The Effects And Mechanisms Of The HIF-1α Inhibitor On Systemic Sclerosis

Objective:Systemic sclerosis(SSc)is a diffuse connective tissue disorder of unknown etiology characterized by clinically progressive tissue and organ fibrosis,vascular lesions,and immune system disorders.According to the extent of fibrosis involvement,it is mainly divided into limited systemic sclerosis(l SSc)and diffused systemic sclerosis(d SSc).The pathophysiology of SSc is a progressive process of self-amplification involving microvascular damage first,followed by autoimmune and inflammatory responses,and finally characterized by diffuse fibrosis.In the early stage of SSc,damage and apoptosis of endothelial cells can lead to perivascular inflammation,oxidative stress,and tissue hypoxia.Hypoxia induced factor 1α(HIF-1α)plays an important role in cellular adaptation to hypoxia.There have been studies showing that HIF-1αinvolved in the development of fibrotic lesions through vascular remodeling,epithelial mesenchymal transformation and extracellular matrix over-deposition.However,the role and mechanism of HIF-1αin SSC has not been fully elucidated,and it is still unclear whether HIF-1α-targeted can effectively interfere the SSC process.Therefore,we hypothesize that HIF-1αis a key regulator of microvascular injury,autoimmunity and inflammation in the early stage of SSC,and may be a noval target for SSC therapy.Here,we firstly constructed two fibrosis mice models of SSc mediated by hypochlorous acid(Hypochlorous)and bleomycin(BLM)respectively,and observed the fibrotic and immunological characteristics of HOCl-induced mouse models for the first time,providing a solid foundation for subsequent studies;then,HIF-1α-specific inhibitors(PX-478)was used in fibrosis mouse models to observe its effect on fibrosis.Differential expression of mRNA spectra and functional enrichment in skin and lung tissues were analyzed by whole transcriptome sequencing to investigate the mechanisms of HIF-1αinhibitors in SSc.To clarify the regulatory effect of HIF-1αon immune cells in SSc,the distribution of T cells and macrophage subsets in diseased tissues and major immune organs were investigated by flow cytology.Methods:The subcutaneous injection of bleomycin(BLM)and hypochlorite(HOCl)was used to simulate the skin or lung fibrosis model of SSC in mice,and the degree of fibrotic lesions and inflammatory cell infiltration changes in mice were assassed by HE staining,immunohistochemistry,and flow cytology of spleen cells.After the use of HIF-1αinhibitors in BLM and HOCl fibrotic mice,the anti-fibrosis effect of HIF-1αinhibitor was determined by HE staining and immunohistochemistry.The whole-transcriptomic sequencing,functional enrichment(GO and KEGG)analysis were performed on the tissues of the fibrosis mice,to initially explore the mechanism of HIF-1αinvolvement in the pathogenesis of SSc.To clarify the effect of HIF-1αon T cell subsets,flow cytology was performed to detect the changes of T cell subsets derived from the diseased tissues(skin and lungs)and major immune organs(spleen and thymus)respectively.Results:1.Establishment of HOCl-induced SSc mouse model:based on the existing defects of the classical BLM-mediated SSc model,we supplemented with a HOCl-induced SSc mouse model.Compared with the localized fibrotic lesions in skin and lung tissue caused by subcutaneous injection BLM,the HOCl-induced mouse model was closer to the clinical features of SSc,showing diffused skin and pulmonary fibrosis,vascular lesions,and a large number of inflammatory cell infiltraton.2.Immunological characteristics of HOCl-induced fibrotic mouse model:We observed the distribution of immune cells in HOCl mice lesions by flow cytometric analysis for the first time.The results showed that CD4~+T cells,CD8~+T cells,and CD19~+B cells were increased significantly in.of the spleen revealed an increase in CD19+B cells,a decrease in skin and lung lesions induced by HOCl than those in the PBS and BLM-treated groups.Spleen cytology analysis showed CD19~+B cells were increased and CD4~+,CD8~+T cells,macrophages,monocytes,and neutrophil were decreased,when compared with the PBS treated group.3.HIF-1αaggravated