Preliminary study on the protective effect of remazolam against sepsis-induced acute respiratory distress syndrome (ARDS)

Introduction

Sepsis is a kind of fatal organ failure that induced by severe infection. One of the most common complications is acute respiratory distress syndrome (ARDS) (Dombrovskiy et al., 2007), which is the leading cause of death in the intensive care units (ICUs).

It has been reported that the serum levels of IL-1β and TNF-α in some sepsis patients are significantly increased, which is positively correlated with the inflammatory reaction and the severity of the disease (Zhen, Li & Wang, 2013). Although invasive mechanical ventilation, continuous renal replacement therapy (CRRT), extracorporeal membrane oxygenation (ECMO), and broad-spectrum powerful antibiotics are all used to sustain organ function in patients with sepsis and ARDS, their clinical efficacy is not promising. Currently, it is believed that ARDS is induced by excessive and unchecked inflammatory reactions in the lung tissue (Zhang et al., 2013).

Based on the pathophysiological basis of massive neutrophil exudation, ARDS is clinically characterized by pulmonary edema and refractory hypoxemia, resulting in extensive lung tissue damage (Mondrinos et al., 2013). Therefore, one of the essential treatments for the prevention and treatment of ARDS is to suppress proinflammatory and anti-inflammatory responses  (Kiemer, Muller & Vollmar, 2002). Remazolam, a brand-new ultrashort-acting benzodiazepine and water-soluble, has been extensively utilized to numb the pain and induce drowsiness in ICU. Remazolam has been reported to reduce inflammatory stress, but the potential mechanism remains unknown. In order to understand the protective effects of remazolam on lung tissue in septic rats with ARDS, we measured the levels of inflammatory markers and oxidative stress indicators in the lung tissue of septic rats.

Materials and Methods

Animal grouping

Forty rats were divided into four groups according to the random number table as follows (Fig. 1): the sepsis + saline group (n = 10), sham operation + saline group (n = 10), sham operation + remazolam group (n = 10), and sepsis + remazolam group (n = 10). All animal experimental protocols were approved by the Medical Ethics Committee of Hainan General Hospital (No. Med-Eth-Re[2022]630).

Results

After CLP, serum inflammatory factors were detected at 6, 12, 24 and 48 h. The levels of inflammatory factors began to increase after 6 h and gradually increased by time. The levels of inflammatory factors were obviously decreased in the sepsis + remazolam group in comparison with the sepsis + saline group. The W/D and lung injury scores of rats were increased in the sepsis + saline group, but decreased in the sepsis + remazolam group. There was no significant change in the levels of inflammatory factors between the sham operation + saline and sham operation + remazolam groups (Figs. 2, 3B and 3C, Tables 1 and 2).

Under the light microscope, the alveolar structure of rats were normal, and there was no inflammatory cell infiltration in the sham operation + saline and sham operation + remazolam groups. In the sepsis + saline group, the alveolar structure was destroyed, the alveolar wall was broken, pulmonary interstitial edema was obvious, and there were a large number of inflammatory cells, such as neutrophils, infiltrated. In the sepsis + remazolam group, the alveolar wall was relatively intact, the degree of pulmonary interstitial edema was reduced, and there were a small number of inflammatory cells infiltrated (Fig. 3A).

Compared with the sepsis + saline group, the oxygen partial pressure of blood gas analysis was increased, and carbon dioxide partial pressure of blood gas analysis was reduced in the sepsis + remazolam group (Figs. 3D–3G and Table 3).

Compared with the sepsis + remazolam group, the contents of CD4+ and CD8+ T cells in the sepsis + saline were decreased, but there was no significant change between the sham operation + saline and sham operation + remazolam groups (Fig. 4 and Table 4).

Compared with the sham operation + saline and sham operation + remazolam groups, the levels of MDP and MPO were significantly higher in the sepsis + saline group, while ATP level was lower, and the opposite was true in the sepsis + remazolam group (Figs. 3D–3G, Table 3).

The HMGB1 protein expression was significantly increased in the sepsis + saline group but decreased in the sepsis + remazolam group (Fig. 5).

