The Efficacy And Mechanisms Of DsRNA Targeting TGF-β₂ On Conjunctival Filtering Bleb Scarring In Rats

PurposeTo investigate the inhibitory effect of small interference RNA targeting TGF-β₂ on conjunctival filtering bleb scarring in rat model,and its mechanism.Materials and MethodsSeventy two healthy and adult(about 3 monthes) SD rats were enrolled to established conjunctival filtering bleb scarring models with full-thickness filtering operation under general anesthesia.These 72 rats were divided into four groups: group PT,MMC,PBS and PSH according to their different managements.There were 18 rats in each group.Full-thickness filtering procedures were performed on both eyes of all the rats.Left eyes of all the rats were taken as an experimental eye and applied PT,MMC,PSH or PBS respectively during procedure.The contralateral eyes of all the rats were only performed filtering operation as self-control.General status, anterior segments of the rats were observed after surgery.More attention was paid to observe the morphous and existing time of conjunctival filtering blebs in these rats. Six rats were sacrificed each group and enucleated their eyes on 7 days,14 days and 28 days after surgery.Then paraffin imbedding and sectioning were procedured.The quantity and activity of fibroblast,deposition of collagen fibers,expression level of TGF-β₂ protein and TGF-β₂ mRNA were detected by HE staining,PCNA immunohistochemistry staining,VG staining,TGF-β₂ immunohistochemistry staining and TGF-β₂ hybridization in situ respectively.The staining results were analysed by semi-quantity analysis.These data was analyzed by the software SPSS for windows 13.0.Results1.Conjunctival filtering bleb scarring animal models with full-thickness filtering operation in rats were established.2.No apparent adverse effects of PT,MMC,PSH and PBS on the general status and anterior segments of the rats were found.3.One week after surgery,the rate of existed blebs in group PT,MMC,PSH and PBS was 100%(18/18),100%(18/18),72.2%(13/18),66.7%(12/18) respectively. The rate of existed bleb between group PT and PSH(P=0.023),MMC and PSH(P=0.023),PT and PBS(P=0.01),MMC and PBS(P=0.01) was significant statistical different.There was no difference in the rate of existed bleb between group PT and MMC.The rate of existed bleb in group PBS and PSH was not significant statistical different(P=0.500).Two weeks after surgery,the rate of existed blebs in group PT,MMC,PSH and PBS was 91.7%(11/12), 83.3%(10/12),8.3%(1/12) and 16.7%(2/12) respectively.The rate of existed bleb between group PT and PSH(P=0.000),MMC and PSH(P=0.000),PT and PBS(P=0.000),MMC and PBS(P=0.001) was significant statistical different. The rate of existed bleb between group PT and MMC,PBS and PSH was not significant statistical different(P=0.500).Four weeks after surgery,the rate of existed blebs in group PT,MMC,PSH and PBS was 50.0%(3/6),33.3%(2/6), 0%(0/6) and 0%(0/6) respectively.The rate of existed bleb between group PT and PBS(P=0.091),PT and PSH(P=0.091),MMC and PBS(P=0.227) MMC and PSH(P=0.227) was not significant statistical different.The rate of existed bleb between group MMC and PT(P=0.500) was not significant statistical different too.4.Quantity of fibroblast was evaluated according to the result of HE staining.One week after surgery,there was significant statistical difference in quantity of fibroblast in four experimental groups(P=0.000).There was significant statistical difference in quantity of fibroblast between group PT and PSH,PT and PBS,MMC and PSH,MMC and PBS(P=0.002).The quantity of fibroblast in group MMC was lower than group PT(P=0.002).There was not significant statistical difference in quantity of fibroblast between group PBS and PSH (P=0.699).Two weeks after surgery,there was significant statistical difference in quantity of fibroblast in four experimental groups(P=0.000).There was significant statistical difference in quantity of fibroblast between group PT and PSH(P=0.009),PT and PBS(P=0.002),MMC and PSH(P=0.002),MMC and PBS(P=0.002).The quantity of fibroblast in group MMC was lower than group PT(P=0.002).There was not significant statistical difference between group PBS and PSH(P=0.093).Four weeks after surgery,there was significant statistical difference in quantity of fibroblast in four experimental groups(P=0.000).There was significant statistical difference in quantity of fibroblast between group PT and MMC,MMC and PSH,MMC and PBS(P=0.002).There was not significant statistical difference in quantity of fibroblast between PT and PSH(P=0.093),PT and PBS(P=0.699),group PBS and PSH(P=0.093) respectively.5.Activity of fibroblast was evaluated according to the result of PCNA immunohistochemistry staining.One week after surgery,there was significant statistical difference in activity of fibroblast in four experimental groups(P=0.000).There was significant statistical difference in activity of fibroblast between group PT and PSH,PT and PBS,MMC and PSH,MMC and PBS(P=0.002).The activity of fibroblast in group MMC was lower than group PT(P= 0.002).The was not significant statistical difference between group PBS and PSH(P=0.180).Two weeks after surgery,there was significant statistical difference in activity of fibroblast in four experimental groups(P=0.000).There was significant statistical differencein activity of fibroblast between group PT and PSH,PT and PBS,MMC and PSH,MMC and PBS(P=0.002).There