Pharmacological Effects Of RNA Polymerase Ⅰ Inhibitor CX-5461 On Psoriasis And Related Pathological Angiogenesis

BackgroundPsoriasis is a common chronic immunoinflammatory proliferative skin disease with a long course and easy recurrence.The clinical manifestations are mainly erythema,scales and itching.The main histopathology of psoriasis is hyperkeratosis of epidermal cells,inflammatory cell infiltration,and capillary proliferation.The etiology and pathogenesis of psoriasis are not fully understood.However,it is currently believed that the occurrence of psoriasis is not the result of a single factor,and may involve multiple aspects,such as genetic background,infection,immune abnormalities,endocrine factors,and mental stress.At present,the drugs used clinically for the treatment of psoriasis have a large side effect and cannot achieve the purpose of radical cure.Therefore,it is of great significance to find new drug targets.CX-5461 is a novel inhibitor of specific RNA polymerase Ⅰ(Pol Ⅰ).Previous studies have confirmed that CX-5461 can inhibit tumor cell proliferation and show good therapeutic effects on certain leukemias and solid tumors.In our previous study,our team found that CX-5461 significantly inhibited arterial intimal hyperplasia and restenosis caused by mechanical injury,and also inhibited the development of neovascular intimal hyperplasia and vascular inflammation after transplantation,and CX-5461 can effectively inhibit the activation and phenotypic transformation of cardiac fibroblasts.Whether CX-5461 has an inhibitory effect on the development of psoriatic lesions has not been studied.ObjectiveIn this study,we used a mature psoriasis model and an angiogenesis model to explore the pharmacological effects of the Pol Ⅰ specific inhibitor CX-5461 on psoriasis and endothelial angiogenesis.MethodsA psoriasis model was established by treating the skin of C57 BL/6 mice with imiquimod cream.CX-5461 is dissolved in DMSO and applied topically to the skin.The mice were randomly divided into 4 groups:normal control group(sham),model+ DMSO group(solvent control),model + CX-5461 low dose group(2 μM),model +CX-5461 high dose group(5 pM).After 1 week of topical application,histopathology and molecular biology were performed.HE staining was used to observe changes in the thickness of the stratum corneum.The number of CD68 positive cells in inflammatory cells was observed by immunohistochemical staining.Lectin and CD31 staining were used to observe angiogenesis.The expression of tumor necrosis factor(TNF-a),interleukin(IL)-17,IL-23,IL-6,IL-8,IL-10 and IL-1βwas detected by real-time quantitative PCR.Human keratinocytes(Hacat)were cultured in vitro,and cell cycle was detected by flow cytometry.An angiogenesis model was established by subcutaneously implanting a sponge pad and a subcutaneous injection of the Matrigel method.C57 BL/6 mice implanted with sponge mats were randomly divided into 4 groups:saline control group,vascular endothelial growth factor(VEGF)stimulation group,CX-5461 group,CX-5461 +VEGF group.The C57 BL/6 mice injected subcutaneously into Matrigel were randomly divided into three groups:Matrigel group,CX-5461 low dose group(2μM)group,and CX-5461 high dose group(5μM).After 2 weeks,the material was taken for histological examination.immunohistochemistry and Lectin staining were used to observe changes in the number of new blood vessels.Human capillary endothelial cells were cultured in vitro,cell proliferation ability was detected by CCK-8 method,cell migration ability was detected by scratch test,tube formation ability was detected by small tube forming experiment,and CX-5461 cell cycle was detected by flow cytometry.Impact.Detection of protein levels of p53 and phosphorylated p53 in endothelial cells by Western blot.ResultsIn the psoriasis model,CX-5461 significantly improved the skin symptoms of psoriasis(the surface of the diseased skin was reduced in scale,the skin folds were reduced,and the redness was significantly reduced).HE staining showed that CX-5461 significantly inhibited keratinocyte proliferation compared with the vehicle control group.Immunohistochemical staining showed that the number of new blood vessels in the lesions of the CX-5461 treatment group was significantly reduced compared with the vehicle control group.In addition,we found that CX-5461 can reduce the inflammatory response in the lesion.The results of immunohistochemistry showed that the number of CD68+ cells in the CX-5461 treatment group was significantly reduced compared with the control group.Real-time quantitative PCR results showed that the expression levels of TNF-α,IL-17,IL-23 and IL-1β were decreased in CX-5461 treatment group,while the expression of IL-6,IL-8 and IL-10 was increased.In the subcutaneous implanted PVA sponge angiogenesis model,CD31 staining and Lectin staining showed that the number of blood vessels was significantly increased in the VEGF group compared with the control group;CX-5461 treatment significantly reduced basal levels and angiogenesis after VEGF stimulation.In the Matrigel angiogenesis model,CD31 immunochemical staining showed that CX-5461 treatment significantly reduced the number of angiogenesis.In vitro cultured cells,CX-5461 has a dose-dependent inhibition of vascular endothelial cell proliferation.At the same time,CX-5461 can significantly inhibit the migration and tube formation of vascular endothelial cells.Furthermore,CX-5461 inhibits the cell cycle of keratinocytes and endothelial cells and blocks cells in the G2/M phase.In vascular endothelial cells,CX-5461 had no significant effect on the total amount of p53 protein,but significantly increased p53 phosphorylation.Pretreatment with p53 inhibitor PFT-α can attenuate the inhibitory effect of CX-5461 on endothelial cell migration,but has no effect on endothelial cell tube formation.ConclusionsTopical application CX-5461 can effectively inhibit the occurrence and development of psoriatic lesions,reduce inflammatory cell infiltration and angiogenesis in the lesion.The inhibitory effect of CX-5461 on angiogenesis may be related to activation of the p53 pathway.Therefore,specific Pol Ⅰ inhibitors may provide clues for the development of novel pharmaceutical agents for the treatment of psoriasis.