Cadmium induced mitochondria apoptosis via the reduction of Th2 cells in pig thyroids

doi:10.21203/rs.3.rs-2085246/v1

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Cadmium induced mitochondria apoptosis via the reduction of Th2 cells in pig thyroids

https://doi.org/10.21203/rs.3.rs-2085246/v1

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Cadmium, as an environmental pollution element, is considered as a strong toxic substance of organisms. High doses of cadmium have serious cytotoxicity and induces programmed cell necrosis, autophagy, and apoptosis. Th1/Th2 balance is involved in regulating the dynamic balance of cytokine networks and is closely related to the occurrence and development of a variety of diseases. The purpose of this study was to investigate the relationship between Th1/Th2 balance and cadmium-induced apoptosis of thyroid cells. Cadmium poisoning pig model was established in which healthy 6-week-old pigs were exposed to CdCl2 for 40 days (with a dietary cadmium content of 20mg/kg). First, apoptosis was detected by TUNEL staining, and Th1/Th2 equilibrium shift was detected by immunofluorescence. Secondly, the mRNA and protein expression of related proteins were detected by qRT-PCR and Western blot. The results showed that the Th1/Th2 balance shifted towards Th1 and Th2 cells decreased；and the expression levels of Ras/Raf/MEK/ERK pathway related proteins were inhibited in group Cd. In addition, Bax, CytC, Caspase9, and Caspase3 expression increased and Bcl2 expression decreased after cadmium exposure. In brief, our experiments suggested that the pro-apoptosis-related genes expression upregulates and apoptosis induces by activating the mitochondrial pathway signaling pathway in thyroid cells after Cadmium exposure. These negative effects are in connection with the Th1/Th2 imbalance and the reduction of Th2 cytokines.

Cadmium

Pig

Thyroid

Th1/Th2 balance

Mitochondria apoptosis

Cadmium (Cd) is widely distributed in the environment as heavy metal element. When it accumulates in the body, it will damage the health of the humans and animals. Studies have shown that the thyroid is also one of the targeted organs for Cd, and Cd poisoning causes thyroid injury (Buha et al. 2018). For example, Cd causes thyroid dysfunction by altering the gene expression on the HPT axis of minnow larvae (Li et al. 2014). Cd induces apoptosis in humans and animals. Cd induces apoptosis bronchial epithelial cells by activating the mitochondrial intrinsic apoptosis pathway in human(Cao et al. 2021), and can also induce apoptosis testicular stromal cells in mouse(Ren et al. 2020). Mitochondria-mediated apoptosis plays an important role in apoptosis induced by Cd poisoning. Cd induces apoptosis of rat osteoblasts by activating tumor protein p53 (P53) protein in mitochondrial apoptotic pathway (Zheng et al. 2020). Inhibition of mitochondria-mediated apoptosis has a strong protective effect on Cd-induced testicular injury in mice (Han et al. 2020). Helper T cells (Th cells) are classified into two subgroups, Th1 cell and Th2 cell, according to the cytokines they secrete. Cytokines secreted by Th1 and Th2 cell not only promote their own proliferation, but also inhibit each other's proliferation. There is a relatively balanced state in healthy animals of Th1 and Th2 cell. Cd poisoning also induces Th1/Th2 cell immune drift. For example, Cd poisoning causes Th1/Th2 balance to shift to Th1 in the pancreas of pigs (Wu et al. 2021). IL-6 and interferon-γ (IFN-γ) as Th1 cell markers expression is increased, and IL-10 as Th2 cell markers expression is decreased in Cd poisoning rat testis (Sivaprakasam and Nachiappan 2016). Body could be damaged when Th1/Th2 balance is deviated. Fluoride and manganese, for example, interfere with the Th1/Th2 cell balance, thereby damaging the spleen of mice (Li et al. 2021) and the nervous system of chickens (Miao et al. 2021). Immune drift of Th1/Th2 cell also induces thyroid damage. For example, Th1/Th2 imbalance mediates DINP-induced thyroid injury in rats (Duan et al. 2019), while ATMSC reduces thyroid inflammatory damage in rats by down-regulating Th1 cytokines and improving Th1/Th2 balance (Choi et al. 2011).

