Protective effect of equol intake on bladder dysfunction in a rat model of bladder outlet obstruction.

OBJECTIVES: This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we investigate its potential as a neuroprotective agent for the obstructed bladder and discuss its applicability in managing overactive bladder (OAB). METHODS: Eighteen male Sprague-Dawley rats were divided into three groups (six rats per group) during the rearing period. The Sham and C-BOO groups received an equol-free diet, while the E-BOO group received equol supplementation (0.25 g/kg). At 8 weeks old, rats underwent BOO surgery, followed by continuous cystometry after 4 weeks of rearing. The urinary oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) were measured, and the bladder histology was analyzed using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining (neurofilament heavy chain for myelinated nerves, peripherin for unmyelinated nerves, and malondialdehyde). RESULTS: Equol reduced BOO-induced smooth muscle layer fibrosis, significantly prolonged the micturition interval (C-BOO: 193 s, E-BOO: 438 s) and increased the micturition volume (C-BOO: 0.54 mL, E-BOO: 1.02 mL) compared to the C-BOO group. Equol inhibited the increase in urinary and bladder tissue malondialdehyde levels. While the C-BOO group exhibited reduced peripherin alone positive nerve fibers within the smooth muscle layer, equol effectively attenuated this decline. CONCLUSIONS: Equol reduces lipid peroxidation and smooth muscle layer fibrosis in the bladder and exhibited neuroprotective effects on bladder nerves (peripheral nerves) and prevented the development of bladder dysfunction associated with BOO in rats. Consumption of equol is promising for the prevention of OAB associated with BOO.

Equol exerts a protective effect on postmenopausal osteoporosis by upregulating OPG/RANKL pathway.

BACKGROUD: Estrogen deficiency is the leading cause of postmenopausal osteoporosis(PMOP) and phytoestrogens soy isoflavones (SI) have been shown to improve PMOP. Equol (Eq), an in vivo metabolite of phytoestrogens soy isoflavones (SI), has a more stable structure and stronger biological activity than its parent compound and has the greatest estrogenic activity. However, there are few studies on the therapeutic effect of Eq on PMOP. PURPOSE: To explore the therapeutic effect and mechanisms of Eq on POMP. METHODS: Osteoblast-like cells ROS1728 were cultured with different doses of Eq, estradiol (E2), separately. The effect of Eq on the proliferation, apoptosis, cell cycle of osteoblasts were detected by CCK-8 and flow cytometry, and the expression of OPG/RANK/RANKL signaling pathway of osteoblasts was detected by Quantitative real-time PCR (qRT-PCR) and Western blot (WB), and RNA silencing technology were carried out to explore the receptors through which Eq plays a role. Then PMOP rat model was established and treated by Eq or E2 to further verification of the effect and mechanism of Eq on PMOP. RESULT: Eq promoted the proliferation and inhibited the apoptosis of osteoblasts and increased the proportion of osteoblasts in the S phase and G2/M phase in a dose-dependent manner. Mechanistically, Eq treatment upregulated the expression of OPG and OPG/RANKL ratio in osteoblasts and this regulatory effect was mainly mediated through the ERß receptor. Furthermore, in vivo study, Eq improved microstructure and BMD of the femur of PMOP rat model, which imitated the osteoprotective effect of E2. Moreover, the Eq or E2 treatment increased serum levels of Ca, 1,25(OH)2D3, bone Gla-protein(BGP), and Type I procollagen (PC1), and reduced serum levels of phosphorus (P), parathyroid hormone(PTH), pyridinol (PYD), tartrate-resistant acid phosphatase (TRAP) and urinary level of deoxypyridinoline (DPD) in the treatment OVX group compared with the untreated OVX group. Meanwhile, Eq or E2 markedly induced the mRNA and protein expression of OPG and OPG/RANKL ratio. CONCLUSION: Eq can combine with ERß and exert a protective effect on PMOP by upregulating OPG/RANKL pathway.

