Co-exposure to cadmium and microplastics promotes liver fibrosis through the hemichannels -ATP-P2X7 pathway

doi:10.21203/rs.3.rs-2333129/v1

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Co-exposure to cadmium and microplastics promotes liver fibrosis through the hemichannels -ATP-P2X7 pathway

https://doi.org/10.21203/rs.3.rs-2333129/v1

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Background

The widespread use of plastic products and the imperfection of plastic recycling systems have led to a continuous increase in microplastics (PS) in the environment. Microplastics have an adsorption effect and can act as carriers for other pollutants in the environment. Cadmium (Cd) is a heavy metal that interacts with microplastics. However, the potential toxicity of co-exposure of cadmium and microplastics to the body is not clear. This study focuses on the effects of co-exposure to cadmium and microplastics on liver fibrosis and its mechanism.

Results

In this study investigated, Cd+PS exposure increased superoxide anion production and promoted extracellular ATP release compared with exposure to Cd or PS alone. Cd+PS increased inflammatory cell infiltration, activated the P2X7-NLRP3 signaling pathway, and promoted inflammatory factor release. Cd+PS aggravated Cd- or PS-induced liver fibrosis and induced liver inflammation. In AML12/HSC-T6 cell in vitropoisoning model, exposure of AML12 cells to Cd+PS increased the opening of connexin hemichannels and promoted extracellular ATP release. Treatment of HSC-T6 cells with the supernatant of AML12 cells exposed to Cd+PS significantly promoted HSC-T6 cell activation. Treatment of HSC-T6 cells with different concentrations of ATP produced similar results. TAT-Gap19TFA, an inhibitor of connexin hemichannels, significantly inhibited the ATP release and activation of Cd+PS-treated HSC-T6 cells. Finally, the expression of the ATP receptor P2X7 was silenced in HSC-T6 cells, which significantly inhibited their activation.

Conclusion

Cadmium and microplastics have a synergistic toxic effect on the liver, destroy the microenvironment in the liver, and promote the development of liver fibrosis through the hemichannel-ATP-P2X7 signaling pathway. Our study reveals the impact of co-exposure to cadmium and microplastics on chronic liver diseases, providing a theoretical basis for disease prevention and treatment.

Cadmium

Microplastics

hemichannel

ATP

P2X7

Cadmium and microplastics have combined toxicity and promote the development of hepatic fibrosis.
Cadmium and microplastics interact to enhance hemichannel function and promote the extracellular release of ATP.
ATP acts as a signaling molecule, activating stellate cells and promoting the development of hepatic fibrosis.
ATP activates hepatic stellate cells by interacting with P2X7, promoting the development of hepatic fibrosis.

The increasing detection of microplastics(PS)in organisms indicates PS has become an important environmental pollutant in today's society. The idea for PS comes from a paper published in the journal Science. Plastic fragments and particles less than 5mm in diameter are collectively referred to as PS[1]. Metals with a density above 4.5g/cm3 are called heavy metals. Cadmium (Cd) is a heavy toxic metal with unknown biological functions in body. Over time, Cd accretion in the different visceral organs is said to impair the function of visceral organs, which is associated with a relatively long biological half-life and very low rate of excretion[2]. Both Cd and PS have an enrichment effect and can enter the biological chain and accumulate in the organism[3]. The liver is an important target organ of Cd accumulation and main efficient organ of Cd toxicity[4]. Cd can induce oxidative stress in liver cells and promote liver cell death[5]. PS can not only cause damage to biological organisms alone but also act as a carrier of other environmental pollutants[6]. The smaller the volume of PS, the larger the specific surface area and the stronger the ability to absorb environmental pollutants[7]. Co-exposure to PS and other environmental pollutants increases the bioavailability of pollutants and their adverse effects on organisms[8]. The environment is full of persistent organic pollutants, such as bisphenol A (BPA) and polychlorinated biphenyls (PCBs), which combine with PS to develop new pollutant complexes[9]. The combination of nanoparticles and perfluorooctane sulfonic acid (PFOS) can aggravate oxidative stress[10].

