| Background:Scleroderma (systemic sclerosis, SSc) is an autoimmune connective tissue disease characterized by small vessel vasculopathy, autoantibody production, and excessive fibrosis of the skin and internal organs. Although the exact mechanisms involved in the pathogenesis of SSc are not well understood, but excessive deposition of extracellular matrix components, especially collagens, in skin and internal organs is one of the important features responsible for the clinical and pathologic manifestations of the disease. During tissue fibrosis, the increased synthesis and/or reduced degradation of collagen results in the destruction of the balance may be one of the important reasons for excessive collagen deposition in fibrosis tissues and organs. Myofibroblasts are alpha smooth muscle actin positive cells that are responsible for increased production of extracellular matrix proteins. The main myofibroblast progenitor after injury of different tissues seems to be the locally residing fibroblast, which transiently differentiates into a protomyofibroblast after the role of endogenous and exogenous factors. The protomyofibroblast is a stable phenotype, representing an intermediate step in most conditions where it proceeds toward the "differentiated myofibroblast" that is characterized by expression of alpha smooth muscle actin. However, as we know, myofibroblasts may have very heterogeneous origins. Recent literature has provided evidence suggesting that the epithelial-mesenchymal transition (EMT) process is a more general biological process that is also involved in several pathophysiological conditions, including organ fibrosis. Several lines evidence suggest that myofibroblasts have been shown to derive from epithelial cells that undergo EMT associated with fibrosis occurring in kidney, liver, lung, and intestine. Initiation of the EMT process obligatory requires proper signals originating from outside and/or inside the epithelial cells that, in turn, involve an extensive intracellular machinery of signal transduction pathways, transcription factors, target genes, and other regulatory mediators. Recently, more and more evidence have shown that reactive oxygen species (ROS)-mediated oxidative stress and endoplasmic reticulum stress involved in epithelial cell undergo EMT process, but whether and how EMT occurs in skin fibrosis remain unknown.Objective:In the present study, we conducted a study on the bleomycin-induced mouse model of scleroderma was established using subcutaneous repeated local treatment of bleomycin in BALB/C mice. The objective of this study was to examine follow:(1) whether bleomycin induced skin epithelial cells occurring EMT which lead to myofibroblasts increase is responsible for increased production of ECM proteins participation in skin dermal tissue;(2) whether ROS-mediated oxidative stress play a role in percutaneous injection of bleomycin-induced skin epithelial cell occurring EMT and tissue fibrosis;(3) whether ROS is involved in the regulation of endoplasmic reticulum stress and the unfolded protein response which trigger skin epithelial cell occurring EMT and its molecular mechanism.Methods:(1) To establish mouse skin fibrosis model of scleroderma, bleomycin was injected subcutaneously into the shaved back of BALB/C mice for1,2,3, and4weeks. The skin specimens was cut and stained with H&E or Sirius red to evaluate dermal thickness and collagen deposition etc histopathological changes. In the fresh skin tissue, the content of skin hydroxyproline was measured according to alkaline lysis method to reflect skin fibrosis.(2) To examine skin epithelial cell undergo EMT process, the skin specimens was stained with H&E and observed skin epithelial cell morphology changes in high magnification optical microscope. The fresh skin specimens were snap-frozen in OCT compound, the preparation of frozen sections were detected E-cadherin expression, the characteristics protein of epithelial cell, and Vimentin expression, the characteristics protein of mesenchymal cells, using immunofluorescence technology. To gain the molecular insight, the protein and mRNA expression of E-cadherin and vimentin were further measured by Western blotting and RT-PCR.(3) To examine whether ROS cause oxidative stress and the change of antioxidant enzymes activity in bleomycin induced skin fibrosis. Malondialdehyde (MDA) content reflects the degree of lipid peroxidation, which was detected using a thiobarbiturate colorimetric method. The content of Carbonyl contents was measured to reflect the degree of oxidative damage of proteins, which was assessed by determining carbonyl groups based on a reaction with dinitrophenylhidrazine (DNPH). Total superoxide dismutase (SOD) activity was measured by using a xanthine and xanthine oxidase system to produce superoxide, and superoxide oxidizes hydroxylamine to nitrite to form a carmine color agent. Catalase (CAT) activity was measured by the slightly yellow complexation, produced by the reaction between the resolvation of H2O2by catalase and molybdenum-acid-nedymium. Glutathione peroxidase (GPX) activity was measured by GPX catalyzes the reduction of organic peroxide in the presence of glutathione (GSH) to H2O and oxidized glutathione (GSSG). Glutathione reductase (GSR) activity was measured by GSR catalyzes the reduction of glutathione in the presence of NADPH, which is oxidized to NADPH+.(4) To examine whether endoplasmic reticulum (ER) stress and the unfolded protein response occur in in bleomycin induced skin fibrosis. Western blotting detects ER stress characteristic protein Bip and activation of the unfolded reactive protein pIRE1α expression in the skin tissue. In addition, transmission electron microscopy observed the ultrastructure of the endoplasmic reticulum in the basal layer of epithelial cells in tissue of the skin fibrosis.(5) To explore the role of Smad and Src kinase signaling pathway in ER stress induced skin epithelial cells undergo EMT process, the animals were administrated with4-phenyl butyric acid (PBA) and western blotting detects the protein of pSmad2/3and pSrc expression in the tissue of skin fibrosis.