Effect Of Rapamycin On Regulating Tropomyosin(TPMs) In Psoriasis In Vitro And In Vivo

Background and purpose:Psoriasis is a kind of chronic inflammatory skin disease mediated by immunity.Psoriasis can be induced by many factors such as infection,trauma,medicine and environment.The main pathological features of psoriasis are the hyperplasia and abnormal differentiationof keratinocyte cell(KC).Psoriasis is a systemic disease with a long course,easy recurrence and difficult to cure.The exact etiology and pathogenesis of psoriasis have not been fully elucidated.Although many clinical treatment methods for psoriasis can effectively alleviate the disease,the recurrence of psoriasis can not be prevented.Therefore,to explore the pathogenesis of psoriasis and to search new and effective targets for psoriasis have become a hot and difficult field of dermatology in the world.The etiopathogenisis and pathogenesis of psoriasis are extremely complex.Studies have shown that the pathophysiological process of psoriasis involves inflammation,immune disorders,abnormal cell proliferation and apoptosis,activation of intracellular signal transduction pathways and other factors.Immune cells,including T cells,KC,y8T cells,dendritic cells(DC),neutrophils andnatural killer T cells are important factors in the pathogenesis of psoriasis,which secrete a series of cytokines and chemokines to mediate the activation of intracellular signal transduction pathways,resulting in cascade amplification effect and the proliferation and abnormal differentiation of KC cells.Tropomyosins(TPMs),play an important role in the muscle cell contraction,are a subtype of cytoskeletal proteins that stabilize microfilaments by binding to actin proteins.It consists of four different genes(TPM1-4)and produces more than 20 TPM isoforms via alternate splicing of mRNAs.Previously studies have shown that TPMs may participate in the pathogenesis of diverse human diseases,such as cancers,autoimmunity,and muscle diseases.Recent study showed that TPMs were downregulated in psoriatic lesions when compared with normal adult skin tissues,suggesting that TPMs may be involved in the pathogenesis of psoriasis.Mammalian target of rapamycin(mTOR)is an important downstream target protein in the Ras-phosphatidylinositol 3-kinase(PI(3)K)signaling pathway.Studies have shown that mTOR signaling pathway is involved in regulating cell proliferation,differentiation and apoptosis,and mTOR signaling pathway is activated in psoriatic lesions.Rapamycin(mTOR inhibitor),also known as sirolimus,was initially used as an antifungal agent and immunosuppressive agent.In recent years,more and more findings showed that Rapamycin was used in treatment of diabetes mellitus and neuron protection.Rapamycin has been reported to be effective in the treatment of psoriasis,which is related to the mTOR pathway.However,the specific mechanism needs further study.Both mTOR and TPMs are regulated by the Ras-PI(3)K signaling pathway and are involved in the proliferation of tumor cells.However,the roles of mTOR andTPMs in the pathogenesis of psoriasis are still unknown.In order to explore the role of rapamycin on regulating TPMs in psoriasis,the expression of TPMs in normal skin,psoriatic lesions and non-lesional skin tissues was detected by real-time PCR and Western blot.Current studies have shown that epigenetic alterations,including DNA methylation and histone modification may play an important roles in psoriasis.Studies have found that psoriatic lesions are accompanied by DNA methylation alterations,which are involved in the epigenetic process of psoriasis,and these differences mainly occur in the formation of cutin lesions.In this study,we firstly examined the expression and methylation levels of TPMs in the normal skin,lesional skin,non-lesional skin,and TNF-α induced cell model of psoriasis.Then,we assessed the effects of RAPA on TPMs expression,cell proliferation,apoptosis,cell cycle,cell skeleton and phosphorylation levels of ERK1/2 and mTOR.Finally,we established an animal model of psoriasis inducing by imiquimod and also for further studied of the effection of RAPA on TPMs expression,methylation.while providing the basic research on clinical treatment of Rapamycin in psoriasis.Part 1 The expression of TPMs in