| Background:Vitiligo is an immune disease characterized by selective destruction and progressive loss of melanocytes,which is easy to diagnose and difficult to treat.The mechanism of the destruction of melanocyte at the localized skin of Vitiligo patient is complicated,and further studies confirmed that oxidative stress is the initial factor of the pathogenesis of vitiligo.The level of oxidative stress in both localized skin lesion and througout the body in vitiligo patients is higher than healthy individuals.However,the generally thought key role of the autoimmune factors could be the secondary phenomenon after the melanocytes were damaged by oxidative stress.When the oxidative stress occurs,the Nrf2 through combined with sequence ARE further promote the expression of its downstream of HO-1,CAT and SOD Ⅱ detoxifying enzymes and antioxidant enzymes,which play a role of anti-oxidation,anti-inflammatory and immune regulation,so as to protect cells against oxidative stress damage.However,excessive ROS is not only an activator of other stress response systems including Nrf2,but also an activator of autophagy.Recent studies have shown that there is an ARE binding site in the promoter region of p62,which is an important participant in autophagy.Nrf2 binds to p62 promoter ARE and up-regulates the expression of p62 to participate in autophagy.Relevant studies have shown that the dysfunction of autophagy caused by abnormal Nrf2/p62 pathway increases the sensitivity of melanocytes of vitiligo to oxidative stress,thus promoting the development of vitiligo.Clinically,we observed that Buwu Decoction has a good therapeutic effect on vitiligo.Therefore,we proposed the following hypothesis:Buwu decoction of Traditional Chinese medicine regulates melanocyte autophagy to protect the damage of melanocyte anti-oxidative stress by activating Nrf2/p62 signaling pathway.Objective:1.In vivo research:Taking patients with vitiligo as the research object,taking Buwu decoction combined with phototherapy as the intervention method,taking Nrf2/p62 pathway as the entry point.Focus on regulating oxidation-antioxidant balance and immunity to inhibit the killing effect of CD8+cells on melanocytes,and explore the preliminary mechanism of Buwu Decoction in the treatment of advanced vitiligo.2.1n vitro experiment:To further explore the protective mechanism of Buwu decoction on melanocyte under oxidative stress.Taking normal humen melanocyte cell line HEM-a under oxidative stress model as the research object and Nrf2/p62 pathway as the entry point,to investigate the protective mechanism of HEM-a cells under H2O2-induced oxidative stress conditions by the serum containing BuWu decoction.Methods:In vivo research:Seventy patients were selected in accordance with the diagnosis criteria with advanced vitiligo,and ten healthy volunteers were selected.Patients with vitiligo were randomly divided into observation group(35 cases)and treatment group(35 cases).Five patients were lost to follow-up.Thus,The control group(32 cases)was treated with 308 excimer light therapy,while the observation group(33cases)was treated with Buwu decoction combined with the 308 excimer light.After the course of treatment(12 weeks),the clinical efficacy,VASI score and pigment score of the two groups were compared.The levels of SOD,MDA,CAT and GSH-Px in serum were biochemically tested before and after the treatment,and the level of HO-1 in serum were detected by ELISA.The levels of chemokines CXCL10 and CXCL16 in peripheral blood were detected by ELISA.The T cell subsets and the proportion of CD4+CD25+CD127LOW/-T cells in the peripheral blood lymphocytes were tested by flowcytometry.And the expressions of Nrf2 and p62mRNA were observed by RT-qPCR.In vitro experiment:Normal human melanocyte cell line HEM-a were treated with different concentrations(0.1mm,0.25mm,0.5mm,1.0mm,2.0mm)and times(0h,24h,48h)of H2O2-Cell activity was detected by CCK-8 method to determine the optimal H2O2 concentration in the model of oxidative stress of human melanocyte.Different concentrations(5%,10%,20%,40%)of the BWJJ-containing serum were selected,and the proliferation activity of melanocyte was detected by CCK-8 method to determine the optimal concentration of the drug serum.Experiments were divided into control group(HEM-a cell+ normal rat serum),model group(HEM-a cell+H2O2+normal rat serum),BWJJ group(HEM-a cell+H2O2+20%BWJJ-containing serum),’autophagy accelerator rapamycin group(HEM-a cell+H2O2+RAP+normal rat serum),RAP+BWJJ group(HEM-a cell+H2O2+RAP+20%BWJJ-containing serum),autophagy inhibitor chloroquine