(S)-Equol Is More Effective than (R)-Equol in Inhibiting Osteoclast Formation and Enhancing Osteoclast Apoptosis, and Reduces Estrogen Deficiency-Induced Bone Loss in Mice.

BACKGROUND: Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol. OBJECTIVES: We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo. METHODS: Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues. RESULTS: (S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively). CONCLUSIONS: These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.

6-methoxydihydroavicine, the alkaloid extracted from Macleaya cordata (Willd.) R. Br. (Papaveraceae), triggers RIPK1/Caspase-dependent cell death in pancreatic cancer cells through the disruption of oxaloacetic acid metabolism and accumulation of reactive oxygen species.

BACKGROUND: Many extracts and purified alkaloids of M. cordata (Papaveraceae family) have been reported to display promising anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis in many cancer types. However, no evidence currently exists for anti-pancreatic cancer activity of alkaloids extracted from M. cordata, including a novel alkaloid named 6­methoxy dihydrosphingosine (6-Methoxydihydroavicine, 6-ME) derived from M. cordata fruits. PURPOSE: The aim of this study was to investigate the anti-tumor effects of 6-ME on PC cells and the underlying mechanism. METHODS: CCK-8, RTCA, and colony-formation assays were used to analyze PC cell growth. Cell death ratios, changes in MMP and ROS levels were measured by flow cytometry within corresponding detection kits. A Seahorse XFe96 was employed to examine the effects of 6-ME on cellular bioenergetics. Western blot and q-RT-PCR were conducted to detect changes in target molecules. RESULTS: 6-ME effectively reduced the growth of PC cells and promoted PCD by activating RIPK1, caspases, and GSDME. Specifically, 6-ME treatment caused a disruption of OAA metabolism and increased ROS production, thereby affecting mitochondrial homeostasis and reducing aerobic glycolysis. These responses resulted in mitophagy and RIPK1-mediated cell death. CONCLUSION: 6-ME exhibited specific anti-tumor effects through interrupting OAA metabolic homeostasis to trigger ROS/RIPK1-dependent cell death and mitochondrial dysfunction, suggesting that 6-ME could be considered as a highly promising compound for PC intervention.

Soy isoflavone metabolite equol inhibits cancer cell proliferation in a PAP associated domain containing 5-dependent and an estrogen receptor-independent manner.

Isoflavone is a species of polyphenol found mainly in soy and soy products. Many studies have demonstrated its estrogen receptor (ER)-dependent action. Equol is an intestinal metabolite of a major soy isoflavone daidzein. We aimed to elucidate the mechanism for ER-independent actions of equol. Equol has been shown to inhibit proliferation of HeLa human cervical cancer cells and mouse melanoma B16 cells in an ER-independent manner. Using functional genetic screening, PAP associated domain containing 5 (PAPD5), which is a non-canonical poly(A) polymerase, was identified as an essential molecule in the ER-independent action. While peroral administration of equol inhibited tumor growth of control B16 cells subcutaneously inoculated in mice, it had little effect on the growth of PAPD5-ablated B16 cells. Intriguingly, equol progressed tumor growth of the PAPD5-ablated human breast cancer MCF-7 cells, which have high ERα expression. Equol has been found to induce polyadenylation of snoRNAs in a PAPD5-depdendent manner. Furthermore, peroral equol administration increased microRNA miR-320a expression in tumors. Together, these results suggest that equol may have a dual effect on ER-positive cancer cells, acting with, antiproliferative activity through PAPD5 and exhibiting proliferative activity via ERα and the former could be associated with miR-320a.

Potential Protective Effects of Equol (Soy Isoflavone Metabolite) on Coronary Heart Diseases-From Molecular Mechanisms to Studies in Humans.

