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. 2022 Mar;12(3):1322-1338.
doi: 10.1016/j.apsb.2021.09.009. Epub 2021 Sep 17.

Anthelmintics nitazoxanide protects against experimental hyperlipidemia and hepatic steatosis in hamsters and mice

Fengfeng Li  1   2 , Man Jiang  1   2 , Minghui Ma  1   2 , Xuyang Chen  1   2 , Yidan Zhang  1   2 , Yixin Zhang  1   2 , Yuanyuan Yu  1   2 , Yunfeng Cui  1   2 , Jiahui Chen  1   2 , Hui Zhao  1   2 , Zhijie Sun  3 , Deli Dong  1   2   3
Affiliations

Anthelmintics nitazoxanide protects against experimental hyperlipidemia and hepatic steatosis in hamsters and mice

Fengfeng Li et al. Acta Pharm Sin B. 2022 Mar.

Abstract

Lipid metabolism disorders contribute to hyperlipidemia and hepatic steatosis. It is ideal to develop drugs simultaneous improving both hyperlipidemia and hepatic steatosis. Nitazoxanide is an FDA-approved oral antiprotozoal drug with excellent pharmacokinetic and safety profile. We found that nitazoxanide and its metabolite tizoxanide induced mild mitochondrial uncoupling and subsequently activated AMPK in HepG2 cells. Gavage administration of nitazoxanide inhibited high-fat diet (HFD)-induced increases of liver weight, blood and liver lipids, and ameliorated HFD-induced renal lipid accumulation in hamsters. Nitazoxanide significantly improved HFD-induced histopathologic changes of hamster livers. In the hamsters with pre-existing hyperlipidemia and hepatic steatosis, nitazoxanide also showed therapeutic effect. Gavage administration of nitazoxanide improved HFD-induced hepatic steatosis in C57BL/6J mice and western diet (WD)-induced hepatic steatosis in Apoe -/- mice. The present study suggests that repurposing nitazoxanide as a drug for hyperlipidemia and hepatic steatosis treatment is promising.

Keywords: AMPK; Autophagy; Hepatic steatosis; Hyperlipidemia; Mitochondrial uncoupling; Nitazoxanide; SQSTM1/P62; Tizoxanide.

