Repurposing nitazoxanide as a novel anti‐atherosclerotic drug based on mitochondrial uncoupling mechanisms

Abstract

Background and Purpose The anthelmintic drug nitazoxanide has a mitochondrial uncoupling effect. Mitochondrial uncouplers have been proven to inhibit smooth muscle cell proliferation and migration, inhibit NLRP3 inflammasome activation of macrophages and improve dyslipidaemia. Therefore, we aimed to demonstrate that nitazoxanide would protect against atherosclerosis. Experimental Approach The mitochondrial oxygen consumption of cells was measured by using the high‐resolution respirometry system, Oxygraph‐2K. The proliferation and migration of A10 cells were measured by using Edu immunofluorescence staining, wound‐induced migration and the Boyden chamber assay. Protein levels were measured by using the western blot technique. ApoE (−/−) mice were fed with a Western diet to establish an atherosclerotic model in vivo. Key Results The in vitro experiments showed that nitazoxanide and tizoxanide had a mitochondrial uncoupling effect and activated cellular AMPK. Nitazoxanide and tizoxanide inhibited serum‐ and PDGF‐induced proliferation and migration of A10 cells. Nitazoxanide and tizoxanide inhibited NLRP3 inflammasome activation in RAW264.7 macrophages, the mechanism by which involved the AMPK/IκBα/NF‐κB pathway. Nitazoxanide and tizoxanide also induced autophagy in A10 cells and RAW264.7 macrophages. The in vivo experiments demonstrated that oral administration of nitazoxanide reduced the increase in serum IL‐1β and IL‐6 levels and suppressed atherosclerosis in Western diet‐fed ApoE (−/−) mice. Conclusion and Implications Nitazoxanide inhibits the formation of atherosclerotic plaques in ApoE (−/−) mice fed on a Western diet. In view of nitazoxanide being an antiprotozoal drug already approved by the FDA, we propose it as a novel anti‐atherosclerotic drug with clinical translational potential.

Anthelmintic nitazoxanide protects against experimental pulmonary fibrosis

“…Previous studies reported that nitazoxanide and tizoxanide could stimulate autophagy responses (Fan et al, 2019; Lam et al, 2012; Shou et al, 2020). Our previous works found that nitazoxanide and tizoxanide induced autophagy in A10 cells, RAW264.7 macrophages and HepG2 cells (F.Li, Jiang, et al, 2022 ; Ma et al, 2023). Here, we found that both nitazoxanide and tizoxanide treatment increased LC3 II and LC3 II/LC3 I ratio in MRC‐5 cells (Figure S2a–d).…”

“…Our previous studies had shown that nitazoxanide and its active metabolite tizoxanide induced mitochondrial uncoupling and activated AMPK in human hepatocellular carcinoma cells (HepG2 cells) and vascular smooth muscle cells (A10 cells) (F. Li, Jiang, et al, 2022; Ma et al, 2023); here, we examined the effect of nitazoxanide and tizoxanide in human embryonic lung fibroblasts (MRC‐5 cells). Reducing the membrane potential and increasing oxygen consumption were characterizations of mitochondrial uncoupling effect.…”

“…Based on the evidence that AMPK activation and/or STAT3 inhibition are approaches for treating pulmonary fibrosis and further, based on our previous findings that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signal (Li, Jiang, et al, 2022; Ma et al, 2023), we hypothesized that nitazoxanide would inhibit experimentally induced pulmonary fibrosis. In the present study, first, the in vitro experiments showed that nitazoxanide and tizoxanide induced mitochondrial uncoupling, activated cellular AMPK, inhibited proliferation and migration of lung fibroblast cells (MRC‐5 cells), inhibited collagen‐I expression and secretion of MRC‐5 cells and inhibited epithelial–mesenchymal transition (EMT) of lung epithelial cells (MLE‐12 cells).…”

“…Recombinant human TGF-β1 was purchased from Proteintech Group The induction of mild mitochondrial uncoupling may subsequently activate AMPK (Jian et al, 2020;Li, Jiang, et al, 2022;Ma et al, 2023). Then we examined the effect of nitazoxanide and tizoxanide on AMPK activity in MRC-5 cells.…”

“…Oral nitazoxanide treatment of Syrian hamsters inhibited SARS‐CoV‐2‐driven weight loss, inflammation and viral dissemination, and syncytia formation in the lungs (Miorin et al, 2022). Our previous studies found that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 in vascular smooth muscle cells, inhibited the NLRP3 inflammasome activation induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and oral nitazoxanide protected against experimental hyperlipidaemia, hepatic steatosis and atherosclerosis (Li, Jiang, et al, 2022; Ma et al, 2023). As AMPK activation and/or STAT3 inhibition are approaches for treating pulmonary fibrosis, we hypothesized that nitazoxanide would be therapeutic for experimental pulmonary fibrosis.…”