Equol inhibits proliferation of human gastric carcinoma cells via modulating Akt pathway

AIM: To investigate the anti-tumor effects of equol in gastric cancer cells and the underlying molecular mechanisms.METHODS: MGC-803 cells were employed for in vitro experiments in this study. Cells were treated with control(vehicle,0.1% DMSO) or equol under specified dose titration or time courses. Cell viability was examined by MTS assay,and the levels of Ki67 were determined by q PCR and immunofluorescent assay. Changes in cell cycle distribution and apoptosis rate were detected by flow cytometry. The m RNA expression of cyclin E1 and P21WAF1 was determined by q PCR. The protein levels of cell cycle regulators,PARP and Caspase-3 cleavage,and the phosphorylation of Akt were examined by Western blot. In addition,to characterize the role of elevated Akt activation in the anti-tumor effect exerted by equol,Ly294002,a PI3K/AKT pathway inhibitor,was used to pretreat MGC-803 cells. RESULTS: Equol(5,10,20,40,or 80 μmol/L) inhibited viability of MGC-803 cells in a dose- and timedependent manner after treatment for 24,36,or 48 h(P < 0.05 for all). Equol also decreased the m RNA(P <0.05 for 12 and 24 h treatment) and protein levels of Ki67. Equol treatment significantly induced G0/G1 cell cycle arrest(P < 0.05),with the percentages of G0/G1 cells of 32.23% ± 3.62%,36.31% ± 0.24%,45.58% ± 2.29%,and 65.10% ± 2.04% for equol(0,10,20,or 30 μmol/L) treatment,respectively,accompanied by a significant decrease of CDK2/4(P < 0.05 for 24 and 48 h treatment) and Cyclin D1/Cyclin E1(P < 0.05),and an increased level of P21WAF1(P < 0.05). A marked increase of apoptosis was observed,with the percentages of apoptotic cells of 5.01% ± 0.91%,14.57% ± 0.99%,37.40% ± 0.58%,and 38.46% ± 2.01% for equol(0,5,10,or 20 μmol/L) treatment,respectively,accompanied by increased levels of cleaved PARP and caspase-3. In addition,we found that equol treatment increased P-Akt(Ser473 and Thr308) at 12 and 24 h compared to vehicle-treated control; longer treatment for 48 h decreased P-Akt(Ser473 and Thr308). P-Akt at Thr450,however,was decreased by equol treatment at all time points examined(P < 0.05 for all). Moreover,Akt inhibition by Ly294002 could not prevent but led to enhanced G0/G1 arrest and apoptosis. CONCLUSION: Equol inhibits MGC-803 cells proliferation by induction of G0/G1 arrest and apoptosis. Its anti-cancer effects are likely mediated by dephosphorylation of Akt at Thr450.

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3β-hydroxysteroid de- hydrogenase （3β-HSD） and 17β-hydroxysteroid dehydrogenase 3 （17β-HSD3） in human and rat testis microsomes. These enzymes （3β-HSD and 17β-HSD3）, along with two others （cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17α-hydroxylase/17-20 lyase）, catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3β-HSD activity （0.2 μmol L^-1 pregnenolone） with half-maximal inhibition or a half-maximal inhibitory concentration （IC50） of 87 ± 15 （human） and 636 ± 155 nmol L^-1 （rat）. Genistein＇s mode of action on 3β-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD＋. There was no difference in genistein＇s potency of 3β-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3β-HSD, genistein had lesser effects on human and rat 17β-HSD3 （0.1 μmol L^-1 androstenedione）, with an IC50 〉 100μmol L^-1. On the other hand, equol only inhibited human 3β-HSD by 42%, and had no effect on 3β-HSD and 17β-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3β- HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

<i>In Vitro</i>Effect of Soy Isoflavone and Equol on Soluble CD40L Release Stimulated by Ristocetin in Platelets from Postmenopause Women

