Study On Anti-diabetic Ulcer Effect And Molecular Mechanism Of Huangbai Liniment And Its Component Berberine

Background and objectiveThe incidence of diabetes is on the rise all over the world,and its complication diabetic ulcer is the main cause of limb loss of diabetic patients,causing serious disability rate and mortality,which has become a global public health problem.Non-healing of diabetic wound makes it difficult for the treatment of diabetic ulcer and it is an urgent to find effective and theraputeic medicine for diabetic ulcer management.Huangbai liniment(HB)is a standardized drug for the treatment of ulcer and wound healing after ulcer,but its molecular me chanism is not clear.Berberine,has been proved to have good hypoglycemic and lipid-lowering effect in a large mount of studies.Therefore,in this study,streptozotocin(STZ)-induced diabetic rat model combined with full-skin resection was used to explore the anti-diabetic ulcer effect of HB and berberine and the molecular mechanism was revealed.The effect of berberine on diabetic ulcer and its molecular mechanism were further investigated at the cellular and molecular levels by using the high glucose(HG)induced Human immortalized keratinocytes(HaCaT)cell injury model.This research provides theoretical basis for the clinical application of HB and berberine,also benefits the development of new drugs for the treatment of diabetic ulcer and the formulation of protective strategies against diabetic ulcer.MethodsStudy on anti-diabetic ulcer effect and molecular mechanism of HB1.The effect of HB on wound healing in diabetes was investigated by STZ-induced diabetic rat model combined with full-skin resection.Normal saline,high and low dose of HB and rhEGF were treated for 13 days,photos were taken on days 6,9 and 13 to calculate the healing rate.HE staining,Masson staining and two-photon microscopy was used to observe the effect of HB on wound healing in diabetes.2.RNA-seq based transcriptome analysis were performed to evaluate the gene expression of skin tissues in normal control(Con group),diabetic ulcer group(STZ group)and HB intervention group(STZ+HB group).Bioinformatics was used for Enrichment analysis and cluster analysis was conducted and the network was generated by using cytoscape.3.Based on the results of transcriptome analysis and bioinformatics analy sis,the expression level of MDA was determined by using the kit.The expression of 8-OHdG was detected by immunofluorescence staining and ELISA.Immunofluorescence staining was used to evaluate the expression of caspase-3 and TGF-β1.The content of MMP9 was determined by ELISA.Western blotting and immunofluorescence staining were used to detect the expression of Nrf2 and its downstream molecule NQO1,so as to elucidate the molecular mechanism of compound phellodendrite solution in the treatment of diabetic ulcer.By using network pharmacology,through BATMAN-TCM、String and other databases,the components of HB were correlated with Nrf2 and its downstream differential antioxidant genes,and a network of "drug-component-target-differentially expressed genes" was constructed.Study on anti-diabetic ulcer effect and molecular mechanism of berberine1.The effect of berberine on wound healing in diabetes was investigated by STZ-induced diabetic rat model combined with full-skin resection.After intervention with normal saline,berberine and rhEGF for 12 days,the wound was photographed on days 3,6,9 and 12 to calculate the healing rate,and the effect of berberine on wound healing was detected by HE staining and Masson staining.2.To explore the molecular mechanism of berberine in treating diabetic ulcer by means of biochemistry and molecular biology,the levels of SOD,MDA and T-AOC were detected with the kit.The level of Caspase-3 was detected by immunohistochemical staining combined with a kit.Expression of TrxR1,MMP9 and TGF-β1 was detected by immunohistochemical staining combined with ELISA.Immunohistochemical staining was used to detect the expression of TIMP1 by 8-OHdG and ELISA,and Western blotting was used to detect the expression of p-JNK/JNK,so as to elucidate the molecular mechanism