healing of DM group was significantly delayed(P<0.05),with the characteristics of decreased angiogenesis,delayed dissipation of inflammation and decreased deposition of matrix.BBR can effectively promote the proliferation and maturation of collagen fibers,and accelerate the coverage of new wound epithelium.At the same time,BBR can promote the growth of granulation tissue,improve the healing rate and shorten the healing time.2.AGEs was constantly generated and accumulated during diabetic wound healing.BBR can inhibit the accumulation of AGEs and reduce the overexpression of RAGE in diabetic wound.At the same time,BBR can increase the expression of Collagen I in diabetic wound and promote the deposition of extracellular matrix.In addition,BBR can increase the proliferation of fibroblasts and reduce the apoptosis level of fibroblasts in diabetic wound.3.HDFs were well separated.AGEs-HSA within 150?g/mL could induce fibroblasts apoptosis in a dose-dependent manner,while HSA had no significant effect on apoptosis at the same concentration.AGEs-HSA could up-regulate the expression level of RAGE protein and increase the expression of cleaved Caspase-9.Further experiments showed that AGEs-HSA increased the ratio of Bax/Bcl-2,caused the loss of mitochondrial membrane potential,and promoted the release of Cytochrome c,suggesting that AGEs-HSA mainly promotes apoptosis through mitochondrial signaling pathway.At the same time,AGEs-HSA increased the levels of ROS in fibroblasts.4.BBR under the concentration of 75?g/mL could antagonize the effects of AGEs-HSA in a dose-dependent manner.BBR promoted cell proliferation,inhibited cell apoptosis and reduced cytotoxicity of AGEs-HSA.BBR did not affect RAGE protein expression.BBR down-regulated the levels of ROS in cells,suggesting that BBR may exert the antagonistic effect of AGEs-HSA by regulating ROS.BBR can regulate the related indicators of mitochondrial apoptosis signal pathway,including increasing mitochondrial membrane potential,reducing Cytochrome c released to cytoplasm,and down-regulating cleaved Caspase-9 and Caspase-3 levels.Conclusion:1.In animal experiments,Berberine hydrochloride can effectively promote wound healing in diabetic rats.The effect of high concentration of BBR is more significant.BBR can inhibit the generation of advanced glycation end products in diabetic wounds,increase the proliferation of fibroblasts,and reduce the apoptosis level of cells.2.AGEs-HSA can induce apoptosis of fibroblasts by binding to RAGE and activating the mitochondrial apoptotic signaling pathway of ROS/cytochrome C/Caspase-9/Caspase-3,thus affecting the healing of diabetic wounds.BBR can inhibit the production of ROS in cells,block the activation of mitochondrial apoptosis signal pathway by AGEs-HSA,and reverse the effect of AGEs-HSA on fibroblasts.