When the balance of Th1/Th2 shifts to Th1, it also causes apoptosis. Hydrogen sulfide induces apoptosis by inducing Th1/Th2 homeostasis shift towards Th1 in the bursa of Fabricius of broilers (Hu, Chi, et al. 2018). The increased expression of IFN-γ and IL-2 as Th1 cell markers and the decreased expression of IL-6 and IL-10 as Th2 cell markers, accompanied by myocardial apoptosis in the serum of rats with autoimmune myocarditis. However, decreased expressions of IFN-γ and IL-2 and increased expressions of IL-6 and IL-10 inhibit myocardial apoptosis (Xue et al. 2019). Apoptosis could be regulated via rat sarcoma (Ras) /rapidly accelerated fibrosarcoma (Raf) /MAP kinase-ERK kinase (MEK) /extracellular regulated MAP kinase (ERK) cascade pathway. Inhibition of ERK1/2 expression in liver cancer (Tian et al. 2021) and colorectal cancer (Odintsov et al. 2021) cells promotes apoptosis, and inhibition of MEK1/2 expression in melanoma cells also promotes apoptosis (Mielczarek-Lewandowska et al. 2019). Activation of Ras/Raf/MEK/ERK cascade pathway alleviates LPS-induced apoptosis of renal podocytes in mice (Li, Ma, and Liu 2019). As members of the B lymphoma gene family, BCL2 like 11 (Bim), Bcl2 Associated X (Bax), and B-cell lymphoma-2 (Bcl2) play a crucial role in mitochondria apoptosis pathway (Maes, Schlamp, and Nickells 2017). Promotion of ERK-mediated Bim degradation inhibits apoptosis in rat PC12 cells (Kennedy et al. 2017), while inhibition of ERK or pERK induces apoptosis by reducing Bim degradation (Sun et al. 2019). The levels of apoptosis-related cytokines such as mitochondrial cytochrome C (CytC), Caspase9 and Caspase3 all increase at different levels during apoptosis (Kalpage et al. 2020).

Although more and more evidence has shown that Cd poisoning may induce immune drift of Th1/Th2 cell and apoptosis, the relationship of Th1/Th2 balance and apoptosis which is cause by Cd poisoning in pig thyroid cells is still unclear. The levels of Th1/Th2 markers, Ras/Raf/MEK/ERK cascade pathway and mitochondrial apoptotic pathway related factors in were detected by TUNEL, immunofluorescence, qRT-PCR and Western Blot. These data provide toxicological study of Cd and comparative medicine theoretical basis and reference basis.

2.1 Cd poisoning animal model establishment and grouping

All procedures in the experiment were consistent with the animal welfare standards of the Northeast Agricultural University Animal Protection and Utilization Committee. Ten healthy 6-week-old pigs were randomly divided into two groups of 5 pigs each. The control group (group C) was fed normally, while the Cd poisoning group (group Cd) was fed with the diet containing 20mg/kg cadmium chloride (CdCl₂). On the 40th day, the pigs were euthanized, the thyroid tissue was extracted and partially fixed in 10% formalin, and left part was placed in liquid nitrogen for further use.

2.2 TUNEL assay in thyroid tissue

To analyze Cd intoxication-induced apoptosis of thyroid cells, TUNEL analysis was performed using an insitu cell death detection kit (luciferin, Roche, Basel, Switzerland). Tunel staining for DNA fragments is considered a standard technique for detecting apoptosis in tissue sections. The experiment was in accordance with the manufacturer's instructions and the samples were observed under a fluorescence microscope after reverse-fluorescence quenching. Sections were randomly selected from each sample, and used a high-power field of vision (×400) to count the number of positive cells in each thyroid section.

2.3 Immunofluorescence analysis

Frozen sections of 5µm thyroid were taken and stained with DAPI. T lymphocyte specific anti-tumor necrosis factor alpha (TNF-α) antibody (ABCAM) and anti-CC motif chemokine receptor 4 (CCR4) antibody (ABCAM) were used to stain Th1 and Th2 subgroups of frozen thyroid sections, respectively. Use Nikon's fluorescence microscope for observation and image acquisition.

