Mitochondria-mediated Apoptosis and Endoplasmic Reticulum Stress are Involved in the Toxicity Induced by Copper in Porcine Spleen

doi:10.21203/rs.3.rs-1757442/v1

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Mitochondria-mediated Apoptosis and Endoplasmic Reticulum Stress are Involved in the Toxicity Induced by Copper in Porcine Spleen

https://doi.org/10.21203/rs.3.rs-1757442/v1

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Journal Publication

published 05 Aug, 2023

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Copper (Cu) is one of the common heavy metal pollutants in the environment, and its toxic mechanisms have been extensively studied. However, the immunotoxicity induced by Cu remains rarely reported, and the effects of Cu on endoplasmic reticulum stress and mitochondria-mediated apoptosis have been little studied in spleen. In this study, pigs were fed with different contents of Cu (10, 125 and 250 mg/kg Cu) for 80 d to establish a toxicity model. The results showed the Cu exposure triggered endoplasmic reticulum stress in the spleen, as evidenced by increased mRNA and protein levels of GRP94, GRP78, CHOP, XBP1, ATF6, and JNK, the positive rate of GRP78 increased by immunofluorescence analysis. Additionally, mitochondrial fission and fusion homeostasis were disrupted, the expression levels of mitochondrial dynamics related genes Mfn1, Mfn2, and OPA1 decreased, DRP1 increased, and the positive rate of Mfn1 decreased by immunofluorescence analysis. Furthermore, Cu exposure could induce apoptosis, as demonstrated by increased the expression level of related protein and gene Bak, Bax, Caspase-3, P53, and Cytc decreased. In conclusion, these results suggest chronic Cu exposure can lead to endoplasmic reticulum stress and imbalance in mitochondrial dynamics, induced apoptosis of pig spleen, provided new insights into the underlying mechanism of Cu exposure caused splenic toxicity, which has public health implications where humans and animals are exposed to copper contamination.

Copper

Pig

Spleen

Endoplasmic reticulum stress

Mitochondrial dynamics

apoptosis

As is known to all, heavy metals are widely distributed in the world, mainly concentrated in industrial, agricultural and environmental pollution. Copper (Cu), one of the most common metal contaminants, has been recognized as a toxic substance(Duan et al. 2016). The existence of excess Cu in the environment poses a major threat to the ecological environment, food safety and sustainable development of agriculture through various pathways. It is reported that Cu exposure can induce toxic effects, including liver damage, intestinal flora dysbiosis and metabolic disturbance (Dai et al. 2020). Mechanically, excess Cu can inhibit the mTOR signaling pathway and adjust the expression of autophagy-related genes and proteins, thereby increasing the level of autophagy(Liu et al. 2021). Moreover, Cu also disrupts the mitochondrial respiratory enzyme complex's redox balance, resulting in the accumulation of massive reactive oxygen species (ROS) and generating an oxidative stress response, which can cause apoptosis (Chen et al. 2022).

The endoplasmic reticulum (ER) is a significant organelle in eukaryotic cells that plays a crucial part in regulating cellular homeostasis(Zhao et al. 2021). Imbalance in ER homeostasis is caused by a variety of factors, which further leads to ER stress and unfolded protein responses (UPR). In the UPR, the three of the main sensors inositol-requiring enzyme 1 (IRE1), PERK (protein kinase r-like ER kinase), and ATF6 (activating transcription factor 6) are activated, which reduce the endoplasmic reticulum damage and remove the excessive accumulation of unfolded proteins to maintain ER homeostasis(Zhang et al. 2018). Studies indicated that excessive Cu exposure triggered endoplasmic reticulum stress through activation of the PERK/ATF4/CHOP pathway and exacerbated Cu-induced apoptosis in duck renal tubular epithelial cells(Wang et al. 2021).

