| PurposeThyroid-associated ophthalmopathy(TAO)is an autoimmune disease associated with thyroid dysfunction.Although the pathogenesis and etiology of TAO are far away from fully illuminated,it has been demonstrated that the transforming growth factor(TGF-?)-induced fibrosis of orbital fibroblasts(OFs)plays a critical role in the diplopia and ocular motility disorder of patients with inactive TAO.In our previous research,we selected a differential expressed protein called pentraxin 3(PTX3)between TAO group and the control group,according to the second generation high-throughput sequencing result.Despite the crucial role of PTX3 in several fibrotic diseases,as demonstrated by many studies,it is still unknown if the interaction between PTX3 and TGF-? is exist and whether PTX3 has a place in TGF-b-induced fibrosis in the orbit of patients with TAO.This study established several strains of the primary cultured OFs as the research objects.In the first part,we plan to cultivate OFs with or without TGF-?1 in vitro and detect the change of PTX3 expression in the level of protein and gene respectively,which could investigate the exact effect of TGF-?1 on PTX3.We also intend to figure out if this effect has a TAO-specific character by comparing the result between the TAO and control group.Next is to use specific inhibitors and si RNA which can block the critical receptors and signaling molecules through the classical TGF-?/Smad signaling pathway.The subsequent change of PTX3 expression could reveal the potential mechanism of PTX3 modulation by TGF-?.In the second part,we plan to utilize the recombinant human PTX3 protein(rhPTX3)to interfere with TGF-?-induced fibrosis in OFs and detect the expression of fibrosis-related proteins and cellular morphologic changes.The aim of this part is to explore the role of PTX3 in TGF-?-induced fibrotic process in the orbit.Methods1.The cellular and intercellular protein expression of PTX3 response to TGF-?1 induction were detected by Western Blot and ELISA respectively.2.After induced by TGF-?1,the expression of PTX3 mRNA in OFs from TAO patients and the control subjects were detected by qRT-PCR.3.The specific inhibitors of TGF-? receptor,called SB431542 and recombinant human TGF-? RII Fc Chimera Protein(rT?RII-Fc),were used to intervene the TGF-?-induced bioreaction in OFs and the expression of PTX3 mRNA was detected by qRT-PCR.4.The phosphrylation of Smad2/3 can be blocked by ITD-1.ITD-1 were utilized to identify the role of this phosphylation in the disincentive effect.OFs were transfected with Smad4 si RNA and the expression of Smad4 mRNA was detected to verify the efficiency of the knockdown.OFs with si Smad4 transfection were cultured with TGF-?1 and the expression of PTX3 mRNA was detected by qRT-PCR.5.The rhPTX3 and TGF-?1 were added to the culture medium and the anti-?-SMA specific antibody with fluorescence was utilized to mark the fibrotic protein.The fibrosis of OFs was observed by fluorescence microscope.6.The rhPTX3 and TGF-?1 were used to induce the OFs and the expressions of fibrotic protein(?-SMA,Col I?1,IL-6)mRNA were detected by qRT-PCR.Results1.TGF-?1 suppressed the protein expression of PTX3 in a time-dependent manner in OFs.2.The expression of PTX3 mRNA was inhibited by TGF-?1 in a time-dependent and concentration-dependent manner.And the magnitude of suppression was not significantly different in OFs from TAO group and control group.3.The treatment of OFs with SB431542 totally eliminated suppression of PTX3 expression by TGF-?1,while rT?RII-Fconly partially reversed the effect.4.ITD-1 abolished the TGF-?-induced disincentive effect on PTX3 mRNA expression,conspicuously.The expressions of Smad4 mRNA were substantially decreased after the si RNA transfection.The transfected OFs was cultured with TGF-?1 and the levels of PTX3 mRNA had no response to TGF-?1 stimulation.5.TGF-?1 promoted the fibrosis of OFs and the level of ?-SMA protein was increased by TGF-?1,while the fibrosis of OFs with rhPTX3 and TGF-?1 collectively treatment did not seem to be significantly different from the TGF-?1-only group.6.The expressions of fibrotic protein(?-SMA,Col I?1,IL-6)mRNA were upregulated by TGF-?1 in OFs.The treatment with just rhPTX3 increased the expression of Col I?1 and IL-6 and had no effect on ?-SMA expression,but it enhanced the TGF-?1-induced upregulation of ?-SMA and IL-6,while the expression of Col I?1 was stable response to rhPTX3.Conclusions1.The expression of PTX3 in OFs was suppressed by TGF-?1 in a time-dependent and concentration-dependent manner.And this suppression was identical in TAO-OFs and the control OFs.It may indicate that there is no TAO-specific character in this suppression.2.SB431542,rT?RII-Fc,ITD-1 and Smad4 si RNA can block the inhibitory effect mentioned above,which could demonstrate the potential role of T?RI-Smad2/3-Smad4 signaling pathway in the suppression of PTX3 by TGF-?1.3.The rhPTX3 could promote the TGF-?1-induced fibrosis in OFs and this indicates that PTX3 may have a positive role in pathogenesis of diplopia in stable phase of TAO. |
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