The Efficacy Of Adipose-derived Mesenchymal Stem Cells Combined With Narrow-band Ultraviolet On Vitiligo Mice Model And Its Action On Endoplasmic Reticulum Stress Signaling Pathway

Objective:Vitiligo is a clinically common depigmentation disease,mainly involving functional epidermal pigment cells and hair follicle melanocytes.The main clinical features of vitiligo are milky spots or patches of the skin,often involving multiple parts of the head,face,neck,trunk,limbs and so on.The pathogenesis of vitiligo is still unclear.There are several related hypotheses,among which the most common ones include autoimmune regulation,oxidative stress,genetic variation and cytotoxic effects.Studies have shown that ER stress can lead to calcium overload of melanocytes,and abnormal or persistent oxidative stress will lead to a large number of reactive oxygen species(ROS)and accumulation of oxidation products,stimulate the disorder of ER internal environment,the imbalance of oxidation and anti-oxidation,and lead to the damage of melanocytes.Narrow-spectrum medium-wave ultraviolet(NB-UVB)irradiation is currently the preferred treatment for vitiligo.NB-UVB irradiation can inhibit the oxidative stress response and promote the recovery of melanocyte function in patients with vitiligo.But there has been a consensus about the possible carcinogenic effects of intense ultraviolet radiation over the decades.Recent studies have found that the proliferation,differentiation and migration of melanocytes can be induced by adipose derived stem cells through co-culture,which is better than that of melanocytes cultured alone.In addition,Nrf2/HO-1 signaling pathway is an important pathway for cells to resist exogenous substances and oxidative damage.Oxidative stress induced by excessive ROS accumulation can activate Nrf2 activity.Nrf2 is uncoupled with cytoplasmic chaperone protein Keapl,transferred to the nucleus under the phosphorylation of a variety of protein kinases,binds to nuclear antioxidant elements ARE,and activates downstream antioxidant molecules such as HO-1 and Trx-1 to play an antioxidant role.Therefore,in this study,we first observe the efficacy of NB-UVB,adipose mesenchymal stem cells transplantation and their combination in the treatment of vitiligo mice,detect the changes of oxidative stress and endoplasmic reticulum oxidative stress,and explore the action of Nrf2/HO-1and provide experimental basis for clinical prevention and treatment of vitiligo.Research methods:1.Observation on the therapeutic effect of adipose mesenchymal stem cells combined with NB-UVB on mice with vitiligo induced by monobenzone.50 C57BL/6 mice were randomly divided into 5 groups:blank control group(control group),vitiligo model group(VIT group),medium-wave narrow-band ultraviolet irradiation group+vitiligo model group(VIT+NB-UVB group),stem cell transplantation+vitiligo model group(VIT+AMSCs group),stem cell transplantation+narrow-band ultraviolet irradiation+vitiligo model group(VIT+AMSCs+NB-UVB group)with 10 mice in each group.C57BL/6 mice were shaved locally on the back with an electric shaver,the size of which was about 2cm×2cm².The blank control group was smeared with glycerine outside the hair preparation area,25mg dosage each time,twice a day.The vitiligo model group,medium wave narrow spectrum ultraviolet irradiation group+vitiligo model group and stem cell transplantation+medium wave narrow spectrum ultraviolet irradiation+vitiligo model group were smeared with 40%monobenzene,25mg each time,twice a day.The local area was cleaned with sterile water before 40%monobenzene was smeared,and the administration time was fixed at 9o'clock and 17:00 per day for 50 days(17:00 on the 0th day,9am and 17:00 on the first day).At 9:00 on the second day,17:00 on the second day,and 9:00 on the 50th day),the mice were shaved every 3 days until the skin was depigmented.The therapeutic effect of experimental vitiligo in each group was observed with naked eye,the histopathological changes of skin were observed by HE staining The stem cells were injected subcutaneously at a concentration of 5×10⁶/ml per square centimeter,not penetrating the skin.Ultraviolet irradiation:wave peak 311nm.0.5J/cm²as the starting dose;Each dose was increased by 0.05-0.1J/cm²,3 times/week,and the intervention was carried out from 30 days to 50 days after modeling.The therapeutic effect of vitiligo in each group was observed by naked eye.He staining was used to observe the changes of skin histopathology in mice.2.Effects of adipose mesenchymal