Monday, December 22, 2008
Wednesday, May 14, 2008
Garlic and Osteoporosis
(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
INTRODUCTION
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.
METHODS
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).
RESULTS
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).
CONCLUSIONS
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.
REFERENCES
1. Ferguson N (2004). Osteoporosis in focus. Pharmaceutical Press: London UK.
2. Riggs BL and Melton LJ (1995).The worldwide problem of osteoporosis: insights offered by epidemiology. Bone (NY) 17:505S-511S.
3. Melton LJ III, Thamer M, Ray NF et al (1997). Fractures attributable to osteoporosis: report from the National Osteoporosis Foundation. J Bone Miner Res,12: 16-23.
4. Wark JD (1996). Osteoporosis: the emerging epidemic. Med J Aust 164:327-328.
5. Hasan N, Yusuf N, Toossi Z and Islam N. (2006). Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF-alpha mRNA expression in human monocytes by allicin. FEBS Letters 580: 2517-2522.
6. Mates JM, Segura JM, Perez-Gomez C et al (1999). Antioxidant enzymatic activities in human blood cells after an allergic reaction to pollen or house dust mite. Blood Cells Mol Dis,25 (7): 103-09.
7. Hasan, N., Siddiqui, M. U., Toossi, Z., Khan, S., Iqbal, J. and Islam, N. (2007) Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes. Biochem. Biophys. Res. Comm., 355(2): 471-476
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
INTRODUCTION
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.
METHODS
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).
RESULTS
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).
CONCLUSIONS
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.
REFERENCES
1. Ferguson N (2004). Osteoporosis in focus. Pharmaceutical Press: London UK.
2. Riggs BL and Melton LJ (1995).The worldwide problem of osteoporosis: insights offered by epidemiology. Bone (NY) 17:505S-511S.
3. Melton LJ III, Thamer M, Ray NF et al (1997). Fractures attributable to osteoporosis: report from the National Osteoporosis Foundation. J Bone Miner Res,12: 16-23.
4. Wark JD (1996). Osteoporosis: the emerging epidemic. Med J Aust 164:327-328.
5. Hasan N, Yusuf N, Toossi Z and Islam N. (2006). Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF-alpha mRNA expression in human monocytes by allicin. FEBS Letters 580: 2517-2522.
6. Mates JM, Segura JM, Perez-Gomez C et al (1999). Antioxidant enzymatic activities in human blood cells after an allergic reaction to pollen or house dust mite. Blood Cells Mol Dis,25 (7): 103-09.
7. Hasan, N., Siddiqui, M. U., Toossi, Z., Khan, S., Iqbal, J. and Islam, N. (2007) Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes. Biochem. Biophys. Res. Comm., 355(2): 471-476
Natural antioxidants and Combating Disease
(Extracts of Invited talk given by Dr. Najmul Islam at ACIBICON Dec. 2007, New Delhi, India)
Natural Antioxidants – A New Hope for Military and Civilian Applications
Najmul Islam and Nazarul Hasan.
Department of Biochemistry, Faculty of Medicine, J. N. Medical College, A. M. U., Aligarh
Objective: Arrays of reasons contribute to the generation of free radicals in vivo, including oxidative stress, infections, etc. thereby resulting in disease proliferation. Far more troops have to be removed from field operations due to diseases than from war-related wounds. Troops who served in the Middle East came back home with Gulf War Syndrome. A mycobacterium is believed to have caused as many as 400,000 cases of Gulf War Syndrome. Because of antibiotic resistance (MDR-TB), antibiotic drugs as prophylaxis cannot be given. Thus, we explored the possible use of a natural antioxidant, namely allicin from garlic as a safe and economical adjunct in tuberculosis management.
Materials and Methods: Blood of TB patients and healthy subjects were collected for PBMC isolation. Monocytes obtained from it were cultured and subjected to treatment with 0-500 ng/ml of allicin for 24 hrs. Levels of TNF-α mRNA and MTB 85B mRNA was determined by RT-PCR. Intramonocyte glutathione (GSH) levels were also measured. Secreted IFN-γ levels were assessed in TB monocyte culture supernatants using ELISA.
