Emission Factors of PAHs Components and Bioreactivity in PM 2.5 from Biomass Burning

Biomass burning releases fine particulate matter (PM 2.5 ), water-soluble inorganic ions (WSIs), metal elements, polycyclic aromatic hydrocarbons (PAHs) and other compounds, and it is one of the largest sources of carbonaceous aerosols. The lungs experience negative health impacts from exposure to PM 2.5 ; however, it is uncertain how PM 2.5 emitted from biomass burning affect the human lung alveolar epithelium. This study investigated emission factors of PM 2.5 from biomass burning and PM 2.5 bioreactivity in human alveolar epithelial A549 cells. Emission factors were measured from biomass samples included maize straw (MS), wheat straw (WS), wood branches (WBs), MS briquettes (MSBs), MSB charcoal (MSC), WS briquettes (WSBs), WSB charcoal (WSC), WB briquettes (WBBs), and WBB charcoal (WBC). A549 cells were exposed to biomass PM 2.5 at 0 and 50 µ g mL –1 for 24 h, and the expression of Yes-associated protein (YAP), phosphorylated (p)-YAP, transcription coactivator with a PDZ-binding motif (TAZ), p-TAZ, E-cadherin, and high mobility group box 1 (HMGB1) proteins were assessed by Western blotting. We found that MSC, WSC, WSBs, and WBs had higher PM 2.5 emission factors. MS has the highest emission factors of polycyclic aromatic hydrocarbons (PAHs) among all the biomass PM 2.5 , especially FLU (26.46 mg kg –1 ) and PYR (26.93 mg kg –1 ). There were 48.30% of PM 2.5 was able to deposit in the alveolar area with a concentration of 32.25 µ g m –3 estimated by a multiple-path particle dosimetry (MPPD) model. We observed decreases in p-YAP/YAP and HMGB1 expressions after biomass PM 2.5 exposure. YAP were positively correlated with ANT, PHE, 1-MP, FLU, PYR, 3,6-DP, BaA, CHR, BbF, BkF, BaP, BeP, PER, IcdP, BghiP, and DahA ( p < 0.05). In conclusion, PAHs in biomass PM 2.5 contribute to cytotoxicity on A549 cells. PAHs in PM 2.5 with high emission factors from biomass burning could cause significant human pulmonary deteritious health effects after inhalation.


