Mechanism by Which Alcohol Extract from Gymnadenia orchidis Delays Aging in Caenorhabditis elegans via the Antioxidant Pathway

To evaluate the anti-aging activity and mechanism of alcohol extract from Gymnadenia orchidis (GoAE), we prepared GoAE from G. orchidis tubers from Jiulong County, Sichuan Province and investigated the effects of different concentrations of GoAE (0, 0.5, 1.0, and 2.0 mg/mL) and its active component gastrodin (Gas) on the lifespan, pharyngeal pumping rate, locomotion frequency, body size (length and width), and fecundity of Caenorhabditis elegans (strain N2). A nematode model of mitochondrial oxidative damage was established using 20 μmol/L rotenone (RO). Physiological and biochemical techniques, fluorescence microscopy, transmission electron microscopy (TEM), and polymerase chain reaction (PCR) were used to investigate the changes in the antioxidant system, mitochondrial number, morphology, membrane potential, membrane proteins, and related gene expression in oxidatively damaged nematodes following 2.0 mg/mL GoAE intervention. The results showed that GoAE and Gas significantly extended nematode lifespan and markedly increased pharyngeal pumping rate, locomotion frequency, body length, and body width (except for body width in the Gas treatment group), while they minimally affected fecundity; the effects of GoAE were significantly superior to those of Gas. Under GoAE intervention, the excessive levels of reactive oxygen species (ROS) and malondialdehyde (MDA) content induced by RO significantly decreased, superoxide dismutase (SOD) and catalase activities significantly increased, and the expression levels of SOD-3 and heat shock protein (HSP)-16.2, as well as those of the genes daf-16, sod-1, sod-3, ctl-1, and hsp-16.2, were significantly upregulated. Furthermore, RO-induced mitochondrial dysfunction was significantly ameliorated, as evidenced by increased mitochondrial number, decreased mitochondrial vacuolation, increased membrane potential, and upregulated expression levels of mitochondrial membrane-related genes (gas-1, clk-1, isp-1, and atp-2) as well as inner and outer membrane proteins. Additionally, the expression levels of mitochondrial fusion genes (eat-3 and fzo-1) and autophagy genes (unc-51, atg-7, bec-1, lgg-1, and vps-34) were upregulated, while the expression of the fission gene drp-1 was downregulated. These results indicate that the GoAE extract can delay aging in C. elegans by activating the DAF-16/SOD-3 antioxidant pathway and ameliorating mitochondrial dysfunction.