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Research Paper Volume 12, Issue 22 pp 22744-22758
Geniposide ameliorated sepsis-induced acute kidney injury by activating PPARγ
Relevance score: 7.648968Jinhong Liu, Ning Zhao, Guiling Shi, Hai Wang
Keywords: geniposide, sepsis, acute kidney injury, PPARγ, inflammation
Published in Aging on November 10, 2020
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Research Paper Volume 12, Issue 14 pp 14897-14917
Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways
Relevance score: 3.7556822Haiyun Chen, Jie Cao, Ling Zha, Peile Wang, Zheng Liu, Baojian Guo, Gaoxiao Zhang, Yewei Sun, Zaijun Zhang, Yuqiang Wang
Keywords: tetramethylpyrazine derivative T-006, Parkinson’s disease, transcriptional factor myocyte enhancer factor 2D, peroxisome proliferator-activated receptor γ (PPARγ) co-activator 1α, adult neurogenesis
Published in Aging on July 24, 2020
T-006 improves motor behavior, protects nigrostriatal neurons, and suppresses disease progression in mice subjected to MPTP. (A) Schedule for T-006 treatment of a chronic MPTP mice PD model. (B) The changes of body weight in different groups (n=14-16 per group). (C) Time spent on pole. (D) Time taken for the left forelimb to grip wire. (E) Distance between hind paws’ markings on white absorbing paper. (F) Distance travelled (cm) in open field. (G) Immunohistochemistry for TH in the substantia nigra (SN; upper panel) and striatum (ST; lower panel) of MPTP mice. (H) Stereological counting of TH-positive DA neurons from SN. (I) Relative density of TH-positive neuronal fibers in ST. (J) Representative Western blots illustrating the expression of TH in SN and ST. (K) Densitometric analysis of TH/β-actin of treatment with T-006 at the indicated concentrations in SN and ST. The results are shown as the mean±SEM (n=6-8 per group). (L) Striatal dopamine and its metabolites DOPAC and HVA were analyzed by electrochemical HPLC with 6 to 8 mice per group. Data are expressed as mean±SEM. #P<0.05, ##P<0.01, ###P<0.001 vs. sham group, and *P<0.05, **P<0.01 and ***P<0.001vs. MPTP group.
T-006 improves motor behavior and prevents dopaminergic neurodegeneration in rats subjected to 6-OHDA. (A) Schedule for T-006 treatment of a progressive 6-OHDA rat PD model. (B) The changes of body weight in different groups (n=12-16 per group). (C) Turns initiated by apomorphine-induced 6-OHDA rats. (D) Time spent on the rotarod. (E) Distance travelled (cm) in open field. (F) Discrimination index in new objective test. (G) Immunohistochemistry for TH in SN. (H) Stereological counting of TH-positive DA neurons from SN. (I) Representative Western blots illustrating the expression of TH in SN. (J) Densitometric analysis of TH/β-actin of treatment with T-006 at the indicated concentrations in SN. (K–M) Striatal dopamine and its metabolites DOPAC and HVA were analyzed by electrochemical HPLC with 6 to 8 mice per group. The results are shown as the mean±SEM. #P<0.05, ##P<0.01, ###P<0.001 vs. sham group, and *P<0.05, **P<0.01 and ***P<0.001vs. 6-OHDA group.
T-006 stimulates MEF2D/PGC1α/Nrf2 signal pathway through regulation of the Akt/GSK3β pathway in PD animal models. (A) Representative Western blots and densitometric analysis the expression of CDK5, p-MEF2D, MEF2D and PGC1α. (B, E) Representative Western blots and densitometric analysis of the expression of p-Akt and p-GSK3β. (C, F) Representative Western blots and densitometric analysis of the expression of Nrf2, HO-1 and TFAM. (D) Representative Western blots and densitometric analysis the expression of MEF2D and PGC1α. Data are expressed as mean±SEM (n=3 per group). #P<0.05, ##P<0.01, ###P<0.001 vs. sham group, and *P<0.05, **P<0.01 and ***P<0.001vs. MPTP or 6-OHDA group.
T-006 prevents the loss of SN DA neuron loss by activating MEF2D/PGC1α/GSK3β signal pathway. Representative images of middle brain sections co-stained with antibodies against (A, D) TH (green) and p-GSK3β (red); (B, E) TH (red) and MEF2D (green); (C, F) TH (green) and PGC1α (red). DAPI (blue) indicates nucleus. (G, H) Quantitative analysis of immunofluorescence intensity in TH-positive cells. Data are expressed as mean±SEM (n=3 to 4 per group). #P<0.05, ###P<0.001 vs. sham group, and *P<0.05, **P<0.01 vs. MPP+ or 6-OHDA group.
