抑制Sonic Hedgehog信号能抑制脑缺血性损伤后纤维瘢痕形成且不利于神经功能恢复

Inhibition of Sonic Hedgehog signaling inhibits fibrous scar formation and adversely affects functional outcome after ischemic brain injury in rats

目的探讨抑制Sonic Hedgehog(Shh)信号对脑缺血性损伤后纤维瘢痕形成及神经功能的影响。方法构建体内成年SD大鼠大脑中动脉闭塞/再灌注(MCAO/R)模型和体外新生24 h SD大鼠脑膜原代成纤维细胞氧糖剥夺/再复氧(OGD/R)模型。成年SD大鼠随机分成假手术组、缺血/再灌注组(I/R)、缺血再灌注腺病毒空载(rAd-NC)组和缺血再灌注腺病毒敲低Shh(rAd-ShShh)组。SD大鼠脑膜原代成纤维细胞随机分成正常组、缺血对照组、肿瘤生长因子-β1预处理组(TGF-β1)、TGF-β1预处理加环王巴明组(TGF-β1+CYC)。每组细胞被预处理24 h经OGD/R 150 min后复氧72 h。采用改良神经严重程度评分(mNSS)、改良Bederson评分评估神经功能缺损。CCK-8法测定细胞增殖,划痕实验检测细胞迁移,免疫荧光法检测缺血中心纤维连接蛋白(Fn)、α-平滑肌肌动蛋白(α-SMA)、Shh的表达,TUNEL染色检测缺血中心细胞凋亡,实时荧光定量PCR(qPCR)法检测缺血中心Fn、α-SMA及Shh mRNA的表达,WB法检测OGD/R后成纤维细胞Shh、α-SMA蛋白的表达。结果体内实验中免疫荧光法及qPCR法结果显示与假手术组相比SD大鼠MCAO/R后可诱导缺血侧大脑半球Shh、α-SMA、Fn蛋白和mRNA的表达升高(P<0.05)。而与MCAO/R组相比,rAd-Shshh组缺血侧大脑半球Shh、α-SMA、Fn蛋白和mRNA的表达降低(P<0.05),缺血侧组织细胞的凋亡增加(P<0.05),改良神经严重程度评分(mNSS)、改良Bederson评分增高(P<0.05)。体外研究中CCK-8实验显示与正常组和缺血对照组相比TGF-β1组A值升高(P<0.05),而TGF-β1+CYC组与TGF-β1组相比A值没有明显差异(P>0.05)。细胞划痕实验结果显示与正常组和缺血对照组相比在24 h内TGF-β1组成纤维细胞的迁移距离增加(P<0.05),而TGF-β1+CYC组与TGF-β1组相比成纤维细胞的迁移距离减少(P<0.05)。免疫荧光实验、Westernblot及qPCR显示与正常组和缺血对照组相比TGF-β1组细胞Shh、α-SMA和Fn的表达升高(P<0.05),而TGF-β1+CYC组与TGF-β1组相比Shh、α-SMA和Fn的表达降低(P<0.05)。结论抑制Shh信号能抑制脑缺血性损伤早期纤维瘢痕的形成且不利于神经功能的恢复。

Objective To investigate the effects of inhibiting Sonic Hedgehog(Shh)signaling on fibrous scar formation and functional outcome after ischemic brain injury.Methods Adult SD rats were randomized into sham-operated group,middle cerebral artery occlusion(MCAO)and reperfusion(I/R)group,I/R with intraventricular empty adenoviral vector(rAd-NC)injection group,and I/R with adenovirus-mediated Shh knockdown(rAd-ShShh)group.After the treatments,the neurological deficits of the rats were assessed,and the protein and mRNA expressions of fibronectin(Fn),α-SMA,and Shh in the ischemic hemisphere were detected with immunofluorescence assay and qPCR;TUNEL staining was used for detecting neural cell apoptosis.In the cell experiment,primary meningeal fibroblasts isolated from neonatal SD rats were pretreated for 24 h with TGF-β1 or TGF-β1 plus cyclopamine(CYC)before oxygen-glucose deprivation for 150 min followed by reoxygenation for 72 h(OGD/R).CCK-8 assay and scratch test were performed to examine the changes in cell proliferation and migration,and immunofluorescence assay,qPCR and Western blotting were used for detecting cell transformation and the expressions of Shh,α-SMA,and Fn.Results Cerebral I/R injury significantly increased the protein and mRNA expressions of Shh,α-SMA,and Fn in the ischemic hemisphere of the rats,but their expression levels were significantly lowered by intraventricular injection of rAd-Shshh(P<0.05),which obviously increased cell apoptosis in the ischemic hemisphere(P<0.05)and improved modified mNSS and modified Bederson scores of the rats(P<0.05).In the cell experiment,pretreatment with TGF-β1 and TGF-β1+CYC both increased the viability of the primary meningeal fibroblasts after OGD/R.TGF-β1 significantly enhanced the migration ability and induced obvious transformation of the exposed cells(P<0.05),but these effects were significantly attenuated by co-treatment with CYC(P<0.05).The expressions of Shh,α-SMA and Fn in the TGF-β1 group were all significantly higher in TGF-β1-treated cells(P<0.05)and were obviously lowered by co-treatment with CYC(P<0.05).Conclusion Inhibition of Shh signaling may inhibit fibrous scar formation and functional recovery in rats after ischemic brain injury.