Chemico-Biological Interactions. 2015 Oct;240:12-21.

Hydrogen-rich water attenuates amyloid β-induced cytotoxicity through upregulation of Sirt1-FoxO3a by stimulation of AMP-activated protein kinase in SK-N-MC cells.

Lin CL, Huang WN, Li HH, Huang CN, Hsieh S, Lai C, Lu FJ

Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan.



Amyloid β (Aβ) peptides are identified in cause of neurodegenerative diseases such as Alzheimer’s disease (AD). Previous evidence suggests Aβ-induced neurotoxicity is linked to the stimulation of reactive oxygen species (ROS) production. The accumulation of Aβ-induced ROS leads to increased mitochondrial dysfunction and triggers apoptotic cell death. This suggests antioxidant therapies may be beneficial for preventing ROS-related diseases such as AD. Recently, hydrogen-rich water (HRW) has been proven effective in treating oxidative stress-induced disorders because of its ROS-scavenging abilities. However, the precise molecular mechanisms whereby HRW prevents neuronal death are still unclear. In the present study, we evaluated the putative pathways by which HRW protects against Aβ-induced cytotoxicity. Our results indicated that HRW directly counteracts oxidative damage by neutralizing excessive ROS, leading to the alleviation of Aβ-induced cell death. In addition, HRW also stimulated AMP-activated protein kinase (AMPK) in a sirtuin 1 (Sirt1)-dependent pathway, which upregulates forkhead box protein O3a (FoxO3a) downstream antioxidant response and diminishes Aβ-induced mitochondrial potential loss and oxidative stress. Taken together, our findings suggest that HRW may have potential therapeutic value to inhibit Aβ-induced neurotoxicity.

PMID: 26271894



AD is the most common neurodegenerative disease characterized by progressive cognitive impairment and neuronal loss. It has been well established that Aβ deposition may play a pathogenic role in age-associated AD pathogenesis. As a result, anti-aging manipulations have the potential to prevent or treat AD. Although the exact sequence of events whereby Aβ causes neurodegeneration in AD is not fully known, there is significant evidence that links mitochondrial dysfunction, accumulation of ROS, and oxidative damage to AD development and progress. As a result, oxidative stress is supposed to play a central role in Aβ-related neurotoxicity. In response to ROS damage, some defense mechanisms have involved in removing of excessive ROS. These survival pathways are controlled in part by the Sirt1, which are known to regulate the expression of several genes involved in cell survival, senescence and redox balance. As a result, Sirt1 is known as a conserved pathway that helps cell survival in the face of oxidative stress. Particularly, the mitochondrial damage correlates with increased intracellular production of oxidants, which is implicated that maintain a healthy mitochondrial function within the neuronal cells eventually play a protective role. Interestingly, Sirt1 is proposed as one of anti-aging key proteins, implicating it may be a potential therapeutic target for AD. Mammalian Sirt1 is a protein deacetylase related to an increase in mitochondrial function and antioxidant protection. Recent evidence suggests that Sirt1 interacts with and deacetylates PGC1α at multiple lysine sites, which increases PGC1α activity and leading to the induction of downstream genes. In our previous studies, we had demonstrated Sirt1 is the key effector against Aβ neurotoxicity by stimulating endogenous antioxidant defense system. Sirt1 and PGC1α can co-regulate expression of genes encoding enzymes involved in the ROS defense system including SODs and catalase, leading to alleviate mitochondrial oxidative stress in neuronal cells.




Figure 1. The hydrogen-rich water generator designed by our study group. HRW was easily produced by placing a metallic magnesium stick in distilled water [Mg + 2H2O → Mg (OH)2 + H2] at the rate of about 200 mL/min. The magnesium stick contained 99.99% pure metallic magnesium in a polypropylene and ceramic container. Generally, the final hydrogen concentrations of fresh HRW can up to over 500 ppb (approximately 30% saturation) by this machine.


However, there are seven known enzymes that activate Sirt1 and its downstream signaling. It has been demonstrated that AMPK, a crucial cellular energy sensor, is an important contributing factor leading to activation of Sirt1. In general, AMPK maintains the energy balance of cells by sensing an increase in the ratio of AMP: ATP and coordinating cellular metabolic activity to supply energy in response to demand. It was also recognized that AMPK was itself regulated by phosphorylation, it is in this catalytic domain where AMPK becomes activated when phosphorylation takes place at threonine-172. Interestingly, we recently reported that signaling is an important contributing factor leading to alleviate Insulin resistance and mitochondrial dysfunction, two common features both in AD and type 2 diabetes. As previous mentioned, mitochondrial dysfunction leads to impairment of insulin sensitivity by reduced activity of AMPK. Aβ was found to cause ROS accumulation and oxidative damage in the brain, which believed to play a pivotal role in the development of insulin resistance. Our results showed that linagliptin, a kind of dipeptidyl peptidase 4 (DPP-4) inhibitor for the treatment of type 2 diabetes, can counteract impaired insulin signaling transduction, and thus contributes to the alleviation of Aβ-induced AD neurodegenerative markers including GSK3β and tau. Moreover, we also observed that linagliptin protects mitochondrial function and suppresses intracellular ROS accumulation depends on AMPK signaling pathways. This observation was further confirmed by Sirt1, a well-known longevity factor, is in fact upregulated by linagliptin. During linagliptin treatment, AMPK can trigger downstream signaling via Sirt1, which was shown previously to triggers downstream antioxidant enzymes such as SOD. As SIRT1 acts as a transcriptional activator of AMPK gene expression, we therefore proposed this protection appears to be associated with the insulin signaling-dependent AMPK activation by the stimulation of Sirt1-induced antioxidant enzyme. To our knowledge, this is the first report demonstrating the AMPK-Sirt1 molecular mechanism against Aβ-induced mitochondria impairment and oxidative damage. Altogether, the results suggest that impaired AMPK stimulated survival signaling and attendant chronic oxidative stress represent major abnormalities in AD, and thus suggest that AMPK-Sirt1 enhancing agents nay be potential useful therapeutic approaches.