T-006 protects against neurotoxin-induced neurotoxicity in neurons. CGNs or corrected DA neurons were pretreated with T-006 at the indicated concentration for 2 hours followed by 150 μM MPP+ or 30 μM 6-OHDA treatment for 24 hours. (A, I) MTT assays to evaluate the cell viability. (B, J) Measurements of the LDH release. (C) ROS production assessed by DCF fluorescence intensity. (D) Hoechst staining to show the apoptotic cells. (E) Statistical analysis of the number of pykonitic nuclei of (D). (F) Mitochondrial membrane potential tested by JC-1 kit. (G) Complex I activity evaluation. (H) Intracellular ATP content measurement. Results are representative of three independent experiments as the mean±SEM. ###P<0.001 vs. control (Ctrl) group, and*P<0.01, **P<0.01 and ***P<0.001 vs. MPP+ or 6-OHDA group.
T-006 activates the MEF2/PGC1α/Nrf2 pathway through regulation of the Akt-GSK3β pathway. A53T and corrected DA neurons treated with T-006 and the positive drug at the indicated concentration for 24 hours. For MPP+-treatment assay, CGNs were pretreated with or without LY294002 (1 μM), Akt-iv (1 μM), or LiCl (10 μM) for 2 h, incubated with or without T-006 for 2 h, and finally exposed to MPP+. Cell viability was examined using an MTT assay. Luciferase reporter gene assays respectively included MEF2 (A), PGC1α (B), PGC1α-ΔMEF2 (C) and ARE (D). (E) Representative images of neurons co-stained with antibody against Nrf2 (green). DAPI (blue) indicates nucleus. (F) Luciferase reporter gene assays of MEF2 with MPP+ induction. (G–K) respectively represent the fold changes of CDK5, MEF2D, PGC1α, Nrf1 and Nrf2 at mRNA level. (L) Effect of MEF2D reduction on MPP+-induced neurotoxicity in CGNs. (M) Effects of Akt pathway inhibitor LY294002 and GSK3β inhibitor on MEF2 transcriptional activity. (N) Effects of Akt pathway inhibitors LY294002 and Akt-iv, and GSK3β inhibitor on MPP+-induced neurotoxicity in CGNs. Data above are all from three independent experiments, expressed as mean±SEM. #P<0.05, ##P<0.01 and ###P<0.001 vs. control (Ctrl) group, and *P<0.05, **P<0.01, ***P<0.001 vs. MPP+ group.
T-006 enhances neural reconstruction by stimulating neurogenesis in rat model of PD. Immunofluorescence images of ipsilateral hemisphere sections co-stained with antibodies against (A) TH (green, a marker of DA neurons) and BrdU (red, a marker of proliferating cells); (C) DCX (green, a marker of migrating neuroblasts) and BrdU (red); (E) Nestin (green, a marker of NPCs) and BrdU (red) cells. DAPI (blue) indicates nucleus. Insets show a higher magnification view of double-positive cells. Scale bar = 200 μm for whole slice, 50 μm for inset magnification. (B, D and F) Quantitative analysis of newly formed mature neurons (BrdU+/TH+, D), in the SN, migrating neuroblasts (BrdU+/DCX+, E) and proliferating NPCs (BrdU+/Nestin+, F) in the SVZ. (G and I) Immunohistochemical images of ipsilateral hemisphere sections co-stained with antibodies against DCX and Nestin in the SNc. (H and J) Quantitative analysis of DCX and Nestin-positive cells in SNc. Data are expressed as mean±SEM (n=3 to 4 per group). *P<0.05, **P<0.01 and ***P<0.001 vs. 6-OHDA group.
T-006 activates BDNF/CREB signaling pathway to provide neuro-repair in PD rats. Representative immunoblotting (A, C) and quantification of the relative protein level of p-CREB/CREB (B), BDNF (D) and synaptophysin (SYP, E) in the brain infarct region of sham, vehicle or T-006-treatment rats in 6-OHDA-induced PD rats. Data are expressed as mean±SEM (n=3 to 4 per group). ##P< 0.01 vs. sham group, and *P<0.05, **P<0.01, ***P<0.001 vs. 6-OHDA group.
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Research Paper Volume 12, Issue 14 pp 14189-14204
Co-exposure to multi-walled carbon nanotube and lead ions aggravates hepatotoxicity of nonalcoholic fatty liver via inhibiting AMPK/PPARγ pathway
Relevance score: 6.482142Enqin Liu, Xinghui Wang, Xidong Li, Ping Tian, Hao Xu, Zenglian Li, Likun Wang
Keywords: multi-walled carbon nanotube, lead ions, NAFLD, AMPK, PPARγ
Published in Aging on July 17, 2020
NAFLD models were successfully established in mice. (A) The body weight change curve of mice in the normal diet and high-fat diet groups (*P<0.05, compared to normal diet mice). (B) The weight of mice organs including the liver, heart, lung, spleen and kidney in the normal diet and high-fat diet groups (*P<0.05, compared to normal diet mice). (C) The serum levels of lipoproteins (CHOL, TG, HDL and LDL) of mice in the normal diet and high-fat diet groups (*P<0.05 and **P<0.01, compared to normal diet mice). (D) The serum levels of hepatic damage biomarkers (ALT, AST and ALP) in the normal diet and high-fat diet groups.
