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ObjectiveTo investigate the protective effect and regulatory mechanism of berberine （BBR） against the senescence of ovarian granulosa cells. MethodA cell senescence model in the human ovarian granulosa-like tumor （KGN） cell line was induced by H2O2. A control group， a model group， and high-dose （1 μmol·L-1） and low-dose （0.5 μmol·L-1） BBR groups were set up. The cells in the model group and the BBR groups were incubated with 10 μmol·L-1 H2O2 for 40 min. The effect of BBR on KGN cell proliferation was detected by cell counting kit-8 （CCK-8） assay. The effect of BBR on the senescence of KGN cells was detected by β-galactosidase staining. The effects of BBR on the apoptosis and ROS content of KGN cells were detected by flow cytometry. The effects of BBR on the mRNA expression of B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific protease-3 （Caspase-3）， forkhead transcription factor O1 （FoxO1）， and catalase （CAT） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the effects of BBR on protein expression of silent information regulator1 （SIRT1）， superoxide dismutase 2 （SOD2）， c-Jun N-terminal kinase （JNK）， FoxO1， autophagy-associated protein microtubule-associated protein light chain 3Ⅱ （LC3BⅡ）， mammalian ortholog of yeast Atg6 （Beclin-1）， and ubiquitin-binding protein p62. ResultAfter H2O2 induction for 40 min， the cell proliferation rate of the model group decreased compared with that of the control group （P<0.01）， and the cell proliferation rates of the BBR groups increased compared with that of the model group （P<0.05）. The results of β-galactosidase staining showed that the cells of the model group showed significant senescence compared with those of the control group （P<0.01）， and the cellular senescence in the BBR groups was reduced compared with that of the model group （P<0.01）. As revealed by flow cytometry， compared with the control group， the model group showed increased apoptosis rate （P<0.01）， and compared with the model group， BBR groups showed decreased apoptosis rates （P<0.05）. Meanwhile， the ROS content in the model group increased compared with that in the control group （P<0.01）， and compared with the model group， the BBR groups showed reduced cellular ROS content （P<0.01）. The Real-time PCR results showed that compared with the control group， the model group showed decreased mRNA expression of CAT and Bcl-2/Bax in KGN cells and increased mRNA expression of Caspase-3 and FoxO1 （P<0.05）， and compared with the model group， the BBR groups showed increased mRNA expression of CAT and Bcl-2/Bax （P<0.05） and reduced mRNA expression of Caspase-3 and FoxO1 in KGN cells （P<0.05）. As revealed by Western blot results， SIRT1， SOD2， and p62 protein levels decreased in the model group compared with those in the control group （P<0.01）， and JNK FoxO1， LC3BⅡ， and Beclin-1 protein levels increased （P<0.05）. After BBR intervention， SIRT1， SOD2， and p62 protein levels increased （P<0.01）， and JNK， FoxO1， LC3BⅡ， and Beclin-1 protein levels decreased compared with those in the model group （P<0.05）. ConclusionBBR has an inhibitory effect on ovarian granulosa cell senescence， and the mechanism is related to the inhibition of apoptosis and autophagy mediated by the SIRT1/FoxO1 pathway.
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This study was aimed to investigate the effects of Si-Wu mixture on the secretion of E2 and the expression of CYP19al gene in granulosa cells after cell injury.Ovarian granulosa cells of SD rats were treated with cisplatin (CDDP).And the expression levels of E2 and CYP19a1 were determined by different testing methods.The results showed that the level of E2 induced by radioimmunoassay of the Si-Wu group was significantly higher than that of the CDDP group.Meanwhile,the group which added TGF-β3 protein pathway blocker was lower than others.The results of immunohistochemistry and western blotting showed that the expression level of CYP 19a1 of the Si-Wu group was higher than that of the CDDP group.In the western blotting,the group which added blocker was significantly lower than the non-blocking group.It was concluded that the pharmacological serum of Si-Wu mixture can enhance the level of E2 in CDDP cells through TGF-β3 protein pathway.And the effect is accomplished by the intervention of CYP 19a1.
