Gandouling Regulates Ferroptosis and Improves Neuroinflammation in Wilson's Disease Through the LCN2/NLRP3 Signaling Pathway

Purpose: Neuroinflammation is a main cause of neurological damage in Wilson's disease (WD). Ferroptosis is present in the WD pathological process, which is also closely related to the neuroinflammation. LCN2, a ferroptosis-related gene in WD, is linked with the activation of NLRP3 inflammasome. Our group has previously demonstrated that Gandouling (GDL) can effectively improve neuroinflammation in WD. This study aims to investigate the protective effect of GDL on neuroinflammation in animal and cell models of WD, and whether the pharmacological mechanism is related to the LCN2/NLRP3 signaling pathway.

Methods: Toxic milk (TX) mice and HT22 cells stimulated by copper ions were selected as models. The pathology of hippocampal tissues in TX mice were observed by HE staining and transmission electron microscopy. High-throughput Sequencing analysis was conducted to screen ferroptosis-related genes in WD. The expression of LCN2 and GPX4 in hippocampus of TX mice were detected by immunohistochemical. The expression of LCN2, NLRP3, GPX4, and SLC7A11 was determined in TX mice and HT22 cells by Western blotting and RT-qPCR. The levels of Fe²⁺, inflammatory factor indicators TNF-α, IL-1β and IL-6 and oxidative stress indicators 4-HNE, MAD, SOD, GSH and ROS were detected in each group by ELISA.

Results: The results showed that GDL ameliorated pathological and mitochondrial damages in hippocampus of TX mice. The analysis of bioinformatics showed that LCN2 was a differential gene associated with Ferroptosis in WD. The results of Western blotting and RT-qPCR indicated that GDL reduced the expression of LCN2 and NLRP3, and enhanced the expression of GPX4 and SLC711 in TX mice and HT22 cells. The ELISA results showed that GDL decreased the expression of Fe²⁺ and inflammatory factors TNF-α, IL-1β and IL-6 in TX mice with Ferroptosis inducer intervention and copper ion-loaded HT22 cells. GDL decreased the expression of oxidative stress indicators ROS, 4-HNE and MDA, and increased the expression of oxidative stress indicators GSH and SOD in TX mice and copper ion-loaded HT22 cells.

Conclusion: GDL has anti-inflammatory and antioxidant effects. LCN2 is a differential gene associated with Ferroptosis in WD. GDL may alleviate Ferroptosis by inhibiting the LCN2/NLPR3 signaling pathway, thereby improving neuroinflammatory responses and exerting neuroprotective effects in WD.

LCN2; NLRP3; Wilson’s disease; ferroptosis; neuroinflammation.