Acacetin protects against sepsis-induced acute lung injury by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis

Abstract

Acute lung injury (ALI) is the leading cause of death in patients with sepsis syndrome and without effective protective or therapeutic treatments. Acacetin, a natural dietary flavonoid, reportedly exerts several biological effects, such as anti-tumor, anti-inflammatory, and anti-oxidative effects. However, acacetin's effect and underlying mechanism on sepsis-induced ALI remain unclear. Here, the mouse model was established to explore the impact of acacetin on sepsis-induced ALI. Acacetin significantly increased ALI murine survival and attenuated lung injury in histological examinations. Additionally, acacetin down-regulated myeloperoxidase activity, protein concentration, and number of neutrophils and macrophages in bronchoalveolar lavage fluid. Subsequently, inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were examined. Results showed that acacetin dramatically suppressed the production of TNF-α, IL-1β, and IL-6. These above results indicated that acacetin attenuated sepsis-induced ALI by inhibiting the inflammatory response. Moreover, acacetin inhibited the expression of markers for M1-type (iNOS, CD86) macrophages and promoted the expression of markers for M2-type (CD206, Arg1) macrophages by western blot. In addition, acacetin down-regulated the expression TRAF6, NF-κB, and Cyclooxygenase-2 (COX2) by western blot. The high concentration of acacetin had a better effect than the low concentration. Besides, over-expression of TRAF6 up-regulated the expression of COX2, CD86, and iNOS, and the ratio of p-NF-κB to NF-κB increased the mRNA levels of TNF-α, IL-1β, and IL-6, down-regulated the expression of CD206 and Arg1. The effects of TRAF6 were the opposite of acacetin. And TRAF6 could offset the impact of acacetin. This study demonstrated that acacetin could prevent sepsis-induced ALI by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

Keywords

Acacetin lung injury sepsis macrophage polarization TRAF6/NF-κB/COX2 axis

Introduction

Sepsis is a systemic inflammatory response syndrome that damages the body, caused by a dysregulated host response to infection.^1,2 Globally, sepsis has substantially increased, estimated to affect more than 30 million patients yearly, and is responsible for nearly 6 million deaths.³ Sepsis could result in severe immune dysfunction, acute kidney injury, and even death.^4,5 Acute lung injury (ALI) is a common complication of sepsis and the primary health condition that aggravates sepsis mortality and morbidity.⁶ According to relevant epidemiological studies, over 50% of patients with sepsis developed ALI, and the 90-day mortality rate of patients with sepsis combined ALI is as high as 35.5%.^7,8 It has been reported that ALI was closely associated with the inflammatory reaction process. From pathological, protein-rich fluids accrue in alveolar cavities, the permeability of the alveolocapillary membrane increases, and inflammatory chemokine is produced for neutrophils flow from blood to the lung, which could be observed during ALI.⁹ Another work has shown that the infiltration of inflammatory cells and the release of pro-inflammatory mediators play a significant role in ALI development.¹⁰ Macrophages, as the first line of defense of lung immune defense, are broadly distributed on the surface of airways and alveoli. Macrophages play vital roles in ALI via cytokines and chemokines release.^11–13

Macrophages are usually divided into two categories: pro-inflammatory M1 type and anti-inflammatory M2 type. Macrophages can conduct inflammatory signals by various factors to affect their function.^14,15 Macrophages are first polarized to the M1 type to eliminate pathogens in infected tissues. Subsequently, macrophages repair damaged tissue by polarizing to M2 type.^16,17 Many studies reported that macrophage polarization is associated with sepsis-induced ALI. Methylprednisolone could promote M2 polarization, attenuating lung damage in ALI.¹⁸ Also, M2 protected the immunosuppressive function of Tregs, thereby reducing inflammation and repairing tissue.¹⁸ In addition, adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma (PPARγ) significantly promoted polarization to M2 macrophage and decreased inflammatory mediators, thereby restoring the injured lung tissue.¹⁹ Therefore, improving the inflammatory environment by modulating macrophage polarization is an effective method for attenuating ALI.

