Retinoic acid enhances ovarian steroidogenesis by regulating granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway.

INTRODUCTION: Ovarian steroidogenesis not only affects the embryonic development and pregnancy outcome, but also associates with many diseases in mammals and women. Exploring the nutrients and mechanisms influencing ovarian steroidogenesis is critical to maintaining the optimal reproductive performance, as well as guaranteeing body health. OBJECTIVES: This research aimed to explore the effect of retinol metabolism on ovarian steroidogenesis and the underlying mechanisms. METHODS: Comparative transcriptomic analysis of ovaries from normal and low reproductive performance sows were performed to identify the main causes leading to low fertility. The metabolites regulating steroid hormones synthesis were investigated in ovarian granulosa cells. Gene interference, overexpression, dual-luciferase reporter assays, chromatin immunoprecipitation and transcriptome analysis were further conducted to explore the underlying mechanisms of Aldh1a1 mediating ovarian steroidogenesis. RESULTS: Transcriptome analysis of ovaries from normal and low reproductive performance sows showed the significant differences in both retinol metabolism and steroid hormones synthesis, indicating retinol metabolism probably influenced steroid hormones synthesis. The related metabolite retinoic acid was furtherly proven a highly active and potent substance strengthening estrogen and progesterone synthesis in ovarian granulosa cells. For the first time, we revealed that retinoic acid synthesis in porcine and human ovarian granulosa cells was dominated by Aldh1a1, and required the assistance of Aldh1a2. Importantly, we demonstrated that Aldh1a1 enhanced the proliferation of ovarian granulosa cells by activating PI3K-Akt-hedgehog signaling pathways. In addition, Aldh1a1 regulated the expression of transcription factor MESP2, which targeted the transcription of Star and Cyp11a1 through binding to corresponding promoter regions. CONCLUSION: Our data identified Aldh1a1 modulates ovarian steroidogenesis through enhancing granulosa cell proliferation and MESP2/STAR/CYP11A1 pathway. These findings provide valuable clues for improving ovarian health in mammals.

Nanoparticles Targeting Delivery Antagomir-483-5p to Bone Marrow Mesenchymal Stem Cells Treat Osteoporosis by Increasing Bone Formation.

BACKGROUND: Promoting bone marrow mesenchymal stem cell (BMSC) osteoblastic differentiation is a promising therapeutic strategy for osteoporosis (OP). The present study demonstrates that miR- 483-5p inhibits the osteogenic differentiation of BMSCs. Therefore, selectively delivering the nanoparticles carrying antagomir-483-5p (miR-483-5p inhibitor) to BMSCs is expected to become an effective treatment drug for OP. METHODS: Real-time PCR assays were used to analyze miR-483-5p, ALP and Bglap levels in BMSCs of ovariectomized and aged osteoporotic mice. Immunoglobulin G and poloxamer-188 encapsulated the functional small molecules, and a BMSC-targeting aptamer was employed to confirm the direction of the nanoparticles to selectively and efficiently deliver antagomir-483-5p to BMSCs in vivo. Luciferase assays were used to determine the target genes of miR-483-5p. Western blot assays and immunohistochemistry staining were used to detect the targets in vitro and in vivo. RESULTS: miR-483-5p levels were increased in BMSCs of ovariectomized and aged osteoporotic mice. Inhibiting miR-483-5p levels in BMSCs by antagomir-483-5p in vitro promoted the expression of bone formation markers, such as ALP and Bglap. The FAM-BMSC-aptamer-nanoparticles carrying antagomir- 483-5p were taken up by BMSCs, resulting in stimulation of BMSC osteoblastic differentiation in vitro and osteoporosis prevention in vivo. Furthermore, our research demonstrated that mitogen-activated protein kinase 1 (MAPK1) and SMAD family member 5 (Smad5) were direct targets of miR-483-5p in regulating BMSC osteoblastic differentiation and osteoporosis pathological processes. CONCLUSIONS: The important therapeutic role of FAM-BMSC-aptamer-nanoparticles carrying antagomir- 483-5p in osteoporosis was established in our study. These nanoparticles are a novel candidate for the clinical prevention and treatment of osteoporosis. The optimized, targeted drug delivery platform for small molecules will provide new ideas for treating clinical diseases.