Equol, a soy isoflavone-derived metabolite of the gut microbiome, may be the key cardioprotective component of soy isoflavones. Systematic reviews have reported that soy isoflavones have no to very small effects on traditional cardiovascular disease risk factors. However, the potential mechanistic mode of action of equol on non-traditional cardiovascular risk factors has not been systematically reviewed. We searched the PubMed through to July 2021 by using terms for equol and each of the following markers: inflammation, oxidation, endothelial function, vasodilation, atherosclerosis, arterial stiffness, and coronary heart disease. Of the 231 records identified, 69 articles met the inclusion criteria and were summarized. Our review suggests that equol is more lipophilic, bioavailable, and generally more potent compared to soy isoflavones. Cell culture, animal, and human studies show that equol possesses antioxidative, anti-inflammatory, and vasodilatory properties and improves arterial stiffness and atherosclerosis. Many of these actions are mediated through the estrogen receptor ß. Overall, equol may have a greater cardioprotective benefit than soy isoflavones. Clinical studies of equol are warranted because equol is available as a dietary supplement.

Phytoestrogens (Resveratrol and Equol) for Estrogen-Deficient Skin-Controversies/Misinformation versus Anti-Aging In Vitro and Clinical Evidence via Nutraceutical-Cosmetics.

The overarching theme for this review is perspective. Superfoods (a marketing term for fruits and vegetables, etc.) have a positive connotation, while many superfoods contain phytoestrogens, a term that is alarming to the public and has a negative connotation because phytoestrogens are endocrine-disruptors, even though they are strong antioxidants that have many health benefits. To understand phytoestrogens, this paper provides a brief summary of the characteristics of: (a) estrogens, (b) estrogen receptors (ER), (c) estrogen-deficient skin, (d) how perspective(s) get off track, (e) phytoestrogen food sources, and (f) misconceptions of phytoestrogens and food safety, in general, that influence person(s) away from what is true. Finally, a brief history of cosmetics to nutraceuticals is covered plus the characteristics of phytoestrogens, resveratrol and equol on: (g) estrogen receptor binding, (h) topical and oral dosing, and (i) in vitro, molecular mechanisms and select clinical evidence, where both phytoestrogens (resveratrol and equol) demonstrate promising applications to improve skin health is presented along with future directions of nutraceuticals. Perspective is paramount in understanding the controversies associated with superfoods, phytoestrogens, and endocrine-disruptors because they have both positive and negative connotations. Everyone is exposed to and consumes these molecules everyday regardless of age, gender, or geographic location around the world, and how we understand this is a matter of perspective.

S-Equol Protects Chondrocytes against Sodium Nitroprusside-Caused Matrix Loss and Apoptosis through Activating PI3K/Akt Pathway.

Osteoarthritis (OA) is a common chronic disease with increasing prevalence in societies with more aging populations, therefore, it is causing more concern. S-Equol, a kind of isoflavones, was reported to be bioavailable and beneficial to humans in many aspects, such as improving menopausal symptoms, osteoporosis and prevention of cardiovascular disease. This study investigated the effects of S-Equol on OA progress in which rat primary chondrocytes were treated with sodium nitroprusside (SNP) to mimic OA progress with or without the co-addition of S-Equol for the evaluation of S-Equol's efficacy on OA. Results showed treatment of 0.8 mM SNP caused cell death, and increased oxidative stress (NO and H2O2), apoptosis, and proteoglycan loss. Furthermore, the expressions of MMPs of MMP-2, MMP-3, MMP-9, and MMP-13 and p53 were increased. The addition of 30 µM S-Equol could lessen those caused by SNP. Moreover, S-Equol activates the PI3K/Akt pathway, which is an upstream regulation of p53 and NO production and is associated with apoptosis and matrix degradation. As a pretreatment of phosphoinositide 3-kinases (PI3K) inhibitor, all S-Equol protective functions against SNP decrease or disappear. In conclusion, through PI3K/Akt activation, S-Equol can protect chondrocytes against SNP-induced matrix degradation and apoptosis, which are commonly found in OA, suggesting S-Equol is a potential for OA prevention.

Roles of Phytoestrogen in the Pathophysiology of Intracranial Aneurysm.

