| Vitiligo is a common depigmented skin disease caused by the destruction of melanocytes,which prevalence rate is 0.5%-2%.The decrease or disappearance of local melanocytes in the skin lesions and their dysfunction are the main reasons for the loss of pigment.The pathogenesis of vitiligo is complex.At present,most of the views believe that vitiligo is the result of the damage of melanocyte due to the combination of environmental factors,immunological factors,neuropsychiatric factors and other factors in the genetic background.In recent years,there are opinions that oxidative stress plays a key role in the occurrence and development of vitiligo and may be the earliest event that induces vitiligo lesions.Under normal physiological conditions,a small amount of ROS can be degraded into a low-toxic or non-toxic substance by the body' s antioxidant system,so it will not cause harm to the human body.However,vitiligo lesions and body are in a state of high oxidative stress and antioxidant system disorder,which caused oxidant-antioxidant imbalance and excessive accumulation of ROS,thereby attacking melanocytes,interfering their normal metabolism,function and differentiation,Which induces the body's autoimmune response,causing irreversible damage to the epidermal melanocytes,resulting in the decrease in the number of melanocytes in skin and hair follicles.In addition oxidative stress can also cause melanin production and conduction disturbances.Together these effects result in localized or generalized skin depigmentation.The main antioxidant enzymes includes superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx),heme oxygenase(HO-1)and so on.The SOD enzyme can convert the superoxide anion(O2-)into H2O2,which is lower toxic,by the disproportionation reaction;CAT and GPx-1 can decompose H2O2 into O2 and H2O,thereby protecting cells from oxidative damage.HO-1 belongs to microsomal enzyme system and is the rate-limiting enzyme of heme catabolism.All of these enzymes can protect melanocytes from oxidative damage.There is still no effective treatment for vitiligo.Looking for effective anti-oxidants for the treatment of vitiligo is a new direction of research.Geniposide is the main active ingredient of Eucommia ulmoides and wolfberry fruit and has high anti-oxidant activity.A number of studies have demonstrated that Geniposide can protect neurons,vascular endothelial cells,hepatocytes,and other cells from oxidative damage.However,there is no report on the oxidation protection effect of melanocytes.The protective effect of geniposide on cells is partly related to the PI3K-Akt signaling pathway and the regulation of antioxidant enzymes.PI3K-Akt signaling pathway is one of the main pathways of cell survival and the Bcl-2 family and the Caspase family are two important groups of proteins in the downstream of PI3K-Akt.Studies have found that Bcl-2 family,and Caspase family all play an important role in the survival and apoptosis of melanocytes.In addition,PI3K-Akt pathway can regulate the activity of SOD,CAT,GPx and HO-1 by activating Nrf-2 protein.In order to investigate whether Geniposide can protect oxidative damage of melanocytes,and the roles of PI3K-Akt signaling pathway and its downstream proteins,we isolated human melanocytes from human foreskin tissues and cultured melanocytes with different concentrations of H2O2.The viability of the cells was detected by MTT to select suitable H2O2 concentrations for inducing oxidative damage.Then melanocyte were incubated with different concentrations of Geniposide for 24 hours,and melanocyte oxidative damage was induced by H2O2.We observe the effects of Geniposide preincubation on the viability,apoptosis and related antioxidant enzymes activities of melanocytes under oxidative stress,so as to select suitable Geniposide concentration for protecting melanocytes from oxidative damage.Then we added LY294002,a PI3K inhibitor,before H2O2-induced oxidative damage and detected Akt,p-Akt,Bcl-2,Bax,cleaved-Caspase3,cleaved-Caspase9,HO-1,GPx-1,which are the key factors that regulate the growth,by Western blot.We also detected melanocytes viability,apoptosis,SOD enzyme activity,CAT enzyme activity and ROS content,to explore the related mechanism of Geniposide in the protection of melanocytes against oxidative stress and to provide theoretical basis and new ideas for the use of Geniposide in the treatment of vitiligo.Part1 Protective effect of geniposide on oxidative damage of melanocytesObjective In order to search for the best H2O2 concentration to establish oxidative stress injury model of melanocytes in vitro,different concentrations of H2O2 were used to induce oxidative stress damage of melanocytes,and the cell viability