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Proliferation study and microscopy evaluation on the effects of tannic acid in human fetal osteoblast cell line (hFOB 1.19)

Hapidin H, Romli NAA, Abdullah H

Microsc Res Tech, 2019 Nov;82(11):1928-1940.
PMID: 31423711 DOI: 10.1002/jemt.23361

Tannic acid (TA) is a phenolic compound that might act directly on osteoblast metabolism. The study was performed to investigate the effects of TA on the proliferation, mineralization, and morphology of human fetal osteoblast cells (hFOB 1.19). The cells were divided into TA-treated, untreated, and pamidronate-treated (control drug) groups. Half maximal effective concentration (EC50 ) values for TA and pamidronate were measured using MTT assay. The EC50 of hFOB 1.19 cells treated with TA was 2.94 M. This concentration was more effective compared to the pamidronate (15.27 M). Cell proliferation assay was performed to compare cell viability from Day 1 until Day 14. The morphology of hFOB 1.19 was observed via inverted microscope and scanning electron microscope. Calcium (Ca) and phosphate (P) were assessed using energy-dispersive X-ray (EDX) analysis. Furthermore, the mineralization of hFOB 1.19 was determined by von Kossa staining (P depositions) and Alizarin Red S staining (Ca depositions). The number of cells treated with TA was significantly higher than the two control groups at Day 10 and Day 14. The morphology of cells treated with TA was uniformly fusiform-shaped with filopodia extensions. Besides, globular-like structures of deposited minerals were observed in the TA-treated group. In line with other findings, EDX spectrum analysis confirmed the presence of Ca and P. The cells treated with TA had significantly higher percentage of both minerals at Day 3 and Day 10 compared to the two control groups. In conclusion, TA enhances cell proliferation and causes cell morphology changes, as well as improved mineralization.
Fulltext The Role of Semipurified Fractions Isolated from Quercus infectoria on Bone Metabolism by Using hFOB 1.19 Human Fetal Osteoblast Cell Model

Abdullah AR, Hapidin H, Abdullah H

Evid Based Complement Alternat Med, 2018;2018:5319528.
PMID: 29861772 DOI: 10.1155/2018/5319528

Background. Quercus infectoria (QI) is a plant used in traditional medicines in Asia. The plant was reported to contain various active phytochemical compounds that have potential to stimulate bone formation. However, the precise mechanism of the stimulation effect of QI on osteoblast has not been elucidated. The present study was carried out to isolate QI semipurified fractions from aqueous QI extract and to delineate the molecular mechanism of QI semipurified fraction that enhanced bone formation by using hFOB1.19 human fetal osteoblast cell model. Methods. Isolation of QI semipurified fractions was established by means of column chromatography and thin layer chromatography. Established QI semipurified fractions were identified using Liquid Chromatography-Mass Spectrometry (LC-MS). Cells were treated with derived QI semipurified fractions and investigated for mineralization deposition and protein expression level of BMP-2, Runx2, and OPN by ELISA followed gene expression analysis of BMP-2 and Runx2 by RT-PCR. Results. Column chromatography isolation and purification yield Fractions A, B, and C. LC-MS analysis reveals the presence of polyphenols in each fraction. Results show that QI semipurified fractions increased the activity and upregulated the gene expression of BMP-2 and Runx2 at day 1, day 3, and day 7. OPN activity increased in cells treated with QI semipurified fractions at day 1 and day 3. Meanwhile, at day 7, expression of OPN decreased in activity. Furthermore, the study showed that combination of Fractions A, B, and C with osteoporotic drug (pamidronate) further increased the activity and upregulated the gene expression of BMP-2 and Runx2. Conclusions. These findings demonstrated that polyphenols from semipurified fractions of QI enhanced bone formation through expression of the investigated bone-related marker that is its potential role when combined with readily available osteoporotic drug.
The effects of acute ethanol administration on ethanol withdrawal-induced anxiety-like syndrome in rats: A biochemical study