The inhibition of specific flavonoid on the in vitro platelet aggregation induced by collagen, arachidonic acid and thromboxane A2 (TxA2) agonist, seems to be related mostly to their ability to compete for binding to the TxA2 receptor (TP). The aim of this study was to analyze the effect of soy isoflavone and equol in terms of inhibiting the platelet aggregation and sCD40L release stimulated by ristocetin, an in vitro-activator of glycoprotein Ib/IX/V, in platelets from postmenopausal women. When platelets were stimulated by 0.75 mg/ml ristocetin, equol (10 μM) exhibited a greater inhibitory activity on platelet aggregation (~68%) than genistein or daidzein. The effect of equol was dependent on the concentration of platelet aggregation agonist. In the presence of ristocetin (0.75 mg/ml, 1.125 mg/ml and 1.5 mg/ml), the inhibitory effect of 10 μM equol was 68% ± 5%, 54% ± 4% and 31% ± 5%, respectively. Equol (10 μM) was a potent inhibitor (~35%) of sCD40L release when stimulated with 1.5 mg/ml ristocetin. However, no significant differences were noted in platelets incubated in the presence of genistein or daidzein and stimulated by ristocetin. On the other hand, SQ29548, a high TP antagonist, also inhibited the sCD40L release stimulated by ristocetin. Finally, 10 μM of genistein, daidzein or equol did not significantly affect the thromboxane B2 production when platelets were incubated with 1.5 mg/ml ristocetin. The relevance of this study was to find that equol exhibits a potent activity by inhibiting ristocetin-induced sCD40L release, suggesting that soy isoflavone has important biological effects on the hemostatic system. However, clinical trials will be necessary to assess the effect of equol on platelet and their impact on inflammatory markers.

Severe &Moderate BPH Symptoms in Mid-Aged Men Improve with Isoflavonoid-Equol Treatment: Pilot Intervention Study

Benign prostatic hyperplasia (BPH) is the pathological cellular progression of glandular proliferation associated with aging. Current available treatment options for BPH have limitations and various adverse effects. Equol is a polyphenolic/isoflavonoid molecule derived from intestinal metabolism, dairy and dietary plant sources. It has the unique characteristic to bind specifically 5α-dihydrotestosterone (5α-DHT) by sequestering 5α-DHT from the androgen receptor, thus decreasing androgen hormone actions to improve prostate health by acting as a selective androgen modulator (SAM). It also has affinity for estrogen related receptor gamma (ERR-γ) and estrogen receptor beta (ER-β) within the prostate that is known to improve male health via selective estrogen receptor modulatory (SERM) activities to decrease inflammation, cellular proliferation and carcinogenesis. We investigated the possible clinical efficacy of equol on the symptoms associated with benign prostatic hyperplasia (BPH) in this study. Materials and Methods: We performed a pilot intervention study evaluating the effects of low dose oral equol supplement (6 mg, twice a day with meals) for 4 weeks in a total of 18 men (49 - 60 years old) with moderate or severe BPH. Subjects included in the study: gave informed consent, underwent a physical examination and verified their BPH symptoms as measured by the International Prostate Symptom Scores (IPSS) and then were assigned to the moderate or severe BPH groups based upon their total IPSS index. All adverse events were reported. The primary efficacy measure was the IPSS parameters comparing baseline to 2 and 4 week IPSS indices. Blood samples were collected at the baseline and 4th week visits that served as secondary efficacy parameters that included testosterone, 5α-DHT and general blood chemistries along with cardiac and hepatic function panels. Results: Low dose equol positively improved moderate to severe BPH symptoms according to the IPSS indices. In moderately symptomatic men (n = 10) 5 out of 7 of the IPSS parameters significantly improved by 4 weeks of equol treatment. In severely symptomatic men (n = 8) all 7 of the IPSS parameters significantly improved with 4 weeks of equol treatment. There were no significant changes in androgen levels, general blood chemistries or cardiac and hepatic function parameters. Although, 5α-DHT levels declined by 21% in severely symptomatic men (from baseline vs. 4 week values). Conclusion: These findings suggest that equol may provide a well tolerated and rapid beneficial therapy for BPH that can be used alone or in combination with current pharmaceutical therapies. The beneficial clinical efficacy of equol observed in this study may be due to the multiple positive biological actions that are not present in current pharmaceutical treatments.