group(HEM-a cell+H2O2+CQ+normal rat serum),CQ+ BWJJ group(HEM-a cell+H2O2+CQ+20%BWJJ-containing serum).The morphology of melanocytes under the oxidative stress model was observed by inverted microscope.The content of HO-1 and GSH-PX in melanocytes and the activities of SOD and CAT in the model of oxidative stress were detected by ELISA.ROS fluorescence intensity,MMP level and cell apoptosis ratio were tested by Flow cytometry.The Autophagosome was observed by transmission electron microscopy.The number of autophagy lysosomes was observed by laser confocal microscopy.Western blot was used to observe the expression of Nrf2,LC3-II/LC3-I and P62 in cells.The expressions of Nrf2 and p62mRNA were observed by RT-qPCR.Results:1.In vivo research:1.1 Comparison of clinical efficacy:After 12 weeks of treatment,the clinical efficacy of the two groups was compared(χ2=6.292,P<0.05).1.2 Comparison of skin lesion area and pigment score between the two groups before and after treatment:Compared with before treatment,VASI score of skin lesion decreased in the two groups after treatment(P<0.01,P<0.05);After treatment,VASI scores were compared between the two groups(P<0.01).(2)Compared with before treatment,the pigment scores of the two groups increased after treatment(P<0.01,P<0.05).After treatment,the pigment scores of the two groups were compared(P<0.01).1.3 Peripheral blood oxidation-antioxidant index expression before and after treatment in the two groups:Before treatment,compared with the healthy group,MDA levels in peripheral blood of patients with vitiligo were significantly increased(P<0.01),and SOD,CAT,GSH-PX and HO-1 levels were significantly decreased(P<0.01).After treatment,MDA in peripheral blood of patients with vitiligo decreased significantly compared with that before treatment,while SOD,CAT,GSH-PX and HO-1 levels increased significantly compared with that before treatment(P<0.01)and after treatment(P<0.01).1.4 Expression of CXCL10 and CXCL16 in serum of patients in the two groups after treatment:Before treatment,compared with the healthy group,the levels of CXCL10 and CXCL16 in peripheral blood of patients with vitiligo were significantly increased(P<0.01).After treatment,peripheral blood CXCL10 and CXCL16 of patients with vitiligo decreased,compared with the same group before treatment(P<0.01),and compared with the two groups after treatment(P<0.01).1.5 Changes of T cell subsets of peripheral lymphocytes in patients with vitiligo before and after treatment:①Before treatment,there was no significant difference in the proportion of CD3+CD4+T cells compared with the healthy group(P>0.05).The proportion of CD3+CD8+T cells was significantly increased compared with the healthy group(P<0.01).The ratio of CD4+/CD8+T cells was significantly lower than that of the healthy group(P<0.01).②After treatment,the proportion of CD3+CD8+T cells decreased compared with that before treatment(P<0.01),the proportion of CD4+/CD8+was significantly higher than that before treatment,compared with that before treatment(P<0.01),and compared with that after treatment(P<0.01).1.6 Proportion of CD4+CD25+CD127LOW/-Treg cells in vitiligo patients before and after treatment:Before treatment,the proportion of CD4+CD25+CD127LOW/-Treg cells in peripheral blood of patients with vitiligo was significantly lower than that of the healthy group(P<0.01).After treatment,the proportion of CD4+CD25+CD127LOW/-Treg cells in peripheral lymphocytes of patients with vitiligo increased,compared-with the same group before treatment(P<0.01).Comparison between the two groups after treatment(P<0.01).1.7 Expression of Nrf2 and p62mRNA in PBMC of patients with vitiligo before and after treatment:Before treatment,the relative expressions of Nrf2 and p62mRNA in patients with vitiligo before and after treatment were significantly lower than those in the healthy group(P<0.01).After treatment,the relative expression.levels of Nrf2 and p62mRNA in patients with vitiligo were increased compared with those before treatment(P<0.01).Comparison between the two groups after treatment(P<0.01).2.In vitro experiment:2.1 In vitro observation showed that the activity of normal human melanocyte cell line HEM-a was decreased in a dose-dependent manner under H2O2 stimulation,According to IC50 value,1mM H2O2 for 24 h was optimal model to induce oxidative stress in melanocytes.2.2 On H2O2-induced oxidative stress,HEM-a cells were treated with different concentrations of BWJJ drug serum at 0h,24h and 48h.Compared with the model group,5%,10%,20%and 40%BWJJ containing serum group showed no significant difference