H2 Protects Against Lipopolysaccharide-Induced Cardiac Dysfunction via Blocking TLR4-Mediated Cytokines Expression

Background and Purpose: Septic cardiomyopathy, which is one of the features of multi-organ dysfunction in sepsis, is characterized by ventricular dilatation, reduced ventricular contractility, and reduction in ejection fraction and, if severe, can lead to death. To date, there is no specific therapy that exists, and its treatment represents a large unmet clinical need. Herein, we investigated the effects and underlying anti-inflammatory mechanisms of hydrogen gas in the setting of lipopolysaccharide (LPS)-induced cardiomyocytes injury. Experimental Approach: Hydrogen gas was intraperitoneally injected to mice in LPS plus hydrogen group and hydrogen group for 4 days. On fourth, LPS was given by intraperitoneal injection to mice in LPS group and to mice in LPS plus hydrogen group. In addition, H9c2 cardiomyocytes were treated with hydrogen-rich medium for 30 min before LPS. The transthoracic echocardiography was performed at 6 h post‐LPS to assess left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (EF%), fractional shortening (FS%), left ventricular mass average weight (LV mass AW), and LV mass AW (Corrected). The histological and morphological analyses of left ventricular were performed by hematoxylin and eosin (H&E) staining and Masson’s trichrome staining. The mRNA levels of ANP and BNP were examined by PCR in vitro. The expression of cytokines were assayed by Enzyme Linked Immunosorbent Assay (ELISA) and PCR. Moreover, Western blotting was performed to examine the expression of TLR4, the activation of ERK1/2, p38, JNK, and the expression of NF-κB in nucleus after 6 h of LPS challenge in vivo and in vitro. Key Results: LPS induced cardiac dysfunction; hydrogen therapy improved cardiac function after LPS challenge. Furthermore, pretreatment with hydrogen resulted in cardioprotection during septic cardiomyopathy via inhibiting the expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-18; suppressing the phosphorylation of ERK1/2, p38, and JNK; and reducing the nuclear translocation of NF-κB and the expression of TLR4 by LPS. Conclusion and Implications: Hydrogen therapy prevents LPS-induced cardiac dysfunction in part via downregulation of TLR4-mediated pro-inflammatory cytokines expression.

