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Cytoprotective and immunomodulating properties of piperine on murine splenocytes: An in vitro study
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European Journal of Pharmacology 576 (2007) 160 – 170 www.elsevier.com/locate/ejphar
Cytoprotective and immunomodulating properties of piperine on murine splenocytes: An in vitro study Neelima Pathak, Shashi Khandelwal ⁎ Industrial Toxicology Research Centre, Mahatma Gandhi Marg, P.Box 80, Lucknow — 226001, India Received 22 February 2007; received in revised form 9 July 2007; accepted 17 July 2007 Available online 25 July 2007
Abstract Piper longum Linn. and Piper nigrum Linn. are conventionally used as immuno-enhancers in Indian system of traditional medicine. The underlying mechanism remains unknown. The present study was therefore, undertaken to delineate the role of piperine (major alkaloid) in cadmium (Cd) induced immuno-compromised murine splenocytes. The various biological determinants such as oxidative stress markers (reactive oxygen species and GSH), Bcl-2 protein expression, mitochondrial membrane potential, caspase-3 activity, DNA damage, splenic B and T cell population, blastogenesis and cytokines (Interleukin-2 and gamma-Interferon) were measured to ascertain its cell protective potential. Cadmium induces apoptosis at 6 h onwards. The oxidative stress markers markedly alter prior to a decline in mitochondrial membrane potential, caspase-3 activation and DNA degradation The splenic cell population was observed to change only at 18 h and the release of two cytokines was affected at 72 h. Addition of piperine in various concentrations (1, 10 and 50 μg/ml) ameliorated the above events. The highest dose of piperine could completely abrogate the toxic manifestations of cadmium and the splenic cells behaved similar to control cells. The reported free radical scavenging property of piperine and its antioxidant potential could be responsible for the modulation of intracellular oxidative stress signals. These in turn appear to mitigate the apoptotic pathway and other cellular responses altered by cadmium. The findings strongly indicate the anti-oxidative, anti-apoptotic and chemo-protective ability of piperine in blastogenesis, cytokine release and restoration of splenic cell population and is suggestive of its therapeutic usefulness in immuno-compromised situations. © 2007 Elsevier B.V. All rights reserved. Keywords: Piperine; Apoptosis; Oxidative stress; Phenotyping; Blastogenesis; Cadmium
1. Introduction Piper longum Linn. and Piper nigrum Linn. are used in Indian traditional medicine (Singh, 1992) and as a spice globally. Piperine, the alkaloid is responsible for much of the taste and smell of black pepper. It exhibits a wide variety of biological effects including anti-metastatic, antithyroid, antidepressant, hepatoprotective and antitumor. The anti-metastatic property of piperine in mice has been shown by reduction in lung collagen hydroxyproline, hexosamine and uronic acid induced by B16F-10 melanoma cells (Pradeep and Kuttan, 2002). Inhibition of Phase I and Phase II enzymes, elevation of GSH metabolizing enzymes, reduction in DNA damage and DNA protein cross-links in benzo(a)pyrene induced lung carcinogenesis has been observed by piperine supplementation ⁎ Corresponding author. Tel.: +91 522 2627586; fax: +91 522 2628227. E-mail address: [email protected] (S. Khandelwal). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.07.033
(Selvendiran et al., 2005a,b). Additionally, the chemopreventive efficacy of piperine relating to a decrease in lipid peroxidation, protein carbonyls, nucleic acid and polyamine synthesis has also been reported (Selvendiran et al., 2004). The bioavailability enhancing activity of piperine with various structurally and therapeutically diverse drugs has been studied by Khanjuria et al. (2002). This was due to alterations in membrane dynamics and permeation characteristics along with induced synthesis of protein associated with cytoskeletal function. The antioxidant efficacy of piperine was shown both in vitro (Mittal and Gupta, 2000) and in vivo (Vijayakumar et al., 2004) and its hepatoprotective ability (Koul and Kapil, 1993) and thyroid hormone lowering efficacy have also been reported. Recently, Li et al. (2007) demonstrated that piperine could reverse the corticosterone induced reduction of brainderived neurotrophic factor mRNA expression in cultured hippocampal neurons. Although, immuno-enhancing ability of Piper sp. has long been known and practiced in Indian
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traditional medicine, sporadic scientific reports are available till date. Piperine was found to be cytotoxic towards Dalton's lymphoma ascites cells and Ehrlich ascites carcinoma cells at a dose of 250 μg/ml (Sunila and Kuttan, 2004). An increase in the circulating antibody titre and antibody forming cells, along with enhanced bone marrow cellularity and α-esterase positive cells was also observed. In addition, we have also studied the cell mediated stimulatory response of piperine in cadmium induced alterations in immuno-compromised murine thymocytes and demonstrated anti-oxidative and anti-apoptotic potential of piperine. Its restorative ability against cell proliferative mitogenic response and phenotypic alterations was also seen (Pathak and Khandelwal, 2006a). Humoral and cell mediated immunity are the two arms of immune system and the latter has been extensively studied by us, we now explored the possible efficacy of piperine in murine splenocytes, since spleen is one of the principle sites for the initiation of most primary immune responses, for B lymphocyte activation and the production of antibodies. We have chosen cadmium (Cd) as an immunotoxicant because, firstly it is classified as a category I human carcinogen (IARC, 1993) and secondly, managing cadmium poisoning with safe and effective compounds has always been a challenge. Towards this goal, we planned the following study incorporating a number of determinants. 1) Oxidative stress and apoptotic hallmarks to ascertain the anti-oxidant and anti-apoptotic efficacy of piperine together with 2) its influence on the splenic population (B and T lymphocytes), cell proliferative mitogenic response and immune function i.e. cytokine release (Interleukin-2 and gamma-Interferon). Our results clearly demonstrated significant immunostimulation by piperine. It exhibited both anti-oxidative and antiapoptotic potential. The suppressed B and T cells by cadmium were reverted back close to the control splenic population and the inhibited splenic cell proliferative response and cytokine release were also restored by piperine. 2. Materials and methods 2.1. Chemicals All the chemicals were of highest grade purity available. Cadmium chloride (CdCl2), RNase A, RPMI 1640, antibiotic– antimycotic solution, Dulbecco's phosphate buffered saline (PBS), fetal bovine serum (FBS), agarose, 3-(4,5-dimethyl-2-yl)2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), piperine, concanavalin A (Con A), FITC-conjugated anti-rabbit Ig G antibody, 7-amino-4-trifluoromethylcoumarin (AFC), benzyloxy-carbonyl-Asp-Glu-Val-Asp7-amino-4-trifluoromethyl-coumarin (DEVD-AFC) and all other chemicals were purchased from Sigma Aldrich, USA. Rhodamine 123 (Rh 123) and 5′-Chloromethylfluorescein diacetate (CMFDA) from Molecular Probes, propidium iodide (PI) from Calbiochem, Annexin V-FITC and rabbit anti Bcl-2 polyclonal antibody from Biovision, [3H] Thymidine from BRIT, Bombay, FITC-conjugated anti-CD3 monoclonal antibody, PE-conjugated anti-CD19 monoclonal antibody and mouse Interleukin-2 and
