Table 2.

Effects of hydrogen treatment on cardiac ischemia − reperfusion injury models: reports from in vivo studies

Study model	Intervention		Major findings					Interpretation	References
	Dose/Duration	Route	Cardiac function/Cardiac injury marker	Oxidative stress	Inflammation	Apoptosis	Autophagy/Mitophagy
Male Wistar rats I/R model 30 min/24 h	Hydrogen-rich saline under 0.4 MPa dissolved in saline for 6 h (0.6 mmol/l)/10 ml/kg/5 min prior to reperfusion	IP	↑ HR ↑ MAP ↑ SBP ↑ DBP ↑ LV + dP/dt ↑ LV − dP/dt ↑ LVEF ↓ LVEDP ↓ Infarct size ↓ CK-MB ↓ cTnI	-	↓ IL-1 $\beta$ ↓ IL-6 ↓ TNF-$\alpha$ ↓ HMGB1	↓ TUNEL ↓ BAX ↓ Caspase 3 ↑ Bcl‑2/Bax ratio	↑ LC3II/I ↑ ATG5 ↑ ATG12 ↑ Beclin 1 ↑ PINK1 ↑ Parkin	Hydrogen-rich saline alleviated myocardial infarct size, reduced inflammation, apoptosis, and promoted autophagy and mitophagy, leading to improved left ventricular function and hemodynamics following cardiac I/R injury	[15]
Male SD rats I/R model 30 min/24 h	Hydrogen-rich saline under 0.4 MPa dissolved in saline for 6 h (0.6 mmol/l)/5 ml/kg/5 min before reperfusion	IP	↑ LVSP ↓ LVDP ↑ LV + dP/dt ↑ LV− dP/dt ↓ Infarct size	↓ MDA in tissue and plasma ↓ 8-OHdG	–	↓ TUNEL ↓ Caspase 3	–	Hydrogen-rich saline improved cardiac function and reduced infarct size from I/R injury by reducing oxidative stress and apoptosis	[5]
Male SD rats I/R model 30 min/24 h	Hydrogen-rich saline under 0.4 MPa dissolved in saline for 6 h (0.6 mmol/l)/10 ml/kg/5 min prior to reperfusion	IP	↑ LVSP ↓ LVEDP ↑ LV + dP/dt ↓ LV − dP/dt ↓ Infarct size ↓ PMN accumulation ↓ CK-MB ↓ cTnI	↓ MPO ↓ 3-nitrotyrosine	↓ IL-1 $\beta$ ↓ TNF-$\alpha$ ↓ ICAM-1	–	–	Hydrogen-rich saline improved cardiac function and reduced infarct size by reducing oxidative stress and inflammation	[20]
Male SD rats I/R model 45 min/3 min, 30 min, or 24 h	Hydrogen-rich saline under 0.4 MPa dissolved in saline for 4 h (60 μL)/NA/at onset of reperfusion	Injected into the myocardial tissue around the infarct zone	↑ LV + dP/dt ↑ LV − dP/dt ↔ Infarct size ↓ CK ↓ CK-MB	↓ MDA ↑ SOD	↓ TNF-$\alpha$	↓ TUNEL ↓ Cyt-c ↓ Caspase-8 ↓ p-p38 ↓ p-JNK ↓ p-ERK	–	Hydrogen-rich saline improved cardiac function from I/R injury by reducing oxidative stress, inflammation, apoptosis, and regulating the MAPK signal pathway	[21]
Male Wistar rats I/R model 30 min/ 24 h	2% H2/at onset of ischemia and continue for 60 min after reperfusion	Inhalation	↓ LVEDP ↓ LVEDd ↓ LVESd ↑ IVS ↑ PW ↑ FS ↑ EF ↓ Infract size	↓ 8-OHdG	–	–	–	Inhalation of H2 improved cardiac function and reduced infarction by reducing oxidative stress after I/R injury	[22]
