Efficacy of hydrogen therapy on hyperlipidemia and related diseases such as nonalcoholic fatty liver disease

【Background Analysis】

As the lightest chemical element and the most abundant chemical element in nature, hydrogen is considered a new antioxidant that can reduce oxidative stress [1].

Since 2007, hydrogen has been at the forefront
of research into therapeutic medical gases.
Many clinical and experimental models, accumulated in the biomedical field, by inhaling hydrogen, or hydrogen-rich aqueous solutions as a mode of administration, hydrogen has shown safe and feasible therapeutic potential
in different disease models.

For example, supplementation with hydrogen-rich aqueous solutions has been shown to have a positive effect on the prevention of type 2 diabetes mellitus and insulin resistance [2], chronic liver inflammation [3], acute oxidative stress, and ischemia of the brain/ Reperfusion injury [1].

Oral administration of hydrogen water for 6 months has been reported to prevent atherosclerosis in apolipoprotein E knockout (apo E−/−) mice, mainly by reducing the level of oxidative stress in vivo [4]
。 Therefore, hydrogen is able to affect plasma lipid and plasma lipoprotein levels, making it possible
to treat hyperlipidemia with hydrogen.

The function and concentration of plasma high-density lipoprotein (HDL) showed a strong negative correlation with the risk of atherosclerotic cardiovascular disease [5, 6].

It is known that HDL undergoes significant changes in structure and composition under the synergistic effect of inflammation and oxidative stress [7, 8], resulting in the gradual loss of normal biological activity
of HDL molecules.
Hydrogen is an electron donor with high reducing capacity
.
Therefore, hydrogen therapy in animals or patients with hyperlipidemia and other cardiovascular diseases may improve HDL damage caused by hyperlipidemia and improve its anti-atherosclerotic function
.

The scientific research team led by Professor Qin Shucun from Shandong First Medical University in China, after 4 years (2012-2016) research, proved that by taking hydrogen-rich drinking water (hereinafter referred to as hydrogen-rich water) and inhaling hydrogen (3L/min).
It has obvious efficacy
in the prevention and treatment of hyperlipidemia in middle-aged and elderly people, as well as related diseases (such as non-alcoholic fatty liver).

【Animal Experiments】

This experiment aims to test the effect of
hydrogen on the functional properties of HDL molecules in golden hamsters.
The experiment used 45 male Syrian golden hamsters, randomly divided into 5 groups, namely the conventional feed group (5 birds, referred to as CD), the high-fat diet group (10 birds, HFD), HFD low-dose hydrogen intervention group (10 animals, referred to as HFD+H2-L, 1ml/KG/d ), HFD moderate-dose hydrogen intervention group (10, HFD+H2-M, 2.
5 ml/KG/d) and HFD high-dose hydrogen intervention group ( 10 pcs, referred to as HFD+H2-H, 5ml/KG/d).

The 5 groups of mice were fed conventional feed (CD) and high-cholesterol feed (15% lard mixed with 0.
2% cholesterol),
respectively.

The experiment was carried out for a total of 8 weeks, and the first 4 weeks were divided into groups and fed conventional feed and high-cholesterol feed.
For the next four weeks, ordinary saline and hydrogen-rich saline
were injected daily according to the group.

After the experiment, mice were anesthetized to collect blood, and plasma total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) were determined enzymatically and the concentration of low-density lipoprotein cholesterol (LDL-C), and the lipoprotein profile is obtained by rapid protein liquid chromatography (FPLC) [9]
。

Result: Hydrogen reduced plasma TC and LDL-C levels

As shown in the figure below, HFD in the high-fat diet group resulted in plasma TC, LDL-C, HDL-C and compared to conventional feed group CD TG levels increased significantly, by 4.
2, 2.
5, 2.
7 and 3.
9 times, respectively, This indicates the successful preparation of a hamster model
of hyperlipidemia.

As shown in the figure below, the results after 4 weeks of hydrogen treatment showed a significant reduction of 28.
3% in plasma TC and LDL-C levels compared to the HFD control group, respectively and 34.
3%.

Result 2: Hydrogen downregulated plasma levels of apoB and apoE proteins

Consistent with the differences observed above with LDL-C, the main proteins on LDL, apoB100 and apoB48, are used in HFD feeding The treatment of hydrogen was significantly reduced in animals, and the apoE protein in the LDL molecule was also downregulated
with the treatment of hydrogen.