Review: Anti-Oxidant and Anti-Aging Properties of Equol in Prostate Health (BPH)

Benign prostatic hyperplasia (BPH) is the pathological cellular progression of glandular proliferation associated with aging. The primary changes in prostate disorders are mediated by the conversion of the principle androgen, testosterone, to its more potent metabolite, 5α-dihydrotestosterone (5α-DHT). However, recent evidence suggests that estrogen hormonal actions via estrogen receptor subtypes also play an important role in BPH. Current pharmaceutical options for BPH have advantages, limitations and adverse effects. Complementary and Alternative Medicine (CAM) treatments for BPH include botanicals such as polyphenols and isoflavones. Equol is a polyphenolic/isoflavonoid molecule derived from intestinal metabolism, dairy and dietary plant sources. Equol has potent anti-oxidant and anti-aging properties to decrease prostatic irritation and potentially neoplastic growth. It has the unique characteristic to bind specifically 5α-DHT by sequestering 5α-DHT from the androgen receptor (AR), thus decreasing androgen hormone actions to improve prostate health by acting as a selective androgen modulator (SAM). It also has affinity for estrogen related receptor gamma (ERR-γ) and estrogen receptor beta (ER-β) within the prostate that is known to improve male health via selective estrogen receptor modulatory (SERM) activities to decrease inflammation, cellular proliferation and carcinogenesis. The possible clinical efficacy of equol on the symptoms associated with BPH is presented and the reviewed findings suggest that equol may provide a well-tolerated and rapid beneficial therapy for BPH that can be used alone or in combination with current pharmaceutical therapies. The beneficial clinical efficacy of equol observed may be due to the multiple positive biological actions that are not present in current pharmaceutical treatments.

Correlation of Equol (7-Hydroxy-3-(4-Hydroxyphenyl)Chroman) in Woman Urine with the Symptoms of Menopause

Objective: In order to identify the correlation between equol excreted through human urine and the symptom of menopause. Methods: The method used is cross sectional study examined to 99 postmenopausal women fulfilling the inclusion criteria from February 2014 to July 2014. This research was taken in the Endocrinology Clinic, Department of Obstetrics and Gynecology, Hasan Sadikin Hospital and Unpad’s Pharmacokinetic Laboratory. All objects were interviewed using menopause rating scale (MRS) questionnaire. Their urine samples were analyzed using high performance liquid chromatography (HPLC). Then the results of questionnaire and HPLC test were evaluated. Results: The result is 97 objects (98%) detected having menopausal symptoms, produced equol. There is significant correlation between the level of equol in post-menopausal women that showed correlation (-0.71) which mean the higher the equol level, the lower the MRS score (p < 0.001) that mean the symptom is milder. Conclusion: Equol level in menopause women urine is a good predictor to identify the level of menopause symptoms.

The Development of S-Equol Diastereoisomer Specific ELISA

Equol is a metabolite of soybean isoflavone, daidzein, and many health benefits are expected. Endogenous equol in urine is S-equol and mostly exists as glucuronate or sulfate conjugate. In this study we preliminary established the simple enzyme-linked immunosorbent assay (ELISA) without deconjugation, then developed the S-equol specific ELISA involves deconjugation showing high stereospecificity to S-equol without using stereospecific antibody. For the simple ELISA, we used a polyclonal antibody that targets the regions not influenced by inhibition by conjugation of glucuronate and sulfate and achieved the correlation coefficient;r = 0.975, but the value was 30 % lower than high performance liquid chromatography (HPLC). Developing upon this we invented the specific ELISA established from S-equol homogeneous combination for the standard and enzyme-labeled antigen to enhance stereospecificity. The correlation with HPLC was favorable: r = 0.986, y = 0.996x – 6. Compared to the previous method using (R,S)-equol combination, cross-reactivity with R-equol was reduced from 65 to 13 %, and that with daidzein from 0.31% to 0.08%, markedly increased in the specificity. This study is expected to be applied for both simple clinical researches, and stereospecific immunoassays in which specific antibody preparation is difficult.