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gamma-Interferon ELISA kits from eBioscience. Cell Death Detection sandwich ELISA kit from Roche, Germany and caspase-3 fluorometric protease assay kit was purchased from Chemicon, USA. 2.2. Preparation of splenocyte suspension The BALB/c mice were maintained in our animal house under standard conditions. They were fed with standard rodent pellet and water ad libitum. Our animal house and breeding facility are registered with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and CPCSEA guidelines were followed (IAEC approval obtained). Spleen was dissected from male BALB/c mice (4–6 weeks old) and single cell suspension prepared under aseptic conditions. The suspension was passed through 100 μm stainless steel mesh, treated with 5.0 ml hypotonic NH4Cl solution (2.42 g Tris and 7.56 g NH4Cl in 1.0 l deionised water, pH adjusted to 7.2) for 5 min at room temperature to lyse red blood cells, centrifuged and washed with FBS free medium. The cells were suspended in complete cell culture medium (RPMI 1640 containing HEPES and 2 mM glutamine, supplemented with 10% FBS and 1% antibiotic–antimycotic solution). The cell density was adjusted to ca. 1.5 × 106 cells/ml and the viability of the freshly isolated cells was always over 95% (trypan blue exclusion test). For the monitoring of various parameters in the present investigation, we have used 25 μM concentration of cadmium and 1, 10 and 50 μg/ml of piperine. The cadmium concentration selected was based on our earlier work (Pathak and Khandelwal, 2006b). 2.3. Assessment of cell viability The cell viability was measured by the MTT reduction method (Mosmann, 1983). Cells in RPMI 1640 were seeded at a density of 1.0 × 104 in 96 well plate. The cells were treated with cadmium along with piperine for 18 h at 37 °C in a CO2 incubator. Ten μl MTT (5 mg/ml PBS) was added to the wells, 4 h prior to the completion of incubation time. The plate was centrifuged at 1200 ×g for 10 min and 100 μl of DMSO was added after removing the supernatant, to dissolve the formazan formed. The absorbance was read at 530 nm after 5 min, in a microplate reader (Synergy HT of BIO-TEK International, USA). 2.4. Measurement of caspase-3 activity A population of 3.0 × 106 cells/ml was incubated with cadmium and piperine for 1.5, 3 and 6 h at 37 °C in a CO2 incubator. The cells were scraped and lysed on ice for 10 min using cell lysis buffer. The reaction buffer (10 mM Tris, 1 mM EDTA, 10 mM dithiothretol, 5% glycerol) and DEVD-AFC substrate (50 μM final concentration) was then added and further incubated at 37 °C in dark for 1 h. AFC was used as standard. Fluorescence was measured at excitation and emission wavelengths of 400 nm and 505 nm respectively, on a microplate reader. The enzyme activity was expressed as nmol AFC/60 min.
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2.5. DNA fragmentation and Cell Death Detection The DNA fragmentation pattern (DNA ladder) was carried out by agarose gel electrophoresis. An aliquot of 2 ml (1.5 × 106 cells/ml) was incubated with cadmium and piperine for 6 and 18 h at 37 °C in a CO2 incubator. At the end of incubation, cells were pelleted by centrifugation at 200 ×g for 10 min and the pellet lysed with 0.5 ml lysis buffer (10 mM Tris, pH 7.5, 20 mM EDTA, 0.5% Triton X-100) on ice for 30 min. The DNA in lysed solution was extracted with phenol/chloroform and precipitated with 3 M sodium acetate (pH 5.2) and cold ethanol. After repeated washings, the DNA was dissolved in Tris–EDTA buffer (10 mM Tris HCl, pH 8.0: 1 mM EDTA). The purity of DNA at 260 and 280 nm absorbance ratio was between 1.7 and 1.9. DNA (2 μg) was then loaded on 0.7% agarose gel and electrophoresis carried out. The bands were visualized by ethidium bromide staining under UV light. The DNA fragmentation (histone associated mono- and oligonucleosomes) was measured using Cell Death Detection Sandwich ELISA kit (Roche, Germany). A sample of 1.5 × 106 cells/ml was incubated with cadmium (25 μM) along with piperine for 6 and 18 h at 37 °C in a CO2 incubator, out of which 1 × 105 cells were transferred to a clear tube. After centrifugation at 200 ×g for 10 min, the cell pellet was suspended in the incubation buffer for 30 min at 15–25 °C for lysis. Following centrifugation at 20,000 ×g for 10 min, the supernatant was diluted 10 folds with incubation buffer and the nucleosomes in the sample solution were measured by ELISA. Modular microtitre plates were incubated overnight with 100 μl of the antihistone antibody (coating solution) under cold conditions. After aspirating the coating solution, 200 μl of incubation buffer was added and allowed to stand for 30 min at 15–25 °C. The plates were then rinsed with washing buffer (200 μl) thrice, 100 μl of the sample solution was added into each well and incubated for 90 min at 15–25 °C. After washing, 100 μl of conjugate solution (anti-DNA-peroxidase) was added to each well and the cells were further incubated for 90 min at 15–25 °C, followed by washing. 100 μl of ABTS (2-2′-azino-di-[3-ethylbenzthiazoline sulfonate]) was then added and incubated for 10 min and the absorbance measured at 405 nm. 2.6. Flow cytometry analysis All of the following assays were carried out on splenocytes treated with cadmium and piperine for different time intervals (60 min, 1.5, 3, 6 and 18 h) at 37 °C in a CO2 incubator. The flow cytometric analysis were done on BD-LSR flow cytometer. Cell debris, characterized by a low FSC/SSC was excluded from analysis. The data was analysed by Cell Quest software and mean fluorescence intensity was obtained by histogram statistics. 2.6.1. Apoptotic DNA analysis The cells with hypodiploid DNA were determined by cell cycle studies. After the treatment period, the harvested cells were washed with PBS and fixed by drop-by-drop addition of ice cold 70% ethanol and stored at 4 °C overnight. The fixed