Meanwhile, SDS polyacrylamide gel electrophoresis (PAGE) of isolated VLDL particles showed a significant reduction in hydrogen to apoE in VLDL of content
.

These data suggest that hydrogen can downregulate the expression of major protein components of LDL and VLDL, which is consistent
with data from FPLC plasma lipoprotein profiles.

Results 3: Hydrogen gas improved the activity of HDL molecules in hyperlipidemic hamsters

Under the synergistic effect of inflammation and oxidative stress, high-density lipoproteins undergo significant changes in structure and activity [7, 8].

Hydrogen acts as a therapeutic medical gas in a variety of disease models by exerting antioxidant and anti-inflammatory effects [2, 4, 10].

Hydrogen intervention improves the activity of HDL molecules, and the verification process is as follows:

First, the research team tested the ability of isolated HDL molecules to
elicit efflux from cholesterol-laden macrophages.

As shown in the figure above, HFD treatment reduced HDL's ability to trigger efflux compared with the conventional CD group, indicating that HDL particle-mediated cholesterol efflux capacity was impaired by hyperlipidemia; However, HDL isolated from hydrogen-treated hamsters exhibited significantly higher outflow characteristics
.

Next, the biological effect of hydrogen on the antioxidant function of HDL, that is, protecting LDL molecules from oxidation
, was tested.

As shown in the figure above, the formation of TBARS after HFD feeding tended to increase compared with the CD group; Different doses of hydrogen treatment significantly inhibited the formation
of TBARS.
These data suggest that HDL function impaired by hyperlipidemia, including the ability to stimulate cholesterol outflow from macrophage foam cells, and the ability to prevent LDL oxidation, was improved
by hydrogen intervention.

However, the levels of the main proteins on HDL, apo A-I and apo A-II (results shown in figure above), were not significantly altered by hydrogen treatment in animals fed HFD.
This is also one of
the results that need further confirmation in this experiment.

The significance of this experiment is that the experimental data provide a clue that hydrogen has the potential to be used as a new type of lipid regulator, and safety is its biggest advantage
compared to other commonly used lipid regulating drugs that have adverse effects on the liver and kidneys.
When translated into clinical practice, inhalation of hydrogen gas may be used in the future to treat patients
with hyperlipidemia.
However, further understanding of hydrogen's ability to influence lipid and cellular metabolism, as well as the underlying mechanisms of the signaling pathways involved, is also needed to take full advantage of inhaled hydrogen as a therapeutic strategy
.

[Clinical Trial - Drinking Hydrogen-Rich Water].

Although animal experiments have revealed that hydrogen interventions can act as novel lipid modulators, they also point out that further understanding of hydrogen's ability to affect lipid and cellular metabolism, as well as the underlying mechanisms of related signaling pathways, is needed to fully utilize inhaled hydrogen as a therapeutic strategy
.
To this end, the research team conducted two more trials
in 2013 and 2015.

This chapter presents a 2015 trial that focuses on the efficacy and mechanism
of action of drinking hydrogen-rich water in patients with hyperlipidemia.
The trial was a double-blind, randomised and placebo-controlled trial
.

A total of 68 participants (35-60 years old) with hypercholesterolemia participated in this trial, and the participants had a plasma TC> of 5.
18 mmol/L, and/or LDL-C > 2.
59 mmol/L [11, 12], did not receive any lipid-lowering drugs
for at least three months before the trial.
At the same time, the research team ruled out any acute and chronic inflammatory processes, stage 2 or 3 hypertension, unstable coronary artery disease, myocardial infarction or stroke, symptomatic congestive heart failure, diabetes, autoimmune diseases, thyroid disease, Participants with chronic pancreatitis, impaired renal or liver function, nephrotic syndrome and body mass index > 35kg/㎡
.

Patients were randomly assigned to either hydrogen-rich water or placebo (purified water) in a 1:1 ratio by computer
.
Drink 0.
9 L (0.
3 L/time, 3 times/d) of hydrogen-rich water every day, hydrogen-rich water and purified water are packed in sealed bags, and are required to drink within 5 minutes after opening
。 The trial lasted for 10 weeks, and the patient's blood was collected before the test and at the end of the test for laboratory tests to compare the results
.

Result: Hydrogen increased pre-β1 high-density lipoprotein (pre-β1-HDL) levels without reducing lecithin cholesterol acyltransferase (LCAT) activity

The role of HDL is related to HDL activity and pre-β1-HDL protein levels [13].

