Combined Negative Effects of Microplastics and Plasticizer DEHP: the Increased Release of Nets Delays Wound Healing


 Introduction: Environmental pollutants microplastics (MPs) and di (2-ethyl) hexyl phthalate (DEHP) can cause damage to multiple organs by causing oxidative stress. Oxidative stress participates in the healing of skin wounds through the release of neutrophil extranets (Nets). Here, we studied the effects of DEHP and MPs on skin wound healing in mice after single and combined exposure for 1 month. Results: The results showed that MPs delayed the healing of skin wounds, and the combination of the two delayed wound healing more significantly. The results of in vivo and in vitro experiments showed that the release of oxidative stress and Nets in the single exposure group increased, and the combined exposure group increased more. Further mechanism studies showed that the skin chemokines of the single exposure group increased, the NF-κB pathway was activated, the Wnt pathway was inhibited, and the epidermal growth factor and fibrosis-related indicators decreased. The combined exposure group showed a more obvious trend.Conclusion: In summary, the above results indicate that DEHP combined with MPs induces an increase in the release of Nets by causing excessive skin ROS production and increases the expression of chemokines and interferes with the expression of healing factors by regulating the NF-κB and Wnt pathways.


Introduction
Plastic products have a sharp increase in the use of plastic products due to their cheapness and convenience. They can be broken down into small fragments through physical and chemical decomposition and become microplastics (MPs) and nanoplastics (NPs) (Yousif and Haddad 2013).
However, due to irresponsible waste recycling and slow biodegradation rates, plastics have accumulated in the global environment. It has been proved that plastics have been detected in surface waters and oceans around the world ( which has caused people to worry about their environmental and human health risks. At the same time, more and more evidence indicate that MPs may cause immune and in ammatory responses, oxidative stress (Shengchen, Jing et al. 2021), abnormal changes in metabolism and even genetic damage (Thomas, Perono et al. 2021). It has been reported in the literature that the use of MPs and tributyltin (TBT) alone or combined may cause an imbalance of the intestinal ora in mice (Jiang, Yuan et al. 2021). Studies have found that PS-MPs cause excessive production of reactive oxygen species (ROS) and inhibit skeletal muscle regeneration, inhibits the myogenic differentiation of C2C12 cells, and promotes fat differentiation through increasing the level of NF-κB pathway (Shengchen, Jing et al. 2021).
Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer. Due to its widespread use, DEHP pollution in the environment seriously threatens the health of humans and animals (Gao, Xu et al. 2019). In the Jiulong River Estuary in the southeastern of China, the annual DEHP concentration in water ranges from 0.12 to 12.4 mg/L. DEHP is one of the main components of PAE distribution in water (Li, Liang et al. 2017).
Although a large number of MPs and DEHP are removed from the nal e uent of the sewage treatment plant, MPs (2.419×10 MP/d) and DEHP still enter the water environment every day, posing a threat to the health of sh and local residents (Takdastan, Niari et al. 2021). Human and animal exposure to DEHP through ingestion, inhalation and skin absorption can cause liver and kidney damage (Camacho, Latendresse et al. 2020), male and female reproductive disorders and developmental toxicity (Wang, Tian et al. 2021). In addition, it is reported that DEHP induces the formation and apoptosis of carp neutrophil extracellular traps (Nets) by promoting ROS burst and autophagy (Yirong, Shengchen et al. 2020). DEHP can cause the production of ROS in HMC-1 cells, the activation of NF-κB pathway, and the regulation of COX-2 and IL-1β expression (Oh, Lim et al. 2010). There are data showing that MPs and DEHP inevitably have the risk of simultaneous exposure to humans and animals in the ecological environment (Takdastan, Niari et al. 2021). Therefore, carrying out relevant research on MPs and DEHP is of great signi cance to the protection of biodiversity and human food security.
