Effects And Mechanism Of Capparis Spinosa On The Expression Of Type I Collagen In Scleroderma Dermal Fibroblasts

Progressive systemic sclerosis(PSS;scleroderma)is a connective tissue disorder characterized by fibrosis of the skin and of visceral organs.The cause of PSS is unknown,but the accumulation of extracellular matrix,collagen in particular,in affected organs results primarily from overproduction of the substance by stromal fibroblasts.Major pro-fibrotic cytokines involved with fibrosis in general is transforming growth factor(TGF-β)and CTGF.Histological studies examining the distribution of CTGF and TGF-βmRNAs in skin sections showed that in diffuse PSS lesions TGF-βis overexpressed in the leading inflammatory edge of the lesion.CTGF expression is abundant in fibrotic lesions. TGF-βand CTGF promotes deposition of the ECM by inducing expression of matrix genes.Matrix metalloproteinases(MMPs),zinc-dependent endopeptidases, are the major enzymes responsible for degradation of ECM components.The activity of MMP is inhibited specifically by tissue inhibitors of metalloproteinases (TIMPs),which bind to activated MMPs with 1:1 molar stoichiometry..A role for reduced ECM turnover in the pathogenesis of fibrosis has been suggested by studies showing that PSS skin fibroblasts in culture produced decreased amounts of MMP-1 andMMP-3,and increased amounts of TIMP-1 and TIMP-3 compared with those produced by control skin fibroblasts.Recently,excessive oxidative stress has been implicated in the pathogenesis of scleroderma.Unstimulated skin fibroblasts and monocyte from PSS patients released more O2^- and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts. reactive oxygen species(ROS)generated in vivo activate the Ha-Ras-ERK1/2-ROS cycle and inducesTGF-β,TGFsynthesis,ultimately results in the stimulation of collagen-gene expression.ROS also upregulate PAI-1 and TIMP-3 mRNA expression and protein secretion and significantly suppress plasmin and MMPs activity.Capparis spinosa L.is a native shrub widely distributed throughout the dry regions in west or central Asia.From ancient times,Capparis spinosa are used in traditional medicine for their diuretic,antihypertensive,poultice and tonic properties.Previous chemical studies on C.spinosa have shown the presence of alkaloids,lipids,polyphenols,flavonoids,indole and aliphatic glucosinolates. These flavonoids display a remarkable role in various pharmacological activities including antifungal,anti-inflammatory,antiallergic,antihepatotoxic and anti-diabetic effects.The methanol extract of C.spinosa showed a noteworth antioxidant/free radical scavenging effectiveness in diverse in vitro tests.It has been demonstrated that C.spinosa posses a noteworthy anti-fibrotie effectiveness in local sclerodera when topically applied.On the basis of the previous considerations and evaluating the interesting properties of the active compounds contained in C.spinosa,In this study,we investigated the effects of the ethanol extract of C.spinosa(ECS)on the expression of type I collagen from cultured PSS fibroblasts and analyzed the mechanisms underlying these effects.[Objective]Our aim in this paper is to investigate the effects of ECS on the fibroblast proliferation and type I collagen production in progressive systemic sclerosis(PSS), and investigate whether ECS suppress deposition of the ECM by decrease expression of TGF-β1 and CTGF or increase ECM turnover by increasing expression of MMPs and reducing TIMPs.Furthermore,we will investigate the role of ECS on ROS production and excessive oxidative stress in PSS fibroblast. [Methods]1.MTT assays were used to determine the effect of different concentrations of ECS on the proliferation of normal and PSS fibroblasts.Apoptosis was detected by flow cytometry analysis of AnnexinV-stained cells when fibroblasts were pretreated with 100μg/ml ECS.RT-PCR and Western-blot were used to determine the effect of different concentrations of ECS on the mRNA expression and protein synthesis of the type I collagen in normal and PSS fibroblasts.2.RT-PCR were used to determine the effect of different concentrations of ECS on the mRNA expression of TGF-β1,CTGF,MMP-1,TIMP-1 in normal and PSS fibroblast.Western-blot and immunofluorescent staining were used to determine the effect of different concentrations of ECS on the protein expression of TGF-β1, CTGF in normal and PSS fibroblast.3.The effect of different concentrations of ECS on the Levels of O2^- and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively.The effect of different concentrations of ECS on the Levels of reactive oxygen species(ROS)in living fibroblasts was estimated by FACS analysis using the fluorescent probe 2V-7V-dichlorodihydrofluorescin diacetate(DCFH-DA).In order to estimate protective effect of Capparis spinosa on oxidative stress-induced apoptosis in normal and PSS fibroblasts,Normal and PSS fibroblasts were stimulated for 2 h with 2 or 6 Mm H2O2 after pretreated with different concentrations of ECS.Cllular activity was then determined by MTT method and apoptosis was detected by FACS analysis of annexin V-C-stained cells.[Results]1.ECS could inhibit the proliferation of PSS fibroblast in a dose-dependent manner.The highest concentration of ECS examined in these experiments(100μg·mL^-1)did not increase apoptosis and did not cause morphologic changes.ECS did not affect the proliferation of fibroblast in normal human.2.ECS could significantly reduced the expression ofα₂(I)collagen mRNA and type I collagen protein in PSS fibroblast in a dose-and time-dependent manner. ECS did not affect theα₂(I)collagen mRNA and type I collagen protein in normal human.3.ECS could significantly reduced the expression of CTGF mRNA and protein in PSS fibroblast in a dose-and time-dependent manner.ECS did not affect the expression of TGF-β1 mRNA and protein in PSS fibroblast.4.ECS could significantly enhanced the expression of MMP-1mRNA and down-regulate the TIMP-3 mRNA in PSS fibroblast in a dose-and time-dependent manner.5.ECS could significantly reduced the release of O2^- and H2O2 from PSS fibroblast in a dose- dependent manner.ECS could significantly reduced the level of ROS in living PSS fibroblast in a dose- dependent manner.6.ECS could significantly reduced the cell death and apoptosis induced by H2O2.[Conclusions]1.The ethanol extract of Capparis spinosa effectivly reduce skin sclerosis by inhibiting fibroblast proliferation,reducing the expression ofα₂(I)collagen mRNA and production of type I collagen protein in PSS.100μg·mL^-1ECS didn't exert Cytotoxicity.2.ECS suppress deposition of the ECM by decrease mRNA and protein expression of CTGF,increase ECM turnover by increasing mRNA expression of MMPs and reducing mRNA expression of TIMPs.3.ECS effectively down-regulate excessive oxidative stress in PSS fibroblast.