The liver is a huge complex of more than 2.5 billion cells, and these cells interact with each other through a variety of channels. One of the important ways is the gap junction intercellular communication(GJIC), which is formed by the polymerization of two and a half channels between cells to allow the transfer of small molecules between cells[11]. The hemichannel is a part of the GJIC, but it acts as the communication bridge between the cell and the outside world when the GJIC is not formed[12]. Studies have shown that the opening of hemichannel may lead to the massive outflow of amino acids and ATP, as well as the intracellular Ca²⁺ overload partially mediated by Ca²⁺ influx[13, 14]. Hemichannel is closely related to the prevalence and development of various diseases[15].

Chronic Hepatic disease is a general term for long-term Hepatic inflammation and necrosis caused by various factors[16]. Hepatic fibrosis is an important link in the development of the disease, and excessive accumulation of extracellular matrix is an important characteristic[17]. Hepatic fibrosis is often accompanied by cirrhosis and Hepatic cancer[18]. Hepatic Stellate Cells (HSCs)are the main source of synthesis and secretion of extracellular mesenchyme and the main driving force of hepatic fibrosis development[19]. The activation of HSCs is mediated by various signaling molecules released by hepatocyte parenchymal cells, hepatic sinusoidal endothelial cells, and macrophages[20].

Therefore, it is particularly important to study the ecotoxic effects of PS, especially the combination of PS with other pollutants such as Cd. Given the potential pro-hepatic fibrotic effect of Cd with PS and the effect on the hemichannels connecting the liver. We speculate that the hemichannel is involved in Cd and PS-induced liver fibrosis as expected. The hemichannels promote the development of liver fibrosis by promoting the extracellular release of ATP.

Co-exposure of Cd and PS promoted the development of hepatic fibrosis and showed a synergistic effect

To investigate the effects of Cd and PS on hepatic fibrosis. We detected hepatic fibrosis levels after Cd and PS exposure in vivo and in vitro. Masson staining and Sirius red staining were used to evaluate the fibrosis level in the liver. Masson staining showed that, compared to control group, the Cd or PS group increased the level of collagen fibers in the liver. While compared with the Cd or PS group, the level of hepatic fibrosis was enhanced in Cd + PS group (Fig. 1A). Similar results were observed in Sirius red staining (Fig. 1B). α-smooth muscle actin (α-SMA) is one of the markers for the activation of Hepatic stellate cell (HSC) cells. The immunohistochemical results showed that compared with the control group, the expression level of α-SMA was increased in Cd group, and the Cd + PS group significantly increased the expression level of α-SMA compared with the Cd group (Fig. 1C). Next, we analyzed the interferon levels of α-SMA, TGF-β, collagen Ⅰ, and collagen Ⅳ. The results showed that, compared with the control group, Cd or PS group did not significantly increase the expression of α-SMA, collagen Ⅰ, and collagen Ⅳ. Only the PS group increased TGF-β expression. However, the expressions of α-SMA, TGF-β, collagen Ⅰ, and collagen Ⅳ were significantly increased in the Cd + PS group (Fig. 1D). In vitro, AML12 cells were treated with Cd or/and PS for 12h, and the supernatant of the cells was added to HSC-T6 cells for further culture for 12h(Fig. 1E). The results showed that the number of cells increased in the Cd and PS treated group, while the Cd + PS group intensified the trend (Fig. 1F). Western Blot (WB) results showed, Cd or PS increased α-SMA expression, and the Cd + PS group intensified this trend. Furthermore, the Cd + PS co-treatment increased the expression level of TGF-β compared to Cd or PS group (Fig. 1G).

Co-exposure to Cd and PS induces liver inflammation

Routine blood results showed that exposure to Cd + PS significantly increased the number of leukocytes, lymphocytes, and neutrophils (Table 1). Light microscopy results showed that exposure of Cd + PS significantly increased the inflammatory cell infiltration (Fig. 2A). The qRT-PCR results showed that exposure to Cd + PS increased the expression of pro-inflammatory factors while inhibiting the expression of anti-inflammatory factors (Fig. 2B-F). WB showed that exposure to Cd + PS increased the levels of NLRP3, ASC, and Caspase-1 (Fig. 2G-J). The immunohistochemistry results also showed that Cd and PS exposure increased the IL-1β and IL-18 levels (Fig. 2K, L).