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ObjectiveTo study the effects of tanshinoneⅡA on proliferation of cervical squamous cancer Siha cells; To discuss its possible molecular mechanism.Methods Cervical squamous cancer Siha cells were treated with different doses of tanshinoneⅡA. The effects of tanshinoneⅡA on proliferation of Siha cells were measured by MTT assay and flow cytometry analysis. The effects of tanshinoneⅡA on expression levels of phospho-extracellular regulate kinase (p-ERK) and Cyclin D in Siha cells were measured by Western blot.Results 1×10-5, 5×10-6, 1×10-6, 5×10-7 mol/L tanshinoneⅡA significantly inhibited Siha cell proliferation and such effect could be enhanced by ERα antagonist MPP and attenuated by ERβ antagonist PHTPP. 1×10-5, 5×10-6, 1×10-6 tanshinoneⅡA could significantly decrease the proliferation index of Siha cells. 1×10-5, 5×10-6, 1×10-6, 5×10-7 mol/L tanshinoneⅡA could significantly reduce the protein expression levels of p-ERK and Cyclin D of Siha cells.ConclusionTanshinoneⅡA can inhibit cervical squamous cancer Siha cell proliferation and such effect is realized via estrogen receptor pathway. TanshinoneⅡA plays anti-proliferation roles by reducing the expression levels of p-ERK and Cyclin D.
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Aim To explore the effects of tanshinone IIA on cell proliferation via G protein-coupled estrogen receptor inductive and regulative pathway in typical es-trogen receptor and G protein-coupled estrogen receptor positive T47D breast cancer cells. Methods The pro-liferation rate of T47 D cells influenced by tanshinone IIA was analyzed by MTT assay. G protein-coupled es-trogen receptor agonist G1 and GPER antagonist G15 were employed as tools. GPER SiRNA was applied to build GPER gene silence T47D cells. GPER expres-sion influenced by tanshinone IIA was measured by Western blot. Results The proliferation rates of T47D cells treated with 1 × 10 -5 mol·L-1 - 1 × 10 -7 mol· L-1 of tanshinone IIA were decreased significantly. Such effects could be attenuated by G1 or enhanced by G15 . Growth of GPER SiRNA transfected T47 D cells were significantly inhibited by 1 × 10 -5 mol·L-1 - 1 × 10 -7 mol·L-1 of tanshinone IIA treating. Result of Western blot showed that tanshinone IIA at 1 × 10 -5 mol· L-1 and 1 × 10 -6 mol · L-1 could induce de-crease of GPER protein expression in T47D cells. Conclusions Tanshinone IIA shows inhibitory effects on proliferation rate of T47 D breast cancer cells via GPER pathway. Tanshinone IIA could perform regula-tive function on GPER expression level in target cells.
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This study was aimed to observe the influence of β-sitosterol (BSS) on estrogen receptor (ER) positive the human breast cancer cell line T47D and to study its mechanisms. ER antagonist ICI182 780 was employed to observe the influence on the proliferation. Proliferations of T47D cells influenced by different concentrations of BSS were analyzed by MTT assay. Cell cycle analyses were examined by flow cytometry. The protein expression of cyclin D1 was measured by western blot analysis and cyclin D1 mRNA was quantified by real-time PCR assay. The results showed that BSS in high dose exhibited significant inhibitory effects that were partly antagonized by ICI182 780 and decreased the proliferative index on T47D cells. However, BSS in low dose obviously promoted the proliferation that was completely inhibited by ICI182 780 and increased the proliferative index on T47D cells. The mRNA and protein levels of cyclin D1 were increased in low-dose BSS. The effect was blocked by ICI182 780. It was concluded that BSS in low concentration had phytoestrogenic effect by up-regulating the expression of cyclin D1 via ER pathway.
