A Glimpse into the Genome-wide DNA Methylation Changes in 6-hydroxydopamine-induced In Vitro Model of Parkinson's Disease.

A cell-based model of Parkinson's disease (PD) is a well-established in vitro experimental prototype to investigate the disease mechanism and therapeutic approach for a potential anti-PD drug. The SH-SY5Y human neuroblastoma cells and 6-OHDA combo is one of the many neurotoxininduced neuronal cell models employed in numerous neuroscience-related research for discovering neuroprotective drug compounds. Emerging studies have reported a significant correlation between PD and epigenetic alterations, particularly DNA methylation. However, the DNA methylation changes of PD-related CpG sites on the 6-OHDA-induced toxicity on human neuronal cells have not yet been reported. We performed a genome-wide association study (GWAS) using Infinium Epic beadchip array surveying 850000 CpG sites in differentiated human neuroblastoma cells exposed to 6-OHDA. We identified 236 differentially methylated probes (DMPs) or 163 differentially methylated regions (DMRs) in 6-OHDA treated differentiated neuroblastoma cells than the untreated reference group with p<0.01, Δbeta cut-off of 0.1. Among 236 DMPs, hypermethylated DMPs are 110 (47%), whereas 126 (53%) are hypomethylated. Our bioinformatic analysis revealed 3 DMRs that are significantly hypermethylated and associated with neurological disorders, namely AKT1, ITPR1 and GNG7. This preliminary study demonstrates the methylation status of PD-related CpGs in the 6-OHDA-induced toxicity in the differentiated neuroblastoma cells model.

Adiponectin as a therapeutic target for diabetic foot ulcer.

The global burden of diabetic foot ulcers (DFUs) is a significant public health concern, affecting millions of people worldwide. These wounds cause considerable suffering and have a high economic cost. Therefore, there is a need for effective strategies to prevent and treat DFUs. One promising therapeutic approach is the use of adiponectin, a hormone primarily produced and secreted by adipose tissue. Adiponectin has demonstrated anti-inflammatory and anti-atherogenic properties, and researchers have suggested its potential therapeutic applications in the treatment of DFUs. Studies have indicated that adiponectin can inhibit the production of pro-inflammatory cytokines, increase the production of vascular endothelial growth factor, a key mediator of angiogenesis, and inhibit the activation of the intrinsic apoptotic pathway. Additionally, adiponectin has been found to possess antioxidant properties and impact glucose metabolism, the immune system, extracellular matrix remodeling, and nerve function. The objective of this review is to summarize the current state of research on the potential role of adiponectin in the treatment of DFUs and to identify areas where further research is needed in order to fully understand the effects of adiponectin on DFUs and to establish its safety and efficacy as a treatment for DFUs in the clinical setting. This will provide a deeper understanding of the underlying mechanisms of DFUs that can aid in the development of new and more effective treatment strategies.

Tocotrienol-Rich Fraction and Levodopa Regulate Proteins Involved in Parkinson's Disease-Associated Pathways in Differentiated Neuroblastoma Cells: Insights from Quantitative Proteomic Analysis.

Tocotrienol-rich fraction (TRF), a palm oil-derived vitamin E fraction, is reported to possess potent neuroprotective effects. However, the modulation of proteomes in differentiated human neuroblastoma SH-SY5Y cells (diff-neural cells) by TRF has not yet been reported. This study aims to investigate the proteomic changes implicated by TRF in human neural cells using a label-free liquid-chromatography-double mass spectrometry (LC-MS/MS) approach. Levodopa, a drug used in the treatment of Parkinson's disease (PD), was used as a drug control. The human SH-SY5Y neuroblastoma cells were differentiated for six days and treated with TRF or levodopa for 24 h prior to quantitative proteomic analysis. A total of 81 and 57 proteins were differentially expressed in diff-neural cells following treatment with TRF or levodopa, respectively. Among these proteins, 32 similar proteins were detected in both TRF and levodopa-treated neural cells, with 30 of these proteins showing similar expression pattern. The pathway enrichment analysis revealed that most of the proteins regulated by TRF and levodopa are key players in the ubiquitin-proteasome, calcium signalling, protein processing in the endoplasmic reticulum, mitochondrial pathway and axonal transport system. In conclusion, TRF is an essential functional food that affects differential protein expression in human neuronal cells at the cellular and molecular levels.

Unravelling the neuroprotective mechanisms of carotenes in differentiated human neural cells: Biochemical and proteomic approaches.