ER and mitochondria are also closely linked in certain biological processes. Mitochondria are key organelles involved in all kinds of cellular processes, including oxidative stress, energy depletion, and apoptosis. The apoptosis mechanism includes endogenous and exogenous pathways(Igney and Krammer 2002). The endogenous pathway called the mitochondrial pathway is one of the key pathways of apoptosis (Gorman et al. 2016). Previous research had shown that Cu-induced mitochondrial dynamics turbulence was closely related to the expression levels of apoptosis-related proteins Bcl-2, Bax, P53, and Caspase-3 which represented by increased mitochondrial fission levels, and reduced fusion levels(Mu et al. 2019a). Spleen is a peripheral organ of the immune system. Furthermore, Heavy metals inhibit the immunomodulatory effects of cytokines in the spleen(Zhu et al. 2012). However, the underlying mechanism of Cu toxicity to the spleen in pigs is little attention. Therefore, the objective of this study was to investigate the mechanisms of Cu-induced toxicity of endoplasmic reticulum stress and mitochondria-mediated apoptosis in the spleen, which provide a research perspective of the spleen toxicity induced by Cu exposure.

2.1. Animals and experimental design

The experimental animal protocols were carried out in accordance with the moral committee of South China Agricultural University. Add up to 60 pigs, 30 days of age, were randomly divided into three groups (a control group and two treatment groups, each group of 20). The ingredient and nutritional levels of the basal diet were displayed in Table S1. Cupric sulfate anhydrous was selected as Cu sources, and the control group was fed a basal diet containing 10 mg/kg Cu, and the Cu content in the treated groups were125mg/kg and 250mg/kg. All pigs were free to drink and feed. The period was 80 days, and spleen tissues were collected after euthanasia with pentobarbital.

2.2. Histopathological studies

Spleen tissues were soaked in 4% paraformaldehyde solvent for 24 h, then the tissues have been trimmed through the slicer and embedded in paraffin. The paraffin blocks were cut and dried, dewaxed in xylene, subjected to gradient alcohol to water, stained nuclei, alcohol separation in hydrochloric acid, blue return in tap water, eosin re-staining, alcohol decolorization and dehydration, xylene transparency, sealing using neutral gum, and finally, pathological changes of spleen tissue were observed under the microscope (Leica, Germany).

2.3. Immuno-histochemical assay

Before the staining, Paraffin sections were baked at 55℃ for 1 h, dewaxed with xylene, antigen repair, antigen blocking, incubation with BCL-2 protein primary antibody for 16 h at 4 ℃, dilute with anti-BCL-2 protein secondary antibody at 1:200 and incubated for 1 h at room temperature, diaminobenzidine (DAB) staining, and hematoxylin staining, finally, the brown part of the tissue was observed under a microscope (Leica, Germany).

2.4. Immunofluorescent analysis

The method of immunofluorescent refers to the previous study(Liao et al. 2019), and the method was the same as immunohistochemistry. The images were captured by a fluorescence microscope, the fluorescence intensity was analyzed by Image J software.

2.5. RNA isolation and real-time PCR

According to the manufacturer's plan, `the total RNA of spleen was extracted with Trizol reagent (Takara, Japan). RNA reverse transcription was performed strictly according to the kit instructions (Vazyme, Nanjing, China). Using GAPDH as an internal reference for standardization, cDNA amplification was performed using qTOWER3G (Analytikjena, Germany). The sequences of primers were provided in Table S2.