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This study was aimed to investigate effects of Er-Xian (EX) decoction in follicular granulosa cell apoptosis in chemotherapy-induced premature ovarian failure (POF) of rats. SD rats were randomly divided into the normal group, model group, positive group and EX decoction group. Intraperitoneal injection of cisplatin was used in the establishment of chemotherapy-induced POF rat model. Intragastric administration of corresponding medication was give to each group for four weeks. The general conditions of rats were observed. The serum contents of E2, FSH, LH and Pro were determined. The ovarian tissue morphology was observed. Granulosa cell apoptosis was analyzed by TUNEL. The expressions of BAX, Bcl-2 and VEGF protein were detected by immunohistochemistry. The results showed that in the model group, the body weight decreased with disordered estrous cycle. After treatment, the weight gained, and the estrous cycle recovered. Compared with the normal group, in the model group, E2 decreased, FSH and LH levels increased. There was no significant difference in the content of progesterone among different groups. Detection of TUNEL showed that the positive expression in the model group was increased compared with the normal group (P < 0.05). The positive expression of EX decoction group was reduced compared to the positive group (P < 0.05). Compared with the normal group, the expression of BAX was obviously increased and Bcl-2 was decreased in the model group. After EX decoction treatment, the expression of BAX was decreased and the Bcl-2 was increased compared to the model group. The expression of VEGF protein in the model group was obviously less than that in the normal group. It was concluded that cisplatin can induce follicular granulosa cell apoptosis, and cause premature ovarian function recession. EX decoction can adjust the content of different hormones in serum, alter expression of apoptosis related protein to reduce the damage of cisplatin on ovarian follicular development, in order to promote follicular development, improve and enhance the ovarian function.
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Aim To isolate and characterize the human circulating fibrocytes from human peripheral blood and explore the effects of curcumin on human circulating fi-brocytes.Methods The cells were isolated and puri-fied by density gradient centrifugation,and identified by flow cytometry and immunocytochemistry .Then , CCK-8 and flow cytometry were used to study the effect of curcumin on the proliferation as well as COL I ex-pression of human circulating fibrocytes,respectively. Results After being isolated the cells expressed CD34,CD45 and COLⅠ,among which 79.7% were both CD45 and collagen I positive,typical of human circulating fibrocytes.Curcumin could exert regulatory effects on proliferation of human circulating fibrocytes. Exposure of the cells to curcumin for as short as 24 hours promoted their growth,while prolonged treatment (72 h ) significantly inhibited cell propagation and downregulated the COLⅠ levels,best manifested at a concentration as high as 20 μmol · L-1 .Conclusion The proliferation of cells and COLⅠexpressions can be effectively inhibited by curcumin with the prolonged action period and high concentrations.
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Postmenopausal osteoporosis is one of the commonest systemic bone metabolism diseases among menopausal women, mainly caused by lowering internal estrogen. Although Hormone Replacement Therapy (HRT) is an effective method in clinical practice for years, it shows side-effect in increasing gynecological carcinoma. It has already been proved by clinical tests that multiple traditional Chinese medicine formulas and their monomer ingredients and phytoestrogen-like active constituents contained in traditional Chinese medicines are effective on treating osteoporosis with relatively less side-effects comparing with HRT. They show protective and therapeutic effects by acting on estrogen receptors of targeted tissues and targeted cells and then affecting expressions of bone metabolism-related regulatory proteins and factors in downstream signal conduct paths. Recent studies on estrogen related receptor (ERR) provide new possibilities and pathways for mechanism of traditional Chinese medicine and their active constituents in osteoporosis.
Subject(s)
