Tiamulin Inhibits TNF-α And Alleviates Psoriasis-like Dermatitis

Background:Psoriasis is a chronic skin disease of all ages which affects about 0.5%of the global population in China.There are four clinical subtypes of psoriasis,including psoriasis vulgaris,psoriasis pustular,psoriasis erythrodermic,and psoriasis arthropathy.Psoriasis vulgaris is the most common subtype of psoriasis,typically characterized by scaly erythema.The lesions of psoriasis can involve the whole skin,seriously impacting the appearance of patients.Moreover,psoriasis is often accompanied by obesity,hypertension,diabetes mellitus,hyperlipidemia,and metabolic syndrome which severely impairs the quality of life of the patients.However,there are some shortcomings in the traditional treatments for psoriasis.Therefore,new effective drugs for psoriasis are needed.TNF-α,a multifunctional cytokine mainly produced by activated macrophages and monocytes,can elicit proliferation and cytokine release in keratinocytes.TNF-α can also exhibit a cascade amplification effect in psoriasis.The development of monoclonal antibodies targeting TNF-α,such as Etanercept and Adalimumab,have been progressed in recent years.However,biological macromolecule drugs have inherent limitations,such as high cost,unavailable oral or topical administration,and severe side effects after long-term use.This dilemma is expected to be solved if efficient small-molecule inhibitors against TNF-α could be developed.However,there are no effective small molecule drugs directly targeting the TNF-α pathway in the clinic now.Objective:Novel TNF-α inhibitor was identified via high-throughput screening.The effect of the novel TNF-α inhibitor was tested by HaCaT cells in vitro and IMQ-induced psoriasis-like mouse model in vivo.Methods:1.HeLa and L929 cell death models induced by TNF-α were established.The CellTiterGlo? Luminescent Cell Viability Assay was used to detect HeLa cell viability,and the Cell-Counting-Kit-8 method was used to detect L929 cell viability.2.A high-throughput screening platform for TNF-α small molecule inhibitors was established based on a compound library with cell viability as readout.3.HeLa cell death model was used for primary screening and L929 cell death model was used for second screening.4.HaCaT cells were stimulated with TNF-α or IL-6 and different concentrations of small molecule,the expression levels of NF-κB,MAPK,and JAK related proteins were detected by Western-blot method,and the expression levels of TNF-α related downstream inflammatory factors were detected by qRT-PCR method;5.Psoriasis-like dermatitis induced by 5%imiquimod(IMQ)cream for 5 days in BALB/c mice.The application of small molecule was divided into two methods:gavage and topical application.The skin lesions were assessed by CSS,qRT-PCR,WB,HE staining,and immunohistochemical staining of Ki67 and Loricrin.Results:1.Thirty-one compounds were found to reverse the Hela cell death induced by TNF-α in primary screening.2.Seven of the thirty-one compounds with different concentrations were used to assess the biological effects in the L929 cell death model.TiamulinFumarate(TF)was selected as a candidate inhibitor.3.TF could effectively inhibit the up-regulation of NF-κB and MAPK inflammatory pathways through Western-blot method analysis in HaCaT cells induced by TNF-α.4.TF could effectively inhibit the up-regulation of JAK inflammatory pathway through Western-blot method analysis in HaCaT cells induced by IL-6.5.TF could effectively inhibit the up-regulation of multiple inflammation factors through qRT-PCR method analysis in HaCaT cells induced by TNF-α.6.TF was applied by gavage in IMQ-induced mice,divided into concentrations of 100mg/kg and 300mg/kg.TF ameliorated psoriasis-like dermatitis in a dose-dependent manner.7.Psoriasis-like dermatitis induced by IMQ was ameliorated by the application of 5%TF cream.Conclusion:In this study,a high-throughput screening platform for TNF-α small molecule inhibitors was established based on a compound library and two cell death models induced by TNFα.Through two rounds of screening,TF was found as a novel TNF-α antagonist.TF significantly blocked the up-regulation of multiple signaling pathways and inflammatory factors in HaCaT cells induced by TNF-α or IL-6.In the IMQ-induced mouse model,systemic application of TF could attenuate psoriasis-like dermatitis,and the efficacy of topical application was also very significant.Our study suggested that TF might represent an effective topical treatment in psoriasis,which provided a new direction for future clinical trials.