cells were harvested, washed with PBS and suspended in 1 ml PBS. Phosphate citrate buffer (200 μl, pH 7.8) was added and the cells incubated for 60 min at room temp. After centrifugation, the cells were resuspended in 0.5 ml of propidium iodide stain (10 mg PI, 0.1 ml Triton X-100 and 3.7 mg EDTA in 100 ml PBS) and 0.5 ml of RNase A (50 μg/ml) and further incubated for 30 min in dark. The propidium iodide (PI) fluorescence was measured through a FL-2 filter (585 nm) and 10,000 events were acquired (Darzynkiewicz et al., 1992). 2.6.2. Assessment of apoptotic and necrotic cells The apoptotic and necrotic cell distribution was analysed by Annexin V binding and PI uptake. Positioning of quadrants on Annexin V/PI dot plots was performed and living cells (Annexin V−/PI−), early apoptotic/primary apoptotic cells (Annexin V+/ PI−), late apoptotic/secondary apoptotic cells (Annexin V+/PI+) and necrotic cells (Annexin V−/PI+) were distinguished (Vermes et al., 1995). Therefore, the total apoptotic proportion included the percentage of cells with fluorescence Annexin V+/PI− and Annexin V+/PI+. Briefly, after the treatment period (6 and 18 h), the harvested cells were suspended in 1 ml binding buffer (1X). An aliquot of 100 μl was incubated with 5 μl Annexin V-FITC and 10 μl PI for 15 min in dark at room temperature and 400 μl binding buffer (1X) was added to each sample. The FITC and PI fluorescence were measured through FL-1 filter (530 nm) and FL-2 filter (585 nm) respectively, and 10,000 events were acquired. 2.6.3. Assessment of T and B lymphocyte population The lymphocyte population of splenocytes was assessed by flow cytometry, based on CD3 and CD19 surface molecules. Briefly, after incubation time (6 and 18 h), the harvested cells were resuspended in 1 ml PBS. An aliquot of 100 μl was incubated with 5 μl FITC-conjugated anti-CD3 monoclonal antibody and 5 μl PE-conjugated anti-CD19 monoclonal antibody for 30 min in dark at room temperature, after which 400 μl PBS was added to each sample. The FITC and PE fluorescence were measured through FL-1 filter (530 nm) and FL-2 filter (585 nm) respectively, and 10,000 events were acquired. 2.6.4. Mitochondrial membrane potential and Bcl-2 protein expression For the detection of mitochondrial membrane potential, the splenocytes were incubated with rhodamine 123 (5 μg/ml final conc) for 60 min in dark at 37 °C, harvested and suspended in PBS. The mitochondrial membrane potential was measured by the fluorescence intensity (FL-1, 530 nm) of 10,000 cells (Bai et al., 1999). For Bcl-2 protein expression, cadmium and piperine (50 μg/ ml) treated cells were harvested and suspended in 1 ml RPMI buffer (containing 2% FBS and 0.1% sodium azide). An aliquot of 200 μl was incubated with 5 μl of 0.2 mg/ml stock solution of rabbit anti Bcl-2 polyclonal antibody for 20 min at 2–8 °C. The excess antibody was removed by washing cells twice with RPMI buffer. After washing, the cells were resuspended in 200 μl RPMI buffer and the binding of antibody was visualized
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Fig. 1. Effect of piperine on cadmium altered viability in murine splenocytes. Freshly isolated splenocytes (1.5 × 104) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 18 h. Absorbance was measured at 530 nm. Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
by adding 5 μl of 50 μg/ml stock solution of FITC-conjugated anti-rabbit Ig G antibody for 20 min at 2–8 °C. After incubation, cells were washed with RPMI buffer, then with PBS and resuspended in 400 μl PBS. The FITC fluorescence was measured through FL-1 filter (530 nm) and 10,000 events were acquired.
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cells in 200 μl of complete medium were seeded in 96-well plate with or without one or more reagents (2.5 μg/ml concanavalin A, cadmium and piperine) and incubated for 72 h at 37 °C in a CO2 incubator. The plate was centrifuged and supernatant was used for measuring cytokine release by ELISA. The ELISA plates were incubated overnight with 100 μl/well of capture antibody in coating buffer under cold conditions. After aspirating the coating solution, the wells were washed 3 times with 300 μl wash buffer and after removing the buffer carefully, 200 μl of blocking solution was added for 1.0 h at 15–25 °C. The plates were washed and the standard/sample solution (100 μl) were added into each well and incubated for 2.0 h at 15–25 °C. After washing the plates 5 times, 100 μl of detection antibody solution was added to each well and the plates were further incubated for 1.0 h at 15–25 °C, followed by washing. 100 μl of Avidin-HRP was then added and incubated for 30 min and the plates were again washed 7 times. The plates were further incubated with 100 μl of substrate solution for 15 min,
2.6.5. Reactive oxygen species measurement The generation of reactive oxygen species was detected by DCF fluorescence. Splenocytes were incubated with cadmium and piperine and DCFH-DA (100 μM final concentration) was added simultaneously to the medium. The cells were harvested, suspended in PBS and reactive oxygen species generation was measured by DCF fluorescence intensity (FL-1, 530 nm) of 10,000 cells (Wang et al., 1996). 2.6.6. Glutathione (GSH) measurement Intracellular GSH in splenocytes was monitored by CMFDA. After treatment, the cells were incubated with CMF-DA (1 μM final concentration) for 30 min in dark at 37 °C. After harvesting, the cells were suspended in PBS and GSH was measured by CMF fluorescence intensity (FL-1, 530 nm) of 10,000 cells (Okada et al., 1996). 2.7. [3H] Thymidine incorporation measurement To measure splenic cell proliferation, 1.0 × 104 cells were seeded in 96-well plates in 200 μl of complete medium with or without one or more reagents {2.5 μg/ml concanavalin A, 5 μg/ml lipopolysaccharide (LPS), cadmium and piperine} and incubated for 24 and 72 h at 37 °C in a CO2 incubator. [3H] Thymidine (2 μCi) was added to the wells, 18 h prior to the completion of incubation time. The cells were collected with cell harvester and incorporated radioactivity was measured in a liquid scintillation counter (Hewett Packard). 2.8. Release of cytokines The release of Interleukin-2 and gamma-Interferon in splenocytes was measured by using Mouse Interleukin-2 and gamma-Interferon ELISA kits respectively. Briefly, 1.0 × 104
Fig. 2. Effect of piperine on cadmium induced reactive oxygen species generation. Freshly isolated splenocytes (1.5 × 106) were incubated with DCFHDA (100 μM), cadmium (25 μM) and piperine (1–50 μg/ml) for 1.5, 3, 6 and 18 h at 37 °C. DCF fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A, 6 h) and mean fluorescence was obtained from the histogram statistics (B, C). Each bar represents mean ± S.D. (n = 3).⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
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piperine (50 μg/ml) restored the viability to near normal (Fig. 1). The 50% loss in cell viability by cadmium was checked even with 1 μg/ml piperine (P b 0.05). Piperine alone did not elicit any cytotoxicity. Following this observation, we next investigated the antioxidative potential of piperine in cadmium induced oxidatively stressed splenocytes. 3.2. Effect of piperine on reactive oxygen species generation and intracellular GSH Reactive oxygen species and GSH are oxidative stress markers and act as early intracellular signals for splenocytes to undergo programmed cell death (Pathak and Khandelwal, 2006b). Cadmium at all time points (1.5–18 h) caused significant reactive oxygen species generation which peaked at 6 h. DCFH-DA, the fluorescent probe is mainly used for the determination of wide range of reactive oxygen species and DCF fluorescence is directly proportional to intracellular