DDIT3 regulates key enzymes in the methionine cycle and flux during embryonic development.

One-carbon metabolism is a key metabolic network that integrates nutritional signals with embryonic development. However, the response of one-carbon metabolism to methionine status and the regulatory mechanisms are poorly understood. Herein, we found that methionine supplementation during pregnancy significantly increased fetal number and average fetal weight. In addition, methionine modulated one-carbon metabolism primarily through 2 metabolic enzymes, cystathionine ß-synthase (CBS) and methionine adenosyltransferase 2A (MAT2A), which were significantly increased in fetal liver tissues and porcine trophoblast (pTr) cells in response to proper methionine supplementation. CBS and MAT2A overexpression enhanced the DNA synthesis in pTr cells. More importantly, we identified a transcription factor, DNA damage-inducible transcript 3 (DDIT3), that was the primary regulator of CBS and MAT2A, which bound directly to promoters and negatively regulated the expression of CBS and MAT2A. Taken together, our findings identified that DDIT3 targeting CBS and MAT2A was a novel regulatory pathway that mediated cellular one-carbon metabolism in response to methionine signal and provided promising targets to improve pregnancy health.

The Combined Use of Medium- and Short-Chain Fatty Acids Improves the Pregnancy Outcomes of Sows by Enhancing Ovarian Steroidogenesis and Endometrial Receptivity.

Fatty acids play important roles in maintaining ovarian steroidogenesis and endometrial receptivity. Porcine primary ovarian granulosa cells (PGCs) and endometrial epithelial cells (PEECs) were treated with or without medium- and short-chain fatty acids (MSFAs) for 24 h. The mRNA abundance of genes was detected by fluorescence quantitative PCR. The hormone levels in the PGCs supernatant and the rate of adhesion of porcine trophoblast cells (pTrs) to PEECs were measured. Sows were fed diets with or without MSFAs supplementation during early gestation. The fecal and vaginal microbiomes were identified using 16S sequencing. Reproductive performance was recorded at parturition. MSFAs increased the mRNA abundance of genes involved in steroidogenesis, luteinization in PGCs and endometrial receptivity in PEECs (p < 0.05). The estrogen level in the PGC supernatant and the rate of adhesion increased (p < 0.05). Dietary supplementation with MSFAs increased serum estrogen levels and the total number of live piglets per litter (p < 0.01). Moreover, MSFAs reduced the fecal Trueperella abundance and vaginal Escherichia-Shigella and Clostridium_sensu_stricto_1 abundance. These data revealed that MSFAs improved pregnancy outcomes in sows by enhancing ovarian steroidogenesis and endometrial receptivity while limiting the abundance of several intestinal and vaginal pathogens at early stages of pregnancy.

Frugoside delays osteoarthritis progression via inhibiting miR-155-modulated synovial macrophage M1 polarization.

OBJECTIVES: Direct inhibition of M1 polarization of synovial macrophages may be a useful therapeutic treatment for OA and OA-associated synovitis. Frugoside (FGS) is a cardiac glycoside compound isolated and extracted from Calotropis gigantea. Cardiac glycosides possess interesting anti-inflammatory potential. However, the corresponding activity of FGS has not been reported. Therefore, our aim was to find direct evidence of the effects of FGS on synovial macrophage M1 polarization and OA control. METHODS: Collagenase was used to establish an experimental mouse OA model (CIOA) with considerable synovitis. Then, FGS was intra-articular administered. The mRNA and protein levels of iNOS were analysed by real-time PCR and Western blotting in vitro. Immunohistochemical and immunofluorescence staining were used to measure the expression of F4/80, iNOS, Col2α1 and MMP13 in vivo. The levels of pro-inflammatory cytokines in FGS-treated M1 macrophage culture supernatants were analysed by flow cytometry. RESULTS: FGS attenuates synovial inflammation and delays the development of OA in CIOA mice. Further results demonstrate that FGS inhibits macrophage M1 polarization in vitro and in vivo, which subsequently decreases the secretion of IL-6 and TNF-α, in turn delaying cartilage and extracellular matrix (ECM) degradation and chondrocyte hypertrophy. FGS inhibits macrophage M1 polarization by partially downregulating miR-155 levels. CONCLUSION: This study demonstrates that intra-articular injection of FGS is a potential strategy for OA prevention and treatment, even at an early stage of disease progression. This is a novel function of FGS and has promising future clinical applications.