雌马酚(S-equol, Eq)对慢性不可预知温和刺激小鼠抑郁样行为的改善作用

为探究雌马酚(S-equol, Eq)对慢性不可预知温和刺激抑郁模型(Chronic Unpredictable Mild Stress, CUMS)小鼠的改善作用,试验设空白组(Con)、模型组(CUMS)、雌马酚低剂量组(Eq-L,10 mg·kg-1)、雌马酚中剂量组(Eq-M,20 mg·kg-1)和雌马酚高剂量组(Eq-H,40 mg·kg-1),在预防给药14 d后,对模型组和雌马酚组进行CUMS造模刺激,造模42 d之后监测体重,依次进行空场、糖水偏爱、悬尾、强迫游泳和新奇抑制摄食5种行为学试验,并检测血清中皮质酮(CORT)水平。行为学结果显示:与空白组相比,CUMS模型组小鼠的自主活动能力未受到显著影响,体重和糖水偏爱指数显著下降,悬尾、强迫游泳中不动时间和新奇抑制摄食潜伏期显著延长;与模型组相比,雌马酚没有影响小鼠的自主活动,显著提高了小鼠的糖水偏爱指数,缩短了悬尾、强迫游泳不动时间和新奇抑制摄食潜伏期。此外,CUMS模型组小鼠血清中CORT显著上调,雌马酚给药显著改善上述异常指标。该结果表明雌马酚可以改善CUMS小鼠抑郁样行为,并通过抑制下丘脑-垂体-肾上腺轴的过度亢进发挥改善抑郁的作用。

S-Equol调节SREBP通路和PPARγ改善大鼠2型糖尿病合并非酒精性脂肪肝的研究

目的分析S-雌马酚(S-Equol)对2型糖尿病(type 2 diabetes mellitus,T2DM)合并非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)大鼠模型糖脂代谢的影响。方法选取6周龄雄性SD大鼠50只,适应性饲养1周,随机分为对照组(n=10)和建模组(n=40)。给予建模组大鼠高脂高糖饮食联合腹腔注射链尿佐菌素(streptozocin,STZ)构建2型糖尿病模型。空腹血糖(fasting blood-glucose,FBG)>11.1 mmol/L的大鼠随机分为模型组(n=9),S-Equol低剂量组(20 mg·kg^(-1)·d^(-1),n=9)、中剂量组(40 mg·kg^(-1)·d^(-1),n=8)和高剂量组(80 mg·kg^(-1)·d^(-1),n=8)。干预满12周后,检测各组大鼠血清总胆固醇(TC)、甘油三酯(TG)、血糖(FBG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、总胆红素(TBIL)、直接胆红素(DBIL)、总蛋白(TP)、白蛋白(Alb)、球蛋白(Glb)。采用ELISA法检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)以及肝脏白介素1β(IL-1β)、白介素6(IL-6)和肿瘤坏死因子-α(TNF-α)。采用Western blot和RT-qPCR法检测肝组织中与脂质代谢相关的过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ),乙酰辅酶a羧化酶(acetyl-CoA carboxylase,ACC),固醇调节元件结合蛋白-1(sterol regulatory element binding protein-1,SREBP-1)和脂肪酸合成酶(fatty acid synthetase,Fasn)表达水平。结果与模型组比较,S-Equol低剂量组胰岛素抵抗水平显著降低(P<0.05),S-Equol中剂量组胰岛素分泌水平显著升高(P<0.05),S-Equol低、高剂量组FBG水平显著降低(P<0.05),S-Equol低剂量组肝指数、血清白球比(A/G)和LDL显著降低(P<0.05),S-Equol中剂量组血清DBIL和ALT显著降低(P<0.05);S-Equol低、中、高剂量组肝脏组织学形态均改善,肝脏脂质沉积面积显著降低(P<0.05),S-Equol中、高剂量组肝组织炎症水平显著降低(P<0.05);Western blot结果显示,中、高剂量的S-Equol干预使大鼠肝组织中PPARγ和ACC的表达显著下调(P<0.05);RT-qPCR结果显示S-Equol的干预使大鼠肝组织中ACC、SREBP-1、Fasn和PPARγ转录水平显著降低(P<0.05)。结论S-Equol能降低T2DM合并NAFLD大鼠的肝脏脂质沉积、脂质代谢相关分子表达和炎症水平,其机制可能与调节SREBP通路和PPARγ分子表达有关。

