| Resveratrol（RES）is a polyphenolic compound,present in present in fruits and plants,that has been shown various medicinal properties,including protection of cardiovascular disease and cancer risk,etc.However,the effects of Res on skin diseases have not been investigated.Recently,it has been reported that resveratrol inhibitsα-melanocyte-stimulating hormone signaling,viability,and migration in melanoma cells.However,these effects have not been confirmed in vivo or in clinical practice.To evaluate the potential of resveratrol as a regulator of melanin for hyperpigmentation therapy,we conducted the following study.Part One Effects of resveratrol on proliferation and melanogenesis ofHT-144 melanoma cells in vitroObjective:To explore the putative effects of resveratrol at different concentrations on the proliferation,tyrosinase activity and melanin formation of human melanoma HT-144 cells in vitro.Methods:MTT colorimetric assay was used for determination of cell survival and IC50 value.Western Blot was used for the evaluation of p-ERK and p-MEK at protein level in HT-144 cells.And the tyrosinase activity and melanin formation were quantitatively determined with colorimetric method.Result:MTT results showed that resveratrol treatment at different concentrations（1-15μM）could inhibit the proliferation of HT-144 cells in a concentrations dependent way.Twenty four hours after resveratrol treatment the expressions of p-Erk at protein level by Western Blot were significantly decreased in HT-144 cells in vitro.The decrease at expression level was more significant as the resveratrol concentration increased.A reverse relation was found between the p-ErK expression and resveratrol concentration in the concentration range from 1-15μM.While at the same time,on the contrary to p-ErK expression,the expressions of p-MEK1/2 were concentration dependently increased as resveratrol concentration increasedin HT-144 cells,suggesting ERK signaling pathway was involved in the proliferation inhibition by resveratrol in HT-144 cells.For tyrosinase activity and melanin formation,as the concentration of resveratrol increased from 1μM,2μM,5μM,10μM to 15μM,tyrosinase activity and melanin formation were decreased by 30%,45.1%,55.2%,79.4%,85.5%and 22.4%,50.9%,77.7%,88.5%,92.5%respectively as compared with the corresponding controls.Thus,the results suggest that resveratrol could concentration-dependently inhibit tyrosinase activity and melanin formation of HT-144 cells in vitro.Conclusion:1 Resveratrol can inhibit the proliferation of HT-144 melanoma cells in vitroin a dose-dependent manner,and ERK signaling pathway may play important role for the inhibitory effect.2 Resveratrol at all concentrations（1-15μM）used in this study could inhibittyrosinase activity and marked inhibition effect could be seen at low concentrations3 Resveratrol inhibits melanin production via inhibition of tyrosinase activity.Part Two Effects of different concentrations of resveratrol on inflammatory hyperpigmentation in guinea pigsObjective:To evaluatethe therapeutic and the putative adverse effects of resveratrol treatment on experimental inflammatory hyperpigmentation skin lesions in guinea pigs and to provide experimental basis for resveratrol treatment of hyperpigmentation skin lesions in clinical practice.Methods:A total of 15 healthy adult guinea pigs were used in this experiment.The establishment of experimental inflammatory hyper-pigmentation skin lesions were conducted,briefly,first shaved off the hair on the back of guinea pig,divided the hair cut skin field into 5 separated regions,exposure to CO2 laser followed by narrow-band ultraviolet（311nm）irradiation,3 times in a rowevery 2 days.Pathological examination and VISIA examination were performed before the treatment.The experimental animals were divided in to five groups:blank control,hydroquinone cream positive control,Q-switch Nd:YAG laser treatment,25%resveratrol treatment and50%resveratrol treatment.No treatment was carried out in blank control group.Animals in hydroquinone cream positive controlwere treated locally by external administration of 2%hydroquinone cream 2 times a day.Animals in Q-switch Nd:YAG laser treatmentgroup were treated by Q-switch laser exposure（energy:4.0J/cm2）one time in every 2 weeks,while those in the two resveratrol treatment groups were treated by external administration of 25%or50%resveratrol solution（3:1 or 1:1 resveratrol stoste:0.9%sodium chloride solution respectively,and 0.5g Laurocapramum in 100ml solution）once a day.Six weeks after treatment,histopathologicalobservation and VISIA examination were used to evaluate the changes of pigmentation,and the therapeutic effect and adverse effect of the different methods on the hyperpigmentation of guinea pigs.Results:The results of VISIA image analysis of brown yellow granules showed that before treatment,the absolute VISIA value of inflammatory hyperpigmentation skin lesions for blank control,hydroquinone cream positive control,Q-switch Nd:YAG laser treatment,25%resveratrol treatment and 50%resveratrol treatmentgroups were 4.5216±0.5374,4.4143±0.5063,4.5063±0.5646,4.5957±0.6615 and 4.4211±0.8270 respectively.There were no differences among the five groups（P>0.05）.Six weeks after treatment,the absolute VISIA values of thetreated area for blank control and Q-switch Nd:YAG laser treatment groups were 4.4331±0.5797 and 4.3617±0.6068,and no differences were found before and after treatment.While the absolute VISIA value for 2%hydroquinone cream,25%resveratrol and 50%resveratrolgroup was 3.1275±0.6400,3.2169±0.7906 and 3.2725±1.5399,which were all significantly decreased as compared with that before treatment and at the same time lower than that of blank control and