MWCNTs and PbAc exposure significantly reduced the body weight of mice. (A, B) The body weight change curve of the control and NAFLD mice upon the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration. (C, D) The impacts of the low dose of PbAc, MWCNTs or MWCNTs + PbAc on the organ index of liver, heart, lung, spleen and kidney of both the control and NAFLD mice.
Combined administration of MWCNTs and PbAc significantly damaged the liver function in NAFLD mice. The effects of low dose of PbAc, MWCNTs or MWCNTs + PbAc on the serum levels of CHOL, TG, HDL, LDL (A, B) and the TG expression levels in liver tissues (C, D) in the control and NAFLD mice. (E, F) Changes in the serum levels of ALT, AST and ALP in the control and NAFLD mice upon the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration (*P<0.05 and **P<0.01, compared to PbAc or MWCNTs).
MWCNTs and PbAc exposure significantly aggravated the nonalcoholic steatohepatitis phenotype in NAFLD mice. (A, B) Histological morphology changes in livers of the control and NAFLD mice exposed to the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration. (C, D) The score of liver damage in the control and NAFLD mice upon the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration (*P<0.05 and **P<0.01, compared to saline water (control) or PbAc).
MWCNTs and PbAc exposure significantly aggravated the hepatic fibrosis and steatosis in NAFLD mice. (A) Upon the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration, Masson staining was conducted to detect the collagen deposition (blue indicates collagen) in liver tissues of control and NAFLD mice. (B) Oil red O staining was performed to detect the lipidoses (red indicates lipid) in liver tissues of control and NAFLD mice.
MWCNTs and PbAc exposure significantly induced apoptosis in primary hepatocytes isolated from NAFLD mice. (A) After treatment with the low dose of PbAc, MWCNTs or MWCNTs + PbAc, apoptotic analyses were performed in primary hepatocytes from control and NAFLD mice using a flow cytometry. (B, C) Apoptotic rates in primary hepatocytes from control and NAFLD mice were statistically analyzed (***P<0.001, compared to saline water).
Combined administration of MWCNTs and PbAc resulted in hepatic lipid peroxidation by inducing antioxidant defense system dysfunction. (A, B) Alterations in SOD, GST, GPx activities as well as GSH, hydrogen peroxide, MDA levels in the control and NAFLD mice livers exposed to the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration (*P<0.05, compared to saline water).
Combined administration of MWCNTs and PbAc significantly enhanced inflammatory cytokine expressions in NAFLD mice livers. (A, B) The effects of the low dose of PbAc, MWCNTs or MWCNTs + PbAc on the expressions of IL-6, IL-1β and TNF-α in the control and NAFLD mice livers (*P<0.05, compared to saline water).
Combined administration of MWCNTs and PbAc may exert its hepatotoxicity to NAFLD mice via inhibiting AMPK/PPARγ pathway. Western blot analysis of PPARγ and p-AMPKα expressions in NAFLD mice livers (A) and in primary hepatocytes from NAFLD mice (C) upon the low dose of PbAc, MWCNTs or MWCNTs + PbAc administration. (B, D) PPARγ and p-AMPKα expression levels normalized to GAPDH (*P<0.05, **P<0.01 and ***P<0.001, compared to saline water). (E) After treatments with DMSO or two AMPK activators (0.5 μM PF-06409577 and 10 μM A-769662) in addition to MWCNTs + PbAc, PPARγ expressions in primary hepatocytes from NAFLD mice were tested using western blot analysis. (F) PPARγ expression levels normalized to GAPDH (***P<0.001, compared to DMSO). (G) Cell viability of primary hepatocytes from NAFLD mice upon the administration of saline water or MWCNTs + PbAc (***P<0.001, compared to saline water). (H) In addition to MWCNTs + PbAc, primary hepatocytes from NAFLD mice were also incubated with DMSO, PF-06409577 (0.5 μM), A-769662 (10 μM) or T0070907 (a selective PPARγ antagonist, 50 μM), then the cell viability was measured (***P<0.001, compared to DMSO or T0070907).
Intervening AMPK/PPARγ pathway significantly changed the hepatotoxicity of MWCNTs + PbAc to NAFLD mice. (A) Upon the administration of MWCNTs + PbAc, NAFLD mice were simultaneously treated with vehicle, PF-06409577 and PF-06409577 + T0070907. Masson staining and Oil red O staining were performed to detect the hepatic fibrosis (blue indicates collagen) and steatosis (red indicates lipid) in NAFLD mice, respectively. (B) The expression levels of IL-6, IL-1β and TNF-α in NAFLD mice livers were measured (**P<0.01 and *P<0.05, compared to PF-06409577).