2.4 Real-time quantitative PCR analysis

Total RNA was extracted from thyroid tissue by using Trizol reagent according to the manufacturer's instructions, and the reverse transcription step for cDNA was also based on the manufacturer's instructions (Roche, Shanghai, China). qRT-PCR was performed using the 480 system (Roche, Basel, Switzerland) and Fast Universal SYBR Green Master Mix (Roche, Basel, Switzerland). The primers used in our experiment were showed in Table 1. Only the peak of each PCR product was shown in the melting curve analysis. The relative gene abundance of mRNAs was calculated by 2^−ΔΔCT method, and the gene-specific efficiency was considered, and normalized to the mean value of the above indexes.

Table 1

The primers used in the present study
Target gene	Forward Primer (5′→ 3′)	Reverse Primer (5′→ 3′)
β-actin	AATCCTGCGGCATCCACGAAAC	CAGCACCGTGTTGGCGTAGAG
IFN-γ	GTGCGACGACCCTGATGCTG	CTGACGGATTCACTGCTGCTCTTC
TNF-α	GCACTGAGAGCATGATCCGAGAC	CGACCAGGAGGAAGGAGAAGAGG
CCR4	ACGAGAAGAAGAACAAGGCAGTGAAG	CCAGGAAGAGCACCACATTGTAAGG
IL-4	GCTTCGGCACATCTACAGACACC	TCTTGGCTTCATGCACAGAACAGG
SHC1	AGGAAGAGCCGCCTGACCATC	TCCCGAAGCCTCATGTCCACTAC
GRB2	GGACATAGAACAGGTGCCACAGC	ACCAGTTGGGGTCCGAGTTATCC
SOS1	TCCTCCTGCTTCTGGTGCTTCTAG	AAAGACGGTATCGCTGCTTGAGTG
KRas	AAAGACGGTATCGCTGCTTGAGTG	TACTCCTCTTGACCTGCTGTGTCG
Raf-1	AATGCGTCGGATGCGAGAATCTG	AGAGGAAGGGCTGGAGGTGTTG
MEK-1	TCGATGAACAGCAGCGGAAGC	ACCTTGAACACCACACCTCCATTG
MEK-2	CGCTCACCATCAACCCTACCATC	TTCTTCTGCTGCTCGTCAAGTTCC
ERK1	ACCTACTGCCAGCGGACCTTG	AGAATGTCTCGGATGCCAATGACG
ERK2	AACCTTCCAACCTGCTGCTCAAC	CCAACGTGTGGCGACATACTCC
Bim	GCAGGAGTTACGGCGTATTGGAG	AGGGAGGGTGTGAGGGGAAAAG
Bax	TTTGCTTCAGGGTTTCATCCAGGATC	GAGACACTCGCTCAACTTCTTGGTAG
CytC	TTGTTCAGAAGTGTGCCCAGTGC	CTGACCTGTCTTCCGCCCAAAG
Caspase9	TCCTGTGTTCATCTCCTGCTTAG	CCCGCTTTGTTGCTTGTCTAC
Caspase3	CTGTAGAACTCTAACTGGCAAACC	CCCACTGTCCGTCTCAATCC
P53	GCCCATCCTCACCATCATCACAC	GCACAAACACGCACCTCAAAGC
Bcl2	CAGAGGGGCTACGAGTGGGATG	CCGGGCTGGGAGGAGAAGATG

2.5 Western blot analysis

The total protein was electrophoresed in a 12% SDS-polyacrylamide gel, and the separated protein was transferred to a nitrocellulose membrane in a Tris-glycine buffer containing 20% methanol at 4°C. The membrane was covered with 5% skimmed milk and blocked at 37°C for 2 h, and then incubated with diluted anti-rabbit primary antibody at 4°C overnight. the diluted concentration was shown in Table 2. It was then combined with peroxidase-conjugated anti-rabbit secondary antibody IgG (1:5000, Santa Cruz, USA) at 37°C for 50 min. Finally, X-ray film (Transgen Biotech Co., Beijing, China) was used to detect the signal.