Carotenoids, fat-soluble pigments found ubiquitously in plants and fruits, have been reported to exert significant neuroprotective effects against free radicals. However, the neuroprotective effects of total mixed carotenes complex (TMC) derived from virgin crude palm oil have not been studied extensively. Therefore, the present study was designed to establish the neuroprotective role of TMC on differentiated human neural cells against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. The human neural cells were differentiated using retinoic acid for six days. Then, the differentiated neural cells were pre-treated for 24 hr with TMC before exposure to 6-OHDA. TMC pre-treated neurons showed significant alleviation of 6-OHDA-induced cytotoxicity as evidenced by enhanced activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes. Furthermore, TMC elevated the levels of intra-neuronal dopamine and tyrosine hydroxylase (TH) in differentiated neural cells. The 6-OHDA induced overexpression of α-synuclein was significantly hindered in neural cells pre-treated with TMC. In proteomic analysis, TMC altered the expression of ribosomal proteins, α/ß isotypes of tubulins, protein disulphide isomerases (PDI) and heat shock proteins (HSP) in differentiated human neural cells. The natural palm phytonutrient TMC is a potent antioxidant with significant neuroprotective effects against free radical-induced oxidative stress.

6-Hydroxydopamine Induces Neurodegeneration in Terminally Differentiated SH-SY5Y Neuroblastoma Cells via Enrichment of the Nucleosomal Degradation Pathway: a Global Proteomics Approach.

The SH-SY5Y human neuroblastoma cells have been used for decades as a cell-based model of dopaminergic neurons to explore the underlying science of cellular and molecular mechanisms of neurodegeneration in Parkinson's disease (PD). However, data revealing the protein expression changes in 6-OHDA induced cytotoxicity in differentiated SH-SY5Y cells remain void. Therefore, we investigated the differentially regulated proteins expressed in terminally differentiated SH-SY5Y cells (differ-SH-SY5Y neural cells) exposed to 6-hydroxydopamine (6-OHDA) using the LC-MS/MS technology and construed the data using the online bioinformatics databases such as PANTHER, STRING, and KEGG. Our studies demonstrated that the neuronal development in differ-SH-SY5Y neural cells was indicated by the overexpression of proteins responsible for neurite formations such as calnexin (CANX) and calreticulin (CALR) besides significant downregulation of ribosomal proteins. The enrichment of the KEGG ribosome pathway was detected with significant downregulation (p < 0.05) of all the 21 ribosomal proteins in differ-SH-SY5Y neural cells compared with undifferentiated cells. Whereas in the PD model, the pathological changes induced by 6-OHDA were indicated by the presence of unfolded and misfolded proteins, which triggered the response of 10 kDa heat shock proteins (HSP), namely HSPE1 and HSPA9. Moreover, the 6-OHDA-induced neurodegeneration in differ-SH-SY5Y neural cells also upregulated the voltage-dependent anion-selective channel protein 1 (VDAC1) protein and enriched the KEGG systemic lupus erythematosus (SLE) pathway that was regulated by 17 histone proteins (p < 0.05) in differ-SH-SY5Y neural cells. These results suggest that the nucleosomal degradation pathway may have regulated the 6-OHDA induced neurodegeneration in PD cell-based model, which is reflected by increased apoptosis and histone release in differ-SH-SY5Y neural cells.

Tocotrienols protect differentiated SH-SY5Y human neuroblastoma cells against 6-hydroxydopamine-induced cytotoxicity by ameliorating dopamine biosynthesis and dopamine receptor D2 gene expression.

Oxidative stress is a critical factor that triggers a "domino" cascade of events leading to the degeneration of dopaminergic neurons in Parkinson disease. Tocotrienols (T3) have antioxidant effects and can protect neuronal cells against oxidative damage. In the present study, we investigated the neuroprotective effects of different forms of T3 (alpha, delta, gamma) or tocotrienol-rich fraction (TRF) against 6-hydroxydopamine (6-OHDA)-induced oxidative damage in differentiated SH-SY5Y human neural cells. Differentiating the SH-SY5Y cells with retinoic acid and a low-serum culture medium for 6 days allowed development of human dopamine-like neural cells. Subsequently, the differentiated SH-SY5Y neural cells were pretreated with different forms of T3 for 24 hours before these cells were exposed to 6-OHDA. The T3 analogues and TRF displayed neuroprotective effects (P < .05) via restoration of cell viability and activation of antioxidant enzymes (e.g., superoxide dismutase, catalase). Notably, TRF was highly efficient in scavenging reactive oxygen species and upregulating dopamine and tyrosine hydroxylase levels in the differentiated SH-SY5Y cells. Gamma-T3 exhibited the most potent effects in attenuating apoptosis, whereas alpha-T3 was most effective in preventing 6-OHDA-induced leakage of α-Synuclein. Delta-T3 displayed a noticeable effect in upregulating the dopamine receptor D2 gene expression compared with controls. These findings suggest T3 isoforms and TRF demonstrate significant neuroprotective effects in protecting differentiated neural cells against 6-OHDA-mediated oxidative stress.

