SZB120 Alleviate IMQ Induced Psoriasis-like Mouse Model By Th17 Suppression Via Targeting EIF2S1/I?B?

Observations1.To screen for potential small molecule compounds that inhibit Th17 differentiation.2.To investigate the mechanism of Th17 differentiation inhibition by SZB120.3.To find the direct target of SZB120 and verify its function in Th17 differentiation.4.To explore the therapeutic effect of SZB120 on IMQ-induced mouse psoriasis model.Methods:IL-17A-GFP mice were used to compare the differentiation ratio of Thl7 cells treated with AKBA and SZB120.After 72 h of cell culture,cells were collected,and then detected by a BD Fortessa X20 flow cytometer.CCK-8 was used to measure the effect of small molecule compounds on the proliferation of HaCaT cells.Adherent HaCaT cells were treated with small molecules for 24 h,10?1 CCK-8 was added to each well to continue the reaction.After 2 h,the plate absorbance was detected by microplate reader at 450 nm.CTV was used to detect the proliferation of Th17 cells.0.5 ?M,5?M SZB120 was added to the naive CD4+ T cells with the cell tracer dye.After 72h,the ratio of CD4+/IL-17+ cells were tested and the cell proliferation was measured.The Apoptosis kit was used to detect the apoptotic effect of SZB120 on Th17 cells.After 72h incubation,Th17 cells were stimulated with eBioscienceTM Cell Stimulation Cocktail for 4-6 hours,and then stained and analyzed by flow cytometry.When analyzing data,the results were analyzed in CD4+/IL-17+ cells.To detect the inhibition phase of Th17 cells by SZB120.After adding 5?M SZB120 and DMSO at Oh,the ratio of Th17 cells was detected at 24h,48h,72h and 96h.The naive CD4+ T cells were induced under Thl,Th2,iTreg conditions,DMSO or 0.5?M-5?M SZB120 were added.After 72h incubation,the corresponding flow index was detected by flow detection.RNA-seq was used to detect the differential expression genes between SZB120 with DMSO in Th17 condition.Flow cytometry was used to detect the protein expression of ROR ?t after Th17 differentiation.Western Blot was used to detect the expression of phosphorylated STAT3 at 24h,48h and 72h.The target stable based drug affinity responsive targets stability(DARTS)method was used to detect the direct target of SZB120.Protein mass spectrum was used after silver staining.Western Blot verified that EIF2S1 is a direct target of SZB120.Naive CD4+ T cells were sorted for ThO or Th17 cell induction.Protein was extracted at 1h and 4h respectively and used for western blotting.The phosphorylation degree of EIF2S1 after DMSO and SZB120 treatment was compared.GSK2606414 was used in combination with SZB120 on differentiated Th17 cells,the ratio of Th17 cells was measured by flow cytometer after 72h.At the same time,the proportions of Th17 cells in DMSO,SZB120 and GSK2606414+SZB120 were compared.Na'ive CD4+T cells were isolated for ThO or Th17 cell induction,and protein was extracted 4h later.Western blot was used to detect the difference of EIF2S1 phosphorylation under DMSO,SZB120 or GSK2606414+SZB120 administration.Naive CD4+T cells were sorted for Th17 cell induction,adding DMSO,SZB120.Western Blot was used to check the protein expression of I?B? Agilent's Seahorse XF Glycolysis Stress Test Kit was used to detect changes in glycolysis after Th17 differentiation under different concentrations of SZB120 or DMSO.At the same time,6-NBDG was used to detect the change of glucose uptake ability of Th17 cells.GSEA Hallmark data set was used to analyze RNA-seq data.Western Blot was used to detect changes in the expression of glycolytic-related proteins.Mice were randomly divided into three groups:vehicle,DMSO,and SZB120.Mice in DMSO or SZB120 groups were applied imiquimod on shaved back daily to induce psoriasis-like skin disease.Body weight,skin thickness,HE staining,immunohistochemistry were measured to see if there were any changes in the relevant indicators.After 7 days of imiquimod administration,the spleens of mice from each group were