Action Mechanism Of 7,8-Dihydroxyflavone Against Metabolic Syndrome In Female Mice

7,8-Dihydroxyflavone(7,8-DHF),a naturally occurring flavonoid with relatively rare structure,can simulate brain-derived neurotrophic factor(BDNF),specifically bind and activate tropomyosin-related kinase B(TrkB)to trigger BDNF/TrkB signaling cascade and exert corresponding physiological regulatory functions.Currently,the majority of reserches on 7,8-DHF focus on the intervention and mechanism of central nervous system and related diseases,and there are few studies on peripheral tissuerelated diseases.Our previous study found that 7,8-DHF can alleviate high-fat diet(HFD)induced obesity by activating skeletal muscle BDNF/TrkB signaling pathway,and the effect was only obvious in female mice,but the specific mechanism for this sex difference is still unclear.Epidemiological surveyes have shown that following the onset of menopause,women are more vulnerable to developing metabolic-related chronic diseases such as metabolic syndrome(MetS)compared with similarly aged men,which is closely related to the drastic fluctuations in sex hormone levels and changes in estrogen related receptors.In order to clarify the intervention effect of 7,8-DHF on MetS in the aging process of female mice and the reasons for sex differences,a systematic investigation was carried out in this paper by using animal experiments in vivo and cell experiments in vitro.The main results and conclusions are as follows:(1)Long-term intervention of 7,8-DHF effectively prevented HFD and menopause induced MetS in female mice.7,8-DHF can obviously inhibit HFD induced excessive weight gain and visceral fat accumulation without inhibiting appetite in mice,and can simultaneously improve glucose and lipid metabolism disorders,insulin resistance and bone loss in mice fed both LFD and HFD.(2)The regulation and protective effects of 7,8-DHF on the hypothalamicpituitary-ovarian(HPO)axis were further evaluated by analyzing the levels of representative sex hormones and the ovarian reserve.Results showed that long-term intervention of 7,8-DHF can effectively regulate the HPO axis function in mice: protect ovarian reserve function,prevent earlier menopause and maintain the stability of major sex hormone levels;In both LFD and HFD dietary patterns,7,8-DHF can distincly prevent follicular atresia and protect the development of healthy oocytes in the ovary;at the same time,the level of serum estradiol(E2)in peripheral circulation was increased with 7,8-DHF treatment,while the level of follicle stimulating hormone(FSH)was decreased.Based on the analysis of serum lipopolysaccharide(LPS),inflammatory cytokines and serotonin(5-HT),it was speculated that the protective effect of 7,8-DHF on the ovarian reserve function of the HPO axis was presumably achieved by reducing gut microbiota-mediated systemic inflammation.(3)The intervention of 7,8-DHF can affect the diversity and structural composition of gut microbiota in mice,promote the colonization of symbolic beneficial bacteria and suppress the growth of potential pathogenicbacterial species in the intestinal tract,which might act to the improvement of MetS,however,there were great differences in different dietary patterns.For LFD-fed mice,78-DHF significantly reduced the richness and diversity of gut microbiota(p < 0.05),but significantly up-regulated 6 beneficial bacteria species(represented by Faecalibacterium prausnitzii,Bacteroides plebeius and Blautia producta)that produce butyric acid and inhibit inflammation,and downregulated Allobaculum and other bacteria associated with diabetes,which might indirectly contribute to the alleviation of MetS-related phenotypes.In contrast,7,8-DHF significantly(p < 0.05)increased the richness and diversity of gut microbiota in HFD-fed mice,and increased beneficial bacteria such as Oscillospira,Mucispirillum Schaedleri and Dehalobacterium,which are related to butyric acid production and anti-inflammation.In addition,7,8-DHF effectively promoted the production of shortchain fatty acids(SCFAs),especially butyric acid in mouse feces.(4)In this study,we also found that estrogen receptor alpha(ER?)is also indispensable to skeletal muscle energy metabolism just like the activation of the BDNF/TrkB signaling pathway.The results of in vivo animal experiments showed that7,8-DHF could maintain the level of ER? protein in skeletal muscle.Furthermore,in vitro studies in C2C12 myotubes,we found that 7,8-DHF induced activation of BDNF/TrkB signaling pathway related extracellular regulated kinase(ERK)and tyrosine family kinase(Src)signal molecules accordingly transactivated ER? in Ser118(S118)in AF-1 domain and Tyr573(Y537)in AF-2 domain,respectively;Furthermore,it was found that the transactivation role of 7,8-DHF to ER? Y537 was failed after k552 a blocked the activation of BDNF/TrkB signaling pathway;In turn,deletion of ER? also blocks the activation of BDNF/TrkB and downstream Akt signaling pathways,as well as the expression of uncoupled protein 1(UCP1)in skeletal muscle mitochondria.The results show that there is a crosstalk between ER? and BDNF/TrkB signaling pathway,and Src family kinases played a core role in the crosstalk,synergistically activating the signaling pathways related to energy metabolism.In conclusion,this study systematically elucidates the effect and mechanism of7,8-DHF,a natural small molecule mimic of BDNF,on the improvement of MetS induced by menopause and HFD in female mice from three aspects: beneficial effect on gut microbiota,regulation of HPO axis function,and mechanism of crosstalk between muscular ER? and BDNF/TrkB signaling in skeletal muscle.The results clearly indicate that maintaining or enhancing ER? activity in skeletal muscle is crucial for alleviating age-related metabolic disorders in female animals.The results of this study provide a reasonable explanation for this sex difference in 7,8-DHF-mediated alleviation of MetS.