Background and Purpose: Septic cardiomyopathy, which is one of the features of multiorgan dysfunction in sepsis, is characterized by ventricular dilatation, reduced ventricular contractility, and reduction in ejection fraction and, if severe, can lead to death. To date, there is no specific therapy that exists, and its treatment represents a large unmet clinical need. Herein, we investigated the effects and underlying anti-inflammatory mechanisms of hydrogen gas in the setting of lipopolysaccharide (LPS)-induced cardiomyocytes injury.
Experimental Approach: Hydrogen gas was intraperitoneally injected to mice in LPS plus hydrogen group and hydrogen group for 4 days. On fourth, LPS was given by intraperitoneal injection to mice in LPS group and to mice in LPS plus hydrogen group. In addition, H9c2 cardiomyocytes were treated with hydrogen-rich medium for 30 min before LPS. The transthoracic echocardiography was performed at 6 h post-LPS to assess left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (EF%), fractional shortening (FS%), left ventricular mass average weight (LV mass AW), and LV mass AW (Corrected). The histological and morphological analyses of left ventricular were performed by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. The mRNA levels of ANP and BNP were examined by PCR in vitro. The expression of cytokines were assayed by Enzyme Linked Immunosorbent Assay (ELISA) and PCR. Moreover, Western blotting was performed to examine the expression of TLR4, the activation of ERK1/2, p38, JNK, and the expression of NF-κB in nucleus after 6 h of LPS challenge in vivo and in vitro.
Key Results: LPS induced cardiac dysfunction; hydrogen therapy improved cardiac function after LPS challenge. Furthermore, pretreatment with hydrogen resulted in cardioprotection during septic cardiomyopathy via inhibiting the expression of proinflammatory cytokines TNFα, IL-1β, and IL-18; suppressing the phosphorylation INTRODUCTION Sepsis is a clinical syndrome, affecting ∼19 million individuals per year worldwide, characterized by a maladaptive host response with ensuing life-threatening organ dysfunction resulting from infection (Reinhart et al., 2017;Weis et al., 2017). Cardiac involvement frequently complicates to sepsis, which, if severe, can lead to death (Havaldar, 2018;Martin et al., 2019;Ward and Fattahi, 2019). Characteristically, early studies indicate cardiac dysfunction in adequately volume-resuscitated septic patients with increased end-diastolic volume index and decreased ejection fraction, and these acute changes in ejection fraction and end-diastolic volume index, although sustained for several days, were reversible (Weisel et al., 1977;Calvin et al., 1981;Parker et al., 1984). More recently, studies in cellular levels, isolated heart studies, animal models in vivo, and in human studies, have clearly established decreased contractility and impaired myocardial compliance as major factors that cause myocardial dysfunction in sepsis (Merx and Weber, 2007;Martin et al., 2019).
The bacterial endotoxin lipopolysaccharide (LPS), which is a component of the outer membrane of Gram-negative bacteremia, has been regarded as a main culprit responsible for cardiac dysfunction in sepsis (Bai et al., 2016). Mechanistically, LPS associates with its receptor toll-like receptor 4 (TLR4) through the help of LPS-binding protein CD14 and, subsequently, results in the production of inflammatory cytokines, such as TNFα, IL-1β, and IL-18, which might directly disturb cardiac function Raeburn et al., 2002;Zhang et al., 2017a). Although tremendous efforts have been made during the last decades, no specific therapy for sepsis-induced cardiomyopathy exists (Weis et al., 2017). Therefore, there is urgent need of innovative therapeutic options for sepsis-induced cardiomyopathy, which is essential for reducing the mortality of sepsis.
Hydrogen gas (H 2 ), a medical gas that has anti-oxidant, anti-apoptotic, and anti-inflammatory properties (Ohsawa et al., 2007;Yu et al., 2011;Zhai et al., 2013;Zhai et al., 2014;Li et al., 2016;Zhai et al., 2017), has been reported to improve sepsis-induced organ dysfunction, such as lung (Qiu et al., 2011;Liang et al., 2012;Xie et al., 2012;Hattori et al., 2015;Liu et al., 2015;Dong et al., 2017;Dong et al., 2018), liver Iketani et al., 2017), and bowel (Sakata et al., 2017). Recent studies have also indicated that H 2 has strong cardiovascular activities (Zhang et al., 2018). For example, intraperitoneal injection of H 2 can improve isoproterenol (ISO)-induced cardiac hypertrophy in vivo (Zhang et al., 2016a;Zhang et al., 2017b) and inhibit vascular hypertrophy induced by abdominal aortic coarctation in vivo . H 2 -rich medium suppresses ISO-induced H9c2 cardiomyocytes hypertrophy and angiotensin II-induced vascular smooth muscle cells proliferation and migration in vitro . However, the effects of H 2 intraperitoneal injection on sepsis-induced cardiomyopathy and the molecular mechanisms remain unclear. The aims of this study are, therefore, to determine the effect of H 2 intraperitoneal injection on LPSinduced cardiac dysfunction in vivo and the effect of H 2 -rich medium on LPS-induced H9c2 cardiomyocytes injury in vitro, as well as to identify the molecular mechanisms that may be involved in this process.