Table 1

Effects of Cd and PS co-treatment on blood routine in mice
	Control	Cd	PS	Cd + PS
WBC	3.53 ± 0.12	4.07 ± 0.06	4.70 ± 0.40**	6.07 ± 0.35**^##
Lymph#	2.83 ± 0.06	3.20 ± 0.10	3.70 ± 0.36**	4.80 ± 0.40**^##
Mon#	0.10 ± 0.00	0.10 ± 0.00	0.13 ± 0.06	0.13 ± 0.06
Gran#	0.53 ± 0.06	0.83 ± 0.06**	0.93 ± 0.12**	1.17 ± 0.06**^##
Lymph%	76.73 ± 0.99	79.33 ± 0.31**	78.43 ± 0.38	81.57 ± 0.67**^##
Mon%	1.83 ± 0.12	2.10 ± 0.20	2.07 ± 0.06	2.60 ± 0.36**
Gran%	16.83 ± 0.32	18.70 ± 0.00**	19.27 ± 0.12**	19.87 ± 0.75**^##

Co-exposure to Cd and PS enhances hemichannel function

A hemichannel is a bridge between the intracellular and extracellular environment. In this study, we found that the supernatant of AML12 cells treated with Cd or/and PS can induce the activation of HSCs. So, we speculated that the hemichannel may play an important role in this process. First, we detected the expression of CX32, the most important connexin in liver tissue, and we found that Cd or PS did not affect the expression of CX32(Fig. 3A). The localization of CX32 in cells was detected using immunohistochemistry and immunofluorescence. Exposure to Cd or/and PS changed the localization of CX32 in cells from a uniform sheet-like intercellular connection to a single-cell cohesive point-like state (Fig. 3B, C). Furthermore, AML12 cells were exposed to Cd or/and PS for 12h, the scratch assay was used to detect gap junction functions. Exposure to Cd + PS inhibited the diffusion of LY dye (Fig. 3D). EB uptake experiments showed that Cd and PS increased EB uptake (Fig. 3E).

The extracellular release of ATP promotes the activation of hepatic stellate cells

The opening of the hemichannel can promote the extracellular release of intracellular ATP, amino acids, and other macromolecules. ATP is an important energy substance in the cell, and extracellular ATP can also induce the release of inflammatory factors. Therefore, we speculate that ATP can be used as a signaling molecule to induce HSCs activity. To verify the role of ATP in HSCs activation, we first measured the amount of ATP in mouse liver and serum. The result is shown in Fig. 4A,B, the Cd or PS group did not increase the content of ATP in serum, while the Cd + PS group significantly increased the content of ATP in serum, and the changing trend of ATP in the liver was similar to that in serum. In addition, we examined ATP synthetase activity, Cd or/and PS increased the activity of Ca²⁺-Mg²⁺ ATP synthetase but decreased the activity of Na⁺-K⁺ ATP synthetase(Fig. 4C, D). Intracellular ROS is a byproduct of the cellular TCA cycle. ROS levels in the liver were also measured in this study. The result showed that exposure to Cd or PS increased the fluorescence intensity, and exposure to Cd + PS aggravated this trend (Fig. 4E). Subsequently, AML12 cells were treated with Cd or/and PS for 12h, and we found that compared with the control group, the content of ATP in the supernatant of cells in the Cd or PS group increased, and the content of ATP in the supernatant of the Cd + PS group was significantly higher than that Cd or PS group (Fig. 4F). To determine whether ATP can be used as a signaling molecule to induce the activity of hepatic stellate cells, different concentrations of ATP were applied to HSC-T6 cells for 12h. As shown in Fig. 4G, H, ATP increased the number and activity of HSC-T6 cells. WB results showed that ATP increased the expression levels of α-SMA and TGF-β in a concentration-dependent manner(Fig. 4I).

