硫化氢介导TGF-β1信号通路在早产儿视网膜病变血管损伤中的作用

Role of Hydrogen Sulfide Mediated Signaling Pathway of Transforming Growth Factor β1 in Retinopathy of Prematurity

目的探讨硫化氢(H2S)在早产儿视网膜病变(ROP)血管损伤中的作用及其机制。方法建立大鼠高氧/低氧诱导ROP模型,HE染色观察ROP模型视网膜血管组织变化,ELISA法检测ROP模型血管组织胱硫醚γ裂解酶活性变化,免疫组化法及Western blot法检测ROP模型视网膜血管组织TGF-β1表达情况。建立高氧/低氧刺激人脐静脉内皮细胞模型,MTT法检测人脐静脉内皮细胞(HUVEC)增殖情况,Transwell实验检测HUVEC迁移能力,Western blot法检测HUVEC中TGF-β1表达情况。结果体内模型中ROP组HE染色切片中突破视网膜内界膜的血管内皮细胞较对照组多。ROP组视网膜血管组织H2S生成高于对照组,差异具有统计学意义(P<0.05)。免疫组化检测显示ROP组视网膜血管组织TGF-β1表达较对照组少。Western blot法检测示ROP组视网膜血管组织中TGF-β1、pSmad 2/3表达水平较对照组低,差异具有统计学意义(均P<0.05)。ROP组HUVEC生存率低于对照组HUVEC。对照组中采用NaHS处理的HUVEC细胞生存率高于未经处理的细胞,ROP组中采用NaHS处理的HUVEC细胞生存率高于未经处理的细胞,差异具有统计学意义(均P<0.05)。ROP组HUVEC迁移能力低于对照组。对照组中经NaHS处理后的迁移细胞数高于未经处理的细胞数,ROP组中经NaHS处理后的迁移细胞数高于未经处理的细胞,差异具有统计学意义(均P<0.05)。ROP组HUVEC中TGF-β1、pSmad 2/3表达水平低于对照组。对照组中经NaHS处理后的TGF-β1、pSmad 2/3表达水平低于未经处理的细胞,ROP组中经NaHS处理后的TGF-β1、pSmad 2/3表达水平低于未经处理的细胞,差异具有统计学意义(均P<0.05)。结论大鼠ROP模型中血管组织H2S生成量较高,TGF-β1及其下游信号Smad 2/3表达水平降低。体外细胞实验提示外源性H2S对正常及高氧/低氧造成的HUVEC损伤具有一定的保护作用,能够增强正常及高氧/低氧HUVEC的迁移能力,下调正常及高氧/低氧HUVEC中TGF-β1及其下游信号Smad 2/3表达水平,H2S可能通过介导TGF-β1信号通路参与视网膜血管病变早期的血管发育。

Objective The objective of this study is to investigate the effect and mechanism of hydrogen sulfide(H2S)on vascular damage of retinopathy of prematurity(ROP).Methods The hyperoxia/hypoxia induced retinopathy rat model was established.Hematoxylin-eosin(HE)staining was applied to observe the retinal vascular tissue change of the ROP model.The change of cystathionine-gamma-lyase in vascular tissue was determined by using the method of enzyme linked immunosorbent assay(ELISA).The transforming growth factorβ1(TGF-β1)expression was tested with immunohistochemistry and Western blotting in retinal vascular tissues of the ROP model.The hyperoxia/hypoxia human umbilical vein endothelial cells(HUVEC)stimulating model was established.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)was used to determine the proliferation of HUVEC,the immigration ability of HUVEC was tested with Transwell,and the expression of TGF-β1 was tested with immunohistochemistry and Western blotting.Results The vascular endothelial cells that break through the internal limiting membrane of retina in HE staining slice of ROP group in in vivo model were more than those in the control group.In ROP group,H2S production in retinal vascular tissue was higher than that of the control group(P<0.05).The expression levels of TGF-β1 and pSmad 2/3 in ROP group retinal vascular tissue were less than those of the control group(P<0.05).The survival rate of HUVEC in ROP group was less than that of the control group.The survival rate of HUVEC in ROP control group using NaHS was higher than that of the cells untreated(P<0.05).The immigration ability of HUVEC in ROP group was lower than that of the control group.The immigrated cell count in the control group and ROP group treated with NaHS was higher than that of untreated cells(P<0.05).Expression levels of TGF-β1 and pSmad 2/3 of HUVEC in ROP group were lower than those in the control group,the expression levels of TGF-β1 and pSmad 2/3 treated with NaHS in the control group were lower than those of the cells untreated,and the expression levels of TGF-β1 and pSmad 2/3 in ROP group were lower than those of the cells untreated(P<0.05).Conclusion The H2S production in vascular tissue of the ROP rat model is higher,and the expression levels of downstream signal Smad 2/3 and TGF-β1 are lower.In vivo cell experiment indicates that exogenous H2S plays a role in protecting HUVEC from being damaged under normal and hyperoxia/hypoxia circumstances,and can enhance the immigration ability of HUVEC,down-regulate the expression levels of TGF-β1 and Smad 2/3 of HUVEC.H2S may participate in the early pathology stage of vascular development in retina through mediation of TGF-β1 pathway.