Results: We observed that allicin from garlic was a strong anti-oxidant and anti-inflammatory agent having beneficial effects on the immune system as well as in combating tuberculosis. We found that allicin from garlic helped in scavenging the free-radicals generated due to MTB-infection in human monocytes by suppressing TNF-α mRNA and MTB 85B mRNA and ameliorating the levels of intramonocyte glutathione (GSH) and IFN-γ levels.
Conclusions: Allicin from garlic may act as a valuable anti-oxidant and anti-inflammatory agent that may combat TB. It is hoped that allicin may act as a potential stealth molecule helping both to military as well as civilians.
Natural Antioxidants – A New Hope for Military and Civilian Applications
Najmul Islam and Nazarul Hasan.
Department of Biochemistry, Faculty of Medicine, J. N. Medical College, A. M. U., Aligarh
Objective: Arrays of reasons contribute to the generation of free radicals in vivo, including oxidative stress, infections, etc. thereby resulting in disease proliferation. Far more troops have to be removed from field operations due to diseases than from war-related wounds. Troops who served in the Middle East came back home with Gulf War Syndrome. A mycobacterium is believed to have caused as many as 400,000 cases of Gulf War Syndrome. Because of antibiotic resistance (MDR-TB), antibiotic drugs as prophylaxis cannot be given. Thus, we explored the possible use of a natural antioxidant, namely allicin from garlic as a safe and economical adjunct in tuberculosis management.
Materials and Methods: Blood of TB patients and healthy subjects were collected for PBMC isolation. Monocytes obtained from it were cultured and subjected to treatment with 0-500 ng/ml of allicin for 24 hrs. Levels of TNF-α mRNA and MTB 85B mRNA was determined by RT-PCR. Intramonocyte glutathione (GSH) levels were also measured. Secreted IFN-γ levels were assessed in TB monocyte culture supernatants using ELISA.
Results: We observed that allicin from garlic was a strong anti-oxidant and anti-inflammatory agent having beneficial effects on the immune system as well as in combating tuberculosis. We found that allicin from garlic helped in scavenging the free-radicals generated due to MTB-infection in human monocytes by suppressing TNF-α mRNA and MTB 85B mRNA and ameliorating the levels of intramonocyte glutathione (GSH) and IFN-γ levels.
Conclusions: Allicin from garlic may act as a valuable anti-oxidant and anti-inflammatory agent that may combat TB. It is hoped that allicin may act as a potential stealth molecule helping both to military as well as civilians.
Herbals versus Tuberculosis
Extract of Poster presented in ACIBICON, Dec. 2007, New Delhi
Jawed Iqbal, Ajay Kumar and Najmul Islam
(Group leader: Dr. Najmul Islam, Dept. of Biochemistry, J.N.Medical College, A.M.U., Aligarh 202002, U.P., India
Azadirachta indica induced suppression of Mycobacterium tuberculosis secreted proteins in human monocyte
Introduction:
Tuberculosis is the most important mycobacterial infection in humans, being the second major infectious cause of death and killing around two million people every year [1]. With the emergence of multidrug-resistant TB (MDR-TB) as well as AIDS-associated TB, this killer parasite is spiraling out of control [2]. Thus, in view of it, focus has now shifted in exploring new novel natural compounds that may combat tuberculosis [3, 4]. Therefore, in the present study, we probed the anti-tuberculosis effect of Azadirachta indica (Neem), which has attained worldwide prominence in recent years owing to its medicinal properties. Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex having wide range of pharmacological properties. It has been known to possess antioxidant and anti-inflammatory properties. It is hoped that the data of the present study may help in understanding the potentiality of neem extract in employing it as a valuable adjunct in TB treatment.
Methods:
1. Preparation of Mycobacteria:
Virulent laboratory-adapted M. tuberculosis (H37Rv) were grown in Middlebrook 7H9 broth (HiMedia, India) supplemented with Middlebrook ADC enrichment fluid (HiMedia) or modified Souton medium [5] at 37°C in 5% CO2. Midlogarithmic mycobacterial cultures (14 days) were harvested and quantified by use of a colony-forming unit assay, as described elsewhere [6]. Aliquots of the stock were kept at −70°C. The viability of the stock remained >99% at 1 year.