The skin is the body's rst line of defense against injury and infection. When the skin is damaged, a series of complex and coordinated processes will occur in the body, involving various cellular components including in ammatory cells and tissue repair cells. And pathways such as TGFβ and Wnt also promote the healing of skin wounds (Gos, Miłoszewska et al. 2009, Yang, Tsai et al. 2017). Zinc oxide nanoparticles (ZnO NPs) affect psoriasis-like lesions and promote in ammation and keratinocyte apoptosis through p-NFκB p65 (Lai, Wang et al. 2021). As we all know, in ammation is one of the typical features of the wound healing process, and neutrophils are quickly recruited to the wound bed and participate in the early stages of wound healing (Wong, Demers et al. 2015). Some studies have found that neutrophils migrate to the wound site and can form a Net (Martin and Leibovich 2005). It is worth noting that in a variety of pathological conditions, the increase of Nets has been shown to impair wound healing (Wang and Jing 2018). Nets delayed peptidyl arginine deiminase-4 (PAD4)-mediated the healing of skin wound in diabetic mice (Wang and Jing 2018). Nets formation requires the production of ROS.
ROS stimulates Nets to activate PAD4, and then leads to the citrullination of histone H3 (Cit H3), thereby activating neutrophil elastase (NE) and the migration and processing of myeloperoxidase (MPO) (Wang, Zheng et al. 2018. Bongkrekic acid (BKA) increases the production of ROS in neutrophils, and PAD4 and P2X1 receptors also mediate the formation of Nets triggered by BKA (Zhou, Sun et al. 2021). Fumonisin B (FB) induces the formation of Nets, increases ROS levels, and reduces SOD and CAT activities (Wang, Liu et al. 2020). It has been reported that chronic low-dose CdCl 2 exposure reduces neutrophil in ltration by inhibiting the expression of chemokines, and inhibits the expression of pro-in ammatory cytokines, thereby impairing skin wound healing (Mei, Yao et al. 2017). Nutritional de ciency diseases such as selenium de ciency can increase the number of chicken neutrophils by increasing the expression of chemokines, and induce the increase of Nets through ROS bursts, which can cause arteritis (Chi, Zhang et al. 2021). The above studies have shown that the wound healing process involves the recruitment of neutrophils, the burst of ROS, and the formation of Nets, which may be accompanied by in ammation.
In summary, too many Nets in the tissues can cause excessive local in ammation, leading to delayed wound healing. DEHP can lead to the release of Nets caused by ROS bursts (Yirong, Shengchen et al. 2020). Exposure to MPs can cause oxidative stress and in ammation (Shengchen, Jing et al. 2021). Due to the in uence of daily necessities and industry, poisons in the environment often appear at the same time (Zhang, Lin et al. 2021), DEHP and MPs may be exposed at the same time (Takdastan, Niari et al. 2021). Therefore, we have reason to doubt whether the combined exposure of MPs and DEHP has any effect on skin wound healing in mice, and whether ROS/Nets and NF-κB/Wnt pathways are involved in this wound healing process. In this study, mice were used as the research object to establish a skin trauma model after 1 month of MPs exposure, DEHP exposure and dual exposure. Then we used H&E staining, Masson staining and Immuno uorescence to determine the formation of the Nets and wound healing. In addition, we detected the expression levels of wound skin in ammatory factors, Wnt pathway, TGFβ pathway and healing factors. The effects of MPs and DEHP on the formation of Nets were detected in vitro. This study purposes to proclaim the mechanism which synergy of DEHP and MPs to cause delayed wound healing through the increasing release of Nets. It will provide new views into the toxicology research of DEHP and MPs and the treatment of wound healing diseases.