Table 2

The primary antibodies used in present study
Target gene	Dilution ratio	Resource
β-actin	1: 10000	ABclonal Biotechnology
GRB2	1: 500	WanLei Biotechnology, China
KRas	1: 500	WanLei Biotechnology, China
Raf-1	1: 500	WanLei Biotechnology, China
MEK1/2	1: 500	WanLei Biotechnology, China
ERK1/2	1: 500	WanLei Biotechnology, China
pERK1/2	1: 300	WanLei Biotechnology, China
Bax	1: 500	WanLei Biotechnology, China
CytC	1: 500	WanLei Biotechnology, China
Caspase9	1: 1000	WanLei Biotechnology, China
cleaved-Caspase9	1: 1000	WanLei Biotechnology, China
Caspase3	1: 500	WanLei Biotechnology, China
cleaved-Caspase3	1: 500	WanLei Biotechnology, China
Bcl2	1: 300	Laboratory made

2.6 Statistical analysis

GraphPad Prism software (version 8.0, GraphPad Software Inc., San Diego, California, U.S.) was used to analyze the data by t test for each group. All the date presented in this study were collected from at least three independent experiments(n = 3), and the data is reported as the mean standard ± deviation (SD). When p < 0.05, the difference is statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

3.1 The number of apoptotic cells in thyroid tissue increased after Cd exposure

In order to detect Cd-induced apoptosis of thyroid tissue cells, the TUNEL kit was first used to detect the thyroid tissue of group Cd and group C respectively. The results were shown in Fig. 1. Compared with the group C, the number of TUNEL positive cells increased in the group Cd.

3.2 Abnormal levels of cytokines secreted by Th1/ Th2 cell in thyroid tissue

Th1/Th2 cell balance is a basic pathway to regulate immune response. To explore the Cd-induced pig thyroid Th1/Th2 imbalance, we evaluated Th1/Th2 cell markers levels in the thyroid (Fig. 2). As shown in Fig. 2(a), Cd regulated the Th1/Th2 imbalance, Th1 cells number increased while Th2 cells number decreased according to TNF-α and CCR4 staining. In Fig. 2(b), mRNA levels of TNF-α and IFN-γ as Th1 cell markers in the group Cd were all upregulated compared with group C, while mRNA levels of Th2 cell marker IL-4 (interleukin 4), CCR4 were down-regulated in Fig. 2(c) (p < 0.05). In summary, the immune response may be modulated by Cd, thus turning polarization homeostasis into a harmful phase.

3.3 Inhibitions of Ras/RAF/MEK/ERK pathway and related factors expression in thyroid tissue

In order to determine whether IL-4 regulates apoptosis via Ras/Raf/MEK/ERK signaling pathway in thyroid tissue, the expression of this signaling pathway related factors were detected by qRT-PCR and Western Blot (Fig. 3). Compared with the group C, the mRNA expression levels of the downstream cytokines SHC1 (SHC adaptor protein 1), GRB2 (growth factor receptor bound protein 2), SOS1 (SOS Ras/Rac guanine nucleotide exchange factor 1), KRas, Raf-1, MEK1/2, and ERK1/2 of IL-4 in the group Cd were significantly down-regulated (Fig. 3(a)); the protein level was consistent with it, and the protein expression level of pERK1/2 was significantly down-regulated (Fig. 3(b), Fig. 3(c)) (p < 0.05). These data suggest that Ras/Raf/MEK/ERK signaling pathway is inhibited in pig thyroid tissues after Cd exposure.

3.4 Increased expression of mitochondrial apoptotic pathway factors in thyroid

Inhibition of Ras/Raf/MEK/ERK signaling pathway can activate mitochondria apoptosis pathway in vivo. To verify this hypothesis, we detected the expression levels of mitochondria apoptosis pathway related cytokines such as Bim, Bax, CytC, Caspase9, Caspase3, P53 and Bcl2 in thyroid by qRT-PCR and Western Blot (Fig. 4). Compared with the group C, the mRNA levels of Bim, Bax and CytC were significantly up-regulated in the group Cd, and the mRNA level of Bcl2 was significantly down-regulated (Fig. 4(a)); the protein expression level was consistent with that (Fig. 4(b), Fig. 4(c)). This indicates a change in mitochondrial permeability and the release of CytC into the cytoplasm in thyroid. In addition, the mRNA levels of Caspase 9, Caspase 3 and P53 in the group Cd were all up-regulated (Fig. 4(a)), the protein levels were consistent with them; and the protein expression levels of cleaved-caspase3 and cleaved-caspase9 were also up-regulated (Fig. 4(b), Fig. 4(c)) (p < 0.05) This result shows that mitochondria apoptosis occurs after Cd exposure in thyroid.

