Research Of Effect Of JNK Pathway On Mediating TGF-β1-Induced CTGF Expression And Corneal Scar Formation After Wound

PartⅠResearch on the role of TGF-β1 in inducing cornea scar formation via CTGFPurposeThis study was undertaken to investigate the role of TGF-β1 in regulating CTGF expression,to identify effect of CTGF on mediating corneal scar formation induced by TGF-β1.Methods and MaterialsPenetrating injury in cornea was randomly taken in rats (200g-300g).After successful general and local anesthesia,lines for tracting were made both in upper and lower eyelids,and then penetrating injury was made in the 1/2 central cornea and treated with interrupted suture.At different time points after wound,corneas were harvested to examine the clinical changes by slip-lamp microscope and histopathological analysis were performed with HE-staining.Real time RT-PCR was used for detecting mRNA expression of CTGF and TGF-β1.THSF cells were cultured in vitro.The effects of CTGF,TGF-β1 on proliferation of THSF cells were measured by MTT analysis.CTGF neutralizing antibody was used to examine the effect of CTGF on TGF-β1-induced proliferation of THSF cells.THSF cells were stimulated with 3ng/ml TGF-β1 for 24 hours,and then Real time RT-PCR was used to examine changes of CTGF and ColⅠα1 mRNA,while western blot analysis was used to measure CTGF protein expression.ResultsThe wound model of cornea in rats was established successfully,and the penetrating wound of cornea was almost healed over a week.There was almost no expression of CTGF mRNA and few expression of TGF-β1 mRNA examined in normal rat stroma.After exposure to wound,mRNA expression of TGF-β1 and CTGF were induced significantly.Both exogenous CTGF and TGF-β1 induced THSF cell proliferation significantly.Blocking of CTGF significantly inhibited cell proliferation of THSFs induced by TGF-β1.Compared with the control,CTGF,ColⅠα1 mRNA and CTGF protein expression in THSF cells was significantly induced by stimulation of 3ng/ml TGF-β1 for 24 hours.While blocking CTGF with antibody,the induction of ColⅠα1 mRNA was significantly inhibited.ConclusionsTGF-β1 and CTGF are induced in wound cornea,indicating that they are involved in wound healing process in cornea stroma;Both TGF-β1 and CTGF play a role in promoting cornea scar formation,and CTGF is the downstream mediator of TGF-β1 in inducing cornea scar formation.PartⅡStudy of the signal pathway in regulating TGF-β1 induced CTGF expression in cornea scar formationPurposeThis study was undertaken to investigate effect of MAPKs pathways on TGF-β1 regulated CTGF expression and corneal scar formation,to probe into the key point of regulating corneal scar formationand to explore the novel target for regulating cornea scar formation.Materials and MethodsCultured THSF cells were pretreated with inhibitors of ERK,P38 and iNK,and then cells were stimulated with TGF-β1 for 24 hours and collected for detecting CTGF mRNA.The corneal wound model was established in rats in vivo,thirty minutes before and every day after wound,the specific inhibitor of iNK pathway(SP600125,50μM) was treated by using subconjunctival injection once a day,with normal saline as control.At different time points after wound,clinical changes were examined by slip-lamp microscope and the corneal histopathological structures were examined with HE-stained.Protein of CTGF and P-JNK was detected by immunochemistry,while real time RT-PCR was used for detecting mRNA expression of CTGF and TGF-β1.The effects of CTGF,TGF-β1 and JNK inhibitor on proliferation of THSF cells were measured by MTT analysis. Activation of the JNK1/2 pathway in TGF-β1-stimulated THSF cells was determined by immunofluorescence.The effects of SP600125 on migration and wound healing in THSF cells were observed with an inverted microscope. The mRNA expressions of CTGF,Fibronectin and ColⅠα1 in wound and TGF-β1-treated cells were measured by Real-time RT-PCR.ResultsThe inhibitors of MAPKs signal pathway inhibited TGF-β1-induced CTGF expression in THSF cells with different degree,and JNK signal pathway had the most obvious effect on inhibiting the role of TGF-β1.There were few expression of TGF-β1 and almost no expression of CTGF and P-JNK in normal rat stroma.After exposure to wound,expression of TGF-β1,CTGF and P-JNK were induced significantly.SP600125 blocked activation of JNK pathway obviously.In addition to SP600125,no significant changes in TGF-β1 expression were examined,while expression of CTGF mRNA and protein was inhibited significantly.The opacity of cornea in rats treated with SP600125 was slighter compared with the control.Both CTGF and TGF-β1 enhanced THSF cells proliferation.The TGF-β1-induced cell proliferation was clearly inhibited by SP600125.TGF-β1 stimulated activation of JNK1/2 and induced cell migration.SP600125 played a role in inhibiting activation of JNK1/2 and cell migration.The mRNA levels of CTGF,FN and collagenⅠalpal in wound and TGF-β1-treated cells were up-regulated,while SP600125 clearly inhibited the expression of these species.ConclusionsJNK signaling pathway was involved in process of corneal wound healing and might mediate the role TGF-β1 in inducing cornea scar formation via upregulating expression of CTGF;Further study on identifying the role of JNK pathway may contribute to regulating cornea scar formation by inhibiting JNK pathway.PartⅢBlocking JNK pathway by siRNA inhibits TGF-β1 induced CTGF expressionPurposeThe previous study(Part1 and Part2) found that TGF-β1 induced cornal scar formation via upregulating CTGF,and JNK signal pathway may mediate the process of TGF-β1 induced CTGF expression.This study was carried out to identify the role of JNK in mediating TGF-β1 induced CTGF expression and corneal scar formation by using RNA interfering.Material and MethodsSpecific JNK1/2 siRNA was designed according to the cDNA sequence of JNK1/2.The FITC-labeled control siRNA(nonsilencing siRNA) encoded no sequences and have no significant homologization with human and mouse cDNA. THSFs were incubated in 96-well plates and concentration gradients of the transfection reagent from 1/350 up to 4/350 were used for selecting transfection conditions.The transfected cells were observed and photographed using a fluorescence microscope.THSF cells were pretreated with JNK1 and JNK2 siRNA to block the JNK pathway,and were then treated with TGF-β1 or scratching.Activation of the JNK pathway was detected using an immunofluorescence assay.Expression of JNK1,JNK2 and CTGF mRNA was examined using real time RT-PCR analysis,and a western blot was used to detect production of CTGF and collagen I.Wound healing of THSF cells in different time in THSF cells were observed with an inverted microscope.ResultsThe results showed an over 90%positive transfection rate in all cells. However,when the transfection reagent was more than 3/350,parts of the cells died.A transfection efficiency of more than 90%and optimal cell viability were obtained when 2μl trasfection reagents were added to 350μl reaction mixture.Therefore,this dosage of transfection reagent was used in the following transfection experiments.Exposure of TGF-β1 and wound resulted in up-regulation of P-JNK,and induced production of CTGF and collagen I.When THSF cells were treated with JNK1/2 siRNA,mRNA expression of JNK1 andJNK2,activation of JNK1/2,induced production of CTGF and collagen I by TGF-β1 and wound were significantly inhibited. TGF-β1-induced cell migration was inhibited by blocking JNK pathway with siRNA.ConclusionsJNK pathway can be inhibited by transfected with siRNA;JNK1/2 pathway mediates the TGF-β1-induced CTGF expression.