of berberine against diabetic ulcer.3.The protective effect of berberine on cell damage induced by high glucose(HG)was detected by HaCaT model induced by HG,and the effect of inhibition of TrxR1 on berberine mediated protective effect was observed by Auranofin(TrxR1 inhibitor),and cell viability was detected by CCK-8 method.The expressions of SOD、MDA、T-AOC、GSH、ROS and p-JNK/JNK were measured with the kit.Mitochondrial membrane potential was detected by JC-1 staining.Apoptosis was detected by TUNEL staining.The contents of TrxR1,TIMP1 and TGF-β1 were determined by ELISA.Immunofluorescence staining was used to detect the cell positive rate of Ki67 and the expression of p-JNK、MMP9 to verify the protective effect of berberine on cell damage induced by HG.ResultsStudy on anti-diabetic ulcer effect and molecular mechanism of HB1.HB obviously promoted wound healing of diabetic ulcer.Compared with the Con group,the wound healing in the STZ group was significantly decreased,which was obviously increased in the STZ+HB group and STZ+rhEGF group after 6,9,and 13 days.HE staining results showed that ECM secretion was significantly decreased in STZ group,HB and rhEGF treatment significantly increased ECM secretion in diabetic wound.The results of Masson staining and two-photon microscopy showed that the expression of collagen in the wound of rats in the STZ group decreased significantly and the expression of collagen increased after the intervention of HB and rhEGF.Therefore,HB has the obvious effect of repairing diabetic skin ulcer2.Transcriptome sequencing revealed that oxidative stress may be the key biological process of HB against diabetic ulcer.RNA-Seq results showed that,compared with Con group,the STZ group had 8469 differentially expressed genes(DEs),while the STZ+HB group had 6551 DEs.GO and KEGG cluster analysis showed that oxidative stress may be a key process for HB to repair diabetic ulcers.Analysis of Nrf2 downstream differentially expressed genes revealed showed that STZ-induced diabetic ulcer injury significantly inhibited the downstream antioxidant gene of Nrf2,while HB treatment increased the expression of the downstream antioxidant gene of Nrf2.This indicates that HB may form an important antioxidant network by enhancing Nrf2 and its downstream antioxidant genes,thus inhibiting oxidative stress damage.3.HB activated Nrf2 and its downstream antioxidant genes to play an important role in anti-diabetic ulcer.The results showed that the levels of the oxidation products MDA and 8-OHdG were significantly increased in the STZ group,and the staining results of Caspase-3 showed that the apoptosis was significantly increased,the expression of Nrf2 and its downstream molecule NQO1 was inhibited,and the expression of MMP9 and TGF-β1 were significantly increased.HB significantly reduced the levels of the oxidation products MDA and 8-OHdG,reduced the cell apoptosis shown by the staining results of Caspase-3,activated the expression of Nrf2 and its downstream molecule NQO1,significantly reduced MMP9 and increased the level of TGF-β1.It was revealed that HB plays an antidiabetic ulcer effect by activating Nrf2 and its downstream antioxidant gene,and its effect is also related to the improvement of TGF-β1 and the reduction of MMP9.Through networkpharmacology analysis,54 components of HB acted on 169 drug targets,and then regulated Nrf2 and its downstream antioxidant genes,so as to inhibit oxidative stress damage by the synergistic action of multiple components and multiple targets.Study on anti-diabetic ulcer effect and molecular mechanism of berberine1.BBR obviously promoted wound healing of diabetic ulcer.Compared with the Control group,the wound healing in the STZ group was significantly decreased.After berberine and rhEGF treatment for 3,6,9 and 12 days,the wound healing rate became significantly higher than that in the STZ group.HE staining results showed that ECM secretion was significantly decreased in STZ group,berberine and rhEGF treatment significantly increased ECM secretion in diabetic skin ulcer wound.Masson staining results showed that the collagen expression in the front of