Fig. 3. Effect of piperine on cadmium inhibited GSH levels. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (1– 50 μg/ml) for 1.5, 3, 6 and 18 h at 37 °C. CMF-DA was added and incubated for 30 min. CMF fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A, 6 h) and mean fluorescence obtained from the histogram statistics (B, C). Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
50 μl of stop solution was added to each well and the absorbance measured at 450 nm. 2.9. Statistical analysis Significance of mean of different parameters between the treatment groups was analysed using one-way analysis of variance (ANOVA) after ascertaining the homogeneity of variance between the treatments. Pair wise comparisons were done by calculating the least significant difference. 3. Results 3.1. Effect of piperine on Cd induced cell viability Piperine when added to cadmium treated splenocytes could enhance the cell viability to an extend that the highest dose of
Fig. 4. Effect of piperine on cadmium altered apoptotic and necrotic cell distribution. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) and the cell distribution was analysed using Annexin V binding and PI uptake. The FITC and PI fluorescence were measured using flow cytometer with FL-1 and FL-2 filters, respectively. Results were expressed as dot plot representing as one of the three independent experiments. LL — living cells (Annexin V−/PI−), LR — early/primary apoptotic cells (Annexin V+/PI−), UR — late/secondary apoptotic cells (Annexin V+/PI+) and UL — necrotic cells (Annexin V−/PI+).
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Fig. 5. Effect of piperine on cadmium induced apoptotic DNA. Freshly isolated splenocytes (1.5×106) were treated with cadmium (25 μM) and piperine (1–50 μg/ ml) for 18 h. The PI fluorescence was measured using a flow cytometer with FL-2 filter. Results were expressed as histogram representing the sub-G1 population (18 h).
reactive oxygen species. The latter was gradually suppressed with time and dose of piperine (Fig. 2). At initial time point i.e. 1.5 h, piperine (at all doses) exhibited significant inhibition of reactive oxygen species and the maximum effect being exhibited by 50 μg/ml piperine, which could almost completely abolish reactive oxygen species generation at 3 h and beyond. Even, ∼50% lowering of reactive oxygen species was observed with 1 μg/ml piperine at 3, 6 and 18 h. Regarding GSH in cadmium treated splenocytes, the levels remained diminished at all times with maximum depletion at 6 h. A dose dependent increase in GSH was seen when piperine was added to the cadmium treated cells (Fig. 3). Best efficacy was monitored by the highest dose of piperine which raised the GSH levels close to controls from 3 h onwards. Concurrent to reactive oxygen species inhibition, the lowest dose of piperine was equally effective in partially preventing the cells to undergo GSH deprivation even in the presence of cadmium. The results on reactive oxygen species and GSH suggest that the free radical scavenging and antioxidant characteristics (Mittal and Gupta, 2000; Vijayakumar et al., 2004) of piperine appear to play a vital role in maneuvering the oxidative stress scenario. After establishing its anti-oxidative potential, we subsequently explored the influence of piperine on various apoptotic markers. 3.3. Effect of piperine on cadmium induced apoptosis The underlying nature of cadmium toxicity via apoptotic cell death is established in various cell types (Pulido and Parrish,
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2003; Shih et al., 2003; Lemarie et al., 2004), including thymocytes (Pathak and Khandelwal, 2006c) and splenocytes (Pathak and Khandelwal, 2006b). Cadmium causes almost 27– 30% splenic cell death by apoptosis at 18 h. The necrotic cell death being negligible. In our earlier studies, cadmium induced both necrosis and apoptosis in murine thymocytes in vitro as well as in vivo, but only apoptotic cell death of splenocytes in vivo. Our present results on splenic apoptosis corroborated with our previous findings. Piperine could substantially suppress apoptosis both at 6 and 18 h. With the Annexin binding assay, it is possible to identify cells in early and late apoptotic phase. At 18 h, out of the total cells undergoing apoptosis, ∼ 90% appeared normal with the highest dose of piperine and the apoptotic index levelled at 6% compared to 3.8% in controls (Fig. 4). The anti-apoptotic efficacy of piperine was observed even with the minimal dose as early as 6 h. Another assay for determining apoptosis i.e. the hypodiploid DNA (sub G1 population) (Fig. 5, Table 1) correlated with the Annexin binding data. In addition to the flow cytometric analysis of apoptosis, the intranucleosomal fragmentation (DNA ladder) induced by cadmium was observed to be diminished by piperine in a dose related fashion (Fig. 6). The Sandwich ELISA method which measures mono- and oligonucleosomes as shown in Table 2 also demonstrated the dose dependent decrease in absorbance at 405 nm by piperine. The anti-apoptotic nature of piperine was clear from the results of Apoptosis and DNA fragmentation. Piperine, itself did not elicit any adverse influence on the splenic cells as apparent by oxidative stress and apoptotic data. 3.4. Mitochondrial membrane potential, Bcl-2 protein and caspase-3 activity To further evaluate the effect of piperine on mitochondrial apoptosis signaling pathway, we examined whether piperine affects apoptosis by modulating the expression of Bcl-2 and caspase-3. The mitochondrial membrane depolarization being the early marker of apoptosis was monitored at 1.5, 3 and 6 h. Mitochondrial membrane potential substantially lowered by Table 1 Effect of piperine on Cd induced apoptotic DNA % of apoptotic cells Groups
3h
6h
18 h
Control pip 1 pip 10 pip 50 Cd 25 μM Cd + pip 1 Cd + pip 10 Cd + pip 50
1.8 ± 0.5 1.6 ± 0.3 1.9 ± 0.3 2.1 ± 0.7 1.4 ± 0.8 1.9 ± 0.6 1.6 ± 0.4 2.0 ± 0.5
2.6 ± 0.6 2.2 ± 0.8 2.4 ± 0.4 2.8 ± 1.2 16.8 ± 2.6 15.4 ± 2.3 2.6 ± 2.2c 10.5 ± 2.4b
3.7 ± 1.2 4.1 ± 1.9 3.8 ± 1.5 3.7 ± 1.4 26.1 ± 48 18.9 ± 3.5b 11.2 ± 2.8a 6.1 ± 1.8a
Freshly isolated splenocytes (1.5 × 106) were treated with Cd (25 μM) and piperine (1–50 μg/ml) for 3, 6 and 18 h. The propidium iodide fluorescence was measured using a flow cytometer with FL-2 filter. Results were expressed as the percentage of apoptotic cells obtained from the histogram statistics. Each value represents mean ± S.D. (n = 3). aP b 0.001, bP b 0.01,cP b 0.05 as compared to Cd group, using one-way ANOVA.
