The Effects Of IL-13 On Collagen Generation In Fibroblasts And Its Molecular Mechanism

Objective and significanceHypertrophic scar (HSc) and keloids are frequent and severe form of fibrosis of the skin, which limits movement and compromises the cosmetic appearance and function of the skin. Following severe skin injuries such as burns, hypertrophic scar (HSc) and keloids are frequent and severe form of fibrosis of the skin, which limits movement and compromises the cosmetic appearance and function of the skin. hypertrophic scar and keloids are formed, which are After is the skin organization burns, the wound repair, as a result of the whole body and the partial factor influence, from the deposition of causesexcessive extracellular matrix (ECM) to secrete the deposition, the proliferation of massive fiber structuretissues proliferation, the excessively reduction of collagen degeneration excessively reduces, the massive productions of blood vessels massive productions and , the proliferation and differentiation of repairs the cell including fibroblasts organization exceptionally to multiply the differentiation to formbecause of the whole body and the partial factors. In the initial fibrosis outset stage, each kind ofdifferent factors urges stimulate many kinds of cytokine the to release. Increasing evidences indicate that the cytokine network plays central role The role which the cytokine network in lies between leads in the inflammation and after thatthe in the fibrosis process plays to receive day by day takes. Some researches indicate that, there ishas imbalance between T helper type 1 lymphocyte in the fibrosis forming process (Th1) cytokine and T helper type 21 lymphocyte (Th2) cytokine imbalancein the fibrosis forming process, which was namely named the Th1/Th2 immunity is unbalanced., thus It promoted the activation and proliferation of to become the fibroblasts to activate, the proliferation,and the deposition of extracellular matrix deposition. The Th1 cytokines including include IFN-γ, IL-2, IL-12, IL-18 and the tumor necrosis factor - beta (TNF-β), the Th2 cytokines including include IL-4, IL-5, IL-10, IL-13 and monocyte chemotaxin protein - 1(MCP-1). .Th1 and the Th2 cytokine reply (i.e. Th1 cytokine anti-fibrosis effect and Th2 cytokine presses fibrosis effect) in the fibrosis aspect The opposing effects of Th1- and Th2- cytokine (i.e. the anti-fibrosis effect of Th1 cytokine and the fibrosis effect of Th2 cytokine) responses in fibrosis have also been substantiated by recent microarray experiments. Interleukine-13(IL-13) is a pleiotropic immune regulatory cytokine and a novel lymphokine produced by activated Type 2 helper cells. Interleukin (IL)-13 is a key inducer of several type-2 cytokine-dependent pathologies. It regulates inflammation, mucus production, tissue remodeling, and fibrosis. Consequently, IL-13 has been implicated in the pathogenesis of various diseases characterized by fibrosis, including skin fibrosis, liver fibrosis and idiopathic pulmonary fibrosis, and it become an important therapeutic target for a number of diseases in which IL-13 is believed to be overproduced. ForTo will understand well the latent function of IL-13 in in the scar forming process latent function, this research we have observed the effects of IL-13 on the scar to become the fibroblast collagen gene the duplicationtranscription ,the collagen generation and the signaling transduction pathway in the fibroblasts in in vitroraise waywill discuss IL-13 to become the fibroblast to the scar the collagen resultant action mechanism and the signal extension leads the way, will observe IL-13 will scar to become the fibroblast the duplicationwhetherthis process will become in the textile fiber cell membrane through the IL-13 union scar the IL-13 acceptor thus will extension to lead the circuit., We have detected the phosphorylation of STAT6 (signal extension will lead the butchetransducer and activator of transcription r in and copies the activation factor 6) STAT6 protein phosphorylationin response to IL-13., latter willchange overto traunion upward duplicationthe ionMoreover, the observation we have study tyrosine kinasestyrosinase inhibitor leflunomideA77 1726 and and the anti-fibrosis medicine interferon - gamma whether blocks the collagen generation induced by IL-13 to press the scar to becomein the fibroblastfibroblasts. the collagen resultant action, thus The study iswill shed new light on the mechanism of keloid and fibrosis mechanism. As a result, it provides new insight forthe IL-13 fibrosis mechanism research and the scar has the mechanism research to increase the new content, simultaneously uses in the the clinical scar keloid treatment for the new method laying the rationale and providing the experiment to rest on. Research content and method1.I The effect of IL-13 on the collagen generation in to becomes the fibroblasts the collagen generation influence: In vitro culture the The fibroblasts, the cell were divideds into the treatedexperimental group(added with 100μg/L IL-13) and the control group. after affects 24, 48, 72 hours, The cell growth condition was observed by the method of cell growth curve, and the cellular morphology was observed the by HE staining and the collagen fiber special staining were used to observe cellular morphology,. The cell fiber formation between fibroblasts was microscope observesd between the fibroblast cell andby the collagen fiber special staining