The pre-β1-HDL protein is the first receptor for free cholesterol and is also affected by LCAT activity [14].

As shown in the figure above, the hydrogen-rich water group (H2) showed a significant increase in plasma pre-β1-HDL levels (panel A), while LCAT levels were not significantly affected, while LCAT levels were not significantly affected Essential for pre-β1-HDL maturation, suggesting that hydrogen-rich water interventions increased pre-β1-HDL levels without reducing LCAT activity
.

At the same time, the research team evaluated lipid levels in HDL3 (Figure C) and showed that hydrogen-rich water interventions significantly reduced phospholipid levels (phospholipid levels reflect the possibility of having certain chronic diseases, such as cirrhosis, diabetes, essential hypertension, hypothyroidism, etc
.
).
Patients with an overweight base and obesity are more
likely to have excessive phospholipid levels in their bodies.
The normal level of phospholipids is between 130~220mg/dl, and the average value is 176mg/dl); At the same time, there was no significant change in the content of S1P (1-phosphate sphingosine) and ceramide in HDL3.

Results 2: Hydrogen intervention promoted cholesterol outflow by upregulating the expression of ABCA1

To verify whether the hydrogen-rich water intervention promoted cholesterol outflow by upregulating the expression of ABCA1, the research team first measured plasma HDL3-mediated bone marrow macrophage outflow with or without glibenclamide, a commonly used inhibitor of ABCA1
。

The results are shown in Figure A above, the outflow capacity of cellular cholesterol in the hydrogen-rich water group is significantly higher than that of HDL3 in the placebo group.
However, there was no significant difference with glibenclamide (Figure B).

At the same time, using RAW264.
7 macrophages, the same results
of cholesterol outflow were observed.
The total outflow was significantly higher in the hydrogen-rich water group than in the placebo group (Figure C), while RAW264.
7 was stimulated by adenosine-3',5'-cyclic phosphate (cAMP) for ABCA1 The total outflow of macrophages and hydrogen-rich water was significantly higher than in the placebo group (panel D).

Outcome 3: Hydrogen intervention improved the functional properties of HDL3 other than cholesterol outflow

The research team first tested the biological effect of hydrogen on the antioxidant function of HDL3, that is, preventing LDL from being oxidized
.

As shown in Figure A, hydrogen treatment significantly inhibited the formation of thiobarbituric acid reactants, which resulted in reduced plasma ox-LDL levels in patients treated with hydrogen-rich water (Figure B).

Next, the biological effect
of hydrogen's anti-apoptotic function was tested.

As shown in Figure C, treating cells with ox-LDL results in a decrease in cell viability and a sharp increase in the number of apoptotic cells, which can be prevented
by HDL pretreatment.
In addition, HDL isolated from the hydrogen-rich water group had a significantly higher inhibitory effect on apoptosis than HDL isolated from the placebo group (panel D).

Finally, the research team tested the effects of hydrogen on the anti-inflammatory properties of HDL3, including preventing ox-LDL-induced monocyte adhesion to endothelial cells and secreting adhesion molecules and inflammatory factors, intercellular adhesion molecule-1 (ICAM-1).
), vascular cell adhesion molecule-1 (VCAM-1) and IL-6
.

As shown in Figures A and B, HUVECs interact with ox-LDL for 6 hours, and monocytes are associated with Adhesion to HUVECs increased significantly, which was subsequently blocked
by HDL intervention.
HDL3 isolated from the hydrogen-rich water group had a significantly higher effect on the prevention of adhesions than HDL3
isolated from the placebo group.

In addition, ox-LDL-induced ICAM-1 and VCAM-1 compared to HDL3 interventions isolated from the placebo group and HDL3 interventions isolated from the hydrogen-rich water group and significantly reduced IL-6 secretion (Panels C, D, E), which indicate improved hydrogen intervention Anti-inflammatory function
of HDL3.

Risk assessment of hydrogen interventions for ASCVD (atherosclerosis) in patients with hypercholesterolemia

The research team conducted a risk assessment
of the development of atherosclerosis in patients with hydrogen therapy in patients with hypercholesterolemia.

As shown in the figure above, the trend towards a 10-year atherosclerosis risk (percentage) is reduced by hydrogen intervention
.

