Pristane cadmium chloride nanoemulsion accelerates the onset of systemic lupus erythematosus in a C57BL/6 mouse model

Abstract

Objective

To accelerate the onset of systemic lupus erythematosus in C57BL/6 mice by injecting cadmium chloride nanoemulsion and shorten the traditional modeling time.

Methods

Pristane cadmium chloride nanoemulsion was prepared, and 66 C57BL/6 mice were randomly divided into four groups. The pristane group was intraperitoneally injected with 0.6 mL of pristane blank nanoemulsion, the model group was injected with 0.6 mL of pristane cadmium chloride nanoemulsion, the Cadmium chloride control group was injected with 0.6 mL of cadmium chloride nanoemulsion, and the control group was injected with the same amount of 0.9% sodium chloride solution. Urine protein content, anti-dsDNA antibody content, Th1 cell/Th2 cell ratio, and kidney staining were detected in each group.

Results

The model group began to develop disease in the 4th week, the anti-dsDNA antibody level reached 566.71 ± 1.44 ng/L, and the proteinuria reached 245.38 ± 30.54 ng/mL. The model group showed an onset at least 5 weeks earlier than that in the pristane group. There was no significant difference in anti-dsDNA antibody content between Cadmium chloride control group and blank group. At the 12th week, the Th1/Th2 cell ratio in the model group significantly decreased, and the pathological changes in the kidneys were consistent with the typical manifestations of lupus in mouse models.

Conclusion

These results suggest that cadmium chloride promotes earlier onset of pristane-induced systemic lupus erythematosus in a C57BL/6 mouse model.

Keywords

autoantibody cadmium chloride mouse model pristane systemic lupus erythematosus

Introduction

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that occurs primarily in women of childbearing age. The blood and kidneys are most affected by the chronic inflammatory process. If not controlled, inflammation can affect the respiratory and nervous systems, and internal organs, and can even endanger life; the long-term prognosis is extremely poor.^1–4 However, the pathogenesis of SLE is complex and unclear.⁵ Many methods are available to study the development of SLE. The construction of animal models provides an important basis for performing basic research on SLE, exploring its pathogenesis, and screening new drug treatments. Animal models of lupus drive substantial discovery and the development of many potential treatments.⁶ Since Satoh et al.⁷ found that the injection of pristane into non-autoimmune BALB/cByJ mice successfully induces lupus in mice, this technique has become the most commonly used method for inducing lupus in mouse models. Pristane is a cell membrane activator that binds lipid biomolecules and cell membranes, thus causing toxicity to cells at certain concentrations.⁸ However, a period of at least 4–6 months is required to induce SLE-like symptoms and signs in mice after injection of pristane alone, and the model establishment time affects the economics of performing animal experimental research.⁹

Experimental studies have indicated that the pathogenesis of SLE may be caused by an interaction between genetic and environmental risk factors.^10,11 Heavy metals may increase systemic autoimmunity, and exposure to certain heavy metals may increase the risk of disease associated with SLE.¹² Cadmium is a toxic heavy metal that can be absorbed by the human body through exposure routes, including air, food, drinking water, or smoking. Over time, its accumulation in multiple organs and tissues can damage organs, including the ovaries, liver, kidneys, and brain.^13–15 Cadmium chloride has been reported to bind ERα with high affinity,¹⁶ activate it in transient transfection assays, induce the expression of estrogen target genes, and promote estrogen-dependent cell proliferation in vitro.^17,18 The expression of ERα is positively correlated with the disease activity of SLE.^19,20

Therefore, to determine whether an earlier disease onset in C57BL/6 mice might be achieved, in this experiment, we prepared a cadmium chloride nanoemulsion for pristane model establishment, and injected an appropriate amount of cadmium chloride emulsifier into mice. This method provides a new way to shorten the experimental period associated with SLE model establishment.

Materials and methods

Animals

6–8-week-old female mice, with body weights of 20 ± 2 g were purchased from Shanghai SLAC Animal Co., Ltd. The animals were reared in a fully enclosed SPF environment at the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine. Autoclaved drinking water and feed were provided by the Animal Experimental Center of Zhejiang University of Traditional Chinese Medicine. The rearing conditions remained consistent, with a temperature of 20–26°C and humidity of 40%–60%, to ensure the reliability of the results of this experiment. The animals were reared adaptively for 1 week, then divided into cages and grouped.