S-Equol对高糖培养HepG2细胞胰岛素敏感性及IRS-1表达的影响

目的:研究S型雌马酚(S-Equol,S-Eq)对高糖培养HepG2人肝癌细胞株胰岛素敏感性和胰岛素受体底物(insulin receptor substrate,IRS)-1表达的影响并探讨其可能的分子机制。方法:高糖培养HepG2细胞,1、10、100μM S-Eq处理细胞后,MTT法检测细胞活力,硫酸蒽酮比色法检测胰岛素刺激细胞糖原合成量,Realtime PCR和Western blot法分别检测IRS-1 mRNA及蛋白表达变化。结果:S-Eq对HepG2细胞活力无明显影响,但显著改善高糖培养条件下HepG2细胞胰岛素敏感性,其中10μM S-Eq+H组胰岛素刺激后细胞糖原合成量上升最为显著(P<0.01),同时发现,S-Eq能显著上调IRS-1 mRNA和蛋白表达量。结论:S-Eq可能通过调控IRS-1的表达,增强高糖培养HepG2细胞胰岛素敏感性,这可能是S-Eq发挥其抗糖尿病作用的重要理论依据。

臭豆腐中雌马酚的测定及其影响因素

目的:研究臭豆腐中雌马酚的含量及其影响因素,以期为选择良好食物性来源的雌马酚提供理论依据。方法:采用反相-高效液相色谱法(RP-HPLC)检测我国市场上常见的4个品牌臭豆腐中雌马酚的含量(对比其它8种发酵豆制品)及其与大豆异黄酮含量和理化指标等因素的Spearnam相关性,并对卤水及其浸泡和油炸时长沙HSJD臭豆腐中雌马酚的含量进行分析。结果:4种臭豆腐中均含雌马酚,其它发酵豆制品均不含雌马酚。臭豆腐中雌马酚的含量在11.93~24.33μg/g干物质范围,其中,长沙HSJD臭豆腐中的含量最高(24.33μg/g干物质)。雌马酚含量与大豆苷元、染料木苷元含量呈显著正相关关系(P<0.05),相关系数分别为0.335,0.411;与氯化物、氨基酸态氮、总酸含量呈极显著负相关关系(P<0.01),相关系数分别为-0.463,-0.506,-0.674;与p H值呈极显著正相关关系(P<0.01),相关系数为0.539。添加豆腐脑发酵一年(STB1)和发酵二年(STB2)的长沙HSJD臭豆腐卤水中均含雌马酚,含量分别为168.31,309.62μg/g干物质,而未加豆腐脑的卤水(STB3)中不含雌马酚;臭豆腐坯ST5、ST6分别浸泡于STB1、STB2卤水,其雌马酚含量分别为24.33,38.31μg/g干物质,浸泡凉白开水和STB3卤水的臭豆腐坯均未检出雌马酚;长沙HSJD臭豆腐经油炸后,雌马酚含量显著下降,ST5中雌马酚含量由24.33μg/g干物质降到9.84μg/g干物质,ST6由38.31μg/g干物质降到10.27μg/g干物质。结论:臭豆腐为雌马酚良好来源的食物,其浸泡卤水和油炸工艺影响臭豆腐中雌马酚的含量。

雌马酚干预改善油酸钠诱导的HepG2细胞脂肪变性

目的 观察雌马酚(equol, Eq)对油酸钠(sodium oleate, NaOL)诱导的HepG2细胞脂肪变性的影响,并探讨其作用机制。方法 体外培养HepG2细胞,以不同浓度(0、0.12、0.24、0.36、0.48、0.60、0.72 mmol/L)的NaOL处理48 h诱导细胞脂肪变性。油红O染色观察细胞内脂滴蓄积情况,并测定细胞TG含量,采用CCK-8法确定诱导肝细胞脂肪变性的适宜NaOL处理浓度和Eq干预的浓度范围。将细胞分为对照组,NaOL组,NaOL+Eq低、中、高浓度组(10-7、10-6、10-5mol/L),NaOL+雌二醇干预组(estradiol, E2, 10-7mol/L)。Eq干预48 h后检测细胞TG,油红O染色观察细胞内脂滴蓄积情况,qRT-PCR和Western blot检测细胞SREBP-1c、FAS、PPARα及CPT-1A mRNA和蛋白的表达。结果 NaOL浓度为0.24 mmol/L时,对HepG2细胞活力无明显影响,同时细胞内TG含量明显增加,出现大量脂滴聚集;与NaOL组相比,Eq或E2干预可以降低NaOL诱导的HepG2细胞内脂滴蓄积,显著减少细胞内TG含量(P<0.05),显著降低细胞SREBP-1c和FAS的mRNA和蛋白表达水平(P<0.05),显著升高PPARα和CPT-1A的mRNA和蛋白表达水平(P<0.05)。结论 Eq可有效改善NaOL诱导的HepG2细胞脂肪变性,可能与Eq降低SREBP-1c和FAS表达,同时升高PPARα和CPT-1A表达有关。