In summary, the research team concluded that hydrogen-rich water intervention can increase pre-β1-HDL levels and activate the outflow of ABCA1-dependent cholesterol from peripheral macrophages.
At the same time, it improves other HDL functions, including preventing LDL oxidation, inhibiting ox-LDL-induced monocytes adhering to endothelial cells, and protecting endothelial cells from ox-LDL-induced apoptosis
。 In addition, hydrogen-rich water therapy reduces plasma cholesterol levels, plasma oxidative stress, and inflammatory states
.

Therefore, the research team ultimately concluded that oral administration of hydrogen-rich water can prevent or delay the onset and development
of hypercholesterolemia and atherosclerosis.

The figure below records the changes in blood lipid data before and after the trial for all trial participants
.

【Clinical Trial - Inhalation of Hydrogen】

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease [15, 16], and obesity, type 2 diabetes, and hyperlipidemia are the main causes of
induce.
NAFLD has traditionally been considered a Western disease, and its prevalence has been increasing due to dietary habits and sedentary lifestyles [17].

However, a meta-analysis published in 2017 suggested that the prevalence of NAFLD in Asia is approximately 25 percent, similar to that in Western countries, and is likely to continue to increase [18].

NAFLD covers a wide range of clinical subtypes histological spectrum, including simple steatosis (NAFL) and steatohepatitis (NASH) [19].

About 20%–30% of NAFL can progress to inflammatory and fibrotic NASH, of which 37%~41% are NASH Patients develop cirrhosis [20, 21].

There are currently no FDA-approved drugs available, and NAFLD is difficult to cure
.
While lifestyle interventions, including dietary modification and exercise, have proven useful [22], there is an urgent need for alternative therapies to prevent disease progression
.

Molecular hydrogen is considered to be an endogenous regulator of liver homeostasis [23].

At the same time, autophagy (that is, a large increase in autophagic lysosomes, thereby playing a protective role against cell damage) has also been shown to be associated with homeostasis [24].

There is growing evidence that dysphenophagus is one of the causes in the pathogenesis of multiple diseases, including lipid-related metabolic disorders
.

However, whether the role of molecular hydrogen is associated with hepatocyte autophagy in NAFLD has not been determined
.
In this study, the team examined the effects
of hydrogen/oxygen inhalation on blood lipid levels and liver fat deposits in 43 patients with NAFLD in a randomized, placebo-controlled manner.

This study was a randomized, placebo-controlled trial conducted from May to August 2016 at Coal Taishan Sanatorium in Shandong Province and Shandong First Medical University
.
A total of 43 patients, aged 30-70 years, with an average age of 54 years, according to the ratio of 1:1, They were randomly divided into hydrogen and placebo
.
The hydrogen stack uses a hydrogen generator (66% hydrogen, 34% oxygen; 3L/min), continuous inhalation for 1 hour per day; The comfort group used the same shaped device to inhale air, also at 3 L/min
.
The trial was conducted for a total of 13 weeks

Before and after the test, fasting venous blood was drawn from patients for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and liver enzymes.
Serum MDA, SOD (Solarbio), TNF-α, IL-6 ( Mlbio) quantitative detection; The grade
of hepatic steatosis is measured by ultrasonography of the liver (USG).

Basic information of the patients participating in this trial

Result 1: Inhalation of hydrogen alleviated NAFLD in patients

To test whether hydrogen/oxygen inhalation regulates blood lipids, the research team analyzed the lipids of all patients before and after the test
.

After 13 weeks, there was no significant change in TC and TG levels in either group, but LDL-C in the hydrogen group decreased
significantly after the trial.
More importantly, AST and ALT, which were indicators of liver damage, decreased in the hydrogen/oxygen group, indicating that hydrogen inhalation had a hepatoprotective effect; At the same time, the biomarkers of oxidative stress and inflammation of the hydrogen group were significantly reduced (MDA, TNF-α, IL-6), indicating that hydrogen had systemic anti-inflammatory effects
.

To verify the effect of hydrogen inhalation on hepatic steteaosis, the research team performed USG and CT scans
.
Moderate steeatosis was reduced in the hydrogen stack, and USG diagnosis and CTL/S values were improved
.

As shown in the table above, 77.
8% of moderate cases improved to mild cases in the hydrogen group, compared with 38.
5%
in the placebo group.
On the other hand, 15.
4% of moderate patients in the placebo group developed severe cases, while there was no such case
in the hydrogen group.