Q-switch Nd:YAG laser treatment group.There was no difference among 2%hydroquinone cream and the two resveratrol treatment groups（P>0.05）,but skin irritation by2%hydroquinone cream could be found.Conclusion:CO2 laser exposure combined with 311nm narrow spectrum UVB treatment could induce a stable post-inflammatory hyperpigmentation lesion in the back skin of guinea pig.Resveratrol treatment could significantly decrease pigmentation of the experimental skin lesion.The therapeutic effects of resveratrol was the same as that of 2%hydroquinone cream but without the skin irritation of the latter.No therapeutic effect was found with Q-switch Nd:YAG laser treatment.Part Three Efficacy of resveratrol mixed solution in the treatment of facial chloasmaObjective:Facial chloasma is one of the skin pigmentation lesions.To observe the clinical efficacy of resveratrol mixed solution in the treatment of facial chloasma,we evaluated the therapeutic efficacy of resveratrol mixed solution on chloasma by skin mirror and VISIA image analysis,and adverse reactions were also observed during the treatment.Methods:1 Cases:Fifty cases of patients with stable facial chloasma without hormone preparations use in the past one year were included in this study.Cases in pregnant and lactating period,cases with thyroid disease and cases with tumors or other systemic diseases were excluded.Cases included in this study were randomly assigned into resveratrol group and control group by referring to the random number table.Informed consents were signed by all participating patients.The experiment plan was approved by the medical ethics committee of Hebei Provincial Tradicinal Medical Hospital.Finally,50patients were included in the study,including 25 cases in resveratrol group and25 cases in the control group.2 Treatment:The patients in resveratrol group were treated with resveratrol mixed solution by external use,while the patients in control group were treated with 2%Hydroquinone Cream also for external use,and the curative effect was determined after 8 weeks.3 Observation:Dermoscopy subcutaneous vascular density,VISIA value of brown granules,vascular reaction zone and the severity of the lesions were observed and recorded at initial diagnosis and eighth weeks after treatmen.Medication compliance and adverse reactions were inquired.4 Curative Effect Evaluation（1）The scoring standard and method for skin change was conducted according to the Standard of Chinese Association of Integrative Medicine and Dermatology,establishedin 2003.（2）Evaluation criteria of efficacy were carried out according to the four grade curative effect standard（Nimodipine method）.Scoring reduction index=total score before treatment-total score after treatment/total score before treatment.5 Safety Evaluation:The adverse reactions including pruritus,skin irritation and the density of skin vascular hyperplasia were recorded during the whole treatment period.6 Statistics:Comparison between the the changes before and after treatment,the efficacy and adverse reaction score was conducted using t test,Ridit analysis,and while that of the effective rate,using chi square test.The statistic software used is SPSS22.0.Result:1 Results of VISIA image analysis of brown yellow granules before and after treatmentThe absolute value of VISIA image analysis of brown yellow granules before and after treatment in resveratrol mixed solution treatment group were32.68±9.47 and 23.82±10.24 respectively,significant decrease in brown yellow granules was found after treatment（P<0.05）.Decreases in the absolute value of VISIA image analysis were also found after 2%hydroquinone cream treatment（33.65±8.90 vs 24.13±6.46,P<0.05）.There were no significant differences in the decreases of the absolute value of VISIA image analysis between resveratrol mixed solution treatment and 2%hydroquinone cream treatment controls（23.82±10.24 vs 24.13±6.46,P>0.05）,suggesting that both resveratrol mixed solution and 2%hydroquinone cream could effectively decrease the pigmentation of facial chloasma.2 Changes of VISIA valueof red areas of skin lesionsbefore and after treatmentThe absolute values of VISIA image analysis of red areas of the skin lesions before and after treatment in resveratrol and hydroquinone cream control were significantly decreased（26.94±9.56 vs 22.52±4.73,P<0.05）and increased（25.97±6.52vs28.75±5.58,P<0.01）respectively,suggesting that resveratrol treatment be without,whilehydroquinone cream could induce skin irritation and enhance inflammation reaction.3 Changes in scoring reduction index of skin lesion before and after treatmentScoring reduction of facial skin lesion of chloasma could be seen both in resveratrol treatment and hydroquinone cream controls.The scoring reduction index of facial skin lesion was 0.66±0.272,and 0.72±0.259 respectively,being significantly decreased than that before treatment（P<0.01）,without difference between the two groups（P>0.05）.Thus,the results confirmed that both resveratrol treatment and hydroquinone cream were effective in the treatment of facial chloasma.4 Comparison of the safety of resveratrol and hydroquinone cream treatmentThere were 3 cases of pruritus,3 papules and 3 cases of erythema in the resveratrol treatment group while in hydroquinone group,the corresponding cases were 8,10 and 9 respectively.Ridit analysis results showed that the adversereactions in resveratrol group was significantly lower than that in the hydroquinone cream control group（P<0.05）.Conclusion:The efficacy of resveratrol in the treatment of chloasma is definite and the incidence of adverse reactions is lower and the safety is higher than that of hydroquinone cream group. |
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