Inhibition of hemichannel function reduces the extracellular release of ATP and activation of hepatic stellate cells

Next, in order to verify the role of hemichannel in the extracellular release of ATP and activation of hepatic stellate cells, TAT-Gap19-TFA was used in this study to inhibit the function of hemichannel. As shown in Fig. 5A, compared with the Cd + PS group, the content of ATP in the supernatant of cells was significantly reduced in the TAT-Gap19-TFA + Cd + PS group. After the supernatant was applied to HSC-T6 cells, it was found that TAT-Gap19-TFA reduced the increase in the number and activity of HSC-T6 cells induced by Cd + PS(Fig. 5B, C). WB results also showed that the inhibition of hemichannel function also significantly alleviated the increase in the expression of α-SMA and TGF-β induced by Cd + PS(Fig. 5D).

P2X7 interacts with extracellular ATP to promote the development of Cd and PS-induced liver fibrosis

The P2X7 receptor is a purinergic receptor and adenosine triphosphate-gated ion channel that can be activated by high levels of extracellular ATP. Since the co-treatment of Cd and PS increases the extracellular release of ATP and promotes the development of liver fibrosis. We hypothesized that P2X7 can serve as the effector of extracellular ATP to promote the development of fibrosis.WB results showed that Cd or PS group did not change the expression level of P2X7, but the Cd + PS group significantly increased the expression level of P2X7(Fig. 6A). The supernatant treated with Cd and PS alone or in combination for 12h applied to HSC-T6 cells, and the result showed the same change trend as mouse liver tissue (Fig. 6B). We further explored the reasons for the increase of P2X7 induced by Cd and PS. In this study, TAT-Gap19-TFA was co-treated with Cd and PS, and the results were shown in Fig. 6C. Compared with the Cd + PS group, the TAT-Gap19-TAF + Cd + PS group significantly alleviated the increase of P2X7 expression. In order to verify the role of P2X7 in liver fibrosis induced by Cd and PS, the small interference technique was used in this study to reduce the expression of the P2X7. The results showed that the number and activity of HSC-T6 cells induced by Cd and PS were significantly reduced after P2X7 silencing (Fig. 6D, E). Moreover, the expression levels of α-SMA and TGF-β were significantly decreased (Fig. 6F).

Inadequate plastic recycling system results in large amount of plastic debris entering the ecosystem. Plastic debris, through physical or chemical factors, leads to the decomposition to PS[21]. PS persists in the environment, causing widespread impacts on ecosystems. Thus, PS can be considered a major emerging global pollutant. PS has more physical properties than other environmental pollutants. PS act as carriers of other environmental pollutants, expanding and enriching their contaminated areas[22]. Heavy metal pollution mainly refers to heavy elements with significant biological toxicity, such as mercury, cadmium, lead, chromium, and metal-like arsenic[23]. Cd has wide biotoxicity and a long metabolic cycle and has attracted wide attention[24]. It has been reported that PS can absorb heavy metals. The adsorption mechanism analysis of five different types of PS on heavy metals (cadmium, cobalt, copper, chromium, nickel, lead, and zinc) showed that a variety of microplastics could interact with heavy metals[25]. These molecular interactions are influenced by specific surface area, porosity, and morphology. Therefore, the interaction between Cd and PS and its mechanism of toxicity to organisms become particularly important.

As the most important metabolic organ, the liver is the target organ of many environmental pollutants[26]. The epidemiological results show that the Cd concentration in the blood correlates with the development of chronic liver disease[27]. PS has been found in the liver of patients with cirrhosis, and in vitro hepatoorganoids, PS also shows significant hepatotoxicity and lipotoxicity and affects liver fibrosis [28]. Both Cd and PS can affect the development of chronic liver disease. Hepatic fibrosis is a pathological change of chronic liver injury caused by various factors, which can lead to chronic liver inflammation and abnormal wound healing response [29].In this study, we found that exposure to Cd + PS significantly increased the number of white blood cells and lymphocytes in the blood, increased the infiltration of inflammatory cells in the liver, promoted the release of inflammatory factors, and activated the NLRP3 signaling pathway that leads to liver inflammation. Liver inflammation destroys the microenvironment where parenchymal and non-parenchymal cells interact, which can promote the development of liver fibrosis. We found that exposure to Cd + PS activated the TGF-β-α-SMA pathway, promoting hepatic fibrosis development.