Drugs, Antibodies, and ELISA Kits
LPS (cat no. L2880, Sigma-Aldrich; MerekKGaA, Darmstadt, Germany) was dissolved in normal saline (1 mg/ml), under sterile conditions immediately prior intraperitoneal injection, and dissolved in double distilled water (1 mg/ml) as for cell culture study. H 2 (99.999%; Guang Zhou Guang Qi Gas Co., Ltd., Guangzhou, China) was stored in a seamless steel gas cylinder. H 2 was injected into a vacuumed aseptic soft plastic infusion bag (100 ml; CR Double-Crane Pharmaceuticals Co., Ltd, Anhui, China) under sterile conditions immediately prior to intraperitoneal injection for animal study. For cell culture study, the same vacuumed bag was injected with 20-ml Dulbecco's modified Eagle medium (DMEM; Gibco, New York City, NY, USA), which was supplemented with 100 U/ml penicillin/ streptomycin (Hyclone); then, the bag with DMEM was bubbling by H 2 until this bag was full of H 2 with no dead volume. The bag was maintained at 4°C for >6 h prior to use, and the concentration of H 2 was measured as our previously described Zhang et al., 2017b).

Animal Model of LPS-Induced Cardiac Dysfunction and Treatment Protocol
Male C57BL/6J mice (8 to 10 weeks of age) were used in this study. All animals were housed in a temperature-controlled animal facility with a 12-h light-dark cycle and allowed to obtain rodent chow and water ad libitum. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals published by the NIH (8 th Edition, Revised 2011) (Polhemus et al., 2017). The Institutional Animal Care and Use Committee (Zhongshan School of Medicine, Sun Yat-sen University) approved all animal procedures.

Cell Culture and Treatment
H 2 -rich medium was prepared as previously described (Zhang et al., 2017b). H9c2 cardiomyocytes were grown in DMEM containing 5.5-mM glucose (Zhang et al., 2017b). Cells were serum starved for 18 h in DMEM containing 1% FBS and then treated with H 2 -rich medium for 30 min before LPS; finally, the medium was added 1 μg/ml LPS. The expression or activation of kinases, NF-κB, TLR4, and cytokines were examined after 6 h of LPS challenge.

The Enzyme-Linked Immunosorbent Assay
The levels of TNFα, IL-18, and IL-1β in serum of mice were quantified by the commercial ELISA kits following manufacturer's instructions. The plates were read on a TECAN infinite F200 Plate Reader, measuring absorbance at 450 nm.

Quantitative Real-Time PCR (qRT-PCR)
Total mRNA was extracted from H9c2 cardiomyocytes using TRIZol reagent (Invitrogen) as previously described (Zhang et al., 2016a). The oligo (dT) primers with the Transcriptor First Strand cDNA Synthesis Kit (PrimeScript ™ RT Master Mix, Takara) were used to synthesize cDNA. qRT-PCR was performed using SYBR green (SYBR ® Premix Ex Taq ™ II, Takara) on a BIO-RAD CFX96 Touch ™ Real-time PCR Detection System. GAPDH was used as an internal control. The relative expression level of target genes was calculated using the 2 −ΔΔCt method. The primers for qRT-PCR are shown in Table 1.

Histological and Morphological Analyses
Hearts were harvested for observing histological and morphological alterations by hematoxylin and eosin (H&E) staining and Masson's trichrome staining as previously revealed (Zhang et al., 2016a;Xu et al., 2019).

Western Blotting
Western blotting was performed as we have previously described (Zhang et al., 2017b). The nuclear protein were isolated by the NE-PER ™ Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), which were incubated with primary and secondary antibodies by standard techniques. The enhanced chemiluminescence (ChemiDoc XRS+ System, Bio-Rad, Hercules, CA, USA) was used to accomplish immunodetection.

Statistical Analysis
Data are expressed as mean ± SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. A value of P < 0.05 was considered as significantly different.