Discussion

ARDS is an acute inflammatory injury that causes by extrapulmonary or intrapulmonary factors, and the morbidity and mortality increase significantly belong the aggravation of sepsis (Bellani et al., 2016). The symptoms of ARDS patients will be more serious if associated with sepsis, and have poor prognosis and higher mortality (Sheu et al., 2010). At present, the pathogenesis of ARDS remains unclear, but more and more studies believe that systemic inflammatory response plays a key role at the beginning and development process of ARDS (Jiang et al., 2018; Yu et al., 2015). In conclusion, inhibiting the production of inflammatory factors may be an effective way to cure ARDS. From our experiment, we found that proinflammatory factors, such as IL-1 β, IL-6, and TNF-α, in peripheral serum of septic rats were obviously increased at 6 h, and anti-inflammatory factor, IL-10, was decreased, which further confirming that proinflammatory and anti-inflammatory systems of the body are in an unbalanced state during sepsis. Under the light microscope, a large number of inflammatory cells infiltrated into the alveolar wall and interstitial lung of rats in the sepsis + saline group, and the alveolar structure was broken and destroyed. Combined with W/D of lung tissue and lung injury score, these results proved that the ARDS model induced by cecal perforation was successful. By comparing the serum inflammatory factors in rats in each group, we found that the levels of inflammatory mediators in the circulating serum of rats decreased obviously, the structure of lung tissue, infiltration of inflammatory cells also improved obviously, partial pressure of arterial oxygen in arterial blood gas increased, and partial pressure of carbon dioxide decreased after drug intervention with remazolam. It indicated that remazolam might reduce lung tissue damage, improve oxygenation capacity and alleviate ARDS induced by sepsis while inhibiting systemic inflammatory reactions, but specific mechanism is unclear.

Immune regulation dysfunction also be one of the important mechanisms leading to the progression of sepsis. Important immune cells in sepsis patients are missing  (Tinsley et al., 2001; Hotchkiss & Tinsley, 2002). Types of missing immune cells include CD4 T and CD8 T cells. CD8+ T lymphocytes regulate activation and proliferation of CD4+ T lymphocytes in cell–cell contact-dependent manner (Elrefaei et al., 2007). In the advanced stage of sepsis, CD4+ T cells, CD8+ T cells and monocytes are reduced to varying degrees, and immunosuppression is aggravated (Riedemann, Guo & Ward, 2003; Barochia et al., 2010), which increases the severity of the disease (Xiufen, Shunyao & Qingming, 2018). By measuring CD4+ and CD8+ T lymphocytes in serum of rats, we found that the number of these lymphocytes in septic rats decreased obviously, and the downward trend was more obvious by increasing inflammatory factors, which may lead to the apoptosis of immune cells and inhibit immune function.

In the process of sepsis, oxidative stress injury is another aspect that needs to be considered. The imbalance of oxidation/antioxidation in the lung plays an important role in ARDS (Barabas et al., 2013; Jing, Cuili & Zhiguo, 2014; Qin et al., 2018). The contents of MDA and MPO can predict the level of lipid peroxide (Tariq et al., 2003). We found that contents of MDA and MPO in the sepsis + saline group were significantly higher than the sepsis + remazolam group, suggesting that remazolam reduces the degree of lipid peroxidation and production of oxygen free radicals. In addition, the ATP level in the sepsis + saline group was lower than the sepsis + remazolam group, indicating that all tissues and organs in the whole body are in a state of high metabolism during sepsis, but there is ischemia and hypoxia due to circulatory stagnation and hypoperfusion, which leads to disorder of cellular energy metabolism. The mechanism may be related to sodium-potassium pump that relies on ATP for energy supply. After the intervention of remazolam, ATP synthesis increased and lung tissue structure damage was improved, indicating that it has a regulatory effect on the balance of oxygen supply and high metabolism in lung tissue.

In addition, we also observed the HMGB1 protein expression in each group. HMGB1 is an important late inflammatory mediator (Yang, Wang & Tracey, 2001), and it plays a key regulatory role in the inflammatory response network of sepsis. Fang et al. (2002) found that compared with TNF-α, IL-1β and other relatively early cytokines, HMGB1 appeared later but lasted longer. The rat model of sepsis established by CLP found that increasing serum HMGB1 level was related to organ dysfunction (Ma et al., 2009). Our results showed that HMGB 1 was expressed in each group, but the expression in the sepsis + saline group was increased, because HMGB1 is a ubiquitous protein that can be slightly expressed in normal cells, and can be upregulated by infection. In our experiment, we also found that severe sepsis in rats is in direct proportion to the HMGB1 expression, which became weakened after treatment with remazolam. It shows that remazolam has a certain inhibitory effect on HMGB1, playing an anti-inflammatory and antioxidative stress role and improving cellular immune function. However, the deficiency of the study is that we did not observe the concentration gradient of remazolam, nor did we observe multiple time points during sepsis. We will design future research base on different doses of remazolam and more intervention time points for comparative experiments to explore the protective mechanism of remazolam on septic ARDS, and potential signal pathways and target genes are also needed to explore to provide more experimental basic data and theoretical basis for clinical application.

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