Instruments and reagents

An anti-dsDNA antibody detection kit, Shanghai Bohu Biotechnology Co., Ltd.; urine protein determination kit, Ningbo Medical system Biotechnology Co., Ltd.; pristane, Sigma, USA; cadmium chloride, Hangzhou Haoda Chemical Technology Co., Ltd.; Tween 80, Beijing Yancheng Technology Co., Ltd.; Span 80, HangZhou Nuoyang Biotechnology Co., Ltd.; Mouse Th1/Th2 Staining Kit, HangZhou MULTISCIENCES Co., Ltd.; Anti-Mouse CD3ε, HangZhou MULTISCIENCES Co., Ltd.; Anti-Mouse CD4, HangZhou MULTISCIENCES Co., Ltd.; Anti-Mouse IFN-γ, HangZhou MULTISCIENCES Co., Ltd.; Anti-Mouse IL-4, HangZhou MULTISCIENCES Co., Ltd.; a multifunctional centrifuge, Eppendorf, Germany; laser nano particle size analyzer, Malvern Instruments Ltd, UK; Flow cytometer, Beckman, USA.

Preparation of cadmium chloride pristane nanoemulsion

A 0.06 mL volume of Span 80 was added to 1 mL of pristane and mixed to form solution A. Then, 10 mL of cadmium chloride with a concentration of 0.1 mg/mL and 0.4 mL of Tween 80 were mixed to form solution B. Subsequently, 1 mL of solution A was placed in an ultrasonic machine, and 0.5 mL of solution B was slowly added dropwise to obtain 1.5 mL of emulsion, which was ultrasonicated for 1 h.

Preparation of pristane blank nanoemulsion

A 0.06 mL volume of Span 80 was added to 1 mL of pristane and mixed to form solution A. Then, 10 mL of double distilled water and 0.4 mL of Tween 80 were mixed to form solution B. The remaining operations are the same as above.

Preparation of cadmium chloride nanoemulsion

10 mL of cadmium chloride with a concentration of 0.1 mg/mL and 0.4 mL of Tween 80 were mixed to form solution B. Take 0.5 mL of B solution and add 0.06 mL of Span 80 to mix. The remaining operations are the same as above.

Measurement of particle size and zeta potential

A 1 mL volume of emulsified cadmium chloride pristane nanoemulsion was injected, and the particle size and zeta potential of the emulsion were measured with a laser nanoparticle particle size analyzer.

Storage stability test

The cadmium chloride pristane nanoemulsion were stored at 4°C, 25°C, or 60°C in the dark, and their particle sizes were detected on the 5th, 10th, 15th, 20th, and 25th days of storage, with the method described above.

Experimental grouping

The experimental mice were randomly divided into four groups: 20 mice in the pristane group, 20 mice in the model group, 20 mice in the Cadmium chloride control group, and 6 mice in the control group. The mice in the pristane group were given a single intraperitoneal injection of 0.6 mL of pristane blank nanoemulsion; the model group was given a single intraperitoneal injection of 0.6 mL of cadmium chloride pristane nanoemulsion; the Cadmium chloride control group was injected with 0.6 mL of cadmium chloride nanoemulsion, and the control group was injected with the same amount of 0.9% sodium chloride.

Detection of anti-dsDNA antibody in mouse serum

At 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 weeks after injection, blood was collected from the tail veins of mice in different groups, and serum was separated and stored at −80°C for later use. The serum anti-dsDNA antibody content was detected with ELISA according to the kit instructions, and the OD value of each well was measured sequentially with a microplate reader at a wavelength of 450 nm.

Urine protein content determination

Before model establishment, and at 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 weeks after model establishment, the bladders of mice were massaged to stimulate urination. The urine of mice in each group was collected, and the content of protein in the urine was detected with a kit.

Th1/Th2 flow cytometry detection

Spleen mononuclear cells were isolated and prepared with lymphocyte separation medium. The pellet was resuspended in medium containing 10% fetal bovine serum, to a cell concentration of 1 × 10⁷/mL. Then, 250 μL of cell suspension was placed into a flow tube, and 1 μL of phorbol ester/ionomycin mixture and 1 μL of brefeldin A/monensin mixture were added and incubated at 37°C for 6 h. Subsequently, 100 μL of cell suspension was transferred to a new flow tube, and Th1/Th2 flow cytometry was performed according to the instructions.

Mouse kidney pathology detection

The mice were sacrificed 3 months after model establishment, and the kidneys were removed, fixed with 10% formaldehyde, dehydrated in ethanol, embedded in paraffin, sectioned, and stained with PAS and Masson stains. The pathological characteristics of the kidneys were observed under a light microscope. Kidney tissue changes were scored using the Austin Scale (Table 1). Mesangial cell count method: The mean number of cells in three random positive cross sections of glomerulus was taken as the number of cells to reflect the degree of mesangial cell proliferation.

Table 1.

Austin score of renal pathological activity in lupus nephritis.