Background and Purpose: The incidences of intracranial aneurysm and aneurysmal subarachnoid hemorrhage are high in postmenopausal women. Although population-based studies suggest that hormone replacement therapy is beneficial for postmenopausal women with intracranial aneurysms, estrogen replacement may no longer be recommended for the prevention of chronic diseases given its association with adverse outcomes, such as cancer and ischemic stroke. The isoflavone daidzein and its intestinal metabolite equol are bioactive phytoestrogens and potent agonists of estrogen receptors. Given their estrogenic properties, we investigated whether the isoflavones daidzein and equol are protective against the formation and rupture of intracranial aneurysms in a mouse model of the postmenopausal state. Methods: We induced intracranial aneurysms in ovariectomized adult female mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. We fed the mice with an isoflavone-free diet with/without daidzein supplementation, or in a combination of intraperitoneal equol, or oral vancomycin treatment. We also used estrogen receptor beta knockout mice. Results: Both dietary daidzein and supplementation with its metabolite, equol, were protective against aneurysm formation in ovariectomized mice. The protective effects of daidzein and equol required estrogen receptor-ß. The disruption of the intestinal microbial conversion of daidzein to equol abolished daidzein's protective effect against aneurysm formation. Mice treated with equol had lower inflammatory cytokines in the cerebral arteries, suggesting that phytoestrogens modulate inflammatory processes important to intracranial aneurysm pathogenesis. Conclusions: Our study establishes that both dietary daidzein and its metabolite, equol, protect against aneurysm formation in ovariectomized female mice through the activation of estrogen receptor-ß and subsequent suppression of inflammation. Dietary daidzein's protective effect required the intestinal conversion to equol. Our results indicate the potential therapeutic value of dietary daidzein and its metabolite, equol, for the prevention of the formation of intracranial aneurysms and related subarachnoid hemorrhage.

S-Equol mitigates motivational deficits and dysregulation associated with HIV-1.

Motivational deficits (e.g., apathy) and dysregulation (e.g., addiction) in HIV-1 seropositive individuals, despite treatment with combination antiretroviral therapy, necessitates the development of innovative adjunctive therapeutics. S-Equol (SE), a selective estrogen receptor ß agonist, has been implicated as a neuroprotective and/or neurorestorative therapeutic for HIV-1 associated neurocognitive disorders (HAND); its therapeutic utility for motivational alterations, however, has yet to be systematically evaluated. Thus, HIV-1 transgenic (Tg) and control animals were treated with either a daily oral dose of SE (0.2 mg) or vehicle and assessed in a series of tasks to evaluate goal-directed and drug-seeking behavior. First, at the genotypic level, motivational deficits in HIV-1 Tg rats treated with vehicle were characterized by a diminished reinforcing efficacy of, and sensitivity to, sucrose. Motivational dysregulation was evidenced by enhanced drug-seeking for cocaine relative to control animals treated with vehicle. Second, treatment with SE ameliorated both motivational deficits and dysregulation in HIV-1 Tg rats. Following a history of cocaine self-administration, HIV-1 Tg animals treated with vehicle exhibited lower levels of dendritic branching and a shift towards longer dendritic spines with decreased head diameter. Treatment with SE, however, led to long-term enhancements in dendritic spine morphology in HIV-1 Tg animals supporting a potential underlying basis by which SE exerts its therapeutic effects. Taken together, SE restored motivated behavior in the HIV-1 Tg rat, expanding the potential clinical utility of SE to include both neurocognitive and affective alterations.

S-equol, a metabolite of dietary soy isoflavones, alleviates lipopolysaccharide-induced depressive-like behavior in mice by inhibiting neuroinflammation and enhancing synaptic plasticity.

