The Effect And Mechanism Of Equol On Liver Oxidative Stress And Bone Mineral Density In Ovariectomized Rats

Nonalcoholic Fatty Liver Disease（NAFLD）is a chronic liver disease characterized by the accumulation of triglyceride（TG）in the liver,and its incidence has gradually increased in recent years.Estrogen levels in women drop after menopause,and the incidence of NAFLD is significantly higher than that in premenopausal women.Oxidative stress is a pathological state produced when the production of reactive oxygen species in the body exceeds the ability of antioxidant detoxification,which is one of the important links in the pathogenesis of NAFLD.NAFLD patients have increased free fatty acids,which leads to increased lipid oxidation and tricarboxylic acid cycle in the liver,and produces excessive reactive oxygen species（ROS）.At the same time,the antioxidant system is broken,the activity of antioxidants is reduced,and ROS cannot be effectively removed,leading to oxidative damage of a variety of biomolecules,cell structure destruction,and then cause cell apoptosis and inflammation.Nuclear Factor Erythroid-2-Related Factor 2（Nrf2）is a major regulator of the body’s anti-oxidative stress response.Upon stimulation,Nrf2 is transferred from the cytoplasm to the nucleus,binds to antioxidant elements,and promotes the expression of downstream antioxidant genes.It can be used as a potential therapeutic target for the prevention and treatment of NAFLD.Osteoporosis（OP）is a bone disease caused by imbalance of bone metabolism,which leads to deterioration of bone mass and bone microstructure.It is characterized by bone fragility and increased risk of fracture.The incidence of OP in postmenopausal women is also significantly higher than that in premenopausal women,and decreased estrogen levels can lead to disorders of bone metabolism.There is a certain correlation between NAFLD and OP in postmenopausal women.Aging and sedentary lifestyle are common risk factors for NAFLD and OP.The average Bone mineral density（BMD）of postmenopausal women with NAFLD is lower than that of postmenopausal women without NAFLD.The occurrence and development of NAFLD can promote the increase of bone marrow adipocytes and the decrease of osteoblasts.Increased inflammatory response and decreased IGF-1 levels can induce the occurrence of OP.The levels of inflammatory factors are increased in postmenopausal NAFLD patients,presenting a chronic inflammatory state,and the level of IGF1 is decreased,which increases the risk of OP.Exogenous estrogen supplementation can improve the symptoms of NAFLD and OP after menopause,but there are some health risks.Equol（Eq）is a natural plant compound.Its structure is very similar to that of estrogen,and its ester-like effect is clear.Eq is a metabolite of Soy Isoflavone（SIF）in the intestine,which has strong biological activities such as anti-inflammation,anti-oxidation,anti-tumor,and improvement of glucose and lipid metabolism.Studies have found that exogenous SIF supplementation can have beneficial effects on postmenopausal NAFLD and postmenopausal OP.Compared with other isoflavones,Eq has the advantages of more stable structure,easy absorption and low clearance rate,but the effect of Eq on postmenopausal NAFLD combined with OP is still unclear.In this study,the effects and mechanisms of Eq on postmenopausal NAFLD,liver oxidative stress and bone metabolism were investigated.Exogenous estrogen supplementation can improve the symptoms of NAFLD and OP after menopause,but long-term use has certain health risks.Equol（Eq）is a natural plant compound,which is produced by the metabolism of soy isoflavone（SIF）in the intestine.Its structure is very similar to that of estrogen,and its estrogen-like effect is clear.It has strong biological activities such as anti-inflammatory,anti-oxidation,anti-tumor,and improvement of glucose and lipid metabolism.Studies have found that exogenous supplementation of SIF can have beneficial effects on postmenopausal NAFLD and OP.Compared with other isoflavones,Eq has the advantages of more stable structure,easy absorption,and low clearance rate,but the effect of Eq on postmenopausal NAFLD combined with OP is still unclear.In this study,the effects and mechanisms of Eq on liver oxidative stress and bone metabolism in postmenopausal NAFLD were investigated.Objective:This study aims to investigate the liver and bone protective effect of Eq in postmenopausal NAFLD,explore the possible mechanism of Eq regulating liver oxidative stress response and bone metabolism,thus to provide new ideas and experimental basis for clinical prevention and treatment of postmenopausal NAFLD.Methods:1.Ovariectomy（OVX）and high fat diet（HFD）were used to establish an animal model of postmenopausal NAFLD,and different doses of Eq and Estradiol（E2）were administered gavage.The animals were divided into 6 groups（n=8 each）,which were control group（Con,sham operation）,model group（Mod）,Eq low-dose group(Eq-L,20 mg·kg^-1·d^-1),Eq medium-dose group(Eq-M,40 mg·kg^-1·d^-1),Eq high-dose group(Eq-H,80 