Kumar J, Hapidin H, Get Bee YT, Ismail Z

Alcohol, 2016 Feb;50:9-17.
PMID: 26626323 DOI: 10.1016/j.alcohol.2015.10.001

Withdrawal from long-term ethanol consumption results in overexcitation of glutamatergic neurotransmission in the amygdala, which induces an anxiety-like syndrome. Most alcoholics that suffer from such symptoms frequently depend on habitual drinking as self-medication to alleviate their symptoms. Metabotropic glutamate receptor subtype 5 (mGlu5) and protein kinase C (PKC) epsilon have been reported to mediate acute and chronic effects of ethanol. This study explores the changes in mGlu5 and PKC epsilon in the amygdala following acute administration of ethanol during ethanol withdrawal (EW) induced anxiety. Male Wistar rats were fed a modified liquid diet containing low-fat cow milk, sucrose, and maltodextrin, with a gradual introduction of 2.4%, 4.8% and 7.2% ethanol for 20 days. Six hours into EW, the rats were intraperitoneally injected with normal saline and ethanol (2.5 g/kg, 20% v/v), and exposed to open-field and elevated plus maze tests. Then, amygdala tissue was dissected from the rat brain for Western blot and gene expression studies. EW-induced anxiety was accompanied by a significant increase in mGlu5, total PKC epsilon, and phosphorylated PKC epsilon protein levels, and also of mRNA of mGlu5 (GRM5) in the amygdala. Acute administration of ethanol significantly attenuated EW-induced anxiety as well as an EW-induced increase in GRM5. The acute challenge of ethanol to EW rats had little effect on the phosphorylated and total protein levels of PKC epsilon in the amygdala. Our results demonstrate that amygdala PKC epsilon may not be directly involved in the development of anxiety following EW.
Fulltext Effects of the mGluR5 antagonist MPEP on ethanol withdrawal induced anxiety-like syndrome in rats

Kumar J, Hapidin H, Bee YT, Ismail Z

Behav Brain Funct, 2013;9:43.
PMID: 24279870 DOI: 10.1186/1744-9081-9-43

Abstinence from chronic ethanol consumption leads to the manifestation of a variety of symptoms attributed to central nervous system hyperexcitability, such as increased irritability, anxiety, and restlessness. Recent studies have demonstrated the importance of metabotropic glutamate receptor 5 (mGluR5) in addictive behaviours. This study investigates the effects of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) on ethanol withdrawal induced anxiety using two behavioural paradigms. Male Wistar rats were fed a Modified Liquid Diet (MLD) containing low fat cow milk, sucrose, and maltodextrin with a gradual introduction of 2.4%, 4.8% and 7.2% ethanol for 20 days. Six hours into ethanol withdrawal, the rats were intraperitoneally injected with normal saline and MPEP (2.5, 5.0, 10, 20, 30 mg/kg) and were assessed for ethanol withdrawal induced anxiety-like syndrome using an automated elevated plus maze and an open field. MPEP at 10 mg/kg significantly attenuated ethanol withdrawal induced anxiety without any compromising effects on locomotor activities. Despite reversing several indices of ethanol withdrawal induced anxiety in both the elevated plus maze and the open field, low doses of MPEP (2.5, 5 mg/kg) significantly compromised the locomotor activities of ethanol withdrawn rats. High doses of MPEP (20 and 30 mg/kg) significantly attenuated withdrawal anxiety when tested in the elevated plus maze but not in the open field. Administration of MPEP (2.5, 5, 10, 20, 30 mg/kg) has no significant compromising effect on the locomotor activities of ethanol naïve rats. Despite significantly reducing withdrawal anxiety in both behavioural paradigms at 10 mg/kg, the compromising effects of low and high doses of MPEP must be further explored along with the therapeutic efficiency of this drug for relieving withdrawal induced anxiety.
Effects of nicotine administration and nicotine cessation on bone histomorphometry and bone biomarkers in Sprague-Dawley male rats

Hapidin H, Othman F, Soelaiman IN, Shuid AN, Mohamed N

Calcif. Tissue Int., 2011 Jan;88(1):41-7.
PMID: 20953592 DOI: 10.1007/s00223-010-9426-4