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Research Paper Volume 11, Issue 23 pp 10992-11009
Mangiferin promotes macrophage cholesterol efflux and protects against atherosclerosis by augmenting the expression of ABCA1 and ABCG1
Relevance score: 7.1229014Kun Ren, Heng Li, Hui-Fang Zhou, Yin Liang, Min Tong, Lu Chen, Xi-Long Zheng, Guo-Jun Zhao
Keywords: mangiferin, ABCA1/G1, LXRα, PPARγ, cholesterol efflux
Published in Aging on December 2, 2019
Mangiferin reduces atherosclerotic lesion formation in apoE-/- mice. Eight-week-old male apoE-/- mice were intraperitoneally injected with PBS or mangiferin (15 mg/kg) every day for 12 weeks. (A–B) Representative image of Oil Red O staining of an aortic lesion. Original magnification: 40×. (C) Quantification of the lesion areas of mice (n=15/group). *P < 0.05 vs. control group. Values are expressed as the mean ± SEM (n =15/group). (D–E) Representative HE staining of an aortic lesion in apoE-/- mice. Original magnification: 40×.
Mangiferin promotes macrophage cholesterol efflux and enhances RCT in apoE-/- mice. (A) [3H]-cholesterol-labeled RAW264.7 cells were intraperitoneally injected into apoE-/- mice. The amounts of [3H]-tracer in the liver, feces and plasma were assessed by LSC. *P < 0.05 vs. control group. (B–D) RAW264.7 macrophage-derived foam cells were treated with mangiferin at different concentrations (0, 5, 10, and 20 μM) for 24 h. Then, the percent cholesterol efflux to apoA-1 (B) or HDL (C) was analyzed by LSC. Lipid droplet content was assessed using Oil Red O staining (D). All results are presented as the mean ± SEM from three independent experiments, each performed in triplicate. *P < 0.05 vs. 0 μM group.
Mangiferin promotes the expression of ABCA1 and ABCG1 in RAW264.7 macrophages and in the aortas of apoE-/- mice. (A–D) RAW264.7 macrophage-derived foam cells were exposed to different concentrations of mangiferin (0, 5, 10, and 20 μM) for 24 h. Then, the protein (A, B) and mRNA (C, D) levels of ABCA1 and ABCG1 were determined by western blot and RT-qPCR analyses, respectively. *P < 0.05 vs. 0 μM group. (E–F) The mice were divided and treated as described above. The protein levels of ABCA1 and ABCG1 in the homogenate of the aortic arch were detected by western blotting. *P < 0.05 vs. control group. Data are presented as the mean ± SEM (n =3/group).
Mangiferin enhances the expression of PPARγ and LXRα in RAW264.7 macrophages and in the aortas of apoE-/- mice. (A–F) After RAW264.7 cells were fully differentiated, the cells were exposed to different concentrations of mangiferin (0, 5, 10, and 20 μM) for 24 h. Then, RT-qPCR and western blot analyses were performed to detect the mRNA (A, D) and protein (B, C, E, F) levels, respectively, of PPARγ and LXRα. *P < 0.05 vs. 0 μM group. (G, H) Protein levels of PPARγ and LXRα in the homogenate of the aortic arch were assessed by western blotting. *P < 0.05 vs. control group. All results were collected from three independent experiments, each performed in triplicate. Data are presented as the mean ± SEM (n =3/group).
Mangiferin promotes ABCA1 and ABCG1 expression by upregulating LXRα levels. RAW264.7 macrophage-derived foam cells were pretreated with 2 μM T0901317 (A–C) or transfected with 20 nM LXRα siRNA (D–F) and then incubated with mangiferin (20 μM) for 24 h. RT-PCR and western blot analyses were performed to assess the mRNA and protein levels, respectively, of ABCA1/G1 and LXRα. All results were obtained from three independent experiments, each performed in triplicate. Data are expressed as the mean ± SEM (n =3/group). *P < 0.05 vs. control group; #P < 0.05 vs. mangiferin only group.
PPARγ mediates the positive effects of mangiferin on the expression of ABCA1/G1 and LXRα. RAW264.7 macrophage-derived foam cells were pretreated with 25 μM rosiglitazone (A–C) or transfected with 20 nM PPARγ siRNA (D–F) and then incubated with mangiferin (20 μM) for 24 h. RT-PCR and western blot analyses were performed to assess the mRNA and protein levels, respectively, of ABCA1/G1, LXRα and PPARγ. All results were obtained from three independent experiments, each performed in triplicate. Data are expressed as the mean ± SEM (n =3/group). *P < 0.05 vs. control group; #P < 0.05 vs. mangiferin only group.
Mangiferin promotes cellular cholesterol efflux through the upregulation of PPARγ and LXRα expression. RAW264.7 macrophage-derived foam cells were pretreated with LXRα siRNA (A, B) or PPARγ siRNA (C, D) and subsequently treated with 20 μM mangiferin for 24 h. LSC assays were performed to detect apoA-1- or HDL-mediated [3H]-cholesterol efflux. All results were obtained from three independent experiments, each performed in triplicate. Data are presented as the mean ± SEM (n =3/group). *P < 0.05 vs. control group; #P < 0.05 vs. mangiferin only group.
