| Systemic sclerosis(SSc)is an autoimmune connective tissue disease characterized by progressive fibrosis of the skin and internal organs.Among connective tissue diseases,SSc is second only to SLE and rheumatoid arthritis in terms of incidence,and the quality of life and survival prognosis of patients are not optimistic if vital organs such as lung,kidney and heart are involved.However,the pathogenesis of SSc is still unclear and immunosuppressive therapy is clinically ineffective.In the pathogenesis of SSc,microvascular injury and tissue fibrosis are the two most important pathological changes,among which microvascular lesions can directly cause fibrotic injury through endothelial-to-mesenchymal transition(Endo MT)of endothelial cells.At the same time,activated fibroblasts are also key effector cells that drive the progression of SSc fibrosis.Therefore,elucidating the specific mechanisms of endothelial and fibroblast dysfunction is of great theoretical significance and application for studying the pathogenesis and treatment of SSc.NAD(P)H: quinone oxidoreductase 1(NQO1)is a common oxidoreductase in the body and an important factor in protecting cells against chemical damage.It has been shown that NQO1 has a role in promoting cell proliferation and active function,especially in malignant neoplastic diseases,where NQO1 is a key factor in ensuring the growth,division,and proliferation of tumor cells.Specifically,NQO1 maintains cell survival through timely removal of chemical toxicants or reduction of oxidative stress damage,in which autophagy plays a key regulatory role.Autophagy is a lysosomal degradation pathway that removes and metabolizes excess intracellular proteins and senescence-damaged organelles,and facilitates the recycling of some nutrients,which is essential for maintaining intracellular homeostasis and is a major protective mechanism in cells.Thus,NQO1 exerts multiple biological effects,either by influencing cell proliferation and differentiation or by interfering with autophagy as a metabolic pathway that ultimately mediates the pathogenesis of proliferative diseases.The pathogenesis of SSc also involves excessive proliferation and hyperfunction of fibroblasts,and whether NQO1 is involved in the pathogenesis of SSc has not yet been investigated.Through in vivo and in vitro experiments,we analyzed for the first time the differences in NQO1 expression in skin fibroblasts and vascular endothelial cells of normal subjects and SSc patients,and revealed the specific molecular mechanism by which NQO1 affects the fibrotic process through regulating the level of cellular autophagy,which provides important clues for further elucidation of the pathogenesis of SSc and the search of intervention targets.Objective:In this project,we firstly examined the expression level of NQO1 in skin fibroblasts of SSc patients,and then used NQO1 si RNA to intervene in skin fibroblasts of SSc patients and NQO1 overexpression plasmid to intervene in normal human skin fibroblasts to investigate the in vitro role and possible mechanism of NQO1 in the process of SSc fibrosis;secondly,we used NQO1 inhibitor bicoumarin to treat mouse model of systemic sclerosis to explore the possible pathways of NQO1 involvement in the fibrosis process in vivo;meanwhile,we investigated the role of NQO1 in SSc vascular injury through SSc vascular endothelial cell model to reveal the potential mechanism of action that NQO1 downregulation can alleviate SSc endothelial mesenchymal transition;finally,we explored the specific regulation of NQO1 expression upregulation in SSc skin tissue mechanism.The aim of this study was to verify the important role of NQO1 in SSc fibrosis and vascular injury in a multidimensional manner,and to provide a new theoretical basis for the exploration of the pathogenesis of SSc and the development of therapeutic tools.Methods:1.Skin fibroblasts from normal human and SSc patients were obtained by tissue apposition method,and NQO1 gene expression levels in skin fibroblasts from normal human and SSc patients were detected by real-time quantitative PCR,Western blot and immunofluorescence staining.2.NQO1 si RNA was transfected in SSc patient skin fibroblasts,and the expression levels of COL1A1,COL3A1 and α-SMA in SSc patient skin fibroblasts after knocking down NQO1 were firstly detected by real-time quantitative PCR,Western blot and ELISA.Next,flow cytometry,electron microscopy,laser confocal technique and Western blotting were used to detect the changes in the levels of apoptosis and autophagy in skin fibroblasts of SSc patients after knockdown of NQO1.Meanwhile,Baf-A1 and rapamycin treatment were given to inhibit and enhance the level of autophagy respectively to observe the alteration of fibrotic phenotype and to explore the possible