Nicotine is a major alkaloid of tobacco, which can increase free radical formation, leading to osteoporosis. The effects of nicotine administration and cessation on bone histomorphometry and biomarkers were studied in 28 Sprague-Dawley male rats. Rats aged 3 months and weighing 250-300 g were divided into four groups: control (C, normal saline for 4 months), nicotine for 2 months (N2), nicotine for 4 months (N4), and nicotine cessation (NC). The NC group was given nicotine for the first 2 months and then allowed to recover for the following 2 months without nicotine. Histomorphometric analysis was done using an image analyzer. ELISA kits were used to measure serum osteocalcin (bone formation marker) and pyridinoline (PYD, bone resorption marker) levels at month 0, month 2, and month 4. All test groups showed a significant decrease in BV/TV, Ob.S/BS, dLS/BS, MAR, BFR/BS, and osteocalcin levels and an increase in sLS/BS and PYD levels compared to group C. No significant differences were observed in all parameters measured among the test groups, except for MAR and BFR/BS. In conclusion, nicotine administration at a dose of 7 mg/kg for 2 and 4 months has detrimental effects on bone metabolism. Nicotine administration at 7 mg/kg for 2 months is sufficient to produce significant effects on bone histomorphometric parameters and biomarkers. In addition, prolonging the treatment for another 2 months did not show any significant differences. Cessation of nicotine for 2 months did not reverse the effects.
Fulltext Nicotine-prevented learning and memory impairment in REM sleep-deprived rat is modulated by DREAM protein in the hippocampus

Abd Rashid N, Hapidin H, Abdullah H, Ismail Z, Long I

Brain Behav, 2017 06;7(6):e00704.
PMID: 28638710 DOI: 10.1002/brb3.704

INTRODUCTION: REM sleep deprivation is associated with impairment in learning and memory, and nicotine treatment has been shown to attenuate this effect. Recent studies have demonstrated the importance of DREAM protein in learning and memory processes. This study investigates the association of DREAM protein in REM sleep-deprived rats hippocampus upon nicotine treatment.
METHODS: Male Sprague Dawley rats were subjected to normal condition, REM sleep deprivation and control wide platform condition for 72 hr. During this procedure, saline or nicotine (1 mg/kg) was given subcutaneously twice a day. Then, Morris water maze (MWM) test was used to assess learning and memory performance of the rats. The rats were sacrificed and the brain was harvested for immunohistochemistry and Western blot analysis.
RESULTS: MWM test found that REM sleep deprivation significantly impaired learning and memory performance without defect in locomotor function associated with a significant increase in hippocampus DREAM protein expression in CA1, CA2, CA3, and DG regions and the mean relative level of DREAM protein compared to other experimental groups. Treatment with acute nicotine significantly prevented these effects and decreased expression of DREAM protein in all the hippocampus regions but only slightly reduce the mean relative level of DREAM protein.
CONCLUSION: This study suggests that changes in DREAM protein expression in CA1, CA2, CA3, and DG regions of rat's hippocampus and mean relative level of DREAM protein may involve in the mechanism of nicotine treatment-prevented REM sleep deprivation-induced learning and memory impairment in rats.
Negative effects of nicotine on bone-resorbing cytokines and bone histomorphometric parameters in male rats

Hapidin H, Othman F, Soelaiman IN, Shuid AN, Luke DA, Mohamed N

J. Bone Miner. Metab., 2007;25(2):93-8.
PMID: 17323178

The effects of nicotine administration on bone-resorbing cytokines, cotinine, and bone histomorphometric parameters were studied in 21 Sprague-Dawley male rats. Rats aged 3 months and weighing 250-300 g were divided into three groups. Group 1 was the baseline control (BC), which was killed without treatment. The other two groups were the control group (C) and the nicotine-treated group (N). The N group was treated with nicotine 7 mg/kg body weight and the C group was treated with normal saline only. Treatment was given by intraperitoneal injection for 6 days/week for 4 months. The rats were injected intraperitoneally with calcein 20 mg/kg body weight at day 9 and day 2 before they were killed. ELISA test kits were used to measure the serum interleukin-1 (IL-1), interleukin-6 (IL-6), and cotinine (a metabolite of nicotine) levels at the beginning of the study and upon completion of the study. Histomorphometric analysis was done on the metaphyseal region of the trabecular bone of the left femur by using an image analyzer. Biochemical analysis revealed that nicotine treatment for 4 months significantly increased the serum IL-1, IL-6, and cotinine levels as compared to pretreatment levels. In addition, the serum cotinine level was significantly higher in the N group than in the C group after 4 months treatment. Histomorphometric analysis showed that nicotine significantly decreased the trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), double-labeled surface (dLS/BS), mineralizing surface (MS/BS), mineral appositional rate (MAR), and bone formation rate (BFR/BS), while causing an increase in the single-labeled surface (sLS/BS), osteoclast surface (Oc.S/BS), and eroded surface (ES/BS) as compared to the BC and C groups. In conclusion, treatment with nicotine 7 mg/kg for 4 months was detrimental to bone by causing an increase in the bone resorbing cytokines and cotinine levels. Nicotine also exerted negative effects on the dynamic trabecular histomorphometric parameters.
Tannic acid enhances cisplatin effect on cell proliferation and apoptosis of human osteosarcoma cell line (U2OS)