INTRODUCTION
It is known that particulate matter with an aerodynamic diameter of < 2.5 µm (PM2.5)increases the risk of lung diseases, such as lung cancer and chronic inflammatory pulmonary disease (He et al., 2017;Li et al., 2018).PM2.5 is released into both indoor and outdoor surroundings when biomass is burned, resulting in poor air quality (Jayarathne et al., 2014).Exposure to PM2.5 from burning biomass fuels is considered to cause pulmonary dysfunction and inflammation of the airways (Oluwole et al., 2013).Epidemiological evidence indicated that the risk of respiratory illnesses increases when people are exposed to PM2.5 from biomass burning (Regalado et al., 2006;Rylance et al., 2020;Zhou et al., 2014).Research revealed the association between PM2.5 and chronic obstructive pulmonary disease (COPD).A 1% increase of PM2.5 would increase the risk in COPD cases by 0.25% (p < 0.10) (Singkam et al., 2022).A meta-analysis reported the risk factors between exposure to biomass burning emissions and COPD in a Chinese population (Chen et al., 2021).An increase in 1 µg m -3 PM2.5 produced by biomass burning increased all respiratory emergency department outcomes by 0.4% (Pennington et al., 2019).Therefore, exposure to PM2.5 from biomass burning is an important public health issue.
Organic matter is one of the significant emission constitutions in fine particular matter and some research analysed the source appointment of organic substances (Zhang et al., 2021a;Zhang et al., 2007).Brown carbon, one of the organic aerosols, was estimated to have emission factors of 15-47 m 2 kg -1 for biomass burning in China (Tian et al., 2019).Other organic chemicals, for example, polycyclic aromatic hydrocarbons (PAHs), might be useful in identifying the emission profile of different biomass fuels such as wood and straw as well.A study in Italy displayed the emission characteristics and concentrations of PAHs measured in PM2.5 samples.The concentrations of PAHs were around 6.58 ± 1.03 ng m -3 (Winter) and 3.16 ± 0.53 ng m -3 (Spring), together with the trend of the PM2.5 mass concentration (Pietrogrande et al., 2022).In Guangzhou, southern China, a study investigated the source profile of PAHs in PM2.5 from emissions in vehicles and biomass burning with the source emission variations.Results showed that biomass burning accounted for 31 (±4)% of the total PAHs while vehicular emissions factored in 11 (±2)% of the total PAHs (Gao et al., 2013).The volatility profiles of semi-volatile species including PAHs and relationships to the oxidative potential of airborne particles were explored in California, implying PAHs might be an important tracer of biomass burning (Pirhadi et al., 2020).Moreover, another study in Thailand showed that the relatively homogeneous organic functional compositions of PM2.5 from crop residue burning can be an important indicator of air quality (Pongpiachan and Paowa, 2015).Potential air pollution source such as agricultural waste burnings is often considered in decreasing air pollution and governing air quality (Pongpiachan et al., 2021).Analysing the emission factors of the organic constituents from burning agricultural or crop residue assists in categorizing and exploring biomass-burning-related aerosols (Siwatt et al., 2022).
Particle toxicity is related to its chemical composition.After being directly exposed to PM2.5 samples for 48 h, cell viability of human epithelial lung cells (A549) was significantly reduced (Pavagadhi et al., 2013).Exposure to PM2.5 from coal burning, vegetable debris, biomass burning, and cooking induced considerable oxidative stress and systemic inflammation in mice (Chen et al., 2013).Also, exposure to PM2.5 from biomass burning caused an increase in cytotoxic lactate dehydrogenase (LDH) in the lungs of mice (Wang et al., 2021).The proinflammatory interleukin (IL)-6 increased after exposure to PM2.5 from major industrial emissions (Chuang et al., 2018).According to a previous report, road dust elemental components were linked to particle toxicity of human upper airway pharyngeal epithelial cells (Tung et al., 2021).Secondary organic aerosols enhanced cytotoxicity, reactive oxygen species, and IL-6 in A549 cells while decreasing cell viability (Laiman et al., 2022b).Accordingly, it is important to further understand the bioreactivity and mechanisms involved after biomass-related PM2.5 exposure.
Human lung epithelial cells have physiologic and pathologic roles in the lungs.They are critical components of lung homeostasis, contributing to alveolar epithelial repair and regeneration (Guillot et al., 2013;Ruaro et al., 2021;Wygrecka and Schaefer, 2020).The Hippo pathway mediators, yes-associated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (TAZ), control the regeneration of alveolar epithelial cells and decelerate lung inflammation (LaCanna et al., 2019).
Associations between PM2.5 from biomass burning and adverse health effects were reported.However, the underlying mechanisms of lung toxicity due to PM2.5 from biomass burning remain unclear.In this study, we investigated emission factors of PAHs components in PM2.5 from biomass burning and their bioreactivity in human A549 cells.

PM2.5 Collection
A fully enclosed combustion chamber that was set up in a lab at the Institute of Earth Environment, Chinese Academy of Sciences (IEECAS; Xi An, China), which was used to burn biomass.Characteristics of the combustion chamber were detailed previously (Tian et al., 2015), and another study demonstrated the combustion conditions (Sun et al., 2018).The weight of the raw biofuels was 0.1-0.2kg for each test with the moisture ranging from 35% to 45% before burning.Combustion emissions were collected using a specially manufactured dilution sampler with a 5-15-fold dilution rate, which was connected to three parallel pipes with a flow rate of 5 L min -1 , and the burn emissions were placed downstream of the residence chamber.To remove the absorbed organic vapours, the quartz filters were pre-fired at 900°C for 3 hours.Afterward, the filters were stored at approximately 4°C to reduce the amount of volatile compounds that evaporate.After being collected using quartz filters (2.2-µm pore size, 47 mm in diameter, Whatman, Maidstone, UK) and Teflon filters (3 µm, 47 mm, Pall Life Sciences, Ann Arbor, MI, USA), PM2.5 samples were obtained utilizing a microbalance (±1 ppm °C-1 sensitivity drift, ±1 µg readability, Sartorius AG MC5, Göttingen, Germany) to determine the PM2.5 mass.To deduct any passive gas adsorption artifacts, a field blank was subtracted to calculate the biomass concentration of each filter.

