Table_9_TGF-β2 Induces Gli1 in a Smad3-Dependent Manner Against Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.XLS

Isoflurane (ISO) post-conditioning attenuates cerebral ischemia/reperfusion (I/R) injury, but the underlying mechanism is incompletely elucidated. Transforming growth factor beta (TGF-β) and hedgehog (Hh) signaling pathways govern a wide range of mechanisms in the central nervous system. We aimed to investigate the effect of the TGF-β2/Smad3 and sonic hedgehog (Shh)/Glioblastoma (Gli) signaling pathway and their crosstalk in the hippocampus of rats with ISO post-conditioning after cerebral I/R injury. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), 1.5 h occlusion and 24 h reperfusion (MCAO/R). To assess the effect of ISO after I/R injury, various approaches were used, including neurobehavioral tests, TTC staining, HE staining, Nissl staining, TUNEL staining, immunofluorescence (IF), qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot. The ISO post-conditioning group (ISO group) received 1 h ISO post-conditioning when reperfusion was initiated, leading to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons. IF staining, qRT-PCR and Western blot showed high expression levels of TGF-β2, Shh and Gli1 in the hippocampal CA1 of the ISO group. Phosphorylated Smad3 (p-Smad3), Patched (Ptch), and Smoothed (Smo) were also increased at protein level in the ISO group, whereas total Smad3 expression did not change in all groups. When TGF-β2 inhibitor, pirfenidone, or Smad3 inhibitor, SIS3 HCl, were administered, the expression levels of p-Smad3 and Gli1 were reduced, and surviving pyramidal neurons decreased. By contrast, the expression levels of TGF-β2 and p-Smad3 did not change significantly after pre-injection of Smo inhibitor cyclopamine, but reduced the expression levels of Shh, Ptch, and Gli1. Moreover, Gli showed the lowest expression levels with pirfenidone combined with cyclopamine. These findings indicate that the TGF-β and hedgehog signaling pathways mediate the neuroprotection of ISO post-conditioning after cerebral I/R injury, and crosstalk between two pathways at the Gli1 level.

异氟醚（ISO）的后期处理能够减轻脑缺血/再灌注（I/R）损伤，但其潜在的机制尚不明确。转化生长因子β（TGF-β）和 hedgehog（Hh）信号通路在中央神经系统中调控着一系列机制。本研究旨在探讨TGF-β2/Smad3和 sonic hedgehog（Shh）/胶质母细胞瘤（Gli）信号通路及其在脑缺血/再灌注损伤后异氟醚后期处理大鼠海马中的相互作用。成年雄性Sprague-Dawley大鼠接受中脑动脉闭塞（MCAO）手术，进行1.5小时的闭塞和24小时的再灌注（MCAO/R）。为评估异氟醚在I/R损伤后的效果，采用了多种方法，包括神经行为测试、TTC染色、HE染色、尼氏染色、TUNEL染色、免疫荧光（IF）、定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹（Western blot）。异氟醚后期处理组（ISO组）在再灌注启动时接受1小时的异氟醚后期处理，结果显示梗死体积和神经功能障碍评分降低，存活神经元增多，受损和凋亡神经元减少。IF染色、qRT-PCR和Western blot显示，ISO组海马CA1区TGF-β2、Shh和Gli1的表达水平升高。磷酸化Smad3（p-Smad3）、Patched（Ptch）和Smoothed（Smo）在ISO组蛋白质水平上也有所增加，而总Smad3表达在所有组别中均无变化。当给予TGF-β2抑制剂吡非尼酮或Smad3抑制剂SIS3 HCl时，p-Smad3和Gli1的表达水平降低，存活锥体神经元减少。相反，在预先注射Smo抑制剂环孢素后，TGF-β2和p-Smad3的表达水平没有显著变化，但Shh、Ptch和Gli1的表达水平降低。此外，在吡非尼酮与环孢素联合使用时，Gli的表达水平最低。这些发现表明，TGF-β和hedgehog信号通路介导了异氟醚后期处理在脑缺血/再灌注损伤后的神经保护作用，并且两通路在Gli1水平上存在相互作用。