The Effects of One-lung Ventilation on the Production of Endogenous Melatonin and NLRP3 Inflammasome-Related Inflammatory Cytokines in Patients Undergoing Laparoscopic Esophagectomy: A Prospective Randomized Double-Blind Controlled Study

Background: NLRP3 inflammasome has been confirmed to play a pivotal role in ventilator-induced lung injury (VILI), while exogenous melatonin can attenuate VILI by inhibiting NLRP3 inflammasome activation in mouse model. However, the relationship between endogenous melatonin and the NLRP3 inflammasome-related inflammatory cytokines in VILI induced by one-lung ventilation (OLV) remains unknown. In this study, we aimed to reveal the relationship between the NLRP3 inflammasome-related inflammatory cytokines: interleukin (IL)-1β, IL-18 and endogenous melatonin in OLV-induced VILI during esophageal cancer surgery. Methods: Twenty-eight patients were randomized to receive “conventional” ventilation (Vt=8 mL/kg) or lung protective ventilation (Vt=5 mL/kg along with 5 cm H 2 O positive end-expiratory pressure (PEEP)). Respiratory variables were evaluated. IL-1β, IL-18 and melatonin in bronchoalveolar lavage fluid (BALF) and plasma were measured. Results: we found that lung protective ventilation during OLV decreased peak airway pressure (Ppeak), plateau airway pressure (Pplat) and driving pressure (ΔP) compared with that in “conventional” ventilation group. Moreover, lung protective ventilation inhibited polymorphonuclear (PMN) cells invasion into BALF. Likewise, lung protective ventilation not only suppressed alveolar and plasma IL-1β secretion, but also reduced increased production of IL-18 in both BALF and plasma after OLV. Furthermore, we found that both alveolar and plasma endogenous melatonin levels in “conventional” ventilation group were lower than that in lung protection group. Conclusion: Taken together, the present study suggested that lung protective ventilation during OLV prevented VILI via suppressing NLRP3 inflammasome-related inflammatory cytokines secretion and restoring the level of endogenous melatonin in patients.

Background One-lung ventilation (OLV) is required for esophageal cancer and can contribute to the surgical field [1]. However, inappropriate ventilation modes may cause or augment acute lung injury, which is known as ventilator-induced lung injury (VILI) [2]. Lung-protective ventilation [low tidal volume + positive end-expiratory pressure (PEEP)] in OLV was shown to achieve good clinical effects and protect against VILI [3,4]. Furthermore, clinical studies have demonstrated that lung-protective ventilation induced an immune response with lower concentrations of inflammatory mediators than that of "conventional" ventilation [5]. Therefore, further study of the pulmonary immune response is essential for the prevention of VILI.
Increasing studies have shown that OLV may lead to proinflammatory cytokine release and inflammatory signaling pathway activation [5][6][7][8]. Overdistension in ventilated lungs followed by compression of alveolar vessels initiates a robust release of proinflammatory cytokines, such as interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-a, in bronchoalveolar lavage fluid (BALF) [5,9]. These proinflammatory cytokines are important chemotactic factors for polymorphonuclear (PMN) cells [10]. Excessive PMN cell aggregation will amplify the inflammatory cascade. Furthermore, a recent study showed that in mouse alveolar macrophages, Nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome activation contributes to the development of VILI [11].
However, whether OLV activates the NLRP3 inflammasome during esophageal cancer surgery remains unknown.
Early preliminary studies have shown that exogenous MT ameliorates VILI by increasing 5 the anti-inflammatory response [14]. Recently, Zhang et al. demonstrated that exogenous MT inhibited NLRP3 inflammasome activation in mice with acute lung injury [15]. However, the crosstalk between endogenous MT and the NLRP3 inflammasome in VILI induced by OLV remains unknown.
Our study aimed to investigate the effects of different ventilation strategies on NLRP3 inflammasome-related inflammatory cytokines secretion and reveal the relationship between NLRP3 inflammasome-related inflammatory cytokines and endogenous MT in OLVinduced VILI during esophageal cancer surgery. In addition, the impact of different ventilation strategies during OLV on postoperative complications was investigated.

Study Design
Patients scheduled for elective laparoscopic esophagectomy at the First Affiliated Hospital

Study Population
Patients with esophageal cancer in our hospital were considered for enrollment. The inclusion criteria were as follows: American Society of Anesthesiologists' (ASA) physical status I or II, requirement for OLV during operation, and 49-77 years old. Exclusion criteria were preexisting hypoxemia, diagnosed major obstructive or restrictive pulmonary disease [preoperative forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) <70% of the predicted value], pulmonary infection before surgery, body mass index (BMI) of less than 20 or more than 35, and use of immune modulators.