Chemical Analysis
The concentrations of 17 PAHs and other organics were determined, including Anthracene (ANT), Phenanthrene (PHE), 1-Methylphenanthrene (1-MP), Fluoranthene (FLU), Pyrene (PYR), 3,6-Dimethylphenanthrene (3,6-DP), Benz(a)anthracene (BaA), Chrysene (CHR), Retene (RET), Dibenz[a,h]anthracene (DahA), arbaitol, Hexamethylbenzene (HMB), levoglucosam, D-glucose, inositol, mannitol and saccharose.One half of the filter was extracted with high-purity dichloromethane and methanol (2:1, v/v) under ultrasonication for 15 min for each filter.The extraction procedure was repeated in triplicate to ensure complete removal of sample.Water and impurities in the extracts were further removed by pipettes filled with sodium sulfate (NaSO4) and glass wool.The extracts were concentrated to 2 mL by a rotary evaporator under vacuum condition and then separated into two portions.One of them was analyzed by a gas chromatography/electron ionization mass spectrometry (GC/EI-MS; Agilent Technologies, USA).GC/EI-MS analysis was performed using an Agilent 7890A GC coupled with an Agilent 5975C MSD (Santa Clare, CA, USA).The biomass sample solution was injected at 275°C to a GC injection port and the compounds separation was performed in a DB-5MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, Agilent Technology).Another half was reacted with N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) at 70°C for 3 h, the derivatives were determined using GC/EI-MS also.These target organic compounds were identified by retention times and ratios of qualifier ions in standards.An internal standard (IS) compound was added to the samples for target compounds quantification.Calibration curves (10-1000 ng mL -1 ) were plotted for all of the organic compounds.The data analysis was completed by Agilent MSD ChemStation software package.More details description on the analysis was described in (Ho et al., 2008;Niu et al., 2021).

Multiple-Path Particle Dosimetric (MPPD) Model
The MPPD model (vers.3.04), created by Applied Research Associates, was used to estimate the amount of inhaled PM2.5 that was deposited in the lungs (Miller et al., 2013).The model provides a widely used approach for assessing the proportion of particles that deposit in the human respiratory system.Compared to previous models, the model offers a more-precise and authentic dose evaluation (Asgharian, 1998;Asgharian et al., 2001;Bergmann, 2008;Miller et al., 2016).Briefly, each of the individual mode size distributions was combined into a single size distribution for use in MPPD.A single multimodal size distribution was then constructed computationally to reflect the overall distribution of particle sizes.The size range of this multimodal particle distribution was then subdivided into logarithmically spaced intervals, and the total number of particles in each size interval was calculated in order to assist MPPD calculations of deposition and clearance.Each of the formulas was specified for diffusion, sedimentation, impaction, and deposition (Asgharian, 1998;Bergmann, 2008).In our study, we used the human symmetric model with the number of segments of 24 by default.A multiple-path technique with a size range of 0.01-1 µm was selected to calculate the number of particles deposited in all respiratory tract airways.The default model parameters with a density of 1.0 g cm -3 were assumed to determine the quantity of particles of a certain size that are deposited in designated areas including the head, tracheobronchial (TB), and pulmonary alveoli.In an exposure situation, the concentration of aerosol was 4.31 mg m -3 .The breathing frequency was set to 16 breaths min -1 with 500 mL of tidal volume and 50 mL of nasopharyngeal dead space.

Cell culture
Human lung alveolar epithelial cells (A549 cells) (American Type Culture Collection; Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium under 5% CO2 at 37°C.Fetal bovine serum (10%) and 1% dual antibiotics (penicillin and streptomycin) were added to the medium to nourish cells and protect them from bacterial invasion.Cells underwent starvation for 24 h at a cell density of 10 6 cells mL -1 before exposure.
2.5.2PM2.5 preparation and exposure Biomass PM2.5 samples collected onto Teflon filters were extracted using two-stage sonication in a methanol solution.A blank Teflon filter that underwent the same process was used as a filter control.Under a nitrogen stream, samples were desiccated by a high-efficiency particulate air (HEPA) filter.Dimethyl sulfoxide (DMSO) was added to PM2.5 samples to resuspend the particles.Then, samples were prepared at a concentration of 0 (control) and 50 µg mL -1 in RPMI 1640 medium (< 0.01% vol DMSO) for 24 h of cell exposure (n = 6 per group).An earlier study mentioned the concentration used (Chuang et al., 2018).