Apoptosis is one of the main forms of body damage caused by Cd. Cd poisoning can induce apoptosis in organs such as kidney (Chen et al. 2021) and liver (Yiming et al. 2021). Cd exposure causes the imbalance of Th1/Th2 and the changes in Th1/Th2 cell markers (Zheng et al. 2021). This study explores the relationship between Th1/Th2 imbalance and Cd-induced apoptosis in thyroid of pig. Our results show that the mitochondrial apoptotic pathways related factors expression increased significantly, such as Bim, Bax, CytC, Caspase9 and Caspase3. In the group Cd, the markers of Th1 cell IFN-γ and TNF-α increased, the markers of Th2 cell CCR4 and IL-4 decreased, and shifted the Th1/Th2 balance to Th1. ERK signaling pathway related factors were significantly down-regulated.

Th1 cell markers such as TNF-α, IL-2, IFN-γ, etc. and Th2 cell markers such as IL-4, IL-10, etc. play an important role in the regulation of apoptosis (Feng et al. 2016). TNF-α and IFN-γ expression is increased, while IL-4 expression is decreased in Cd poisoning chicken neutrophils(Chen et al. 2017). The expression of TNF-α is also increased in Cd poisoning rat testis and the expression of IL-4 is also decreased in the same way (Al-Azemi et al. 2010). Moreover, the experiments of Wu et al. have also shown that TNF-α and IFN-γ expression is increased and IL-4 and CCR4 expression is decreased, causing Th1/Th2 imbalance in Cd poisoning pig pancreas (Wu et al. 2021). In this experiment, we detected the Th1 cell markers TNF-α, IFN-γ and Th2 cell markers IL-4 and CCR4 expression levels. The results showed that Th1 cell markers TNF-α and IFN-γ expression levels increased significantly in the group Cd, and Th2 cell markers CCR4 and IL-4 expression levels decreased significantly. This result indicates that Th1/Th2 cell balance shifts and Th2 cells are reduced in the thyroid of Cd poisoning pigs. These results suggest that Cd exposure causes Th1/Th2 cell imbalance and Th2 cell reduction in pig thyroid tissue. In macrophages, lead treatment leads to imbalance of Th1/Th2 cell and induces expression of Th1 cell markers such as IL-2 and IFN-γ (Krocova et al. 2000). The Th1/Th2 cytokine ratio in serum and liver of African green frog exposed to Cu, Cr, Pb and other heavy metals is more inclined to Th1, and the levels of IL-6 and IFN-γ in serum and TNF-α and IFN-γ in liver are significantly increased (Jayawardena et al. 2016).