mg·kg^-1·d^-1)and E2 intervention group(E2,0.25 mg·kg^-1·d^-1)for 16 weeks.The effects of Eq on lipid metabolism and oxidative stress in liver tissues of postmenopausal NAFLD were investigated by detecting serum lipids,liver lipid accumulation and oxidative stress indexes in rats.Western blot and RT-PCR were used to detect the influence of Eq on the expression of Nrf2 signaling pathway related proteins and genes,and to explore the potential mechanism of Eq regulating oxidative stress.2.Sodium oleate was used to induce HepG2 cells to establish oxidative stress cell injury model in vitro,and different concentrations of Eq and E2 were used to observe the effects of Eq on lipid accumulation and oxidative stress response in cells.Western blot and RT-PCR were used to detect the effects of Eq on Nrf2 signaling pathway-related protein and gene expression.3.In order to further elucidate the molecular mechanism of Eq regulating oxidative stress,the Nrf2 gene was silenced by siRNA to verify the role of Nrf2 in Eq regulating oxidative stress.Furthermore,the role of PI3K and its downstream AKT and GSK3βpathways in the regulation of Nrf2 signaling by Eq was verified by using PI3K specific inhibitors.In order to verify the estrogen-like effect of Eq,the molecular docking between Eq and G protein-coupled estrogen receptor（GPER）protein was simulated to verify the binding between Eq and GPER protein.Furthermore,the effects of Eq on oxidative stress,PI3K pathway and Nrf2 protein expression were observed using estrogen receptor inhibitors and GPER receptor inhibitors.4.BMD and bone microstructure were detected in the postmenopausal NAFLD rats.HE staining and oil red O staining were used to observe the histological morphological changes of the femur to verify the bone protective effect of Eq.To explore the possible mechanism of Eq on bone protection,the serum levels of estrogen,tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）were detected,and the expression of insulin-like growth factor-1（IGF-1）in liver and bone tissues was detected by Western blot and qRT-PCR.Results:1.Compared with the Con group,the blood lipids and liver lipid accumulation in the Mod group were increased,indicating the postmenopausal NAFLD animal model was successfully established.At the same time,the levels of TAOC,SOD and CAT in the liver were decreased,the content of MAD was increased,and the level of liver oxidative stress was increased.Eq and E2 intervention reduced liver lipid accumulation and oxidative stress levels.2.Eq and E2 could increase the phosphorylation levels of PI3K,AKT and GSK3βproteins and promote the expression of Nrf2 and its downstream antioxidant genes and proteins in the liver.3.Eq and E2 intervention could improve the lipid accumulation and oxidative stress level in HepG2 cells induced by sodium oleate,promote the phosphorylation of PI3K,AKT and GSK3βproteins,and activate the Nrf2 pathway and the expression of downstream antioxidant genes and proteins.4.When Nrf2 were silenced by siRNA,the improvement of Eq on oxidative stress in hepatocytes and the activation of Nrf2 and its downstream antioxidant proteins were inhibited.After inhibition of PI3K signaling pathway,the phosphorylation levels of AKT and GSK3βdecreased,the expression of Nrf2 and its downstream antioxidant protein decreased,and the oxidative stress induced by Eq was inhibited.The simulation of molecular docking showed that Eq could form stable binding with GPER protein.After estrogen receptor inhibitor or GPER inhibitor,the phosphorylation level of PI3K/AKT/GSK3βpathway was decreased,the expression of Nrf2 and its downstream antioxidant proteins was decreased,and Eq’s improvement of oxidative stress was inhibited.5.Compared with the control group,the BMD in Mod group was significantly decreased,the bone microstructure was seriously damaged,and the accumulation of fat in bone tissue was obvious,suggesting that postmenopausal NAFLD was associated with bone injury.Eq and E2 intervention can significantly increase BMD,improve bone microstructure,reduce lipid accumulation in bone tissue,presenting a bone protective effect.Eq and E2 can increase circulating estrogen levels,decrease serum TNF-αand IL-6 levels,and increase IGF-1 protein and mRNA expression levels in liver and bone tissue.Conclusion:1.Eq can reduce liver lipid accumulation in postmenopausal NAFLD rats and up-regulate the expression of Nrf2 and its downstream antioxidant genes through PI3K/AKT/GSK3βaxis,thereby improving liver oxidative stress response;2.Eq could improve lipid accumulation and oxidative stress in HepG2 cells induced by sodium oleate;The regulatory mechanism of Eq on oxidative stress may be mediated by estrogen receptor and GPER,thereby promoting the phosphorylation of PI3K/AKT/GSK3βand activating Nrf2 signaling pathway.3.Eq can improve the decrease of bone mineral density in postmenopausal NAFLD rats,and has a bone protective effect.The mechanism may be related to the reduction of inflammatory response and the promotion of IGF-1 expression in liver and bone tissues.