the wound was less in STZ group,but was significantly increased after berberine and rhEGF treatment.In addition,Auranofin(TrxR1 inhibitor)significantly delayed berberine-mediated wound healing at day 3,6,9,and 12,and reduced ECM secretion and collagen production.This indicated that berberine showed obvious effect in promoting diabetic wound healing,but the effect can be cancelled by Auranofin.2.Berberine played an anti-diabetic role by regulating TrxRl/JNK signaling pathway.The results of animal experiments showed that the activity of antioxidant enzymes T-AOC and SOD in STZ group was significantly reduced,MDA content and 8-OHdG level were significantly increased,TUNEL staining and caspase-3 results showed a significant increase in apoptosis,the expression of antioxidant protein TrxR1 was inhibited,and the expression of p-JNK/JNK was increased,while the expression of MMP9 was significantly increased,and the levels of TGF-β1 and TIMP1 were decreased.After berberine intervention,the activity of T-AOC and SOD was increased,MDA content and 8-OHdG level were decreased,cell apoptosis shown by TUNEL staining and caspase-3 results was reduced,the antioxidant protein TrxR1 was activated,and the expression of p-JNK/JNK was decreased,meanwhile,MMP9 was significantly reduced,and TGF-β1 and TIMP1 levels were increased.In addition,compared with the STZ+BBR group,the activity of T-AOC and SOD were significantly reduced and the levels of MDA and 8-OHdG were significantly increased after intervention with the inhibitor kinofen.The results of TUNEL staining and caspase-3 showed a significant increase in apoptosis,and the expression of TrxR1 was significantly downregulated while the expression of p-JNK/JNK was significantly up-regulated.Meanwhile,the expression of MMP9 was significantly increased and the levels of TGF-β1 and TIMP1 were significantly decreased.This results suggested that berberine repaired diabetic ulcer wounds by reducing oxidative damage and apoptosis by restoring the TrxR1/JNK signaling pathway,and its repair effect was related to the reduction of MMP9 and the increase of TGFβ1 and TIMP1 levels.3.Berberine protected HaCaT cells from HG induced damage by regulating the TrxR1/JNK signaling pathway.The results of in vitro studies showed that HG significantly reduced the activity of HaCaT cells,increased ROS and MDA levels,decreased GSH level,T-AOC and SOD activity in HaCaT cells.HG induced increased Caspase-3 activity,decreased mitochondrial membrane potential,increased TUNEL-positive cell rate,and decreased Ki67-cell positive rate.In addition,HG induction reduced TrxR1 expression in HaCaT cells and increased its downstream p-JNK/JNK levels.At the same time,the level of MMP9 in HaCaT cells significantly increased,and the level of TGF-β1 and TIMP1 significantly decreased.Berberine significantly alleviated the damage of HaCaT cells induced by HG,reduced ROS and MDA levels,increased GSH level,T-AOC and SOD activity,decreased Caspase-3 activity,inhibited the decline of mitochondrial membrane potential,decreased the TUNEL-positive cell rate and increased the Ki67-cell positive rate.Berberine intervention activated TrxR1 and inhibited the expression of its downstream molecule p-JNK/JNK,promoted the expression of TGF-β1 and TIMP1,and inhibited the expression of MMP9.Notably,TrxR1 inhibitor(Auranofin)increased ROS and MDA levels,decreased GSH level,T-AOC and SOD activity,increased Caspase-3 activity,decreased mitochondrial membrane potential and increased TUNEL-positive cell rate and Ki67-cell positive rate,depriving berberine of its protective effect on HaCaT cells.In addition,Auranofin inhibited the expression of TrxR1 and activated the expression of its downstream molecule p-JNK/JNK,resulting in the increased expression of MMP9 and decreased expression of TGF-β1 and TIMP1.These results suggested that berberine reduced cell oxidative damage,inhibited cell apoptosis,and promoted cell proliferation by restoring the TrxR1/JNK signaling pathway.Meanwhile,berberine promoted the expression of TGF-β1 and TIMP1,and inhibited the expression of MMP9,protecting HaCaT cells from damage induced by HG.