TRAF6 (TNF receptor-associated factor 6) is a member of the TRAF protein family, which is a critical component and participates in the toll-like receptor-induced NF-κB signaling pathway in immune responses.²⁰ It has been proven that TRAF6-mediated signals play crucial roles in the development, homeostasis, and activation of innate immune cells, including macrophages.²¹ Besides, the absence of TRAF6 suppresses inflammation and restores organ function by facilitating M2 macrophage polarization.^22,23 However, the molecular mechanism of TRAF6 in M2 macrophage polarization is still poorly understood.

Acacetin (5,7-dihydroxy-4′-methoxyflavone), an O-methylated flavone monomer, is naturally present in several plants, such as Sparganii rhizoma, Sargentodoxa cuneata and Patrinia scabiosifolia.^24,25 Acacetin possesses anti-peroxidative, anti-inflammatory, and anti-plasmodial activity.^26,27 For example, acacetin has been proven to inhibit the expression of pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS) and COX-2, in LPS-induced RAW 264.7 cells.²⁸ However, the underlying molecular mechanism of acacetin in inhibiting inflammation during sepsis remains unclear. In this study, we investigated acacetin's effect and underlying molecular mechanism on sepsis-induced ALI.

Material and methods

Animal model and treatment

Male C57BL/6 mice (aged 8–9 weeks) were obtained from the laboratory animal center of Ningbo University. The Ethics Committee of The General Hospital of Western Theater Command approved this animal study and all animal experiments. Animals were maintained in pathogen-free cages at 21 ± 2 °C temperature and 55 ± 10% humidity under a 12 h light/dark cycle with food and water provided ad libitum.

The mice were randomly divided into three groups (n = 6): sham group, model group, and treatment group. The sepsis-induced ALI model was established by Cecal Ligation and Puncture (CLP) as described in a previous study.²⁹ Briefly, mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium at 100 µL/10 g. A middle abdominal cavity incision was performed to expose the cecum. Then, we ligated the cecum 1 cm from the end and punctured it twice with a no.21 needle. Subsequently, a small amount of stools were squeezed into the abdominal cavity. Finally, the cecum was returned, and the abdominal wall was sutured. The mice in the sham group performed no other surgical procedures and only had a laparotomy to expose the cecum. Then, we administered mice with a postoperative subcutaneous injection of 200 µL normal saline for fluid replacement. The mice in the CLP + acacetin group were given a gavage of acacetin (80 mg/kg, Sigma-Aldrich Corp) 2 days before CLP. After 24 h of CLP administration, mice were sacrificed, and the lung tissues were collected for further analysis. In addition, 10 mice from each group were selected randomly to observe the survival rate every 12 h for the following 4 days.

Histology and immunohistochemistry

The lung tissue samples were fixed in 10% paraformaldehyde at room temperature for 24 h. After dehydrating in graded ethanol solutions, the lung tissue cleared with xylene and embedded into paraffin wax. Next, paraffin blocks were cut into 5 μm sections and dried for later use. For hematoxylin and eosin staining (HE staining), lung tissue samples were performed using the HE staining kit (Solarbio, China) according to instructions.³⁰ Then, the results were observed with a microscope (magnification, ×400). For immunohistochemistry examination of F4/80 expression, lung tissue slides were performed according to the instructions of the DAB substrate kit (CST, #8059). Briefly, the sections were first incubated for 1 h with primary antibody against F4/80 (CST, #70076). Then, slides were incubated for 20 min with a secondary antibody. Finally, The slides were colored with diaminobenzidine and observed with a microscope.

Detection of lung wet-to-dry weight ratio (W/D ratio)

We use filter paper to remove the moisture on the surface of lung tissues. To evaluate lung edema, we weighed the lung tissues to get wet weight. Then, the lung was placed in an oven at 70 °C for 48 h. Subsequently, the lung was immediately weighed to obtain dry weight. The lung wet-to-dry (W/D) weight ratio was calculated.

Collection and analysis of bronchoalveolar lavage fluid (BALF)

BALF was harvested according to a previously described.³¹ Briefly, mice were first euthanized. Then, a tracheal cannula slowly instilled 1 ml of phosphate-buffered saline (PBS) into the lungs thrice. The recovered BALF was centrifuged at 450×g for 10 min at 4 °C. The supernatant was discarded, and the sedimented cells were re-suspended in PBS. Bicinchoninic Acid Protein Assay Kit (Beyotime, China) was used to measure the protein level in BALF according to the instructions. Wright-Giemsa staining was used to subject the cells to the count of macrophages and neutrophils. Then, the cells were observed with a bright-filed microscope.

