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Pumpkin (Cucurbita ficifolia Bouche) extract attenuate the adipogenesis in human mesenchymal stem cells by controlling adipogenic gene expression.

ABSTRACT: Prevention and management of obesity through dietary modification is one of the top way to trim down its consequences. Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs), into adipocytes. Since food constituents play a major role in the cell differentiation and proliferation, we sought to determine if various extracts of Cucurbita ficifolia (C. ficifolia), could affect the adipogenic differentiation of hMSCs. Flow cytometry analysis with quantitative and qualitative Nile red, and quantitative PCR methods were employed to evaluate the C. ficifolia effect on hMSCs adipogenesis. Results revealed that, chloroform extract exhibits significant adipogenic inhibition than that of hexane and methanol extracts. Chloroform extract treated cells display the down-regulation of ADIPOQ, FABP4, PPARGC1A, CEBPB & LPL and up-regulation of ACACB & CEBPA genes. Further, various phytoconstituents present in the chloroform extract of C. ficifolia were analyzed though LC-MS and GC-MS. Our results indicates that chloroform extract of C. ficifolia might be used as a food supplement to control obesity and its related consequences.

SUBMITTER: Alshammari GM

PROVIDER: S-EPMC6486525 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Pumpkin (Cucurbita ficifolia Bouché) extract attenuate the adipogenesis in human mesenchymal stem cells by controlling adipogenic gene expression.

Alshammari Ghedeir M GM Balakrishnan Aristatile A

Saudi journal of biological sciences 20181002 4

Prevention and management of obesity through dietary modification is one of the top way to trim down its consequences. Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs), into adipocytes. Since food constituents play a major role in the cell differentiation and proliferation, we sought to determine if various extracts of Cucurbita ficifolia (C. ficifolia), could affect the adipogenic differentiation of ...[more]

PMID: 31048999

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Project description:The microenvironment plays a vital role in both the maintenance of stem cells in their undifferentiated state (niche) and their differentiation after homing into new locations outside this niche. Contrary to conventional in-vitro culture practices, the in-vivo stem cell microenvironment is physiologically crowded. We demonstrate here that re-introducing macromolecular crowding (MMC) at biologically relevant fractional volume occupancy during chemically induced adipogenesis substantially enhances the adipogenic differentiation response of human bone marrow-derived mesenchymal stem cells (MSCs). Both early and late adipogenic markers were significantly up-regulated and cells accumulated 25-40% more lipid content under MMC relative to standard induction cocktails. MMC significantly enhanced deposition of extracellular matrix (ECM), notably collagen IV and perlecan, a heparan sulfate proteoglycan. As a novel observation, MMC also increased the presence of matrix metalloproteinase -2 in the deposited ECM, which was concomitant with geometrical ECM remodeling typical of adipogenesis. This suggested a microenvironment that was richer in both matrix components and associated ligands and was conducive to adipocyte maturation. This assumption was confirmed by seeding undifferentiated MSCs on decellularized ECM deposited by adipogenically differentiated MSCs, Adipo-ECM. On Adipo-ECM generated under crowding, MSCs differentiated much faster under a classical differentiation protocol. This was evidenced throughout the induction time course, by a significant up-regulation of both early and late adipogenic markers and a 60% higher lipid content on MMC-generated Adipo-ECM in comparison to standard induction on tissue culture plastic. This suggests that MMC helps build and endow the nascent microenvironment with adipogenic cues. Therefore, MMC initiates a positive feedback loop between cells and their microenvironment as soon as progenitor cells are empowered to build and shape it, and, in turn, are informed by it to respond by attaining a stable differentiated phenotype if so induced. This work sheds new light on the utility of MMC to tune the microenvironment to augment the generation of adipose tissue from differentiating human MSCs.

