In WD patients, the loss of function of copper transporter ATP7B can impair the excretion of copper in bile, which leads to a large amount of copper accumulation and oxidative stress in the liver[22, 23]. Due to the difficulty of diagnosis and treatment of WD, liver transplantation is one of the few reasonable options for patients. At present, the main treatment strategy for the disease is to reduce the level of copper in patients and restore the balance of copper in the body[24]. However, these strategic approaches do not address potential mutations in the ATP7B of the disease. Therefore, patients are expected to need lifelong treatment, so the focus is on avoiding the side effects of treatment. Existing treatments include treatment with copper chelating agents or zinc salts, which are sufficient to control the disease. However, chelating agents may cause a variety of serious toxicities, including obvious myelosuppression, hypersensitivity, degeneration of skin autoimmune diseases, and nephrotoxicity[25]. As a first-line treatment, zinc acetate is the best option. Although the toxicity of zinc acetate is lower than that of chelating agents, its copper excretion effect is not good, so it is only suitable for patients in the early stage of WD. The expression of metallothionein in intestinal cells reduces the intestinal absorption of copper, and then traps these two metals, causing them to be excreted into feces as mucosal cells fall off[26]. However, zinc acetate cannot remove copper from overloaded tissues, although it is less toxic than chelating agents. In addition, a study showed that more than 36 percent of Italian WD children responded to existing drugs, but liver enzymes did not return to normal. New treatment strategies are needed to treat WD.
Copper is a trace element, which exists mainly in the form of the oxidized state (Cu2+) or reduced state (Cu+) in the biological system[27, 28]. In addition, the transition among these states produces hydroxyl radicals, which are essential for the activities of different cellular enzymes involved in energy metabolism, respiration, and DNA synthesis. Nonetheless, redox reactions generate free radicals that attack biological macromolecules, which is why excess copper is toxic. New antioxidants can be obtained from isolated compounds in natural products that have fewer side effects. In patients with hepatolenticular degeneration, the accumulation of copper in the body and progressive liver damage caused by oxidative stress is the most important clinical symptoms. In the liver, copper ions cannot continually accumulate through biliary excretion, causing WD liver fibrosis. The fibrosis process will gradually progress, and eventually lead to liver failure. Injuries to the liver are believed to be caused mainly by oxidative stress.
Several clinical and theoretical studies have demonstrated that GSH is effective in treating WD. GSH is a strong antioxidant in the body, which has a good effect on eliminating free radicals. Among the non-protein sulfhydryl compounds contained in the body, it contains the highest number of sulfhydryl groups and the largest amount of sulfhydryl groups. Sulfhydryl groups can scavenge a variety of free radicals, participate in cell defense mechanisms, including cell detoxification and cell damage and repair, and maintain the stability of the internal environment[29]. GSH can be used as the substrate of GSH peroxidase, inhibit lipid peroxidation, protect cell membrane and restore cell function. Reductive GSH can prevent and treat stomatitis caused by radiotherapy, and it can also be used after chemotherapy and tumor chemoembolization to protect the liver. GSH has a good effect on acute hepatorenal damage and even hepatorenal dysfunction[30].
Furthermore, we investigated whether GSH protects the liver against excessive copper damage by inhibiting oxidative stress. In this study, TX mice were studied from two aspects of liver protection and antioxidant stress. The TX mice used in this study is a natural mutant strain produced by homologous breeding to F68 DL mice, and its pathogenic gene has 82% homology with the ATP7B gene, so it is an ideal WD model animal. The results indicated that GSH could reverse liver injury in TX mice. Elevated serum ALT, AST, and AKP activities are usually used to indicate the occurrence of hepatocyte injury[31]. Thus, we examined the typical biomarkers of the above-mentioned liver injury. GSH significantly reduced serum ALT, AST, and AKP levels in TX mice (Fig. 2). Oxidative stress can lead to the production of many reactive oxygen species (ROS). Antioxidant enzymes (such as T-SOD) and non-enzymatic antioxidants (such as MDA) are important factors of oxidative stress. Superoxide dismutase can reduce the production of free radicals and lipid peroxides, and even accelerate the clearance of these molecules, thus reducing the damage to hepatocytes[32]. MDA content is an important parameter to reflect the potential antioxidant capacity of the body, can reflect the rate and intensity of lipid peroxidation, and indirectly reflect the degree of tissue peroxidation damage. Therefore, maintaining appropriate levels of CAT, MDA and T-SOD is deemed to be the key to reducing oxidative damage. In this study, the levels of CAT, MDA, and T-SOD in the liver of the model group changed, which is a sign of oxidative stress. High-dose GSH can significantly increase the level of CAT in model mice; low-and medium-dose GSH can significantly reduce the level of MDA in model mice; high-dose GSH can significantly increase the level of T-SOD in model mice (Fig. 3). These results suggest that GSH has a protective effect on oxidative stress induced by copper in the liver. At the same time, the pathological changes of liver tissue stained by HE further confirmed the protective effect of GSH on liver injury in TX mice.
In conlusion, we proved that glutathione for the treatment of hepatolenticular degeneration effectively reduced the accumulation of copper in the body and reduced oxidative stress caused by the copper disorder in this study. According to the related indexes of liver injury and the results of liver pathological staining, GSH, as an important intracellular regulatory metabolite, can have a potential liver protective effect in patients with hepatolenticular degeneration. Alternatively, copper excretion and anti-oxidation are important mechanisms of GSH in the treatment of TX mice. This study highlights the noteworthy value of GSH treatment for WD. Therefore, GSH has a strong potential to become a safe and effective medication to treat WD patients.