clinical samples of psoriasisObjective:To observe the expression level of TPMs mRNA in skin tissue,psoriasis lesion and non-lesion tissues of healthy controls,and detect the protein expression of TPMs and the promoter methylation level of TPMs.Methods:1.Specimen collection:20 cases of plaque psoriasis and 20 cases of non-lesion tissues were collected from June 2015 to June 2016 in the Department of Dermatology.Qianfoshan Hospital,Shandong University.Diagnostic criteria are based on the Chinese Psoriasis Therapeutic Guidelines(Psoriasis Group,2008 Edition,Branch of Dermatology and Venereal Diseases,Chinese Medical Association).All patients met the inclusion criteria(see the main text).In addition,20 healthy skin specimens from the plastic surgery department were selected and matched with the plaque psoriasis group in age,sex and location.2.The expression of TPMs in psoriasis samples was detected by real-time PCR;the skin epidermis thickness and inflammation of psoriasis and control samples were determined by H&E staining.3.The protien expression of TPMs was detected by immunohistochemistry;4.The methylation PCR was used to detect the methylation level of TPMs promoter.Results:1.The results of real-time PCR showed that the mRNA expression levels of TPM1 and TPM2 were significantly down-regulated in psoriatic lesions and non-lesional tissues as compared with healthy controls(P<0.001),and the expression levels of TPM1 and TPM2 in psoriatic lesions were also significantly down-regulated(P<0.01),while the mRNA expression levels of TPM3 and TPM4 did not show significantly different.2.H&E staining showed that the epidermis of psoriatic lesions was thicker than that of healthy controls and non skin lesions.3.Immunohistochemical results found thatcomparing with healthy controls,the expression of TPM 1 and TPM2 was decreased in psoriatic lesions and non-lesional.4.Methylation-specific PCR showed that the promoter methylation levels of TPM1 and TPM2 in psoriatic lesions were significantly higher than those in healthy controls and non-lesion groups.Conclusion:1.The expression of TPM1 and TPM2 decreased significantly in psoriatic tissues.2.The promoter of TPM 1 and TPM2 was methylated in psoriasis.Part2 Effect of Rapamycin on the expression of TPMs and proliferation of keratinocytes in the cell model of psoriasis in vitroObjective:To demonstrate the inhibitory effects of rapamycin.a mTOR inhibitor,on cell models of psoriasis in vitro.Methods:1.In vitro psoriasis cell model was induced by TNF-a.Hacat and HEK cells were seeded onto 24-well plates at a concentration of 1×104 cells and cultured overnight at 37℃.Then,TNF-α(10ng/ml)was added to each well to stimulate cells.2.Rapamycin was used to treat the psoriatic cell model,and 5-azacytidine(5-Aza)was used as the positive control.1)Hacat cells(blank control group,without any drugs).2)Hacat cell +TNF-α(10ng/ml,1 hours)3)Hacat cell +TNF-α(10ng/ml,1 hours)+ rapamycin(50nM/L,1 hours)4)Hacat cell +TNF-α(10ng/ml,1 hours)+5-Aza(positive control,5M/L,1 hours)5)HEK(blank control group,without any drugs).6)HEK+TNF-α(10ng/ml,1 hours)7)HEK+TNF-α(10ng/ml,1 hours)+ rapamycin(50nM/L,1 hours)8)HEK+TNF-α(10ng/ml,1 hours)+5-Aza(positive control,5M/L,1 hours)3.The mRNAexpression levels of TPM 1 and TPM2 was detected by real-time PCR,and the expression levels of TPM1 and TPM2 protein were detected byimmunofluorescence with anti-TPM1 and anti-TPM2 antibodies.4.CCK8 was used to detect the proliferation of each group at 24,48 and 72 hours,and EdU staining was used to observe the proliferation of each group.5.Phalloidin and DAPI staining were used to detect the cytoskeleton distribution in each group.6.Flow cytometry was used to detect the distribution of cell cycle in each group,and three experimental results were statistically analyzed.7.The protein expression of TPM1 and TPM2 was detected by Western blot.and the phosphorylation levels of ERK1/2 and mTOR were detected.8.The methylation PCR was used to detect the methylation level of TPM 1 and TPM2 promoter in each group.Results:1.After TNF-α treatment,the expression levels of TPM1 and TPM2 decreased significantly(P<0.001),while rapamycin reversed the expression of TPM1 and TPM2.2.Immunofluorescence showed that the expression of TPM1 and TPM 2 decreased significantly