stem cells combined with NB-UVB on endoplasmic reticulum stress in vitiligo mice.The experimental animals were divided into groups with the same as in Method 1 the content of oxidative stress factor in serum of mice in each group was detected by Elisa;the release of ROS in tissue was detected by immunofluorescence;Western blot and PCR to detect the expression of endoplasmic reticulum stress-related proteins GRP78,caspase-12 and SERCA2a;immunofluorescence labling was used to observe calciumion.3.Adipose mesenchymal stem cells combined with NB-UVB activate Nrf2/HO-1 signal pathway to improve the stress injury of endoplasmic reticulum in vitiligo.The experimental animals were divided into groups with the same method 1,In order to investigate the mechanism of Nrf2/HO-1 signaling pathway,human epidermal melanocytes were selected and treated with 500?mmol/L H₂O₂for 24h to simulate vitiligo model.The cells were divided into I)control group(CONTROL),II)model group(VIT),III)model+adipose mesenchymal stem cell transplantation+UV irradiation group(VIT+AMSCS+NB-UVB group),IV)model+adipose mesenchymal stem cell transplantation+UV irradiation group+NRF2 inhibitor ML385 group(VIT+AMSCS+NB-UVB+ML385).4.Western blot was used to detect the expression of proteins related to Nrf2/HO-1signal pathway in skin tissue of mice in each group.H₂O₂was used to induce melanocytes to establish oxidative stress injury cell model.ELISA method was used to detect the contents of SOD,MDA,MPO,TYR,MIF and MAO in the supernatant of each group.;Western blot was used to detect the expression of endoplasmic reticulum stress-related proteins GRP78,caspase-12 and SERCA2a,and immunofluorescence was used to observe calcium ion.The composition of CD4 and CD8T subsets in peripheral blood lymphocytes of mice was detected by flow cytometry.Results:1.adipose stem cells culture and curative effect observation:cultivation of AMSCs cells after 7 days,there are a large number of adherent cell growth,cell colony formation sizes,cells are long spindle,via immunofluorescence appraisal results showed that stem cell surface marker CD90 and CD45 were positive,induced into fat and oil red O staining identified cells have a lot of fat droplets.Effect evaluation of the focal area of mice in each group was carried out.No significant changes were observed in the focal area of mice in the blank control group,and the evaluated effective rate was 0.The lesion area of VIT group was pale and leuk-like,and the total effective rate was 10%.Compared with VIT group,the total effective rate of VIT+NB-UVB group and VIT+AMSCs group was 50%and 40%,respectively.The total effective rate of the combined treatment group was 70%.2.HE staining results:the infiltration of lymphocytes in the decolorization area and surrounding skin tissue in the VIT group was the most obvious,the infiltration of VIT+NB-UVB group and VIT+AMSCs group was reduced,while the infiltration of lymphocytes in the VIT+AMSCs+NB-UVB group was only a few.Flow cytometry analysis was also consistent with the above performance.Using CD3T as the reference,the proportion of CD4T in VIT group was the lowest(26.776±4.783),and the proportion of CD8T was the highest(18.106±4.562,P<0.05).There was no significant difference in the proportion of CD4T and CD8T between the VIT+NB-UVB group and the VIT+AMSCs group.The proportion of CD4T in VIT+AMSCS+NB-UVB group was 34.183±0.635 and the proportion of CD8T was the lowest(14.383±1.557,P<0.05)compared with the other four groups.3.Plasma tyrosinase(TYR),macrophage mobility inhibitory factor(MIF)and monoamine oxidase(MAO)levels were detected by ELISA:compared with blank control group,plasma MAO(25.94±3.41)and MIF(28.41±4.47)expressions were increased in VIT group,while TYP expression was decreased(157.18±19.64,P<0.05).Compared with VIT group,plasma MAO and MIF expressions were significantly decreased in VIT+NB-UVB group and VIT+AMSCs group,while plasma TYP expression was significantly increased(P<0.05),but there was no statistical difference between the two groups.Compared with the other three groups(except the control group),the plasma expressions of MAO(12.49±2.51)and MIF(16.79±2.28)in VIT+AMSCs+NB-UVB group were significantly decreased,while the expression of TYP was significantly increased(302.84±44.01,P<0.05).4.The expressions of superoxide dismutase(SOD),malondialdehyde(MDA)and myeloperoxidase(MPO)in serum of mice were detected by ELISA:compared with blank control group,the expressions of MDA(14.91±0.86)and MPO(1120.65±131.11)in VIT group