Lung myeloperoxidase (MPO) activity determination

The MPO activity of lung tissue samples was determined using an MPO assay kit purchased from Jiancheng Bioengineering (Nanjing, China). The lung tissues were homogenized in 0.5% HTAB buffer. Then, the homogenates were centrifuged at 13,000× g for 10 min at 4 °C. The supernatant was used to calculate MPO activity according to the instructions.

RNA extraction and real-time PCR

Total RNA was extracted from the lung tissue and cells using TRIzol reagent (Invitrogen, USA) by the manufacturer's instructions. The total was reverse transcribed into cDNA using a PrimeScript RT reagent kit (Tiangen, China). The mRNA expression was determined using the SYBR@ Green reagent (Roche, Indianapolis, IN, USA) and normalized using GAPDH as an internal control.

ELISA assay

ELISA was performed to detect the concentrations of TNF-α, IL-1β, and IL-6 in BALF following the kit's instructions (R&D Systems, Inc., Minneapolis, MN). The OD value of each hole at 450 nm with a microplate reader (TECAN-GENious, Tecan) was measured. The standard OD value is calculated according to the standard blank OD.

Western blot analysis

The protein sample was isolated from lung tissue and cells with RIPA buffer (Beyotime, China). BCA kit (Thermo Fisher Scientific, USA) measured protein concentration. An equal amount of denatured protein sample was separated onto 10–12.5% SDS-PAGE gel and subsequently electro-transferred to the FVDF membrane (EDM Millipore, Billerica, MA). The membranes were blocked with 5% non-fat dry milk in TBST at room temperature for 1 h. Then, the membranes were incubated with primary antibodies at 4 °C overnight and subsequently incubated with secondary antibodies at 37 °C for 1 h. Protein blots were visualized using Chemiluminescent Substrate. Image software was used for protein semi-quantitative analysis. Densitometric values were normalized to GAPDH.

Statistical analysis

Every experiment has been performed at least triplicates repeated. Means ± standard deviation (SD) was expressed as the value. The data were processed by SPSS statistical software using one-way ANOVA for three or more groups and a two-tailed t-test for two groups. The value of p < 0.05 was considered a statistically significant difference.

Results

The effect of acacetin pre-treatment on sepsis-induced ALI mice

To assess whether acacetin could mitigate sepsis-induced ALI, mice were subjected to the CLP administration and gavage of acacetin (80 mg/kg). As shown in Figure 1(a), the 96-h survival rate of mice in the CLP group was significantly decreased (p < 0.01) compared with sham group mice. However, treatment with acacetin significantly reduced the mortality of sepsis-induced ALI model mice (p < 0.01). Histopathological examination of lung tissues was performed to evaluate the impact of acacetin on sepsis-induced lung injury tissue by HE staining. Compared with the sham group, the lung sections of sepsis model mice showed that the alveolar septum was broken and many inflammatory cells infiltrated into the alveolar septum and cavity. However, administration of acacetin significantly ameliorated these histological changes (Figure 1(b)). Besides, pulmonary edema was examined by measuring the lung W/D ratio and BALF protein concentration. As shown in Figure 1(c), the lung W/D ratio and BALF protein concentration were significantly increased in the CLP group (p < 0.01). Still, treatment with acacetin reduced the lung W/D ratio and BALF protein concentration (p < 0.01).

Figure 1.

The effect of acacetin pre-treatment on sepsis-induced ALI mice. (a) The 96-h survival rate of mice in sham, CLP, and CLP + acacetin groups, respectively (n = 10). (b) Lung tissue histopathology by HE staining and lung injury score. (c) Pulmonary edema was assessed by the lung W/D ratio and the protein level in BALF. (d) The number of neutrophils and macrophages in BALF. (e) Effect of acacetin on lung MPO activity. At least three repeats were carried out, and the mean ± SD was presented, n = 6, ** p < 0.01 vs. Sham; # p < 0.05, ## p < 0.01 vs. CLP.

