Double edged roles of endogenous PRDX6 in ventilator-induced lung injury via modulating oxidative stress and macrophages activation

doi:10.21203/rs.3.rs-3971634/v1

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Double edged roles of endogenous PRDX6 in ventilator-induced lung injury via modulating oxidative stress and macrophages activation

https://doi.org/10.21203/rs.3.rs-3971634/v1

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PRDX6 is highly expressed in lung tissues and shows an important biological significance. However, its specific roles in ventilator-induced lung injury (VILI) have not been clearly explored. The present study investigated the roles of endogenous PRDX6 in VILI, focusing on its overall and the iPLA2 function on lung injury, oxidative stress (OS), macrophages activation and polarization. Broncho-alveolar lavage fluid (BALF) from patients with mechanical ventilation was acquired. Both wild-type (WT) Sprague-Dawley rats and PRDX6 gene-knock out (PRDX6^-/-) rats were used to set up VILI model. Biological and histopathological analyses were performed. Our results showed that OS and PRDX6 expression were up-regulated after mechanical ventilation. PRDX6 deficiency led to lung inflammation and VILI aggravation, and the OS was further elevated and the activation and M1 polarization of lung macrophages were obviously increased in PRDX6^-/- rats. The iPLA2 inhibitor MJ33 alleviated VILI in rats and reversed the increased OS and M1 macrophages activation and polarization. The present study showed the double-edged roles of endogenous PRDX6 in VILI, including its overall protective role and the role of promoting VILI via iPLA2 by producing the excessive OS and subsequent increase of M1 macrophages polarization.

Biological sciences/Molecular biology

Health sciences/Diseases

Health sciences/Molecular medicine

Lung injury

mechanical ventilation

oxidative stress

PRDX6

Macrophages

Mechanical ventilation (MV) is an important method of life support for seriously-ill patients, and also a key part of respiratory management strategy for surgical patients. However, mechanical ventilation can cause lung tissue injury, namely mechanical ventilator-induced lung injury (VILI) [1]. So far, the specific molecular mechanism of VILI remains unclear, and there is no effective prevention measure for VILI in clinical practice. Studies in vivo have shown that VILI is associated with inflammatory cell infiltration and increases the secretion of pro-inflammatory cytokines in addition to the pathological changes of lung tissue edema and alveolar hemorrhage [2–5]. Hyperventilation directly causes lung tissue damages. The specific cellular and molecular mechanisms involved in VILI-induced lung inflammation are still undefined. Aseptic inflammatory responses induced by mechanical ventilation are likely to play an important role in initiating and mediating VILI and deserve further investigation.

Oxidative stress (OS) is a state of excessive production and accumulation of Oxidative intermediates represented by ROS caused by oxidation-antioxidant imbalance [6]. OS is related to the occurrence and development of various diseases [7, 8]. Recent studies have shown that the excessive oxidative stress may be the underlying cause of VILI-associated lung inflammation and injury. Oxidative stress in lung tissues occurs in the early stage of mechanical ventilation. PRDX6 (Peroxiredoxin 6) has both glutathion peroxidase (GSH-PX) and phospholipase A2 (iPLA2) activity [8], which is highly expressed in lung tissues and shows an important biological significance. It plays a crucial role in maintaining redox balance of lung tissue, repairing membrane damages caused by oxidative stress, and participating in the regulation of lung inflammation and injury [9, 10]. A recent report has suggested that phospholipase activity of PRDX6 might mediates VILI pathogenesis [11]. However, PRDX6 is a dual-enzyme protein, and its overall changes and phospholipase role in mechanical ventilator-related inflammation have not been clearly investigated, which raises our concerns. As the important immune cells in lung, alveolar macrophages activation and polarization are closely related to a variety of lung inflammation and injuries [12, 13].Therefore, PRDX6’s effect on alveolar macrophages activation during mechanical ventilation also arouse our interest.

In this study, BALF from patients with mechanical ventilation was acquired, and wild-type (WT) and PRDX6 gene knockout (PRDX6^−/−) rats were used to establish VILI model. We detected PRDX6 expressions and oxidative stress in human and rat lungs with VILI. We investigated the effect of PRDX6 deficiency on VILI, the oxidative stress and macrophages activation in rat lung. We investigated the effects of PRDX6 deficiency on VILI, oxidative stress and macrophages activation in rat lungs. We also explored the effects of iPLA2 inhibitor MJ33 on oxidative stress and macrophages activation in rat lungs with VILI.