2.6. Western bolting analysis

The protein stock solution was detached by gel electrophoresis and then transferred to the polyvinylidene fluoride (PVDF) membrane. Protein strips were blocked with 5% skim milk in TBST(Tris, Buffered, Saline, Tweeen) solution for 1 h at room temperature and incubated with the corresponding protein primary antibody for 16 hours at 4°C. anti-glucose-regulated protein 94 (GRP94) antibody (1:1000, rabbit, Beijing Biosynthesis Biotechnology, China), anti-glucose-regulated protein 78 (GRP78)antibody (1:1500, rabbit, Sigma, USA), anti-CHOP antibody (1:2000, rabbit, Beijing Biosynthesis Biotechnology, China), anti-X-box binding protein 1 (XBP1) antibody (1:2000, rabbit, Nanjing Bioworld Biotechnology, China), anti-JNK antibody (1:2000, rabbit, Nanjing Bioworld Biotechnology, China), anti-mitofusin 1 (MFN1) antibody (1:1000, rabbit, Cell Signaling Technology, USA), anti-mitofusin 2 (MFN2) antibody (1:1000, rabbit, Cell Signaling Technology, USA), anti-DRP1 antibody (1:1000, rabbit, Cell Signaling Technology, USA), anti-optic atrophy 1 (OPA1) antibody (1:1000, rabbit, Abcam, China), anti-Bcl2 antibody (1:1000, rabbit, Abcam, USA), anti-bcl-2-antagonist/killer protein(Bak) antibody(1:2000, rabbit, Abcam, China), anti-bcl-2-associated X protein (Bax) antibody (1:5000, mouse, Sigma, USA), anti-caspase-3 antibody (1:1000, rabbit, Beijing Biosynthesis Biotechnology, China), anti-cytochrome C (Cytc), antibody (1:1000, rabbit, Cell Signaling Technology, China), anti-p53 antibody(1:2000, Bioss,China), anti-GAPDH antibody (1:5000, mouse, Beijing Biosynthesis Biotechnology, China), anti-Tubin antibody(1:5000, mouse, Beijing Biosynthesis Biotechnology, China). The protein strips were incubated with the corresponding rabbit or mouse antibodies for 1 h at room temperature, washed with buffer solution, and displayed with ECL solvent (NCM Biotech, China). The optical density of protein bands was analyzed by Image J software.

2.7. Statistical analysis

Statistical images were created by Graph-Pad Prism 8.0.1 (GraphPad Software, Inc., USA). One-way analysis of variance was used to test for comparison between groups, and a t-test was used to analyze the significance of differences. Data analysis P < 0.05 indicates a significant difference.

3.1 Effect of Cu exposure on ER stress levels in spleen

The results were obtained from Fig. 1A&B, the fluorescence intensity of GRP78 was remarkably elevated in the 250mg/kg Cu-treated group compared with the control group (P < 0.01). In addition, the expression levels of ER-related genes GRP94, GRP78, CHOP, XBP1, and ATF6 were increased to different extents in the Cu-treated group compared with the control group (P < 0.05 or P < 0.01). The results of the quantitative analysis were demonstrated in Fig. 2A&D. Further examination of the expression levels of ER stress-related proteins manifested that the expression levels of GRP94, GRP78, CHOP, XBP1, and JNK were significantly elevated at day 80 after Cu treatment compared with the control group (Fig. 2E) (P < 0.05 or P < 0.01), and the results of the quantitative analysis were demonstrated by Fig. 2B&C.

3.2 Effect of Cu exposure on mitochondrial dynamics in the spleen

The immunofluorescent results showed that the expression of MFN1 was remarkably decreased with increasing Cu levels (P < 0.05 or p < 0.01) (Fig. 3). In addition, mRNA and protein levels of Drp1 were remarkably increased (P < 0.05 or P < 0.01) in the 125 mg/kg Cu-treated group and 250 mg/kg Cu-treated group compared to the control group (Fig. 4A-E), and mfn1, Mfn2, and OPA1 mRNA levels showed a decreasing trend (Fig. 4A&D), OPA1, Mfn1, and Mfn2 protein expression was down-regulated with the Cu levels elevated (P < 0.05 or P < 0.01) (Fig. 4B&E).

