青光安缓释剂对抗青光眼术后滤过道瘢痕化TGF-β/Smad信号传导通路相关mRNA的影响

Effect of Qingguang'an Slow-release Agent to Related mRNA of TGF-β/Smad Signal Transduction Pathway in Fltering Scarring after Glaucoma Filtering Surgery

目的:观察青光安缓释剂对抗青光眼术后滤过道瘢痕化过程中TGF-β/Smad信号传导通路相关mRNA的影响,探讨青光安缓释剂对滤过道瘢痕化的效果及作用机制,并判断其安全性。方法:通过高通量中药筛选体系提取4种青光安有效组份,并制成缓释药膜。将48只实验用兔分为8组,每组6只。分别为:A:空白组;B:模型组;C:MMC对照组;D:有效组份1组;E:有效组份2组;F:有效组份3组;G:有效组份4组;H:安慰剂组。除A组外,其余各组兔眼行常规小梁切除+虹膜根部切除术;C组在术中联合应用MMC;D、E、F、G 4组分别在结膜下植入相对应的青光安缓释药膜;H组植入安慰剂膜片。术后各组进行眼部常规检查。用药4周后处死实验用兔,取术眼滤过道巩膜组织,进行HE染和Real-time PCR法检测,并进行统计学分析。结果:(1)术后C、E两组眼压回升缓慢,第4周时两组眼压低于其他各组(P<0.05)。(2)C、E组术后都能维持有效滤过泡,B组滤过泡在术后两周内基本都变为无功能滤过泡或消失,D、F、G、H组在术后第4周都有少量功能性滤过泡存在。(3)HE染色见C、E两组滤过道轮廓清晰,纤维细胞及炎症细胞少,其他各手术组滤过道轮廓消失,被大量纤维细胞和炎症细胞填充。(4)C组Smad3、Smad4的mRNA表达值明显低于其他各组(P<0.01);C组和E组CollagenⅠ的mRNA表达值明显低于其他各组(P<0.01)。结论:青光安有效组份2可能与TGF-β竞争性结合TGF-β受体,或者可能通过影响炎症因子的生成减少TGF-β的产生,使TGF-β/Smad细胞信号传导通路的信号传导减弱,抑制滤过道瘢痕形成。且通过局部给药其不良反应小,安全性优于MMC。

Objective: To observe the effect of Qingguang’an slow-release agent to the related mRNA of TGF-β/Smad signal transduction pathway in the filtering scarring after glaucoma filtering surgery. To study the mechanisms of Qingguang’an slow-release agent and preliminarily determine their safety. Method: By using high throughput drug screening system,we screened out 4 effective elements from Qingguang’an. And 4 effective elements were made into sustained-release film. Forty-eight rabbits were randomly divided into 8 groups, 6 rabbits in each group. Group A: blank group, Group B: model group, Group C: MMC control group, Group D: effective component 1, Group E: effective component 2, Group F: effective component 3, Group G: effective component 4, Group H: the placebo group. The rabbits of Group A were without any treatment and all eyes of rabbits from group B to H were underwent routine trabeculectomy and iridotomy. In Group C, combined with MMC in operation,corresponding Qingguang’an release membrane was implanted under conjunctvia of eyes from group D to G. Group H received placebo release membrane in conjunctiva. All rabbits were measured intraocular pressure in postoperative to observe the shape of filtering bleb and we checked the wound healing and general case of eyes. After 4 weeks, all rabbits were sacrificed to separate the scleral tissue of operated eyes. We observed the histomorphology of the scleral tissue after HE staining and measured the mRNA of TGF-β2,Smad3,Smad4 and collagenⅠin the scleral tissue by Real-time PCR, and analyzed statistically. Results:(1) Intraocular pressure of Group C and E increased slowly after operation, and at the 4 th week, IOP in both groups was lower than that in other groups(P<0.05).(2)All eyes in Group C and E were able to maintain effective bleb after operation. In Group B, filtration blebs were basically not functional or disappeared within two weeks after operation. There were a few functional blebs in Group D, F, G and H at 4 weeks after operation.(3) HE staining showed that the outline of the filtering passage was clear, and the fibroblasts and inflammatory cells were less in the Group C and E. In other groups, the filter corridor disappeared and was filled with fibroblasts and inflammatory cells.(4)The mRNA expression of Smad3 and Smad4 in group C was significantly lower than that in other groups(P<0.01). The mRNA expression of Collagen Ⅰ in group C and E was significantly lower than that in other groups(P<0.01). Conclusions:The effective component 2 of Qingguang’an may bind TGF-β receptor competitively with TGF-β. Or it may decrease the production of TGF-β by affecting the production of inflammatory factors, and weaken the signal transduction of TGF-β/Smad cell signal transduction pathway and inhibit the formation of scar in filter passage. The MMC, effective component 2 of Qingguang’an,is more safe with less side-effect.