2. Preparation of PBMC, cell culture and infection:
Peripheral blood mononuclear cells (PBMCs) from blood were isolated as described by us previously [3]. PBMC (0.5x106 cells/well) were added onto 12-well tissue culture plates in complete RPMI-1640 medium, and were subsequently incubated at 37°C, 5% CO2 for 1-2 hrs for adherence. Thereafter, non-adherent cells were removed and the adherent monocytes were rested overnight at 37°C, 5% CO2. Thereafter, monocytes were infected with M. tuberculosis at 1:1 (bacteria: cell) in 30% autologous unheated serum for 90 min. at 37°C, 5% CO2, and subsequently washed for 4 times complete medium. Cells harvested at this time point were considered as time zero after infection (t0). Other cultures received RPMI-1640 medium with 2% autologous serum. As per experimental design, cultures immediately after infection received varying concentrations of neem extract (0-1000 ng/ml)., NAC (10 mM), SN50 (100 ug/ml) or SN50M (100 ug/ml). Cultures were then harvested after 24 hrs and cell protein lysates were prepared as described earlier [3,4].
3. Measurement of secreted TNF-α and MTB 85B protein in culture supernatant by ELISA:
The amount of soluble secreted TNF-α protein in various culture supernatants were determined by use of a commercial ELISA Kit (R & D systems), according to the manufacturer’s specifications. The cut off or lower limit of sensitivity was 4.4 pg/ml. ELISA was carried out to determine secreted MTB 85B protein in culture supernatants as described by us previously [4].
4. Glutathione peroxidase assay:
The activity of glutathione peroxidase (GPx) was measured as described elsewhere [13]. Briefly, MTB-infected monocytes were co-cultured for 24 h with varying concentrations of neem extract (0-1000 ng/ml). Thereafter, cells were scrapped, sonicated and centrifuged as described earlier [3 and references within], 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 37oC. The conversion of NADPH to NADP was evaluated using UV absorbance at 340 nm [3]. GPx activity was calculated after subtraction of the blank value, as lmol of NADPH oxidized/min/mg protein (U/mg protein).
Results:
Dose response effect of neem extract on soluble TNF-alpha and MTB Ag85B protein expressions in monocyte cultures infected with MTB
We observed neem extract-induced suppression in secreted TNF-α and MTB Ag85B protein expressions in MTB-infected monocyte cultures were dose dependent. Neem extract at a maximum dose of 1 ug/ml exhibited a down-regulation in secreted TNF-α and 85B proteins to the extent of 4.7-folds and 4.3-folds respectively.
Glutathione peroxidase activity:
Healthy control cultures showing appreciably high GPx activity was suppressed in MTB-infected monocytes (Fig. 3, P<0.001). Interestingly, with neem extract treatment of MTB infected monocytes, GPx activity ameliorated appreciably in a dose-dependent manner. Regulation studies by employing SN50 exhibited the above suppression to be mediated via NF-kB. These results clearly point to neem extract as a potent natural enhancer of GPx levels in monocytes infected with MTB.
Conclusion:
1. The neem extract-induced suppression in secreted TNF-alpha and MTB Ag85B proteins in 24 hr culture supernatants of MTB-infected human monocytes was found to be dose-dependent.
2. Glutathione peroxidase activity was decreased after M. tuberculosis infection of monocytes.
3. Interestingly, glutathione peroxidase activity was augmented significantly with neem extract treatment in a dose-dependent manner.
4. The above-said suppression was mediated via NF-kB.
5. The results are indicative of neem extract- a natural antioxidant and an anti-inflammatory agent, may act as an adjunct in combating tuberculosis infection.
References:
1. WHO (2001) Global Tuberculosis Control, Geneva, Switzerland, Report no. WHO/CDS/TB/2001.287.
2. Toossi, Z; Wu, M; Islam, N; Teixeira-Johnson, L; Hejal, R and Aung, H. (2004) Transactivation of human immunodeficiency virus-1 in T-cells by Mycobacterium tuberculosis-infected mononuclear phagocytes. J. Lab. Clin. Med., 144(2):108-15.