The liver plays an important role in maintaining the balance of various systems in the body[30]. The liver is mainly composed of hepatocyte parenchymal cells and non-parenchymal cells. The spatial distribution characteristics of non-parenchymal cells affect the function and renewal of hepatocyte parenchymal cells[31]. The GJIC is an important channel for material and information exchange between cells. Hemichannel enables inorganic ions and molecules to flow between the intracellular and extracellular spaces[15]. GJIC is determined by the vigorous metabolic activity occurring in liver cells, which is another key mechanism for maintaining liver tissue homeostasis[32]. However, the effects of Cd and PS on hepatic GJIC and hemichannel are not clear. In this study, we found that Cd and PS altered the intracellular localization of CX32, but not its levels. Subsequent in vitro experiments showed that Cd + PS significantly reduced the function of intercellular GJIC, but increased the function of hemichannel. The opening of the hemichannel can promote the outflow of substances in hepatocytes and affect the stability of the microenvironment in the liver. Studies have shown that hemichannel opening is positively correlated with the development of chronic liver disease, and inhibiting hemichannel opening can improve liver injury[33]. Overactive hemichannel has also been detected in a variety of diseases, such as cataracts, skin diseases, and deafness[34, 35]. Therefore, we speculated that the hemichannel might be involved in developing liver fibrosis. In this study, TAT-Gap19-TFA was used to inhibit the function of the hemichannel, and the results showed that the inhibition of the hemichannel function significantly reduced the extracellular release of ATP, and alleviated the Cd + PS-induced increases in α-SMA and TGF-β, and the activation of HSC-T6 cells.

ATP is the most direct energy source in living organisms and is usually present in every living cell of the human body. ATP can leak into the extracellular environment in a variety of ways during periods of cellular injury, and ATP in the extracellular compartment is thought to help regulate a variety of other biological processes, including neurotransmission, liver glycogen metabolism, and inflammation[36, 37]. In this study, we also found that Cd + PS significantly disrupted mitochondrial function and increased the extracellular release of ATP. Extracellular nucleotides (such as ATP) may function as endogenous signaling molecules that control inflammation and immune responses. Therefore, we speculate that the release of extracellular ATP may be related to liver injury caused by cadmium and microplastic co-treatment. ATP was applied to HSC-T6 cells, and the results of extracellular ATP could increase the levels of TGF-β and α-SMA and activate HSC-T6 cells.

Purinergic receptors are a class of ion channels that can interact with ATP and adenosine and can be mainly divided into G-protein-coupled P2Y receptors and ligand-gated ion channels P2X receptors[38]. The P2X7 receptor is a class of non-specific cationic channels which can promote the inflow of Na⁺ and Ca²⁺ and the efflux of K⁺, and activate the downstream signaling pathway[39]. The P2X receptor is mainly activated and opened by extracellular ATP[40]. Purinergic receptors are widely distributed in tissues throughout the body, and the role of extracellular ATP in immunity and inflammation depends especially on the expression of purinergic receptors in various cell types[41]. We found that Cd + PS significantly increased P2X7 expression in the liver. This corresponds to the level of liver inflammation seen in this study. Previous studies have shown that the P2X7 receptor is associated with diseases such as high blood pressure, arteriosclerosis, and enteritis. The role of chronic liver disease remains unclear. In this study, we reduced the expression of P2X7 using a siRNA which also alleviated the Cd + PS-induced elevation of α-SMA and TGF-βand the activation of HSC-T6 cells. The results showed that P2X7 mediated the activation of HSCs through its interaction with extracellular ATP.