3.3 Cu exposure induces mitochondria-mediated apoptosis in the spleen

Immunofluorescence analysis of Cytc positive expression showed that the level of Cytc expression was increased in two treatment groups (P < 0.05) (Fig. 5A&B). In addition, the immunohistochemical positive rate of Bcl-2 was detected, the results showed that the positive rate of Bcl-2 was reduced in treatment groups, especially in the 250mg/kg Cu-treated group(P < 0.01) (Fig. 5C&D). Furthermore, the expression levels of Bak, Caspase-3 and Cytc mRNA were remarkably elevated in the 250 mg/kg Cu-treated group compared to the control group (P < 0.01), BCL-2 mRNA showed a downtrend in the 250mg/kg group compared to the 125mg/kg Cu-treated group (Fig. 6A&D). In addition to this, the protein expression levels of Bak, Bax, Caspase-3, Cytc, and p53 were remarkably elevated in the Cu-treated groups compared to the control group, Bcl-2 protein levels were consistent with gene expression levels (P < 0.05 or P < 0.01) (Fig. 6E).

3.4 Correlation analysis of Cu exposure on ER stress, mitochondrial function and mitochondria-mediated apoptosis

To investigate the correlation of proteins between endoplasmic reticulum stress and mitochondria-mediated apoptosis in the spleen, a numerical analysis of correlation coefficients was performed. After excessive Cu-treatment, MFN1, MFN2, OPA1, and Bcl-2 showed intense inverse correlation with Bak, Bax, Caspase-3, Cytc, and p53 proteins. Furthermore, there was a strong positive correlation between GRP94, GRP78, XBP1, JNK, CHOP proteins and Caspase-3, Cytc, Bax, Bak, and p53 proteins (Fig. 7A). Meanwhile, Protein interaction diagram analysis showed that Bcl-2, Cytc and JNK proteins had intense connectivity with other proteins associated with ER stress, mitochondrial dynamics and apoptosis (Fig. 7B).

Excessive heavy metals residue in the environment can result in threats to ecological security, ultimately endangering humans, animals, and plants(Kumar et al. 2019). Related studies have shown that cadmium can cause renal apoptosis through a nuclear receptor (AHR/CAR/PXR)-mediated xenobiotic detoxification pathway(Ge et al. 2022). Moreover, it has been reported that heavy metals can cause immunotoxicity to the spleen, DNA damage, and apoptosis(Li et al. 2010). Meanwhile, Guo et al. have manifested that excessive Cu exposure can cause oxidative stress and inflammatory responses in the spleen, which also suggested that Cu can induce immunotoxicity in the spleen(Guo et al. 2021). Nevertheless, the influences of Cu on ER stress and mitochondria-mediated apoptosis in the spleen have been less studied. In this study, mitochondria-mediated apoptosis and ER stress were investigated in pig spleen under Cu exposure, which provides a novel insight into Cu toxicology.

The ability to properly fold and modify proteins in ER is impeded, leading to a prolonged accumulation of misfolded proteins in the endoplasmic reticulum, this phenomenon is known as endoplasmic reticulum stress(Wang et al. 2021). many investigations indicate that ER stress is adjusted by three major signal pathways: the PERK-eIF2α pathway, the IRE1α-XBP1 pathway, and the ATF-6 pathway. When stimulated by adverse external factors, three transmembrane proteins (ATF6, PERK, and IRE1) activate, initiating an ER-mediated unfolded protein response leading to the binding of the ER molecular chaperone GRP94 and GRP78(Song et al. 2016). Wang et al. showed that arsenic induces ER stress through the PERK/IRE1 pathway in the spleen, ultimately leading to apoptosis in the spleen(Wang et al. 2021a). In addition, ER stress is a toxicological target of Cu toxins, high Cu induces ER stress which leads to duck renal tubular epithelial cells death(Wang et al. 2021b). Our results showed that the transmembrane protein ATF6 was remarkably elevated under Cu treatment, and the fluorescence intensity of GRP78 protein was remarkably increased in the 250 mg/kg Cu-treated group, which is consistent with the results of related studies(Li et al. 2020). In addition, the expression levels of endoplasmic reticulum stress-related genes were examined, which showed that the mRNA expression levels of GRP94, GRP78, XBP1, CHOP, and ATF6 were upregulated in Cu groups. Our results show that Cu induced endoplasmic reticulum stress in the pig spleen by regulated ER stress-related proteins and genes.