Systemic injection with lipopolysaccharide can lead to depressive-like behavior in experimental animals by inducing neuroinflammation and is considered to be a classic model of depression. S-equol is a major metabolite of dietary soy isoflavones with antioxidant and anti-inflammatory effects, and it has many beneficial effects on human health, including alleviation of menopausal symptoms, osteoporosis, cancer, obesity, chronic kidney disease, and cognitive dysfunction. A recent study reported that S-equol inhibited lipopolysaccharide-stimulated neuroinflammation in astrocytes. However, there is no research on the antidepressant-like effects of S-equol. Therefore, the present study was conducted to evaluate the antidepressant-like effects of S-equol in a lipopolysaccharide-induced depression model in mice and explore its underlying mechanisms. Our results demonstrated that treatment with S-equol (10, 20 and 40 mg kg-1) for 19 days markedly reversed the behavior of acute LPS (1.0 mg kg-1) treated mice in sucrose preference, tail suspension and forced swimming tests, exerting antidepressant-like effects. In addition, S-equol administration significantly decreased the levels of pro-inflammatory cytokines (tumor necrosis factor, interleukin-6, interleukin-10, interleukin-1ß), increased the levels of 5-hydroxytryptamine and norepinephrine, and normalized the release of tryptophan and kynurenine in the hippocampi of lipopolysaccharide-treated mice. Moreover, treatment with S-equol significantly up-regulated the expression of synaptic plasticity-related proteins (phospho synapsin, synapsin, postsynaptic density-95) and down-regulated the toll-like receptor 4/nuclear factor kappa B signaling pathway in the hippocampi of lipopolysaccharide-treated mice. These findings demonstrated that S-equol significantly alleviated the depressive-like behavior induced by acute systemic injection of LPS, and its antidepressant action was related to mediation of neuroinflammation via the TLR4/NF-κB signaling pathway, normalization of the monoamine neurotransmitter levels, reversal of tryptophan metabolism dysfunction, and enhancement of synaptic plasticity. The current study provides insight into the potential of S-equol in the prevention of depression.

A Systematic Review of the Effects of Equol (Soy Metabolite) on Breast Cancer.

Equol is a soy isoflavone metabolite that can be produced by intestinal bacteria. It is lipophilic and resembles natural oestrogens with an affinity to oestrogen receptors. This review is focused on how equol affects breast cancer, as evidenced by in vivo and in vitro studies. Equol is considered chemoprotective in specific endocrine-related pathologies, such as breast cancer, prostate cancer, cardiovascular diseases, and menopausal symptoms. In humans, not everyone can produce equol from gut metabolism. It is postulated that equol producers benefit more than non-equol producers for all the endocrine-related effects. Equol exists in two enantiomers of R-equol and S-equol. Earlier studies, however, did not specify which enantiomer was being used. This review considers equol's type and concentration variations, pathways affected, and its outcome in in vivo and in vitro studies.

Estrogen receptor beta signaling in CD8+ T cells boosts T cell receptor activation and antitumor immunity through a phosphotyrosine switch.

BackgroundThe non-overlapping functions of the two estrogen receptor subtypes, ERα (Estrogen Receptor α)and ERß (Estrogen Receptor ß), in tumor cells have been studied extensively. However, their counterparts in host cells is vastly underinterrogated. Even less is known about how ERα and ERß activities are regulated in a subtype-specific manner. We previously identified a phosphotyrosine residue (pY36) of human ERß that is important for tumor ERß to inhibit growth of breast cancer cells in vitro and in vivo. A role of this ERß phosphotyrosine switch in regulating host ERß remains unclear.Conventional gene editing was used to mutate the corresponding tyrosine residue of endogenous mouse ERß (Y55F) in mouse embryonic stem cells. The derived homozygous mutant Esr2Y55F/Y55F mouse strain and its wild-type (WT) counterpart were compared in various transplant tumor models for their ability to support tumor growth. In addition, flow cytometry-based immunophenotyping was carried out to assess antitumor immunity of WT and mutant hosts. Adoptive transfer of bone marrow and purified CD8+ T cells were performed to identify the host cell type that harbors ERß-dependent antitumor function. Furthermore, cell signaling assays were conducted to compare T cell receptor (TCR)-initiated signaling cascade in CD8+ T cells of WT and mutant mice. Lastly, the ERß-selective agonist S-equol was evaluated for its efficacy to boost immune checkpoint blockade (ICB)-based anticancer immunotherapy.Disabling the ERß-specific phosphotyrosine switch in tumor-bearing hosts exacerbates tumor growth. Further, a cell-autonomous ERß function was defined in CD8+ effector T cells. Mechanistically, TCR activation triggers ERß phosphorylation, which in turn augments the downstream TCR signaling cascade via a non-genomic action of ERß. S-equol facilitates TCR activation that stimulates the ERß phosphotyrosine switch and boosts anti-PD-1 (Programmed cell death protein 1) ICB immunotherapy.Our mouse genetic study clearly demonstrates a role of the ERß phosphotyrosine switch in regulating ERß-dependent antitumor immunity in CD8+ T cells. Our findings support the development of ERß agonists including S-equol in combination with ICB immunotherapy for cancer treatment.

