Metallothionein-3-mediated intracellular zinc mediates antioxidant and anti-inflammatory responses in the complete Freund's adjuvant-induced inflammatory pain mouse model

Abstract

Chronic inflammatory pain is often caused by peripheral tissue damage and persistent inflammation. This disease substantially affects patients' physical and social well-being. We investigated the role of metallothionein-3 (MT3) in modulating complete Freund's adjuvant (CFA)-induced intracellular Zn²⁺ activity in an MT3 knockout mouse model of inflammatory pain in the hind paw. The results demonstrated that increasing intracellular Zn²⁺ levels ameliorate deficits in motor behavior, as well as inflammation in the paw, spleen, and thymus. Furthermore, intracellular Zn²⁺ was crucial in regulating oxidative stress markers (glutathione, superoxide dismutase, catalase, and malondialdehyde) and inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, in MT3 knockout mice induced with CFA. This study highlights the critical role of MT3 in coordinating the intracellular interaction with Zn²⁺, which is vital for the immune systems's protective functions. These interactions are fundamental for maintaining metal ion homeostasis and regulating the synthesis of various biomolecules in the body.

Fig. 1. Genotyping and schematic diagram of drug treatment in complete Freund’s adjuvant (CFA)-induced inflammatory pain in mice.

A Genotyping analysis of MT3 to identify three genotypes: MT3 wild-type (WT), MT3 knockout (KO), and MT3 heterozygous (Hetero). B Behavioral assessments of CFA-induced inflammatory pain mouse model. Behavioral tests were conducted from day 9 (D9) to day 14 (D14), with experimental animals sacrificed on day 15 (D15). The mice were divided into eight experimental groups as follows: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), and (8) MT3-KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as the mean (n = 8) ± SD. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 2. Involvement of Zn²⁺ and MT3 in paw swelling and arthritis score in complete Freund’s adjuvant (CFA)-induced inflammatory pain mouse model.

A Assessment of paw edema and swelling in treatment groups of MT3 WT and KO mice, following treatment with various drug combinations. B Paw edema measurements recorded on days 0, 3, 6, 9, 12, and 15, expressed as foot breadth of the ankle using a Vernier caliper. C Arthritis index was calculated on days 0, 3, 6, 9, 12, and 15 as the mean value of the left and right hind paws in each group. The mice were assigned to the following eight experimental groups: (1) MT3 WT (control group) (n = 8) receiving 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 3. Effects of Zn²⁺ and MT3 on sensorimotor deficits in complete Freund’s adjuvant (CFA)-induced inflammatory pain mouse model.

A Schematic description of the experiment. B Total distance traveled, C Number of foot slips, and D Number of turns as measured by the walk beam test (WBT). Mice were divided into the following eight groups: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as the mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 4. Effects of Zn²⁺ and MT3 on antinociceptive activity and mechanical hyperalgesia in complete Freund’s adjuvant (CFA)-induced inflammatory pain.

A Schematic representation of the behavioral test designed to measure mean latency time. B–D Mean latency times on days 1 (B), 7 (C), and 14 (D), assessed using the hot plate test. This test was conducted once per week to evaluate the degree of pain in the hind paws of each group. E Schematic overview of the mechanical hyperalgesia test. F Quantitative measurement of the nociceptive responses to pain. The nociceptive response to the application of a clip on the inflamed hind paw in CFA mice, serving as a model for mechanical hyperalgesia, was evaluated. Mice were divided into the following eight groups: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as the mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 5. Protective role of Zn²⁺ and MT3 on spleen and thymus indices in complete Freund’s adjuvant (CFA)-induced inflammatory pain in mice.

A Abnormal enlargement of the spleen due to edema and swelling in the different treatment groups. B Spleen index measurements in the treatment groups. C Abnormal enlargement of the thymus due to edema and swelling in the treatment groups. D Thymus index measurements in the treatment groups. Mice were divided into eight experimental groups as follows: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 6. Changes in Zn²⁺ concentration in complete Freund’s adjuvant (CFA)-induced inflammatory pain in MT3 knockout mouse model.

The Zn²⁺ concentration changes in the A paw, B thymus, and C spleen of MT3 knockout mice with CFA-induced inflammatory pain. Zn²⁺ levels were measured using a kit. Mice were divided into the following eight experimental groups: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as the mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 7. Effects of Zn²⁺ and MT3 on the activities of antioxidant-associated molecules in complete Freund’s adjuvant (CFA)-induced inflammatory pain mice.

Significant changes in A, E glutathione (GSH), B, F superoxide dismutase (SOD), C, G catalase (CAT), and D, H malondialdehyde (MDA) in the paw (A–D) and spleen (E–H) tissues. Mice were divided into eight experimental groups as follows: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as the mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to CFA-treated group; ns, not significant.

Fig. 8. Role of Zn²⁺ and MT3 on cytokine levels in the paw, spleen, and thymus in complete Freund’s adjuvant (CFA)-induced inflammatory pain in mice.

Levels of cytokines, TNF-$\alpha$ and IL-6 in A, B the paw, C, D spleen, and E, F the thymus tissues of inflammatory pain mice induced by CFA. Mice were divided into eight experimental groups as follows: (1) MT3 WT (control group) (n = 8) received 0.9% saline and 2% dimethyl sulfoxide (DMSO), (2) MT3 KO (control group) (n = 8), (3) MT3 WT + 10 mg/mL CFA (n = 8), (4) MT3 KO + 10 mg/mL CFA (n = 8), (5) MT3 WT + 10 mg/mL CFA + 1 mg/kg TPEN (n = 8), (6) MT3 WT + 10 mg/mL CFA + 10 mg/kg TPEN (n = 8), (7) MT3 KO + 10 mg/mL CFA + 1 mg/kg ZnCl₂ (n = 8), (8) MT3 KO + 10 mg/mL CFA + 10 mg/kg ZnCl₂ (n = 8). Results are presented as the mean (n = 8) ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0^.001 compared to CFA-treated group; ns, not significant.