was detected.The effects of Geniposide on the proliferation,apoptosis,SOD and CAT enzyme activity and ROS content of melanocytes were observed in order to screen the suitable concentration of Geniposide in the pretreatment of melanocytes.Method 1.Melanocytes were cultured in 96-well plat in the density of 5×103/well.They were cultured with 0? mol/L,62.5 u mol/L,125? mol/L,250?mol/L,500?mol/L,1000 ? mol/L H2O2 respectively for 4h.Melanocytes viability was measured by MTT assay.2.The melanocytes were incubated with 0 ?mol/L(H2O2 group),5 ?mol/L,25 ? mol/L,125 ?mol/L,and 625?mol/L Geniposide for 24h.Then,according to the above results,250 ?mol/L H2O2 was used to induce melanocyte oxidant damage.To observe the protective effect of geniposide on oxidative stress injury of melanocytes,the cell viability rate was measured by MTT assay,the activity of SOD and CAT was detected by biochemical method,the apoptotic rate and ROS content were detected by flow cytometry.Results 1.After incubation melanocytes with H2O2 in low concentration group(25 ?mol/L?125 ? mol/L),their activity did not change significantly(cell viability were 91.31±10.41%and 85.82± 12.17%respectively,all P>0.05),H202 in high concentration group(500 ? mol/L?1000 ? mol/L)could cause melanocyte viability to decrease significantly(cell viabiligy were 40.211 ±4.43%? 10.19 ±10.96%respectively,all P<0.01),and too much cell death.250 ? mol/L H202 not only caused a significant decrease in cell viability but did not cause too much cell death(cell viability was 60.09±5.09%,p<0.01).2.Geniposide pretreatment could improve the oxidative damage induced by H202 in melanocytes.The cell viability rate and apoptotic rate of 5 ? mol/L and 25 P mol/L Geniposide group were not statistically different from that of H202 group(cell viability of H202 group?5 ?mol/L and 25 ? mol/L Geniposide group were 58.92 ± 4.16%.72.75±4.42%?73.45±6.10%respectively,apoptosis rate were 17.82 ± 2.27%?17.91 ±3.29%?15.66±1.48%respectively,all P>0.05),while 125 ?mol/L and 625 ? mol/L Geniposide group cell viability rate increased(cell viability were 84.23±9.94%82.01 ±6.64%,all P<0.05)and apoptotic rate was significantly decreased(apoptosis rate were 7.74±0.98%?9.60±0.66%,all P<0.01).However,there was no significant difference in the rate of viability and apoptosis between 125 ? mol/L and 625 ? mol/L Geniposide groups(P>0.05).3.Compared with control group(SOD activity was 7.77 ±1.54 units/mg and CAT activity was 55.94 ± 5.74 units/mg),H202 can significantly reduce the SOD activity(1.78±0.19 units/mg,P<0.01)and CAT activity(20.99 ± 3.83 units/mg,P<0.01)in melanocytes.Compared to H2O2 group,there was no significant difference in SOD and CAT enzyme activity between 5?mol/L and 25? mol/L Geniposide groups(SOD activity were 2.21 ± 0.39 units/mg?3.82±0.30 units/mg respectively and CAT activity were 24.36±4.29 units/mg?34.07 ± 4.61 units/mg respectively,all P>0.05).while the activity of SOD and CAT in 125 mol/L and 625 mol/L Geniposide groups increased significantly(SOD activity were 6.06±0.95 units/mg?6.66 ± 0.34 units/mg respectively and CAT activity were 50.37±6.27 units/mg?47.15±4.96 units/mg respectively,all P<0.01),but there was no significant difference between 125?mol/L and 625?mol/L Geniposide(P>0.05).Compared with the H2O2 group(ROS was 2086±110.81),the ROS content in 5?mol/L Geniposide group had no significant change(ROS was 1758.33±130.12,P>0.05),while the ROS content in 25pmol/L,125?mol/L,and 625?mol/L Geniposide group was significantly decreased(ROS was 1399±106.52?1105±?121.31?983.33±93.67 respectively,all P<0.01).There was no significant difference in ROS between 25 ?mol/L and 125 ?mol/L,125 ? mol/L and 625 ?mol/L Geniposide groups(P>0.05),while the difference between 25?mol/L and 625? mol/L Geniposide groups was statistically significant(P<0.05).Conclusion 1.250 ? mol/L H2O2 could induce oxidative damage of melanocytes and successfully construct the oxidative stress model.2.Geniposide can relieve H2O2-induced melanocyte oxidative damage and 125? mol/L is a suitable concentration to alleviate the oxidative damage of melanocytes.Part 2 Effects of geniposide on PI3K-Akt signaling pathway and apoptosis-related proteins in downstreamObjective To detect the changes of PI3K-Akt signaling pathway and its downstream proteins including Bcl-2,Bax,cleaved-Caspase3 and cleaved-Caspase9 in the protection of Geniposide on oxidative damage in melanocytes.Methods Geniposide preincubated melanocytes for 24 hours,added or did not add PI3K inhibitor LY294002,and then induced oxidative stress reaction of melanocytes with H202.The melanocytes were divided to control group,Geniposide group,LY294002 group,H202 group,Geniposide+H2O2 group,and