Randomization and Blinding
The randomized numbers were generated by a research coordinator using block sizes on a 1 : 1 ratio. This ensured that each group had an equal number of subjects. Then, the research coordinator sealed the numbers in opaque envelopes. During OLV, the anesthesia assistant opened the envelopes, set the breathing parameters and covered the breathing parameters using opaque paper. The anesthesia assistant did not participate in the next study. One anesthesiologist collected the specimens, and another anesthesiologist recorded the breathing parameters. Both physicians were blinded to the allocation.

Study Protocol
Standard monitoring devices were applied after admission to the operating room. Before induction of anesthesia, an artery catheter was inserted into the left radial artery. inspiratory to expiratory ratio of 1:2, 100% oxygen, and an I respiratory rate to maintain an end-tidal CO 2 (ETCO 2 ) of 35-40 mmHg.

Observational Indexes
Peak airway pressure (Ppeak), plateau airway pressure (Pplat) and blood gas analyses were evaluated at three stages: during TLV before surgery, 30 minutes after OLV and during TLV at the end of surgery. Furthermore, the driving pressure (ΔP) was recorded. ΔP was defined and calculated as follows: ΔP=Pplat-PEEP [16]. The primary outcomes were pulmonary complications, including pulmonary infection, acute lung injury or acute respiratory distress syndrome and reintubation or invasive mechanical ventilation. In addition, secondary outcomes, including anastomotic fistula, incision infection, ICU stay and death before hospital discharge, were recorded.

Statistical Aanalysis
According to previous studies, the cell numbers in the BALF increased more than 30% 8 after OLV [5].We assumed that the cell numbers in the BALF increased at least 25% with a power of 80% and an α of 5%, and thus, 12-14 patients in each group were needed. Data are presented as the mean ± SD or number of patients. One-way ANOVA with a post hoc Bonferroni test was used to analyze normally distributed data. Non-normally distributed data were analyzed by chi-square tests. All statistical analyses were performed with SPSS 19, and a P value of <0.05 was considered significant.

Baseline Parameters of Patients
In total, 34 patients were included and assessed. 4 patients did not meet the criteria, and 30 were included in this study. However, 2 patients withdrew for technical reasons, and at last 28 completed the study (Fig. 1). The patient characteristics and preoperative details showed no significant differences between the groups (Table 1).

Changes in the Respiratory Parameters
The respiratory and gas exchange variables are presented in Table 2. During OLV, an increase in Ppeak and Pplat was observed in all patients. ΔP in the control group increased substantially compared with that at baseline. Furthermore, our readings for Ppeak, Pplat and ΔP during OLV in the control group were higher than those in the lung-protective ventilation group. Additionally, in both groups, the oxygenation index decreased during OLV compared with that at baseline; however, there was no difference between the two groups.

Changes in the Number of Cells in the BALF
The cells in the BALF were counted after Wright-Giemsa staining. The number of total cells and PMN cells in the BALF was substantially increased during mechanical ventilation (Fig.   2). However, in the lung-protective strategy group, the total cells and PMN cells in the BALF were significantly reduced compared to those in the control group (Fig. 2).

Changes in IL-1β and IL-18 in the BALF and Plasma
Recently, researchers demonstrated that NLRP3 inflammasome activation is important for exacerbating VILI [17,18]. The activation of the NLRP3 inflammasome produces IL-1β and IL-18 [19]. Therefore, commercial ELISA kits were used to detect the production of both IL-1β and IL-18 in the BALF and plasma. As expected, the IL-1β and IL-18 levels in the BALF showed an increased trend after OLV, but the increase in IL-1β was not significant in the lung-protective strategy group (Fig. 3A, B). In addition, a significant reduction in the IL-1β and IL-18 levels in the BALF was observed in the lung-protective strategy group compared with the control group (Fig. 3A, B).
In all patients, the plasma IL-1β and IL-18 concentrations were significantly increased after OLV (Fig. 3D, E). However, lung-protective ventilation resulted in a significant decrease in the plasma IL-1β and IL-18 concentrations after OLV compared to those in the control group (Fig. 3D, E).