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3.5. Effect of piperine on cadmium altered splenic cell population
Fig. 6. Effect of piperine on cadmium induced DNA fragmentation by (0.7%) agarose gel electrophoresis. Freshly isolated splenocytes (3.0 Effect of piperine on cadmium induced DNA fragmentation by (0.7%) agarose gel electrophoresis. Freshly isolated splenocytes (3.0 × 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 18 h.
To address whether the cell protective efficacy of piperine is predominant with B or T cells, we determined their population with the help of cell surface markers: CD3 and CD19 for T and B cells, respectively. Alterations in T and B cell percentage was seen not earlier than 18 h. The CD3 population fell from 39.8 to 24.2% by cadmium and in the presence of piperine, the CD3 cells were restored in a dose dependent manner. With 50 μg/ml piperine, the number of T cells became very close to control cells i.e. 41.5%. Similarly, the suppressed B cell population (from 54.8 to 31.5%) was also observed coming close to the control with piperine. The highest dose of piperine, completely restored B cells to 55.2% (Fig. 9).
cadmium at 3 and 6 h was significantly reverted by piperine (Fig. 7) and with the highest concentration, the depolarization effect of cadmium was completely nullified. Similar was the case with caspase-3 activation by cadmium and its amelioration by piperine (Table 3). The higher the dose of piperine, the greater was the inhibition of caspase-3 activity, being maximum at 3 h. The Bcl-2 family proteins control the rate of apoptosis. Members of the Bcl-2 family have been demonstrated to be associated with the mitochondrial membrane and regulate its integrity (Adams and Corey, 1998; Hengartner, 2000). In the mitochondrial death pathway, the ratio of expression of the proapoptotic Bax protein and the anti-apoptotic Bcl-2 or Bcl-xl protein ultimately determine the fate of the cell. The reduced expression of Bcl-2 by cadmium was substantially upregulated by piperine (50 μg/ml) and the alkaloid alone had no influence on the protein (Fig. 8). The outcome of above results is a clear indication of chemopreventive efficacy of piperine (anti-oxidative and antiapoptotic). As strongly suggested for the anti-apoptotic potential of most herbal antioxidants, modulation of reactive oxygen species and GSH at early time points may lead to the amelioration of cadmium induced lymphocyte apoptosis.
Table 2 Effect of piperine on Cd induced DNA fragmentation (mono- and oligonucleosomes) Groups
O.D. 405 (6 h)
O.D. 405 (18 h)
Control Cd 25 μM Pip 1 Pip 10 Pip 50 Cd + Pip 1 Cd + Pip 10 Cd + Pip 50
100 ± 8.6 130 ± 8.6 106 ± 4.8 98 ± 6.5 105 ± 10.5 122 ± 4.6 115 ± 5.7b 106 ± 12.6a
100 ± 6.5 183 ± 18.8 105 ± 16.5 102 ± 11.2 96 ± 10.5 159 ± 8.6b 126 ± 12.4a 109 ± 7.8a
Freshly isolated splenocytes (1.5 × 106) were treated with Cd (25 μM) and piperine (1–50 μg/ml) for 6 and 18 h at 37 °C. DNA fragments were determined by Cell Death Detection ELISA. Each value represents mean ± S.D (n = 3). a P b 0.001, bP b 0.01 as compared to Cd group, using one-way ANOVA.
Fig. 7. Effect of piperine on cadmium induced mitochondrial membrane depolarization. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 1.5, 3 and 6 h at 37 °C. Rh 123 was added and incubated for 60 min. The fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A, 3 h) and mean fluorescence obtained from the histogram statistics (B, C). Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
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Table 3 Effect of piperine on Cd induced caspase-3 activity Groups
1.5 h
3.0 h
6.0 h
Control Cd 25 μM Pip 1 Pip 10 Pip 50 Cd + Pip 1 Cd + Pip 10 Cd + Pip 50
0.06 ± 0.01 0.11 ± 0.02 0.06 ± 0.01 0.07 ± 0.01 0.06 ± 0.02 0.12 ± 0.01 0.10 ± 0.03 0.09 ± 0.01
0.08 ± 0.02 0.34 ± 0.06 0.07 ± 0.01 0.09 ± 0.03 0.10 ± 0.02 0.29 ± 0.04c 0.27 ± 0.01c 0.12 ± 0.01a
0.09 ± 0.01 0.42 ± 0.01 0.12 ± 0.01 0.11 ± 0.01 0.12 ± 0.01 0.32 ± 0.01b 0.20 ± 0.01a 0.13 ± 0.01a
Freshly isolated splenocytes (3.0 × 106) were treated with Cd (25 μM) and piperine (1–50 μg/ml) for 1.5, 3 and 6 h at 37 °C. The enzyme activity was determined by Fluorometric Assay using AFC as standard and the activity was expressed as nmol AFC/60 min. The fluorescence was measured at Ex: 400 nm and Em: 505 nm. aP b 0.001, bP b 0.01, cP b 0.05 as compared to cadmium group, using one-way ANOVA.