Long time mechanical ventilation induced up-regulated oxidative stress in lung tissues of human patients

The total cell number in human patients’ BALF after at least 3 hours’ mechanical ventilation (PMV) was significantly higher compared to that at the initiation of mechanical ventilation (Baseline) (Figure.1A). The protein levels of pro-inflammatory cytokines IL-1β and MCP-1 in BALF were significantly higher at the time point of PMV than those at baseline (Figure.1B, C). Interestingly, levels of ROS and PRDX6 were also higher at the time point of PMV than those at baseline (Figure.1D, E).

High tidal volume ventilation induced oxidative stress and up-regulated PRDX6 expression in rat lungs

High tidal volume mechanical ventilation resulted in the increased inflammatory cell infiltration in the lung tissue and higher lung damage score as compared to those of vehicle-treated rats (Figure. 2A, B). Also, high tidal volume mechanical ventilation significantly induced the increased inflammatory cells in BALF and lung W/D weight ratio (Figure.2C, D). Strikingly, H₂O₂ and NO levels in the lung tissues were significantly increased compared to that of control rats (Figure.2E, F).

Furthermore, PRDX6 expression in the lung tissue of rats was determined. Results showed that PRDX6 mRNA and protein levels were remarkably up-regulated in the VILI rats’ lung as compared to those of control rats (Figure.3A). Results from immunohistochemistry (Figure.3B, C) and western blot (Figure.3D, E) also showed a significant increase of PRDX6 in the lung VILI rats’ lung.

PRDX6 deletion induced lung injury and inflammation in rats to be more susceptible to VILI

To further study the role of PRDX6 in the VILI model, we first observed the effect of PRDX6 deletion on lung inflammation. H-E staining showed that knockout of PRDX6 induced inflammatory cells infiltration and alveolar wall thickening spontaneously (Figure.4A). The lung injury score was significantly increased in PRDX6^−/− rats compared to WT control rats (Figure.4B). Besides, the lung wet/dry mass ratio (Figure.4C), IL-1β and MPO levels (Figure.4D, E; Supplemented Figure.1) in lung were all increased by RPDX6 knockout. In addition, PRDX6 deletion also increased NO in lung tissue (Figure.4F).

To understand the role of PRDX6 in VILI, the effects of PRDX6 knockdown on VILI were explored. Results from HE staining revealed a significant inflammatory cells infiltration and tissue injury in lung of PRDX6^−/− rats after mechanical ventilation when compared to that of WT-VILI group (Figure.5A, B). Similarly, PRDX6 deletion induced inflammatory cells infiltration into BALF after mechanical ventilation (Figure.5C). Moreover, IL-1β and TNF-α levels in lung was elevated by PRDX6 knockout after mechanical ventilation (Figure.5D, E). These results implied that PRDX6 deficiency is able to exacerbate VILI.

PRDX6 deletion aggravated oxidative stress and M1 macrophages polarization in rat lungs with VILI

Next, we examined whether the endogenous PRDX6 could affect VILI via modulating OS and macrophages M1 polarization. PRDX6 knockout significantly increased OS-related markers (H₂O₂) in lung after mechanical ventilation compared with those of WT VILI rats (Figure.6A). Results from immunohistochemistry staining showed that both CD68-positive cells (Figure.6B;Supplemented Figure.2A) and iNOS -positive cells (Figure.6C; Supplemented Figure.2B) were increased in lung tissues of PRDX6^−/− VILI rats, compared with those of WT VILI rats. In addition, the mechanical ventilation-induced increases of MCP-1, MIP-2 and MPO in lung tissues were greater in PRDX6^−/− rats than those in WT VILI rats (Figure.6D-F; Supplemented Figure.2C-F).

iPLA2 inhibitor MJ33 alleviated VILI, modulated oxidative stress and macrophage polarization in rat lungs with VILI

To further clarify the exact effect of endogenous PRDX6 on VILI, we used MJ33, a specific inhibitor of PRDX6 acid phospholipase, to treat rats. After MJ33 treatment, mechanical ventilation-induced inflammatory cells infiltration and lung damage were attenuated in lung tissues (Figure.7A, B). Likewise, mechanical ventilation-induced increase of lung W/D weight ratio (Figure.7C), cell count (Figure.7D) in BALF, IL-β and TNF-α in lung tissues (Figure.7E, F) were all decreased by MJ33.