Changes in MT in the BALF and Plasma
Endogenous MT levels in both the BALF and plasma were also detected. In contrast to the IL-18 and IL-1β levels, the plasma MT levels in both groups were significantly decreased after OLV, as was the BALF MT level in the control group (Fig. 3C, F). Additionally, both the BALF and plasma MT concentrations in the control group were lower than those in the lung protection group (Fig. 3C, F).

The Incidence of Complications
As shown in Table 3, there was no difference in the incidence rates of complications between the two groups.

Discussion
Our study indicated that the lung-protective ventilation improved respiratory variables, including Ppeak, Pplat and ΔP. The lung-protective ventilation not only inhibited PMN cell 10 invasion but also suppressed IL-1β and IL-18 secretion. In addition, we found that lungprotective ventilation resulted in decreased inhibition of endogenous MT production compared to "conventional" ventilation.
OLV is an established procedure during laparoscopic esophagectomy. However, clinical studies have shown that the extended use of OLV is an independent risk factor for postoperative pulmonary dysfunction [20]. Excessive stretching or repeated opening of lung tissues is an important cause of VILI during OLV [21]. A lung-protective strategy using low Vt along with PEEP during OLV was confirmed to improve postoperative pulmonary dysfunction [6]. In our study, we found that the lung-protective strategy notably decreased Ppeak and Pplat, indicating that the shear force was reduced via a lung-protective strategy. Meanwhile, we also observed a substantial decrease in ΔP with the lung-protective strategy during OLV, which suggested that the lung-protective strategy was associated with a reduced incidence of postoperative pulmonary complications [16]. However, our research failed to find a difference in the incidence of complications between the two groups, which may be related to the small sample size.
Increased mechanical strain further activating the inflammatory response is a key event during the development of VILI [5]. The results from previous and recent studies have shown that IL-1β is a special proinflammatory cytokine that promotes VILI in animal models and patients [22][23][24][25]. Regulation and inhibition of IL-1β can finally achieve organ protection because blockade of the IL-1 receptor has been demonstrated to inhibit neutrophil sequestration and edema formation in VILI [26]. In our study, we clearly showed that OLV increased the concentration of IL-1β in the plasma as well as the alveolar PMN cell counts in the BALF. However, lung-protective ventilation blocked the elevated IL-1β level and PMN cell recruitment. Most interestingly, we observed a dramatic increase in both the alveolar and plasma concentrations of IL-18 after OLV, while lung-protective ventilation resulted in a profound reduction in IL-18. IL-1β and IL-18 were confirmed to be products of NLRP3 inflammasome activation [19]. Furthermore, current studies have demonstrated that NLRP3 inflammasome activation plays a key role in the pathogenesis of VILI in a mouse model [17,18]. Therefore, lung-protective ventilation may inhibit inflammatory responses by inhibiting activation of the NLRP3 inflammasome during OLV.
For the first time, our results provide evidence that NLRP3 inflammasome activation may be involved in the initiation and development of VILI during OLV.
As discussed above, the precise mechanism of how OLV activated the NLRP3 inflammasome during laparoscopic esophagectomy was unclear. In recent years, the antiinflammatory effects of both exogenous and endogenous MT have been observed in many conditions [27,28]. Paula et al. demonstrated that the exogenous addition of MT protected against VILI through decreasing the levels of inflammatory cytokines in a mouse model [14]. Further research confirmed that exogenous replenishment of MT alleviated lipopolysaccharide (LPS)-induced acute lung injury by inhibiting NLRP3 inflammasome activation [15]. Therefore, we hypothesized that endogenous MT may mediate VILI through inhibiting NLRP3 inflammasome activation. As expected, we found that OLV substantially reduced the production of endogenous MT in patient plasma and the BALF level of MT in the control group. Surprisingly, pulmonary protective ventilation significantly inhibited the reduction of endogenous MT. Accordingly, our results suggested that NLRP3 inflammasome activation regulated by endogenous MT may be involved in the pathogenesis of VILI.

Conclusion
In conclusion, our study suggested that pulmonary protective ventilation during OLV improved lung function and suppressed NLRP3 inflammasome-related inflammatory cytokines secretion in patients undergoing laparoscopic esophagectomy. These effects may be attributable to pulmonary protective ventilation-mediated restoration of the LXW, JL and YTH collected the data, and drafted the manuscript, they contributed equally as co-first authors; YZ performed the statistical analysis; QYS and HYZ revised the manuscript critically for important intellectual content. All authors were responsible for the conception and design of the trial, and approved the final manuscript.

Supplementary Files
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