Effect And Mechanism Of Tropical Mycophenolate Mofetil On Experimental Depigmented Models Induced By Hydroquinone

OBJECTIVE: Vitiligo is a depigmentation disease whose specific pathogenesis is unknown.The autoimmune theory explains one of the existing hypothesis.Mycophenolate mofetil(MMF)is a new type of immunosuppressant drug that selectively inhibits inosine 5-monophosphate dehydrogenase(IMPDH)from synthetizing DNA in lymphocytes.In view of its mild adverse effects,MMF has been used for dermatological immune diseases such as psoriasis and systemic lupus erythematosus.Recent studies have reported the effective treatment of vitiligo with MMF;however,the specific mechanism has not yet been illustrated.The objective of this study was to investigate the therapeutic effect and mechanism of topical MMF in the treatment of guinea pigs and mice following melanin reduction with hydroquinone(HQ).METHOD: 1.The model of vitiligo was established by using chemical decolorization.The influences of hydroquinone on the distribution of melanin and the expression of melanin-related genes was observed.2.The guinea pigs and the mice were respectively dived into three groups:the first group was the control group、the second was treated with hydroquinone alone,and the third group was treated with hydroquinone in combination with MMF.Clinical changes were evaluated by the Image-Pro Plus on the 30 days,40 days,and 50 days after treatment.Then they were visualized by haematoxylin and eosin(H&E)and Masson-Fontana(M-F)staining to calculate the melanin-containing hair follicles and the basal layer melanocytes on the 50 th day,observing the influence of MMF on the quantity and the distribution of melanocytes.Immunofluorescence staining of melanin protein was performed in the MMF group.Real-time PCR was applied to measure the m RNA expression of melanin-related genes in the three groups.3.Try to explore the therapeutic mechanism of MMF in the vitiligo guinea pig models: the expression of lymphocytes in each group was compared by the account of lymphocytes in the peripheral blood and HE staining;Wnt pathway-related protein β-catenin,GSK3β and genes such as Axin2,Dkk,Dact2,TGF-β,IFN-γ were detected by Western Blotting and RT-PCR.4.The vitiligo mouse models were constructed and the above experiments were repeated to confirm the reliability of MMF for the treatment of vitiligo.RESULT: 1.Hydroquinone decolorization method can successfully establish vitiligo guinea pig models(1)H&E staining and Masson-Fontana staining showed that the melanin-containing hair follicles and the basal layer melanocytes disappeared substantially at the depigmentation spots of the hydroquinone-coated side compared with the self-control side;(2)Immunofluorescence staining showed that the expression of melanin-related protein TYR in the hydroquinone side was decreased;(3)Hydroquinone significantly reduced the expression of melanin genes of TYR and TYRP-1(P < 0.05).2.MMF has a therapeutic effect on vitiligo guinea pig models(1)Image-Pro Plus showed that MMF significantly reduced hydroquinone-induced white spot area compared to the hydroquinone group;(2)Compared with the hydroquinone group,the number of melanin-containing hair follicles and the number of basal layer melanocytes in the MMF group were significantly increased(P < 0.05);(3)After the application of MMF,immunofluorescence staining showed significant expression of TYR protein in the MMF group;(4)MMF increased TYR and TYRP1 m RNA expression(P < 0.05).3.MMF counteracts IFN-γ-induced hypopigmentation by activating the Wnt/β-catenin pathway(1)Both blood routine and H&E staining showed that MMF could inhibit hydroquinone-induced vitiligo-like lymphocytes increase and elevated IFN-γ and TGF-β inflammatory factors;(2)By Western Blotting,MMF increased β-catenin and decreased GSK3β expression compared with the hydroquinone group(P < 0.05);(3)Downstream genes such as Axin2,Dkk,and Dact2 in the Wnt/β-catenin pathway were significantly decreased after MMF application.4.The therapeutic effect of MMF on the hair follicle melanin loss was verified in vitiligo mouse model(1)By visual observation of hair pigmentation,H&E staining and Masson-Fontana staining,MMF had a therapeutic effect on the pigmentation of hydroquinone,and the number of melanin-containing hair follicles increased significantly(P < 0.05).(2)Immunofluorescence staining showed that the expression of TYRP-1 protein in the MMF group was higher than that in the hydroquinone group.(3)Compared with the hydroquinone group,the expression of TYRP-1,TYR,MITF,and SILV m RNA in the MMF group was significantly increased(P < 0.05).CONCLUSION: MMF can significantly improve the pigment-loss effect of hydroquinone by activating the Wnt/ β-catenin pathway.Therefore,MMF is a promising drug for reducing skin pigmentation diseases.