Then, we examined whether the effect of MJ33 on VILI was mediated by inhibiting OS and M1 macrophages polarization in lung. The results showed that MJ33 treatment significantly suppressed mechanical ventilation-induced upregulation of H₂O₂ and NO in lung tissues (Figure.8A, B). Also, the immunofluorescence results showed that MJ33 treatment inhibited CD86-positive M1 macrophages infiltration (Figure.8C) and promoted CD206-positive M2 macrophages accumulation (Figure.8D) in lung tissues after mechanical ventilation.

In this study, we observed a significant increase of oxidative stress response and PRDX6 expression in both rats’ and human patients’ lung tissues after long time mechanical ventilation. We found that PRDX6 knockout attenuated OS and pro-inflammatory macrophages polarization in rat lungs with VILI. Besides, we also verified the protective role of MJ33 on VILI and its role in lung macrophages activation.

OS is an important regulatory way for organisms to respond to the external environmental changes [7]. However, the excessive OS responses will cause tissue damages. Studies have reported that OS is involved in lung injury induced by a variety of factors[18, 19]. Results from experiments associated with VILI have shown that the prolonged or high-volume ventilation is associated with the excessive OS [20, 21]. OS plays a pivotal role in the early stage of VILI [22]. The increased ROS activates immune cells in lung tissues by activating NLRP3 inflammasome and other signal transductions, thus initiating lung inflammation [23, 24]. Our study also confirmed the OS response induced by high-volume mechanical ventilation. In this study, we found that the tissular H₂O₂ and NO were significantly increased in lungs of VILI rats. Meanwhile, we found the increased M1 macrophages activation and polarization in lung tissues of rats with VILI. Both our results and data from other researchers strongly suggest that targeting OS may be a promising approach for VILI prevention.

ROS inhibition is an important method for OS regulation in diseases and inflammatory responses [25, 26]. PRDX6 plays an important role in maintaining REDOX equilibrium and scavenging the excessive ROS [27]. In this study, we found that PRDX6 expression was increased in lung tissues of VILI rats, suggesting that it may also play a role in VILI. PRDX6 knockout not only caused inflammation in lungs, but also aggravated VILI. These results show that PRDX6 has a protective effect on VILI.

The phospholipase activity is a key function of PRDX6 and is involved in many lung diseases [28]. It has been reported that PLA2 activity of PRDX6 activates NOX₂ under pathological conditions to be a main source of ROS [29]. Studies show that inhibiting PLA2 activity protects lungs of mice against multiple types of acute lung injuries caused by OS [30]. In order to clarify whether the phospholipase activity of PRDX6 is involved in mediating lung injury, we examine the effect of MJ33 on VILI in this study. We found that lung damages and OS level were significantly lower after MJ33 intervention than those of the group without MJ33 treatment. This suggests that the phospholipase activity of PRDX6 is involved in the occurrence of VILI, which is in accordance with a recently research from Fisher et al [29]. Peroxidase activity is another important function of PRDX6 to play a pivotal role in scavenging ROS. We suspect that PRDX6 may exert its protective effect by its peroxidase activity.

Macrophages are the main members of the natural immune cells in lung tissues [31]. Both the resident and recruited macrophages of lung tissues can be activated by various stimulations, including mechanical ventilation [32]. Macrophages are divided into M1 and M2 phenotypes due to the different functional polarization states, which respectively dominate the pro-inflammatory and anti-inflammatory repair effects [33–35]. M1 macrophages are also the main cell source of ROS that in turn causes pro-inflammatory macrophages polarization [32]. Studies have found that macrophages are activated and mainly polarized to M1 phenotype during mechanical ventilation, and TLR4 signaling pathway is activated to release pro-inflammatory factors that subsequently result in lung inflammation and injury [32, 36]. Here, we observed the obvious increase of OS in the VILI group, accompanied by the increased macrophages activation and M1 polarization. Besides, we found that deletion of PRDX6 gene, an important protein regulating OS response, led to the elevated OS, increased macrophage activation and aggravated VILI. Additionally, we found that the phospholipase inhibitor MJ33 inhibited the excessive OS response, macrophage activation and M1 polarization in VILI. These results suggest that the excessive OS response is possible to be an important factor in mediating macrophage activation, as well as initiation and expansion of pulmonary inflammatory response during VILI.

