川芎嗪在高血糖加重脑缺血损伤和诱发卒中后癫痫发作中的治疗作用和分子机制研究

It’s well documented that hyperglycemia exacerbates ischemic brain damage and induces post-ischemic epileptic seizures. The mechanisms are not fully understood. Our previous and preliminary studies have revealed that hyperglycemic ischemia activated mTOR signaling pathway, increased Toll-like Receptor4(TLR4), and inhibition of mTOR by rapamycin significantly reduced hyperglycemia-exacerbated ischemic brain damage and prevented post-ischemia seizures. These effects were associated with inhibition of TLR4 and increase of Kv4.2, a subtype of potassium out flow ion channel that inhibits the neuronal excitability. We hypothesize that the mTOR, which is activated by hyperglycemic ischemia, further activates TLR4. TLR4 causes increased releases of inflammation cytokines and suppression of Kv4.2, which eventually lead to increased brain damage and development of post-ischemic seizures. We propose to study the role of TLR4 and Kv4.2 in cerebral ischemia under hyperglycemic condition by employing neuro-behavioral studies, histopathology, immunohistochemistry, cytokine array， patch-clamp on cultured brain slides and specific inhibitors that target the TLR4. We also examine the effects of Chinese herbal medicine-Chuanxiong extract Tetramethylpyraziue(TMP) on ischemic brain damage and post-ischemic seizures in hyperglycemic animals and its effects on TLR4 and Kv4.2..This study will define the role of TLR4-Kv4.2 in hyperglycemia-enhanced ischemic brain damage and post-ischemic seizures. We will also explore the effects of TMP on ischemic brain damage and post-ischemic seizures and its mechanisms of action.

高血糖加重脑缺血损伤并诱发卒中后癫痫。前期实验发现mTOR激活介导高糖诱发的脑缺血损害及卒中后癫痫，但mTOR抑制导致胰岛素分泌减少，故明确mTOR下游分子靶标并干预非常必要。mTOR能激活TLR4，后者促进NF-κB核转移，增加TNF-α、IL-1β等释放。TLR4激活、Kv4.2功能异常介导癫痫发作。TNF-α、IL-1β等下调海马神经元Kv4.2表达。前期实验发现大鼠高糖脑缺血后海马TLR4上调、Kv4.2下调；抑制mTOR则TLR4下调、Kv4.2上调。本课题验证以下假说：高糖通过激活mTOR进而激活TLR4，诱发炎症,加重脑缺血损伤；mTOR、TLR4激活导致Kv4.2下调参与卒中后癫痫。我们将采用大鼠高糖脑缺血再灌模型，结合药物干预、病理、炎性因子阵列、蛋白印迹、膜片钳等验证以上假说，并检验川芎嗪对TLR4和Kv4.2的影响及对高糖脑缺血的作用。深化高糖加重脑缺血损伤机制研究。

我们在前期研究中发现：1. 哺乳动物雷帕霉素靶蛋白(mTOR)通路的激活在糖尿病加重脑缺血损伤和脑卒中后癫痫发作的过程中起重要作用。2. 高血糖通过引起缺血区域炎症因子的释放而加重脑缺血再灌注(ischemia/reperfusion, I/R)损伤。3. 川芎嗪(Tetramethylpyrazine, TMP)具有抗炎作用。因此，我们推测高血糖激活mTOR信号通路，进而激活TLR4-NF-κB炎症通路，加重大鼠脑I/R损伤及介导大鼠脑缺血后癫痫发作；TMP可能通过抑制炎症来缓解脑缺血损伤。为了证实这一假设，我们完成了以下研究: 1. 建立大鼠大脑中动脉脑I/R模型，给予TMP干预，通过测定脑含水量及病理、Nissl染色和TUNEL染色等方法，发现糖尿病大鼠脑I/R后脑损伤明显加重，而TMP治疗后神经元受损减轻。行全长转录组测序并对其结果进行GO和KEGG富集分析后使用免疫组化、RT-PCR和WB等对测序结果进行验证，发现TLR2、MyD88、p-NF-κB、p65、IL-1β、IL-6的mRNA及蛋白表达水平及Cleaved-Caspase-3、Bcl-2的mRNA及蛋白表达水平在糖尿病大鼠脑I/R后增高，而川芎嗪干预可逆转以上分子的表达，提示川芎嗪可能通过抑制TLR2/MyD88/NF-κB炎症通路及降低凋亡水平发挥神经保护作用。2. 采用高糖干预HT22细胞并构建细胞缺氧／复氧模型（OGD/R），结果发现高糖可显著加重神经元缺氧再灌注损伤，破坏细胞线粒体功能，激活细胞线粒体自噬；Sirt3可通过抑制PINK1/Parkin通路介导的线粒体自噬减轻神经元高糖缺氧再灌注损伤。3. 成功建立糖尿病大鼠前脑I/R模型及稳定的缺血后癫痫发作模型，通过采用雷帕霉素干预、脑电监测以及形态学检测、全长转录组测序、分子生物学等方法，发现糖尿病大鼠在脑I/R后痫性发作率达到100%，cEEG脑电波形图可见明显棘波出现，磷酸化mTOR及其下游分子P70S6K和S6的磷酸化水平在脑I/R后显著升高，而雷帕霉素干预后，上述指标磷酸化水平显著下降，同时脑组织损伤显著减轻，痫性发作被明显抑制。对转录组结果进行分析和验证，发现糖尿病大鼠卒中后癫痫发作可能与mTOR/MAPK通路和钙离子通道蛋白Cav2.3激活有关，而雷帕霉素处理可抑制以上通路及分子的表达。