in relative cell activity at 0h(P>0.05).The relative cell activity in HEM-a increased after the treatment of 5%,10%,and 20%BWJJ serum for 24h and 48h,compared with the model group(P<0.01).Compared with 20%BWJJ,40%BWJJ gradually decreased relative cell activity in HEM-a with the increase of drug concentration or prolonged action time.Therefore,the 20%BWJJ for 24h was selected as the optimal concentration and time of the drug-containing serum.2.3 An inverted microscope was used to observe the effect of BWJJ-containing serum on HEM-a H2O2-induced oxidative stress:Compared with the control group,the dendritic cells of the model group were significantly shortened and detached after 24h of stimulation with 1.0mm hydrogen peroxide.The dendrites of melanocytes were prevented from retracting at 20%BWJJ concentration.The results showed that the concentration of 20%serum containing aconite decoction could prevent the shortening of melanocyte dendritic process under oxidative pressure.2.4 Effect of 20%BWJJ on the activity of SOD,CAT and the contents of HO-1,GSH-PX in HEM-a cells under H2O2-induced oxidative stress model:①Compared with the control group,the activity of SOD,CAT and the content of HO-1,GSH-PX decreased in the model group(P<0.01).②Compared with the model group,the activity of SOD,CAT and the content of HO-1,GSH-PX in the BWJJ group increased(P<0.01).The activity of SOD,CAT and the content of HO-1,GSH-PX in the CQ group decreased(P<0.01,P<0.05).The activity of SOD,CAT and the content of HO-1,GSH-PX in the RAP group increased(P<0.01).③Compared with the CQ group,the activity of SOD,CAT and the content of HO-1,GSH-PX in the CQ+BWJJ group increased(P<0.01).④Compared with the RAP group,there was no significant change in the activity of SOD,CAT and the content of HO-1,GSH-PX in the RAP+BWJJ group(P>0.05).2.5 Effect of 20%BWJJ on the ROS fluorescence intensity in HEM-a cells under H2O2-induced oxidative stress model:①Compared with the control group,the level of intracellular ROS in the model group increased significantly(P<0.01).②Compared with the model group,the level of intracellular ROS in the BWJJ group decreased(P<0.01).The level of intracellular ROS in the CQ group increased(P<0.01).The level of intracellular ROS in the RAP group decreased(P<0.01).③Compared with the CQ group,the level of intracellular ROS in the CQ+BWJJ group decreased(P<0.01).④Compared with the RAP group,there was no significant change in the level of intracellular ROS in the RAP+BWJJ group(P>0.05).2.6 Effect of 20%BWJJ on the level of mitochondrial membrane potential in HEM-a cells under H2O2-induced oxidative stress model:①Compared with the control group,the level of MMP in the model group significantly decreased(P<0.01).②Compared with the model group,the level of MMP in the BWJJ group increased obviously(P<0.01).The MMP in the CQ group decreased(P<0.01).The level of MMP in the RAP group increased(P<0.01).③Compared with the CQ group,the level of MMP increased in the CQ+BWJJ group(P<0.01).④Compared with the RAP group,the level of MMP in the RAP+BWJJ group increased(P<0.05).2.7 Effect of 20%BWJJ on the percentage of cell apoptosis in HEM-a cells under H2O2-induced oxidative stress model:①Compared with the control group,the proportion of cell apoptosis in the model group increased significantly(P<0.01).②Compared with the model group,the percentage of apoptosis in the BWJJ group was significantly decreased(P<0.01).The percentage of apoptosis in the CQ group was significantly increased(P<0.01).The percentage of apoptosis in the RAP group was significantly decreased(P<0.01).③Compared with the CQ group,the proportion of apoptosis in CQ+BWJJ group decreased(P<0.05).④Compared with the RAP group,there was no significant difference in the proportion of apoptosis in the RAP+BWJJ group(P>0.05).2.8 Effect of 20%BWJJ on autophagosomes in HEM-a cells under H2O2-induced oxidative stress model:Compared with the control group,the number of mitochondria in the model group was reduced,and a large number of vacuolated mitochondria and a few autophagosomes were observed.Compared with the model group,in the BWJJ group and the RAP group,typical autophagosomes formed by a double-layer membrane wrapped part of the cytoplasm were seen near the nucleus.In the CQ group,a large number of vacuolated mitochondria were observed,and the integrity of the cell membrane was destroyed.The number of vacuolated mitochondria in the CQ+BWJJ group decreased,and a small number of autophagosomes were observed.2.9 Effect of 20%BWJJ on the autophagy flux in HEM-a cells under H2O2-induced oxidative