Plasmon-activated water effectively relieves hepatic oxidative damage resulting from chronic sleep deprivation

Abstract

The role of the hepato-protective agent plasmon-activated water (PAW) as an innovative anti-oxidant during chronic sleep deprivation (SD) is realized in this study. PAW possesses reduced hydrogen-bonded structure, higher chemical potential and significant anti-oxidative properties. In vitro tests using rat liver cell line (Clone-9) have demonstrated that PAW is non-cytotoxic and does not change the cellular migration capacity. The in vivo experiment on SD rats suffering from intense oxidative damage to the liver, an extremely common phenomenon in the present-time with deleterious effects on metabolic function, is performed by feeding PAW to replace deionized (DI) water. Experimental results indicate that PAW markedly reduces oxidative stress with enhanced bioenergetics in hepatocytes. PAW also effectively restores hepatocytic trans-membrane ion homeostasis, preserves membranous structures, and successfully improves liver function and metabolic activity. In addition, the hepato-protective effects of PAW are evidently demonstrated by the reduced values of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) and the recovery of total protein and albumin levels. With clear evidences of PAW for protecting liver from SD-induced injury, delivering PAW as a powerful hepato-protective agent should be worthy of trailblazing new clinical trials in a healthier, more natural, and more convenient way.

Fig. 1. Schematic diagram showing the experimental design and the proposed mechanism(s) of the current study. CSD was processed for three cycles (with 5 days of total sleep deprivation followed by a 2 day break in each cycle). During the entire CSD period, rats were drank either DIW or PAW. Sleep deprivation was achieved by the disc-on-water method. When sleep onset was detected by the electroencephalography (EEG) machine in a sleep-deprived rat, the disc was slowly rotated at a moderate speed of 3.5 rpm by a computerized monitoring system, forcing the rat to remain awake and walk against the direction of the disc rotation to avoid being forced into the water. When a sleep-deprived rat was spontaneously awoken, the disc became stationary. During this process, the sleep-deprived rats drank either DIW or PAW. PAW was produced by treating DIW with excited AuNP-absorbed ceramic particles. PAW with weak hydrogen bonds (demonstrated by deconvoluting Raman spectra) exhibited efficient anti-oxidative and anti-inflammatory properties. The underlying mechanism(s) of the hepato-protective effects of PAW was proposed to be resulting from the intrinsic anti-oxidative activity of PAW that protects liver from CSD-induced oxidative damage.

Fig. 2. Line chart (a) and histograms ((b) and (c)) showed the anti-oxidative effects of PAW on liver as determined by both (a) in vitro and ((b) and (c)) in vivo assessments. Please note that PAW significantly decreased the H₂O₂ level of rat liver Clone-9 cells (a). Also note that drinking PAW successfully reduced the CSD-induced hepatic oxidative stress (c) by effectively suppressing hepatic H₂O₂ levels (b). ^#p < 0.05 when compared with that of CSD + DIW group, and **p < 0.01 when compared with that of untreated group.

Fig. 3. Effects of PAW on preserving Na⁺/K⁺ ATPase function and restoring the trans-membrane ionic gradient following CSD injury. Positive spectra/ionic images showed that in the normal untreated rats, most of the Na⁺ signals were localized to the extracellular portion of the hepatocytes [arrows in (a)]. Following CSD, strong Na⁺ signals were detected in the cytoplasmic portion of the hepatocyte (b), indicating the impairment of trans-membrane ionic regulation (b). However, in animals supplied with PAW everyday during the entire CSD period, the distribution pattern of Na⁺ was much similar to that of normal untreated ones in which the majority of Na⁺ were localized to the extracellular sinusoid space [arrows in (c)]. The corresponding data of the normalized spectral intensities for a–c (d). Biochemical data coincided well with ionic imaging findings in which PAW effectively preserved hepatic Na⁺/K⁺ ATPase expression (f) and improved Na⁺/K⁺ ATPase activity (e).

Fig. 4. Scanning electron microscopy showed morphological changes in oxidative stress in hepatocytes following CSD injury. The detrimental effects of oxidative stress were evidently demonstrated by the significant lipid peroxidation of plasma membranes [arrows in image (e)]. Note that daily drinking of PAW (when compared with drinking of DIW) during CSD successfully preserved the integrity of hepatocytic membranous structures from oxidative damage ((c) and (f)). Scale bar = 120 μm in ((a)–(c)); scale bar = 20 μm in ((d)–(f)).

Fig. 5. Photomicrographs ((a)–(c)) and histograms ((d) and (e)) showed the hepato-protective effects of PAW on preserving the cellular bioenergetics [as determined by cytochrome oxidase (COX) reaction] ((a)–(d)), and reducing the stress level [as expressed by heat shock protein-27 (HSP-27) immunoblots] (e) following CSD injury. Note that CSD significantly depressed the cellular bioenergetics and increased hepatic stress level as shown by decreased COX staining [arrows in (b)] and enhanced HSP-27 activity (e). However, in case of animals drinking PAW during the entire CSD period, effective increment in COX staining [arrows in (c)] combined with reduced HSP-27 expression (e) was detected in the hepatic tissues. V: central vein. Scale bar = 100 μm in ((a)–(c)).

Fig. 6. Histograms showed the hepato-protective effects of PAW on increasing the activities of anti-oxidant enzymes following CSD injury. Please note that daily drinking of PAW during the entire CSD period would significantly preserve the hepatic superoxide dismutase (a), catalase (b) as well as glutathione peroxidase (c) activities.

Fig. 7. Histograms showing the serum level of biochemical markers related to liver (a) and metabolic (b) functions. Note that CSD contributes to severe liver and metabolic deficiencies. However, drinking of PAW successfully exerts beneficial effects on liver and metabolic function in which almost all biochemical markers were noticeably returned to nearly normal values.