(Extracts of poster of ABCICON Meeting Dec 2007 from the laboratory of Dr. Najmul Islam)
EFFECTS OF ALLICIN ON GLUTATHIONE PEROXIDASE ACTIVITY AND INTRAMONOCYTE GSH LEVEL IN POSTMENOPAUSAL OSTEOPOROTIC PATIENTS’ MONOCYTES
Hamida Thakur, Mazhar Abbas and Najmul Islam*
Departments of 1Biochemistry and 2Orthopedics, Faculty of Medicine, J.N. Medical College, A.M.U., Aligarh, 202002, U.P., India
Augmented bone resorption is a major mechanism contributing to bone loss in postmenopausal women. Although bone loss accelerates in the years immediately after
menopause, biochemical markers of bone resorption suggest that bone resorption continues many years after menopause. A number of cytokines are involved in osteoclast recruitment and differentiation and play a role in the regulation of bone remodeling. Estrogen-deficient bone loss may be related to modulation of local bone resorbing factors in the bone microenvironment, such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a. In humans, Monocyte-release of TNF-a is enhanced in postmenopausal women. Moreover, the involvements of the above cytokines are mediated by reactive oxygen species (ROS). Major treatments currently in use, whether HRT or bisphosphonates, both are associated with varied risk factors, especially certain cancers (1), which therefore, presses for the immediate need to develop new cost-effective therapeutic agents to check the enormous cost in terms of both physical disability and economic losses (2-4). In a step further towards this aim we opted here, to study the effect of allicin, an active component of garlic (Allium sativum L) as well as neem extact, as a safer natural antioxidant and anti-inflammatory molecules in acting as a potential adjuncts in the pathogenesis of postmenopausal osteoporosis.
1. Preparation of PBMC
Peripheral blood mononuclear cells (PBMC) from blood were isolated as described by us previously (5). Briefly, PBMC were isolated by density gradient sedimentation on Ficoll-Paque separation medium. The cells were centrifuged at 1,500 rpm, at 4°C for 10 min. Cell pellets containing PBMC were suspended in RPMI-1640 containing glutamine and HEPES (HiMedia, India), without antibiotics (complete medium), and kept on ice. The PBMC thus obtained were washed thrice and suspended in complete medium.
2. Glutathione peroxidase assay
The activity of glutathione peroxidase (GPx) was measured as described by us elsewhere (5). Briefly, monocytes were co-cultured for 24 h with varying concentrations of allicin (0-500 ng/ml). Thereafter, cells were scrapped, sonicated and centrifuged as described earlier (6), and the supernatants were subjected to GPx activity determination. The GPx activity was quantified in 100 μl of each sample, with continuous photometric monitoring of oxidized glutathione (GSSG) at 37° C. The conversion of NADPH to NADP was evaluated using UV absorbance at 340 nm (10). GPx activity was calculated after subtraction of the blank value, as μmol of NADPH oxidized/min/mg protein (U/mg protein).
3. GSH assay and TNF-alpha mRNA expression quantitation.
As described by us previously (7), GSH levels in treated or control monocytes were assayed by spectrophotometry, using a GSH assay kit (Calbiochem). Monocytes were mixed with equal volume of ice cold 5% metaphosphoric acid and centrifuged at 3000 rpm for 15 min. Supernatants were used for GSH assay, as per the manufacturer’s instruction. TNF-alpha mRNA expression was determined by employing ‘real time’ RT-PCR as described by us earlier (5).
1) Glutathione peroxidase activity.
GPx activity was determined in supernatants of 24 h cultured monocytes from postmenopausal osteoporotic patients and compared with that of postmenopausal non-porotic subjects and premenopausal non-porotic healthy individuals. GPx activity was suppressed in monocyte culture supernatants of postmenopausal osteoporotic patients by ~ 44% (p<0.001) and 46% (p<0.001) when compared to premenopausal non-porotic healthy subjects and also suppressed relative to postmenopausal non-porotic subjects (Fig 1).
2) Dose response effect of allicin on GPx activity in cultured monocyte supernatants.
We investigated the effect of varying doses of allicin, on GPx activity in 24 h cultured monocyte supernatants of postmenopausal osteoporotic patients which revealed a dose-dependent response. The GPx activity in untreated monocyte culture supernatants was 33.88 U/mg protein which increased to 43.27, 52, 66.5 and 71.58 U/mg protein when cultures received 50, 100, 250 and 500 ng/ml allicin respectively (p<0.001, Fig 2).
3) Modulation of intramonocyte GSH levels.
Intramonocyte levels of GSH were determined in supernatants of 24 h cultured monocytes from postmenopausal osteoporotic patients which were found to be of the order of 148.37 pg/ml. Next, in comparison to above untreated control cultures, monocytes when co-cultured with 10 mM NAC, 100 g/ml SN50, 100 g/ml SN50/M, and 500 ng/ml allicin, supernatants revealed enhanced intramonocyte GSH levels; 243.67, 269.37, 143.75 and 286.20 pg/ml respectively (p<0.001 in all cases, Fig 3). These results clearly point to allicin as a potent natural enhancer of GSH levels in postmenopausal osteoporosis.
4) Effect of Allicin and Neem extracts on the expression of TNF-alpha mRNA
Quantitative ‘real time’ RT-PCR data showed that the augmented TNF-alpha mRNA expressions in monocytes of patients with post menopausal osteoporosis were appreciably down-regulated by allicin from garlic as well as by neem extracts in a dose-dependent manner (Figs. 4 and 5).
1) Glutathione peroxidase activity was much lowered both in sera and supernatants of 24 h cultured monocytes from postmenopausal osteoporotic patients.
2) GPx activity was appreciably and dose-dependently augmented with allicin treatment.
3) Intramonocyte GSH level in supernatants of 24 h cultured monocytes of postmenopausal osteoprotic patients was suppressed and this suppression was NFB mediated.
4) Interestingly, allicin proved to be potent natural antioxidant capable of efficient reversal of suppressed GSH levels in postmenopausal osteoporosis.
5) The suppression of TNF-alpha mRNA by allicin and neem extract was dose-dependent.
6) Thus, allicin and neem extact may act as potential adjuncts in the pathogenesis of postmenopausal osteoporosis.
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