Hydrogen is a biological antioxidant and anti-inflammatory substance. Inhalation of hydrogen significantly inhibits ischemia/reperfusion injury of multiple organs by buffering oxidative stress. Drinking hydrogen water may help alleviate neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease, thus improving the quality of life of the elderly. In animal models, hydrogen can inhibit inflammatory non-communicable diseases induced by lipopolysaccharide, concanavalin A and sodium dextran sulfate, accompanied by the decrease of pro-inflammatory cytokines. However, the anti-oxidation and anti-inflammatory therapeutic effects of hydrogen can’t be explained by scavenging reactive oxygen species, because hydrogen can’t directly reduce reactive oxygen species. On the other hand, animal studies have shown that pre-administration of hydrogen can also prevent inflammatory diseases. Considering that hydrogen can’t stay in the body for a long time, it further shows that hydrogen has indirect effects. Further, we found that hydrogen induced the cultured cells to produce mild oxidative stress in mitochondria, and then induced the expression of antioxidant enzymes. These results indicated that hydrogen could trigger the protective adaptive response of mitochondrial mediated antioxidant stress-mitochondrial toxic excitatory effect. In order to determine the precise molecular mechanism of the biological effect of hydrogen, it is necessary to determine the target molecules of the biological effect of hydrogen.

Custom hydrogen alkaline water machine

Many gas molecules, including hydrogen, are easier to dissolve in lipids than in water, and dissolved gases in lipids can affect cell and physical functions. High concentration of nitrogen (N2) increases the bending modulus and stability of cellular lipid bilayer, and hinders the phase separation of ternary lipid bilayer. Although the precise molecular mechanism of anesthetic action is largely unknown, Meyer-Overton correlation provides the relationship between the efficacy of anesthetic gas and its solubility in lipid nonpolar media. Anesthetic gas dissolved in lipid changes cell membrane structure through hydrophobic interaction. Inhalation of sevoflurane, an anesthetic, can cause brain damage in mice during development, which is a side effect of narcotic drugs. Interestingly, the nervous system damage of narcotic drugs can be alleviated by inhaling hydrogen at the same time.

Many gas molecules, including hydrogen, are easier to dissolve in lipids than in water, and dissolved gases in lipids can affect cell and physical functions. High concentration of nitrogen (N2) increases the bending modulus and stability of cellular lipid bilayer, and hinders the phase separation of ternary lipid bilayer. Although the precise molecular mechanism of anesthetic action is largely unknown, Meyer-Overton correlation provides the relationship between the efficacy of anesthetic gas and its solubility in lipid nonpolar media. Anesthetic gas dissolved in lipid changes cell membrane structure through hydrophobic interaction. Inhalation of sevoflurane, an anesthetic, can cause brain damage in mice during development, which is a side effect of narcotic drugs. Interestingly, the nervous system damage of narcotic drugs can be alleviated by inhaling hydrogen at the same time.

The membrane can quickly respond to various environmental disturbances by changing its composition. For example, exposure to halothane can reduce the synthesis of phosphatidylcholine (PC) in alveolar cells, which is the main lipid component of pulmonary surfactant. Recently, it has been reported that hydrogen reduces the cytotoxicity of tert-butyl hydroperoxide by inhibiting fatty acid peroxidation and membrane permeability, indicating that hydrogen affects membrane environment and lipid composition.

Lipid bilayer is the main component of all cell membranes, mainly formed by phospholipids. Phosphatidylcholine, phosphatidylinositol (phosphatidylinositol) and phosphatidylserine (phosphatidylserine) mainly promote the formation of lipid bilayers, while phospholipids, cardiolipin (CL) and phosphatidylethanolamine (PE) formed by non-bilayers play a specific role in the assembly and activity of mitochondrial respiratory chain complexes, which indicates that the changes of phospholipid components have a significant impact on cell metabolism, especially energy production. In addition, phospholipid is not a passive structural bystander of cell membrane, but has an active role in regulating physiological events, such as cytokinesis, exocytosis and endocytosis. Complex endosome processes are only partially understood. However, it is clear that phospholipid plays a key role in each stage of this process. Endophagous vesicles usually fuse with early endosomes (EEs). Early endosomes mature into late endosomes (LEs), which fuse with lysosomes to degrade their contents. Various endocytosis pathways have been identified. Some contents, such as transferrin (Tfn) receptor, were sorted into circulating endosomes (REs) by early endosomes and returned to the cell membrane. Cholera toxin B (CTxB) was transported from circulating endosomes to Golgi apparatus retrograde.

The purpose of this study is to study the effect of short-term hydrogen exposure on lipid composition of cultured human neuroblastoma SH-SY5Y cells, because pre-culture in the presence of hydrogen for only 3 hours can reduce cell death induced by oxidative stress.

We analyzed the cells by liquid chromatography-high resolution mass spectrometry to determine the changes of lipid composition. Lipid analysis of cells exposed to hydrogen for 1 hour showed that glycerophospholipids (including phosphatidylethanolamine, phosphatidylinositol and cardiolipin) increased instantaneously. Metabonomics analysis also showed that hydrogen exposure for 1 hour instantly inhibited the total energy metabolism, accompanied by the decrease of glutathione. Through specific antibody staining, we further observed the morphological changes of endosomes. In cells exposed to hydrogen, the transport of cholera toxin B to circulating corpuscles around Golgi apparatus was delayed. We speculate that the change of lipid composition induced by hydrogen will inhibit the energy production and endosome transportation, accompanied by the enhancement of oxidative stress, thus temporarily stimulating the stress response pathway to protect cells.