DEHP combined with MPs delays the healing of skin wounds
According to the pictures of wound healing, it can be directly observed that DEHP and MPs can cause the delay of wound healing compared with the control group. Surprisingly, the wound healing was the worst in the DEHP+MPs group (Fig. 1a). In addition, the wound regeneration rate further showed that the control group healed 90%, MPs and DEHP healed about 40% and 60%, while the DEHP+MPs group only healed less than 20% (Fig. 1b) (p<0.05). In addition, the weight ratio before and after the establishment of the trauma model was analyzed, and it was found that the ratio of the DEHP+MPs group increased (Fig. 1c) (p<0.05), indicating that the skin trauma caused the mice to lose weight when DEHP and MPs were exposed together.
H&E staining showed that the wound in the control group was basically healed, the boundary between the disease and healthy was gradually blurred, and the epidermal hair follicle was regenerated well (red arrow) (Fig. 1d). Masson staining also con rmed that the healed wound was lled with a large amount of collagen bers, and under a 10X microscope, it was also found that the muscle layer of the wound was regenerated from the edge (yellow arrow) (Fig. 1e). In the DEHP group, the boundary between disease and health was obvious, with slight in ammatory cell in ltration, but the skin wound was basically healed, hair follicles began to form, and the magni cation revealed that the muscle tissue was not signi cantly regenerated (Fig. 1d). Combined with Masson staining, it was found that most of the wound was lled with brotic collagen bers (black arrow) (Fig. 1e). A large crust was seen in the MPs group, the wound was in the initial stage of healing, and the bottom of the wound was regenerated obviously (green arrow) ( Fig. 1d). And Masson staining also found that there is slight brosis here, and brosis and in ammatory cell in ltration are obvious at the junction of disease and health (Fig. 1e). In the DEHP+MPs group, brosis and in ammatory cell in ltration occurred at the junction of disease and health, and broblasts proliferated, but there was no obvious healing at the skin wound, only a thin layer of crust (Fig. 1d, 1e). In short, H&E and Masson staining show that, compared with the control group, DEHP mainly delayed the muscle regeneration in the healing wound, and the brosis was no different from the control group. The skin wound regeneration in the MPs group was slow, and the brosis was milder than that in the control group and the DEHP group. The skin wounds in the DEHP+MPs group did not heal, and the degree of brosis was the lowest among the four groups.

Effects of DEHP and MPs on skin oxidative stress and neutrophil ROS production
Since oxidative stress can induce the formation of Nets, we rst texted the superoxide production and antioxidant enzyme activities during skin wound healing under DEHP and MPs exposure. As shown in These results indicate that DEHP and MPs can induce overproduction of ROS, and the combination of the two makes ROS reach higher levels.

Effects of DEHP and MPs on the recruitment of neutrophils and the formation of Nets in mouse skin
Then in order to detect the formation of Nets, we detected the expression of Nets markers. The results showed that the uorescence intensity of MPO and Cit H3 in mouse skin wounds increased signi cantly with the exposure of DEHP or MPs (Figure 3a, 3b), and the DEHP+MPs group made the uorescence intensity reach another height. In view of the net appearance of Nets, we counted Merged graphs and found that the number of Nets in the DEHP group and MPs group increased, while the DEHP+MPs group further increased (Figure 3c) (p<0.05). We also found that the exposure of DEHP or MPs induced an up-regulation of Cit H3, NE, MPO and PAD4 mRNA levels, and the combined treatment of DEHP and MPs further up-regulated ( Fig. 3d) (p<0.05). The western blot results also added that the DEHP and MPs combined treatment group Nets marker NE, Cit H3 and MPO protein expression levels were the highest ( Fig. 3e) (p<0.05). The above results indicate that DEHP promotes the increase in the formation of Nets in the wounded skin of mice induced by MPs.

Effect of DEHP and MPs on the formation of Nets of isolated neutrophils in vitro
In order to verify the effect of DEHP and MPs on the formation of Nets in vitro, we extracted neutrophils from the peripheral blood of mice through a kit. Compared with the control group, we observed that the DEHP group had very few Nets formation through scanning electron microscopy ( Fig. 4a). In the MPs group, we not only saw the basic morphology of MPs (1-10µm in diameter), but also observed that neutrophils adhered to the surface of MPs and formed obvious Nets. It is worth noting that DEHP+MPs were showing more Nets release (Fig. 4a). In addition, we performed Sytox Green staining on the neutrophils and found that compared with the control group, there was very little green lamentous uorescence (red arrow) in the DEHP group (Fig. 4b). The green lamentous uorescence in the MPs group was relatively more, and the DEHP+MPs group showed a lot of green lamentous uorescence, which was comparable to the positive control group (PMA group). By detecting the expression levels of Nets components (NE, Cit H3, MPO and PAD4) in neutrophils, we found that DEHP and MPs induced a signi cant rise in the levels of Cit H3 and NE (p<0.05), while the expression changes of MPO and PAD4 were not signi cantly different in Fig. 4c (p>0.05). It is worth noting that the mRNA and protein standards of NE, H3, MPO and PAD4 in the DEHP+MPs group were signi cantly increased (p<0.05), which were at least two times higher than the control group (Fig. 4d). The above results indicate that single exposure induces an increase in the release of Nets, but the combined treatment of DEHP and MPs makes the release of Nets more signi cantly increased.