the cell fiber formation,. the The activity of proliferation rate of fibroblasts with different concentration of IL-13in both groups was investigated and compared by MTT method. After 24, 48 and 72 hours in treated group, the total collagen content of the culture supernatant, was measured by Hydroxyproline(Hyp) release assay. The expression level of collagen Type I alpha 1 gene (COL1A1) mRNA in fibroblast was determined by the hydroxyproline examines IL-13 to become the fibroblast secretion collagen the influence, RT-PCR observes IL-13 to become the fibroblast Type I collagenα1 gene (Type I procollagen alpha 1, COL1A1) the mRNA level expression influence,, the level of collagen type I protein induced by IL-13 was analyzed by WWestern-Blotting was used to analyze the level of collagen type I induced by IL-13 in fibroblast. 2. The research of the signaling transduction pathway of IL-13 to becomesin the fibroblastfibroblasts function the signaling transduction pathway to lead the circuit the research: The expression of IL-13 receptorα1RT-PCR was examines examined by the fibroblast RT-PCR in fibroblastsIL-13 acceptorα1 expression. In addition, phosphorylation protein and the non-phosphorylation protein of transcription factor STAT6 were dectected in the treated group by Western-Blotting , after Western-blotting observes IL-13 to affect the fibroblastfibroblasts was induced by IL-13 for 1, 2, 4 and 8 hours1, 2, 4, 8 hours transcription factor STAT6 phosphorylation protein and the non-phosphorylation protein expression situation. 3. Tyrosine kinases inhibitor leflunomideA77 1726 to blockinhibites the collagen generation in fibroblasts induced by IL-13 to become the fibroblast to press the collagen resultant action: The inhibition rate of fibroblast proliferation with different concentration of A77 1726 was observed by MTT means. Becomes tThe fibroblasts were to divided into the experimental group(A77 1726 50μM and IL-13 100μg/L) and the experimental control group(IL-13 100μg/L).experimental joins sleflunomide, aAfter affects 24, 48 and 72 hours, the MTT means observes leflunomide to promote the fibroblast proliferation influence to IL-13, the inhibition effects of A77 1726 on collagen secretion of fibroblasts investigated the by hydroxyproline release assayto examine leflunomide to promote the fibroblast. The inhibition effects of A77 1726 on collagen type Iα1 gene mRNA expression in fibroblasts secretion collagen influence to IL-13, RT-PCR were to examinated leflunomide to promote the fibroblast type I collagenα1 gene mRNA level expression by RT-PCRinfluence to IL-13., The influence of A77 1726 on collagen type I synthesis in fibroblasts was analyzed Western-blotting to observe leflunomide influence IL-13 to promote the fibroblast to secrete type I collagenby Western-Blotting.4. Interferons - gamma suppress collagen protein synthesis in fibroblasts induced by IL-13:to become the fibroblast the fibrosis function The inhibition rate of fibroblast proliferation with different concentration of interferon–γwas observed by MTT means. The fibroblasts were divided into the experimental group(interferon–γ4×105U/L and IL-13 100μg/L) and the control group. After 24, 48 and 72 hours, the inhibition effects of interferon–γon collagen secretion of fibroblasts were investigated by hydroxyproline release assay. The inhibition effects of interferon–γon collagen type Iα1 gene mRNA expression in fibroblasts induced by IL-13 were examinated by RT-PCR. The influence of interferon–γon collagen type I synthesis in fibroblasts induced by IL-13 was analyzed by Western-Blotting.Becomes the fibroblast to divide into the experimental group and the blank control group, the experimental group joins the interferon–γ4×105U/L and IL-13 (100μg/L), after affects 24, 48, 72 hours, hydroxyproline examination interferon - gamma whether suppresses IL-13 to promote the fibroblast secretion collagen, RT-PCR observation interferon - gamma whether suppresses IL-13 to promote the fibroblast type I collagenα1 gene mRNA level expression, Western-blotting observation interferon - gamma whether suppresses IL-13 to promote the fibroblast to secrete type I collagen.Results1. The effect of IL-13 on the collagen generation: IL-13 to becomes the fibroblast the collagen resultant action influence: The rapid growth of fibroblasts, the arrangement multipolarization, the obvious overlap phenomenon, as well as richer cytoplasma were found in the treated group by HE staining observes the experimental group cell growth to be exuberant, the arrangement multipolarization, has the obvious overlap phenomenon, the cytoplasma is alternately richer, but in the treated group roughly the cell shape is roughly the same as in and the control group cell not obvious difference,. the The more collagen fibers were observed in the experimental group by collagen fiber special staining microscope observes the experimental group cell collagen fiber synthesis to increase,. The proliferation rate of fibroblasts treated with IL-13 were increased obviously. the hydroxyproline examination experimental group cell raise supernate secretes the The total collagen secretion from fibroblasts induced by IL-13 for 48h or 72h was content obviously to be higher than that of the control group by hydroxyproline release assay (P<0.05); RT-PCR examines becomes the fibroblast to The expression of collagen type I collagenα1 gene in fibroblasts treated with IL-13 for 48h or 72h were