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Abstract：
Gastrodin (GAS) is the main chemical component of the traditional Chinese herb Gastrodia elata (called \"Tianma\" in Chinese), which has been used to treat neurological conditions, including headaches, epilepsy, stroke, and memory loss. To our knowledge, it is unclear whether GAS has a therapeutic effect on Huntington\'s disease (HD). In the present study, we evaluated the effect of GAS on the degradation of mutant huntingtin protein (mHtt) by using PC12 cells transfected with N-terminal mHtt Q74. We found that 0.1-100 μM GAS had no effect on the survival rate of Q23 and Q74 PC12 cells after 24-48 h of incubation. The ubiquitin-proteasome system (UPS) is the main system that clears misfolded proteins in eukaryotic cells. Mutated Htt significantly upregulated total ubiquitinated protein (Ub) expression, decreased chymotrypsin-like, trypsin-like and caspase-like peptidase activity, and reduced the colocalization of the 20S proteasome with mHtt. GAS (25 μM) attenuated all of the abovementioned pathological changes, and the regulatory effect of GAS on mHtt was found to be abolished by MG132, a proteasome inhibitor. The autophagy-lysosome pathway (ALP) is another system for misfolded protein degradation. Although GAS downregulated the expression of autophagy markers (LC3II and P62), it increased the colocalization of LC3II with lysosomal associated membrane protein 1 (LAMP1), which indicates that ALP was activated. Moreover, GAS prevented mHtt-induced neuronal damage in PC12 cells. GAS has a selective effect on mHtt in Q74 PC12 cells and has no effect on Q23 and proteins encoded by other genes containing long CAGs, such as Rbm33 (10 CAG repeats) and Hcn1 (>30 CAG repeats). Furthermore, oral administration of 100 mg/kg GAS increased grip strength and attenuated mHtt aggregates in B6-hHTT130-N transgenic mice. This is a high dose (100 mg/kg GAS) when compared with experiments on HD mice with other small molecules. We will design more doses to evaluate the dose-response relationship of the inhibition effect of GAS on mHtt in our next study. In summary, GAS can promote the degradation of mHtt by activating the UPS and ALP, making it a potential therapeutic agent for HD.
摘要：
天麻素（GAS）是传统中草药天麻（中文称为“天麻”）的主要化学成分，用于治疗神经系统疾病，包括头痛,癫痫,中风,和记忆丧失。据我们所知,目前尚不清楚GAS是否对亨廷顿病（HD）有治疗作用。在本研究中,我们使用N端mHttQ74转染的PC12细胞评估了GAS对突变亨廷顿蛋白（mHtt）降解的影响。我们发现0.1-100μMGAS在孵育24-48小时后对Q23和Q74PC12细胞的存活率没有影响。泛素-蛋白酶体系统（UPS）是清除真核细胞中错误折叠蛋白的主要系统。突变的Htt显著上调总泛素化蛋白（Ub）表达，减少胰凝乳蛋白酶样，胰蛋白酶样和半胱天冬酶样肽酶活性，并降低了20S蛋白酶体与mHtt的共定位。GAS（25μM）减弱了所有上述病理变化，发现GAS对mHtt的调节作用被蛋白酶体抑制剂MG132消除。自噬-溶酶体途径(ALP)是另一个错误折叠蛋白降解系统。尽管GAS下调了自噬标志物（LC3II和P62）的表达，它增加了LC3II与溶酶体相关膜蛋白1（LAMP1）的共定位，这表明ALP被激活。此外，GAS可预防mHtt诱导的PC12细胞神经元损伤。GAS对Q74PC12细胞中的mHtt具有选择性作用，对Q23和含有长CAGs的其他基因编码的蛋白质没有影响，例如Rbm33(10个CAG重复)和Hcn1(>30个CAG重复)。此外，口服100mg/kgGAS可增加B6-hHTT130-N转基因小鼠的握力并减弱mHtt聚集体。当与具有其他小分子的HD小鼠的实验相比时,这是一个高剂量(100mg/kgGAS)。我们将在下一个研究中设计更多的剂量来评估GAS对mHtt抑制作用的剂量-反应关系。总之,GAS可以通过激活UPS和ALP来促进mHtt的降解，使其成为HD的潜在治疗剂。
