Abstract

Introduction

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are a series of pulmonary pathological changes arising from a wide variety of lung injuries, which have high morbidity and mortality (Butt et al., 2016), characterized by disruption of endothelial barrier integrity and diffuse lung damage. It can cause an imbalance between coagulation and inflammation to induce inflammation (Frantzeskaki et al., 2017). Besides, macrophages, neutrophils inflammatory cells and their pro-inflammatory products can destroy pulmonary epithelial cells, increase pulmonary microvascular permeability, produce pulmonary edema, damage gas exchange, and lead to respiratory failure (Butt et al., 2016). Therefore, how to maintain the integrity of the endothelial barrier through the regulation of the microenvironment in the inflammatory state is critical to the treatment.

ECs dysfunction and inflammation contribute to the occurrence and development of lung injury. Thus, vascular endothelial repair is an integral part of lung injury repair (Zhao et al., 2020). EPCs are a kind of progenitor cells that can differentiate into vascular ECs (Asahara et al., 1997), which can migrate to the damaged area and differentiate into ECs to participate in angiogenesis or repair, and promote the improvement of endothelial functions (Li et al., 2017). In addition, EPCs can repair the vascular injury and alleviate LPS-induced lung injury, reduce inflammation, and promote bacterial clearance of pneumonia (Mao et al., 2010), which has a broad prospect in the treatment of lung injury.

Hydrogen (H₂) is an important physiological regulatory factor that has protective effects of anti-oxidation, anti-inflammation, and anti-apoptosis on cells and organs (Huang et al., 2010b). H₂ can reduce oxidative stress (Song et al., 2011), promote the scavenging of free radicals, and inhibit vascular aging (Iketani et al., 2018). A multicenter, open-label clinical trial showed that hydrogen/oxygen mixed gas inhalation improved disease severity and dyspnea in patients with Coronavirus disease 2019 (COVID-19) (Guan et al., 2020). In addition, H₂ has an excellent therapeutic effect on inflammation, ischemia-reperfusion injury, diabetes, cancer, atherosclerosis, and other diseases (Li et al., 2013Lee et al., 2015Shimada et al., 2016Li et al., 2019). It can reduce the levels of serum Low-Density Lipoprotein Cholesterol (LDL-C) and apolipoprotein- B (Apo-B), improve the high-density lipoprotein (HDL) functions damaged by dyslipidemia (Song et al., 2013), reduce the formation of neointima after vein transplantation in rats (Sun et al., 2012), and decrease hypertension, angiogenesis imbalance and oxidative stress caused by placental ischemia (Ushida et al., 2016). It also can protect the pulmonary microvessels of mice from the endothelial function damage induced by septicemia, maintain the consistency of pulmonary endothelium (Li Y. et al., 2020), and improve microvascular ECs viability in traumatic brain injury by inhibiting autophagy (Wang Y. et al., 2020). Animal experimental studies have shown that H₂ inhalation can provide protection in animal models of lung injury caused by mechanical ventilation, sepsis, ischemia-reperfusion, LPS and hyperoxia, seawater infusion, etc. (Ohsawa et al., 2007Xie et al., 2010Chen et al., 2015Diao et al., 2016Audi et al., 2017). Clinical studies show that the inhalation of H₂ by pregnant women can also inhibit the LPS-induced apoptosis and oxidative damage of fetal lung cells (Hattori et al., 2015). Previous studies have found that EPCs can repair lung injury induced by LPS (Yang et al., 2019), and H₂ has the same effect (Hattori et al., 2015). We speculate that H₂ may have a protective effect on EPCs, and even repair LPS-induced lung injury by improving the functions of EPCs. In this study, the injury model of EPCs was established by LPS treatment, and the cell viability, migration, angiogenic ability, and related protein expression of EPCs were measured. The molecular mechanisms of H₂ on the functional damage and repair of EPCs induced by LPS were discussed.

Materials and Methods

Results

LPS Impaired EPCs Functions

MTT assay results showed that, compared with the control group, LPS reduced the viability of EPCs in a concentration-dependent and time-dependent manner. We used 20 μg/ml LPS to induce 72 h for the follow-up experiments (Figure 2). In addition, we also found that LPS significantly reduced the ability of migration and tube formation of EPCs (Figures 3B–E).

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FIGURE 2

EPCs viability was reduced after being induced by LPS. EPCs were treated with different concentrations (2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml) LPS for different time (24, 48, 72 h). Cell viability was measured by MTT assay. Data are presented as mean ± SD (n = 6); **p < 0.01.

