Evaluation of the effects of hydrogen combination drugs on diabetes and its complications (e.g., diabetic heart disease).

【Background】

Diabetes can cause a variety of complications, and diabetic heart disease is one of the leading causes of death in people with diabetes in middle-aged and elderly people, especially in
patients with type 2 diabetes.

Diabetic heart disease in a broad sense includes coronary atherosclerotic heart disease (coronary heart disease), diabetic cardiomyopathy, and diabetic cardiac autonomic neuropathy.

Compared with non-diabetic patients, diabetic heart disease often has a relatively early onset, and diabetic patients with coronary heart disease often manifest as painless myocardial infarction, the infarction area is relatively large, there are more perforating infarctions, the disease is more serious, the prognosis is relatively poor, and the mortality rate is higher; Such as coronary angiography and clinical exclusion of coronary artery disease, diabetic patients with severe arrhythmia, cardiac hypertrophy, pulmonary congestion and congestive heart failure, especially refractory heart failure, can be considered clinically diabetic cardiomyopathy
.

Among them, diabetic cardiomyopathy (DCM) is a more representative disease
.
Since it only occurs in patients with diabetes, it cannot be explained by hypertensive heart disease, coronary atherosclerotic heart disease, and other cardiac changes
.

It is due to extensive focal myocardial necrosis on the basis of metabolic disorders and microangiopathy, and the cause is related to cardiomyocyte metabolism disorders and calcium transport defects of cardiomyocytes [1].

If not treated properly, cardiac fibrosis and hypertrophy can lead to heart failure and even death [2].

【Pathogenesis】

Cytopyrosis (cellular inflammatory necrosis) is a programmed cell death that occurs in the presence of inflammation [3] characterized by the continuous enlargement of cells until the cell membrane ruptures, triggering a pronounced inflammatory response
.
In response to different types of stimuli, such as a hyperglycemic environment, cells produce various NRL receptors, such as NLRP3 inflammasomes
.
It then activates the lysis of caspase-1 (cl-Caspase-1, a proteolytic enzyme).

cl-Caspase-1 lyses the pore-forming cell death execution factor gasdermin-D (GSDMD), GSDMD translocates to membrane-forming pores, Leads to the release of interleukin (IL)-18 and IL-1β [3, 4].
】
。

Cytopyrosis involves a variety of cardiovascular diseases, such as myocardial infarction [5, 6], ischemia-reperfusion injury [7], and cardiomyopathy [8, 9].

。 All of these studies suggest that finding an intervention that targets DCM cell pyroptosis is an effective way
to prevent and treat diabetic cardiomyopathy.

Another pathogenesis of DCM is fibrosis, marked primarily by ventricular remodeling, resulting in ventricular wall stiffness and diastolic dysfunction, ultimately inducing heart failure
.
Hyperinsulinemia and hyperglycemia stimulate activation of the reninangiotensin-aldosterone pathway in DCM, leading to activation of transforming growth factor-β1 (TGF-β1).
10】
。

TGF-β1 is a profibrotic cytokine that phosphorylates
Smad.
Thus, the TGF-β1/Smad signaling pathway acts as a downstream effector, modifying target gene expression and ultimately leading to cardiac fibrosis and systolic/diastolic dysfunction[11].
TGF-β1 has become one of the most important targets of antifibrotic therapy [12], which is of great significance
for delaying disease progression and reducing the occurrence of heart failure.

Therefore, finding effective interventions that can selectively inhibit cell pyrosis and fibrosis is of great clinical significance
for the prevention of DCM.

[Feasibility analysis of hydrogen for DCM treatment].

Hydrogen acts as a therapeutic antioxidant by selectively reducing cellular oxygen radicals, especially the most harmful reactive oxygen species (ROS) [13].

In recent years, hydrogen has been shown to have anti-inflammatory, antioxidant and anti-fibrotic properties [14, 15].

There is growing evidence that hydrogen is effective for ischemic heart disease [16], ischemia-reperfusion injury of the small intestine and lungs [17, 18], and hemorrhagic shock [19
。

As a first-line drug for the treatment of diabetes [20], metformin has been shown to have hypoglycemic effects and cardioprotective effects [21].

However, hepatic and renal insufficiency limits its use in high doses [22].

Hydrogen inhalation has multiple mechanisms of action, has no side effects, is safe, and is easy to use [23, 24], so hydrogen combined with metformin may be a new therapeutic strategy
.