3.6. Effect of piperine on cadmium altered splenocyte proliferation Another aspect of piperine studied in the present study is its influence on mitogenic stimulated splenic cell proliferation. Piperine effectively mitigated the inhibitory effect of cadmium studied by thymidine uptake in concanavalin A and lipo polysachharide stimulated T and B cells, respectively. The mitogenically stimulated and non-stimulated splenic cells, incorporated 50% less thymidine, on cadmium treatment. Piperine, alone did not influence blastogenesis. As seen in
Fig. 9. Effect of piperine on cadmium altered splenic cell population. Freshly isolated splenocytes (1.5× 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 18 h and the cells were stained with FITC-conjugated anti-CD3 monoclonal antibody for T cells and PE-conjugated anti-CD19 monoclonal antibody for B cells. The FITC and PE fluorescence were measured independently, using flow cytometer with FL-1 and FL-2 filters, respectively. Results were expressed as dot plot representing as one of the three independent experiments.
Fig. 10, with the highest dose of piperine, the cadmium induced inhibited mitogenic proliferative response was almost abolished and the thymidine uptake both in concanavalin A and lipo polysachharide stimulated cells reached to near normal values. A similar activity of piperine was also observed in nonstimulated splenic cells. After establishing significant protection by piperine against cadmium induced apoptosis, oxidative stress, splenic phenotypes and blastogenesis, we further explored the role of piperine in modulation of immune function i.e. the release of cytokines (Interleukin-2 and gamma-Interferon). 3.7. Effect of piperine on the release of cytokines Fig. 8. Effect of piperine on cadmium lowered Bcl-2 protein. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (50 μg/ ml) for 18 h at 37 °C. Bcl-2 protein was measured using rabbit anti Bcl-2 polyclonal antibody and FITC-conjugated anti-rabbit Ig G antibody. FITC fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A) and mean fluorescence obtained from the histogram statistics (B). Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001 as compared to cadmium group, using one-way ANOVA.
Cadmium caused ∼ 2 fold suppression of Interleukin-2 and gamma-Interferon in concanavalin A stimulated splenic cells (Fig. 11). With piperine addition, the level of Interleukin-2 and gamma-Interferon continued to rise with increase in dose and with the highest dose of piperine, the cytokines were close to normal. In non-stimulated cells, there was no influence of
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piperine. The above results suggest the enhanced efficacy of piperine in the release of cytokines by splenic cells. 4. Discussion
Fig. 10. Effect of piperine on cadmium altered blastogenesis. Freshly isolated splenocytes (1.0 × 104) were treated with cadmium (25 μM), piperine (1– 50 μg/ml), concanavalin A (2.5 μg/ml) and LPS (5 μg/ml) for 72 h at 37 °C. [3H] Thymidine (2 μCi) was added, 18 h prior to completion of incubation time. The radioactivity was measured in a liquid scintillation counter and the results were expressed as cpm (A) concanavalin A, lipopolysachharide and piperine, (B) concanavalin A, lipopolysachharide, cadmium and piperine. Each bar represents mean ± S.D. (n = 3). aP b 0.001, bP b 0.01, cP b 0.05 as compared to Cd, ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01 as compared to cadmium + concanavalin A, x P b 0.001, yP b 0.01 as compared to cadmium + lipopolysachharide group, using one-way ANOVA.
Plant alkaloids like berberine, tetrandrine, vinblastin, taxol, piperine etc. are recognized as chemopreventive agents and are believed to be pharmacologically harmless (Henderson et al., 2003; Ho et al., 2004; Sikorska et al., 2004; Hwang et al., 2006). Piperine with a long history of medicinal use, has been experimentally evaluated for its multiple biological activities, such as anti-metastatic, anti-inflammatory, hepatoprotective, antithyroid, antitumor and immunomodulatory (Koul and Kapil, 1993; Pradeep and Kuttan, 2002; Panda and Kar, 2003; Sunila and Kuttan, 2004). The mechanism of immunomodulation by piperine, has been studied by us in the present investigation. Oxidative stress and apoptosis, the two closely interlinked phenomena (Pulido and Parrish, 2003; Lemarie et al., 2004; Pathak and Khandelwal, 2006b) have been successfully proven in cadmium exposed rodent models as well as various cell types (Hart et al., 1999; Fujimaki et al., 2000; Pathak and Khandelwal, 2007). There are reports that thiol antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate reduce apoptosis by modulation of oxidative stress markers (GSH and reactive oxygen species) (Aruoma et al., 1989; Schreck et al., 1991; Shih et al., 2003). Recently, Pathak and Khandelwal (2006c) have also demonstrated oxidative stress followed by apoptosis leading to altered immune function and cell proliferative mitogenic response in murine thymocytes. The same group further reported that piperine supplementation effectively restored immune function
Fig. 11. Effect of piperine on cadmium altered cytokine levels. Freshly isolated splenocytes (1.0 × 104) were treated with cadmium (25 μM), and concanavalin A (2.5 μg/ml) for 72 h at 37 °C. The cytokines (Interleukin-2 and gamma-Interferon) were measured by ELISA kits. Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
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and proliferation notably by curbing oxidative stress damage to murine thymocytes (Pathak and Khandelwal, 2006a). Similarly, splenocytes comprising of both B and T cells respond favourably to piperine, resulting in complete abrogation of the influence of cadmium and leading to restoration of the B and T cell population in this study. Piperine by itself remains non cytotoxic (MTT assay). As reported by Vijayakumar et al. (2004), the antioxidant efficacy of black pepper and piperine in rats with high fat diet induced oxidative stress, was shown by lowering TBARS and Conjugated dienes. The SOD, CAT, GPX, GST and GSH levels were maintained close to controls. The anti-oxidative efficacy of piperine observed in our study could be attributed to its free radical scavenging property (Mittal and Gupta, 2000). Piperine (50 μg/ml) could effectively enhance the GSH levels at 3 h and lower the reactive oxygen species production as early as 1.5 h and may account for corrective signalling by these critical cellular biomarkers. These in turn could lead to apoptotic attenuation and at 18 h almost all the splenic cells undergoing apoptosis behaved as normal. Similar to the T cell phenotypic alterations (Pathak and Khandelwal, 2006a), cadmium also alters the splenic cell population to a significant extend. The anti-apoptotic nature of piperine appears quite evident in the normalization of B and T splenic population. Considering that viable cells are responsible for any immune response and function, we observed that piperine appears to mitigate the adverse effects of cadmium on cytokines (Interleukin-2 and gamma-Interferon) and cell proliferative mitogenic response. The data on cadmium exposed splenic cells and piperine revealed normal immune responsive behaviour of viable cells and could explain the chemopreventive action of piperine. Changes in reactive oxygen species and GSH by cadmium may be associated with alterations in the expression of several antioxidant and apoptotic genes and interaction of piperine through redox sensitive pathways is quite plausible, since Li et al. (2007) observed that piperine could upregulate the mRNA level of brain-derived neurotrophic factor in hippocampus of chronic mild stressed mice. It is unclear at present whether the anti-oxidative and free radical scavenging property of piperine is wholly responsible for its immuno-compromised restorative ability or it is the additive influence of its multiple cellular activities which remain undetermined so far. We can therefore, conclude that immunomodulation by piperine may be clearly attributed to its multi faceted activities such as anti-oxidative, anti-apoptotic and restorative ability against cell proliferative mitogenic response, splenic B and T cell population and cytokine release. These cytoprotective properties of piperine are suggestive of its therapeutic usefulness in immuno-compromised situations. Acknowledgements Authors are grateful to Director, ITRC for his keen interest in this work and to Dr. Y. Shukla for providing flow cytometer facility. The secretarial assistance of Mr. R.S. Verma is acknowledged.