after treatment with TNF-α,while the expression of TPM1 and TPM2 returned to the control level when rapamycin was added to TNF-α.3.After treatment with TNF-α for 24,48 and 72 hours,the proliferation of HEK and Hacat cells increased significantly(P<0.001),while the proliferation of HEK and Hacat cells was reversed by treating with rapamycin.4.We found that cytoskeleton was destroyed in both TNF-α treated Hacat and HEK cells,but rapamycin treatment restored cytoskeleton structure,indicating that rapamycin restored cytoskeleton structure in psoriatic cell model.5.TNF-α induced S-phase arrest in cell cycle,while rapamycin treatment restored S-phase distribution,indicating that rapamycin regulated cell cycle distribution in psoriatic cell model.6.The expression levels of TPM1 and TPM2 decreased significantly after TNF-αtreatment,but the expression levels of TPM1 and TPM2 returned to the control level after rapamycin treatment.7.TNF-α significantly increased the expression of phosphorylated ERK1/2 and phosphorylated mTOR,while rapamycin reversed these effects.8.The promoters of TPM1 and TPM2 were methylated after TNF-α treatment,while the methylation of TPM1 and TPM2 was significantly weakened after rapamycin treatment,suggesting that rapamycin inhibited the expression of TPM1 and TPM2 in psoriatic cell model in vitro by decreasing the methylation of TPM 1 and TPM2.Conclusion:1.In psoriatic cell models,the expression of TPM 1 and TPM2 was regulated by rapamycin.2.Rapamycin inhibits the proliferation and S phase arrest of psoriatic cell models in vitro.3.Rapamycin restored the distribution of cytoskeleton.4.TNF-α not only activates the mTOR signaling pathway,but also increases the activity of ERK1/2 signaling pathway.indicating that ERX1/2 signaling pathway is also involved in the pathogenesis of psoriasis.Part3 Regulatory effect of Rapamycin on TPMs in animal models of psoriasis in vivoObjective:To further verify of the role of rapamycin in the regulation of TPMs in mouse psoriasis models.Methods:1.IMQ was used to make mouse psoriasis model.(1).Normal control group(NSgroup,saline,6 mice);(2).Psoriasis model group(IMQ group,5%IMQ,62.5mg/cm2 after hair removal,once a day.continuous administration for 10 days,6 mice);(3)Rapamycin treatment group(IMQ+RAPA group,3.0mg/kg/body weight,intraperitoneal injection,once a day for 10 days,6 mice);(4)5-azacytidine control group(IMQ+5-Za group,3.0mg/kg/body weight,intraperitoneal injection,once a day for 10 days,6 mice);2.PASI score was used to evaluate the mouse model.3.H&E staining was used to evaluate the skin phenotype and pathological characteristics of the model and the treatment group.4.Immunohistochemistry and Western blot were used to detect the expression of TPM1 and TPM2.5.The expression level of phosphorylated ERX1/2 and phosphorylated mTOR in skin tissue was detected by Western blot.6.Methylation PCR was used to detect the methylation level of TPM1 and TPM2 in skin tissue.Results:1.PASI score confirmed that the mouse psoriasis model induced by IMQ was successfully produced.2.The results of H&E staining showed that the epidermis of IMQ mice was significantly thickened,while rapamycin treatment was significantly recovered compared with IMQ mice,suggesting that rapamycin protected IMQ-induced psoriasis.3.The expression of TPM1 and TPM2 decreased significantly in IMQ treated mice,while the expression of TPM1 and TPM2 was equivalent to that in control mice treated with rapamycin and IMQ at the same time.4.The phosphorylation levels of ERK1/2 and mTOR were up-regulated in psoriatic animal models,while the phosphorylation levels of ERK1/2 and mTOR were inhibited by rapamycin treatment.5.In IMQ group,the methylation levels of TPM1 and TPM2 increased significantly,while rapamycin inhibited the methylation of TPM1 and TPM2,suggesting that rapamycin restored the expression of TPM 1 and TPM2 in psoriatic animal models by inhibiting the methylation of TPM 1 and TPM2.Conclusion:1.Rapamycin protects mice from psoriasis induced by IMQ as in cell model induced by TNF-a.2.The expression of TPM1 and TPM2 in skin tissue of psoriatic animal models was decreased.3.Rapamycin reversed the expression of TPM1 and TPM2 in psoriatic animal models by decreasing the promoter methylation of TPM1 and TPM2.