Exocarpium Citri Grandis formula granules alleviate fatty liver disease in Zebrafish by maintaining iron homeostasis and suppressing lipid peroxidation and ferroptosis

doi:10.12122/j.issn.1673-4254.2024.12.01

Abstract

Abstract:

Objective To investigate the therapeutic effect of Exocarpium Citri Grandis formula granules (ECGFG) on fatty liver disease (FLD) in zebrafish and explore the underlying mechanism. Methods Nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (ALD) models were established in zebrafish larvae at 3 days post fertilization (dpf), in which the treatment efficacy of 16, 32, or 64 μg/mL ECGFG was evaluated by examining zebrafish survival and liver pathologies and using whole-fish oil red O staining and RT-qPCR. The therapeutic mechanism of ECGFG for FLD was investigated using Prussian blue staining, DCFH-DA probe, MDA content detection, RT-qPCR assay and immunohistochemical staining for CAV1. Results In zebrafish models of NAFLD and ALD, treatment with ECGFG significantly reduced lipid accumulation and the expression levels of FASN, SREBP1, HMGCRA, TNF-α and IL-6, increased the expressions of Apoa1 and PPARα, and reduced iron deposition and the contents of MDA and ROS in the liver. In zebrafish models of NAFLD, treatment with ECGFG at the 3 doses significantly increased hepatic expressions of Tf, TfR, FPN and SLC7A11, and at the doses of 32 and 64 μg/mL, ECGFG obviously increased hepatic expression of GPX4. ALD fish models showed significantly increased hepatic expressions of Tf, TfR and FPN, which were effectively lowered by treatment with ECGFG at the 3 doses. ECGFG did not obviously affect the expression of SLC7A11, but its high dose (64 μg/mL) caused significant elevation of GPX4 expression. Both zebrafish models of NAFLD and ALD showed obviously increased CAV1 expression level in the liver, which was significantly reduced by treatment with 32 and 64 μg/mL ECGFG. Conclusion In zebrafish models of NAFLD and ALD, ECGFG can alleviate lipid accumulation and inflammatory response and lower the expression level of CAV1 to restore iron homeostasis and suppress lipid peroxidation and ferroptosis in the liver.

Key words: fatty liver disease, Exocarpium Citri Grandis, formula granules, zebrafish, iron homeostasis

Yuxue ZAHNG, Jieying LAN, Xinyi MA, Qiong ZHOU, Mengchen QIN, Lei GAO. Exocarpium Citri Grandis formula granules alleviate fatty liver disease in Zebrafish by maintaining iron homeostasis and suppressing lipid peroxidation and ferroptosis[J]. Journal of Southern Medical University, 2024, 44(12): 2265-2275.

Figures/Tables 7

Tab.1 Primer sequences for RT-qPCR

Primer	Primer sequence (5'-3')	Primer sequence (5'-3')
β-actin	ATGGATGAGGAAATCGCTGCC	CTCCCTGATGTCTGGGTCGTC
FASN	GAGAAAGCTTGCCAAACAGG	GAGGGTCTTGCAGGAGACAG
SREBP1	CATCCACATGGCTCTGAGTG	CTCATCCACAAAGAAGCGGT
HMGCRA	CCTGTTAGCCGTCAGTGGA	TCTTTGACCACTCGTGCCG
PPARα	TGCTGGACTACCAGAACTGTGACA	TGCTGGCTGAGAACACTTCTGAG
Apoa1	GCACTAAGCTGACCGAGCGT	GGAGGTCCTGGGTGTGTGGA
TNF-α	GCTTATGAGCCATGCAGTGA	TGCCCAGTCTGTCTCCTTCT
IL6	CCTCAAACCTTCAGACCGCT	GAACAGGATCGAGTGGACCG
Tf	GTTGATGGTGGCCAGGTGTA	AGCGTAGTAACTTGCGGTCC
TfR	CCGCTCATACTCGCGGTTTA	TGGTTCAGAACGACCTGTGG
FPN	GCCAACGTCACCGTTTTTGA	CGGTCAAGTCGAAGGACCAA
GPX4	GAGGTTTACGCATCCTGGCT	GGCTGATCCTTCAGCCACTT
SLC7A11	TGTGGGAATGTCACTGGTGG	ACGCCTCCAGAATGTACGTG

Fig.1 Results of acute toxicity test of Exocarpium Citri Grandis (ECG) in zebrafish larvae. A: Development of zebrafish larvae 96 h after ECG administration. B: Effects of ECG granules on survival of zebrafish. C: Effects of ECG granules on heart rate of zebrafish at 96 h after administration.

Fig.2 Efficacy evaluation of ECG decoction tablets and ECG granules for treatment of NAFLD in zebrafish larvae. A: HE staining of the liver of zebrafish larvae in each group. B: Whole fish oil red O staining in different groups. C: Effects of ECG decoction tablets and ECG granules on lipid metabolism-related genes and inflammatory factors. ECGFG:Exocarpium Citri Grandis formula granules.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.3 Efficacy evaluation of ECG decoction tablets and ECG granules for treatment of ALD in zebrafish larvae. A: HE staining of the liver of zebrafish larvae in each group. B: Whole fish oil red O staining in different groups. C: Effects of ECG decoction tablets and ECG granules on lipid metabolism-related genes and inflammatory factors. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.4 Effect of ECG granules on iron metabolism and ferroptosis in zebrafish larvae models of NAFLD. A: Prussian blue staining of the liver of zebrafish larvae in each group. B: DCFH-DA probe detection of zebrafish larvae in each group. C: Detection of MDA content in zebrafish larvae in each group. D: Effects of ECG granules on iron metabolism- and ferroptosis-related genes. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.5 Effect of ECG granules on iron metabolism and ferroptosis in zebrafish larvae models of ALD. A: Prussian blue staining of the liver of zebrafish larvae in each group. B: DCFH-DA probe detection of zebrafish larvae in each group. C: Detection of MDA content in zebrafish larvae in each group. D: Effects of ECG granules on expressions of iron metabolism- and ferroptosis-related genes. ***P<0.001, ****P<0.0001.

Fig.6 ECG granules regulate hepatic iron metabolism and ferroptosis possibly through CAV1 in zebrafish larvae. A: Liver CAV1 immunohistochemical staining in zebrafish larvae models of NAFLD. B: Effect of ECG granules on CAV1 expression in zebrafish larvae models of NAFLD. C: Liver CAV1 immunohistochemical staining in zebrafish larvae models of ALD. D: Effect of ECG granules on CAV1 expression in zebrafish larvae models of ALD. ****P<0.0001.

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