Abstract: BACKGROUND: Xanthohumol is a natural polyphenol that exhibits biological activities such as antioxidant and anti-inflammatory properties. Recently, it has been found to potentially improve lipid metabolism disorders. As an aerobic exercise, swimming can effectively regulate body energy metabolism and reduce hepatic fat accumulation. However, the intervention effect and mechanism of their combined application on metabolic associated fatty liver disease remain unclear.
OBJECTIVE: To investigate the effect of xanthohumol combined with swimming on the ferroptosis pathway mediated by nuclear factor erythroid 2-related factor 2 in rats with metabolic associated fatty liver disease.  
METHODS: Rats were randomly divided into seven groups: control group, model group, exercise group, low-, medium-, and high-dose xanthohumol, and combination groups, with 12 rats in each group. The rats in control group were fed with normal feed, while the rats in other groups were used to prepare metabolic associated fatty liver disease models. Rats in the exercise and combination groups received swimming training once a day, 6 days per week, for a total of 8 weeks. Rats in other groups were raised quietly. Rats in the low-, medium-, and high-dose xanthohumol groups were intragastrically administered 2 mL of xanthohumol at 25, 50, and 100 mg/kg/d, respectively. Rats in the combination group were intragastrically administered 2 mL of xanthohumol at 100 mg/kg/d. Rats in other groups were intragastrically administered 2 mL of 0.3% sodium carboxymethylcellulose. The intragastric administration lasted for 8 weeks. After the treatment, the serum contents of alanine transaminase, aspartate transaminase, and free fatty acids in each group were detected. Hepatic lipid accumulation was observed by hematoxylin-eosin staining. The hepatic levels of malondialdehyde and reduced glutathione were detected according to the kit instructions. The hepatic ferrous ion content was measured by microassay. The protein expression levels of nuclear factor erythroid 2-related factor 2, Kelch-like ECH-associated protein 1, and glutathione peroxidase 4 in liver tissues were detected by Western blot assay. The mRNA expression levels of ferroptosis-related genes (glutathione peroxidase 4, solute carrier family 7 member 11, ferritin heavy chain 1, ferroportin 1, and ChaC aspartate-specific cysteine peptidase 1) were detected by reverse transcription-quantitative polymerase chain reaction.
RESULTS AND CONCLUSION: (1) Compared with the exercise and high-dose xanthohumol groups, the combination group showed decreased serum levels of alanine transaminase, aspartate transaminase, and free fatty acids, significant improvement in liver morphology, reduced hepatic malondialdehyde level, increased hepatic reduced glutathione level, decreased hepatic ferrous ion content, increased protein expression levels of nuclear factor erythroid 2-related factor 2 (in cell nucleus) and glutathione peroxidase 4 in liver tissues, decreased protein expression of Kelch-like ECH-associated protein 1, increased mRNA expression levels of glutathione peroxidase 4, solute carrier family 7 member 11, ferritin heavy chain 1, and ferroportin 1, and decreased mRNA level of ChaC aspartate-specific cysteine peptidase 1 in liver tissues (P < 0.05). (2) These results suggest that xanthohumol combined with swimming may ameliorate liver injury in rats with metabolic associated fatty liver disease by regulating the nuclear factor erythroid 2-related factor 2-mediated ferroptosis pathway.

Key words: metabolic associated fatty liver disease, xanthohumol, swimming, ferroptosis, nuclear factor erythrocyte 2 related factor 2