Butyrate drives the acetylation of histone H3K9 to activate steroidogenesis through PPARγ and PGC1α pathways in ovarian granulosa cells.

Maintaining ovarian steroidogenesis is of critical importance, considering that steroid hormones are required for successful establishment and maintenance of pregnancy and proper development of embryos and fetuses. Investigating the mechanism that butyrate modulates the ovarian steroidogenesis is beneficial for understanding the impact of lipid nutrition on steroidogenesis. Herein, we identified that butyrate improved estradiol and progesterone synthesis in rat primary ovarian granulosa cells and human granulosa KGN cells and discovered the related mechanism. Our data indicated that butyrate was sensed by GPR41 and GPR43 in ovarian granulosa cells. Butyrate primarily upregulated the acetylation of histone H3K9 (H3K9ac). Chromatin immune-precipitation and sequencing (ChIP-seq) data of H3K9ac revealed the influenced pathways involving in the mitochondrial function (including cellular metabolism and steroidogenesis) and cellular antioxidant capacity. Additionally, increasing H3K9ac by butyrate further stimulated the PPARγ/CD36/StAR pathways to increase ovarian steroidogenesis and activated PGC1α to enhance mitochondrial dynamics and alleviate oxidative damage. The improvement in antioxidant capacity and mitochondrial dynamics by butyrate enhanced ovarian steroidogenesis. Collectively, butyrate triggers histone H3K9ac to activate steroidogenesis through PPARγ and PGC1α pathways in ovarian granulosa cells.

CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

OBJECTIVES: Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected. MATERIALS AND METHODS: We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine ß-synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells. RESULTS: We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine-mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway-associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S-adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus. CONCLUSIONS: Taken together, these studies demonstrate that CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

MicroRNA-9 overexpression suppresses vulnerable atherosclerotic plaque and enhances vascular remodeling through negative regulation of the p38MAPK pathway via OLR1 in acute coronary syndrome.

Acute coronary syndrome (ACS) is characterized by atherosclerotic plaque rupture with a high incidence of recurrent ischemic events. Several microRNAs are found to be aberrantly expressed in atherosclerotic plaques. This study aims to investigate the effects of microRNA-9 (miR-9) on vulnerable atherosclerotic plaque and vascular remodeling in ACS and underlying mechanisms. Microarray-based gene expression profiling was used to identify differentially expressed genes related to ACS and regulatory miRNAs. Oxidized low-density lipoprotein (lectin-like) receptor 1 (OLR1) was identified to be aberrantly activated in ACS and regulated by miR-9. OLR1 was verified as a target gene of miR-9 by bioinformatics prediction and dual luciferase reporter gene assay. The atherosclerotic models were induced in ApoE-/- mice, in which the agomir or antagomir of miR-9, or small interfering RNA (siRNA) against OLR1 were separately introduced. Serum lipid levels and expression of vascular remodeling and inflammatory response-related factors were determined, respectively. On the basis of the obtained results, in the atherosclerosis mice treated with the agomir of miR-9 and siRNA against OLR1, the p38-mitogen-activated protein kinase (p38MAPK) pathway was inhibited; levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol, tumor necrosis factor-α, interleukin-6, and vascular endothelial growth factor were reduced, but the high-density lipoprotein cholesterol level was increased, along with decreased vulnerable atherosclerotic plaque area and enhanced vascular remodeling. Taken together, these findings suggested an inhibitory role miR-9 acts in the formation of vulnerable atherosclerotic plaques in ACS mice, along with a promoted vascular remodeling, via a negative feedback regulation of OLR1-mediated p38MAPK pathway.

Maternal short and medium chain fatty acids supply during early pregnancy improves embryo survival through enhancing progesterone synthesis in rats.