As we all know, mitochondria produce most of the energy of eukaryotic cells and play a leading role in many metabolic processes(Orrenius et al. 2015). Under normal conditions, mitochondria maintain a balance between fusion and division to retain their normal structure(Guo et al. 2018). When stimulated by various external factors, mitochondrial dysfunction is induced. Cu exposure caused mitochondria-mediated apoptosis by disrupting the homeostasis of mitochondrial kinetics(Lei et al. 2021). However, there are few reports of Cu effects on mitochondrial dynamics in splenic injury. Our results showed that the expression level of MFN1 decreased, but the expression level of fission protein Drp1 increased, indicated that the normal mitochondrial metabolic function was disturbed, the results are consistent with previous study(Zhao et al. 2019). In addition, in the process of apoptosis, the permeability of the outer mitochondrial membrane is controlled by the Bcl-2 family of apoptosis proteins(Renault and Manon 2011), Bax could translocate to the outer mitochondrial membrane, which results in oligomerization, leading to the entry of apoptotic factors such as Cytc into the cytoplasm, and signal transduction activates the apoptotic effector molecule Caspase 3, which activates the Caspase cascade reaction, finally leading to apoptosis(Mu et al. 2019b), our results showed that Bcl-2, Cytc, caspase-3 and other apoptosis-related proteins and genes were increased to varying degrees, which was accord with the results of other research(Zheng et al. 2020), it was further confirmed that Cu could induce mitochondrial division and mitochondrial damage and activate apoptosis.

In summary, Cu exposure could induce ER stress, mitochondrial dynamics disorder, and mitochondria-mediated apoptosis in the spleen. This study provides new understanding into the toxicity of Cu on pig spleen, and the scientific data can be used to assess the potential risk of copper-induced immune system diseases.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32072930 and No. 31572585) and the National Key R & D Program of China (No. 2016YFD0501205 and No. 2017YFD0502200).

Author information

Kai Zhang and Jianzhao Liao made equal contributions to this work.

Authors and Affiliations

College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, Guangdong, P.R. China.

Kai Zhang, Jianzhao Liao, Zhuoying Hu, Quanwei Li, Lianmei Hu, Jianying Guo, Ying Li, Hui Zhang, Jiaqiang Pan, Zhaoxin Tang

Author contribution statement

Zhang Kai did the data analysis and wrote the manuscript. Experimental design, material preparation and data collection were completed by Kai Zhang, Jianzhao Liao, Zhuoying Hu, and Quanwei Li. The manuscript was revised by Dr. Jianzhao Liao. Manuscripts reviewed by Lianmei Hu, Jianying Guo, Ying Li, Hui Zhan, Jiaqiang Pan. Professor Zhaoxin Tang directed the research. All authors have read and approved the submitted version.

Corresponding author

Correspondence to [email protected]

Ethics approval

The procedures of animal experiments were authorized and performed by the Ethics Committee of South China Agricultural University (approval number: No. 2019A015).

Consent to participate

Not applicable.

Consent for Publication

All the authors have consented for publication of this research.

Data Availability

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

Conflict of interest statement

The authors declare that there is no conflict of interest among all participants in this study.

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You are reading this latest preprint version

Mitochondria-mediated Apoptosis and Endoplasmic Reticulum Stress are Involved in the Toxicity Induced by Copper in Porcine Spleen

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Animals and experimental design

2.2. Histopathological studies

2.3. Immuno-histochemical assay

2.4. Immunofluorescent analysis

2.5. RNA isolation and real-time PCR

2.6. Western bolting analysis

2.7. Statistical analysis

3. Results

3.1 Effect of Cu exposure on ER stress levels in spleen

3.2 Effect of Cu exposure on mitochondrial dynamics in the spleen

3.3 Cu exposure induces mitochondria-mediated apoptosis in the spleen

3.4 Correlation analysis of Cu exposure on ER stress, mitochondrial function and mitochondria-mediated apoptosis

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

Status:

Journal Publication

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