Collectively, Cd and PS can damage the liver environment and mitochondrial function and inhibit the gap junction function in the liver. Cd and PS increased the gap link hemichannel function and the extracellular release of ATP and induced liver inflammation through P2X7, thereby promoting the development of liver fibrosis. Our study reveals the underlying mechanisms of Cd and PS-induced liver fibrosis and provides new insights into the prevention and treatment of Cd and PS-induced hepatotoxicity

Chemicals and antibodies

The following antibodies and chemicals were used: Cadmium chloride (CdCl₂), Lucifer Yellow CH dilithium salt (LY), and Rhodamine B isothiocyanate-Dextran (RD) (Sigma-Aldrich, St. Louis, MO, USA). Microplastics (Basesula Chromatography Technology Development Center, Tianjin, China). Ethidium bromide (EB) and TAT-Gap19-TFA were obtained from Aladdin (Shanghai, China). Anti-connexin 32 (CX32) (Santa Cruz, CA, USA). Anti-NLR family pyrin domain containing 3 (NLRP3), anti-apoptosis-associated Speck-like protein containing a CARD (ASC), anti-Caspase1, anti-interleukin (IL)-1β, anti-IL-18, anti-alpha smooth muscle actin (α-SMA), anti-transforming growth factor beta (TGF-β), and anti-β-actin antibodies( Cell Signaling Technology, Danvers, MA, USA). Anti-purinergic receptor P2X7 (P2X7) antibodies were purchased from Proteintech (Wuhan, China). Dihydroethidium (DHE) and the ATP content assay kit were bought from Beyotime (Shanghai, China). The Micro Na⁺-K⁺ ATPase Assay Kit and Micro Ga²⁺-Mg²⁺ ATPase Assay Kit were purchased from Solarbio Beijing (Beijing, China). The Cell Counting Kit-8 (CCK-8) was purchased from Vazyme (Nanjing, China).

Animals and Treatment

Thirty-two 6-week-old male C57BL/6 mice were obtained from the Experimental Animal Center of Jiangsu University (Jiangsu, China). This study was approved by the Institutional Animal Care and Use Committee of Yangzhou University and carried out by the Guide for the Care and Use of Laboratory Animals of the National Research Council (approval ID: SYXK (Su) 2017- 0044). The mice were randomly divided into four groups: (1) The control group (receiving double distilled water (DDW)); (2) the PS group (administered with DDW containing 10 mg/L PS with a particle size of 5 µm); (3) the Cd group (administered with 50 mg/L Cd), and (4) the PS and Cd co-treatment group (Cd + PS; administered with purified water containing 50 mg/L Cd and 10 mg/L MPs with a particle size of 5 µm). All groups had free access to water. After 3 months exposure, all mice were anaesthetized using 2% sodium pentobarbital and then sacrificed by cervical dissection.

Cell culture and processing

The alpha mouse liver 12 (AML12) cell line was purchased from the American Type Culture Collection (ATCC CRL-2254, Manassas, VA, USA). The cells were cultured in DMEM/F-12 supplemented with 0.6% Insulin-Transferrin-Selenium-Sodium Pyruvate (ITS-A), 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in the presence of 95% air and 5% CO₂. The HSC-T6 cell line was purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). The cells were cultured in DMEM supplemented with 10% FBS and incubated at 37°C in 5% CO₂ air

Histopathology

The fixed tissue was embedded in a wax block then, the tissue was cut into 5µm sections using a microtome. The paraffin section slides were stained with hematoxylin and eosin (HE) for examination by light microscopy.

Immunochemistry

The embedded tissue was cut into 2 µm sections. The sections underwent dewaxing, hydration, and antigen retrieval. Then, the slides were incubated with CX32, IL-1β, IL-18andα-SMA primary antibodies (1:200, 12 h, 4℃), then incubated with secondary antibody (1:500, 90 mins, RT), and observed using a confocal microscope.

Immunofluorescence

A part of the slides uses sodium citrate for antigen retrieval. After antibody incubation, stained with DAPI, rinse with running water for 1 min, then the slides were observed under a fluorescence microscope (TCSSP8STED, Leica, Wetzlar, Germany).

Detection of the ATP content and ATP production-related enzyme activities

According to the manufacturer's instructions, appropriate amounts of tissue and serum were taken to measure the ATP content using specific commercial kits (Beyotime). AML12 cells were treated with Cd and/or PS, and the supernatant and cell pellet were collected to measure their ATP contents. The appropriate amount of tissue was taken to measure the enzymatic activity of Na⁺-K⁺ ATPase, and the enzymatic activity of Ga²⁺-Mg²⁺ ATPase using specific commercial kits (Solarbio).