雌马酚对高糖高脂诱导的成骨细胞线粒体损伤的保护作用

目的 观察雌马酚(equol, Eq)干预对高糖高脂诱导的成骨细胞的影响,并探讨其作用机制。方法 体外培养ROS1728成骨细胞,以不同浓度的Eq、棕榈酸钠(sodium palmitate, PA)和葡萄糖(glucose, GLU)处理48 h,通过HE染色观察细胞核凋亡表现及CCK-8法检测细胞存活率确定GLU、PA及Eq干预的适宜浓度。将细胞分为对照(Control)组(GLU,5.5 mmol/L)、模型(Model)组(GLU,30 mmol/L+PA,250μmol/L)、LEq组(Model+1×10^(-8)mol/L Eq)、MEq组(Model+1×10^(-7)mol/L Eq)、HEq组(Model+1×10^(-6)mol/L Eq)。CCK8检测细胞存活率,流式细胞术检测细胞凋亡率,Hoechst33342染色和HE染色观察细胞核凋亡表现,透射电镜观察线粒体结构,Mito-Tracker Red CMXRos(线粒体红色荧光探针)染色检测细胞线粒体膜电位(mitochondrial membrane potential, MMP)变化情况,Western blot检测Sirt1、PGC-1α、Bcl-2、Bax和Cleaved caspase-3蛋白表达。结果 与对照组相比,当GLU和PA浓度分别为30 mmol/L和250μmol/L时细胞存活率显著降低(P<0.05),凋亡率升高,确定为诱导成骨细胞高糖高脂环境的适宜浓度;与模型组相比,Eq干预后细胞存活率显著升高、凋亡率显著降低(P<0.05),线粒体结构受损明显改善、MMP显著升高(P<0.05),同时ROS显著减少(P<0.05);细胞Sirt1、PGC-1α、Bcl-2表达水平显著升高,Bax、Cleaved caspase-3表达水平显著降低(P<0.05)。结论 Eq干预可有效减少高糖高脂诱导的成骨细胞线粒体损伤、氧化应激和细胞凋亡,其部分机制可能与Eq促进Sirt1、PGC-1α表达有关。

进料浓度对模拟移动床雌马酚对映体分离效能的影响

通过调节模拟移动床(SMB)进料口处样品初始进样浓度,可以有效提高目标产物的单位产量。以分离雌马酚对映体作为研究对象,在一定的纯度约束条件下,同时对产品纯度和单位产量进行多目标优化。基于多目标优化所获得非劣解,比较和分析了5个进样浓度对SMB生产性能的影响。

雌马酚通过PI3K/AKT通路对高糖高脂环境下成骨细胞凋亡及增殖的影响

目的探讨雌马酚(equol,Eq)对高糖高脂环境下成骨细胞增殖和凋亡的影响及其可能机制。方法建立ROS17/2.8成骨细胞高糖高脂损伤模型,设立对照组(C组)、模型组(M组)、不同浓度梯度Eq干预组(Eq^(-7)组:1×10^(-7)mol/L Eq,Eq^(-6)组:1×10^(-6)mol/L Eq,Eq^(-5)组:1×10^(-5)mol/L Eq)。药物处理24 h后,采用CCK-8、HE染色检测成骨细胞活性及形态结构变化,流式细胞术检测细胞凋亡及周期改变,Western blot检测细胞PI3K/AKT通路磷酸化程度及相关蛋白表达变化,并联合PI3K抑制剂验证PI3K/AKT通路在其中的作用。结果与C组比较,M组成骨细胞存活率降低、细胞抑制率增加(P<0.05),细胞形态结构明显损伤,细胞凋亡率增加(P<0.05),细胞周期G1期比例增加、S期比例降低(P<0.05),PI3K/AKT通路磷酸化程度降低、Cyclin D1、Bcl-2表达减少、Bax、cleaved caspase-3表达增加(P<0.05);与M组相比,各Eq干预组细胞存活率增加、细胞抑制率降低(P<0.05),细胞形态结构损伤减轻,细胞凋亡减少(P<0.05),细胞周期G1期比例降低、S期比例增加(P<0.05),PI3K/AKT通路磷酸化程度提高、Cyclin D1、Bcl-2表达增加、Bax、cleaved caspase-3表达减少(P<0.05),且Eq的干预效应随着浓度升高而增强;联合PI3K抑制剂后,Eq的上述效应显著降低。结论Eq可通过激活PI3K/AKT通路减少高糖高脂环境下成骨细胞凋亡、促进成骨细胞增殖。