stress model:After mRFP-GFP-LC3 adenovirus was transfected into HEM-a cells,laser confocal microscopy observed the change of 20%BWJJ on the autophagosomes(yellow fluorescent spots)and autolysosomes(red fluorescent spots)in HEM-a cells under H2O2-induced oxidative stress model.Compared with the control group,the number of autolysosomes in the red fluorescent spots of the model group is reduced;Compared with the model group,the number of autolysosomes in the red fluorescent spots of the BWJJ group and the RAP group was significantly increased,and the number of autolysosomes in the red fluorescent spots of the autophagy inhibitor CQ group was significantly reduced.Compared with the CQ group,the number of red fluorescent spots in the CQ+BWJJ group increased.Compared with the RAP group,the number of autolysosomes in the red fluorescent spots in the RAP+BWJJ group decreased.2.10 Effect of 20%BWJJ on the expression level of Nrf2,LC3 Ⅱ/Ⅰ,and P62 protein in HEM-a cells under H2O2-induced oxidative stress model:①Compared with the control group,The relative expression level of Nrf2 and LC3II/I protein in the model group were down-regulated,and the relative expression level of P62 protein was up-regulated(P<0.01).②Compared with the model group,The relative expression level of Nrf2 and LC3Ⅱ/Ⅰ protein in the BWJJ group were up-regulated,and the relative expression level of P62 protein was down-regulated(P<0.01).The relative expression level of Nrf2 and LC3Ⅱ/Ⅰ protein in the CQ group were down-regulated,and the relative expression level of P62 protein was up-regulated(P<0.01).The relative expression level of Nrf2 and LC3Ⅱ/Ⅰ protein in the RAP group were up-regulated,and the relative expression level of P62 protein was down-regulated(P<0.01).③Compared with the CQ group,The relative expression level of Nrf2 and LC3II/I protein in the CQ+BWJJ group were up-regulated,and the relative expression level of P62 protein was down-regulated(P<0.01).④Compared with the RAP group,The relative expression level of Nrf2 and P62 protein in the RAP+BWJJ group were up-regulated,and the relative expression level of LC3Ⅱ/Ⅰ protein was down-regulated(P<0.01).2.11 Effect of 20%BWJJ on the expression level of Nrf2 and p62 mRNA in HEM-a cells under H2O2-induced oxidative stress model:①Compared with the control group,the relative expression level of Nrf2 and p62mRNA in the model group was down-regulated(P<0.01).②Compared with the model group,the relative expression of Nrf2 and p62mRNA in the BWJJ group was up-regulated(P<0.01),the relative expression of Nrf2 and p62mRNA in the CQ group was down-regulated(P<0.01),the relative expression of Nrf2 and p62mRNA was up-regulated in the RAP group(P<0.01).③Compared with the CQ group,the relative expression of Nrf2 and p62mRNA in the CQ+BWJJ group was up-regulated(P<0.01).④Compared with the RAP group,the relative expression of Nrf2mRNA in the RAP+BWJJ group was up-regulated and the relative expression of p62mRNA was down-regulated(P<0.01).Conclusion:1.BWJJ combined with 308 excimer light can significantly reduce the VASI score of vitiligo to control the progression of the disease and increase pigment integral score to promote the repigmentaion in the treatment of advanced vitiligo.2.BWJJ combined with 308 excimer light can increase the level of HO-1,SOD,CAT and GSH-PX in serum,reduce the content of MDA in serum,and up-regulate the ratio of CD4+/CD8+and proportion of CD4+CD25+CD127LOW/-Treg cells in peripheral blood lymphocytes,showing good anti-oxidative stress and immune regulation effects.3.BWJJ combined with 308 excimer light can up-regulate the expression of Nrf2 and p62mRNA in peripheral blood PBMC.The mechanism of Buwu decoction combined with the 308 excimer light in the treatment of advanced vitiligo may be related to the activation of the Nrf2/p62 antioxidant pathway.4.BWJJ can increase the activity of SOD,CAT and the content of HO-1,GSH-PX of antioxidant enzymes,reduce the level of intracellular ROS in HEM-a cells under H2O2-induced oxidative stress,increase the level of MMP,and reduce the proportion of cell apoptosis.BWJJ has an anti-oxidative stress effect.5.BWJJ can up-regulate the autophagy level of HEM-a cells under H2O2-induced oxidative stress model,and reduce the autophagy level under the action of the accelerator rapamycin RAP,which has a two-way regulation effect.6.BWJJ can protect melanocytes against oxidative stress damage,and its mechanism may be related to the up-regulation of the autophagy level of cells activated by the Nrf2/p62 signaling pathway. |
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