RGD多肽水凝胶对Tenon囊成纤维细胞活化和YAP信号的影响

Effects of arginine-glycine-aspartic acid peptide hydrogels on the activation of Tenon capsule fibroblasts and Yes-associated protein signaling

目的 探究不同浓度的精氨酸-甘氨酸-天冬氨酸(RGD)多肽水凝胶,对Tenon囊成纤维细胞(HTF)活化和Yes相关蛋白(YAP)表达的影响。方法 制备0.5%、1.0%、2.0%三种浓度RGD多肽水凝胶,透射电子显微镜观察其内部微观结构,流变学检测弹性模量(E)。构建SD雄性大鼠结膜损伤模型后,分为空白组(不作处理)、低浓度RGD组(结膜下注射0.5%RGD多肽水凝胶)、中浓度RGD组(结膜下注射1.0%RGD多肽水凝胶)、高浓度RGD组(结膜下注射2.0%RGD多肽水凝胶),观察1周后取材,Masson染色观察大鼠结膜处胶原纤维增生情况,免疫组织化学检测大鼠结膜YAP、平滑肌肌动蛋白-α(α-SMA)表达情况。利用转化生长因子-β2(TGF-β2)刺激HTF活化构建瘢痕化细胞模型,按照实验要求分为对照组、 0.5%RGD水凝胶组、1.0%RGD水凝胶组、2.0%RGD水凝胶组、正常组,培养24 h后,Western blot检测HTF中YAP、结缔组织生长因子(CTGF)、α-SMA、纤连蛋白(FN)和I型胶原蛋白(Col I)的蛋白相对表达量;激光共聚焦显微镜观察细胞骨架蛋白(F-actin)和YAP蛋白表达定位。结果 电镜下观察可见,RGD多肽水凝胶内部纳米纤维交联密度随多肽浓度增加而升高,流变学测得0.5%、1.0%及2.0%RGD多肽水凝胶弹性模量(E)依次约为0.067 kPa、0.150 kPa、2.170 kPa。Masson染色结果显示,除中浓度RGD组大鼠结膜胶原纤维增生面积比明显低于空白组外(P<0.05),低浓度组、高浓度组与空白组差异均无统计学意义(均为P>0.05)。免疫组织化学结果显示,与空白组相比,低浓度RGD组、中浓度RGD组、高浓度RGD组大鼠YAP及α-SMA蛋白相对表达量均下降,中浓度RGD组下降最明显,差异均有统计学意义(均为P<0.05)。细胞免疫荧光染色结果显示,与对照组相比,0.5%RGD水凝胶组、1.0%RGD水凝胶组、2.0%RGD水凝胶组HTF的F-actin绿色荧光减弱,YAP红色荧光从细胞核转位于细胞质表达。Western blot检测结果显示,与对照组相比,0.5%RGD水凝胶组、1.0%RGD水凝胶组、2.0%RGD水凝胶组HTF中YAP、CTGF、α-SMA、FN及Col I蛋白相对表达量均下降,1.0%RGD水凝胶组下降最明显,差异均有统计学意义(均为P<0.05)。结论 RGD多肽水凝胶的最佳作用浓度为1.0%,其通过抑制YAP蛋白的表达水平及其核转位,减少HTF的活化及其纤维化蛋白的表达,从而产生抗瘢痕效果。

Objective To investigate the effects of arginine-glycine-aspartic acid(RGD)peptide hydrogels at different concentrations on the activation of Tenon capsule fibroblasts(HTF)and the expression of Yes-associated protein(YAP).Methods Three concentrations(0.5%,1.0%,2.0%)of RGD peptide hydrogels were prepared.Their internal microstructures were observed under a transmission electron microscope,and their elastic moduli(E)were measured using the rheological test.SD male rat models with conjunctival injury were established and divided into a blank group(no treatment),a low-concentration RGD group(subconjunctival injection of 0.5%RGD peptide hydrogel),a medium-concentration RGD group(subconjunctival injection of 1.0%RGD peptide hydrogel),and a high-concentration RGD group(subconjunctival injection of 2.0%RGD peptide hydrogel).After one week,tissues were collected,and collagen fiber proliferation in the rat conjunctiva was observed after Masson staining.Immunohistochemistry was performed to detect the expression of YAP andα-smooth muscle actin(α-SMA)in the rat conjunctiva.Scar formation cell models were constructed by stimulating HTF activation with transforming growth factor-β2(TGF-β2).According to experimental requirements,the cells were divided into a control group,a 0.5%RGD hydrogel group,a 1.0%RGD hydrogel group,a 2.0%RGD hydrogel group,and a normal group,and co-cultured for 24 hours.The relative expression levels of YAP,connective tissue growth factor(CTGF),α-SMA,fibronectin(FN),and type I collagen(Col I)proteins in HTF were detected by Western blot.The localization of cytoskeletal protein(F-actin)and YAP protein expression was observed under a confocal laser scanning microscope.Results Electron microscopy showed that the crosslinking density of the nanofibers inside the RGD peptide hydrogel increased with the rise of the peptide concentration.Rheological measurements indicated that the E values of the 0.5%,1.0%,and 2.0%RGD peptide hydrogels were approximately 0.067 kPa,0.150 kPa,and 2.170 kPa,respectively.Masson staining results revealed that the area of collagen fiber proliferation in the conjunctiva of rats in the medium-concentration RGD group was significantly lower than that in the blank group(P<0.05),while it showed no significant difference in the low-concentration and high-concentration RGD groups compared with the blank group(both P>0.05).Immunohistochemistry results showed that the relative expression levels of YAP andα-SMA proteins in the conjunctiva of rats in the low-,medium-,and high-concentration RGD groups decreased compared to the blank group,and the decrease was the most significant in the medium-concentration RGD group(all P<0.05).Cell immunofluorescence staining results indicated that the green fluorescence of F-actin was attenuated,and the red fluorescence of YAP was translocated from the nucleus to the cytoplasm in the 0.5%,1.0%,and 2.0%RGD hydrogel groups compared to the control group.Western blot results showed that the relative expression levels of YAP,CTGF,α-SMA,FN,and Col I proteins in HTF were lower in the 0.5%,1.0%,and 2.0%RGD hydrogel groups compared to the control group,with the 1.0%RGD hydrogel group showing the most significant reduction(all P<0.05).Conclusion With the optimal concentration of 1.0%,the RGD peptide hydrogel can inhibit YAP protein expression and nuclear translocation and reduce HTF activation and the expression of fibrotic proteins,thus alleviating scar formation.