There is increasing interest for the role of molecular hydrogen which exhibits potent systemic anti-free radical activities. Molecular hydrogen is soluble easily in water, and can rapidly penetrate across most cell membranes in human tissues. Although hydrogen gas is hard to store and handle, HRW is relatively easy to use, safe, and economical for use against oxidative stress-induced damage in many in vitro and in vivo models. Accordingly, previous studies have shown that HRW is suitable for application in a number of oxidative stress-related disorders, including metabolic syndrome, neurological disorders, and ischemia/reperfusion injuries. Most importantly, some studies have also reported that HRW significantly reduces several neurodegenerative diseases associated with oxidative stress. This indicates molecular hydrogen may have significant neuroprotective properties. In the present study, we found that HRW effectively scavenges hydroxyl radicals and suppresses Aβ-induced oxidative damage and protected mitochondrial function. Moreover, we observed that AMPK was activated by HRW treatment, which subsequently blocked FoxO3a degradation and increased downstream FoxO-targeted antioxidative genes, such as SOD1. These reports suggest that the molecular targets of HRW not only capture ROS directly, but also activate the cellular detoxification system. In addition, we found that the activity of AMPK could be reduced during the incubation of cells with Aβ, and this inhibition was prevented by HRW treatment. This outcome is consistent with the concept that HRW may be an important mediator against Aβ-induced neuronal senescence and with the results of previous studies indicating that molecular hydrogen acts as an AMPK activator. This observation was further confirmed by the demonstration in the present study that Sirt1, a well-known longevity factor, is in fact upregulated by HRW. During HRW treatment, AMPK can trigger downstream signaling via Sirt1, which was shown previously to reduce the Aβ-induced cellular oxidative stress burden through the formation of a complex with FoxO3 proteins in the nucleus. Moreover, HRW also significantly improved mitochondrial membrane potential. This can be explained by noting that HRW stimulates AMPK activity and subsequently enhances Sirt1-PGC1α or Sirt1-FoxO3a activity, which triggers downstream mitochondrial antioxidant enzymes and preserves mitochondrial activities. Because the Sirt1–FoxO axis is an evolutionarily well conserved survival pathway, pharmacological targeting of AMPK–Sirt1–FoxO3a signaling by HRW treatment may have potential therapeutic implications for AD and similar age-related diseases. In conclusion, our data provide evidence for the view that HRW inhibits intracellular ROS production induced by Aβ. This protection appears to be associated with the preservation of mitochondrial function and the stimulation of intracellular antioxidant defense system. We hope that these new insights into the protective function of HRW will provide a better understanding of AD.




Figure 2. Proposed mechanism how HRW prevents Aβ-induced neurotoxicity. The schematic diagram illustrates a sequence of events in the proposed cell death pathway evoked by Aβ-induced ROS accumulation, which results in the reduction of nuclear FoxO3a-targeted SOD1 and culminates in apoptotic cell death. However, HRW can enhance antioxidative systems in cells under Aβ-stimulated oxidative stress by the upregulation of the AMPK–Sirt1–FoxO3a signaling pathway. This protection is associated with the preservation of mitochondrial function and the stimulation of cellular antioxidant defenses.



  1. Li HH, Lu FJ, Hung HC, Liu GY, Lai TJ, Lin CL 2015 Humic acid increases amyloid β-induced cytotoxicity by induction of ER stress in human SK-N-MC neuronal cells. Int. J. Mol. Sci. 16: 10426-10442
  2. Kornelius E, Lin CL, Chang HH, Li HH, Huang WN, Yang YS, Lu YL, Peng CH, Huang CN 2015 DPP-4 inhibitor linagliptin attenuates Aβ-induced cytotoxicity through activation of AMPK in neuronal cells. CNS Neurosci. Ther. 21:549-557


Acknowledgements:  This work was supported by grants from the Chung Shan Medical University Industry-Academia Cooperation project (CSMU-E101-N0088), and from the Ministry of Science and Technology of Taiwan (MOST 101-2320-B-040-015-MY3).


fig3Contact: Fung-Jou Lu, Chair Professor Institute of Medicine, Chung Shan Medical University No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwan E-mail:


Chih-Li Lin, Associate Professor Institute of Medicine, Chung Shan Medical University No. 110, Sec. 1, Jianguo N. Rd., Taichung City 402, Taiwan E-mail:



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