Index of activity
Capillary hyperplasia	0–3
Infiltration of white blood cells	0–3
Endosubcutaneous transparent material deposition	0–3
Cellulosic necrosis/nuclear fragmentation	0–6
Cellular crescent body	0–6
Formation of interstitial crescent bodies	0–3

Statistical analysis

SPSS 20.0 was used for statistical analysis, and one-way analysis of variance was used for data analysis. Comparisons between groups were analyzed with two-way Anova. When p < 0.05, the difference was considered statistically significant.

Results

Results of cadmium chloride pristane nanoemulsion preparation

The prepared cadmium chloride pristane nanoemulsion was analyzed with a particle size and zeta potential detection analyzer. The particle size of the nanosolution was 1.630 ± 0.23 nm, the polydispersity coefficient (PDI) was 0.227 ± 0.01, and the zeta potential was 31.1 ± 0.54 mV. The particle size of the cadmium chloride pristane nanoemulsion was below 300 nm, and its PDI value was less than 0.3, thus indicating that the particle dispersion of the modified nanoemulsion was relatively uniform. The zeta potential reflects the electrostatic repulsion of the nanoemulsion system, and a larger absolute value corresponds to better emulsion stability. The results indicated that the absolute value of the zeta potential of the cadmium chloride pristane nanoemulsion was greater than 30 mV, and the emulsion system was thus considered completely stable (Figure 1(a)–(b)).

After storage at 4°C and 25°C in the dark for 25 days, the cadmium chloride pristane nanoemulsion of pristane essentially maintained a concentration of approximately 1.73 nm, and no significant changes were observed. However, on the 25th day, the nanoemulsion stored at 60°C showed delamination, and the average particle size increased to 2.27 nm (Figure 1(c)). In the dark, the PDI of the nanoemulsion showed an increasing trend, which was slow at 4°C and significantly faster at 25°C and 60°C (Figure 1(d)). Thus, the stability of the cadmium chloride pristane nanoemulsion was higher at lower temperatures.

Cadmium chloride accelerates the onset of SLE in a C57BL/6 mouse model

In the 4th week of model establishment, the anti-dsDNA antibody level reached 566.71 ± 1.44 ng/L (Figure 2(a)), the proteinuria level reached 245.38 ± 30.54 ng/mL (Figure 2(b)), and the spleens of the mice were significantly larger (Figure 2(c)) than those in the control group (p < 0.01). In the 10th week of model establishment, in the pristane group, the dsDNA antibody level reached 633.247 ± 3.48 ng/L, and the proteinuria reached 289.62 ± 28.58 ng/mL, which increased each week; the spleens of the mice were significantly more swollen than those in the control mice. Spleen tissue size of Cadmium chloride control group was normal, dsDNA antibody level and urine protein content were not significantly different from blank group (p > 0.05). Compared with the pristane group, the model group had an SLE onset at least 5 weeks earlier (Figure 2).

Cadmium chloride accelerates pathological changes in a C57BL/6 mouse model of SLE

No pathological lymph nodes were found in the blank control group and the cadmium chloride control group. Compared with those in the control group, the mice in the model group showed sparse hair loss in the 12th week of model establishment, and many lymph nodes with a diameter of 1–3 mm were seen in the body (Figure 3(a)). Masson staining showed that the renal vascular endothelial cells and the mesangial area contained distributed rose red precipitates and blue collagen deposition; the glomeruli showed segmental sclerosis; extensive cell proliferation was observed in the renal tubules; and many transparent capillary lumen thrombi were present in the model group (Table 2). In addition, in the model group, compared with the control group, PAS staining was positive, significantly more mesangial cells and stroma were observed (Table 3), and many lymphocytes had infiltrated in the glomerular mesangium, basement membrane, and renal tubules (Figure 3(b)); the number of Th1 cells in the spleen tissue was significantly lower, the number of Th2 cells was significantly higher (p < 0.01), and the ratio of Th1 cells/Th2 cells was significantly lower (Figure 3(c)). These findings are typical pathological manifestations of SLE in mice. However, the general lymphatic lesions, kidney staining results, and Th1/Th2 cell ratios were essentially the same in the pristane group in the 20th week of model establishment and the model group in the 12th week of model establishment (Figure 3(a)–(c)) (Table 4). Thus, injection of cadmium chloride can shorten the SLE model onset time in mice. No significant changes in renal tissue structure were observed in the cadmium chloride control group. There was no significant difference in Th1/Th2 ratio between the cadmium chloride control group and the blank control group. It is concluded that a small amount of Cadmium chloride does not induce the onset of SLE, but the same dose can accelerate the onset and aggravate the course of pristane-induced lupus. Therefore, it is speculated that a small amount of heavy metals in the environment will not induce the development of SLE, but even a small amount of heavy metals will accelerate the development of SLE in the presence of high-risk populations or other precipitating conditions.