Exploring strategies to prevent miscarriage in women or early pregnancy loss in mammals is of great importance. Manipulating maternal lipid metabolism to maintain sufficient progesterone level is an effective way. To investigated the embryo loss and progesterone synthesis impacts of short and medium chain fatty acids on the lipid metabolism, pregnancy outcome and embryo implantation were investigated in rats fed the pregnancy diets supplemented without or with 0.1% sodium butyrate (SB), 0.1% sodium hexanoate (SH), or 0.1% sodium caprylate (SC) during the entire pregnancy and early pregnancy, respectively, followed with evaluation of potential mechanisms. Maternal SB, SH, or SC supply significantly improved live litter size and embryo implantation in rats. Serum progesterone, arachidonic acid, and phospholipid metabolites levels were significantly increased in response to maternal SB, SH, and SC supply. The expression of key genes involved in ovarian steroidogenesis and granulosa cell luteinization were elevated in ovaries and primary cultured granulosa cells, including cluster of differentiation 36 (CD36), steroidogenic acute regulatory protein (StAR), and cholesterol side-chain cleavage enzyme (CYP11A1). Additionally, the expression of lysophosphatidic acid receptor 3 (LPA3) and cyclooxygenase-2 (COX2) related with phospholipid metabolism were enhanced in uterus in vivo and in in vitro cultured uterine tissue. In conclusion, maternal SB, SH and SC supply reduced early pregnancy loss through modulating maternal phospholipid metabolism and ovarian progesterone synthesis in rats. Our results have important implications that short or medium chain fatty acids have the potential to prevent miscarriage in women or early pregnancy loss in mammals.

Metabolic disorder of amino acids, fatty acids and purines reflects the decreases in oocyte quality and potential in sows.

Oocyte quality is closely related to female fertility. Nevertheless, core nutritional metabolites influencing oocyte quality are unclear. Herein, comprehensive metabolomics analysis of follicular fluid, serum, and urine from low reproductive performance (LRP) and normal reproductive performance (NRP) sows was conducted. Twenty-seven, fourteen and sixteen metabolites (involved in metabolism of amino acids, fatty acids, purine and pyrimidine) were altered in follicular fluid, serum and urine, respectively, in LRP compared with NRP sows, and could decrease oocyte quality and developmental potential, ultimately leading to low fertility. Deoxyinosine, guanidine acetate, thymidine, 5,6-epoxy-eicosatrienoic acid, carnosine, docosahexaenoic acid and carbamoyl phosphate in follicular fluid, cysteine, carnitine, serotonin, hypoxanthine, valine and arginine in serum, as well as carnitine, phenyl glycine, N-acetyl glutamine, propionyl carnitine and choline in urine could be selected as diagnostic markers to indicate oocyte quality. Consistent with metabolomics data, we confirmed changes in concentrations of fatty acids and amino acids in follicular fluid. Targeting purine metabolism, elevating levels of deoxyinosine in in-vitro maturation medium of porcine oocyte significantly promoted the blastocyst rate. Collectively, this study provided new information of potential targets for predicting oocyte quality and developmental potential, and may help with strategies for early diagnosis or therapeutic/dietary intervention in improving reproductive outcomes.

Artesunate suppresses RANKL-induced osteoclastogenesis through inhibition of PLCγ1-Ca2+-NFATc1 signaling pathway and prevents ovariectomy-induced bone loss.

Bone lytic diseases including osteoporosis, rheumatoid arthritis, and bone metastatic tumors affect hundreds of millions people worldwide. Targeting over-activated osteoclasts as an anti-resorptive treatment becomes an important strategy to treat osteolytic diseases. Artesunate is a compound derived from artemisinin (qinghaosu) and has been used to treat malaria and rheumatoid arthritis clinically in China, but its role in osteolysis is unknown. Here, we found that artesunate could suppress RANKL-induced osteoclastogenesis and bone resorption from 1.56 to 12.5µM. Artesunate obviously reduced RANKL-induced NF-κB-luc activity at 50µM, but had no effects on RANKL-induced NF-κB activation (NF-κB luciferase activity, IκB-α degradation and nuclear NF-κB p65 protein level) from 3.125 to 12.5µM in pre-osteoclastic RAW264.7 cells. Interestingly, artesunate could significantly inhibit RANKL-induced NFATc1 activation measured by NFAT luciferase activity, NFATc1 mRNA and nuclear NFATc1 protein levels from 3.125 to 12.5µM. Further study revealed that artesunate inhibited RANKL up-regulated PLCγ1 activation, intracellular calcium, and calcineurin (PP2B-Aα) protein expression from 3.125 to 12.5µM. In addition, the NFATc1 targeted osteoclast-specific genes expression including cathepsin K, MMP-9, and TRAP was reduced by artesunate. Finally, we showed that artesunate was able to reverse the bone loss in an ovariectomized mouse model in vivo accompanied with reduced RANKL, RANKL/OPG, and TRAP-5b levels. This study indicates that artesunate inhibits RANKL-induced osteoclastogenesis and bone loss by inhibiting PLCγ1-Ca2+-calcineurin-NFATc1 pathway. Collectively, our data suggest that artesunate is a potential treatment option against RANKL-mediated osteolytic bone disease.