【Research Experiment】

A team from the Department of Cardiology and Laboratory Medicine of the Fourth Affiliated Hospital of Harbin Medical University in China conducted this research, and the results were published in Free Radical Biology and Medicine
.

In the experiment, 4-week-old male C57BL/6 mice were selected and randomly divided into five groups: control group (n = 10) and diabetes mellitus group (DM, n = 10), inhaled hydrogen treatment group (DM + H2, n = 10), metformin treatment group (DM + Met, n = 10).
and metformin and hydrogen treatment groups (DM+Met+H2, n=10).

All mice are captive and acclimatized under standard conditions and fed normal food until the end of
the study.

Then, a diabetes model was established for mice in the diabetic group (blood glucose value ≥16.
7 mmol/L confirmed that the diabetes model was successfully established) [25,26].

Next, the groups were treated
as follows.
inhalation of hydrogen therapy group, inhalation of 2% hydrogen per day for 3 hours; In the metformin treatment group, metformin (200 mg/kg/d, Sigma) was added to drinking water [25, 27]; Metformin and hydrogen treatment groups, receiving combination therapy with hydrogen and metformin
.
All mice were housed for 8 weeks, after which they were tested
.

【Experimental Results】

Experimental result 1: Hydrogen gas improved cardiac dysfunction and abnormal morphological structure in diabetic mice

The research team used echocardiography to assess changes in
heart function.
Mice in the diabetes group had significant cardiac systolic and diastolic dysfunction, a change that was greatly reduced
in the hydrogen finger treatment group.

These changes were shown in data comparisons for left ventricular ejection fraction (EF%), left ventricular short axis shortening rate (FS%), end-diastolic left ventricular diameter (LVIDd), and end-systolic left ventricular diameter (LVIDs).

。

Myocardium of mice in the diabetes group showed severe sarcomere disorder and mitochondrial (M) swelling, which was not seen in the control group, and sarcomere disorder and mitochondrial swelling were significantly reduced
in the hydrogen treatment group.

Brain natriuretic peptide (BNP) concentrations were significantly elevated in the diabetes group, a change reversed in the hydrogen treatment group
.
Taken together, these data suggest that hydrogen can alleviate heart dysfunction and abnormalities
.

Results 2: Hydrogen gas reduced the expression of cellular pyrozotic protein mediated by NLRP3 in diabetic mice

To determine the efficacy of hydrogen on cytopyrosis in diabetic mice, the team looked at the levels
of proteins associated with cell pyrosis.

It was found that NLRP3, cl-Caspase-1, ASC, IL-18, IL-1β and GSDMD-N levels were elevated in mice in the diabetic group; Hydrogen inhalation therapy effectively restored the levels of
these inflammatory cytokines.

In addition, compared with mice in the diabetes group, the levels of NLRP3, cl-Caspase-1 and IL-1β were lower
in the hydrogen treatment group.
The above results show that hydrogen can reduce pyroptosis
of cardiac NLRP3-associated cells in diabetic mice.

Results 3: Hydrogen reduced the expression of TGF-β1-mediated fibrotic protein in diabetic mice

Excessive myocardial fibrosis is another important feature of dilated cardiomyopathy that makes treatment difficult
.
The TGF-β1/SMAD signaling pathway plays a crucial role in the process of myocardial fibrosis [10, 11].

To verify the efficacy of hydrogen on myocardial fibrosis in diabetic mice, the team looked at fibrosis-related protein levels
.

TGF-β1, p-smad3, p-smad2, COL-I, COL-III of mice in the diabetic group compared with the control group and α-SMA levels were significantly elevated, while levels of these proteins were significantly reduced
after inhaled hydrogen therapy.

Immunohistochemical staining tests, which also confirmed these findings, showed that hydrogen inhalation reduced levels of TGF-β1, COL-I, and COL-III in the diabetes group
.
The above results show that hydrogen can reduce cardiac fibrosis
in diabetic mice.

Results 4: Inhalation of hydrogen under hyperglycemic (HG) conditions inhibited cell pyroptosis by reducing the AMPK/mTOR/NLRP3 signaling pathway

After confirming the role of hydrogen in mitigating cell pyroptosis in diabetic mice, the next step is to elucidate its underlying mechanism
.
Previous studies have confirmed that hydrogen-rich saline can reduce endotoxin-induced acute lung injury through the AMPK/mTOR signaling pathway [28], and metformin alleviates diabetes through the AMPK/mTOR pathway [25
。 It is hypothesized that hydrogen may also inhibit cell pyroptosis
by mediating the AMPK/mTOR/NLRP3 pathway in the diabetes group.