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FIGURE 3

H₂ alleviated EPCs dysfunctions induced by LPS. EPCs treated with LPS induced EPCs damage, and then EPCs were treated with different concentrations (20%, 40%, 60%) for different time (24, 48, 72 h). The viability of EPCs was assessed by MTT assay (A). EPCs migration was measured by transwell assay (B,C). Tube formation was measured by a Tube formation assay (D,E). Data are presented as mean ± SD (n = 6); **p < 0.01.

Discussion

EPCs were first initially discovered in 1997 by Asahara et al. EPCs are defined as a cell population capable of differentiating into mature ECs and have vasculogenic potential, which contributes to vasculogenic, wound healing (Asahara et al., 1997) and repair of ischemic tissue damage (Fan et al., 2014). As a potential therapeutic agent, EPCs have attracted attention for a variety of diseases including cerebral ischemia (Zhou et al., 2021), diabetes (Wang K. et al., 2020), ALI etc. (Asahara et al., 1997). However, due to differences in the isolation, amplification and identification of EPCs, as well as controversy over therapeutic function, the further development and clinical application of EPCs were limited. In this study, we successfully isolated the mouse bone marrow-derived EPCs for subsequent research of the repair mechanism of H₂ on EPCs dysfunctions induced by LPS. This study is the first report of the effect of H₂ on EPCs.

H₂ is an odorless, colorless, tasteless, and insoluble multifunctional medical gas. It can cross the cellular membranes and has the functions of anti-oxidation, anti-inflammatory and anti-apoptosis (Hong et al., 2010). In 1975, Dole et al. found that high-pressure hydrogen gas has an antioxidant effect and can inhibit the growth of tumors in mice (Dole et al., 1975). However, the study was not taken seriously because of the limitations of the experiment and the difficulty of reproducibility. In recent years, research on H₂ in the medical field has gradually widened. Clinical studies have found that H₂ plays a therapeutic role in diseases which related to the respiratory system (Guan et al., 2020), nervous system, cardiovascular (Camara et al., 2019), digestive system (Eryilmaz et al., 2020), reproductive system, urinary system (Gokalp et al., 2017), and metabolic exercise (Qiu et al., 2020). It is recommended to inhale a mixture of H₂ and oxygen (O₂) (33.3% O₂ and 66.6% H₂) in the “Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment (Trial Version 7)” issued by the China National Health Commission. The recommendation recognized the importance of H₂ in contemporary medical gas research.

In this study, LPS (20 μg/ml) significantly reduced the proliferation, migration, and tube formation of EPCs. This is consistent with Yu et al. research, which found that LPS (10 μg/ml) impaired the viability, migration, adhesion abilities, and tube formation of late EPCs (Yu et al., 2017). Compared with the control group, the proliferation and adhesion activities of bone marrow-derived EPCs were impaired in LPS (100 ng/ml) induced group in dose and time dependence (Li et al., 2014). Our previous study also demonstrated that LPS (30 μg/ml) inhibited the proliferation, migration, and tube formation of EPCs (Yang et al., 2019). Furthermore, western blot results showed that LPS reduced the expression levels of p-AKT, eNOS, and p-eNOS in EPCs. LPS had no effect on AKT expression. Our previous research proved that LPS (30 μg/ml) decreases the levels of p-AKT, eNOS, and p-eNOS in EPCs (Yang et al., 2019). Yang et al. found that LPS (10 μg/ml) reduced the expression of p-eNOS in human pulmonary microvascular endothelial cells (HPMECs) (Yang et al., 2018). Liu et al. found that p-AKT was markedly suppressed in H9c2 cells after treatment with LPS (1 μg/ml) for 24 h (Liu et al., 2021). The effect of LPS on AKT expression in out study was consistent with the study of Li et al. and Zhan et al. Li et al. showed that there was no significant change in AKT expression of LPS-induced (20 μg/ml) mouse microvascular ECs (Li H. et al., 2020). Zhan et al. reported that the AKT level of HPMECs did not change markedly under LPS (1 mg/L) induction (Zhan et al., 2020). In addition, Wang et al. found that p-AKT protein expression was significantly increased in LPS-induced (100 ng/ml) rat microvascular ECs (Wang et al., 2013). Fan et al. found that LPS (100 ng/ml) stimulation significantly increased the phosphorylation of both AKT and eNOS in HPMECs (Fan et al., 2020). Taken together, we speculate that the inconsistent study results may be due to the differences in LPS concentration. In addition to toxic effects, LPS also has extensive biological activity. Low doses of LPS have immune-activating effects (Morris and Li, 2012) (Lin et al., 2009). Therefore, there are differences in the functional gene expression and cell functions under different concentrations LPS treatment. However, the relevant mechanism still needs further confirmation.

