| BackgroundNon-alcoholic fatty liver disease（NAFLD）has become the most common cause of chronic liver disease worldwide,affecting more than 25%of the world’s population.Estrogen plays an important protective role in hepatic lipid homeostasis,and the incidence of NAFLD is increased in postmenopausal women due to reduced estrogen levels.Unhealthy lifestyles such as unreasonable dietary structure,high energy intake,sedentary and inactive lifestyles,and increased life expectancy have increased the incidence of NAFLD in postmenopausal women.NAFLD is closely related to cardiovascular diseases,osteoporosis,malignant tumors and various endocrine diseases.The increasing prevalence of this disease and its possible serious complications will bring huge economic burden and poor health-related quality of life to patients.However,due to the complex pathogenesis,the current treatment for NAFLD is not ideal.Soy isoflavone（SI）is a class of natural phytoestrogens,its molecular structure and biological activity are similar to the estrogens synthesized in the body,which is abundant in legumes,with anti-inflammatory,antioxidant,improve insulin resistance,alleviate lipid metabolism disorders and other biological effects,while avoiding the occurrence of adverse reactions of synthetic estrogen.The biological effects of SI are closely related to its intestinal metabolite equol（Eq）,which has a wide range of biological effects,but its role and mechanism on postmenopausal NAFLD are unclear.ObjectivesThe postmenopausal NAFLD model rats were used as the research object to explore the intervention effect of Eq from the aspects of inflammation,insulin resistance and lipid metabolism,and the potential molecular mechanism of Eq alleviating postmenopausal NAFLD was further explored with the steatosis cell model,providing new ideas for the exploration of safe and effective treatment for postmenopausal women with NAFLD.Methods1.Five-week-old female SD rats constructed a model of postmenopausal NAFLD by high-fat diet（HFD）combined with bilateral ovarietomized（OVX）,followed by 12 weeks of low-,medium-,and high-dose[20,40,80 mg/（kg·d）]doses of Eq or estradiol[E2,0.25mg/（kg·d）]gavage intervention,and rat body weight and feed intake were recorded weekly.2.Fasting plasma glucose（FPG）,lipids,liver function,and liver lipid levels were measured at the end of the intervention;ELISA was used to detect serum estrogen,fasting insulin（FINS）,serum and liver inflammatory factor levels in rats.The liver index and homeostasis model assessment-insulin resistance（HOMA-IR）were calculated in rats,and lipid deposition and morphological changes in liver were observed by oil red O and hematoxylin-eosin（HE）staining,respectively.Quantitative Real-time PCR and Western blot detected nuclear factor kappa-B（NF-κB）p-p65,tumor necrosis factor-alpha（TNF-α）,interleukin-6（IL-6）and lipid synthesis-related gene liver X receptorr-alpha（LXRα）,sterol regulatory element binding protein-1c（SREBP-1c）,acetyl-coa carboxylase 1（ACC1）and fatty acid synthase（FAS）expression levels.3.Hep G2 cells were cultured in vitro,sodium oleate（Na OL）at different concentrations（0.12～0.72）mmol/L were used for intervention for 48h,and the appropriate Na OL concentration to induce steatosis of Hep G2 cells were determined by detecting intracellular TG level,oil red O staining to observe intracellular lipid droplet accumulation and CCK-8 method to detect cell viability.4.Interventions were performed with different concentrations of Eq(10-7mol/L,10-6mol/L,10-5mol/L)or E2(10-9mol/L,10-8mol/L,10-7mol/L);The content of triglyceride（TG）was detected,and the changes of lipid deposition were observed by oil red O staining.Quantitative Real-time PCR and Western blot were used to detect the expression levels of cellular estrogen receptor-alpha（ERα）,estrogen receptor-beta（ERβ）,LXRα,SREBP-1c,ACC1,and FAS genes.5.The expression of ERαand ERβwas inhibited by the estrogen receptor inhibitor Fulvestrant;The effects of Eq on cell TG content,lipid deposition,and expression levels of ERα,ERβ,LXRα,SREBP-1c,ACC1 and FAS genes under the action of Fulvestrant group were detected.6.SPSS 