Depletion of dietary phytoestrogens reduces hippocampal plasticity and contextual fear memory stability in adult male mouse.

Introduction: Phytoestrogens are non-steroidal estrogen analogues and are found primarily in soy products. They have received increasing attention as dietary supplements for estrogen deficiency and as modulators of endogenous estrogen functions, including cognition and emotion. In addition to modifying the levels of circulating sex hormones, phytoestrogens also exert direct effects on estrogen and androgen receptors in the brain and thus effectively modulate the neural circuit functions.Objective: The aim of this study was to investigate the long-term effects of low phytoestrogen intake (∼6 weeks) on the hippocampal plasticity and hippocampus-dependent memory formation in the adult C57BL/6 male mice.Methods and Results: In comparison to mice on a diet with normal phytoestrogen content, mice on low phytoestrogen diet showed a significant reduction in the phosphorylation of NR2B subunit, a molecular correlate of plasticity in the Schaffer collateral-CA1 synapse. We observed a profound decrease in long-term potentiation (LTP) in the ventral hippocampus, whereas no effect on plasticity was evident in its dorsal portion. Furthermore, we demonstrated that acute perfusion of slices with an estrogen analogue equol, an isoflovane metabolized from daidzein produced by the bacterial flora in the gut, was able to rescue the observed LTP deficit. Examining potential behavioral correlates of the plasticity attenuation, we found that mice on phytoestrogen-free diet display decreased contextual fear memory at remote but not at recent time points after training.Conclusions: Our data suggests that nutritional phytoestrogens have profound effects on the plasticity in the ventral hippocampus and ventral hippocampus-dependent memory.

Therapeutic Potential of Equol: A Comprehensive Review.

Equol (4',7-isoflavandiol), is a phytoestrogenic compound, which is synthesized from parent molecule diadzein by intestinal bacterial flora. It is among one of the most extensively researched molecule due to its high affinity towards estrogen receptors. Its enantiomeric form S-equol has been explored in the treatment of estrogen/androgen mediated diseases. Various therapeutic applications such as anti-cancer, cardioprotective, antidiabetic, antiosteoporosis, anti-ageing, and neuroprotective efficacy are attributed to it. This review explored major studies related to biochemistry and pharmacological applications of equol for human health.

Equol, a Blood-Brain Barrier Permeable Gut Microbial Metabolite of Dietary Isoflavone Daidzein, Exhibits Neuroprotective Effects against Neurotoxins Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells and Caenorhabditis elegans.