Effect of excessive release of Nets on in ammation
We further tested the expression changes of NF-κB in ammation pathway genes (NF-κB, COX-2, TNF-α, iNOS and PTGEs) and ILs (IL-1β, IL-6, IL-8 and IL-10) in ammatory factors, which re ects the in uence of in ammatory factors released by Nets on the NF-κB in ammatory pathway. We found that DEHP or MPs caused in ammation in the wounds of mice, which was manifested by the upregulation of NF-κB pathway related genes, IFN-γ, IL-1β, IL-6 and IL-8 in the DEHP and MPs groups, while the downregulation of IL-10 ( Figure 5a, 5c) (p<0.05). However, the protein level of IL-10 in the MPs group did not change signi cantly (p>0.05). In addition, under the co-stimulation of DEHP and MPs, the level of in ammation further increased, manifested by changes in the mRNA levels ( Figure 5a) and protein levels (Figure 5b, 5c) of the interleukin and NF-κB pathway related genes (p<0.05). The above results indicate that DEHP or MPs can cause Nets-mediated skin wound in ammation in mice, and DEHP can exacerbate the in ammatory effect of MPs.
2.6 Effect of excessive in ammation on Wnt pathway, TGFβ pathway and EGF expression Due to the important role of Wnt pathway in brosis healing, we analyzed the mRNA and protein levels of Wnt pathway (Wnt, GSK-3β and β-catenin). We discovered that DEHP or MPs treatment signi cantly reduced the levels of Wnt and β-catenin, and signi cantly increased the level of GSK-3β (Fig. 6a, 6b) (p<0.05). The DEHP+MPs dual treatment group exacerbated the changes in these genes. Then, we detected the expression level of TGFβ pathway, which is closely related to brosis and healing.
Immuno uorescence results showed that compared with the control group, DEHP or MPs treatment reduced the uorescence intensity of TGFβ and α-SMA to varying degrees, while the uorescence intensity of the combined treatment group further decreased (Fig. 6c). The mRNA and protein results of TGFβ and α-SMA also corresponded to the immuno uorescence results (Fig. 6d, 6g). In addition, we also found growth promoting factor 1 (IGF-1), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), basic broblast growth factor (bFGF), type I collagen (Col1) and type III collagen (Col3) levels were signi cantly lower than the single exposure group (Figure 6e) (p<0.05). Then we also found that single exposure resulted in the downregulation of epidermal growth factor (EGF) (Figure 6c, 6f, 6g). DEHP combined with MPs reduced the expression of EGF to the lowest value among the four groups, which coincided with the appearance of skin healing. The expression level of bFGF also corresponded to the level of EGF (Fig. 6e, 6g). Moreover, we also compared the mRNA levels of two EGFs (vEGF and bEGF) and found that DEHP and MPs have a signi cant effect on the expression of vEGF (Fig. 6f) (p<0.05).
These ndings indicate that DEHP or MPs inhibit the TGFβ and Wnt pathway and reduce the level of EGF. The combined treatment of DEHP and MPs will exacerbate this phenomenon.

