Copper exposure induces inflammation and PANoptosis through the TLR4/NF-κB signaling pathway, leading to testicular damage and impaired spermatogenesis in Wilson disease.

Copper is a toxic heavy metal that causes various damage when it accumulates in the body beyond the physiological threshold. Wilson disease (WD) is an inherited disorder characterized by impaired copper metabolism. Reproductive damage in male patients with WD is gradually attracting attention. However, the underlying mechanisms of copper toxicity are unclear. In this study, we investigated the role of inflammation and PANoptosis in testicular damage and impaired spermatogenesis caused by copper deposition using the WD model toxic milk (TX) mice. Copper chelator-penicillamine and toll-like receptor 4 (TLR4) inhibitor-eritoran were used to intervene in TX mice in our animal experiment methods. Testis samples were collected from mice for further analysis. The results showed that the morphology and ultrastructure of the testis and epididymis in TX mice were damaged, and the sperm counts decreased significantly. The TLR4/nuclear factor kappa-B (NF-κB) signaling pathway was activated by copper deposition, which led to the upregulation of serum and testicular inflammatory factors in TX mice. Meanwhile, pyroptosis, apoptosis, and necroptosis were significant in the testis of TX mice. Both chelated copper or inhibited TLR4 expression markedly suppressed the TLR4/NF-κB signaling pathway, thereby reducing the expression of inflammatory factors. PANoptosis in the testis of TX mice was also reversed. Our study indicated that pathological copper exposure induces inflammation and PANoptosis through the TLR4/NF-κB signaling pathway, leading to toxic testicular damage and impaired spermatogenesis in WD.

Berberine alleviates ovarian tissue damage in mice with hepatolenticular degeneration by suppressing ferroptosis and endoplasmic reticulum stress.

OBJECTIVE: Hepatolenticular degeneration (HLD) is an autosomal recessive disorder that manifests as multiorgan damage due to impaired copper (Cu) metabolism. Female patients with HLD often experience reproductive impairments. This study investigated the protective effect of berberine against ovarian damage in toxic-milk (TX) mice, a murine model for HLD. METHODS: Mice were categorized into control group, HLD TX group (HLD group), penicillamine (Cu chelator)-treated TX group and berberine-treated TX group. Body weight, ovary weight and the number of ovulated eggs were recorded. Follicular morphology and cellular ultrastructure were examined. Total iron, ferrous iron (Fe2+) and trivalent iron (Fe3+) levels, as well as malondialdehyde (MDA), glutathione (GSH) and oxidized glutathione (GSSG), were measured in the ovaries. Western blot analysis was used to analyze the expression of proteins related to ferroptosis and endoplasmic reticulum (ER) stress. RESULTS: Ovarian tissue damage was evident in the HLD group, with a significant increase in ferroptosis and ER stress compared to the control group. This damage was inhibited by treatment with penicillamine, a Cu chelator. Compared with the HLD group, berberine increased the number of ovulations, and improved ovarian morphology and ultrastructure. Further, we found that berberine reduced total iron, Fe2+, MDA and GSSG levels, elevated GSH levels, decreased the expression of the ferroptosis marker protein prostaglandin-endoperoxide synthase 2 (PTGS2), and increased glutathione peroxidase 4 (GPX4) expression. Furthermore, berberine inhibited the expression of ER stress-associated proteins mediated by the protein kinase RNA-like ER kinase (PERK) pathway. CONCLUSION: Ferroptosis and ER stress are involved in Cu-induced ovarian damage in TX mice. Berberine ameliorates ovarian damage in HLD TX mice by inhibiting ferroptosis and ER stress. Please cite this article as: Liu QZ, Han H, Fang XR, Wang LY, Zhao D, Yin MZ, Zhang N, Jiang PY, Ji ZH, Wu LM. Berberine alleviates ovarian tissue damage in mice with hepatolenticular degeneration by suppressing ferroptosis and endoplasmic reticulum stress. J Integr Med. 2024; Epub ahead of print.