was not significant statistical difference in activity of fibroblast between group MMC and PT(P=0.052),group PBS and PSH(P=0.180).Four weeks after surgery, there was not PCNA positive cell in all experimental groups.6.Deposition of collagen fiber was evaluated according to the result of VG staining.One week after surgery,there was significant statistical difference in depositon of collagen fiber in four experimental groups(P=0.001).There was significant statistical difference in depositon of collagen fiber between group PT and PSH(P=0.002),PT and PBS(P=0.002),MMC and PSH(P=0.009),MMC and PBS(P=0.004) respectively.There was not significant statistical difference in depositon of collagen fiber between group MMC and PT(P=0.394),group PBS and PSH(P=0.589) respectively.Two weeks after surgery,there was significant statistical difference in depositon of collagen fiber in four experimental groups(P=0.000).There was significant statistical difference in depositon of collagen fiber between group PT and PSH,PT and PBS,MMC and PSH,MMC and PBS(P=0.002) respectively.There was not significant statistical difference in depositon of collagen fiber between group MMC and PT(P= 0.485),group PBS and PSH(P=0.394) respectively.Four weeks after surgery, there was significant statistical difference in depositon of collagen fiber in four experimental groups(P=0.000).There was significant statistical difference in depositon of collagen fiber between group PT and PSH,PT and PBS,MMC and PSH,MMC and PBS(P=0.002) respectively.There was not significant statistical difference in depositon of collagen fiber between group MMC and PT(P= 0.132),group PBS and PSH(P=0.394) respectively.7.Expression level of TGF-β₂ protein was detected according to the result of TGF-β₂ immunohistochemistry staining.One week after surgery,there was significant statistical difference in expression level of TGF-β₂ protein in four experimental groups(P=0.000).There was significant statistical difference in expression level of TGF-β₂ protein between group PT and PSH,PT and PBS, MMC and PSH,MMC and PBS(P=0.002) respectively.The expression level of TGF-β₂ protein in group MMC was lower than group PT(P= 0.002).There was not significant statistical difference in expression level of TGF-β₂ protein between group PBS and PSH(P=0.180).Two weeks after surgery,there was significant statistical difference in expression level of TGF-β₂ protein in four experimental groups(P=0.000).There was significant statistical difference in expression level of TGF-β₂ protein in group PT and PSH(P=0.009),PT and PBS(P=0.015),MMC and PSH(P=0.00),MMC and PBS(P=0.002) respectively. The expression level of TGF-β₂ protein in group MMC was lower than group PT (P= 0.002).There was not significant statistical difference in expression level of TGF-β₂ protein between group PBS and PSH(P=0.240).Four weeks after surgery,there was not significant statistical difference in expression level of TGF-β₂ protein in four experimental groups(P=0.073).8.Expression level of TGF-β₂ mRNA was evaluated according to the result of TGF-β₂ hybridization in situ.One week after surgery,there was significant statistical difference in expression level of TGF-β₂ mRNA in four experimental groups(P=0.000).There was significant statistical difference in expression level of TGF-β₂ mRNA between group PT and PSH,PT and PBS,MMC and PSH, MMC and PBS(P=0.002) respectively.The expression level of TGF-β₂ mRNA in group MMC was lower than group PT(P=0.002).There was not significant statistical difference in expression level of TGF-β₂ between group PBS and PSH(P=0.260).Two weeks after surgery,there was significant statistical difference in expression level of TGF-β₂ mRNA in four experimental groups(P=0.001).There was significant statistical difference in expression level of TGF-β₂ mRNA between group MMC and PT,MMC and PSH,MMC and PBS(P=0.002) respectively.There was not significant statistical difference in expression level of TGF-β₂ mRNA between group PT and PSH(P=0.093),PT and PBS(P=0.054),group PBS and PSH(P=0.394) respectively.Four weeks after surgery,there was significant statistical difference in expression level of TGF-β₂ mRNA in four experimental groups(P=0.758).Conclusions 1.The rat conjunctival filtering bleb scarring animal model with full-thickness filtering operation was successfully achieved,with the advantages of normalization of procedure,longer existing time of bleb and less interfere factors.2.Application of PT in glaucoma filtering surgery was safe and tolerable in rat.3.Both PT and MMC could prolong the existing time of conjunctival filtering bleb. The difference of the existing time between group PT and MMC was no significant.4.In vivo,PT and MMC could degrade the expression level of TGF-β₂ mRNA and TGF-β₂ protein,depress the quantity and activity of fibroblast,decrease the deposition of collagen fiber.5.By interfering the expression of TGF-β₂ mRNA,PT could degrade the synthesis of TGF-β₂ protein,then depress the quantity and activity of fibroblast,decrease the deposition of collagen fibers,at last inhibit the scarring reaction of conjunctival filtering bleb and improve operation result.6.The anti-proliferation effect of PT was lower than MMC in our experiment,but the effect of PT is more similar to physiological condition without histological and ultrastructural abnormality.So PT could partly inhibit the proliferative response of filtering bleb.