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European Journal of Pharmacology 576 (2007) 160 – 170 www.elsevier.com/locate/ejphar
Cytoprotective and immunomodulating properties of piperine on murine splenocytes: An in vitro study Neelima Pathak, Shashi Khandelwal ⁎ Industrial Toxicology Research Centre, Mahatma Gandhi Marg, P.Box 80, Lucknow — 226001, India Received 22 February 2007; received in revised form 9 July 2007; accepted 17 July 2007 Available online 25 July 2007
Abstract Piper longum Linn. and Piper nigrum Linn. are conventionally used as immuno-enhancers in Indian system of traditional medicine. The underlying mechanism remains unknown. The present study was therefore, undertaken to delineate the role of piperine (major alkaloid) in cadmium (Cd) induced immuno-compromised murine splenocytes. The various biological determinants such as oxidative stress markers (reactive oxygen species and GSH), Bcl-2 protein expression, mitochondrial membrane potential, caspase-3 activity, DNA damage, splenic B and T cell population, blastogenesis and cytokines (Interleukin-2 and gamma-Interferon) were measured to ascertain its cell protective potential. Cadmium induces apoptosis at 6 h onwards. The oxidative stress markers markedly alter prior to a decline in mitochondrial membrane potential, caspase-3 activation and DNA degradation The splenic cell population was observed to change only at 18 h and the release of two cytokines was affected at 72 h. Addition of piperine in various concentrations (1, 10 and 50 μg/ml) ameliorated the above events. The highest dose of piperine could completely abrogate the toxic manifestations of cadmium and the splenic cells behaved similar to control cells. The reported free radical scavenging property of piperine and its antioxidant potential could be responsible for the modulation of intracellular oxidative stress signals. These in turn appear to mitigate the apoptotic pathway and other cellular responses altered by cadmium. The findings strongly indicate the anti-oxidative, anti-apoptotic and chemo-protective ability of piperine in blastogenesis, cytokine release and restoration of splenic cell population and is suggestive of its therapeutic usefulness in immuno-compromised situations. © 2007 Elsevier B.V. All rights reserved. Keywords: Piperine; Apoptosis; Oxidative stress; Phenotyping; Blastogenesis; Cadmium
1. Introduction Piper longum Linn. and Piper nigrum Linn. are used in Indian traditional medicine (Singh, 1992) and as a spice globally. Piperine, the alkaloid is responsible for much of the taste and smell of black pepper. It exhibits a wide variety of biological effects including anti-metastatic, antithyroid, antidepressant, hepatoprotective and antitumor. The anti-metastatic property of piperine in mice has been shown by reduction in lung collagen hydroxyproline, hexosamine and uronic acid induced by B16F-10 melanoma cells (Pradeep and Kuttan, 2002). Inhibition of Phase I and Phase II enzymes, elevation of GSH metabolizing enzymes, reduction in DNA damage and DNA protein cross-links in benzo(a)pyrene induced lung carcinogenesis has been observed by piperine supplementation ⁎ Corresponding author. Tel.: +91 522 2627586; fax: +91 522 2628227. E-mail address: [email protected] (S. Khandelwal). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.07.033
(Selvendiran et al., 2005a,b). Additionally, the chemopreventive efficacy of piperine relating to a decrease in lipid peroxidation, protein carbonyls, nucleic acid and polyamine synthesis has also been reported (Selvendiran et al., 2004). The bioavailability enhancing activity of piperine with various structurally and therapeutically diverse drugs has been studied by Khanjuria et al. (2002). This was due to alterations in membrane dynamics and permeation characteristics along with induced synthesis of protein associated with cytoskeletal function. The antioxidant efficacy of piperine was shown both in vitro (Mittal and Gupta, 2000) and in vivo (Vijayakumar et al., 2004) and its hepatoprotective ability (Koul and Kapil, 1993) and thyroid hormone lowering efficacy have also been reported. Recently, Li et al. (2007) demonstrated that piperine could reverse the corticosterone induced reduction of brainderived neurotrophic factor mRNA expression in cultured hippocampal neurons. Although, immuno-enhancing ability of Piper sp. has long been known and practiced in Indian
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traditional medicine, sporadic scientific reports are available till date. Piperine was found to be cytotoxic towards Dalton's lymphoma ascites cells and Ehrlich ascites carcinoma cells at a dose of 250 μg/ml (Sunila and Kuttan, 2004). An increase in the circulating antibody titre and antibody forming cells, along with enhanced bone marrow cellularity and α-esterase positive cells was also observed. In addition, we have also studied the cell mediated stimulatory response of piperine in cadmium induced alterations in immuno-compromised murine thymocytes and demonstrated anti-oxidative and anti-apoptotic potential of piperine. Its restorative ability against cell proliferative mitogenic response and phenotypic alterations was also seen (Pathak and Khandelwal, 2006a). Humoral and cell mediated immunity are the two arms of immune system and the latter has been extensively studied by us, we now explored the possible efficacy of piperine in murine splenocytes, since spleen is one of the principle sites for the initiation of most primary immune responses, for B lymphocyte activation and the production of antibodies. We have chosen cadmium (Cd) as an immunotoxicant because, firstly it is classified as a category I human carcinogen (IARC, 1993) and secondly, managing cadmium poisoning with safe and effective compounds has always been a challenge. Towards this goal, we planned the following study incorporating a number of determinants. 