Discussion
Although some studies have found that exposure to MPs and DEHP can cause immune dysfunction, most studies have focused on aquatic animals or exposure alone. It is well known that the healing of skin wounds is also closely related to immune function. These results re ect that DEHP combined with MPs exposure rst recruits more neutrophils to the wound by up-regulating the level of chemokines, while dual exposure stimulates the release of Nets by aggravating oxidative stress. Therefore, the two pathways working at the same time allow us to discover that the dual exposure of DEHP and MPs leads to excessive activation of Nets in the skin wound.
Recently, the downside of excessive activation of Nets, that Nets may be the center of the vicious circle of in ammation when in ammation occurs, has gradually been revealed ( In this experiment, we rst found through immuno uorescence staining that DEHP or/and MPs reduced the expression of EGF, a factor that re ects epidermal regeneration. And the expression of TGFβ, α-SMA and healing-related factors also changed accordingly. It shows that the Wnt pathway is involved in regulating the expression of IGF-1, EGF, TGFβ, KGF, PDGF, bFGF and other growth factors to affect the healing of skin wounds. It is worth noting that the combined exposure of DEHP and MPs has a more signi cant decrease in expression level than other groups. The above results indicate that excessive in ammatory response inhibits the Wnt pathway is the mechanism of delayed skin wound healing. In conclusion, we found that the combined effects of DEHP and MP caused delayed skin wound healing in mice. In terms of mechanism, DEHP promotes MPs-induced overproduction of ROS and increased levels of chemokines in wound tissues, thereby stimulating increased Nets release, and interferes with the University Experimental Animals. The indoor air is fresh, 75%±5% relative humidity, suitable temperature, and free supply of water and feed (Shenyang Changsheng provides standard pellet feed). Observe all experimental animals for one week and carry out follow-up tests after there is no death or mental abnormality. Forty 6-week-old mice were stochastically separated into Control group, DEHP group, MPs group and DEHP+MPs group (n=10). The control group had normal diet and drinking water. MPs (Mingshuo Chemical Company, China) were added to the drinking water (0.1 g/L) of the MPs group and DEHP+MPs group, and the water was mixed 3 times a day. In the DEHP group and the DEHP+MPs group, DEHP (Solarbio, SD9580) was added to the feed at a dose of 200 µM/kg, while the control and MPs group were given the same amount of corn oil (solvent). All groups of food and drinking water were free to eat. The whole process lasted for four weeks and then the mouse skin injury model was constructed.
Use iso urane (EZVET, China) to perform inhalation anesthesia on mice, and the anesthesia time does not exceed 30 s. We shaved off the hair on the back of the mouse and used a skin punch and scissors to cut out a round full-thickness skin with a diameter of 6 mm ± 1 mm. One week after the wound was healed, the mice were euthanized by iso urane inhalation. One portion was fastened in 4% neutral formalin (Biosharp, China) for H&E staining, Masson staining and Immuno uorescence. Another portion of the skin wound was taken out and stored at -80°C for future use.

Cell separation, extraction and treatment
The mice were inhaled anesthetized with iso urane for 30 s, then opened the chest cavity of the mice with clean scissors and tweezers and used a 5 mL syringe (pre-lled with 0.6 mL anticoagulant, TBDTM-0050) for cardiac blood sampling. Then, we used the mouse peripheral blood neutrophil extraction kit (TBD, China) to isolate neutrophils according to the instructions. The medium ratio of neutrophils is 90% RPMI-1640 (Gibco, USA), 10% fetal bovine serum (FBS; BI, Italy) and 1% penicillin-streptomycinamphotericin B (Beyotime, China). The neutrophils were cultured in a humidi ed 37°C incubator with 5% CO 2 . We used the trypan blue method to determine the survival rate of isolated neutrophils, which exceeded 95%. In addition, we made cell smears and checked the purity of neutrophils with Giemsa staining, which exceeded 99%.
Freshly isolated primary neutrophils were cultured for 2 h, then DEHP and MPs were added to the DEHP group, MPs group and DEHP+MPs group at 400 µM and 0.1 mg/L and cultured for 6 h. If PMA is added for stimulation, we need to add 500 nM PMA for 3 h after adding DEHP or MPs for 3 h.