20.0 software was used for statistical analysis.Measurement data of normal distribution were expressed as±s,the one-way analysis of variance was used for comparison between groups,and the LSD test was used for pairwise comparison.Measurement data of non-normal distribution was expressed as M（P25,P75）,the comparison between multiple groups uses non parametric test（Kruskal Wallis H test）,and the pairwise comparison was performed by Mann-Whitney U test.P<0.05 was considered statistically significant.Animal experiment results1.The serum estrogen level of rats in the model group was significantly lower than that in the sham surgery group,and a large number of lipid droplet vacuoles and inflammatory cell infiltration appeared in liver cells,indicating that the postmenopausal NAFLD model was successfully established;After the intervention of Eq or E2,the serum estrogen level of rats was significantly higher than that in the model group,the lipid droplet vacuole and inflammatory cell infiltration of hepatocytes were reduced,and the steatosis of liver tissue was improved to varying degrees.2.After Eq or E2 intervention,rats had significantly higher serum high-density lipoprotein cholesterol（HDL-c）levels than those in the model group,body weight,liver index,serum TG,total cholesterol（TC）,low-density lipoprotein cholesterol（LDL-c）,The level of free fatty acid（FFA）and the level of TG and TC of liver tissue were significantly lower than those in the model group,the accumulation of red stained granules and lipid droplets in liver tissue was reduced to varying degrees,and the whole body lipid metabolism disorder of rats was significantly improved.3.After Eq or E2 intervention,the levels of FPG,FINS,HOMA-IR,alanine transaminase（ALT）,aspartate amino transferase（AST）,serum and liver tissue TNF-αand IL-6 were significantly reduced compared with the model group,reducing inflammation,insulin resistance and improving liver function.4.After the intervention of Eq or E2,the expression levels of NF-κB p-p-p65,TNF-α,IL-6,LXRα,SREBP-1c,ACC1 and FAS genes in rat liver tissue were significantly downregulated compared with the model group,and the inflammatory signaling pathway and lipid synthesis pathway of liver tissue were significantly inhibited.Cell experiment results1.Compared with the control group without Na OL treatment,when the Na OL concentration was 0.24 mmol/L,the cell viability did not change significantly,while the TG content increased significantly,and a large number of red-stained granules and lipid droplets infiltrated the cells.In subsequent experiments,0.24 mmol/L Na OL was selected as the condition for inducing steatosis of Hep G2 cells.2.After the intervention of Eq or E2,the TG content of cells was significantly lower than that in the model group,and the infiltration of intracellular red stained granules and lipid droplets decreased to varying degrees,which effectively alleviated the lipid deposition of Hep G2 cells induced by Na OL.At the same time,the expression levels of LXRα,SREBP-1c,ACC1 and FAS were down-regulated,the lipid synthesis pathway was significantly inhibited,and the expression levels of ERαand ERβwere up-regulated.3.After the intervention of Fulvestrant,the effect of Eq up-regulating ERα,ERβand down-regulation of LXRα,SREBP-1c,ACC1 and FAS were weakened,and the ability of Eq to alleviate fat deposition in Hep G2 cells was reduced,indicating that Eq inhibited the LXRα/SREBP-1c signaling pathway through estrogen receptors,reduced hepatocyte lipid synthesis,and effectively improved hepatocyte steatosis.Research conclusionThe above results suggest that Eq dietary intervention could effectively alleviate NAFLD by high-fat diet-induced in ovariectomized rats through reducing inflammation and insulin resistance,improving systemic lipid metabolic disorder and abnormal liver function.The mechanism was related to Eq inhibited liver inflammation by down-regulating NF-κB signal pathway and inhibited LXRα/SREBP-1c signaling pathway through estrogen receptor pathway to reduce lipid synthesis in hepatocytes. |
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