Humans , Chemistry, Pharmaceutical , Drugs, Chinese Herbal , Chemistry , Pharmacology , Therapeutic Uses , Medicine, Chinese Traditional , Methods , Osteoporosis, Postmenopausal , Drug Therapy , Receptors, Estrogen , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate phytoestrogenic effects of ferulic acid in ER-positive T47D and ER-negative MDA-MB231 cells in culture.</p><p><b>METHOD</b>T47D and MDA-MB231 human breast cancer cells were treated with ferulic acid and examined cell proliferation by means of MTT assay. Cell cycle distribution, ERalpha and ERbeta expression were treated by flow cytometer. The pS2 mRNA expressions were detected by real-time fluorescence quantitative PCR.</p><p><b>RESULT</b>The proliferations were enhanced significantly by treatment with ferulic acid on T47D cells and the proliferation effects were inhibited by adding Faslodex (1 x 10(-8) mol x L(-1)). However, there was no significant difference on the proliferation in MDA-MB-231 cells compared with solvent control group by both treatment with ferulic acid and co-treatment with Faslodex (1 x 10(-8) mol x L(-1)). Ferulic acid stimulated the amount of T47D cells in phase S and proliferation index increased significantly. The effects were inhibited by treatment with Faslodex (1 x 10(-8) mol x L(-1)), and the amount of cells in phase S and proliferation index decreased, the amount of cells in G0/G1 phase increased, cell cycle of T47D was arrested in G0/G1 phase. Ferulic acid up-regulated pS2 mRNA expressions and increased the level of ERalpha protein expression in T47D cells. Ferulic acid did not show remarkable effect to the level of ERbeta protein expression in T47D cells.</p><p><b>CONCLUSION</b>Ferulic acid possessed phytoestrogenic effect by up-regulating pS2 gene expression and the receptor subtype of ERalpha.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Chemistry , Pathology , Cell Line, Tumor , Cell Proliferation , Coumaric Acids , Pharmacology , Estrogen Receptor alpha , Estrogen Receptor beta , Gene Expression Regulation, Neoplastic , RNA, Messenger , Trefoil Factor-1 , Tumor Suppressor Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>Research on the phytoestrogenic effect and its possible mechanism of formononetin.</p><p><b>METHOD</b>To evaluate the estrogenic effect and mechanisms of formononetin through the test of its influence on proliferation and ER subtype expression of T47D cells.</p><p><b>RESULT</b>The proliferation rates of T47D cells treated with 1 x 10(-7) -1 x 10(-6) mol x L(-1) formononetin were not increased. On the influence of ICI182, 780, the proliferation rates of T47D cells treated with 1 x 10(-7) 1 x 10(-6) mol x L(-1) formononetin were decreased. Formonenetin could induce the augment of ERalpha expression significantly of T47D.</p><p><b>CONCLUSION</b>Formonenetin has phytoestrogenic effect Formonenetin can not accelerate ER(+) T47D cell proliferation. But the expression level of ERalpha subtype in T47D cells change significantly with certain concentrations of formonenetin.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Drug Therapy , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Isoflavones , Pharmacology , Phytoestrogens , Pharmacology , Receptors, Estrogen , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the phytoestrogenic effects and their possible mechanisms of Jiaoai tang and Shenqi Jiaoai tang through the tests in mice and ER (+) MCF7 cells.</p><p><b>METHOD</b>Sixty kunming mice weighing 9-12 g were randomly divided into 6 groups: solvent control group (administrated equal dose of diswater), diethylstilbestrol control group (administrated diethylstilbestrol at a dose of 0.35 mg x kg(-1) x d(-1) and 4 Chinese medicine treated groups (administrated low or high doses of Jiaoai tang and Shenqi Jiaoai tang at 2.5 g x kg(-1) x d(-1) or 5.0 g x kg(-1) x d(-1) respectively). After administration for 4 days, the mice were sacrificed; uterus was removed and weighed, uterus rate was calculated. The blood serum was also separated. The proliferation rate of MCF7 cells influenced by Jiaoai tang and Shenqi Jiaoai tang was determined by MIT assay. PS2, ERalpha and ERbeta mRNA expression was quantified by Real-time PCR assay. Estrogen receptor antagonist ICI182, 780 was employed as a tool.</p><p><b>RESULT</b>Administration of Jiaoai tang and Shenqi Jiaoai tang at high dose significantly increased uterus rate in mice (P < 0. 05). The pharmacological serum from two high-dosage groups of Chinese herbal medicine decoction significantly enhanced proliferation rate of MCF7 cells (P < 0.05 or 0.01), while their effects were blocked by ICI182, 780 (P < 0.05 or 0.01). The pharmacological serum could cause elevation of pS2 level (P < 0.01) which would be obviously inhibited by ICI182, 780 (P < 0.01). ERalpha and ERbeta mRNA levels were also elevated significantly (P < 0.05 and P < 0.01 respectively).</p><p><b>CONCLUSION</b>Jiaoai tang and Shenqi Jiaoai tang have phytoestrogenic effects, which were attained via ER pathway. They can also increase the mRNA levels of estrogen receptor subtypes, especially ERbeta.</p>