H 2 Alleviates LPS-Induced Cardiac Dysfunction
We attempted to use intraperitoneal injection of H 2 , to test whether H 2 can suppress LPS-induced cardiac dysfunction. As previously described (Drosatos et al., 2013), 6 h after LPS challenge, mice exhibited cardiac dysfunction compared with the control group, as indicated by decreasing percent fractional shortening (FS%) and percent ejection fraction (EF%) and increasing LVESD, while

Genes
Species left ventricular end-diastolic diameter (LVEDD) was not affected (Figures 1A-E). H 2 injection alleviated the impaired cardiac function by LPS, as evidenced by increasing FS% and EF% and decreasing LVESD (Figures 1A-E). Therefore, intraperitoneal injection of H 2 can improve LPS-induced cardiac dysfunction.  (Liu et al., 2008). In our study, the upregulation of mRNA levels of ANP and BNP by LPS was suppressed by H 2 -rich medium pretreatment (Figure 2A). LPS slightly increased LV mass AW and LV mass AW (Corrected), and the mean of LV mass AW and LV mass AW (Corrected) were lower in H 2 plus LPS group than that in LPS group, although there was no statistical difference between groups ( Figure 2B). Similar to the increased in LVESD in LPS group (Figure 1C), H&E staining showed that LPS induced left and right ventricular cavities enlargement, decreased the thickness of interventricular septum, and H 2 improves these phenotype ( Figure 2C). Moreover, no obvious cardiac fibrosis was induced by LPS, H 2 , and H 2 plus LPS as determined by Masson's trichrome staining ( Figure 2D). As previously discussed (Liang et al., 2012;Xie et al., 2012), the formation of lung edema and the influx of immune-competent cells into the lung tissue by LPS were reduced by H 2 (Figure 2E). Collectively, these data suggested that H 2 has the potential ability to improve LPS-induced left ventricular structure injury in these mice.

H 2 suppresses TLR4-Mediated Innate Immune signaling
The increased cytokines previously discussed in LPS-challenged mice or in H9c2 cardiomyocytes are primarily induced by TLR4-mediated innate immune signaling, such as TRAF6-TAK1-IKKβ-NF-κB and TRAF6-MAPK-AP-1 signaling (Zhang et al., 2017a). Thus, we investigate whether the inhibition of H 2 on LPS-induced inflammatory cytokines production is related to TLR4-mediated innate immune signaling in cardiomyocytes. The immunoblotting indicated that the increased phosphorylation of p38MAPK, JNK1/2, and ERK1/2 (Figures 4A-F) and the nuclear translocation of NF-κB (Figures 5A, B) by LPS were suppressed by H 2 in vivo and in vitro. Moreover, the expression of TLR4 was increased after LPS stimulation, which was also blocked by H 2 in vivo and in vitro (Figures 6A, B). Collectively, TLR4-mediated innate immune signaling in cardiomyocytes can be inhibited by H 2 in vivo and in vitro.