Project description:BACKGROUND:Mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate via osteogenesis and adipogenesis. The mechanism underlying MSC lineage commitment still remains incompletely elucidated. Understanding the regulatory mechanism of MSC differentiation will help researchers induce MSCs toward specific lineages for clinical use. In this research, we intended to figure out the long noncoding RNA (lncRNA) that plays a central role in MSC fate determination and explore its application value in tissue engineering. METHODS:The expression pattern of lncRNAs during MSC osteogenesis/adipogenesis was detected by microarray and qRT-PCR. Lentivirus and siRNAs were constructed to regulate the expression of lncRNA repressor of adipogenesis (ROA). MSC osteogenesis/adipogenesis was evaluated by western blot and alizarin red/oil red staining. An adipokine array was used to select the paracrine/autocrine factor PTX3, followed by RNA interference or recombinant human protein stimulation to confirm its function. The activation of signaling pathways was also detected by western blot, and a small molecule inhibitor, SCH772984, was used to inhibit the activation of the ERK pathway. The interaction between ROA and hnRNP A1 was detected by RNA pull-down and RIP assays. Luciferase reporter and chromatin immunoprecipitation assays were used to confirm the binding of hnRNP A1 to the PTX3 promotor. Additionally, an in vivo adipogenesis experiment was conducted to evaluate the regulatory value of ROA in tissue engineering. RESULTS:In this study, we demonstrated that MSC adipogenesis is regulated by lncRNA ROA both in vitro and in vivo. Mechanistically, ROA inhibits MSC adipogenesis by downregulating the expression of the key autocrine/paracrine factor PTX3 and the downstream ERK pathway. This downregulation was achieved through transcription inhibition by impeding hnRNP A1 from binding to the promoter of PTX3. CONCLUSIONS:ROA negatively regulates MSC adipogenesis through the hnRNP A1-PTX3-ERK axis. ROA may be an effective target for modulating MSCs in tissue engineering.

Project description:BackgroundAdipogenesis is the developmental process by which mesenchymal stem cells (MSC) differentiate into pre-adipocytes and adipocytes. The aim of the study was to analyze the developmental strategies of human bone marrow MSC developing into adipocytes over a defined time scale. Here we were particularly interested in differentially expressed transcription factors and biochemical pathways. We studied genome-wide gene expression profiling of human MSC based on an adipogenic differentiation experiment with five different time points (day 0, 1, 3, 7 and 17), which was designed and performed in reference to human fat tissue. For data processing and selection of adipogenic candidate genes, we used the online database SiPaGene for Affymetrix microarray expression data.ResultsThe mesenchymal stem cell character of human MSC cultures was proven by cell morphology, by flow cytometry analysis and by the ability of the cells to develop into the osteo-, chondro- and adipogenic lineage. Moreover we were able to detect 184 adipogenic candidate genes (85 with increased, 99 with decreased expression) that were differentially expressed during adipogenic development of MSC and/or between MSC and fat tissue in a highly significant way (p < 0.00001). Subsequently, groups of up- or down-regulated genes were formed and analyzed with biochemical and cluster tools. Among the 184 genes, we identified already known transcription factors such as PPARG, C/EBPA and RTXA. Several of the genes could be linked to corresponding biochemical pathways like the adipocyte differentiation, adipocytokine signalling, and lipogenesis pathways. We also identified new candidate genes possibly related to adipogenesis, such as SCARA5, coding for a receptor with a putative transmembrane domain and a collagen-like domain, and MRAP, encoding an endoplasmatic reticulum protein.ConclusionsComparing differential gene expression profiles of human MSC and native fat cells or tissue allowed us to establish a comprehensive differential kinetic gene expression network of adipogenesis. Based on this, we identified known and unknown genes and biochemical pathways that may be relevant for adipogenic differentiation. Our results encourage further and more focused studies on the functional relevance of particular adipogenic candidate genes.

Dataset Information

Pumpkin (Cucurbita ficifolia Bouche) extract attenuate the adipogenesis in human mesenchymal stem cells by controlling adipogenic gene expression.

Publications

Pumpkin (<i>Cucurbita ficifolia Bouché</i>) extract attenuate the adipogenesis in human mesenchymal stem cells by controlling adipogenic gene expression.

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