Celastrus orbiculatus Thunb. extracts and celastrol alleviate NAFLD by preserving mitochondrial function through activating the FGF21/AMPK/PGC-1α pathway

Abstract

Objective: Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease globally, characterized by the accumulation of lipids, oxidative stress, and mitochondrial dysfunction in the liver. Celastrus orbiculatus Thunb. (COT) and its active compound celastrol (CEL) have demonstrated antioxidant and anti-inflammatory properties. Our prior research has shown the beneficial effects of COT in mitigating NAFLD induced by a high-fat diet (HFD) in guinea pigs by reducing hepatic lipid levels and inhibiting oxidative stress. This study further assessed the effects of COT on NAFLD and explored its underlying mitochondria-related mechanisms.

Methods: COT extract or CEL was administered as an intervention in C57BL/6J mice fed a HFD or in HepG2 cells treated with sodium oleate. Oral glucose tolerance test, biochemical parameters including liver enzymes, blood lipid, and pro-inflammatory factors, and steatosis were evaluated. Meanwhile, mitochondrial ultrastructure and indicators related to oxidative stress were tested. Furthermore, regulators of mitochondrial function were measured using RT-qPCR and Western blot.

Results: The findings demonstrated significant reductions in hepatic steatosis, oxidative stress, and inflammation associated with NAFLD in both experimental models following treatment with COT extract or CEL. Additionally, improvements were observed in mitochondrial structure, ATP content, and ATPase activity. This improvement can be attributed to the significant upregulation of mRNA and protein expression levels of key regulators including FGF21, AMPK, PGC-1α, PPARγ, and SIRT3.

Conclusion: These findings suggest that COT may enhance mitochondrial function by activating the FGF21/AMPK/PGC-1α signaling pathway to mitigate NAFLD, which indicated that COT has the potential to target mitochondria and serve as a novel therapeutic option for NAFLD.

Keywords: Celastrus orbiculatus Thunb.; FGF21/AMPK/PGC-1α; NAFLD; celastrol; mitochondrial function.

FIGURE 1

HPLC chromatogram of ethanolic extract of Celastrus orbiculatus Thunb. (A) CEL; (B) COT extract.

FIGURE 2

COT extract and CEL attenuated hepatic steatosis and metabolic dysfunction in HFD-fed mice. (A) Experiment design used to evaluate the role of COT extract and CEL against NAFLD; (B) Body weight recorded every week; (C) Liver weight and (D) visceral adiposity index at the end of the experiment; (E) Representative images of HE- and ORO-stained liver sections (×400 magnification, scale bar = 50 μm); (F–I) AST and ALT levels in livers and serum; (J–L) Levels of TG, TC and LDL-C in livers and/or serum (n = 8); (M) Blood glucose levels in OGTT (n = 6); (N) Summary of area under the curve (AUC) of the OGTT. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the CON group; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 vs. the MOD group; ^& P < 0.05 and ^&&& P < 0.001 vs. the CL group; ^▽ P < 0.05 and ^▽▽ P < 0.05 vs. the EL group.

FIGURE 3

COT extract and CEL improved the structure and function of liver mitochondria during NAFLD induced by HFD feeding in mice. (A) Representative TEM images of mouse hepatocytes, M represents the mitochondria, while N represents the nucleus (×2 k magnification, scale bar = 2 μm); Activity of (B) Na⁺/K⁺ ATPases (C) Mg²⁺ ATPases and (D) Ca²⁺ ATPases in liver tissue (n = 8); (E) liver ATP content (n = 3). Data are represented as mean ± SD. ***P < 0.001 vs. the CON group; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 vs. the MOD group; ^& P < 0.05, ^&& P < 0.05, and ^&&& P < 0.001 vs. the CL group.

FIGURE 4

COT extract and CEL mitigated the oxidative stress and inflammation caused by HFD in mice. Changes in the levels of oxidative stress-related markers: (A) MDA (B) SOD (C) GSH, and (D) GSH-Px in liver tissue (n = 8); Levels of inflammatory factors (E) TNF-α and (F) IL-6 in liver tissue (n = 3). Data are represented as mean ± SD. ***P < 0.001 vs. the CON group; ^### P < 0.001 vs. the MOD group; ^& P < 0.05 and ^&&& P < 0.001 vs. the CL group; ^▽ P < 0.05 and ^▽▽ P < 0.05 vs. the EL group.

FIGURE 5

COT extract and CEL regulated FGF21/AMPK/PGC-1α signal pathway in HFD-fed mice. The mRNA expression of (A) PPARγ (B) FGF21 (C) p-AMPK (D) PGC-1α, and (E) SIRT3 in liver tissue by RT-qPCR analysis; The abundance of target protein involved in lipid metabolism and mitochondrial dysfunction: (F) PPARγ (G) FGF21 (H) p-AMPK (I) PGC-1α, and (J) SIRT3 in liver tissue. Experiments were repeated in triplicates. Data are represented as mean ± SD, n = 3. **p < 0.01 and ***p < 0.001 vs. the CON group; ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. the MOD group; ^& p < 0.05 vs. the CL group.

FIGURE 6

COT extract and CEL alleviated NaOL-induced intracellular lipid accumulation in HepG2 cells. (A) Representative images of ORO-stained HepG2 cells treated with NaOL with/without COT extract or CEL for 24 h (×400 magnification); (B) Quantitative analysis of ORO intensity; (C, D) Analysis of TG and TC content in HepG2 cells. Data are represented as mean ± SD, n = 3. ***P < 0.001 vs. the CON group; ^### P < 0.001 vs. the MOD group; ^& P < 0.05 vs. the CL group.

FIGURE 7

COT extract and CEL attenuated NaOL-induced oxidative stress and inflammation in vitro. Levels of (A) MDA (B) SOD (C) GSH (D) GSH-Px (E) TNFα, and (F) IL-6 in HepG2 cells. Data are represented as mean ± SD, n = 3. ***P < 0.001 vs. the CON group; ^### P < 0.001 vs. the MOD group; ^& P < 0.05 and ^&& P < 0.05 vs. the CL group; ^▽ P < 0.05 vs. the EL group.

FIGURE 8

COT extract and CEL protected against mitochondrial dysfunction by regulating FGF21/AMPK/PGC-1α signal pathway in HepG2 cells. (A) ATP content in HepG2 cells. The mRNA expression of (B) PPARγ, (C) FGF21, (D) p-AMPK, (E) PGC-1α, and (F) SIRT3 in HepG2 cells; The abundance of target protein involved in lipid metabolism and mitochondrial dysfunction: (G) PPARγ, (H) FGF21, (I) p-AMPK, (J) PGC-1α, and (K) SIRT3. The experiments were repeated in triplicates. Data are represented as mean ± SD, n = 3. *P < 0.05 and ***P < 0.001 vs. the CON group; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 vs. the MOD group; ^& P < 0.05 and ^&& P < 0.05 vs. the CL group; ^▽ P < 0.05 and ^▽▽ P < 0.01 vs. the EL group.

FIGURE 9

Schematic diagram showing the effects of COT extract and CEL on improving mitochondrial function in NAFLD via activating the FGF21/AMPK/PGC-1α signal pathway.