Statistical Analysis
Data of emission factors are expressed as the mean with the standard deviation (SD).A oneway analysis of variance (ANOVA) with Tukey's post-hoc test was used for comparisons.Pearson's correlation coefficient was used to examine relationships of emission factors of chemical compounds in biomass PM2.5 with YAP, TAZ, HMGB1, and E-cadherin.p < 0.05 was set as the level of significance.Visualization of correlation data was performed using RStudio vers.4.1.1 for macOS.Statistical analyses were performed using GraphPad Prism vers.7 for Windows (San Diego, CA, USA).

Emission Factors of PM2.5
Fig. 1 shows emission factors of PM2.5 for different types of burning biomass.The PM2.5 emission factors varied from 1.22 to 11.28 g kg -1 .MSC and WSC had higher emission factors of PM2.5 than other biomass samples.MS, WS, MSBs, WBBs, and WBC had relatively lower emission factors.Data displayed that the PM2.5 emission factors of PAHs components were decreased after carbonizations.The emission factors of PM and PAHs in biomass pellets were lower than in raw fuels burning (Shen et al., 2012) and average PM2.5 emission factors of raw biomass were reduced from 50 ± 28 mg kJ -1 to 10 ± 5 mg kJ -1 (carbonized) after carbonizations (Li et al., 2019).The discrepancy in our study may come from combustion conditions such as confined spaces with controlled airflow or open-air can affect combustion efficiency and the emissions produced.Different burning sources, for example, wood logs and incense will also result in different compounds, contributing to variations in emissions.A previous study showed that emission factors from indoor source combustion (environmental tobacco smoke and incense burning) varied from 55.6 to 253.7 mg g -1 (Niu et al., 2021).Another survey also showed that emission factors of two types of incense burning ranged 290.1-417.2mg g -1 (Chuang et al., 2012).A study conducted in three pizzerias in São Paulo City, Brazil showed that the average indoor PM2.5 emission factor from burning logs was 0.38 g kg -1 (Lima et al., 2020).This suggests that the PM2.5 generated from biomass burning in our study was much lower than emission factors of other emission sources.Furthermore, environmental settings in which sources operate influence air exchange rates and air quality, which in turn impact emissions.Converting from raw solid fuel to carbonised solid fuel will help minimize the emissions.The use of alternative energy sources (carbonized) in regions where biomass burning is prevalent might improve air quality.

Emission Factors of Chemical Components in PM2.5
Table 1 shows emission factors of 24 detected organic components in PM2.5 released by different types of burning biomass.The lowest average emission factor of polycyclic aromatic hydrocarbons (PAHs) was 0.03 mg kg -1 in 3,6-DP and the highest average emission factor was 3.8 mg kg -1 in PYR.For other non-PAHs organic compounds, the average emission factors ranged from 0.06 mg kg -1 (inositol) to 14.16 mg kg -1 (levoglucosan), which is higher than the emission factors of PAHs.MS has the highest emission factors of all PAHs.The emission factors of FLU and PYR are especially high in MS, which is 26.46 mg kg -1 and 26.93 mg kg -1 respectively.MSC has higher emission factors in non-PAHs organic compounds than other biomass samples.Previous study included 14 different fuel-stove combinations from indoor emission.Based on the fuel mass, the total emission factors of 28 PAHs varied from 20.7 mg kg -1 to 535 mg kg -1 (Du et al., 2021).In our study, the emission factors of PAHs are far less than the emission factors of PAHs from indoor solid fuel combustion.Another study manifested that substituting raw biomass fuels used in conventional cooking stoves with granules combusted in modern pellet burners reduced the total emissions of PAHs by 71% (Shen et al., 2012).Thus, emission factors might be easily influenced by the conditions of combustion as well as the burners.Moreover, emission factors of the total PAHs ranged from 84.5 mg kg -1 to 344 mg kg -1 for residential biomass burning and emissions of the benzo[a]pyreneequivalent (BaP) factor for biomass were in the range of 2.79-11.3mg kg -1 (Zhang et al., 2021b), while the emission factors of BaP from different biomass samples were from 0.01 mg kg -1 to 5.36 mg kg -1 in our study.Previous studies have proved that PAHs from biomass burning increase the risk of lung cancer and respiratory infections (Kim et al., 2011;Sarigiannis et al., 2015).With source appointment of 230 daily PM2.5 samples from outdoor urban and sub-urban sites of Shanghai, China, results show that the contribution of traffic from outdoor collections increased from 18.3% to 31.3% when the annual concentrations of PAHs were above 6.9 ng m -3 (Wang et al., 2016a).In 2012-1013, a yearlong sampling campaign in six major cities of Italy found that total PAHs concentrations ranged from 0.19 to 70.4 ng m -3 and BaP accounted for 17.4% of the total concentration of PAHs, where biomass burning was the main reason for PAHs increasing (Khan et al., 2018).The findings of this study suggest that identifying the sources of PAHs and designing different combustion conditions is important to reduce organic pollutant emissions.Combining practical recommendations with policy implications might be useful in mitigating the health risks associated with PAHs exposure.This approach involves reducing individuals' exposure to PAHs by avoiding or minimizing contact with PAH sources such as cigarette smoke and industrial air pollution, as well as implementing regulations to limit PAH emissions from industrial process and other sources.