DISCUSSION
The current studies were initiated to determine the effect of H 2 intraperitoneal injection on LPS-induced cardiac dysfunction and its immune mechanism. We find that H 2 protects against LPS-induced cardiac dysfunction via blocking TLR4 signalingmediated cytokines expression.
Being traditionally recognized as a biologically inert gas, recent studies suggest that H 2 can act as a biomolecule and has the potential ability to inhibit oxidative stress, inflammation, and apoptosis, thus, manifests cardioprotective effects or safeguards against tissue injury (Ostojic, 2017a;Ostojic, 2017b;Zhang et al., 2018). Supplements of exogenous H 2 by inhalation or intraperitoneal injection of H 2 -rich saline attenuates myocardial ischemia/reperfusion (I/R) injury and improves cardiac function through anti-oxidative, anti-apoptotic, and antiinflammatory effects (Hayashida et al., 2008;Sun et al., 2009;Zhang et al., 2011). Our previous studies indicated that H 2 protects against isoproterenol (ISO)-induced cardiac dysfunction and cardiomyocyte hypertrophy in vivo or in vitro (Zhang et al., 2016a;Zhang et al., 2017b). Akiko Noda group indicated that chronic H 2 inhalation prevents left ventricular hypertrophy in hypertensive Dahl salt-sensitive rats (Matsuoka et al., 2019). We reveled that H 2 protects against LPS-induced cardiac dysfunction.
LPS (1 μg/ml) can upregulate pathologic hypertrophy marker ANP and BNP in H9c2 cardiomyocytes from 2 to 24 h and induce cellular hypertrophy in 3, 6, 12, and 24 h through calcineurin/ NFAT-3 signaling pathway in H9c2 cardiomyocytes (Liu et al., 2008). In our study, H 2 -rich medium inhibits the upregulation of ANP and BNP by LPS in vitro. Left ventricular wall edema occurred during sepsis (Smeding et al., 2012;Castanares-Zapatero et al., 2013), and LPS induced enlargement in the cell size of cardiomyocytes in vivo . In our study, left and right ventricular cavities were enlarged by LPS, LPS decreased the thickness of interventricular septum, and these phenotypes were improved by H 2 . The LV mass AW and LV mass AW (Corrected) were slight increased by LPS, and H 2 decreases the upregulation of LV mass AW and LV mass AW (Corrected), although there were no statistical differences between groups. Drosatos et al. has also revealed that heart:body weight and lung:body weight ratios were not increased by LPS (Drosatos et al., 2013). The animal strains, animal sources, feeding environment, and the batch and dosage of LPS used might be responsible for these differences.
The endogenous H 2 is produced by the bacterial species present in human gut, mouth and pharynx, the respiratory system, vagina, and skin (Ostojic, 2017b). Thus, given antibiotics for 4 days by mixing penicillin and streptomycin in the drinking water can reduce the H 2 concentration below the 1-ppm detection limit in the expired air of male C57BL/6J mice, and the infarct size was significantly higher in mice administered with antibiotics than that in antibiotics nontreated mice (Shinbo et al., 2013). Another clinical investigation recently revealed overnight change in H 2 concentration (ΔH 2 ) was significantly lower in patients with chronic heart failure compared with controls and was positively correlated with cardiac index (Shibata et al., 2018). Ostojic also proposed an idea that an impaired production of endogenous H 2 by intestinal August 2019 | Volume 10 | Article 865 Frontiers in Pharmacology | www.frontiersin.org  (Ostojic, 2018). The key question that needs to be resolved is what is the function of the endogenous H 2 in cardiovascular homeostasis and whether the endogenous H 2 levels are related to the individual difference in the resistance of sepsis-induced cardiomyopathy.
It is well established that NF-κB and AP-1 are mainly responsible for TLR4 to induce the production of proinflammatory cytokines, such as TNFα, IL-1β, and IL-18 (Zhang et al., 2017a;Zhang et al., 2017c). The nuclear translocation of NF-κB and the phosphorylation of AP-1 upstream kinases ERK1/2, p38MAPK, and JNK by LPS in the heart are suppressed by H 2 . The suppression of H 2 on NF-κB and MAPK exists widely in many kinds of animal models, such as isoproterenol-induced cardiac hypertrophy (Zhang et al., 2016a), LPS-induced acute lung injury (Xie et al., 2012), and intimal hyperplasia in arterialized vein grafts (Sun et al., 2012). Besides TLR4 signaling activation, LPS also enhanced TLR4 expression (Fallach et al., 2010), and the increased TLR4 was blocked by H 2 in cardiomyocytes. However, the precise molecular targets of H 2 in TLR4-mediated innate immune signaling still need further investigation. H 2 protects against LPS-induced cardiac dysfunction via suppressing the excessive production of pro-inflammatory cytokines in the parenchymal cells of the heart by blocking TLR4-MAPKs/NF-κB signaling. Therefore, H 2 is a promising natural agent for the prevention of LPS-induced cardiac dysfunction.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
Male C57BL/6J mice (8 to 10 weeks of age) were used in the study. All animals were housed in a temperature-controlled animal facility with a 12-h light-dark cycle, and allowed to obtain rodent chow and water ad libitum. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals published by the NIH (8th Edition, Revised 2011) (Polhemus et al., 2017). The Institutional Animal Care and Use Committee (Zhongshan School of Medicine, Sun Yat-sen University) approved all animal procedures.