The number of inflammatory cells in BALF, including neutrophils and macrophages, was measured to determine the effect of acacetin on pulmonary inflammation. The results suggested that the number of neutrophils and macrophages in BALF was significantly increased in the CLP group (p < 0.01). Still, administration of acacetin markedly reduced the cell number of neutrophils and macrophages (p < 0.05) (Figure 1(d)). Besides, MPO is a kind of hemoprotein secreted from activated phagocytes. MPO and MPO-derived oxidants could cause tissue damage. As shown in Figure 1(e), the lung MPO activity in the CLP group displayed an obvious increase, but treatment with acacetin inhibited CLP-induced MPO activity (p < 0.01). These findings indicated that acacetin could attenuate sepsis-induced ALI mice.

Acacetin regulated the inflammatory cytokine concentration in the BALF of sepsis-induced ALI mice

It was reported that inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are involved in the pathogenesis of ALI. Thus, the inflammatory cytokine levels in BALF were determined by ELISA and qRT-PCR. The results showed that TNF-α, IL-1β, and IL-6 levels in sepsis-induced ALI model mice significantly increased (p < 0.01). In contrast, treatment with acacetin inhibited the expression of TNF-α, IL-1β, and IL-6 (p < 0.01) (Figure 2(a) and (b)). Moreover, F4/80 immunohistochemistry results suggested that administration with acacetin significantly decreased the number of F4/80 positive cells (Figure 2(c)).

Figure 2.

Acacetin regulated the inflammatory cytokine concentration in the BALF of sepsis-induced ALI mice. (a) The protein levels of TNF-α, IL-1β, and IL-6 were examined by ELISA. (b) The mRNA levels of TNF-α, IL-1β, and IL-6 were detected by qRT-PCR. (c) Immunohistochemistry was used to determine the expression of F4/80. The error bars are presented as mean ± SD, n = 6, ** p < 0.01 vs. Sham; ## p < 0.01 vs. CLP.

Acacetin-regulated macrophage polarization by TRAF6/Nf-κB/COX2 axis

To investigate the potential mechanism of acacetin on the regulation of inflammation, the expression of TRAF6, NF-κB, p-NF-κB, and Cyclooxygenase-2 (COX2) was examined by western blot. In addition, the M1 phenotype macrophage is capable of expressing cell-surface markers such as inducible nitric oxide synthase (iNOS) and CD86, whereas the M2 phenotype is anti-inflammatory with expressing CD206 and Arginase-1 (Arg-1).¹² Thus, the expression of iNOS, CD86, CD206, and Arg1 was measured by western blot. The results showed that the expression of TRAF6, COX2, iNOS, CD86, CD206, and Arg1 was up-regulated, and the ratio of p-NF-κB to NF-κB increased in the CLP group, compared with the sham group (p < 0.01) (Figure 3). After acacetin treatment, the expression of TRAF6, COX2, iNOS, and CD86 was down-regulated, and the ratio of p-NF-κB to NF-κB decreased, compared with the CLP group. However, acacetin further increased the expression of CD206 and Arg1 (Figure 3). These results showed that acacetin could modulate macrophage polarization by suppressing the TRAF6/NF-κB/COX2 axis expression.

Figure 3.

Acacetin regulated macrophage polarization by TRAF6/NF-κB/COX2 axis. The expression of TRAF6, p-NF-κB, NF-κB, COX2, CD86, iNOS, CD206 and Arg1 were determined by western blot. Data were normalized to the control and presented as mean ± SD, n = 6, ** p < 0.01 vs. Sham; # p < 0.05, ## p < 0.01 vs. CLP.

Acacetin-modulated LPS-induced macrophage polarization

To further determine acacetin's effect on macrophage polarization, the mouse macrophage RAW264.7 cells were induced with 1 μg/mL LPS to mimic inflammatory conditions cellularly. LPS up-regulated the mRNA level of TNF-α, IL-1β, and IL-6 (p < 0.01). Acacetin decreased the mRNA level of TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells, and a high concentration of acacetin had a better effect than a low concentration (Figure 4(a)). In addition, the expression of iNOS, CD86, CD206, and Arg1 was up-regulated when treated with LPS (p < 0.01). Acacetin prevented the increase of LPS-induced M1 polarized macrophages (iNOS and CD86) (p < 0.01). On the contrary, acacetin further promoted M2 polarized macrophages (CD206 and Arg1) (p < 0.01). Also, the effect of high-concentration acacetin was more evident than that of low-concentration (Figure 4(b)). These above results showed that acacetin could regulate RAW264.7 macrophage polarization, and a higher concentration of acacetin would result in better regulation of macrophage polarization.