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Research Paper Volume 11, Issue 12 pp 4125-4144
Altered FoxO1 and PPARγ interaction in age-related ER stress-induced hepatic steatosis
Relevance score: 6.427692Dae Hyun Kim, Sugyeong Ha, Yeon Ja Choi, H. Henry Dong, Byung Pal Yu, Hae Young Chung
Keywords: aging process, FoxO1, PPARγ, ER stress, lipid accumulation
Published in Aging on June 25, 2019
Changes of aging-related serum parameter in insulin resistance and lipogenesis. (A) Fasting glucose levels (B) insulin levels (C) triglycerides (D) FFA level in the serum of aging rats (each n = 6). Results of one- factor ANOVA: *p < 0.05, ***p < 0.001 vs. 6-months old rats.
Aging-related increase in ER stress and insulin signaling. Western blotting was performed to detect the protein level of factors involved in ER stress, insulin signaling, and Akt signaling. (A) ER stress markers (p-IRE, IRE, p-PERK, PERK, p-JNK, and JNK) (B) insulin signaling factors (pSer-IRS1, pTyr-IRS1, IRS1) (C) aging-related increase in phospho-Akt level. β-actin was the loading control of the cytosolic fractions. Results of one-factor ANOVA: #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. 6 months.
Aging-related increase in FoxO1-induced lipid accumulation. (A) Western blotting was performed to examine the protein levels of p-FoxO1 and FoxO1 in the liver of aging rats. Results of one-factor ANOVA: ##p < 0.01, and ###p < 0.001 vs. 6 months. (B) Immunohistochemical staining for FoxO1 in aging liver. Scale bar: 200 μm. (C) Western blotting analysis of PPARs in the nuclear of aging liver. TFIIB was the loading control of the nuclear fraction. One representative result of the three experiments for each protein is shown. Results of one-factor ANOVA: #p < 0.05 vs. 6 months. (D) Western blotting showed that immunoprecipitated FoxO1 and PPARγ were physically associated with PPARγ and FoxO1, respectively. (E) Hepatic TGs in aging rats. Results of one-factor ANOVA **p < 0.01, and ***p < 0.001 vs. 6 months. (F) Aging livers were stained with Oil red O to visualize lipid accumulation. Scale bar: 100 μm. Representative H&E staining shows increased vacuoles in liver tubules during aging. Scale bar: 300 μm. (G) Real-time PCR analyses was performed for measuring the mRNA levels of SREBP-1c, PPARγ, FASN, SCD, PPARα, CPT1α, and ACOX. The data are expressed as a mean ± SEM. *p < 0.05, **p < 0.01, and ***p<0.001 vs. 6 months. (H) FoxO1 binds to the PPARγ promoter in aging livers. The livers were subjected to ChIP assay by using rabbit pre-immune IgG and an anti-FoxO1 antibody. Immunoprecipitates were subjected to PCR by using rat PPARγ promoter DNA.
High glucose induced ER stress-mediated lipid accumulation. (A) Western blot was used to detect p-IRE, total-IRE, p-PERK, total-PERK, p-JNK, and total-JNK in cytoplasmic extracts (20 μg protein) after treatment of AC2F cells with glucose (30 mM) for 6 h. β-actin was the loading control of the cytosolic fractions. (B) Cellular triglyceride concentration after treatment with glucose (30 mM) for 36 h was measured by a colorimetric assay. The data are expressed as a mean ± SEM. Three independent experiments were performed and similar results were obtained. *p < 0.05 vs. non-treated cells. (C) Real-time PCR analyses was performed for measuring the mRNA levels of lipogenesis genes (SREBP-1c, PPARγ, FASN, SCD) and β-oxidation genes (PPARα, CPT1α, and ACOX). The data are expressed as a mean ± SEM. Three independent experiments were performed and similar results were obtained. *p < 0.05, and **p < 0.01 vs. non-treated cells. (D) After stimulation with glucose (30 mM) for 2 h with insulin (100 nM) for 10 min in the absence (-) or presence (+) of JNK inhibitor (PD98059, 20 μM) for 1 h, cells were lysed and analyzed by western blotting. β-actin was the loading control of the cytosolic fractions.
FoxO1 regulates ER stress through PPARγ in FoxO1-virus treated cells. (A) Activation of PPARγ by FoxO1. AC2F cells were grown to 80% confluence in 100 mm dishes in DMEM, and then stimulated with 100 and 200 MOI FoxO1 and analyzed by western blotting using the appropriate antibody. (B) FoxO1-induced activation of ER stress genes. Western blot was used to detect p-IRE, total-IRE, p-PERK, and total-PERK in cytoplasmic extracts (20 μg protein) from AC2F cells. (C) AC2F cells were grown to 80% confluence in 100-mm dishes containing DMEM, pretreated (one day) with or without PPARγ-siRNA (10 nM), then stimulated with the FoxO1 virus (200 MOI) for 1 days, and Western blot was used to detect PPARγ, p-IRE, total-IRE, p-PERK, and total-PERK in cytoplasmic extracts (20 μg protein) by using the β-actin as a control from AC2F cells.