To better understand the interactions between hydrogen, AMPK, and NLRP3, the team extracted primary cardiomyocytes and used hydrogen to intervene in a high-glucose environment (labeled HG+H2) and without hydrogen in a high-sugar environment (tagged HG) treated with 5.
0 mmol/L (control) and 30 mmol/L glucose
.
Compound C (labeled CC) is a selective, ATP-competitive AMPK inhibitor that has been shown to worsen high glucose-induced cell damage and act as a blocker Hydrogen was used to intervene in a high-glucose environment, and Compound C (labeled HG+H2+CC) was added to administer high-glucose treated cardiomyocytes
.

Hoechst33342/PI double fluorescence staining was used to detect high glucose induced cell death, and the results showed that the increase in high glucose induced primary cardiomyocyte death was reversed by hydrogen, and this protective effect was destroyed
by CC intervention.

The activity and degree of damage of cardiomyocytes were detected by CCK8 assay and LDH release assay, and the results were consistent with the results of Hoechst33342/PI double fluorescent staining, indicating CC Destroys the protective effect
of hydrogen.

Subsequently, Western blot testing showed a decrease
in p-AMPK levels in the HG group.
After hydrogen treatment, HG+H2 group mTOR, IL-18, IL-1β, cl-Caspase-1, The levels of ASC, GSDMD-N and NLRP3 were significantly lower than those in the HG group.
This protection is destroyed by CC (HG+H2+CC).

These findings suggest that hydrogen inhibits cell pyroptosis via the AMPK/mTOR/NLRP3 axis.

Results 5: Inhalation of hydrogen in a high-glucose environment alleviated fibrosis by inhibiting the TGF-β1/Smad pathway

After confirming that hydrogen reduced myocardial fibrosis in diabetic mice and that hydrogen reduced levels of TGF-β1, p-smad3, and p-smad2, the research group explored the relationship between hydrogen and TGF-β1
。

Take cardiac fibroblasts and treat them in a high-glucose environment (HG+H2) or a high-glucose environment (HG) without hydrogen intervention, treated at 5.
5 mmol/L (control) and Incubate in 30 mmol/L glucose solution; Exogenous TGF-β1 is placed in a high glucose environment (labeled HG + H2 + TGF-β1) in a high glucose environment intervened with hydrogen.

According to western blotting, TGF-β1, p-smad3, p-smad2, COL-I, COL-III and α-SMA levels were significantly higher in high-sugar environments without hydrogen intervention; After hydrogen intervention in a high sugar environment (HG + H2) treatment, the level was significantly reduced; With the addition of exogenous TGF-β1, the protective effect of hydrogen is destroyed
.

Immunofluorescence detection of TGF-β1 confirmed this finding

Cardiac fibroblast (CFs) migration is another cause
of pathological fibrosis.
Transwell (for detecting cell migration) is used to assess the effect of
hydrogen on the migration of CFs.

The results showed that the migration of CFs in the heart increased in the high-sugar environment (HG) without hydrogen intervention, and hydrogen intervention (HG+H2) effectively prevented migration, but this protective effect was affected by exogenous TGFβ1 Sabotage
.

Results 6: Metformin + hydrogen treatment inhibited diabetes-induced histopathological changes in mice

Although metformin is a first-line agent, high-dose use in patients with hepatic and renal insufficiency has historically been contraindicated due to concerns about the increased risk of side effects [22].

Hydrogen is a physiologically inert gas that does not react with any active compounds [29] and is very easy to use
.
Next, the research team analyzed the response
of diabetic mice to the combination administration of metformin and hydrogen.

The experimenters divided the mice into 5 groups, control group (n=20), diabetes mellitus (DM, n=20), inhaled hydrogen therapy group (DM+H2).
, n = 20), metformin treatment group (DM + Met, n = 20), and metformin and hydrogen treatment group (DM+Met+H2 ，n=20）
。 After 2 months, the number of survivors in each group was 20, 10, 14, 13, and 13 17
。

Both metformin and hydrogen treatment improved the survival of mice, while metformin and hydrogen combined administration showed more significant protection, in addition, metformin alone (DM + Met) or in combination with hydrogen (DM + Met + H2).
) significantly reduces fasting blood glucose levels
.
Fasting blood glucose levels were slightly lower
in the DM+H2 group compared with the DM group.