Male Wistar rats I/R model 1 h/2 h	2% H2/5 min before reperfusion until 2 h after reperfusion	Inhalation	↓ Infarct size ↓ TnI	↓ 8-OHdG ↓ MDA ↓ ROS ↓ TRAF2 ↓ GRP78	–	↓ p-Bcl-2/Bcl2	↓ LC3II/I ↓ Beclin 1	Inhalation of 2% H2 gas attenuated myocardial injury by attenuating ER stress, oxidative stress, apoptosis and autophagy	[23]
	Postischemic conditioning treatment (Four cycles of 1 min reperfusion/1 min ischemia (total time, 8 min) was given at the end of 1 h coronary occlusion)		↓ Infarct size ↓ TnI	↓ 8-OHdG ↓ MDA ↓ ROS ↓ TRAF2 ↓ GRP78	–	↓ p-Bcl-2/Bcl2	↓ LC3II/I ↓ Beclin 1
	2% H2 combined with postischemic conditioning treatment		↓ Infarct size ↓ TnI	↓ 8-OHdG ↓ MDA ↓ ROS ↓ TRAF2 ↓ GRP78	–	↓ p-Bcl-2/Bcl2	↓ LC3II/I ↓ Beclin 1
Swine I/R model Myocardial stunning 12 min/90 min	2% H2/during and after ischemia	Inhalation	↓ Incidence of VF, VT ↑ SS	–	–	–	–	Inhalation of 2% H2 gas during I/R improved cardiac function and reduced VF/VT incidence from myocardial stunning, while inhalation of 4% H2 gas during I/R reduced infarct size	[24]
Myocardial infarction 40 min/120 min	4%H2/during and after ischemia	Inhalation	↓ Infarct size	–	–	–	–
Male SD rats CBP model (1 h)	Hydrogen-rich water under 0.8 MPa dissolved in saline for 24 h/6 ml/kg/prior to hypoxia and during reoxygenation	IV injection via tail vein	↑ MAP ↑ LV + dP/dt max ↓ LDH ↓ CK-MB	↓ MDA ↓ MPO ↑ SOD	↓ IL-1 $\beta$ ↓ IL-6 ↓ TNF$\alpha$	↓ TUNEL ↓ BAX ↓ caspase 3 ↑ Bcl-2	-	Hydrogen-rich water improved cardiac function and reduced cardiac injury by reducing oxidative stress, inflammation, and apoptosis	[6]
Male Lewis rats Heterotopic heart transplantation (I/R model) 6 or 18 h/6 h	1%, 2%, 3%H2/1 h before ischemia and 1 h after reperfusion 1% H2 2% H2 3% H2	Inhalation	↔ CPK ↓ CPK ↓ CPK	–	–	–	–	The combination of hydrogen and CO therapy reduced infarct size, cardiac injury, and enhanced cardiac graft survival by decreasing oxidative stress, inflammation, and apoptosis	[11]
	CO (After 6 h cold ischemia) – CO: 50 ppm – CO: 250 ppm	Inhalation	↔ CPK ↓ CPK	–	–	–	–
	Inhaled gas (After 18 h cold ischemia) Mixed H2 and CO		↓ Infarct size ↓ Macrophage ↓ CPK ↓ cTnI ↑ Transplantation score (3 h after storage) ↑ Graft survival after 7 days	3 h after perfusion ↓ MDA ↓ MPO 6 h after perfusion ↓ MPO	3 h after perfusion ↓ IL-1 $\beta$ ↓ IL-6 ↓ TNF $\alpha$ ↓ iNOS ↓ HMGB1	↓TUNEL ↓ ED1 ↓ cleaved caspase 3	–
	H2 alone	Inhalation	↑ Transplantation score (3 h after storage) ↑ Graft survival after 7 days	3 h after perfusion ↓ MDA 6 h after perfusion ↔ MPO	3 h after perfusion ↔ IL-1 $\beta$ ↔ IL-6 ↔ TNF $\alpha$ ↔ iNOS ↓ HMGB1	↔ TUNEL ↔ ED1 ↔ cleaved caspase 3	–