Figure 4.

Acacetin modulated LPS-induced macrophage polarization. (a) The mRNA levels of TNF-α, IL-1β, and IL-6 were examined by qRT-PCR in mouse macrophage RAW264.7 cells treated with different concentrations of acacetin. (b) The protein levels of CD86, iNOS, CD206, and Arg1 were analyzed by western blot in mouse macrophage RAW264.7 cells treated with different concentrations of acacetin. Data were normalized to the control and presented as mean ± SD, ** p < 0.01 vs. Control; ## p < 0.01 vs. LPS; ^ p < 0.05, ^^ p < 0.01 vs. LPS + Acacetin(30).

Acacetin promoted M2 polarization by TRAF6/Nf-κB/COX2 axis

To explore the specific roles of acacetin in inflammatory responses, the expression of TRAF6, NF-κB, p-NF-κB, and COX2 were examined by western blot. The results showed that LPS up-regulated the expression of TRAF6 COX2 and significantly increased the ratio of p-NF-κB to NF-κB (p < 0.01). Acacetin effectively down-regulated TRAF6 and COX2 protein expression and decreased the ratio of p-NF-κB to NF-κB in LPS-induced RAW264.7 macrophage cells (p < 0.01) (Figure 5(a)).

Figure 5.

Acacetin promoted M2 polarization by TRAF6/NF-κB/COX2 axis. (a) The protein levels of TRAF6, p-NF-κB, NF-κB, and COX2 were detected by western blot in mouse macrophage RAW264.7 cells treated with different concentrations of acacetin. ** p < 0.01 vs. Control; ## p < 0.01 vs. LPS; ^ p < 0.05, ^^ p < 0.01 vs. LPS + Acacetin(30). (b) The TRAF6, p-NF-κB, NF-κB, and COX2 protein levels were determined by western blot in mouse macrophage RAW264.7 cells treated with acacetin and TRAF6-OE. (c) The mRNA levels of TNF-α, IL-1β, and IL-6 were examined by qRT-PCR in mouse macrophage RAW264.7 cells treated with acacetin and TRAF6-OE. (d) The CD86, iNOS, CD206, and Arg1 protein levels were analyzed by western blot in mouse macrophage RAW264.7 cells treated with acacetin and TRAF6-OE. At least three repeats were carried out, and the mean ± SD is presented, ** p < 0.01 vs. LPS; ## p < 0.01 vs. LPS + Acacetin; ^^ p < 0.01 vs. LPS + TRAF6-OE.

For more insights into the molecular mechanisms of acacetin on the regulation of macrophage polarization, we constructed TRAF6 overexpression (TRAF6-OE) vector by cloning the ORF of TRAF6 cDNA into pcDNA3.1 vector (Invitrogen, USA). The results showed that TRAF6-OE prevented the inhibitory effect of acacetin on the expression of TRAF6, COX2, TNF-α, IL-1β, and IL-6 and the ratio of p-NF-κB to NF-κB (Figure 5(b) and (c)). Moreover, TRAF6-OE increased the expression of iNOS and CD86 and inhibited the expression of CD206 and Arg1 in acacetin-treated LPS-induced RAW264.7 cells (p < 0.01) (Figure 5(d)). These results indicated that acacetin could enhance M2 polarization in LPS-treated RAW264.7 cells via the TRAF6/NF-κB/COX2 axis.

Discussion

Sepsis is a dysregulated host response to infection, resulting in life-threatening organ dysfunction.³² The lung is the most vulnerable and important organ during the pathogenesis of sepsis, and ALI is one of the most critical factors for mortality in sepsis.^33,34 In this study, our results showed that acacetin administration obviously inhibited sepsis-induced ALI. Moreover, acacetin decreased inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and promoted M2 polarized macrophages. Furthermore, our finding demonstrated the involvement of acacetin in macrophage M2 polarization, and the anti-inflammatory response was related to TRAF6/NF-κB/COX2 axis.