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

AIM: Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. METHODS: The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. RESULTS: Aconine (0.125, 0.25 µmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, ß3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. CONCLUSION: These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Sinomenine induces apoptosis in RAW 264.7 cell-derived osteoclasts in vitro via caspase-3 activation.

AIM: Sinomenine (SIN) is an alkaloid found in the roots and stems of Sinomenium acutum, which has been used to treat rheumatic arthritis in China and Japan. In this study we investigated the effects of SIN on osteoclast survival in vitro and the mechanisms of the actions. METHODS: Mature osteoclasts were differentiated from murine monocyte/macrophage cell line RAW264.7 through incubation in the presence of receptor activator of NF-κB ligand (RANKL, 100 ng/mL) for 4 d. The cell viability was detected using the CCK-8 method. The survival and actin ring construction of the osteoclasts were scored using TRACP staining and phalloidin-FITC staining, respectively. The apoptosis of the osteoclasts was detected by DNA fragmentation and Hoechst 33258 staining, and the cell necrosis was indicated by LDH activity. The activation of caspase-3 in osteoclasts was measured using Western blotting and the caspase-3 activity colorimetric method. RESULTS: SIN (0.25-2 mmol/L) inhibited the viability of mature osteoclasts in dose-dependent and time-dependent manners, but did not affect that of RAW264.7 cells. Consistently, SIN dose-dependently suppressed the survival of mature osteoclasts. The formation of actin ring, a marker associated with actively resorbing osteoclasts, was also impaired by the alkaloid. SIN (0.5 mmol/L) induced the apoptosis of mature osteoclasts, which was significantly attenuated in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. SIN increased the cleavage of caspase-3 in mature osteoclasts in dose-dependent and time-dependent manners. Furthermore, SIN dose-dependently enhanced caspase-3 activity, which was blocked in the presence of Ac-DEVD-CHO. CONCLUSION: Sinomenine inhibits osteoclast survival in vitro through caspase-3-mediated apoptosis, thus it is a potential agent for treating excessive bone resorption diseases.

Sinomenine suppresses osteoclast formation and Mycobacterium tuberculosis H37Ra-induced bone loss by modulating RANKL signaling pathways.

Receptor activator of NF-κB ligand (RANKL) is essential for osteoclastogenesis. Targeting RANKL signaling pathways has been an encouraging strategy for treating lytic bone diseases such as osteoporosis and rheumatoid arthritis (RA). Sinomenine (SIN), derived from Chinese medicinal plant Sinomenioumacutum, is an active compound to treat RA, but its effect on osteoclasts has been hitherto unknown. In the present study, SIN was found to ameliorate M. tuberculosis H37Ra (Mt)-induced bone loss in rats with a decreased serum level of TRACP5b and RANKL, and an increased level of osteoprotegerin (OPG). In vitro study also showed that SIN could inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, MMP-9, TRACP were inhibited by SIN in a dose dependent manner. Signal transduction studies showed that SIN could obviously reduce the expression of RANK adaptor molecule TRAF6 and down-regulate RANKL-induced NF-κB activation. It decreased the RANKL-induced p38, JNK posphorylation but not ERK1/2 posphorylation. SIN could also reduce RANKL-mediated calcium influx which is associated with TRAF6/c-Src complex. Finally, SIN suppressed RANKL induced AP-1 and NFAT transcription, as well as the gene expression of NFATc1 and AP-1 components (Fra-1, Fra-2, c-Fos). The protein expression of c-Fos and TRAF6 were also inhibited by SIN after RANKL stimulation. Taken together, SIN could attenuate osteoclast formation and Mt-induced bone loss by mediating RANKL signaling pathways.