Histologic preparation and assessments
In order to reveal the internal mechanism of the pathological changes and delayed healing of MPs and DEHP on the wound healing, we performed the following histological examination on the wound site of mice. The skin of mice was xed with 4% paraformaldehyde for at least 24 h. Then, the skin tissues of each group were evenly embedded in 3 parallel specimens in liquid para n, and after cooling, 3 sections (4 µm) (n=3) were made on a microtome (Leica, Germany). As the method described by the predecessors

Scanning electron microscopy
Neutrophils were inoculated on polylysine-coated cell slides (Liangyi Biotech, China), treated according to the dose in section 2.2, and nally xed with 2.5% glutaraldehyde at 4°C overnight. According to the way described by the predecessors, we used the scanning electron microscope to observe the formation of the mesh (Yin, Yang et al. 2019). We used a scanning electron microscope (SU-8010, Hitachi Ltd., Tokyo, Japan) to magnify 8000× to take pictures.

Detection of oxidative stress in mice skin
The skin tissues of each group were homogenized in 0.9% saline, and the supernatant was centrifuged for the detection of the oxidative stress kit. We used Coomassie Brilliant Blue Method to determine protein concentration. MDA, GSH, H 2 O 2 , GSH-Px, SOD, CAT and T-AOC detection kits were all purchased from Nanjing Jiancheng Institute of Biological Engineering. SOD activity, xanthine oxidase method; GSH-PX and GSH activity, 5'5-dithio-nitrobenzoic acid color method; MDA content, thiobarbituric acid method: CAT activity, molybdate ammine complex Compound method; T-AOC, phenanthline complex method.

ROS detection
According to the method stated in Song's paper (Song, Li et al. 2021), we used a reactive oxygen kit (Nanjing Jiancheng, China) to uorescently label the respiratory bursts of neutrophils in the four treatment groups to analyze the generation of ROS. Transfer 100 µL of PBS containing neutrophils to a 96-well plat and use an In nite M200® plate reader (TECAN, Switzerland) to detect the uorescent signal.
Then we obtained ROS images of neutrophils through a uorescence microscope (Thermo Fisher Scienti c, USA).

Fluorescence microscopy assessment of NETs
We rst put poly-L-lysine-coated glass slides in a 24-well plate, planted neutrophils (10 5 cells), and then let it stand at 37°C for 2 h. Then perform grouping processing as described in section 2.2. Washing twice with PBS, and then incubate with 5 µM Sytox Green stain (Invitrogen, USA) and 5 µM DAPI stain (Beyotime, China) for 30 min. After washing twice with PBS, it was covered with anti-uorescence mounting tablets (Beyotime, China). A uorescence microscope (EVOS FL Auto, Life Technologies, MD, USA) was used to evaluate the formation of Nets.

RNA preparation and RT-PCR in tissue and cell
We use RT-PCR to detect the mRNA expression of target genes in skin tissues and neutrophils. As mentioned before (Shengchen, Jing et al. 2021), Trizol (Takara, Japan) was used to extract total RNA from mouse skin tissues and neutrophils according to the manufacturer's instructions. When the A260/A280 of total RNA is between 1.9-2.1 and the concentration is >350 ng/µL, we used cDNA rststrand synthesis kit (Bioer, China) and Real Time PCR Kit (Bioer, China) for reverse transcription and uorescence quanti cation of total RNA from wounded skin and neutrophils. The mRNA primer sequence used in the experiment was synthesized by Shanghai Shenggong (Table 1). The GAPDH sequence was used as a standardized endogenous control. The 2 −ΔΔCT method was used to calculate the expression level of target mRNA relative to GAPDH.

Statistical analyses
All data were submitted to one-way analysis of variance, and the Tukey method was used for multiple comparisons. GraphPad Prism (version 9.1, USA) was used to draw graphs. The results of this experiment were shown as mean ± standard error of mean (SEM). As shown in the legend, n represents the number of replicates of a single mouse or a single experiment. We performed the Shapiro-Wilk normality test and found that the data in this article passed the normality test (alpha=0.05). Declarations Ethics approval and consent to participate All procedures were approved by the Northeast Agricultural University Animal Management and Use Committee (SRM-11).

Consent for publication
Not applicable.

Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declaration of Competing Interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or notfor-pro t sectors.
CRediT authorship contribution statement