背景:大建中汤(DJZ)是中医治疗阳虚证的经典名方,记载于东汉朝的金奎要略。认知衰老可以表现出与阳虚的临床特征相似的线粒体能量缺乏特征。然而,对DJZ治疗对认知衰老中线粒体自噬的影响了解甚少。
目的:这项工作的目的是破译DJZ对抗认知衰老的有效性和机制,专注于线粒体自噬。
方法:YFP-ParkinHeLa细胞,D-半乳糖(D-gal)诱导小鼠(500mg/kg,持续35d,s.c.)和SH-SY5Y细胞(80mg/ml,持续6h)。首先,利用YFP-ParkinHeLa细胞中YFP-Parkinpuncta(一种众所周知的线粒体自噬标记)的形成来发现DJZ的线粒体自噬诱导。此外,在体内用DJZ处理后评估与PINK1/Parkin途径和线粒体功能相关的基因和蛋白(3.5g/kg或1.75g/kg,i.g,35d)和体外(0.2、2和20μg/ml,12h).此外,DJZ的有效性(3.5g/kg或1.75g/kg,在D-gal小鼠中测量用于减轻认知老化和神经损伤的i.g)。最后,siPINK1用于DJZ的体外反向验证。
结果:DJZ以剂量依赖性方式显著诱导YFP-ParkinHeLa细胞中YFP-Parkin点的形成。DJZ治疗后D-gal小鼠Parkin免疫荧光强度和线粒体膜Parkin蛋白表达均显著升高。DJZ显著激活了D-gal诱导的小鼠和SH-SY5Y细胞中PINK1/Parkin通路的抑制作用。同时,DJZ明显逆转了D-gal诱导的线粒体功能损伤。此外,DJZ可显着改善D-gal小鼠的神经病理学损伤和认知能力下降。最后,PINK1在体外被siPINK1敲低后,DJZ的神经保护作用和DJZ的Parkin增强作用明显逆转。
结论:我们的发现首先表明DJZ可以通过促进PINK1/Parkin介导的线粒体自噬来缓解认知老化,从而保护线粒体功能,表明DJZ可能被认为是认知衰老的有希望的干预措施。
BACKGROUND: Dajianzhong decoction (DJZ) is a classical famous formula for treating yang-deficiency-syndrome in traditional Chinese medicine and recorded in Jin-Kui-Yao-Lue in Dynasty of Dong Han. Cognitive aging can present similar features of mitochondrial energy deficits to the clinical features of Yang deficiency. However, there is poor understanding of the effects of DJZ treatment on mitophagy in cognitive aging.
OBJECTIVE: The aims of this work were to decipher the effectiveness and mechanism of DJZ against cognitive aging, focusing on mitophagy.
METHODS: YFP-Parkin HeLa cells, D-galactose (D-gal) -induced mice (500 mg/kg for 35 d, s. c.) and SH-SY5Y cells (80 mg/ml for 6 h) were established. Firstly, the formation of YFP-Parkin puncta (a well-known mitophagy marker) in YFP-Parkin HeLa cells was employed to discover the mitophagy induction of DJZ. Moreover, the genes and proteins related to PINK1/Parkin pathway and mitochondrial functions were evaluated after treatment with DJZ in vivo (3.5 g/kg or 1.75 g/kg, i. g, 35 d) and in vitro (0.2, 2 and 20 μg/ml, 12 h). Furthermore, the effectiveness of DJZ (3.5 g/kg or 1.75 g/kg, i. g) for alleviating cognitive aging and nerve damage was measured in D-gal mice. Finally, siPINK1 was applied to reverse validation of DJZ in vitro.
RESULTS: The formation of YFP-Parkin puncta in YFP-Parkin HeLa cells was markedly induced by DJZ in a dose-dependent manner. The immunofluorescence intensity of Parkin and the protein expression of Parkin in mitochondrial membrane in D-gal mice were significantly increased after treatment of DJZ. The inhibition of PINK1/Parkin pathway in D-gal-induced mice and SH-SY5Y cells was significantly activated by DJZ. Simultaneously, the impairment of mitochondrial functions induced by D-gal were markedly reversed by DJZ. In addition, DJZ significantly ameliorated the neuropathological injury and cognitive declines in D-gal mice. Finally, after PINK1 was knocked down by siPINK1 in vitro, the neuroprotective effects of DJZ and the Parkin enhancement effect of DJZ were markedly reversed.
CONCLUSIONS: Our findings firstly showed DJZ could relieve cognitive aging through facilitating PINK1/Parkin-mediated mitophagy to protect against mitochondrial functions, indicating DJZ may be regarded as a promising intervention in cognitive aging.