Kasiram MZ, Hapidin H, Abdullah H, Hashim NM, Azlina A, Sulong S

Pharmacol Rep, 2022 Feb;74(1):175-188.
PMID: 34652600 DOI: 10.1007/s43440-021-00330-3

BACKGROUND: The increase in cases of chemoresistance of cisplatin for osteosarcoma treatment has called for the need to establish a new treatment regime. Tannic acid (TA) possesses a potent antiproliferative effect against various cancers. Therefore, this study investigated the effect of TA combined with cisplatin on human osteosarcoma cell lines (U2OS).
METHODS: MTT assay was used to determine the half-maximal inhibitory concentration (IC50), while the combination index (CI) value was utilized to analyze the interaction within each combination. The antiproliferative effect of the treatment was evaluated by trypan blue exclusion assay. The morphological changes of cells were observed under a phase-contrast inverted microscope. The nuclear morphology and percentage of apoptosis cells were evaluated by using the Hoechst 33258 staining and annexin V/PI assay, respectively.
RESULTS: The U2OS cells showed cytotoxic effect when treated with TA and cisplatin, with IC50 at 4.47 µg/mL and 16.25 µg/mL, respectively. The TA demonstrated no significant inhibition effect on the normal human fetal osteoblast cells (hFOB 1.19); yet, interestingly, a potent proliferative effect was indicated. Synergistic interaction was triggered when TA was combined with cisplatin at percentage ratios of 90:10 and 85:15. Meanwhile, antagonistic interaction was induced in the combination at percentage ratios of 75:25 and 50:50. On the other hand, a significant antiproliferative effect with prominent morphological alteration was detected in the cells treated with a combination of TA and cisplatin at the percentage ratio of 90:10. Additionally, combination-treated cells demonstrated the highest percentage of apoptosis cells, with distinct chromosomal condensation, nuclear fragmentation, reduction of nuclear volume, and notable apoptotic body.
CONCLUSION: Therefore, there is a high potential for the inclusion of TA in the cisplatin-based chemotherapeutic regimen of osteosarcoma.
Fulltext Quercus infectoria Gall Extract Enhanced the Proliferation and Activity of Human Fetal Osteoblast Cell Line (hFOB 1.19)

Hapidin H, Rozelan D, Abdullah H, Wan Hanaffi WN, Soelaiman IN

Malays J Med Sci, 2015 Jan-Feb;22(1):12-22.
PMID: 25892946 MyJurnal

The present study investigated the effects of Quercus infectoria (QI) gall extract on the proliferation, alkaline phosphatase (ALP), osteocalcin, and the morphology of a human fetal osteoblast cell line (hFOB 1.19).
Alcohol Addiction- Metabotropic Glutamate Receptor Subtype 5 and its Ligands: How They All Come Together?

Kumar J, Ismail Z, Hatta NH, Baharuddin N, Hapidin H, Get Bee YT, et al.

Curr Drug Targets, 2018;19(8):907-915.
PMID: 28494749 DOI: 10.2174/1389450118666170511144302

In the past decade, many studies have highlighted the role of metabotropic glutamate receptor subtype 5 (mGlu5) modulators in attenuating alcohol-related biological effects such as alcohol consumption, alcohol-seeking and relapse-like behaviors. Taken together, these findings suggest that pharmacological agents acting at mGlu5 could be promising tools in curbing inebriation. mGlu5s are present abundantly in brain regions known to be involved in emotion regulation, motivation and drug administration. On a cellular level, they are primarily located at the postsynaptic part of the neuron where the receptor is functionally linked to various downstream proteins that are involved in cell signaling and gene transcription that mediate the alcohol-induced neuroplasticity. As well, the discovery of a functional link between mGlu5 and a specific isozyme, Protein Kinase C epsilon (PKCε) in mediating the attenuating effects of selective negative allosteric modulators of mGlu5 such as methyl- 6(phenylethynyl)pyridine (MPEP) and 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP) has sparked interesting speculations. In this article, we shall review the following: the effects of acute and chronic alcohol intake on mGlu5 signaling; the effects of mGlu5 ligands on alcohol-related neurobehavioral changes that are currently being studied both at pre-clinical and clinical stages; and the mechanisms underlying the pharmacological effects of these drugs.

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