Scrape Loading/Dye Transfer Assay

AML12 cells were scraped, incubated with PBS containing LY (0.5 mg/ml) and RD (2.5 mg/ml) at 37°C for 3 mins, and fixed with 4%paraformaldehyde. Finally, the distance from the scraped edge to the neighboring cells was measured by fluorescence microscopy (Leica DMI3000 B, Solms, Germany).

Ethidium bromide uptake Assay

AML12 was incubated with PBS containing EB at 37°C for 30 mins. Then, observe the fluorescence intensity under a fluorescence microscope.

Cellular viability detection

Cells were seeded into a 96-well plate and grown to 70% confluence; the viability of AML12 was assayed using the CCK-8 assay.

Superoxide anion detection

Fresh liver tissue is made into frozen sections. The slices were treated with DHE dye for 20 min and rinsed using running water. The sections on slides were covered with a coverslip and observed under a fluorescence microscope.

Western blotting analysis

Weigh a certain amount of fresh tissue and add protein lysate in a 1:9 ratio. The protein is then ultrasonicated and centrifuged. Protein concentration is determined by the BCA method. The proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After incubating with primary antibodies and secondary antibodies, the densities of the immunoreactive protein bands were measured using Image Laboratory software (Bio-Rad, Hercules, CA, USA).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Trizol extracts RNA from liver tissue, further reverse-transcribes RNA into cDNA and then uses cDNA as a template to build a reaction system. The reaction was placed in 7500 to detect mRNA expression. The ^2−ΔΔCt formula was used to calculate relative mRNA expression.

Statistical analysis

The data from at least three independent experiments was analyzed statistically and expressed as the mean ± standard deviation (SD). GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA USA) was used to analyze the data using one-way analysis of variance (ANOVA) (Scheffe's SF test). P values less than 0.05 were considered to indicate significant differences.

Cadmium

Microplastics

ATP

Adenosine triphosphate

AML12

alpha mouse liver 12

P2X7

P2X purinoceptor 7

NLRP3

NOD-like receptor thermal protein domain associated protein 3

HSC-T6

Rat hepatic stellate cells

BPA

Bisphenol A

PCBs

Polychlorinated biphenyls

PFOS

Perfluorooctane sulfonic acid

GJIC

Gap junction intercellular communication

HSCs

Hepatic Stellate Cells

α-SMA

α-Smooth Muscle Actin

TGF-β

Transforming growth factor beta

ASC

Apoptosis-associated speck-like protein

IL-1β

Interleukin-1β

IL-18

Interleukin-18

CX32

Connexin32

Ethics approval and consent to participate

Every procedure and protocol involving animals were permitted by the Yangzhou University Comparative Medical Center (Jiangsu Province, China). The study was based on the Guide to moral Control and Supervision in Animal Conservation and use.

Consent for publication

Not applicable

Credit authorship contribution statement

Jian Sun , Huayi Qu and Hui Zou: Conceptualization, Methodology, Software. Yan Chen and Tao Wang :Data curation, Writing- Original draft preparation. Yonggang Ma, Yan Yuan, Jianhong Gu: Visualization, Investigation. Jianchun Bian ,Waseem Ali and Zongping Liu: Writing- Reviewing and Editing.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 31702305, 31872533,31802260, and 31772808]

Availability of data and material

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgments

Not applicable

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Co-exposure to cadmium and microplastics promotes liver fibrosis through the hemichannels -ATP-P2X7 pathway

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Abstract

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Background

Results

Co-exposure to Cd and PS induces liver inflammation

Co-exposure to Cd and PS enhances hemichannel function

The extracellular release of ATP promotes the activation of hepatic stellate cells

P2X7 interacts with extracellular ATP to promote the development of Cd and PS-induced liver fibrosis

Discussion

Conclusion

Experimental

Chemicals and antibodies

Animals and Treatment

Cell culture and processing

Histopathology

Immunochemistry

Immunofluorescence

Detection of the ATP content and ATP production-related enzyme activities

Scrape Loading/Dye Transfer Assay

Ethidium bromide uptake Assay

Cellular viability detection

Superoxide anion detection

Western blotting analysis

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Statistical analysis

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