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 (Cincinelli, Scopetani et al. 2017, Koongolla, Lin et al. 2020). Moreover, MPs particles ingested have been texted in various tissues of mammals and aquatic such as mice, fish, rats, etc. (Feng, Zeng et al. 2021, Shengchen, Jing et al. 2021, Zhang, Sun et al. 2021), even threatening the integrity of the food chain and the ultimate ability of ecosystem restoration (Thomas, Perono et al. 2021), 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 inflammatory 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 flora 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 final effluent 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 fish 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 significance to the protection of biodiversity and human food security.
The skin is the body's first 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 inflammatory 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 inflammation and keratinocyte apoptosis through p-NFκB p65 (Lai, Wang et al. 2021). As we all know, inflammation 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, Zheng, Wang et al. 2020). 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 CdCl2 exposure reduces neutrophil infiltration by inhibiting the expression of chemokines, and inhibits the expression of pro-inflammatory cytokines, thereby impairing skin wound healing (Mei, Yao et al. 2017). Nutritional deficiency diseases such as selenium deficiency 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 inflammation.
In summary, too many Nets in the tissues can cause excessive local inflammation, 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 inflammation (Shengchen, Jing et al. 2021). Due to the influence of daily necessities and industry, poisons in the environment often appear at the same time (Zhang, Lin et al. 2021, Zhang, Wang 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 Immunofluorescence to determine the formation of the Nets and wound healing. In addition, we detected the expression levels of wound skin inflammatory 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.