increased significantly, compared with the control by RT-PCR after also IL-13 affects 24h, 48h, 72h, the expression strengthens (P<0.05); The expression of collagen type I protein in fibroblasts treated with IL-13 for 48h or 72h were also increased significantly, compared with the control by Western Blotting (P<0.05)Western blotting examines becomes the fibroblast to express I collagen, after also IL-13 affects 24h, 48h, 72h, the expression strengthens (P<0.05).2. IL-13 induces the phosphorylation of STAT6 inIL-13 to becomes the fibroblastfibroblasts function the signaling transduction pathway to lead the circuit the research: RT-PCR examines The expression of IL-13 receptorα1mRNA was detected inbecomes the fibroblastfibroblasts by RT-PCR.to express the IL-13 acceptorα1, The expression of non-phosphorylation STAT6 protein was detected after Western-blotting observes IL-13 to affectafter the fibroblastfibroblasts were induced by IL-13 for 0, 1, 2, 4 and 8 hours by Western-Blotting has the non-phosphorylation STAT6 protein expression, and the expression of phosphorylation STAT6 protein was stronger after IL-13 affects in 2h the phosphorylation STAT6 protein expression to be strongest, and became weaker after 4h weakens.3. Tyrosine kinases inhibitor A77 1726 inhibites the collagen generation in fibroblasts induced by IL-13: 3. Tyrosine kinases inhibitor leflunomide to block IL-13 to become the fibroblast to press the collagen resultant action: The proliferation of The fibroblasts was inhibited by A77 1726 MTT means observes the experiment to compose the fibroblast the proliferation obviously to be lower than the experimental control groupusing MTT means (P<0.05),. Total collagen generation was down-regulated after 48h or 72h stimulation of A77 1726 the hydroxyproline examines various time section experiment to compose the fibroblast secretion collagen the content obviously to be lower than the experimental control group by hydroxyproline release assay (P<0.05). The expression level of collagen type Iα1 gene mRNA was obviously lower in the experimental group than in the control group after 48h or 72h stimulation of A77 1726 by RT-PCR (P<0.05), RT-PCR observes various time section experiment to compose the fibroblast type I collagenα1 gene mRNA expression level obviously to be lower than the experimental control group (P<0.05). C, ollagen type I production of fibroblasts treated with A77 1726 for 48 and 72 hours was decreased obviously in the experimental group than in the control group byW Western-Blotting observes various time section experiment to compose the fibroblast to secrete type I collagen the level obviously to be lower than the blank control group (P<0.05).4. Interferons - gamma suppress collagen protein synthesis in fibroblasts induced by IL-13: The proliferation of fibroblasts was inhibited by interferon - gamma by MTT means. Total collagen generation was down-regulated of fibroblasts treated with interferon - gamma for 48h or 72h by hydroxyproline release assay (P<0.05). The expression level of collagen type Iα1 gene mRNA was obviously lower in the experimental group than in the control group after 48 or 72hours stimulation of interferon - gamma by RT-PCR (P<0.05). Collagen type I production of fibroblasts treated with interferon - gamma in 48 or 72 hours was decreased obviously in the experimental group than in the control group by Western-Blotting (P<0.05).4. Interferon-γsuppresses IL-13 to become the fibroblast the fibrosis function: The hydroxyproline examines various time section experiment to compose the fibroblast secretion collagen the content obviously to be lower than the blank control group (P<0.05), RT-PCR observes various time section experiment to compose the fibroblast type I collagenα1 gene mRNA expression level obviously to be lower than the blank control group (P<0.05), Western-blotting observes various time section experiment to compose the fibroblast to secrete type I collagen the level obviously to be lower than the blank control group (P<0.05).ConclusionIL-13 may promote promotes the proliferation of the fibroblastfibroblasts proliferation, to be possible to increases the total collagen generation promoteof the fibroblastfibroblasts secretion collagen content, upregulates to be possible to promotethe expression of mRNA and protein of collagen type Iα1 gene the fibroblast I collagenα1 gene mRNA level and the protein level expression. IL-13 receptorα1 was expressed in human keloid fibroblastBecomes the fibroblast to express the IL-13 acceptorα1., The expression of phosphorylated STAT6 protein was strongest in fibroblasts after stimulation with IL-13 for 2h. after also IL-13 affects the fibroblast for 2 hours to transcription factor STAT6 phosphorylation protein the expression enhancement. Tyrosine kinases inhibitor leflunomideA77 1726 has the inhibitory effect on proliferation and total collagen generation induced in fibroblasts by IL-13to be possible to block IL-13 to become the fibroblast to proliferation function, it also blocks decreasedIL-13 to become the fibroblast to press the collagen resultant action, blocks IL-13 to facilitate the expression of c the fibroblastollagen type I collagenα1 gene mRNA level and collagen type I the protein level expressionin fibroblasts induced by IL-13. The interferon - gamma may suppresshas the inhibitory effect on collagen protein synthesis in fibroblasts induced by IL-13to become the fibroblast the fibrosis function.