enriched and stimulated in vitro.After staining,cells were detected by flow cytometry and the expression of IL-17A was measured.The back skin of the mice was extracted,and the expression of EIF2S1 phosphorylation in the spleen of each group was measured.Results:Combined CCK-8 experiment with Th17 differentiation experiment,SZB120 was selected for subsequent studies.SZB120 inhibits the proliferation of HaCaT cells while inhibiting the proliferation of Th17 cells in a dose-dependent manner(P<0.001).CTV experiments showed that the proliferation of Thl7 cells were not affected by SZB120.Apoptosis experiments showed that when gate to CD4+/IL-17+ cell population,we found that SZB120 has an apoptotic trend effect from 0.5 ?M.SZB120 had a strong inhibitory effect on the protein expression of IL-17A.SZB120 was able to maintain stable inhibition efficiency both during initial Th17 differentiation and subsequent development phases.SZB120 has no significant effect on Thl,Th2 or iTreg differentiation at working concentration influential for Th17 cells(P>0.05).SZB120-treated T cells stimulated for 72 hours under Th17 polarization showed different expression of 279 annotated genes compared to DMSO,of which more than half were down-regulated.Compared to DMSO,SZB120-treated cells reduced mRNA expression of IL17A,IL17F,IL22,IL23r,CCL20,and CCR6.SZB120 affects the phosphorylation of the transcription factor STAT3 but does not affect the protein expression of ROR?t.With DARTS,we obtained the differential bands visible to the naked eye after silver staining.The interaction between SZB120 and EIF2S1 was identified by mass spectrometry and Western Blot.SZB120 promoted the phosphorylation of EIF2S1 at 4 h.At the same time,we found that under Th0 induction condition,SZB120 can also promote the phosphorylation level of EIF2S1.After adding different concentrations of GSK2606414,the inhibitory effect ability of SZB120 on Th17 differentiation was significantly impaired(P<0.001).We found that when GSK2606414 was added with SZB120,the phosphorylation level of EIF2S1 was lower than SZB120 added only.We found that SZB120 can promote the phosphorylation of EIF2S1 and down-regulate the expression of I?B? at the same time.We found the glycolytic abilities of Thl7 cells were significantly reduced when treated with SZB120.In addition,glucose uptake was downregulated after activation and this inhibition effect was most profound at 48 hours(P<0.001).GSEA was used to analyze RNA-seq data,we found that SZB120 down-regulated the MYC-targeted genome set as well as the gross hypoxia genome caused by HIF1 a,but up-regulated the P53 signaling pathway.At the same time,we examined the protein expression levels of several genes related to glycolysis and found the expression of PFKFB3 and HIF1 a was decreased.SZB120 significantly relieved the morphological characteristics of IMQ mice.Acanthosis was significantly reduced in mice treated with SZB120(P<0.001).Immunohistochemistry also showed a significant reduction in CD4+T cell and Ki67+ cells in mice treated with SZB120.We measured the proportion of IL-17 positive cells in CD4+ cell population of IMQ-induced mice spleen by flow cytometer analysis.We found that the proportion of IL-17 expressing cells was low in SZB120-treated mice.Simultaneously,SZB120 could up-regulate the phosphorylation level of EIF2S1 of spleen cells.Conclusion:In this study,we found that SZB120 can inhibit Th17 cell differentiation by interacting with EIF2S1,thereby attenuating the imiquimod-induced mouse-like psoriasis model.The study clarified that SZB120 can inhibit Th17 differentiation and explored its mechanism.The expression of I?B? was reduced after elevated phosphorylation of EIF2S1.I?B? is an important transcription factor for Th17 differentiation.SZB120 can significantly improve the phenotypic characteristics associated with psoriasis on mouse model.In conclusion,SZB120 can be a potential compound for the treatment of psoriasis.