Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts

<p>Abstract</p> <p>Background</p> <p>Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H₂O₂-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.</p> <p>Results</p> <p>HDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal) activity. However the expression level of GAPDH was consistent in all treatment groups.</p> <p>Conclusion</p> <p>The study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.</p

The effects of various growth factors on the proliferation of human nasal septum chondrocytes and reconstruction of human cartilage via tissue engineering technology

Tissue loss and organ failure are one of the costly health care management in all over the world. One of the recent strategies for the treatment of organ failure and damaged tissues is via tissue engineering technology. The primary goal of all approaches in tissue engineering is to repair and restore tissue functions through the delivery of living elements which become integrated into the patient (Langer and Vacanti, 1993)

Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells

OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were divided into four groups: control, treatment with 180 &#956;M hydrogen peroxide (H2O2), treatment with 150 &#956;g/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs

Osteogenic potential of human adipose derived stem cell co-culture with human osteoblast on titanium dioxide nanofibrous surface

The study aims to evaluate the osteogenic potential of human adipose derived stem cell (HADSC) co-culture with human osteoblast (HOB) using selected HADSC/HOB ratio of 2:1, 1:1 and 1:2, respectively. The HADSC/HOB was seeded on Titanium dioxide (TiO2) coated with or without nanofibre substrate. The non-coated TiO2 was used as control. The effects of TiO2 based scaffolds on cell adhesion were characterized by scanning electron microscopy (SEM). Cell viability, differentiation and mineralization were assessed by Alamar Blue, alkaline phosphatase (ALP) and Alizarin Red assays, respectively. The combination of HADSC/HOB, 2:1 ratio, seeded on nanofibrous-coated TiO2 showed better cell adhesion, viability, differentiation and mineralization than the other groups. This study offers opportunity to assess in vitro cellular development of HADSC through direct cell to cell contact with HOB. This study indicates that the co-cultured HADSC/HOB seeded on TiO2 based scaffolds may serve as a promising approach to facilitate osteogenic differentiation activity

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction

Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of &#947;-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with &#947;-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with &#947;-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. &#947;-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: &#947;-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression

Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts

This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G0/G1 phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G0/G1 phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence

Pharmacotherapy of alzheimer’s disease: seeking clarity in a time of uncertainty

Alzheimer’s disease (AD) is recognized as a major health hazard that mostly affects people older than 60 years. AD is one of the biggest medical, economic, and social concerns to patients and their caregivers. AD was ranked as the 5th leading cause of global deaths in 2016 by the World Health Organization (WHO). Many drugs targeting the production, aggregation, and clearance of Aβ plaques failed to give any conclusive clinical outcomes. This mainly stems from the fact that AD is not a disease attributed to a single-gene mutation. Two hallmarks of AD, Aβ plaques and neurofibrillary tangles (NFTs), can simultaneously induce other AD etiologies where every pathway is a loop of consequential events. Therefore, the focus of recent AD research has shifted to exploring other etiologies, such as neuroinflammation and central hyperexcitability. Neuroinflammation results from the hyperactivation of microglia and astrocytes that release pro-inflammatory cytokines due to the neurological insults caused by Aβ plaques and NFTs, eventually leading to synaptic dysfunction and neuronal death. This review will report the failures and side effects of many anti-Aβ drugs. In addition, emerging treatments targeting neuroinflammation in AD, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and receptor-interacting serine/threonine protein kinase 1 (RIPK1), that restore calcium dyshomeostasis and microglia physiological function in clearing Aβ plaques, respectively, will be deliberately discussed. Other novel pharmacotherapy strategies in treating AD, including disease-modifying agents (DMTs), repurposing of medications used to treat non-AD illnesses, and multi target-directed ligands (MTDLs) are also reviewed. These approaches open new doors to the development of AD therapy, especially combination therapy that can cater for several targets simultaneously, hence effectively slowing or stopping AD

The effect of Stichopus chloronotus aqueous extract on human osteoarthritis articular chondrocytes in three-dimensional collagen type I hydrogel in vitro

Autologous chondrocyte-seeded scaffolds have proved to be one of the most promising alternative therapies for articular cartilage defects. However, the chondrocytes have specific nutritional requirements and risk of dedifferentiation during in vitro expansion. Stichopus chloronotus aqueous extract (SCAE) was investigated as a medium supplement for three-dimensional (3D) collagen type I hydrogel scaffold seeded with chondrocytes to determine whether SCAE is capable of maintaining phenotype and sustaining extracellular matrix synthesis and deposition. Human osteoarthritis articular chondrocytes were isolated from the knee joint cartilage of patients underwent total knee replacement surgery. Human osteoarthritis articular chondrocytes were encapsulated in collagen type I hydrogel and cultured in basic medium with 0, 0.1 and 0.2% of SCAE. The chondrocytes in 3D culture were evaluated by means cell morphology and proliferation, quantitative phenotypic expression of collagen type I, II, aggrecan core protein and sox-9. H&E, toluidine blue staining and sulfated glycosaminoglycan (sGAG) production were analyzed after 7 days in culture. Chondrocytes cultured in 3D with various SCAE concentration appeared with polygonal morphology maintaining their chondrocytes characteristic. SCAE supplementation promoted chondrocytes proliferation and the ability of the cells to express gene encoding collagen type I decreased, whereas their ability to express collagen type II, aggrecan core protein and sox9 increased as compared to control. The cartilaginous matrices were positively stained toluidine blue concomitant with higher sGAG accumulation in SCAE-supplemented culture medium. This study shown that SCAE may be beneficial for in vitro development of 3D chondrocytes culture for use in cartilage tissue engineering therapies

Tocotrienol Attenuates Stress-Induced Gastric Lesions via Activation of Prostaglandin and Upregulation of COX-1 mRNA

The present study aims to distinguish the effect of tocotrienol on an important gastric protective factor, prostaglandin E2 (PGE2), in stress-induced gastric injury. Twenty-eight Wistar rats were divided into four groups of seven rats each. Two control groups were fed commercial rat diet, and two treatment groups were fed the same diet but with additional dose of omeprazole (20 mg/kg) or tocotrienol (60 mg/kg). After 28 days, rats from one control group and both treated groups were subjected to water-immersion restraint stress for 3.5 hours once. The rats were then sacrificed, their stomach isolated and gastric juice collected, lesions examined, and gastric PGE2 content and cyclooxygenase (COX) mRNA expression were determined. Both the regimes significantly attenuated the total lesion area in the stomach compared to the control. Gastric acidity, which was increased in stress, was significantly reduced in rats supplemented with omeprazole and tocotrienol. The PGE2 content was also significantly higher in the rats given tocotrienol supplementation compared to the control followed by an increase in COX-1 mRNA expression. We conclude that tocotrienol supplementation protected rat gastric mucosa against stress-induced lesions possibly by reducing gastric acidity and preserving gastric PGE2 by increasing COX-1 mRNA