Influence of serum concentration in retinoic acid and phorbol ester induced differentiation of SH-SY5Y human neuroblastoma cell line.

Numerous protocols to establish dopaminergic phenotype in SH-SY5Y cells have been reported. In most of these protocols there are variations in concentration of serum used. In this paper, we compared the effects of high (10%), low (3%) and descending (2.5%/1%) serum concentration in differentiation medium containing different proportion of retinoic acid (RA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) or RA-only on the undifferentiated SH-SY5Y cells with regards to cell morphology, biochemical and gene expression alterations. Cells differentiated in culture medium containing low and descending serum concentrations showed increased number of neurite projections and reduced proliferation rates when compared to undifferentiated cells. The SH-SY5Y cells differentiated in culture medium containing 3% RA and low serum or descending (2.5%/1% RA/TPA) were found to be more susceptible to 6-hydroxydopamine (6-OHDA) induced cytotoxicity. Cells differentiated with RA/TPA or RA differentiated showed increased production of the α-synuclein (SNCA) neuroprotein and dopamine neurotransmitter compared to undifferentiated cells, regardless serum concentrations used. There was no significant difference in the expression of tyrosine hydroxylase (TH) gene between undifferentiated and differentiated SH-SY5Y cells. However, the expression of dopamine receptor D2 (DRD2) gene was markedly increased (p<0.05) in differentiated cells with 3% serum and RA only when compared to undifferentiated cells. In conclusion, to terminally differentiate SH-SY5Y cells to be used as a cell-based model to study Parkinson's disease (PD) to investigate molecular mechanisms and drug discovery, the optimal differentiation medium should contain 3% serum in RA-only.

CD40 polymorphism in cervical carcinoma in a subset of Malaysian population.

AIM: The aim of this study was to determine the allelic frequency of single nucleotide polymorphisms (SNPs) in the human CD40 gene in cervical cancer. METHODS: A total of 200 cases were selected from the records of the Department of Pathology, Hospital Tuanku Jaafar, Seremban, Malaysia. The samples were collected in three separate groups: cervicitis (n = 61), cervical intraepithelial neoplasia (n = 69), and cervical carcinoma (n = 70). The patients' demographic data and the respective paraffin-embedded tissue samples from Hospital Tuanku Jaafar, Seremban were obtained upon consent. The sample tissues were submitted for DNA extraction using G-spin Total DNA Extraction Kit. DNA obtained was then submitted for nested PCR before restriction enzyme digestion. RESULTS: SNP rs1883832 showed higher prevalence of T alleles in the cervical carcinoma group compared to the control groups and in rs3765459, a higher prevalence of G alleles in the cervical carcinoma group was noted. The results of rs1800686 and rs4810485 were insignificant. CONCLUSION: The data from our study indicates a potential association between the rs1883832 and rs3765459 CD40 gene polymorphism and susceptibility to cervical cancer.

Ethnic differences in the prognostic utility of rheumatoid factor isotypes and anticyclic citrullinated peptides in rheumatoid arthritis patients: a cross-sectional study.

OBJECTIVES: The prognostic significance of rheumatoid factor (RF) and anticyclic citrullinated peptide antibody (anti-CCP) in rheumatoid arthritis (RA) remains contentious due to the conflicting lines of evidence. This study aims to determine the association between RF isotypes and anti-CCP with disease severity in RA patients from three ethnic groups. METHODS: A total of 147 RA patients from three different ethnic groups (Malays, Chinese, and Indians) who fulfilled the 1987 American College of Rheumatology (ACR) revised criteria for RA were recruited into this study. The seroprevalence of RF isotypes immunoglobulin (Ig)A, IgG, and IgM, as well as anti-CCP was determined using commercial enzyme-linked immunosorbent assay (ELISA) kits. Multinomial regression analysis was performed to assess the independent effects of autoantibody status on the development of deforming and erosive RA and the presence of extra-articular manifestations (EAM). RESULTS: In Chinese patients, we found a significant association (p < 0.05) between IgG RF and anti-CCP and the presence of erosive disease, as well as IgM RF and IgG RF with the presence of joint deformities. In Indian patients, IgM RF was associated with deforming disease, whereas none of the antibodies were associated with disease severity in Malay patients. Multinomial regression analysis revealed that IgG RF was the most important predictor variable for erosive disease in Chinese patients, and IgM RF the only predictor variable associated with deforming disease in both Chinese and Indian RA patients. CONCLUSIONS: There is variability in the phenotypic association of RF isotypes and anti-CCP in relation to disease severity of RA in the three ethnic groups. RF, in particular, IgG and IgM, may be better prognosticators of severe disease in Chinese and Indian patients.