FoxO1-dependent changes in lipid accumulation in liver cells. (A) AC2F cells incubated with or without FoxO1 (200 MOI) for 24 h were subjected to real-time qRT–PCR analysis of different transcripts (FoxO1, PPARα, PPARβ, PPARγ, SREBP-1c, and ChREBP) by using the β-actin gene as a control. Results of one-way ANOVA: **p < 0.01 vs. untreated cells. (B) Cells incubated without or with FoxO1 (200 MOI) for 24 h were subjected to real-time qRT-PCR analysis of different transcripts (ACC, FASH, SCD1, DGAT1, DGAT2, AGPAT1, AGPAT9, FATP1, and lipin1) by using the β-actin gene as a control. Results of one-way ANOVA: *p < 0.05 vs. untreated cells. (C) Cellular triglyceride concentration was measured by a colorimetric assay. The data are expressed as a mean ± SEM. Three independent experiments were performed, providing similar results. ***p < 0.001 vs. untreated cells. (D) Effect of wild-type FoxO1 on the activity of the PPARγ promoter. AC2F cells in 48-well microplates were transduced with AdV-FoxO1 or control AdV-null vectors at a fixed dose (MOI, 200 pfu/cell), followed by transfection with 1 μg of pcDNA and PPARγ DNA in the culture medium. After a 24 h incubation, the cells were harvested. The relative luciferase activity was calculated based on the PPARγ-luciferase/β-galactosidase activity ratio. The data are expressed as a mean ± SEM. Three independent experiments were performed and similar results were obtained. $$$p < 0.001 vs. pcDNA treated cells; ***p < 0.001 vs. PPRE treated cells; ###p < 0.001 vs. PPRE with PPARγ DNA treated cells. (E) FoxO1 transfected cells were stained with Oil red O to visualize lipid accumulation. Scale bar: 100 μm.
Effect of high glucose and FoxO1 deletion on the regulation of lipid metabolism. (A) Western blot was used to detect FoxO1 and PPARγ in FoxO1-siRNA treated liver cells. β-actin was used as a loading control. (B) The expression of SREBP-1c, PPARγ, FASN, SCD, PPARα, CPT1, and ACOX was analyzed by qPCR after treatment with glucose (30 mM) for 24 h in FoxO1-siRNA transfected (200 MOI) cells. The results were normalized based on the actin level. (C) Possible mechanism by which FoxO1 activates ER stress-induced lipogenesis in aging.
Obesity induces hepatic steatosis and insulin resistance through ER signaling. (A) Glucose level (B) insulin level (C) triglyceride level (D) glucose tolerance test in the serum of obese models. The data are expressed as a mean ± SEM (each n=5). The data are expressed as a mean ± SEM. ***p<0.001 vs. db+. Western blot analyses of liver cytosolic (E) p-IRE, IRE, p-PERK, PERK, p-IRS1 (S307), p-IRS1 (T632), IRS1, p-Akt, and total-Akt levels were performed using cytosolic proteins from obese mice (n = 5 in each group). β-actin was the loading control of the cytosolic fractions. (F) Western blotting showed that immunoprecipitated FoxO1 and PPARγ were physically associated with PPARγ and FoxO1, respectively. (G) Hepatic triglyceride concentration was measured by a colorimetric assay. The data are expressed as a mean ± SEM. ***p<0.001 vs. db+. (H) PPARγ, SREBP-1c, Acoca, Fasn, SCD, PPARα, CPT1, and Acox mRNA. Real-time PCR analyses were performed to determine the mRNA levels in liver tissues of db/db mice (n = 5 in each group). The data are expressed as a mean ± SEM. *p < 0.05, and ***p<0.001 vs. db+.
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Research Paper Volume 9, Issue 1 pp 256-285
Melatonin-micronutrients Osteopenia Treatment Study (MOTS): a translational study assessing melatonin, strontium (citrate), vitamin D3 and vitamin K2 (MK7) on bone density, bone marker turnover and health related quality of life in postmenopausal osteopenic women following a one-year double-blind RCT and on osteoblast-osteoclast co-cultures
Relevance score: 6.7477407Sifat Maria, Mark H. Swanson, Larry T. Enderby, Frank D’Amico, Brianna Enderby, Rebekah M. Samsonraj, Amel Dudakovic, Andre J. van Wijnen, Paula A. Witt-Enderby
Keywords: osteopenia, melatonin, osteoblasts, osteoclasts, PPARγ, GLUT4, adipocytes
Published in Aging on January 26, 2017
Flow chart of study subject recruitment and enrollment.
Treatment effects on bone mineral density (BMD) in placebo and MSDK groups. Bone mineral density in the left femoral neck, total left hip and lumbar spine was measured via DXA (n=11 per group). Each bar represents the mean (± S.E.M.) change in bone mineral density (g/cm2) from baseline to month 12 in the (A) left femoral neck, (B) total left hip and (C) lumbar spine (L1-L4) area, respectively for placebo (open bar) and MSDK (closed bars). *p ≤ 0.05 and ***p ≤ 0.001 versus placebo, unpaired one tailed t-test with Welch’s correction.