Deposition of PM2.5 Particles in the Respiratory Tract
Fig. 2 displays deposition percentages of PM2.5 from biomass combustion in the head, TB, and pulmonary alveoli by breathing via the nose.The model shows that the pulmonary alveoli area, at a concentration of 32.25 µg m -3 , had the highest deposition of PM2.5 (48.30%).The TB region had a deposition concentration of 9.15 µg m -3 (13.71%), while the concentration in the head region was 25.36 µg m -3 (37.99%).Another study used the same multiple-path particle dosimetry model to inspect the deposition of PM, collecting PM from an arterial road located in Chennai city of Tamil Nadu state, India with the Grimm portable environmental dust monitor.They found that approximately 45% of PM2.5 was primarily deposited in the head region, 9% was deposited in the TB region, and the rest was mainly deposited in the pulmonary region (28.2%-52.7%)(Manojkumar et al., 2019).By comparison, our study showed a higher percentage of deposition in the pulmonary alveoli region than in previous reports.Another study in urban areas in Taichung City reported that 52.19% of PM2.5 was deposited in the alveolar region followed by the TB (31.22%) and head (16.58%) regions (Laiman et al., 2022b).Together, this suggested that PM2.5 was mainly deposited in pulmonary alveoli and could have an effect on the lungs.In alveoli, PM2.5 penetrates deep into the respiratory system, leading to inflammation, oxidative stress, and potential damage to lung tissues, increasing long-term health risk.

Bioreactivity
Fig. 3 shows alterations in expressions of YAP, p-YAP, TAZ, p-TAZ, due to PM2.5 exposure in A549 cells.We observed that p-YAP/YAP ratios significantly decreased after exposure to MS2 and WBC2 (p < 0.05).Similarly, a previous study showed that air pollution exposure in Sprague-Dawley rats for 6 months caused a decrease in the p-YAP/YAP ratio (Chang et al., 2022).Another study indicated that YAP and TAZ are important downstream signaling pathways in the mammalian Hippo path (Hong and Guan, 2012).YAP/TAZ are well known to regulate cell proliferation, migration, and survival; they subsequently regulate homeostasis and cancer during tissue amplification and regeneration (Piccolo et al., 2014).YAP is a transcriptional coactivator that is part of the Hippo  signaling pathway, which is a regulatory pathway for controlling organ size and tissue growth (Pocaterra et al., 2020).The result suggests that PM2.5 exposure can disrupt the YAP pathway, altering p-YAP/YAP ratios.In Fig. 4, we also found that there was a significant declining pattern in HMGB1 expression observed in cells after exposure.Results showed that HMGB1 expression significantly decreased after exposure to MSC, WBBs, WBC, WS, WSB2, MS2, and WB2 in A549 cells (p < 0.05).HMGB1 is an important inflammatory response modulator, which can trigger chronic inflammation via influencing cellular hyporesponsiveness (Li et al., 2013;Xue et al., 2020).Through HMGB1-driven autophagy, asbestos causes the transformation of mesothelial cells.Moreover, through HMGB1-mediated cancer cell adherence, quasi-ultrafine particles from coal burning facilitate cell metastasis (Gao and Sang, 2020).Taken together, exposure to PM2.5 emitted from biomass can cause alterations in Hippo pathway-related expressions and HMGB1, which can occur when inhaled PM2.5 is deposited in the lung alveoli region.There is potential for therapeutic interventions based on the findings that blocking the HMGB1/YAP pathway by small molecule inhibitors could be developed to mitigate respiratory health effects triggered by PM2.5.Furthermore, advocacy for stricter environmental policies to reduce PM2.5 levels could be informed.