3. Hasan, N., Yusuf, N., Toossi, Z. and Islam, N. (2006) Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF- α mRNA expression in human monocytes by allicin. FEBS Letters 580: 2517-2522.
4. Hasan, N., Siddiqui, M. U., Toossi, Z., Khan, S., Iqbal, J. and Islam, N. (2007) Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes. Biochem. Biophys. Res. Comm., 355(2): 471-476.
5. Islam, N; Kanost, RA; Teixeira-Johnson, L; Hejal, R; Aung, H; Wilkinson, RJ; Hirsch, CS; and Toossi, Z. (2004) The role of cellular activation and tumor necrosis factor alpha (TNF-alpha) in the early expression of M. tuberculosis 85B mRNA in human alveolar macrophages. J. Infect. Dis. 15; 190:341-351.
6. Wilkinson, RJ; DesJardin, LE; Islam, N; Gibson, BM; Wilkinson, KA; Poelman, D; Kanost, RA; Eisenach, KD and Toossi, Z. (2001) An increase in expression of a M. tuberculosis mycolyl gene (fbpB) occurs early after infection of human monocytes. Mol. Microbiol, 39(3): 813-821.
Jawed Iqbal, Ajay Kumar and Najmul Islam
(Group leader: Dr. Najmul Islam, Dept. of Biochemistry, J.N.Medical College, A.M.U., Aligarh 202002, U.P., India
Azadirachta indica induced suppression of Mycobacterium tuberculosis secreted proteins in human monocyte
Introduction:
Tuberculosis is the most important mycobacterial infection in humans, being the second major infectious cause of death and killing around two million people every year [1]. With the emergence of multidrug-resistant TB (MDR-TB) as well as AIDS-associated TB, this killer parasite is spiraling out of control [2]. Thus, in view of it, focus has now shifted in exploring new novel natural compounds that may combat tuberculosis [3, 4]. Therefore, in the present study, we probed the anti-tuberculosis effect of Azadirachta indica (Neem), which has attained worldwide prominence in recent years owing to its medicinal properties. Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex having wide range of pharmacological properties. It has been known to possess antioxidant and anti-inflammatory properties. It is hoped that the data of the present study may help in understanding the potentiality of neem extract in employing it as a valuable adjunct in TB treatment.
Methods:
1. Preparation of Mycobacteria:
Virulent laboratory-adapted M. tuberculosis (H37Rv) were grown in Middlebrook 7H9 broth (HiMedia, India) supplemented with Middlebrook ADC enrichment fluid (HiMedia) or modified Souton medium [5] at 37°C in 5% CO2. Midlogarithmic mycobacterial cultures (14 days) were harvested and quantified by use of a colony-forming unit assay, as described elsewhere [6]. Aliquots of the stock were kept at −70°C. The viability of the stock remained >99% at 1 year.
2. Preparation of PBMC, cell culture and infection:
Peripheral blood mononuclear cells (PBMCs) from blood were isolated as described by us previously [3]. PBMC (0.5x106 cells/well) were added onto 12-well tissue culture plates in complete RPMI-1640 medium, and were subsequently incubated at 37°C, 5% CO2 for 1-2 hrs for adherence. Thereafter, non-adherent cells were removed and the adherent monocytes were rested overnight at 37°C, 5% CO2. Thereafter, monocytes were infected with M. tuberculosis at 1:1 (bacteria: cell) in 30% autologous unheated serum for 90 min. at 37°C, 5% CO2, and subsequently washed for 4 times complete medium. Cells harvested at this time point were considered as time zero after infection (t0). Other cultures received RPMI-1640 medium with 2% autologous serum. As per experimental design, cultures immediately after infection received varying concentrations of neem extract (0-1000 ng/ml)., NAC (10 mM), SN50 (100 ug/ml) or SN50M (100 ug/ml). Cultures were then harvested after 24 hrs and cell protein lysates were prepared as described earlier [3,4].