regulatory pathways.3.NQO1 overexpression plasmid was transfected in normal human skin fibroblasts,and the expression levels of COL1A1 and autophagy-related proteins in normal human skin fibroblasts after overexpression of NQO1 were detected by Western blot.Similarly,Baf-A1 and rapamycin were used to inhibit and enhance the level of autophagy,respectively,to observe the fibrotic phenotype and the alteration of related signaling pathways.4.A mouse model of SSc was constructed by subcutaneous injection of bleomycin,and the expression of NQO1 was reduced by gavage administration of bicoumarin in the SSc mouse model,followed by inhibition and enhancement of autophagy levels by intraperitoneal injection of Baf-A1 and rapamycin,respectively,to verify whether the fibrogenic effects of NQO1 in vivo were dependent on autophagy levels and to explore the possible mechanisms.5.SSc vascular endothelial cell model was established using SSc patient serum combined with hypoxia treatment,and the expression levels of NQO1 in SSc patient skin vascular endothelial cells and SSc vascular endothelial cell model were detected by immunofluorescence staining and Western blot.Secondly,the changes of endothelial mesenchymal transition and autophagy levels in SSc vascular endothelial cell models were detected by cellular immunofluorescence and Western blot,and the expression levels of related signaling pathways were examined.Finally,the expression of NQO1 in SSc vascular endothelial cell model was interfered by NQO1 si RNA to detect whether the extent of endothelial mesenchymal transition was reduced.6.The methylation level of NQO1 promoter region was detected by heavy sulfite sequencing in normal human and SSc patient skin fibroblasts.5-Azacytidine(5-AZA),a DNA methyltransferase inhibitor,was treated with normal human skin fibroblasts to reduce the DNA methylation level,and the expression level of NQO1 was detected by Western blot.The protein expression level of NQO1 was detected after hypoxia treatment of normal human skin fibroblasts,and the change in the level of methylation in the promoter region of NQO1 was also detected.Results:1.The m RNA and protein expression levels of NQO1 were significantly upregulated in skin fibroblasts and lesion tissues of SSc patients compared with normal human skin fibroblasts.2.m RNA expression levels of fibrosis-related genes COL1A1 andα-SMA were significantly downregulated after treatment with NQO1 si RNA in SSc patient skin fibroblasts,and protein levels of COL1A1 were also downregulated,while COL3A1 expression levels were not significantly changed;after knocking down NQO1,autophagy levels in SSc patient skin fibroblasts were significantly increased and The mTOR/STAT3 signaling pathway negatively regulates the autophagic process.3.The dorsal skin of the SSc mouse model was significantly thickened and hardened compared to the control group,with increased COL1A1 and hydroxyproline production.Inhibition of NQO1 expression or increasing autophagy levels by rapamycin could alleviate skin fibrosis in the scleroderma mouse model.In contrast,Baf-A1 treatment exacerbated the fibrotic phenotype in mice by inhibiting autophagy levels.Consistent with the results of in vitro experiments,the activation of mTOR/STAT3 signaling pathway was negatively correlated with the level of autophagy in mice.4.NQO1 expression was significantly upregulated in SSc lesions.SSc patient serum combined with hypoxic treatment mimicked the SSc vascular endothelial microenvironment and successfully induced Endo MT in vascular endothelial cells.knockdown of NQO1 resulted in a diminished Endo MT phenotype in the SSc vascular endothelial cell model,accompanied by enhanced autophagy levels.mTOR/STAT3 phosphorylation Phosphorylation of mTOR/STAT3 is also involved in regulating the level of autophagy in vascular endothelial cells.5.NQO1 promoter region was significantly hypermethylated in skin fibroblasts from SSc patients,and 5-AZA decreased methylation levels in the promoter region of normal human skin fibroblasts resulting in increased protein expression of NQO1.Hypoxia similarly upregulated NQO1 protein expression levels and downregulated NQO1 promoter region methylation levels.Conclusions:1.NQO1 expression is upregulated in skin fibroblasts from SSc patients,and hypomethylation of the NQO1 promoter region may be an important mechanism regulating the upregulation of NQO1 expression,while hypoxia-induced reduction in DNA methylation may be a further explanation for the high NQO1 expression in skin fibroblasts from SSc patients.2.In SSc patient fibroblasts,vascular endothelial cells,and scleroderma mice,NQO1 may promote SSc fibrotic injury in part by inhibiting autophagy levels through activation of the mTOR/STAT3 signaling pathway. |
PDF Full Text Request