Subject(s)
Animals , Female , Mice , Cell Line, Tumor , Cell Proliferation , Drug Evaluation, Preclinical , Drugs, Chinese Herbal , Pharmacology , Estrogen Receptor alpha , Genetics , Metabolism , Estrogen Receptor beta , Genetics , Metabolism , Gene Expression , Phytoestrogens , Pharmacology , Random Allocation , Signal Transduction , Uterus , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the phytoestrogenic-like effects of four kinds of Chinese medicine including Radix rehmanniae preparata, Radix Paeoniae Alba, Radix Angelicae Sinensis, Rhizoma Chuanxiong.</p><p><b>METHOD</b>Sixty immature female SD rats weighting (70 +/- 5) g were randomly divided into six groups: normal control group, positive control group and 4 Chinese medicine groups. The rats in different groups were treated for 4 days. On the fifth day, animals were sacrificed and uteri were separated solely and weighed. The blood was collected, and serum was separated. The effect of the pharmacological serum on proliferation assay with human breast cancer cell line (MCF7) by MTT method. Cell-cycle analyses were carried out with propidium iodide staining by flow cytometer. The expressions of subtypes-estrogen receptor-alpha (ERalpha) were detected by flow cytometry.</p><p><b>RESULT</b>Radix Rehmanniae Preparata, Radix Paeoniae Alba and Radix Angelicae Sinensis could increase the immature rat's uterus wet weight and the ratio of uterus to body weight (P < 0.05). The pharmacological serum of the four kinds of Chinese medicine stimulated proliferation of MCF7 cell respectively compared with normal control group (P < 0.01). The cell cycle was impulsed from G1 to S, DNA synthesizing was inhanced, and PI was also increased. The pharmacological serum of Radix Angelicae Sinensis and Rhizoma Chuanxiong could increase the expressions of (ERalpha) (P < 0.05).</p><p><b>CONCLUSION</b>Radix Rehmanniae Preparata, Radix Paeoniae Alba and Radix Angelicae sinensis have phytoestrogenic effects. But the data werent consistent with in vitro and in vivo assay of Rhizoma Chuanxiong.</p>
Subject(s)
Animals , Female , Rats , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Drugs, Chinese Herbal , Estrogen Receptor alpha , Genetics , Metabolism , Phytoestrogens , Random Allocation , Rats, Sprague-Dawley , Uterus , Cell Biology , MetabolismABSTRACT
Objective To investigate the effects of quercetin on the proliferation of mammary cancer cell in vitro. Methods Effects of quercetin on the cell proliferation was tested in ER-positive MCF-7 cell and ER-negative MDA-MB231 cell by MTT measurement. And to evaluate the estrogen-like effect of quercetin and its relation with the estrogen-receptor by pure estrogen receptor antagonist ICI 182, 780 as a tool. Cell cycle was examined by flow cytometry. Result Quercetin (10～50 ?mol/L) stimulated proliferation of ER-positive MCF-7 cell compared with solvent control, whereas ER-negative MDA-MB231 cell proliferation effect was inhibited, and the cell cycle was impulsed from G1 to S, DNA synthesizing was inhanced, PI was also increased. The above function on boosting MCF-7 cell proliferation can be inhibited by adding estrogen receptor antagonism ICI182, 780. Conclusion Quercetin has the estrogen-like activities through the estrogen response pathway.
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Aim To study the phytoestrogenic effect and its mechanism of isopsoralen in estrogen receptor (ER) positive T47D and ER negative MDA-MB231 breast cancer cell line.Methods The proliferation rate and cell cycle distribution of T47D and MDA-MB231 cells influenced by isopsoralen were analyzed by MTT and flow cytometry assay respectively.PS2 mRNA level in T47D cells was quantified by Real-time RT-PCR assay.PS2 expression was measured by flow cytometry.Estrogen receptor antagonist ICI180,782 was employed as a tool.Results The proliferation rates and indexes of T47D cells treated with 1?10-6 mol?L-1 and 1?10-7 mol?L-1 isopsoralen were increased.These effects could be blocked by ICI182,780.Meanwhile,isopsoralen didn't show stimulative effects on proliferation rate of MDA-MB231 cells.Real-time RT-PCR and flow cytometry results showed that isopsoralen at 1?10-6 mol?L-1 and 1?10-7 mol?L-1 could induce elevation of pS2 mRNA and protein expression level in T47D cells.These effects could be partly blocked by ICI182,780.Conclusion Isopsoralen has phytoestrogenic effect and its effect is attained mainly via ER pathway.