| ObjectiveExtraocular muscle fibrosis of thyroid-associated ophthalmopathy (TAO) is inreversible in pathology,the patients complain of strabisums, diplopia and even lose their sight because of compressed optic neuropathy.Transforming growth factor-β1 (TGF-β1)is one of the most important profibrotic cytokines,it can lead to proliferation of orbital fibroblasts(OF), induce OF transdifferentiation to myofibroblast(MFB)and stimulate OF to express components of extracellular matrix(ECM). Studies have confirmed that TGF-β1 play an important role in the pathogenesis of Extraocular muscle fibrosis of TAO and the abnormally expressive TGF-β1, (connective tissue growth factor, CTGF)and P-Smad2/Smad3 in OF of TAO.However,the molecule mechanism and signal ransduction pathways are uncertain.This research was designed to study the expression ofα-smooth muscle actin(α-SMA),CTGF and TGF-β1/Smad pathway and key enzymes of mitogen- activated protein kinase(MAPK) and phosphatidylinositol 3-kinase(PI3K)signal transduction pathways in orbital fibroblasts after TGF-β1stimulation and the correlation betweenα-SMA,CTGF and the above signal transduction pathways,to investigation the expression changes and significance of TGF-β1 in OF and to explore its downstream signal transduction pathways and their interrelationship in above process.Methods1. To detect the expression of TGF-β1,CTGF,P-Smad2/ Smad3,Smad4,Smad7 with immunohistochemical staining in human extraocular muscle tissues of TAO patients.2. Extraocular muscle fibroblasts were primary cultured by tissue explant culture technique and identified by morphological and immunocytochemistry analysis.3. OF were treated with 10μg·L-1 TGF-β1 at different time points (0 hours,3 hours,6 hours,12 hours,24 hours and 48 hours) and different concentrations(1μg·L-1,5μg·L-1,10μg·L-1 and 20μg·L-1) for 24 hours, and real-time quantitative RT-PCR was performed to observe the effects of TGF-β1 on the expression of TIMP-1, COL-I, COL- III and FN mRNA.Cell immunofluorescence was used to detect expression of TIMP-1, COL-I, COL- III and FN protein.4. MTT was used to assess the proliferation of OF by TGF-β1,real -time PCR and Western blot were used to assess the signal transduction pathways involved in TGF-β1inducedα-SMA and CTGFgene expression in OF cells,various inhibitors(SB431542 for TGF-β1/Smad, PD98059 for ERKl/2 ,SB203580 for p38 ,SP600125 for JNK ,LY294002 for PI3K/AKT,) specific to signal mediators were employed.Activation of Smad3(phosphorylation) was assessed by Western blot using antibodies against the active(phospho)forms . Real -time PCR was used to assess the signal transduction pathways involved in CTGF inducedα-SMA and CTGF gene expression in OF. Cell immunofluorescence was used to detect expression ofα-SMA and CTGF protein.Results1. The positive staining of TGF-β1,CTGF,P-Smad2/Smad3 and Smad4 mainly localized in orbital fibroblasts of TAO and were higher than those in control group,while Smad7 was lower than that in control group.2.The Extraocular muscle fibroblasts were cultured and passaged successfully.The cells showed long spindle appearance. their Vimentin staining were positive,but their Desmin staining, Keratin staining and S-100 staining were all negative by immunocytochemistry analysis,indicating that the cells were mesoderm derived fibroblasts.3. Time effect of OF treated with 10μg·L-1 TGF-β1:TIMP-1 mRNA was 1.5 folds to that of control group at 3 hours and 2.46 folds at 6 hours (both p<0.01);COL-I mRNA was 5.49 folds to that of control group at 24 hours and 3.69 folds at 48 hours,(both p<0.01);COL- III mRNA was 2.14 folds to that of control group at 24 hours and 1.63 folds at 48 hours(both p<0.01);FN mRNA was 2.45 folds to that of control group at 12 hours and 1.53 folds at 24 hours(both p<0.01 );Dose effects of OF treated with different concentrations TGF-β1 for 24 hours :TIMP-1 mRNA was 1.79 folds to that of control group at 5μg·L-1 and 1.46 folds at 10μg·L-1 ( both p<0.01);COL-I mRNA was 1.94 folds to that of control group at 1μg·L-1 and 3.29 folds at 10μg·L-1 (both p<0.01);COL- III mRNA was 1.52 folds to that of control group at 5μg·L-1 and 3.28 folds at 10μg·L-1(both p<0.01);FN mRNA was 1.3 folds to that of control group at 1μg·L-1 and 2.45 folds at 10μg·L-1, both p<0.01). Cell immunofluorescence showed that OF expression of TIMP-1, COL-I, COL- III and FN protein by TGF-β1.4. Real -time PCR and Western blot showed that bothα-SMA ,CTGF mRNA and protein expression were higher than those in control group(p<0.05). SB431542, PD98059 and SB203580 partly attenuated TGF-β1stimulated expression ofα-SMA(p<0.05) ,while the JNKinhibitor SP600125 or the PI3K inhibitor LY294002 had no such impact((p>0.05)).SB431542 and SP60125 partly attenuated TGF-β1stimulated expression of CTGF(p<0.05),while the P38inhibitor SB203580 or the PI3K inhibitor LY294002 or ERK inhibitor PD98059 had no such impact(p>0.05).TGF-β1 induced Smad3 phosphorylation ,SB431542 effectively inhibited the phosphorylation of Smad3 stimulated by TGF-β1but not that of the components of the MAPK pathways.Real -time PCR showed that CTGF can stimulate the expression ofα-SMA in OF in a time and dose-dependent manner, and SP600125 significantly attenuated CTGFstimulated expression of CTGF in OF(p<0.05).Conclusion1. TGF-β1/Smad pathway may play an important role in the pathogensis of extraocular muscle fibrosis of TAO.2. The cells cultured by tissue explant culture technique are consistent with typical extraocular muscle fibroblasts characteristics in morphology and immunocytochemical observation.The extraocular muscle fibroblasts are used as cell model in vitro for further research on the mechanism of extraocular muscle fibrosis of thyroid -associated ophthalmopathy.3.TGF-β1 can stimulate the expression of TIMP-1 mRNA,COL-I mRNA, COL- III mRNA and FN mRNA in OF in a time and dose-dependent manner, it may paly an important role in the pathogenesis of extraocular muscle fibrosis with TAO.4. SB431542 significantly attenuated TGF-β1 stimulated expression of CTGF andα-SMA,While MAPK partly attenuated TGF-β1 stimulated expression of CTGF andα-SMA. CTGF can stimulate the expression ofα-SMA in OF,and SP600125 significantly attenuated CTGF stimulated expression of CTGF in OF.
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