Experimental method

After the cells were cultured, they were exposed to hydrogen. In short, SH-SY5Y cells were preserved in modified Eagle medium containing 10% fetal bovine serum. As a control, N2 gas mixture contains 20% O2, 5% CO2 and 75% N2, which is almost the same as that in the traditional CO2 incubator. The mixed hydrogen gas contains 20% O2, 5% CO2, 1-50% hydrogen, and the rest is N2.

These boxes are at a proper gas mixture flow rate of 1.6 liters/minute for 15 minutes under normal pressure. Sealed, and placed in an incubator at 37°C immediately after incubation, the concentration of hydrogen and O2 in the culture medium was monitored with a specific electrode for 15 minutes. Under 50% hydrogen gas mixture, the hydrogen concentration is 365 5μ m, and the O2 concentration is 245 5μ m. In N2 mixed gas, the concentration of hydrogen cannot be detected, and the concentration of O2 is kept at 250±5 μM m.

After the cultured SH-SY5Y cells were exposed to 50% hydrogen gas at 37°C for 1 and 6 hours, the cell lipids were extracted with methanol and chloroform. In order to determine the changes of major lipid components, we used liquid chromatography-high resolution mass spectrometry (HPLC-MS) to check the lipid profile of biological samples in an unbiased way. At the same time, we quantified 15 kinds of lipids, including phospholipids, ganglioside and diacylglycerol (DAG), and normalized them according to the number of cells. It shows that PCA hydrogen treatment and control cells have obvious lipid species clustering [Figure 1].

The levels of PE, cardiolipin and phosphatidylinositol in cells treated with hydrogen -1 hour were significantly higher than those in control group and cells treated with hydrogen -6 hours [Figure 1]B, but there was no difference in the sum of all lipids in each cell. Phosphatidylserine in hydrogen treated cells also tends to have a temporary increase level. However, hydrogen exposure did not change the proportion of PC, which is the most abundant phospholipid, but phosphatidylcholine increased significantly. In addition, there was no significant difference in free fatty acids or triglycerides between the two groups (data not shown). Cardiolipin is synthesized only in mitochondria, mainly located in the inner membrane of mitochondria (IMM), and cooperates with PE to maintain mitochondrial activity. The level of DAG in cells treated with hydrogen tends to increase [Figure 1]B, DAG is produced by dephosphorylation of phosphatidic acid, which is used to synthesize PC and PE.

Metabonomics changes induced by hydrogen exposure

The composition of mitochondrial phospholipid and PE affects respiratory chain function and changes energy metabolism. In fact, we have previously observed that hydrogen exposure significantly changed mitochondrial membrane potential and cell oxygen consumption, while glutathione decreased. Metabonomics was used to analyze the early hydrogen-induced changes of SH-SY5Y cells after 1 and 6 hours of hydrogen exposure. We selected 116 metabolites (52 cations and 64 anions), which participate in glycolysis system, pentose phosphate pathway, tricarboxylic acid cycle, urea cycle, polyamine and creatine metabolism pathway, purine metabolism pathway, glutathione metabolism pathway, nicotinamide metabolism pathway, choline metabolism pathway, and various amino acid metabolism pathways. Comparing the metabolomic changes of PCA cells with those of cells treated with hydrogen for 1 hour and 6 hours, the results show that hydrogen treatment leads to different metabolomic changes [Figure 3].

As shown in Figure 3, the metabolomics map through hierarchical cluster analysis. Some characteristic changes were found in cells exposed to hydrogen. After hydrogen treatment for 1h, the level of most metabolites decreased. These include glycolytic metabolites (fructose 6- phosphate, fructose 1,6- diphosphate, glyceraldehyde 3- phosphate, dihydroxyacetone phosphate, glycerol 3- phosphate, 3- phosphoglyceric acid, 2- phosphoglyceric acid, pyruvic acid and lactic acid) [Figure 3]C, and cyclic metabolites of tricarboxylic acids (citric acid, 2- oxoglutarate, succinic acid and malic acid). Fructose 1,6- diphosphate was metabolized by aldolase to dihydroxyacetone phosphate and glyceraldehyde 3- phosphate, and the levels of these three metabolites decreased significantly. After hydrogen treatment for 1 hour, the level of nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) in cells decreased significantly [Figure 3]. After 6 hours, the levels of many of these down-regulated metabolites, including reduced nicotinamide adenine dinucleotide and adenosine triphosphate, returned to the levels of control cells. However, the levels of pyruvic acid and tricarboxylic acid circulating metabolites still declined. After hydrogen treatment for 1 hour or 6 hours, the ratio of reduction/oxidation nicotinamide adenine dinucleotide remained unchanged, but the ratios of lactic acid/pyruvic acid and malic acid /Asp decreased significantly. These results indicate that hydrogen temporarily inhibits the overall metabolism, but can continuously inhibit the mitochondrial energy energy metabolism.