Table 2.

Renal tissue injury scores of each group.

Group	Results of AI score in lupus mice
Blank	0
Pristane	14
Model	17
CdCl₂ control	0

Table 3.

Mesangial cell numbers in each group.

Group	Number of mesangial cells (x ± s)
Blank	30.67 ± 2.87
Pristane	51 ± 9.29
Model	59 ± 9.62
CdCl₂ control	35 ± 4.21

Table 4.

Statistical data of Th1/Th2 ratio in each group.

Group	Th1/Th2 ( $\bar{x}$ ± s)
Blank	34.625 ± 19.98
Pristane	2.462 ± 0.57
Model	0.088 ± 0.03
CdCl₂ control	32.57 ± 16.09

Discussion

The SLE animal model induced by injection of pristane, an important tool for experimental research, shows similar pathogenesis to that of human SLE in terms of clinical manifestations, histopathology, laboratory indicators, and other aspects. However, the pristane-induced SLE onset time is long.²¹ In this experiment, after emulsification of pristane and cadmium chloride solution, a cadmium chloride pristane nanoemulsion with uniform particle dispersion and high stability was obtained and analyzed with a particle size and zeta potential detection analyzer. A single injection of cadmium chloride pristane nanoemulsion effectively ameliorated the trauma caused by the second injection in the experimental mice. In the SLE model group with cadmium chloride pristane nanoemulsion injection, the dsDNA increased significantly earlier, and its concentration at 5 weeks did not significantly differ from that in the pristane group at 10 weeks; moreover, these mice showed clear clinical lupus symptoms and a significant increase in urinary protein levels. The results indicated that cadmium chloride accelerated the establishment of the pristane-induced SLE model. Because the pathogenesis of SLE is associated with genetic and environmental factors,²² environmental exposure to the heavy metal ion cadmium simulates endogenous hormones, thus stimulating or inhibiting their biological effects; interfering with biological processes such as hormone synthesis, function, and clearance; and affecting the body’s immune system,²³ thereby accelerating the pathogenesis of SLE.

The kidney is one of the most frequently involved organs in SLE, and most patients with SLE have lupus nephritis, thus, damaging renal function. With the development of SLE, the kidneys of the mice were invaded and gradually developed proteinuria. The urinary protein content in the model group was significantly higher than that in the pristane group. Systemic plaques, a typical pathological change in lupus, appeared in stained kidney sections in the 12-week model group and the 20-week pristane group. Studies have shown that cadmium induces oxidative stress and inflammatory responses by inducing the production of reactive oxygen species.²⁴ When the body is in a state of oxidative stress, large amounts of reactive oxygen species are produced²⁵ and cannot be removed sufficiently rapidly to prevent their accumulation. These reactive oxygen species lead to tissue and cell damage, and induce or aggravate the occurrence and development of SLE.^26,27

Th cells can be divided into Th1 cells and Th2 cells according to their secreted cytokines and functions, and the balance of their secreted cytokines plays an important role in the pathogenesis of SLE.^28,29 Th1 cells secrete IL-2, IFN-γ, and lymphotoxin, thus, promoting macrophage activation and participating in antibody-dependent cell-mediated cytotoxicity and delayed-type hypersensitivity, whereas Th2 cells secrete mainly IL-4, IL-5, IL-6, IL-10, and IL-13, mediating humoral immunity and promoting the production of antibodies.³⁰ Therefore, in this experiment, IL-4 and IFN-γ were used as markers to distinguish Th1 and Th2 cells,³¹ and to reflect the Th1/Th2 ratio in each group of mice. Compared with the 20-week model group, the 12-week model group had significantly fewer Th1 cells, significantly more Th2 cells, and a significantly lower Th1 cell/Th2 cell ratio. The results further confirmed that cadmium chloride aggravates pathogenesis in the SLE model.

Conclusion

Herein, we experimentally demonstrated that injection of cadmium chloride nanoemulsion accelerates the onset of SLE in a C57BL/6 mouse model induced by pristane, thus shortening the time required to construct the model of SLE. This study may guide future studies on the pathogenesis of SLE and the exploration of new treatment methods; it also provides a new experimental method for studying the effects of environmental factors on the pathogenesis of SLE.

Footnotes

Acknowledgments

We are grateful to the Laboratory Animal Unit of Zhejiang Chinese Medical University for performing routine animal husbandry.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (No. 81973778 and 81774179); 2021 Zhejiang Province College Students Science and Technology Innovation Activity Plan (No.2021R410056).

ORCID iDs

Yi-bo Jin

Xing-hong Ding

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