雌马酚通过调控自噬干预棕榈酸诱导的L6细胞肌管萎缩

目的观察雌马酚(equol,Eq)干预对棕榈酸(palmitic acid,PA)诱导的L6大鼠成肌细胞肌管萎缩的影响,并探讨其机制。方法体外培养L6细胞,以不同浓度的Eq、PA处理24 h,通过CCK-8法检测相对细胞活力确定Eq、PA干预的适宜浓度。将细胞分为对照组、模型组(PA:0.25 mmol/L)、干预组(PA:0.25 mmol/L+Eq:1μmol/L)和自噬抑制剂组(PA:0.25 mmol/L+Eq:1μmol/L+3-MA:1 mmol/L)。干预24 h后检测各组细胞葡萄糖摄取能力;HE染色观察细胞肌管生长情况;透射电镜观察细胞自噬小体数量;激光共聚焦显微镜观察经线粒体荧光探针标记的线粒体形态;Western blot检测MyHC、p62蛋白、LC3蛋白、MFN2和DRP1表达水平。结果干预24 h后,与对照组比较,模型组L6细胞葡萄糖消耗明显减低(P<0.05),肌管直径明显减小(P<0.05),自噬小体数量减少,线粒体裂变增加,MyHC、MFN2蛋白表达水平降低(P<0.05),而p62、DRP1表达水平及LC3-Ⅰ/LC3-Ⅱ比值增加(P<0.05);与模型组相比,干预组L6细胞葡萄糖消耗明显增加(P<0.05),肌管直径明显增大(P<0.05),自噬小体数量增加,线粒体裂变减少,MyHC、MFN2蛋白表达水平升高(P<0.05),而p62、DRP1表达水平及LC3-Ⅰ/LC3-Ⅱ比值降低(P<0.05);Eq对PA诱导的L6细胞葡萄糖消耗,肌管直径,自噬小体数量,线粒体裂变,MyHC、p62、MFN2、DRP1蛋白表达水平及LC3-Ⅰ/LC3-Ⅱ比值的影响可被3-MA有效抑制。结论Eq可有效改善PA诱导的L6细胞肌管萎缩,其部分机制与Eq通过促进自噬,减轻线粒体裂变有关。

雌马酚对绝经后非酒精性脂肪肝病大鼠的骨保护作用

目的观察雌马酚(equol,Eq)对绝经后非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)大鼠的骨保护效应,并探讨其作用机制。方法选取6周龄雌性Sprague Dawley(SD)大鼠48只,随机分为对照组、模型组、低剂量Eq组[20 mg/(kg·d)]、中剂量Eq组[40 mg/(kg·d)]、高剂量Eq组[80 mg/(kg·d)]和雌二醇干预组[estradiol,E2,0.25 mg/(kg·d)],每组8只,对照组给予假手术及正常饮食,其余各组给予卵巢切除术(OVX)合并高脂饮食(HFD)。16周后测量大鼠体质量、体长,计算BMI和Lee’s指数,检测各组大鼠的骨密度和骨微结构变化;麻醉后处死大鼠并取材,检测大鼠血清雌激素、TNF-α和IL-6水平,肝脏和股骨组织学检查观察形态学变化;采用Western blot和qRT-PCR检测肝和骨组织中胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)表达水平。结果模型组大鼠BMI和Lee’s指数显著升高(P<0.05),肝组织损伤明显,雌激素和骨密度水平较对照组显著下降(P<0.05),骨显微结构破坏严重,提示绝经后NAFLD合并明显骨损伤;Eq和E2干预能显著升高大鼠血清雌激素和骨密度水平(P<0.05),改善骨显微结构,降低BMI、Lee’s指数和血清TNF-α、IL-6水平(P<0.05),减轻肝和骨组织脂质累积,升高肝和骨组织IGF-1蛋白和mRNA表达水平(P<0.05)。结论Eq能改善绝经后NAFLD大鼠的骨损伤,具有骨保护作用,其部分机制可能与Eq促进IGF-1表达和减轻炎症反应有关。