The Ras/Raf/MEK/ERK signaling pathway can be widely activated as a mitogen-activated protein kinase pathway, which can transfer extracellular signals into cells, thereby regulating cell apoptosis, proliferation and differentiation. The dysregulation of the Ras/Raf/MEK/ERK pathway controls the occurrence and development of apoptosis. For example, catechin-3-gallic acid induces apoptosis of human thyroid cancer cells by down-regulating the expression of the Ras/Raf/MEK/ERK pathway (Wu et al. 2019). And the apoptosis of human liver cancer cells could be induced by inhibiting of RaS/RAF/MEK/ERK signaling pathway (Tian et al. 2020). The Th2 cell markers IL-4 and CCR4 have regulatory effects on the Ras / Raf / MEK / ERK pathway. For example, IL-4 can interact with important upstream proteins of ERK signaling pathway SHC and GRB2 and activate ERK2 (Soon et al. 1999). IL-4 causes phosphorylation of ERK in the rat retina (Goulart et al. 2018). CCR4 can also activate the ERK signaling pathway. For example, in mice, CCR4 activates its downstream ERK signaling pathway and improves hematoma after cerebral hemorrhage (Deng et al. 2020). This result also confirms the above statement. When the Th1/Th2 balance shifts to Th1 cell, the Th2 cell markers IL-4 and CCR4 is decreased, inhibit the activation of the ERK signaling pathway, activate the mitochondrial pathway, and thyroid cells undergo apoptosis. Studies have also shown that the mitochondria apoptosis pathway could be activated by inhibiting the Ras/Raf/MEK/ERK pathway. For example, in mouse endometrial epithelial cells, inhibiting the Ras/Raf/MEK/ERK signaling pathway, the expression levels of Bax, Caspase3 increased and the expression level of Bcl2 decreased, which activates the mitochondria apoptosis pathway (Liu et al. 2020). In addition, Bhalla et al. demonstrated that MEK inhibitors mediated mitochondria apoptosis pathway in diffuse large B-cell lymphoma by inhibiting MEK/ERK pathway activation (Bhalla et al. 2011). And by up-regulating the expression of MEK/ERK in cervical cancer cells, the ratio of Bax to Bcl2 can be inhibited, and the lysis of Caspase9 and Caspase3 can be reduced, thereby inhibiting the mitochondrial apoptotic pathway (Hu, Zhang, et al. 2018). In granulosa cell KGN, Cd causes an increase in Bax level and a decrease in Bcl2 level by inducing mitochondrial dysfunction, leading to apoptosis (Xu et al. 2021). Cd can also activate the mitochondrial apoptotic pathway in rat neural stem cells (Luo et al. 2021). The above evidence validates our results, that is, Cd causes Th1/Th2 cell balance shift, Th2 cells decrease, inhibits Ras/Raf/MEK/ERK signaling pathway, and induces mitochondria apoptosis in pig thyroid.

In conclusion, our study showed that the pig thyroid is one of the important target organs of Cd, and Cd can inhibit the expression of Ras/Raf/MEK/ERK pathway and activate the mitochondrial apoptosis pathway by interfering with Th1/Th2 balance and reducing Th2 cells. These results indicate that Cd induces mitochondria apoptosis in pig thyroid via reducing Th2 cell. This study aims to draw people's attention to the harm of soil environmental damage to humans and animals, and to provide a theoretical basis for the toxicological study of Cd.

Ethics approval and consent to participate

All procedures of this experiment were in accordance with animal welfare standards and the requirements of the Animal Protection and Utilization Committee of Northeast Agricultural University.

Consent for publication

All of authors have read the manuscript and have agreed to submit it in its current form for consideration for publication.

Availability of data and material

The authors claim that none of the material in the paper has been published or is under consideration for publication elsewhere.

Competing interests

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions

Yilei Zhang: Conceptualization, Methodology, Software, Investigation, Writing-original draft, Formal analysis, Visualization. Xiaojing Liu: Validation, Formal analysis, Software. Wenyue Zhang: Supervision, Data curation. Jing Lan and Gang Sun: Writing - review & editing.

Acknowledgements

All authors thank all members of the Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention; Heilongjiang Provincial Animal Disease Prevention and Control Center; Institute of Quality and Safety of Agricultural Products, Heilongjiang Academy of Agricultural Sciences and Treatment in the College of Veterinary Medicine, Northeast Agricultural University, for their help in the experiment.

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No competing interests reported.

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Cadmium induced mitochondria apoptosis via the reduction of Th2 cells in pig thyroids

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Cd poisoning animal model establishment and grouping

2.2 TUNEL assay in thyroid tissue

2.3 Immunofluorescence analysis

2.4 Real-time quantitative PCR analysis

2.5 Western blot analysis

2.6 Statistical analysis

3 Results

3.1 The number of apoptotic cells in thyroid tissue increased after Cd exposure

3.2 Abnormal levels of cytokines secreted by Th1/ Th2 cell in thyroid tissue

3.3 Inhibitions of Ras/RAF/MEK/ERK pathway and related factors expression in thyroid tissue

3.4 Increased expression of mitochondrial apoptotic pathway factors in thyroid

4 Discussion

Declarations

References

Additional Declarations

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