HMGB1在类青光眼引流阀植入术后瘢痕化中的作用机制

Mechanism of HMGB1 in scarring after glaucoma drainage valve implantation

目的:探究高迁移率族蛋白1(HMGB1)在类青光眼引流阀植入术后瘢痕组织中的动态表达,揭示HMGB1在青光眼术后瘢痕化中的作用及其可能的作用机制。方法:将60只新西兰大白兔随机分为空白对照组(n=20)、模型组(n=20,结膜囊下硅胶植入)、模型给药组(n=20,硅胶植入联合5-氟尿嘧啶注射)。分别于术后4、8 wk取球结膜组织,采用HE染色和Masson染色检测结膜组织中成纤维细胞和胶原纤维增生及分布情况;免疫组织化学染色检测结膜组织中HMGB1、转化生长因子(TGF)-β1、Smad3、α-平滑肌肌动蛋白(α-SMA)的分布及变化情况;RT-PCR和Western blot检测结膜组织中HMGB1、TGF-β1、Smad3、α-SMA mRNA及蛋白的表达。结果:HE染色和Masson染色结果显示,4、8 wk模型组较空白对照组结膜组织中炎症细胞、成纤维细胞及胶原纤维增生明显,而4、8 wk模型给药组结膜组织中成纤维细胞及胶原纤维增生相对于同时期模型组明显减少。免疫组织化学染色结果显示,4、8 wk模型组结膜组织中可见HMGB1、TGF-β1、Smad3、α-SMA蛋白表达,呈棕褐色染色,8 wk模型组染色更深,而4、8 wk模型给药组较同时期模型组阳性染色程度下降。4、8 wk模型组结膜组织中HE染色成纤维细胞数与免疫组织化学染色HMGB1的表达水平均呈正相关(r=0.602、0.703,均P<0.05)。RT-PCR和Western blot结果显示,4、8 wk模型组结膜组织中HMGB1、TGF-β1、Smad3、α-SMA mRNA及蛋白表达量显著高于空白对照组(均P<0.05),4、8 wk模型给药组结膜组织中HMGB1、TGF-β1、Smad3、α-SMA mRNA及蛋白表达量显著低于同时期模型组(均P<0.05)。模型组和模型给药组中HMGB1与TGF-β1、Smad3的mRNA表达水平具有正相关性(均P<0.05)。结论:类青光眼引流阀植入术后随时间延长HMGB1表达升高,HMGB1在青光眼术后具有致瘢痕形成的作用,并可能通过TGF-β/Smad信号通路参与瘢痕化的发生发展。

AIM:To explore the dynamic expression of high mobility group box 1(HMGB1)in scar tissues after glaucoma drainage valve implantation,and to further reveal the role and possible mechanism of HMGB1 in scarring after glaucoma surgery.METHODS:A total of 60 New Zealand white rabbits were randomly divided into control group(n=20),model group(n=20,silicone implantation under conjunctival sac)and model with drug administration group(n=20,silicone implantation under conjunctival sac combined with 5-fluorouracil injection).The conjunctival tissues were collected at 4 and 8 wk after surgery.HE staining and Masson staining were used to detect the proliferation and distribution of fibroblasts and collagen fibers in conjunctival tissues.Immunohistochemistry was utilized to detect the distribution and changes of HMGB1,transforming growth factor(TGF)-β1,Smad3 andα-smooth muscle actin(SMA)in conjunctival tissues.RT-PCR and Western blot were adopted to detect the mRNA and protein expression of HMGB1,TGF-β1,Smad3 andα-SMA in conjunctival tissues.RESULTS:HE staining and Masson staining showed that the proliferation of inflammatory cells,fibroblasts and collagen fibers in the model group was significantly higher than that in the control group at both 4 and 8 wk.Meanwhile,the proliferation of fibroblasts and collagen fibers in the model with drug administration group was significantly lower than that in the model group.Immunohistochemical staining showed that the expression of HMGB1,TGF-β1,Smad3 andα-SMA protein was observed in the conjunctival tissues of the model group both 4 and 8 wk,with brown and significantly deeper staining of the model group at 8 wk.Meanwhile,the positive staining in the model with drug administration group at both 4 and 8 wk was significantly lower than that in the model group.There was positive correlations between the number of fibroblasts stained with HE and the expression of HMGB1 in the conjunctival tissue of the model group at both 4 and 8 wk(r=0.602,0.703,all P<0.05).RT-PCR and Western blot revealed that the mRNA and protein expression levels of HMGB1,TGF-β1,Smad3 andα-SMA in the model group were significantly higher than those in the control group at both 4 and 8 wk(all P<0.05).Meanwhile,the mRNA and protein expression levels of HMGB1,TGF-β1,Smad3 andα-SMA in the model with drug administration group were significantly lower than those in the model group(all P<0.05).There was positive correlations between mRNA expressions of HMGB1 and TGF-β1,Smad3 in the model group and the model with drug administration group(all P<0.05).CONCLUSION:The expression of HMGB1 increased at a time-dependent manner after glaucoma valve implantation.HMGB1 acts an indispensable role in the initiation and progression of scar formation after glaucoma surgery,which may be involved in the regulation of TGF-β/Smad signaling pathway.