Treatment effects on serum bone marker turnover in placebo and MSDK groups. Bone formation markers (A) total procollagen type 1 amino-terminal propeptide (P1NP) and (B) osteocalcin (OC; both intact and N-terminal mid-fragments), serum bone resorption marker (C) Collagen Type I C-Telopeptide (CTx) were measured at months 0, 6 and 12. Serum samples were collected from participants (n=11 per group) and bone markers concentrations were measured by sandwich ELISA assay using commercially available specific bone marker kits at all three time points. Each point in the line graph represents the least square mean (± S.E.M.) in the bone markers concentration for placebo (open circle, red) and treatment (closed box, blue). Scatter plots represent the ratio of (D) CTx: P1NP and (E) CTx: OC for each study subject, respectively, where the solid lines indicate the mean (± S.E.M.) value for each group. *p ≤ 0.05 and **p ≤0.01 versus placebo at similar time points. Longitudinal analysis for repeated measures using a generalized linear mixed model (GLMM) approach, considering groups and times as fixed effects and subjects nested within the groups as random.
Treatment effects on urinary nocturnal melatonin, serum vitamin D3 and serum C-reactive protein (CRP) level in placebo and MSDK groups. (A) Nocturnal hourly melatonin secretion in the urine was measured at month 12 (n=10 per group), by collecting all urine samples between 10pm and 6am and measuring using urinary melatonin-sulfate ELISA kit. Each point in the scatter plots represents an individual’s urine melatonin-sulfate level, in placebo (open circle, red) or MSDK (closed circle, blue). *p ≤ 0.05 versus placebo at month 12; Unpaired one-tailed t-test with Welch’s correction. Serum levels of (B) Vitamin D3 and (C) CRP were measured at months 0, 6 and 12, using specific ELISA kits. Each point in the scatter plots represents the concentration for a single subject at a specific time point, in the placebo (open circle, red) or MSDK (closed circle, blue). *p ≤ 0.05 versus placebo at similar time point; Longitudinal analysis for repeated measures using generalized linear mixed model (GLMM) approach, considering groups and times as fixed effects and subjects nested within the groups as random.
Treatment effects on participants' sleep quality, mood, GI upset and general aches/pains in placebo and MSDK groups. (A) Total diary comments made by the participants in each group throughout the study were stratified into four categories: sleep, mood, GI upset and general aches/pains, as illustrated by the four segments in the pie diagram. Each category was sub-stratified as positive (pink), neutral (yellow) and negative (green) comments. Each portion represents the percent of total comments made under each category.
Effect of MSDK on osteoblast-mediated calcium mineralization. On day 21 of MSDK exposure, calcium deposition by differentiated, matured osteoblasts were evaluated via alizarin red staining on the (A) bottom chamber cells of transwell co-culture and (B) layered co-culture. Inset graph represents similar analysis in absence of Os+/MSDK. Each bar represents the mean concentration of alizarin red (μM) for respective groups normalized against Os-/Veh (n=3; Transwell co-culture: ****=p<.0001 vs. all groups, a=p<.01 vs. Os-/Veh, b=p<.05 vs. Os-/MSDK, c=p<.05 vs. Os+/Veh; Layered co-culture: ****=p<.0001 vs. all groups, a=p<.01 vs. Os-/Veh, b=p<.01 vs. Os-/MSDK, c=p<.01 vs. Os-/Veh). Os- =basal media, Os+ =osteogenic media, Veh= vehicle, Mel= melatonin, SC= strontium citrate, D3= vitamin D3 (Cholecalciferol), K2= vitamin K2 (MK7).
Effect of MSDK on osteoclast differentiation. On day 21 of MSDK exposure, TRAP releasing activity by differentiated, mature osteoclasts was evaluated by quantitative Tartrate Resistant Acid Phosphatase (TRAP) assay on the (A) top chamber cells of transwell co-culture and (B) layered co-culture. Each bar represents the mean fluorescence reading of TRAP (at 405nm ex, 515nm em) for respective groups normalized against Os-/Veh (n=3; Transwell co-culture: **=p<.01 vs. Os-/Veh; Layered co-culture: *=p<.05 vs. Os-/Veh). Os- =basal media, Os+ =osteogenic media, Veh= vehicle, Mel= melatonin, SC= strontium citrate, D3= vitamin D3 (Cholecalciferol), K2= vitamin K2 (MK7).
Effect of MSDK on sOPG and sRANKL expression in osteoblasts grown in transwell co-cultures, layered co-cultures and hMSCs monocultures. After 21 days of MSDK exposure, OPG and RANKL expression in osteoblasts was measured by western blot in (A) transwell co-cultures, (B) layered co-cultures and (C) hMSCs monocultures. Cell lysates were prepared on day 21 from the bottom (osteoblasts) and top (osteoclasts) chambers in the transwell co-culture and from the whole plate (both osteoblast and osteoclast) in the layered co-culture. Protein levels were normalized against β-actin and then to Os-/MSDK. Mean expression of (i) OPG: RANKL, (ii) OPG and (iii) RANKL in each culture were analyzed, normalized against Os-/MSDK and compared between groups (Os-/MSDK, Os+/Veh, Os+/MSDK).