雌马醇对映体的高效液相色谱分离

目的:考察色谱条件对 equol 对映体手性分离的影响,建立反相条件下手性流动相添加剂法色谱分离方法。方法:采用高效液相色谱手性流动相法,固定相为 ODS 柱(250mm×4.6mm,5μm),流动相为环糊精水溶液与有机改性剂(甲醇、乙醇、异丙醇、乙腈)的混合溶液,流速1.0mL·min^(-1),检测波长254nm,柱温为室温。结果:首次用β-环糊精作为手性流动相添加剂成功拆分了 equol 对映体,并确立最佳分离条件:30mmol·L^(-1)β-CD 水溶液(pH 6.4)-乙腈(95:5),分离因子(α)可达1.21。结论:本方法简便易行,操作成本低,可直接放大到制备分离。

雌马酚对去卵巢后大鼠骨质疏松症的影响

目的观察雌马酚对去卵巢后大鼠骨质疏松症的作用。方法雌性SD大鼠去卵巢以建立骨质疏松症模型。健康3月龄雌性SD大鼠分为5组:假手术组(sham)、去卵巢组(ovarietomized,OVX)、OVX+雌马酚高剂量组[equol,Eq,0.5 mg/(kg·d)]、OVX+雌马酚低剂量组[equol,Eq,0.25 mg/(kg·d)]、OVX+雌二醇阳性对照组(estradiol,E2)。去卵巢8周后,按以上分组继续给药8周后取右侧股骨检测骨密度(bone mineral density,BMD),眼部取血检测血钙、血磷和骨钙素(bone gla protein,BGP)含量;ELISA检测血清雌激素(estrogen,ES)、成骨细胞护骨素(osteoprotegerin,OPG),破骨细胞分化因子(receptor activator of nuclear factor kappa B ligand,RANKL)水平;qRT-PCR法检测OPG和RANKL mRNA的表达。结果 OVX组与sham组比较,ES值、BMD和血钙水平显著下降(P<0.05),分别为sham组的78.1%、84.4%和83.2%。同时,OVX组大鼠血磷和BGP含量明显升高(P<0.05),表明去卵巢后骨质疏松症模型造模成功;OVX组大鼠与sham组比较,OPG蛋白和OPG mRNA表达量显著下降,RANKL蛋白和RANKL mRNA表达量明显升高(P<0.05),而Eq和E2组处理后能显著增加OPG蛋白和OPG mRNA表达,下调RANKL蛋白和RANKL mRNA表达,同时提高OVX大鼠的BMD和血钙水平,降低血磷和BGP含量(P<0.05)。结论雌马酚能有效改善去卵巢后大鼠骨质疏松症,可能与其能上调OPG而抑制RANKL分泌有关。

雌马酚对PC12细胞缺氧/复氧损伤的保护作用及其机制（英文）

目的探讨雌马酚对PC12细胞缺氧/复氧损伤的保护作用及机制。方法以PC12细胞为研究对象,构建体外神经元缺氧/复氧损伤模型,用雌马酚进行干预。MTT法检测细胞活力,比色法测定细胞培养上清液中乳酸脱氢酶(LDH)活性和丙二醛(MDA)含量,免疫细胞化学法检测活性氧(ROS)含量,Western blotting检测缺氧/复氧损伤的PC12细胞内NADPH氧化酶2(gp91phox)和磷酸化Src(p-Src)激酶的表达。结果雌马酚可有效减轻缺氧/复氧引起的PC12细胞损伤,降低缺氧/复氧损伤细胞培养上清液中LDH的活性和MDA的含量,明显抑制ROS的生成及gp91^(phox)和p-Src的表达。结论雌马酚通过抑制p-Src激酶和gp91^(phox)表达,减少ROS生成,从而对PC12细胞缺氧/复氧损伤产生保护作用。