The effectiveness of molecular hydrogen has been proven in the prevention and treatment of many diseases. H₂ had a protective effect on the rat model of ALI (Jiang et al., 2013Audi et al., 2017) by significantly improving lung endothelial permeability, reducing cell apoptosis and histopathological changes (Diao et al., 2016), and preventing LPS-induced pulmonary ECs dysfunction (Li Y. et al., 2020). Fu et al. demonstrated that hydrogen-rich saline has a protective effect on LPS-induced ALI by regulating cell apoptosis and inhibiting endothelial dysfunction (Fu et al., 2020). Inhalation was the most direct way to administer molecular hydrogen (Huang et al., 2010aKawamura et al., 2010). At the same time, molecular hydrogen could be dissolved in physiological saline to make hydrogen-rich water for intravenous injection, or it could be taken orally (Cardinal et al., 2010He et al., 2013). A hydrogen incubator was used in this study. It could simulate the in vivo environment after inhaling hydrogen, which was more conducive to our research. H₂ has a protective effect on the ECs barrier, mechanism of which has not been fully elucidated, and the effect of H₂ on EPCs has not been reported. Our study indicated that H₂ attenuated the dysfunctions of EPCs induced by LPS, improved EPCs proliferation, migration, tube formation, and restored the expression levels of p-AKT, eNOS, p-eNOS. Recent studies revealed that H₂ could inhibit the expression of inflammatory factors, reduce sepsis-induced endothelial damage and inflammation, improve endothelial dysfunction (Cardinal et al., 2010He et al., 2013).

Previous studies showed that the PI3K/AKT/eNOS pathway is involved in the changes of the LPS-induced ECs barrier function (Zheng et al., 2018), but whether it is involved in the effect of H₂ on EPCs repairment remains unclear. The results of the present study discovered the unique molecular basis for H₂ to inhibit LPS-induced EPCs dysfunctions. After inhibiting the PI3K/AKT/eNOS signaling pathway by LY294002 and L-NAME, the H₂-mediated restoration of EPCs functions was partially prevented. The western blot results demonstrated that H₂ up-regulated p-AKT, eNOS and p-eNOS levels were inhibited by LPS. LY294002 significantly inhibited the increase of p-AKT, eNOS and p-eNOS induced by 60% H₂. L-NAME significantly inhibited the increase of eNOS and p-eNOS induced by 60% H₂, and had no effect on p-AKT levels. PI3K/AKT/eNOS activation plays a crucial role in our study. PI3K/AKT signaling pathway mediates a variety of pathophysiological processes and involves multiple important cellular activities, such as cell proliferation, apoptosis and autophagy (Ravikumar et al., 2010Liby and Sporn, 2012Saiprasad et al., 2014). Additionally, numerous studies show that the PI3K/AKT signaling pathway plays a vital role in the process of EPCs proliferation, migration, homing and tube formation (Everaert et al., 2010Wang et al., 2011). Our previous results demonstrate that Rev-D4F mediates restoration of EPCs functions by PI3K/AKT/eNOS signaling pathway (Yang et al., 2019). The PI3K/AKT/eNOS pathway was involved in restoring the dysfunctions of EPCs in diabetic mice (Cao et al., 2017). Yu et al. suggested that the proliferation, migration and survival of EPCs impaired by LDL cholesterol via the PI3K/AKT signaling pathway (Yu et al., 2010).

In summary, we concluded that the PI3K/AKT/eNOS signaling pathway was contributed to H₂ repairment of EPCs dysfunctions induced by LPS (Figure 8).

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FIGURE 8

H₂-mediated restoration of EPCs dysfunctions is mediated by the PI3K/AKT/eNOS pathway.

Conclusion

Our results showed that H₂ reversed the LPS-induced EPCs dysfunctions. Moreover, H₂ restored the LPS-attenuated levels of p-AKT, eNOS and p-eNOS. Therefore, this study proves that H₂-mediated restoration of EPCs dysfunctions is mediated by the PI3K/AKT/eNOS pathway.

References