the fibrotic lesions of SSc:immunohistochemistry assay showed that HIF-1αwas high expressed in skin tissue from SSc patients,and PCR showed that hif-1αmRNA expression were increased both in BLM and HOCl mouse models;after administration of HIF-1αinhibitor(PX-478),the fibrotic lesions of skin in BLM and HOCl mouse models were significantly alleviated.4.HIF-1αinhibitor allivated fibrotic lesions through multiple biological processes:1)The whole transcriptome sequencing analysis of skin tissue found that compared with the HOCl group,the inhibitor intervention group(HIFi)expressed 408 upregulated genes and 618downregulated genes;compared with the BLM group,the HIFi group specifically expressed 921 upregulated genes and 1103 downregulated genes;84 upregulated genes and 156 downregulated genes were simultaneously expressed in the HOCl/BLM+HIFi group.Through GO analysis and KEGG analysis,it was found that compared with the HOCl group,there were 862(GO)and 46(KEGG)differentiated pathways in the HIFi group,respectively;compared with the BLM group,the differentiated pathways in the HIFi group were 1266(GO)and 41(KEGG)pathways;and the number of pathways associated with the common change of the two intervention groups was 429(GO)and 14(KEGG).The main change pathways involve T cell differentiation,Wnt signaling pathway,Hippo signaling pathway,Hedgehog signaling pathway,etc.2)The whole transcriptome sequencing analysis of lung tissue found that compared with the HOCl group,the HIFi group expressed 849 upregulated genes and 297downregulated genes;compared with the BLM group,the HIFi group expressed 664 upregulated genes and 685 genes expressed downregulated;the HOCl/BLM+HIFi group had 96 upregulated genes and 15 genes expressed down-regulated synchronously.Using GO analysis and KEGG analysis,it was found that compared with the HOCl group,there were 1110(GO)and 57(KEGG)differentiated pathways in the HIFi group,while compared with the BLM group,the HIFi group was 838(GO)and 49(KEGG);the two sets of synchronous changes were 349(GO)and 18(KEGG).The main involved pathways included fatty acid metabolism pathways,immune activation,B cell activation,complement system activation,and IL-17 signaling pathways.5.HIF-1αinhibitor regulates immune cell subsets distribution to alleviate fibrosis:1)After the use of HIF-1αinhibitor,the proportion of Treg cells in skin and lung tissue significantly increased to 13.68%and13.16%,respectively,compared with the HOCl group(3.30%,1.97%)(p<0.001);the proportion of Treg cells in skin and lung tissue in the BLM+HIFi group significantly increased to 16.67%and 16.21%,respectively,compared with the BLM group(3.17%,7.62%)(p<0.01).2)In the skin and lungs of the HOCl+HIFi group,the proportion of M1 cells showed an increasing trend;the proportion of M2 cells decreased to 6.29%and 4.31%,respectively,compared with the HOCl group(14.47%,21.26%),and there was a statistical difference in lung tissue(p<0.001).The proportion of skin and lung M1 cells in the BLM+HIFi group increased to 29.7%and 22.4%,respectively,which was statistically different(p<0.001)compared with the BLM group(2.47%,2.00%),and the proportion of M2 cells in lung tissue decreased(9.18%),compared with the BLM group(15.99%),with a statistical difference(p<0.05).Conclusion:1.Based on oxidative stress theory,a HOCl-induced mouse model of SSc was successfully constructed,showing diffuse skin and pulmonary fibrosis,vascular lesions and inflammatory infiltrates,which were closer to the phenotype of clinical SSc patients.It complements the classical BLM model and provides an important experimental tool for SSc study.2.HIF-1αpromotes fibrosis in SSc mice,and fibrosis can be relieved by inhibiting HIF-1α,and its mechanism is related to multiple biological processes and signaling pathways such as fatty acid metabolism,glycogen metabolism,immune cell differentiation,Wnt signaling pathway,Hippo signaling pathway,hedgehog signaling pathway,etc.3.HIF-1αpromotes fibrosis in SSc mice through altering the Th17/Treg cell balance to Th17 differentiation;and macrophage M1/M2polarization to M2 differentiation.HIF-1αmight be a potential therapeutic target to fibrosis in SSc therapy.