Glutathione improves testicular spermatogenesis through inhibiting oxidative stress, mitochondrial damage, and apoptosis induced by copper deposition in mice with Wilson disease.

BACKGROUND AND OBJECTIVE: There are considerable evidence of reproductive impairment in male organisms with Wilson disease (WD). The purpose of this study was to observe spermatogenesis, mitochondrial damage, apoptosis, and the level of oxidative stress in the testes of Wilson disease model TX mice, and to observe the effect and mechanism of glutathione on testicular spermatogenesis. METHODS: Mice were divided into a normal control group (control group), Wilson disease model TX mice group (WD group), penicillamine-treated TX mice group (penicillamine group) and glutathione-treated TX mice group (glutathione group). Testicular coefficient, histomorphology of testis and epididymis, number of spermatozoa, apoptosis of spermatogenic cells and expression of apoptosis-related proteins were observed. Ultrastructural analysis of mitochondria and mitochondrial membrane potential (MMP) monitored using JC-1 dye were used to detect mitochondrial damage. The levels of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and reactive oxygen species (ROS) in testicular cells were measured to assess oxidative stress. RESULTS: Testicular coefficient did not change in mice with Wilson disease. However, the tissue structure of the testicular seminiferous tubules was damaged, and the number of spermatozoa in the epididymal lumen was significantly reduced in WD group. The apoptosis rate in the testes was significantly increased. The protein expression of the pro-apoptotic proteins Bax and Caspase-3 significantly increased, and the expressions of the anti-apoptotic protein Bcl-2 significantly decreased. The levels of ROS and MDA significantly increased, and the levels of CAT and GSH significantly decreased. Mitochondria with abnormal ultrastructure and the rate of JC-1 positive cells were significantly increased in the WD group. After copper chelation by penicillamine, the structure of the testicular seminiferous tubules and the number of spermatozoa in the epididymal lumen were significantly improved. The number of apoptotic cells was significantly reduced. The levels of Bax and Caspase-3 decreased, and the expression of Bcl-2 increased. The contents of CAT and GSH increased, and the levels of ROS and MDA decreased significantly. The abnormal mitochondria and JC-1 positive cells was significantly decreased. The histomorphology of seminiferous tubules, spermatogenic function, apoptosis rate, apoptosis-related proteins, mitochondrial damage, and oxidative stress in Wilson disease TX mice significantly improved after glutathione treatment. CONCLUSION: Copper deposition in Wilson disease can lead to oxidative stress injury, mitochondrial damage, and apoptosis in the testis, leading to the impairment of spermatogenesis. Glutathione may improve testicular spermatogenesis in male Wilson disease TX mice by inhibiting copper deposition-induced oxidative stress, mitochondrial damage, and apoptosis.

A retrospective cohort study: vaccination status and safety analysis of SARS-CoV-2 vaccine in patients with Wilson's disease.

BACKGROUND: Wilson's disease (WD) is a rare hepatic and neurological disorder, which can dramatically worsen by traumatic injuries, surgeries, and infections. No studies have reported safety data of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in WD patients. We aimed to investigate the SARS-CoV-2 vaccination status and post-vaccination adverse events in WD patients. METHODS: This is a multicenter, retrospective, observational study. We investigated the vaccination rates, the type of vaccine, subjective reasons for non-vaccination, and the adverse events following vaccination. Logistic regression analysis was used to assess the correlation between vaccination status and increased Unified Wilson's Disease Rating Scale (UWDRS) scores. RESULTS: A total of 554 WD patients with a mean (SD) age of 25.3 (10.85) years were included in this study, of whom 336 (60.6%) were males and 218 (39.4%) were females. 368 (66.4%) patients received at least one dose of the SARS-CoV-2 vaccine.186 (33.6%) patients were unvaccinated. Logistic regression analysis showed that vaccination against SARS-CoV-2 was not significantly associated with increased UWDRS scores. The safety analysis demonstrated that 21.2% had post-vaccination adverse events. CONCLUSIONS: In this study, vaccination against SARS-CoV-2 was safe in WD patients, providing evidence for the safety of vaccination in WD patients.