Background The main cause of filtering surgery failure is over proliferation of fibroblasts in filtering channels,leading to excessive fibrosis and scar formation.Researches determined that p38 mitogen-activated protein kinase(MAPK) signaling pathway plays an important role in fibroblast phenotype transition. Objective The present study was to investigate the inhibitory effect of p38 MAPK inhibitor on myofibroblasts transdifferentiation and the extracellular matrix synthesis after filtration surgery in rabbit eyes. Methods Trabeculectomy was performed on 24 eyes of 12 clean New Zealand white rabbits to establish the filtering operative models.The models were randomized into model group,SB203580 group and mitomycin C ( MMC ) group.1 ml SB203580 ( 0.2 g/L) was conjunctively injected at the end of operation in the rabbits of the SB203580 group,and the cotton piece with 0.2 g/L MMC solution was placed on the operative area for 3 minutes intraoperatively in the rabbits of the MMC group.The bleb appearances were examined under the slit lamp microscope,and intraocular pressure(IOP) was measured with Icare tonometer I,3,7,10,14 days after operation.0.2 ml aqueous humor was extracted and the conjunctive tissue at the filtering area was obtained 14 days after operation for the detection of α-smooth muscle actin (α-SMA) and fibronectin protein by ELISA.Expression of ACTA2 mRNA,connective tissue growth factor(CTGF) mRNA and alpha2 chain of type Ⅰ collagen( COL1A2 )mRNA in conjunctive tissue was assayed with fluorescence real-time PCR. Results Vascularization of fibrosis of filtering bleb were obvious in the eyes of the model group,and the bleb was flat and diffuse in the eyes of the SB203580 group and MMC group on 14 days following operation.No significant difference was seen in IOP before trabeculectomy among these three groups( F=0.065,P=0.937 ).IOP was gradually elevated with the increase of time after operation ( F =32.873,P =0.030 ).ELISA assay showed that α-SMA level in conjunctiva was lower in the SB203580 group and MMC group compared with the model group,and that of MMC group was significant lower than the SB203580 group( P＜0.05 ).Fibronectin level in conjunctiva was lower in the SB203580 group and MMC group compared with the model group,and that of MMC group was significant lower than the SB203580 group (P＜0.05).Fluorescence real-time PCR showed that expressions of the ACTA2 mRNA,CTGF mRNA and COL1A2 mRNA were significantly different among the three groups( P＜0.01 ),with the highest expression in model group and the lowest expression in the MMC group ( P ＜ 0.05 ). Conclusions Fibrotic reaction after trabeculectomy can be suppressed by inhibiting p38 MAPK signal pathway.The mechanism of SB203580 is to reduce the synthesis of myofibroblasts transdiffercntiation and extracellular matrix.%背景 各种原因导致滤过手术失败的共同点是滤过通道的成纤维细胞过度增生、分化,导致过度纤维化、瘢痕形成.研究发现,p38丝裂原活化蛋白激酶(MAPK)信号通道在成纤维细胞表型转化过程中发挥重要作用. 目的 探讨p38 MAPK抑制剂SB203580对滤过手术后结膜下纤维化反应的抑制效果及其作用机制.方法 12只清洁级新西兰白兔行常规青光眼滤过手术制作双眼滤过手术模型,模型兔按随机数字表法随机分为单纯滤过手术组、SB203580治疗组和丝裂霉素C(MMC)对照组.SB203580治疗组兔眼滤过术毕立即结膜下注射0.2 g/L SB203580溶液1 ml,MMC对照组兔眼术中用浸泡0.2 g/L MMC的棉片置于术区结膜下及巩膜瓣下3 min.各组兔术后进行裂隙灯显微镜观察,术后1、3、7、10、14d使用Icare回弹式眼压计测量眼压,术后14 d抽取房水0.2ml,并获取手术滤过区结膜下组织标本,用ELISA法检测房水及滤过区结膜组织中的α-平滑肌肌动蛋白( α-SMA)、纤维连接蛋白的表达,用荧光实时定量PGR检测各组兔术眼滤过区结膜组织中ACTA2、结缔组织生长因子(CTGF)和Ⅰ型胶原蛋白α2链(COL1 A2) mRNA的表达. 结果 术后14d,单纯滤过手术组滤过泡血管化、瘢痕形成,SB203580治疗组滤过泡扁平、弥散,MMC对照组滤过泡呈苍白缺血状、扁平弥散.单纯滤过手术组、SB203580治疗组和MMC对照组兔间术前眼压值差异无统计学意义( F=0.065,P=0.937),术后眼压值随着时间的延长逐渐升高,各时间点的总体比较差异有统计学意义(F=32.873,P=0.030).ELISA法检测SB203580治疗组和MMC对照组兔房水及滤过区结膜下组织中的α-SMA质量浓度均明显低于单纯滤过手术组,而MMC组α-SMA质量浓度明显低于SB203580治疗组,差异均有统计学意义(P＜0.05).SB203580治疗组和MMC对照组兔房水及滤过区结膜下组织中的纤维连接蛋白质量浓度均明显低于单纯滤过手术组,而MMC对照组α-SMA质量浓度明显低于SB203580治疗组,差异均有统计学意义(P＜0.05).荧光实时定量PCR检测表明,各组兔术眼滤过区结膜下组织ACTA2、CTGF、COL1A2mRNA表达差异均有统计学意义(P＜0.01),以单纯滤过手术组表达量最高,SB203580治疗组次之,MMC对照组最低,差异均有统计学意义(P＜0.05).结论 p38 MAPK抑制剂SB203580能减少肌成纤维细胞分化及细胞外基质合成,减轻兔眼滤过手术后的组织纤维化反应.
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