Geniposide+LY294002+H2O2 group.Western blot was used to detect the expression of Akt,p-Akt,Bcl-2,Bax,cleaved-Caspase3,and cleaved-Caspase9 proteins in each group.Results Compared with the control group,the expression of p-Akt/Akt,Bcl-2,Bax,cleaved-Caspase9 and cleaved-Caspase3 did not change significantly in 125?mol/L Geniposide(P>0.05).H2O2 can significantly reduce p-Akt/Akt(P<0.01)and Bcl-2(P<0.01),and increase apoptotic protein Bax(P<0.01),cleaved-Caspase3(P<0.01)and cleaved-Caspase9(P<0.05).Compared with H2O2 group,the expression of p-Akt/Akt was increased(q=5.044,P<0.05)and Bcl-2 was upregulated(q= 7.532,P<0.05),while Bax(q=6.099,P<0.05),cleaved-Caspase3(q=6.630,P<0.05),and cleaved-Caspase9(q=4.828,P<0.05)were downregulated in Geniposide+H2O2 group.Compared with Geniposide+H2O2 group,Bax?cleaved-Caspase3 and cleaved-Caspase9 were up-regulated(all P<0.05),and p-Akt/Akt was decreased(q=8.560,P<0.01).Bcl-2 was slightly down-regulated,but there was no significant difference compared with the Geniposide + H2O2 group(q=3.745,P>0.05).Conclusion Under H2O2-induced oxidative stress,pre-cultured with Geniposide can enhance Akt phosphorylation,activate PI3K-Akt signaling pathway,increase Bcl-2 expression,and reduce the expression of apoptotic proteins Bax,cleaved-Caspase3 and cleaved-Caspase9 in melanocytes.PI3K inhibitor LY294002 inhibits geniposide's activation of PI3K-Akt pathway and reverses its downregulation of Bax,cleaved-Caspase3 and cleaved-Caspase9.Part 3 The role of PI3K-Akt signaling pathway in the regulation of antioxidant enzymes by geniposideObjective To study the effects of Geniposide on the expression of HO-1 and GPx-1,the activities of SOD and CAT,and ROS content in melanocytes under H2O2-induced oxidative stress,and to explore the role of PI3K-Akt signaling pathway in this effect.Methods Geniposide preincubated melanocytes for 24 hours,added or did not add PI3K inhibitor LY294002,and then induced oxidative stress reaction of melanocytes with H2O2.Melanocytes were divided to control group,Geniposide group,LY294002 group,H202 group,Geniposi de+H2O2 group,and Geniposide + LY294002 + H2O2 group.Western blot was used to detect HO-1 and GPx-1 protein expression,biochemical methods to detect SOD,CAT enzyme activity and flow cytometry to detect intracellular ROS content.Results The expression of HO-1(P<0.01),GPx-1(P<0.01)protein in H2O2 treated group was down regulated,SOD activity(1.29±0.43 units/mg P<0.01)and CAT activity(46.08±4.16 P<0.05)were lower than those in control group while ROS level was significantly higher(2158 + 222.75 P<0.01).Compared with H2O2 treated group,HO-1(P<0.01)?GPx-1(P<0.01)level,SOD activity(6.82±1.03 units/mg,P<0.05)and CAT activity(46.08±4.16 units/mg,P<0.05)were increased,while intracellular ROS(1284.33±110.64,P<0.01)was decreased in Geniposide + H202 group.Notably,these effects were largely blocked by treatment with LY294002 prior to H202,which was manifested in decreasing HO-1(P<0.05)and GPx-1(P<0.05)expression,lowering SOD(1.31±0.65 units/mg,P<0.05)and CAT activity(23.25±5.56 units/mg,P<0.05),and increasing ROS content(1668±62.03,P<0.05).Conclusion Geniposide can increase the content of HO-1 and GPx-1,increase the activity of SOD and CAT,and reducing ROS content through the PI3K-Akt signaling pathway.Part 4 The role of PI3K-Akt signaling pathway in the regulation of cell viability and apoptosis of melanocytes by geniposideObjective To study the effect of Geniposide on the cell viability and apoptosis of melanocytes under the condition of H2O2 induced by oxidative stress.Methods Geniposide preincubated melanocytes for 24 hours,added or did not add PI3K inhibitor LY294002,and then induced oxidative stress reaction of melanocytes with H2O2,The melanocytes were divided to control group,Geniposide group,LY294002 group,H2O2 group,Geniposide+H2O2 group,and Geniposide+LY294002+H2O2 group.The cell viability of melanocytes was detected by MTT assay.Flow cytometry was used to detect the apoptotic rate.Results The cell viability of H2O2 group(50.53 ± 10.85%)was significantly lower than that of control group and the apoptotic rate was significantly increased(20.99±3.83%,P<0.01).Compared with H2O2 group,the cell viability of the Geniposide + H2O2 group was increased(72.98±8.92%,P<0.05)and the apoptotic rate was decreased(13.82±2.36%,P<0.05).However,this effect of Geniposide was reversed by LY294002,and the cell viability was significantly decreased(44.35±14.85%,P<0.01)and the apoptotic rate was increased(24.55 ± 5.01%,P<0.05).Conclusion The preculture of Geniposide can protect melanocytes against H2O2-induced oxidative damage through the PI3K-Akt signaling pathway. |
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