(6) To investigate whether the ROS mediate endoplasmic reticulum stress in bleomycin induced epithelial cell undergo EMT and skin fibrosis, the animals were administrated with antioxidant N-Acetyl-L-cysteine (NAC) and western blotting detects the protein of Bip, pIRE1α, E-cadherin and Vimentin expression in the tissue of skin fibrosis. Moreover, the skin of treatment groups were stained with H&E and Sirius red staining for pathological observation the histopathological changes of skin thickness and collagen content etc.Results:(1) Morphological characteristics of skin sections were percutaneous injection of bleomycin showed the skin elasticity decreased, the skin thickness and hardness increased, and the hair of skin sections were percutaneous injection of bleomycin showed difficult to growth at4wks. At the same time, the body weight of mice showed significantly decreases after treated with bleomycin for4wks. In addition, the skin sections were stained with H&E and Sirius red staining for observation the histopathological changes, and quantified using digital image analysis of skin thickness and collagen content. Our results showed that there was a time-dependent increase in dermal thickness, collagen staining and extensive inflammatory cell infiltration in bleomycin-treated mice after2wks. The skin tissue hydroxyproline content also was increased in bleomycin-treated mice after4wks. Taken together, these results indicate that the current subcutaneous administration of bleomycin induces collagen deposition and skin fibrosis in BALB/c mice.(2) In the skin biopsies from the mice treated with bleomycin, many of the suprabasal cells were apparently detached from basement membrane zone (BMZ). The scattering of epithelial cells in the hair follicle was most pronounced following bleomycin treatment for3weeks, whereas the scattering of epithelial cells superficial to the BMZ was most pronounced following bleomycin treatment for4weeks. To further characterize EMT in the skin from mice treated with bleomycin, immunofluorescent staining indicated vimentin-positive epithelial cells were frequently found in the thickened epidermis from mice treated with bleomycin for3wks. The protein and mRNA expression of E-cadherin and vimentin, two EMT specific markers, were further measured by Western blotting and RT-PCR. Our results indicated that significantly decreased the expression of E-cadherin after bleomycin treatment for2weeks and increased the expression of vimentin after bleomycin treatment for3weeks. RT-PCR results showed that the mRNA expression of E-cadherin was significantly decreased but the expression of vimentin was significantly increased in the skin from mice treated with bleomycin for3weeks. Taken together, these results demonstrate bleomycin can induce EMT during the development of sclerotic skin of the mice, with specific EMT features at both tissue and molecular levels.(3) In order to monitor the ROS-mediated oxidative damage, we evaluated the content of MDA, protein carbonylation and antioxidant enzyme activity in bleomycin-treated skin tissue. Our results showed that the content of MDA and protein carbonylation significantly increased in bleomycin-treated group, NAC significantly inhibited bleomycin-induced increase of MDA and protein carbonylation. In antioxidant enzyme activity evaluation, we found the skin tissue SOD activity in bleomycin-treated mice was significantly increased, but GSR activity in bleomycin-treated mice was significantly decreased. In addition, CAT activity in bleomycin-treated mice was decreased, GPX activity in bleomycin-treated mice was increased, but when they compared with PBS group, there were not significant difference. Taken together, these results demonstrate bleomycin can induce oxidative stress in tissue of skin fibrosis, and this biology effect might mediate by H2O2.(4) Western blotting detects ER stress characteristic protein Bip and the unfolded reactive protein pIRE1α expression in the skin tissue. Our results indicated that a time-dependent increased the expression of Bip and pIREla since bleomycin treatment for2weeks, and PBA, a chemical chaperone, significantly inhibited the expression of Bip and pIREla in skin tissue of bleomycin treatment. Furthermore, transmission electron microscopy observed the ultrastructure of the endoplasmic reticulum in the basal layer of epithelial cells in tissue of the skin fibrosis. Our results indicated that the rough endoplasmic reticulum of the basal layer of epithelial cells showed a significant expansion since bleomycin treatment for2weeks. In addition, PBA also can significantly inhibit the down-regulation of E-cadherin and the up-regulation of vimentin in skin tissue of bleomycin treatment. Taken together, these results indicated bleomycin can induce skin tissue occurring ER stress, and ER stress might involve in the regulation of epithelial cell undergo EMT process in bleomycin-induced skin fibrosis.(5) Western blotting detects the expression of pSmad2/3and pSrc protein in skin tissue after percutaneous injection of bleomycin for3wks. Our results indicated that the expression pSmad2/3and pSrc protein significantly increased in the skin tissue of bleomycin treatment, and PBA can significantly inhibit the expression pSmad2/3and pSrc protein. These results suggested Smad and Src kinase signaling pathway might involve in ER stress induced skin epithelial cells undergo EMT process.(6) To evaluate the effect of ROS on bleomycin induced ER stress and EMT in the sclerotic skin, we treated mice with NAC, a direct scavenger of oxygen free radicals. Our western blotting results indicated that NAC significantly attenuated bleomycin-induced the increase of Bip and pIRE1α, and inhibit the down-regulation of E-cadherin and the up-regulation of vimentin in skin tissue of bleomycin treatment. Moreover, NAC also significantly attenuated bleomycin-induced skin thickness and collagen deposition. Taken together, these results indicated ROS might be the upstream signaling molecules, which might through ER stress-induced skin epithelial cell EMT and tissue fibrosis.Conclusion:The results of this study indicate that the skin epithelial cells can undergo EMT process in bleomycin-induced skin fibrosis. ROS might be the upstream signaling molecules, which might through ER stress-induced unfolded reactive protein pIREla activation, then pIREla activated Smad and Src kinase signaling pathway induce the down-regulation of E-cadherin and the up-regulation of vimentin in skin tissue of bleomycin treatment. Moreover, myofibroblasts come from EMT origin then increased production of extracellular matrix proteins, which might strengthen the level of skin fibrosis induced by bleomycin. |
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