were significantly increased,while the expression of SOD was significantly decreased(23.25±2.27,P<0.05).Compared with VIT group,the expressions of MDA and MPO in VIT+NB-UVB group and VIT+AMSCs group were significantly decreased,while the expression of SOD was significantly increased(P<0.05).There was no statistical difference between the two groups.Compared with the other three groups(except the control group),the expressions of MDA(8.72±0.42)and MPO(669.52±76.95)in VIT+AMSCs+NB-UVB group were the lowest,and the expression of SOD was the highest(57.74±2.75,P<0.05).5.Immunofluorescence detection of skin tissue ROS detection:ROS fluorescence intensity in VIT group was higher than that in control group;Compared with the VIT group,the ROS fluorescence intensity in the VIT+NB-UVB group and the VIT+AMSCs group was decreased.However,the fluorescence intensity of ROS in VIT+AMSCs+NB-UVB group was the lowest,suggesting that ROS release was significantly inhibited.6.The expression and m RNA expression of ER stress-related proteins GRP78,Caspase-12 and SERCA2a were detected by Western blot and PCR.Compared with the control group,the expression of SERCA2a was decreased in VIT group,while the expression of GRP78 and Caspase-12 were increased(P<0.05).Compared with VIT+NB-UVB and VIT+AMSCs groups,the expression of SERCA2a was increased,and the expression of GRP78 and Caspase-12 were decreased.In the VIT+AMSCs+NB-UVB group,the expression of SERCA2a was increased,while the protein expression of GRP78 and Caspase-12 was decreased,with statistical difference between the two groups(P<0.05).7.Western blot analysis of the expression of Nrf2/HO-1 signaling pathway:The results showed that among the four groups,the expressions of Nrf2,HO-1 and Trx-1 in the control group were the lowest(P<0.05),and the expressions of VIT+NB-UVB and VIT+AMSCs groups were significantly better than those in the VIT group(P<0.05),but there was no statistical difference between the two groups.The expression of VIT+AMSCs+NB-UVB group was the highest compared with the other three groups,and the difference between groups was statistically significant(P<0.05).8.The contents of TYR,MIF,MAO,SOD,MDA and MPO in the supernatant of melanocytes were detected by increasing Nrf2/HO-1 antagonist(ML385)ELISA.Results Compared with the control group,the contents of MAO,MIF,MDA and MPO in the supernatant of melanocytes in VIT group were significantly increased,while the expressions of TYP and SOD were significantly decreased(P<0.05).Compared with VIT group,the contents of MAO(12.29±2.85),MIF(14.76±5.58)MDA(8.59±0.44)and MPO(526.91±61.28)in cell supernatant of VIT+AMSCs+NB-UVB group were significantly decreased,while the contents of TYP(250.67±35.99)and SOD(40.76±3.42)were significantly increased,with statistical differences among groups(P<0.05).Compared with VIT+AMSCS+NB-UVB group,the contents of MAO(18.55±6.13),MIF(25.95±2.94),MDA(13.21±1.22)and MPO(876.75±79.00)in VIT+AMSCS+NB-UVB+ML385 group were increased,while the contents of Typ(132.46±37.40)and SOD(20.39±2.34)were decreased in VIT+AMSCS+NB-UVB+ML385group,with statistical difference(P<0.05),suggesting antagonism.9.Nrf2/HO-1 antagonist(ML385)was added to detect the expression of ER stress-related GRP78,Caspase-12 and SERCA2a in each group by Western blot:the results showed that the expression of SERCA2a in VIT+AMSCs+NB-UVB group was increased and the expression of GRP78 and Caspase-12 were decreased compared with VIT group(P<0.05).However,the expression of SERCA2a in VIT+AMSCS+NB-UVB+ML385 group was significantly lower than that in VIT+AMSCS+NB-UVB group,while the expression of GRP78 and Caspase-12 was increased(P<0.05),suggesting antagonism10.Immunofluorescence detection of calcium ions in cells showed that the fluorescence intensity of calcium ions in VIT group increased and calcium ion overload was serious.Compared with the VIT group,the fluorescence intensity of calcium ions in the VIT+AMSCs+NB-UVB group decreased significantly,and calcium ion overload was inhibited.Compared with the VIT+AMSCs+NB-UVB group,the fluorescence intensity of calcium ions in the VIT+AMSCs+NB-UVB+ML385 group increased.Conclusion:1.The effect of AMSCs combined with NB-UVB on vitiligo mice is better than that of vitiligo alone,and has a certain curative effect on vitiligo.2.AMSCs combined with NB-UVB can inhibit the stress injury of endoplasmic reticulum in vitiligo mice.3.AMSCs combined with NB-UVB can improve vitiligo injury by activating Nrf2/HO1pathway to regulate endoplasmic reticulum stress.