Inbred male LEW (RT1l) and BN (RT1n) rats Heterotopic heart transplantation (I/R model)	Hydrogen-rich water/dose: NA/60 d, 100d	Oral	↑ Viability of cardiac allografts ↑ Tissue ATP ↑ Mito activity ↓ CD3 + T cells ↓ CD68 + macrophages	50 d after transplant ↓ MPO ↓ MDA	↓ IFN $\gamma$ ↓ TNF $\alpha$ ↓ CCL2 ↓ CCL5 ↓ MCP-1	–	–	Hydrogen-rich water enhanced cardiac allograft survival by reducing intimal hyperplasia, inhibition of T cell proliferation, reduction of oxidative stress and increased tissue ATP and mitochondrial activity	[25]
Male LEW (RT1l) and BN (RT1n) rats Orthotopic Aortic transplantation			↓ Intimal hyperplasia in aortic graft

AAR area at risk, Akt protein kinase B, ATG autophagy-related protein, ATP adenosine triphosphate, BAX apoptosis regulator Bax, Bcl-2 apoptosis regulator Bcl-2, BNP brain natriuretic peptide, CBF coronary blood flow, CCL chemokine (C–C motif) ligand, CD cluster of differentiation, CK-MB creatinine kinase-MB, CO carbon monoxide, CPB cardiopulmonary bypass, CPK creatine phosphokinase, cTnI cardiac troponin-I, Cyt-c cytochrome c, DBP diastolic blood pressure, EF ejection fraction, ER endoplasmic reticulum, ERK extracellular signal-regulated kinase, FS fractional shortening, GRP78 glucose-regulated protein 78, HO1 heme oxygenase 1, HMGB1 high mobility group box 1, HRS hydrogen-rich saline, HR heart rate, ICAM-1 intercellular adhesion molecule 1, IFN $\gamma$ interferon $\gamma$, IL interleukin, iNOS inducible nitric oxide synthase, IP intraperitoneal, IVS interventricular septum, JNK c-Jun-N-terminal Kinase, LC3 microtubule-associated protein 1 light chain 3 $\alpha$, LDH lactate dehydrogenase, LV left ventricle, LVAWd LV anterior wall thickness at end-diastole, LVEDd LV endodiastolic diameter, LVEDP left ventricular end-diastolic pressure, LV dp/dt rate of pressure change in left ventricle, LVSP LV systolic pressure, LVDP LV diastolic/developed pressure, LVDP LV developed pressure (LVSP-LVDP), LVEDd LV endodiastolic diameter, LVESd LV endosystolic diameter, LVPWd LV posterior wall thickness in diastole, LVW left ventricular weight, IVS intraventricular septum diameter, MAP mean arterial pressure, MAPK mitogen-activated protein kinase, MCP-1 monocyte chemotactic protein-1, MDA malondialdehyde, Mfn2 mitofusin-2, MPO myeloperoxidase, Nrf2 the nuclear factor erythroid 2-related factor2, ·OH hydroxyl radicals, p phosphorylation, PERK protein kinase-RNA-like endoplasmic reticulum kinase, PI3K phosphatidylinositol 3-kinase, PINK PTEN-induced kinase 1, PMN polymorphonuclear neutrophil, PW posterior wall thickness, p38 38-kDa protein, ROS reactive oxygen species, SBP systolic pressure, SD Sprague Dawley rat, SOD superoxide dismutase, SpO2 pulse oximetry, SS segment shortening, TnI troponin I, TNF-$\alpha$ tumor-necrosis factor-$\alpha$, TRAF2 tumor-necrosis factor-a (TNF-a) receptor-associated factor 2, VF ventricular fibrillation, VT ventricular tachycardia, 8-OHdG 8-hydroxydeoxyguanosine