Emerging data support that plant food based isoflavones have ameliorating effects on a variety of neurodegenerative diseases including Parkinson's disease (PD). Our previous investigation revealed that dietary isoflavones including genistein (GEN), daidzein (DAI), and equol (EQL; a gut microbial metabolite of DAI) showed promising blood-brain barrier permeability and anti-neuroinflammatory activity in murine microglial BV2 cells. However, the neuroprotective effects of EQL against neurotoxins induced toxicity in PD related models remains unclear. Herein, EQL, along with GEN and DAI, were evaluated for their cytoprotective effect in a non-contact co-culture model with LPS-BV2-conditioned media and human neuroblastoma SH-SY5Y cells. In addition, their neuroprotective effects against PD related neurotoxins including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+) induced cytotoxicity were evaluated in SH-SY5Y cells. Furthermore, EQL was evaluated for its neuroprotective effects against MPP+ induced neurotoxicity using in vivo PD model including Caenorhabditis elegans lifespan assay. DAI (10 µM) and EQL (10 and 20 µM) showed cytoprotective effects by decreasing LPS-BV2-conditioned media induced cytotoxicity in SH-SY5Y cells by 29.2, 32.4 and 27.2%, respectively. EQL (10 and 20 µM) also showed neuroprotective effects by decreasing 6-OHDA and MPP+ induced cytotoxicity in SH-SY5Y cells by 30.6-34.5 and 17.9-18.9%, respectively. Additionally, data from the in vivo assay supported EQL's neuroprotective effect as it increases survival of C. elegans exposed to MPP+ from 72 to 108 h. Our findings support a growing body of evidence of the neuroprotective effects of dietary isoflavones and further studies are warranted to elucidate their mechanisms of action.

Selective Estrogen Receptor ß Agonists: a Therapeutic Approach for HIV-1 Associated Neurocognitive Disorders.

The persistence of HIV-1 associated neurocognitive disorders (HAND) in the post-cART era, afflicting between 40 and 70% of HIV-1 seropositive individuals, supports a critical need for the development of adjunctive therapeutic treatments. Selective estrogen receptor ß agonists, including S-Equol (SE), have been implicated as potential therapeutic targets for the treatment of neurocognitive disorders. In the present study, the therapeutic efficacy of 0.2 mg SE for the treatment of HAND was assessed to address two key questions in the HIV-1 transgenic (Tg) rat. First, does SE exhibit robust therapeutic efficacy when treatment is initiated relatively early (i.e., between 2 and 3 months of age) in the course of viral protein exposure? Second, does the therapeutic utility of SE generalize across multiple neurocognitive domains? Treatment with SE enhanced preattentive processes and stimulus-response learning to the level of controls in all (i.e., 100%) HIV-1 Tg animals. For sustained and selective attention, statistically significant effects were not observed in the overall analyses (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). However, given our a priori hypothesis, subsequent analyses were conducted, revealing enhanced sustained and selective attention, approximating controls, in a subset (i.e., 50%, n = 5 and 80%, n = 8, respectively) of HIV-1 Tg animals treated with SE. Thus, the therapeutic efficacy of SE is greater when treatment is initiated relatively early in the course of viral protein exposure and generalizes across neurocognitive domains, supporting an adjunctive therapeutic for HAND in the post-cART era. Graphical Abstract HIV-1 transgenic (Tg) and control animals were treated with either 0.2 mg S-Equol (SE) or placebo between 2 and 3 months of age (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). Neurocognitive assessments, tapping preattentive processes, stimulus response learning, sustained attention and selective attention, were conducted to evaluate the utility of SE as a therapeutic for HIV-1 associated neurocognitive disorders (HAND). Planned comparisons between HIV-1 Tg and control animals treated with placebo were utilized to establish a genotype effect, revealing prominent neurocognitive impairments (NCI) in the HIV-1 Tg rat across all domains. Furthermore, to establish the utility of SE, HIV-1 Tg animals treated with SE were compared to control animals treated with placebo. Treatment with 0.2 mg SE ameliorated NCI, to levels that were indistinguishable from controls, in at least a subset (i.e., 50-100%) of HIV-1 Tg animals. Thus, SE supports an efficacious, adjunctive therapeutic for HAND.

A Novel Mechanism of S-equol Action in Neurons and Astrocytes: The Possible Involvement of GPR30/GPER1.