Left ventricular ejection fraction (EF%), left ventricular short axis shortening rate (FS%), end-diastolic left ventricular diameter (LVIDd) and end-systolic left ventricular diameter (LVIDs) decreased, It has been shown that hydrogen combined with metformin treatment exerts a more significant effect
than monotherapy.

HE staining (hematoxylin-eosin staining) showed structural abnormalities and hypertrophy of myocardium in the diabetic group, which could be alleviated by a single dose of metformin or hydrogen, while the combination of metformin and hydrogen was more effective
.

Masson staining (green fixing method) showed that the degree of interstitial fibrosis in the DM+Met+H2 group was lower than that in the monotherapy group
.
In addition, transmission electron microscopy showed that the diabetic group was deranged, mitochondrial swelling, and nucleus solidification; In the DM+Met and DM+H2 groups, this problem was alleviated; In the DM+Met+H2 group, damage such as sarcomere disorder, mitochondrial swelling, and nuclear contraction was almost completely eliminated
.

Results 7: Compared with the intervention alone, metformin combined with hydrogen intervention reduced cell pyrosis and fibrosis in diabetic mice

Hydrogen inhibits cell pyroptosis and fibrosis in mouse models of diabetes, and metformin has been shown to have anti-cell pyrosis and antifibrosis properties [25, 30].

The research team evaluated the effect
of metformin and hydrogen gas on the expression of pyrozowhosis-associated protein and fibrosis-related protein in diabetic mice by measuring the protein by western blotting and immunohistochemistry.

Both metformin and hydrogen reduced the expression of NLRP3, cl-Caspase-1, and IL-1β, while combination therapy significantly reduced protein expression
.

Western blot detection also found TGF-β1, p-smad3, p-smad2 and in the DM+Met+H2 group α-SMA levels, lower than the DM+H2 and DM+Met groups
.

In addition, immunohistochemical testing has confirmed a similar phenomenon
.
These results suggest that the co-administration of metformin and hydrogen gas shows better anti-pyrozoosis and antifibrosis effects on DCM compared to administration alone
.

Result 8: In the environment of hyperglycemia (HG), metformin combined with hydrogen intervention further reduced cell damage
.

According to the CCK-8 test (cell proliferative toxicity test), hyperglycemia significantly reduces the activity
of cardiomyocytes.

Both metformin and hydrogen increased the activity of cardiomyocytes exposed to hyperglycemia, while co-administration of metformin and hydrogen provided better protection (Figure A); The results of the LDH release test also confirm the above conclusions (Figure B).

The results of western blot analysis showed that hyperglycemia upregulated cell pyrozowhosis-associated proteins, including IL-1β, cl-Caspase-1, ASC, and NLRP3
.

The protein expression levels of metformin and hydrogen co-administration groups were significantly lower than those in the monotherapy group
.
These data suggest that co-administration of metformin and hydrogen can reduce cell pyrosis under hyperglycemic conditions, thereby minimizing cell damage
.

Results 9: In the hyperglycemic environment, metformin combined with hydrogen intervention further alleviated fibrosis
.

Western blot test found that the hyperglycemic environment increased fibrosis-related proteins TGF-β1, p-smad3, p-smad2, COL-I, Protein expression levels
of COL-III, α-SMA.

Combination therapy reduces the expression
of fibrosis-related proteins more than monotherapy.

In addition, the immunofluorescence staining detection of α-SMA also verifies the above conclusions
.

The Transwell experiment (cell invasion experiment) with cardiac fibroblasts (CFs) migration showed that hyperglycemia increased the migration capacity of CFs, while treatment with metformin or hydrogen reduced the migration capacity
of CFs 。 The value of concern is that this migration capacity reaches the lowest water when metformin and hydrogen are administered in combination, indicating a higher
efficacy of combination therapy.

【Result Analysis】

The research team pointed out that this is the first time that inhalation of hydrogen can be shown to effectively reduce heart damage
in diabetic mice by inhibiting cell pyrosis and fibrosis.
Evidence suggests that hydrogen reduces pyroptosis by reducing the AMPK/mTOR/NLRP3 signaling pathway and mitigating fibrosis
by inhibiting the TGF-β1/Smad signaling pathway.

Further studies confirmed that metformin and hydrogen co-administration had a better protective effect against diabetic heart disease (DCM) compared to dosing alone, suggesting that hydrogen can be used in combination with metformin to mitigate DCM
.
These findings provide a candidate strategy
for treating diabetic heart injury.