Furthermore, in the future, the peroxidase activity of PRDX6 will be focused in our research to better verify our hypothesis that PRDX6 plays a protective role in VILI through its peroxidase activity. Taken together, our study suggests that the endogenous PRDX6 acts as a protective factor via modulating OS, macrophages activation and M1 polarization (Figure. 9).

In conclusion, we demonstrated the protective role of PRDX6 in VILI by modulating OS and macrophages activation and M1 polarization. Although phospholipase activity of PRDX6 is involved in mediating VILI, PRDX6 gene deletion attenuated VILI. The elevated OS might contribute to the initiation of inflammatory response in VILI through activating macrophages. Our findings suggest that macrophages activation is involved in the initiation of inflammatory response in VILI and PRDX6 can be a target for VILI prevention.

Methods were reported in accordance with ARRIVE guidelines.

Human BALF collection

11 patients undergoing elective laparoscopic abdominal surgery without pulmonary inflammation were involved for acquisition of bronchoalveolar lavage fluid (BALF) at the time points of initiation of mechanical ventilation and at least more than 3 hours’ mechanical ventilation. All individuals were recruited under an investigational protocol approved by Ethics Committee of Anhui Medical University (PJ2023-08-11; China: ChiCTR2300073376). The detailed information of human subjects is provided in Supplemented Table 1 of the supplementary file. BAL acquisition was performed according to our previous study. Total cell counting was performed using a haemocytometer.

Animal experiments

8 to 10 weeks old WT Sprague Dawley rats were purchased from Vital River Laboratory. PRDX6 knock-out (PRDX6^−/−) rats (PKO) were purchased from Model animal research center of Nanjing University. Rats were acclimatized in animal laboratory, School of Basic Medical Sciences, Anhui Medical University. The animal experimental were approved by the Animal Ethics Committee of Anhui Medical University (LLSC 20190466). This study was performed in accordance with an approved Institutional Animal Care and Use Committee protocol.

VILI rat model was established according to the previous study [14]. Briefly, wild-type rats and PRDX6^−/− rats received high tidal volume (tidal volume of 35ml/kg; PEEP of 0; respiratory rates of 60 breaths/min) ventilation for 3 hours after tracheotomy. Control rats received tracheotomy without mechanical ventilation. Some WT rats were intraperitoneally injected with 0.01µmol/kg MJ33(lithium salt, Sigma-Aldrich, USA)2 h before ventilation as described by others[15]. Experiments were performed with six rats which were sacrificed immediately after mechanical ventilation. The right lung BALF was collected according to our previous report [16]. The collected BALF was centrifuged to separate cellular components (4℃,700g, 5min).The separated cells were resuspended in 1ml PBS, and the total and differential cell counting was performed using a haemocytometer or Wright-Giemsa staining.

Lung histopathology

The left lung was fixed with 4% paraformaldehyde for one week, embedded in paraffin to produce 5 µm sections. Then, the paraffin sections were stained with hematoxylin and eosin (H&E). Lung injury score was calculated according to our previous report [16].

Enzyme linked immunosorbent assay (ELISA)

ELISA kits were used for detecting the human BALF and homogenized rat lung tissues, including IL-1β (Cusabio, CSB-E08053h), MCP-1 (Cusabio, CSB-E04855h), PRDX6 (Cusabio, CSB-EL018859HU), IL-1β (Cusabio, CSB-E08055r) and TNF-α (Cusabio, CSB-E11987r).

Oxidative stress examination

DHE-ROS kit (BB-470516, BestBio) was used to detect ROS level in human BALF. NO and H₂O₂ levels in the homogenized rat lung tissues were measured using the specific kits (A012-1 for NO, A064-1-1 for H₂O₂, Nanjing Jiancheng Bioengineering Institute). The microplate reader (BioTek) was used to detect and calculate OD values.

Real-time quantitative polymerase chain reaction

The total RNA was extracted from lung tissues using Trizol (AG, China). The single-stranded cDNA was synthesized from RNA samples with High-capacity cDNA reverse transcription kit (AG, China). Quantitative real-time PCR analysis was performed using ABI 7500 system with Brilliant II SYBR Green QPCR Master Mix (AG, China). The following gene-specific primers were used: rat PRDX6, 5’-TGACAGCCCGTGTGGTATTC-3’ and 3’-GTCAGCTGGAGGGAGTCAAC-5’. The data output is expressed as a fold-change of control gene expression levels for normalization.