S-equol is a major bacterial metabolite of the soy isoflavone daidzein. It is known to be a phytoestrogen that acts by binding to the nuclear estrogen receptors (ERs) that are expressed in various brain regions, including the cerebellum. However, the effects of S-equol on cerebellar development and function have not yet been extensively studied. In this study, the effects of S-equol were evaluated using a mouse primary cerebellar culture, Neuro-2A clonal cells, and an astrocyte-enriched culture. S-equol augmented the dendrite arborization of Purkinje cells induced by triiodothyronine (T3) and the neurite growth of Neuro-2A cell differentiation. Such augmentation was suppressed by G15, a selective G-protein coupled ER (GPR30) antagonist, and ICI 182,780, an antagonist for ERs in both cultures. On the other hand, in astrocytes, S-equol induced cell proliferation and cell migration with an increase in the phosphorylated extracellular-signal-regulated kinase 1/2 and F-actin rearrangements. Such effects were suppressed by G15, but not by ICI. These findings indicated that S-equol may enhanced cerebellar development by affecting both neurons and astrocytes through several signaling pathways, including GPR30 and ERs. We here report a novel mechanism of S-equol in cerebellar development that may provide a novel possibility to use S-equol supplementation during development.

Equol Pretreatment Protection of SH-SY5Y Cells against Aß (25-35)-Induced Cytotoxicity and Cell-Cycle Reentry via Sustaining Estrogen Receptor Alpha Expression.

ß-amyloid formation in the brain is one of the characteristics of Alzheimer's disease. Exposure to this peptide may result in reentry into the cell cycle leading to cell death. The phytoestrogen equol has similar biological effects as estrogen without the side effects. This study investigated the possible mechanism of the neuron cell-protecting effect of equol during treatment with Aß. SH-SY5Y neuroblastoma cells were treated with either 1 µM S-equol or 10 nM 17ß-estradiol for 24 h prior to 1 µM Aß (25-35) exposure. After 24 h exposure to Aß (25-35), a significant reduction in cell survival and a reentry into the cell cycle process accompanied by increased levels of cyclin D1 were observed. The expressions of estrogen receptor alpha (ERα) and its coactivator, steroid receptor coactivator-1 (SRC-1), were also significantly downregulated by Aß (25-35) in parallel with activated extracellular signal-regulated kinase (ERK)1/2. However, pretreatment of cells with S-equol or 17ß-estradiol reversed these effects. Treatment with the ER antagonist, ICI-182,780 (1 µM), completely blocked the effects of S-equol and 17ß-estradiol on cell viability, ERα, and ERK1/2 after Aß (25-35) exposure. These data suggest that S-equol possesses a neuroprotective potential as it effectively antagonizes Aß (25-35)-induced cell cytotoxicity and prevents cell cycle reentry in SH-SY5Y cells. The mechanism underlying S-equol neuroprotection might involve ERα-mediated pathways.

S-equol inhibits proliferation and promotes apoptosis of human breast cancer MCF-7 cells via regulating miR-10a-5p and PI3K/AKT pathway.

S-equol is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora, which has strong anti-cancer activity. Based on this, the purpose of this study was to investigate the anti-breast cancer mechanism of S-equol. We examined the effects of S-equol on proliferation and apoptosis of MCF-7 cells by cell counting kit-8 assay and flow cytometry. Screening for microRNAs and predicting their target genes using the starBase and Targetscan website, respectively. Protein expression was detected by Western blot. The microRNA level were quantified by real-time PCR. The results showed that S-equol inhibited the proliferation of breast cancer MCF-7 cells in a time- and dose-dependent manner and promoted apoptosis of MCF-7 cells. The expression of miR-10a-5p was significantly decreased in breast cancer tissues and breast cancer cell lines, and the expression of miR-10a-5p was negatively correlated with the proliferation of MCF-7 cells. Luciferase reporter experiments demonstrated that miR-10a-5p directly targets PIK3CA 3'UTR to function. It was further found that S-equol exerts an anti-breast cancer effect by up-regulating miR-10a-5p and inhibiting the PI3K/AKT pathway. Our study revealed the mechanism of S-equol against breast cancer, and miR-10a-5p may be a potential target for the treatment of breast cancer.