Correlations Between Emission Factors and Bioreactivity
Relationships of the organic compositions of emission factors with PM2.5 bioreactivity are shown in Fig. 5. YAP was positively correlated with ANT (p < 0.05), PHE (p < 0.05), 1-MP (p < 0.05), FLU (p < 0.05), PYR (p < 0.05), 3,6-DP (p < 0.05), BaA (p < 0.05), CHR (p < 0.05), BbF (p < 0.05), BkF (p < 0.05), BaP (p < 0.05), BeP (p < 0.05), PER (p < 0.05), IcdP (p < 0.05), BghiP (p < 0.05) and DahA (p < 0.05).Previous study on type II alveolar epithelial cells (AECII) exposed to zinc oxide nanoparticles showed increased nuclear YAP expression while decreasing cytoplasmic YAP expression, resulting in an increase in YAP/TAZ transcriptional activity (Laiman et al., 2022a).However, limited experimental studies were available on the relationships between YAP/TAZ and organic components in PM2.5, suggesting further experiments are necessary to explore the potential biological responses caused by PM2.5 and chemicals in biomass samples.This will help advance our understanding of the molecular mechanisms underlying the health effects associated with PM2.5 exposure.E-cadherin might be a mediater in the inflammatory responses in A549 cells, FaDu cells, as well as in the mouse lungs (Chang et al., 2022;Chuang et al., 2015;Tung et al., 2021).A previous study suggested the elements could disrupt E-cadherin expression after the Fenton reaction (Waisberg et al., 2003).Another study also found negative correlations of variations in E-cadherin with elemental constituents according to a Pearson correlation analysis (Tung et al., 2021).Additionally, E-cadherin was found to be influenced by air pollution, thus facilitating the induction of proliferative inhibition and reductions in p-YAP/YAP expressions in emphysema (Chang et al., 2022).Furthermore, combustion-generated PM--both fine and ultrafine--decreased E-cadherin expression to produce the EMT (Bae et al., 2013).Future studies should focus on exploring correlations of E-cadherin with organic components in biomass emissions.

CONCLUSIONS
Our study delved into the emission factors of PAHs from various biomass fuels and their processed forms, revealing how their bioreactivity affects A549 lung cells.We discovered that exposure to smoke from biomass burning alters the expression of cellular proteins, which could be resulted from PAH compounds in PM2.5, indicating a link between these emissions and their biological impact.The limitations of the study include: 1) minor difference caused by sampling, combustion process and seasonal fluctuation; 2) in-vivo effects of biomass emissions were not measured; 3) inorganic chemicals in PM2.5 can be involved in future exploring in YAP/TAZ expressions; 4) factors including particle size, shape, or chemical composition that can affect deposition patterns were not considered in our MPPD models.This research on the bioreactivity of PM2.5 particles from biomass combustion underscores the urgent need to comprehend these emissions for public health protection and the development of environmental mitigation strategies.

Fig. 2 .
Fig. 2. Deposition fractions of PM2.5 in the head, tracheobronchial (TB), and pulmonary alveoli (P) after inhalation into human lungs by Multiple-Path Particle Dosimetric (MPPD) model.The model used was of human symmetric lung model.

Fig. 5 .
Fig. 5. Correlation of emission factors of organic compounds in PM2.5 with yes-associated protein (YAP), phosphorylated yes-associated protein (p-YAP), p-YAP/YAP, transcriptional coactivator with a PDZ-binding motif (TAZ), phospho-TAZ (p-TAZ), p-TAZ/TAZ, high-mobility group box 1 (HMGB1), and E-cadherin.The size of the point indicates the p value.The depth of the color indicates the strength of the correlation coefficient (red, positive correlation; white, weak correlation; blue, negative correlation).