Due to aging, obesity and diabetes, the incidence of heart failure and its associated morbidity and mortality is increasing
at an alarming rate.
It is well known that when it comes to heart failure, patients with diabetes have worse clinical outcomes than non-diabetic patients [2].

Complications of diabetes, such as dilated cardiomyopathy, diabetic nephropathy and diabetic retinopathy, are the most
harmful to humans.

Hydrogen is a medical gas
found after NO, CO and H2S.
It has a wide range of beneficial properties, including antioxidant, anti-inflammatory, anti-apoptosis, anti-fibrotic, anti-allergic, and energy-metabolic-stimulating properties [14, 15].

Hydrogen improved cardiac dysfunction in diabetic mice, as evidenced by an increase in left ventricular ejection fraction (EF%), left ventricular short axis shortening rate (FS%), and a decrease in brain natriuretic peptide (BNP) concentration.

According to HE and Masson staining tests, symptoms such as myocardial structural abnormalities, hypertrophic phenotype, and collagen deposition are reduced
by hydrogen inhalation.
These data confirm for the first time the cardioprotective role
of hydrogen in DCM.

Hydrogen in combination with metformin exerts cardioprotective effects in DCM

There is increasing evidence that cytopyrosis can cause cardiac damage to DCM, and inhibition of cytopyrosis can greatly improve prognosis [8, 31, 32].

In this study, the levels of cell pyrozoosis-related proteins (including NLRP3, cl-Caspase-1, ASC, IL-1β, etc.
) in diabetic mice were obtained IL-18 and GSDMD-N) were significantly elevated, while these indicators were significantly reduced after hydrogen inhalation, which was associated with
significant improvements in cardiac abnormalities and cardiac dysfunction.
Western blot analysis showed that hydrogen increased the expression of p-AMPK and reversed the expression of pyrozowhosis-associated proteins, including NLRP3, GSDMD-N, and cl-Caspase-1 , ASC, IL-1β and IL-18 once again verified the above experimental results
.
These findings suggest that hydrogen reduces cell pyroptosis by inhibiting the AMPK/mTOR/NLRP3 signaling pathway.

Myocardial fibrosis is another important pathological feature of
diabetes.
Fibrosis is attributed to excess cardiac fibroblasts (CFs).

TGF-β1 is the main player of the fibrosis process of the heart, which is produced
by CFs.
The TGF-β1/Smad signaling pathway was found to be activated in both diabetic mice and CFs treated in hyperglycemic environment (HG), TGF-β1 This is evidenced by the increased production of p-smad3, p-smad2, COL-I, and COL-III
.
Hydrogen inhibited the expression of fibrosis-related proteins in CFs treated in diabetic mice and hyperglycemic environment (HG), indicating that hydrogen prevented fibrosis
by inhibiting the TGF-β1/Smad signaling pathway.

Taken together, these results suggest that hydrogen has anti-pyroptosis and anti-fibrosis properties in DCM, which may provide an explanation
for how hydrogen improves cardiac dysfunction and abnormal morphological structure.

Previous evidence suggests that oxidative stress is strongly associated with cell pyrosis and fibrosis [33, 34].

Excess ROS caused by hyperglycemia destroys mitochondrial and nuclear DNA, promoting the development of DCM [35].

The use of antioxidants to reduce ROS levels in diabetic patients has been shown to minimize myocardial fibrosis and improve myocardial contractility [36].

As a new medical gas, hydrogen was first found to reduce the formation of ROS [13].

Given its different properties, hydrogen may also participate in protecting DCM
by mitigating redox reactions.

【Research Conclusion】

Although metformin is more effective at reducing glycosylated hemoglobin, side effects, including gastrointestinal problems, drug-induced dermatitis, and lactic acidosis, become apparent
after considerable clinical use.
The negative effects of metformin, especially in people with liver or kidney disease, limit its use in high doses [22].

To reduce side effects, metformin-based combination therapies
are now widely used.

In the above study, the co-administration of metformin and hydrogen gas further reduced the expression of cytopyrosis-associated protein and fibrosis-associated protein in diabetic mice compared to a single dose, even in a hyperglycemic environment (HG
).
Hydrogen gas has a similar effect to metformin, which has been confirmed by immunohistochemistry, immunofluorescence, and transwell assays.

Both hydrogen and metformin can reduce cell pyrosis and fibrosis, and they can function
largely in parallel pathways.

Hydrogen can be used in combination with metformin and shows a stronger cardioprotective effect
in dilated cardiomyopathy.
Therefore, the research team recommends a combination of metformin and hydrogen for diabetic heart disease (DCM).

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