The hydrogen group and placebo group compared fractionated CT for steteaosis before and after the trial in 30% ≥ (CTL/S < 0.
8) of patients with hepatic steteaosis

Representative liver CT scans of patients in the hydrogen and placebo groups

As shown in the figure above, after the end of the test, the CTL/S of the hydrogen group after use was improved compared with the placebo group, and the CT scan results were consistent with the ultrasound results, indicating that hydrogen injection alleviated the patient's moderate hepatic steatosis
.

Outcome 2: Hydrogen reduces lipid accumulation in AML-12 cells by promoting autophagy

To explore the mechanism by which hydrogen reduces liver lipid content, the research team used an FFA-loaded in vitro model of hepatocytes
.
In the presence of FFA or PA, keep AML-12 cells in a 20% hydrogen incubator to assess the inhibitory effect
of hydrogen on lipid accumulation.

The results showed that hydrogen-treated cells were exposed to 1 mmol/LFFA and 0.
5 mMPA intracellular compared to the simulated group of AML-12 cells cultured in an eCO2 incubator TG is significantly reduced; Meanwhile, no difference was observed when AML-12 cells were treated with 0.
2 mmol/L FFA or 0.
2 mmol/L PA, meaning that hydrogen had no effect
on lipid accumulation at low concentrations.

Next, the research team explored the association
between hydrogen treatment and autophagy.
These include LC3-II/LC3-I, Beclin1, and autophagy-associated protein 5 (ATG5) associated with FFA-induced autophagy
。

The results showed that hydrogen slightly inhibited autophagy when AML-12 cells were exposed to 0.
2 mmol/L FFA, and slightly promoted autophagy
when cells were exposed to 1 mmol/L FFA.
The effect of hydrogen on autophagy treated by FFA was not significant
.
However, autophagy is significantly promoted
when AML-12 cells are exposed to PA.

The research team then applied TEM from AML-12 cells treated with 0.
5 mmol/L PA to investigate whether hydrogen treatment caused any ultrastructural changes
.

The results showed that AML-12 cells produced bilayer autophagosomes and some autophagic lysosomes after 12 h incubation with 0.
5 mmol/L PA, while hydrogen-treated cells produced larger and more autophagic lysosomes, indicating that autophagy was promoted
.

Finally, the research team added 3-methyladenine (3-MA), a specific inhibitor of macroautophagy, to hydrogen-treated cells to determine whether hydrogen's inhibition of lipid accumulation is dependent on autophagy.

The results showed that intracellular TG could be restored by the addition of 3-MA, indicating that blocking autophagy reversed the inhibitory effect of hydrogen on lipid accumulation
.
Ultimately, the team confirmed that hydrogen molecules inhibit lipid accumulation
in AML-12 cells by promoting autophagy.

The highlight of this study is that, unlike previous population studies, this study is the first report
to describe the effect of molecular hydrogen on autophagy in hepatocytes.

Among the pharmacological treatments for NAFLD, pioglitazone and vitamin E have been the most
studied.
Treatment with pioglitazone improves hepatic steatosis in NASH [25, 26], however, there are side effects, including weight gain, foot edema, bone loss, and heart failure [26].

Vitamin E supplementation is an option for the treatment of non-cirrhosis and non-diabetic NASH [27], but side effects may include death from cardiovascular events, hepatotoxicity, and elevated bilirubin [28].
、29】
。 In hydrogen biomedicine, most clinical trials have found no adverse events or side effects, which is also consistent with the research of Professor Qin's group [30].

The 13-week intervention, although longer than previously reported, was still a short-term treatment
.
A study after 24 weeks of intervention in patients with metabolic syndrome showed that reduced cholesterol and glucose levels and improved biomarkers of inflammation and redox homeostasis were found in patients who drank hydrogen-rich water [31].

Therefore, prolonging the intervention may lead to better improvement
.

In summary, the research team believes that molecular hydrogen has an inhibitory effect on liver fat accumulation and promotes autophagy
as a protective mechanism.
The findings provide evidence
for hydrogen therapy as an adjunct to NAFLD.

【Research Conclusion】

Based on the above animal experiments, as well as hydrogen-rich water and inhaled hydrogen intervention trials, it has been proved that hydrogen can be used as a new lipid regulator to prevent/treat arteriosclerosis, as well as various diseases caused by hyperlipidemia (such as non-alcoholic fatty liver disease).

In terms of hydrogen intervention, for middle-aged and elderly people, it is recommended to drink hydrogen-rich water and inhale hydrogen therapy daily, and adhere to it for a long time (3-6 months), which may have better results
.

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