Accelerated evidence has shown that traditional Chinese medicine and its extracts play an essential role in treating inflammatory diseases.^35,36 It was found that paeoniflorin possessed the function of anti-inflammatory and could inhibit the expression of iNOS and IL-6 in LPS-induced RAW264.7 cells.³⁷ Some studies have reported that Andrographolide could improve ovalbumin-induced lung injury in mice by inhibiting the activation of the NF-κB signaling pathway and NLRP3 inflammasome.³⁸ Acacetin, as a natural compound, has been reported to be able to exert pronounced anti-inflammatory.^28,39 However, few researches have been done on the use of acacetin in the treatment of sepsis-induced ALI. In this study, the sepsis-induced ALI model was established by CLP to explore the effect of acacetin on sepsis-induced ALI. CLP treatment led to significant pathological changes, including alveolar hemorrhage and massive inflammatory cell infiltration, which indicated that the sepsis-induced ALI mice model was constructed successfully. Acacetin increased the 96 h survival rate of ALI mice, improved lung injury and the W/D ratio, and reduced total protein concentration, MPO activity, neutrophils, and macrophages in BALF.

Alveolar macrophages in the alveolar cavity are derived from monocytes with the function of phagocytosis and secretion, forming the first line of defense against foreign pathogens. Alveolar macrophages adjust the balance of pro-inflammatory and anti-inflammatory tissue and tissue repair.^40,41 In sepsis-induced ALI, the number of M1 macrophages in alveoli increases, which promotes the secretion of various inflammatory factors, such as TNF-a, IL-1β, and IL-6. The secretion of these pro-inflammatory cytokines further aggravates lung injury.^42,43 Consequently, we examined the expression of TNF-α, IL-1β, and IL-6. In our results, acacetin showed an inhibition effect on TNF-α, IL-1β, and IL-6 expression in sepsis-induced ALI mice and LPS-induced RAW264.7 cells. Accumulating evidence suggests macrophage polarization is a critical factor in the pathogenesis of ALI.¹⁷ Our results showed that acacetin down-regulated the expression of CD86 and iNOS and up-regulated the expression of CD206 and Arg1 in sepsis-induced ALI mice and LPS-induced RAW264.7 cells. These results suggested that acacetin prevents sepsis-induced ALI by inhibiting the inflammatory factors and promoting M2 polarization.

TRAF6 is essential for M2 macrophage activation. The absence of TRAF6 in macrophages results in conducting macrophage M2 polarization.²³ Our data indicated that acacetin could down-regulate TRAF6 expression and promote M2 polarization. COX2, an NF-kB target gene, is a crucial player in inflammation and an M2 macrophage polarization modulator.^44–46 Our results showed that acacetin could inhibit the expression of COX2 and the ratio of p-NF-κB to NF-κB. These results suggested that acacetin protected against sepsis-induced ALI by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

Given the complex pathogenesis of sepsis-induced ALI, we studied only the effect and mechanism of acacetin on alveolar macrophages. Neutrophils are the important culprit to ALI. The potential role of acacetin on neutrophils needs to be further explored in sepsis-induced ALI.

Conclusion

In conclusion, our study demonstrated for the first time that acacetin increased M2 polarized macrophages by regulating TRAF6/NF-κB/COX2 axis, thereby preventing sepsis-induced ALI. Our data suggested that acacetin might be a candidate anti-inflammatory drug for preventing and treating inflammatory diseases.

Footnotes

Acknowledgments

Not applicable.

Authors Contribution

Binbin Chang and Zhang Wang designed the study and carried them out; Binbin Chang, Zhang Wang, Hui Cheng, Tingyuan Xu, Jieyu Chen, Wan Wu, and Yizhi Li supervised the data collection, analyzed the data, interpreted the data, Binbin Chang, Zhang Wang, and Yong Zhang prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethics approval

Ethical approval was obtained from the Ethics Committee of The General Hospital of Western Theater Command.

Consent to participate statement

Written informed consent was obtained from a legally authorized representative(s) for anonymized patient information to be published in this article.

ORCID iD

Yong Zhang

Data availability

The authors declare that all data supporting the findings of this study are available within the paper, and any raw data can be obtained from the corresponding author upon request.

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