3. Measurement of secreted TNF-α and MTB 85B protein in culture supernatant by ELISA:
The amount of soluble secreted TNF-α protein in various culture supernatants were determined by use of a commercial ELISA Kit (R & D systems), according to the manufacturer’s specifications. The cut off or lower limit of sensitivity was 4.4 pg/ml. ELISA was carried out to determine secreted MTB 85B protein in culture supernatants as described by us previously [4].
4. Glutathione peroxidase assay:
The activity of glutathione peroxidase (GPx) was measured as described elsewhere [13]. Briefly, MTB-infected monocytes were co-cultured for 24 h with varying concentrations of neem extract (0-1000 ng/ml). Thereafter, cells were scrapped, sonicated and centrifuged as described earlier [3 and references within], 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 37oC. The conversion of NADPH to NADP was evaluated using UV absorbance at 340 nm [3]. GPx activity was calculated after subtraction of the blank value, as lmol of NADPH oxidized/min/mg protein (U/mg protein).
Results:
Dose response effect of neem extract on soluble TNF-alpha and MTB Ag85B protein expressions in monocyte cultures infected with MTB
We observed neem extract-induced suppression in secreted TNF-α and MTB Ag85B protein expressions in MTB-infected monocyte cultures were dose dependent. Neem extract at a maximum dose of 1 ug/ml exhibited a down-regulation in secreted TNF-α and 85B proteins to the extent of 4.7-folds and 4.3-folds respectively.
Glutathione peroxidase activity:
Healthy control cultures showing appreciably high GPx activity was suppressed in MTB-infected monocytes (Fig. 3, P<0.001). Interestingly, with neem extract treatment of MTB infected monocytes, GPx activity ameliorated appreciably in a dose-dependent manner. Regulation studies by employing SN50 exhibited the above suppression to be mediated via NF-kB. These results clearly point to neem extract as a potent natural enhancer of GPx levels in monocytes infected with MTB.
Conclusion:
1. The neem extract-induced suppression in secreted TNF-alpha and MTB Ag85B proteins in 24 hr culture supernatants of MTB-infected human monocytes was found to be dose-dependent.
2. Glutathione peroxidase activity was decreased after M. tuberculosis infection of monocytes.
3. Interestingly, glutathione peroxidase activity was augmented significantly with neem extract treatment in a dose-dependent manner.
4. The above-said suppression was mediated via NF-kB.
5. The results are indicative of neem extract- a natural antioxidant and an anti-inflammatory agent, may act as an adjunct in combating tuberculosis infection.
References:
1. WHO (2001) Global Tuberculosis Control, Geneva, Switzerland, Report no. WHO/CDS/TB/2001.287.
2. Toossi, Z; Wu, M; Islam, N; Teixeira-Johnson, L; Hejal, R and Aung, H. (2004) Transactivation of human immunodeficiency virus-1 in T-cells by Mycobacterium tuberculosis-infected mononuclear phagocytes. J. Lab. Clin. Med., 144(2):108-15.
3. Hasan, N., Yusuf, N., Toossi, Z. and Islam, N. (2006) Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF- α mRNA expression in human monocytes by allicin. FEBS Letters 580: 2517-2522.
4. Hasan, N., Siddiqui, M. U., Toossi, Z., Khan, S., Iqbal, J. and Islam, N. (2007) Allicin-induced suppression of Mycobacterium tuberculosis 85B mRNA in human monocytes. Biochem. Biophys. Res. Comm., 355(2): 471-476.
5. Islam, N; Kanost, RA; Teixeira-Johnson, L; Hejal, R; Aung, H; Wilkinson, RJ; Hirsch, CS; and Toossi, Z. (2004) The role of cellular activation and tumor necrosis factor alpha (TNF-alpha) in the early expression of M. tuberculosis 85B mRNA in human alveolar macrophages. J. Infect. Dis. 15; 190:341-351.
6. Wilkinson, RJ; DesJardin, LE; Islam, N; Gibson, BM; Wilkinson, KA; Poelman, D; Kanost, RA; Eisenach, KD and Toossi, Z. (2001) An increase in expression of a M. tuberculosis mycolyl gene (fbpB) occurs early after infection of human monocytes. Mol. Microbiol, 39(3): 813-821.
Wednesday, February 6, 2008
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