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Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Nitazoxanide and tizoxanide induce mild mitochondrial uncoupling and subsequently activate AMPK in HepG2 cells. (A, B) Nitazoxanide and tizoxanide dose-dependently enhanced mitochondrial oxygen consumption rate (OCR) of HepG2 cells. The representative original recordings of OCR are shown in (A) and the analyzed data are shown in (B). FCCP was as positive control. n = 6 in DMSO group, n = 7 in nitazoxanide group, n = 8 tizoxanide group, n = 7 in FCCP group. (C, D) Nitazoxanide and tizoxanide reduced mitochondrial membrane potential of HepG2 cells. HepG2 cells were treated with different concentrations of nitazoxanide and tizoxanide for 24 h, then the mitochondrial membrane potential was assayed by using TMRM staining. The representative images of TMRM staining are shown in (C) and the analyzed data are shown in (D). n = 6 in each group. (E) Nitazoxanide and tizoxanide activated AMPK in HepG2 cells. HepG2 cells were stimulated with free fatty acids (FFA, 250 μmol/L) for 24 h then treated with different concentrations of nitazoxanide and tizoxanide for 24 h n = 6 in each group. (F) Nitazoxanide and tizoxanide increased phosphorylation of ACC in HepG2 cells. HepG2 cells were stimulated with free fatty acids (FFA, 250 μmol/L) for 24 h then treated with different concentrations of nitazoxanide and tizoxanide for 24 h n = 6 in each group. Data are presented as mean ± SEM. Statistical analysis was performed with the randomized block ANOVA (repeated measures ANOVA). ∗∗P < 0.01 vs. CTL (control). Nit, nitazoxanide; Tiz, tizoxanide.
Figure 2
Figure 2
Gavage administration of nitazoxanide prevents HFD feeding-induced hyperlipidemia in hamsters. (A) Schematic illustration of the experiment design. (B) The time course of body weight and average food intake changes. The food intake every three days was averaged. (C) Body temperature of animals. The anus temperature of animals anesthetized with 10% chloral hydrate were measured by using a thermometer. (D) Nitazoxanide reduced the elevated blood lipids induced by HFD feeding in hamsters. TC, total cholesterol; TG, triglyceride. HDL-C, high density lipoprotein cholesterol; LDL-C, high density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Data are presented as mean ± SEM; n = 10, 13, 12, 12, 11, and 9 in ND, HFD, HFD + Nit (50 mg/kg), HFD + Nit (100 mg/kg), HFD + Nit (200 mg/kg), and HFD + Ato (1.2 mg/kg) groups, respectively. Statistical analysis was performed with one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HFD. ND, normal diet; HFD, high-fat diet; Nit, nitazoxanide; Ato, atorvastatin.
Figure 3
Figure 3
Gavage administration of nitazoxanide prevents HFD feeding-induced liver histopathology in hamsters. (A) The representative images of hamster livers from different groups. It was apparent that the liver from HFD group was swollen and pale in color. (B) The body weight, liver weight, and liver weight/body weight of hamsters from different groups. (C) The total cholesterol and triglyceride content of livers. TC, total cholesterol; TG, triglyceride. (D) Hematoxylin and eosin (H&E) staining of hamster livers from different groups. The framed area was enlarged. (E) Oil red O staining of hamster livers from different groups. The framed area was enlarged. Data are presented as mean ± SEM; n = 10, 13, 12, 12, 11, and 9 in ND, HFD, HFD + Nit (50 mg/kg), HFD + Nit (100 mg/kg), HFD + Nit (200 mg/kg), and HFD + Ato (1.2 mg/kg) groups, respectively. Statistical analysis was performed with one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01 vs. ND. #P < 0.05, ##P < 0.01 vs. HFD. ND, normal diet; HFD, high-fat diet; Nit, nitazoxanide; Ato, atorvastatin.
Figure 4
Figure 4
Gavage administration of nitazoxanide reverses HFD feeding-induced decreases of AMPK, phosphorylated AMPK and phosphorylate ACC protein levels in livers of hamsters. Data were presented as mean ± SEM; n = 10 in each group. Statistical analysis was performed with one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HFD. ND, normal diet; HFD, high-fat diet.
Figure 5
Figure 5
Gavage administration of nitazoxanide rescues HFD-induced hyperlipidemia and hepatic steatosis in hamsters. (A) Schematic illustration of the experiment design. (B) The representative images of livers. (C) The analyzed data of body weight, liver weight and liver weight/body weight (LW/BW). (D) The analyzed data of total cholesterol (TC) and triglyceride (TG) content in liver tissues. (E, F) The representative images of H&E and oil red O staining of livers. (G) Nitazoxanide increased phosphorylated AMPK and AMPK protein level in livers from HFD-fed hamsters. (H, I) Nitazoxanide reversed HFD-induced increase of serum cholesterol (TG), triglyceride (TC), LDL-C and HDL-C in hamsters. The serum images are shown in (H) and the quantitative data of serum TC, TG, LDL-C and HDL-C are shown in (I). Data are presented as mean ± SEM; n = 4, 9, and 12 in ND, HFD, and HFD + Nit groups, respectively. Statistical analysis was performed with one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01 vs. ND; ##P < 0.01 vs. HFD. ND, normal diet; HFD, high-fat diet; Nit, nitazoxanide; TC, total cholesterol; TG, triglyceride. HDL-C, high density lipoprotein cholesterol; LDL-C, high density lipoprotein cholesterol.
Figure 6
Figure 6
Gavage administration of nitazoxanide prevents HFD-induced hepatic steatosis in C57BL/6J mice. (A) The representative images of livers. (B) The analyzed data of body weight, liver weight, and liver weight/body weight (LW/BW). (C) The representative images of hematoxylin/eosin (H&E)-stained liver paraffin sections and the analyzed data of lipid deposits. (D) The representative images of oil red O staining of livers and the analyzed data of lipid deposits. (E) The analyzed data of TC and TG content in liver tissues. TC, total cholesterol; TG, triglyceride. (F) Nitazoxanide (100 mg/kg) increased phosphorylated AMPK and phosphorylated ACC protein level in livers. Data were presented as mean ± SEM; n = 10, 10, 10, and 11 in ND, HFD, HFD + Nit (50 mg/kg), HFD + Nit (100 mg/kg) groups, respectively. Statistical analysis was performed with one-way ANOVA. ∗∗P < 0.01 vs. ND; #P < 0.05, ##P < 0.01 vs. HFD. Nit, nitazoxanide.
Figure 7
Figure 7
Gavage administration of nitazoxanide prevents western diet (WD) feeding-induced hepatic steatosis in Apoe–/– mice. (A) Schematic illustration of the experiment design. (B) The time course of body weight. (C) The analyzed data of body weight, liver weight, and liver weight/body weight (LW/BW). (D) The representative images of H&E and oil red O staining of livers. The framed area are enlarged. The analyzed data of oil red O staining was calculated by the area of positive red staining divided by the total area of sections. (E) The analyzed data of TC and TG content in liver tissues. TC, total cholesterol; TG, triglyceride. (F) The serum transaminase level of Apoe–/– mice. ALT, aspartate aminotransferase; AST, alanine aminotransferase. (G) Nitazoxanide increased phosphorylated AMPK protein level in livers of Apoe–/– mice. Data were presented as mean ± SEM; n = 12, 14, 14, 14, and 13 in WT + ND, Apoe–/–+WD, Apoe–/–+WD + Nit (100 mg/kg), Apoe–/–+WD + Nit (200 mg/kg), Apoe–/–+WD + Ato (2 mg/kg) groups, respectively. Statistical analysis was performed with one-way ANOVA. ∗P < 0.05, ∗∗P < 0.01 vs. WT + ND; #P < 0.05, ##P < 0.01 vs. Apoe–/–+WD.
Figure 8
Figure 8
A schematic diagram summarizing the potential mechanisms that nitazoxanide protects against experimental hyperlipidemia and hepatic steatosis.
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