Immunofluorescence and immunohistochemistry

Immunofluorescence was performed according to our previous report [16, 17]. Briefly, 5-µm-thick lung paraffin sections were rinsed in PBS three times for 5 minutes each. Then, the sections were incubated in 0.05M for antigen retrieval. Next, the sections were incubated with primary antibodies at 4°C overnight, including anti-PRDX6 (1:200, Abcam, USA), anti-IL-1β (1:100, Abcam, USA), anti-MPO (1:50, Santa Cruz, CA, USA), anti-CD68 (1:100, Abcam, USA), anti-iNOS (1:50, Santacruz, CA, USA), anti-MCP-1 (1:100, Abcam, USA), anti-MIP-2 (1:100, Abcam, USA), anti-CD86 (1:100, Abcam, USA) and anti-CD206 (1:100, Abcam, USA). For immunohistochemistry staining, HRP-conjugated secondary antibodies (Beijing Golden Bridge Biotechnology, Beijing, China) were used to incubate sections for 30 min. For immunofluorescence staining, FITC-conjugated secondary antibodies were added to incubate sections for staining. DAPI was used to dye the nuclei for 5 min. Confocal laser scanning microscope (Olympus, Tokyo, Japan) was used for image acquisition.

Western blot

Lung tissues were homogenized in RIPA buffer with 1mM protease inhibitor cocktail (MCE, China). Lysates were centrifuged (12000 rpm at 4°C, 15 min), followed by protein concentration using BCA assay (Sigma, USA). The equal protein sample per lane was separated on 8–14% SDS-PAGE gels and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, 0.45 µm, IPVH00010, USA). PVDF membranes were blocked with 5% skimmed milk, incubated with primary antibody of PRDX6 (1:4000, Abcam, USA) overnight at 4°C and then probed with horseradish peroxidase-conjugated secondary antibody (1:4000) for 1 h at room temperature. Results were visualized using ECL reagent (Pierce, USA), detected with gel imager (Tanon, China), and quantified with ImageJ Software (NIH).

Statistical analysis

All data were expressed as means ± SEM and analyzed using SPSS 23.0 (IBM, USA). A preplanned, independent, two-sample t test was used for comparisons between two groups, and one-way ANOVA with post hoc test (Bonferroni test) was used for three or more groups. One-way ANOVA with Bonferroni post hoc test was performed for experiments with one single time point analysis. All tests were two-tailed, and P < 0.05 was considered as the significant change.

Competing interest

The authors have declared that they have no competing interest.

Funding statement

This work was supported by the Natural Science Foundation of China (no.81902003) and Anhui University Scientific Research Project (no. 2022AH051179).

Author Contribution

Q.S. and C. W. designed study; G. C. and W. T. collected the human sample and clinical data; C. L., L. Z., W.T. and W. L. set up animal model, collected sample and performed molecular protein detection and tissue slices staining; G. C. and Q.S. organized the results; G. C. and L. Z. wrote the draft of the paper; Q.S. and C. W. revised and finalized the paper.

Acknowledgments

We would like to thank Professor Yuxian Shen from Anhui Medical University for the kindly provision of PKO rats and the technical assistance. We also thank Professor Yinguang Fan from Public Health College of Anhui Medical University for support in statistical assistant.

Data availability

Data will be made available from Qiying Shen on request if someone wants to request the data from this study.

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Double edged roles of endogenous PRDX6 in ventilator-induced lung injury via modulating oxidative stress and macrophages activation

Status:

Version 1

Abstract

Figures

Introduction

Results

Long time mechanical ventilation induced up-regulated oxidative stress in lung tissues of human patients

High tidal volume ventilation induced oxidative stress and up-regulated PRDX6 expression in rat lungs

PRDX6 deletion induced lung injury and inflammation in rats to be more susceptible to VILI

PRDX6 deletion aggravated oxidative stress and M1 macrophages polarization in rat lungs with VILI

Discussion

Conclusion

Materials and Methods

Human BALF collection

Animal experiments

Lung histopathology

Enzyme linked immunosorbent assay (ELISA)

Oxidative stress examination

Real-time quantitative polymerase chain reaction

Immunofluorescence and immunohistochemistry

Western blot

Statistical analysis

Declarations

Competing interest

Funding statement

Author Contribution

Acknowledgments

Data availability

References

Additional Declarations

Supplementary Files

Status:

Version 1