Effect of MSDK on sOPG and sRANKL expression in osteoblasts grown in transwell co-cultures, layered co-cultures and hMSCs monocultures. Treatments effects on secreted OPG (sOPG) and RANKL (sRANKL) were measured by ELISA in (D) transwells and (E) monocultures. Mean concentrations (pg/mL) of (i) sOPG: sRANKL, (ii) sOPG and (iii) sRANKL in culture media were analyzed, normalized against Os-/MSDK and compared between groups. Treatment effects on RANKL processing were detected via western blot by measuring the mean osteoblast’s expression of (i) 25 KDa and (ii) 24 KDa RANKL fragments in (F) transwells; and (G) 24 KDa RANKL fragment in layered co-cultures. *=p<.05, **=p<.01 and ***=p<.001; One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison t-test (n=6 per group).
Effect of MSDK on MAPKs, ERK1/2 and ERK5. After 21 days of MSDK exposure, western blot was performed to determine (A) ERK1/2 expression of osteoblasts grown as transwell co-cultures, (B) ERK1/2 expression of osteoblasts and osteoclasts grown as layered co-cultures, (C) ERK5 expression of osteoblasts grown as transwell co-cultures.
Effect of MSDK on MAPKs, ERK1/2 and ERK5. (D) ERK5 expression of osteoblasts and osteoclasts grown in layered co-culture, (E) ERK5 expression of osteoblasts in hMSCs mono-culture and (F) phospho-ERK5 expression of osteoclasts grown in transwell co-culture. Cell lysates were prepared on day 21 from the bottom (osteoblasts) and top (osteoclasts) chambers in the transwell co-culture and from the whole plate (both osteoblast and osteoclast) in the layered co-culture. Protein levels were normalized against β-actin and then to Os-/MSDK. Mean expression of (i) phospho-ERK: total-ERK (ii) phospho-ERK (iii) total-ERK in each co-cultures were analyzed and compared between groups (Os-/MSDK, Os+/Veh, Os+/MSDK). *=p<.05, **=p<.01 and **=p<.001; One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison t-test (n=6 per group).
Effect of MSDK on RUNX2, β1 integrin, NFκB and metabolic proteins. After 21 days of MSDK treatment, western blot was performed to determine (A) RUNX2 expression of osteoblasts grown in transwell co-cultures, (B) RUNX2 expression of osteoblasts grown in layered co-culture, (C) β1 integrin expression of osteoblasts grown in transwell co-cultures, (D) β1 integrin expression of osteoblasts and osteoclasts grown in layered co-cultures (E) NFκB expression of osteoclasts grown in transwell co-cultures, and (F) NFκB expression of osteoblasts and osteoclasts grown in layered co-cultures.
Effect of MSDK on RUNX2, β1 integrin, NFκB and metabolic proteins. Metabolic proteins such as PPARγ, GLUT4 and Insulin Rβ expression were also measured in (G) osteoblasts grown in transwell co-cultures and (H) in osteoblasts and osteoclasts grown in layered co-cultures. Cell lysates were prepared on day 21 from the bottom (osteoblasts) and top (osteoclasts) chambers in the transwell co-culture and from the whole plate (both osteoblast and osteoclast) in the layered co-culture. Protein levels were normalized against β-actin and then to Os-/MSDK. Mean protein levels were analyzed and compared between groups (Os-/MSDK, Os+/Veh, Os+/MSDK). *=p<.05, **=p<.01 and **=p<.001; One-way ANOVA followed by Bonferroni’s post-hoc multiple comparison t-test (n=6 per group).
Effect of MSDK on adipose-derived mesenchymal stem cells. Adipose-derived mesenchymal stem cells (AMSCs) were exposed to MSDK for 7 days (A), 8 days (C) or 14 days (B) to assess its effects on alkaline phosphatase (A) or alizarin red staining (B, C). Red color indicates calcium deposition by MSCs. Each bar represents the mean absorbance of alkaline phosphatase activity or alizarin red at 490nm for respective groups Os+/Veh; Os+/GSK126; Os+/MSDK; Os+/GSK126/MSDK repeated twice in triplicate (except for the alizarin red experiment at day 8). GSK 126 = S-adenosyl-methionine-competitive small molecule inhibitor of EZH2 methyltransferase is known to promote osteogenic differentiation of MSCs through effects on the osteoblast epigenome [49,50]. The observed co-stimulatory effects of MSDK and GSK126 suggest mechanistic synergy.
Potential mechanism underlying MSDK effects on bone formation. The diagram illustrates regulatory and biological relationships between the indicated proteins and treatments.