Preclinical Evaluation of Dimethyl Itaconate Against Hepatocellular Carcinoma via Activation of the e/iNOS-Mediated NF-κB–Dependent Apoptotic Pathway

Hepatocellular carcinoma (HCC) is one of the most common tumors affecting a large population worldwide, with the fifth and seventh greatest mortality rates among men and women, respectively, and the third prime cause of mortality among cancer victims. Dimethyl itaconate (DI) has been reported to be efficacious in colorectal cancer by decreasing IL-1β release from intestinal epithelial cells. In this study, diethylnitrosamine (DEN)-induced HCC in male albino Wistar rats was treated with DI as an anticancer drug. The function and molecular mechanism of DI against HCC in vivo were assessed using histopathology, enzyme-linked immunosorbent assay (ELISA), and Western blot studies. Metabolomics using 1H-NMR was used to investigate metabolic profiles. As per molecular insights, DI has the ability to trigger mitochondrial apoptosis through iNOS- and eNOS-induced activation of the NF-κB/Bcl-2 family of proteins, CytC, caspase-3, and caspase-9 signaling cascade. Serum metabolomics investigations using 1H-NMR revealed that aberrant metabolites in DEN-induced HCC rats were restored to normal following DI therapy. Furthermore, our data revealed that the DI worked as an anti-HCC agent. The anticancer activity of DI was shown to be equivalent to that of the commercial chemotherapeutic drug 5-fluorouracil.

Dimethyl itaconate attenuates CFA-induced inflammatory pain via NLRP3/ IL-1β signaling pathway

Background Itaconate plays potent anti-inflammatory effects and has gradually been discovered as a promising drug candidate for treating inflammatory diseases. However, its roles and underlying mechanism on pain remain unknown. Methods In the current work, we investigated the effects and mechanisms of dimethyl itaconate (DI, a derivative of itaconate) in a mouse model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Male/Female C57 BL/6 mice were randomly divided into five groups: a vehicle group, an CFA group ,an CFA+PBS group and an CFA + DI(10mg /d and 20 mg/d) group.DI was performed for 11 consecutive days after CFA models were established.Paw withdrawal frequencies and paw withdrawal latencies were used to Behavioral Tests. The activation of macrophages and microglia, the level of proinflammatory cytokine production, the number of M1/M2 macrophages were evaluated .The possible involvement of the NLRP3/ IL-1β signaling pathway was also investigated. Results DI significantly reduced mechanical allodynia and thermal hyperalgesia, decreased peripheral inflammatory cell infiltration and the expression of pro-inflammatory factors IL-1β and TNF-α, and upregulated anti-inflammatory factor IL-10. Interestingly, DI promoted macrophages at the inflammatory site polarization from M1 into M2 type. Additionally, DI inhibited activation of macrophages in dorsal root ganglion (DRG) and microglia in the spinal cord, exhibiting reduced expressions of pro-inflammatory cytokines. Mechanismly, DI exerts the analgesic action primarily via inhibiting the activation of NLRP3 inflammasome complex and the release of IL-1β in derived and resident macrophages in the hind paw, DRG and spinal cord. Conclusion DI could alleviate the pain-like behavior of CFA mice by inhibiting the infiltration of plantar inflammatory cells and macrophages activation in DRG and microglia in the spinal cord. The analgesic behavior of itaconate was related to the inhibition of NLRP3 inflammasome. This study suggested possible evidence for prospective itaconate utilization in the management of inflammatory pain for the first time.

Dimethyl itaconate alleviates the pyroptosis of macrophages through oxidative stress

Macrophages are involved in the pathophysiology of many diseases as critical cells of the innate immune system. Pyroptosis is a form of macrophage death that induces cytokinesis of phagocytic substances in the macrophages, thereby defending against infection. Dimethyl itaconate (DI) is an analog of itaconic acid with anti-inflammatory effects. However, the effect of dimethyl itaconate on macrophage pyroptosis has not been elucidated clearly. Thus, the present study aimed to analyze the effect of DI treatment on a macrophage pyroptosis model (Lipopolysaccharide, LPS + Adenosine Triphosphate, ATP). The results showed that 0.25 mM DI ameliorated macrophage pyroptosis and downregulated interleukin (IL)-1β expression. Then, real-time quantitative polymerase chain reaction (RT-qPCR) was used to confirm the result of RNA-sequencing of the upregulated oxidative stress-related genes (Gclc and Gss) and downregulated inflammation-related genes (IL-12β and IL-1β). In addition, Gene Ontology (GO) enrichment analysis showed that differential genes were associated with transcript levels and DNA replication. Kyoto encyclopedia of genes and genomes (KEGG) enrichment showed that signaling pathways, such as tumor necrosis factor (TNF), Jak, Toll-like receptor and IL-17, were altered after DI treatment. N-acetyl-L-cysteine (NAC) reversed the DI effect on the LPS + ATP-induced macrophage pyroptosis and upregulated the IL-1β expression. Oxidative stress-related protein Nrf2 is involved in the DI regulation of macrophage pyroptosis. Taken together, these findings suggested that DI alleviates the pyroptosis of macrophages through oxidative stress.