1) Oxidative stress and apoptotic hallmarks to ascertain the anti-oxidant and anti-apoptotic efficacy of piperine together with 2) its influence on the splenic population (B and T lymphocytes), cell proliferative mitogenic response and immune function i.e. cytokine release (Interleukin-2 and gamma-Interferon). Our results clearly demonstrated significant immunostimulation by piperine. It exhibited both anti-oxidative and antiapoptotic potential. The suppressed B and T cells by cadmium were reverted back close to the control splenic population and the inhibited splenic cell proliferative response and cytokine release were also restored by piperine. 2. Materials and methods 2.1. Chemicals All the chemicals were of highest grade purity available. Cadmium chloride (CdCl2), RNase A, RPMI 1640, antibiotic– antimycotic solution, Dulbecco's phosphate buffered saline (PBS), fetal bovine serum (FBS), agarose, 3-(4,5-dimethyl-2-yl)2,5-diphenyl tetrazolium bromide (MTT), 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), piperine, concanavalin A (Con A), FITC-conjugated anti-rabbit Ig G antibody, 7-amino-4-trifluoromethylcoumarin (AFC), benzyloxy-carbonyl-Asp-Glu-Val-Asp7-amino-4-trifluoromethyl-coumarin (DEVD-AFC) and all other chemicals were purchased from Sigma Aldrich, USA. Rhodamine 123 (Rh 123) and 5′-Chloromethylfluorescein diacetate (CMFDA) from Molecular Probes, propidium iodide (PI) from Calbiochem, Annexin V-FITC and rabbit anti Bcl-2 polyclonal antibody from Biovision, [3H] Thymidine from BRIT, Bombay, FITC-conjugated anti-CD3 monoclonal antibody, PE-conjugated anti-CD19 monoclonal antibody and mouse Interleukin-2 and
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gamma-Interferon ELISA kits from eBioscience. Cell Death Detection sandwich ELISA kit from Roche, Germany and caspase-3 fluorometric protease assay kit was purchased from Chemicon, USA. 2.2. Preparation of splenocyte suspension The BALB/c mice were maintained in our animal house under standard conditions. They were fed with standard rodent pellet and water ad libitum. Our animal house and breeding facility are registered with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and CPCSEA guidelines were followed (IAEC approval obtained). Spleen was dissected from male BALB/c mice (4–6 weeks old) and single cell suspension prepared under aseptic conditions. The suspension was passed through 100 μm stainless steel mesh, treated with 5.0 ml hypotonic NH4Cl solution (2.42 g Tris and 7.56 g NH4Cl in 1.0 l deionised water, pH adjusted to 7.2) for 5 min at room temperature to lyse red blood cells, centrifuged and washed with FBS free medium. The cells were suspended in complete cell culture medium (RPMI 1640 containing HEPES and 2 mM glutamine, supplemented with 10% FBS and 1% antibiotic–antimycotic solution). The cell density was adjusted to ca. 1.5 × 106 cells/ml and the viability of the freshly isolated cells was always over 95% (trypan blue exclusion test). For the monitoring of various parameters in the present investigation, we have used 25 μM concentration of cadmium and 1, 10 and 50 μg/ml of piperine. The cadmium concentration selected was based on our earlier work (Pathak and Khandelwal, 2006b). 2.3. Assessment of cell viability The cell viability was measured by the MTT reduction method (Mosmann, 1983). Cells in RPMI 1640 were seeded at a density of 1.0 × 104 in 96 well plate. The cells were treated with cadmium along with piperine for 18 h at 37 °C in a CO2 incubator. Ten μl MTT (5 mg/ml PBS) was added to the wells, 4 h prior to the completion of incubation time. The plate was centrifuged at 1200 ×g for 10 min and 100 μl of DMSO was added after removing the supernatant, to dissolve the formazan formed. The absorbance was read at 530 nm after 5 min, in a microplate reader (Synergy HT of BIO-TEK International, USA). 2.4. Measurement of caspase-3 activity A population of 3.0 × 106 cells/ml was incubated with cadmium and piperine for 1.5, 3 and 6 h at 37 °C in a CO2 incubator. The cells were scraped and lysed on ice for 10 min using cell lysis buffer. The reaction buffer (10 mM Tris, 1 mM EDTA, 10 mM dithiothretol, 5% glycerol) and DEVD-AFC substrate (50 μM final concentration) was then added and further incubated at 37 °C in dark for 1 h. AFC was used as standard. Fluorescence was measured at excitation and emission wavelengths of 400 nm and 505 nm respectively, on a microplate reader. The enzyme activity was expressed as nmol AFC/60 min.
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2.5. DNA fragmentation and Cell Death Detection The DNA fragmentation pattern (DNA ladder) was carried out by agarose gel electrophoresis. An aliquot of 2 ml (1.5 × 106 cells/ml) was incubated with cadmium and piperine for 6 and 18 h at 37 °C in a CO2 incubator. At the end of incubation, cells were pelleted by centrifugation at 200 ×g for 10 min and the pellet lysed with 0.5 ml lysis buffer (10 mM Tris, pH 7.5, 20 mM EDTA, 0.5% Triton X-100) on ice for 30 min. The DNA in lysed solution was extracted with phenol/chloroform and precipitated with 3 M sodium acetate (pH 5.2) and cold ethanol. After repeated washings, the DNA was dissolved in Tris–EDTA buffer (10 mM Tris HCl, pH 8.0: 1 mM EDTA). The purity of DNA at 260 and 280 nm absorbance ratio was between 1.7 and 1.9. DNA (2 μg) was then loaded on 0.7% agarose gel and electrophoresis carried out. The bands were visualized by ethidium bromide staining under UV light. The DNA fragmentation (histone associated mono- and oligonucleosomes) was measured using Cell Death Detection Sandwich ELISA kit (Roche, Germany). A sample of 1.5 × 106 cells/ml was incubated with cadmium (25 μM) along with piperine for 6 and 18 h at 37 °C in a CO2 incubator, out of which 1 × 105 cells were transferred to a clear tube. After centrifugation at 200 ×g for 10 min, the cell pellet was suspended in the incubation buffer for 30 min at 15–25 °C for lysis. Following centrifugation at 20,000 ×g for 10 min, the supernatant was diluted 10 folds with incubation buffer and the nucleosomes in the sample solution were measured by ELISA. Modular microtitre plates were incubated overnight with 100 μl of the antihistone antibody (coating solution) under cold conditions. After aspirating the coating solution, 200 μl of incubation buffer was added and allowed to stand for 30 min at 15–25 °C. The plates were then rinsed with washing buffer (200 μl) thrice, 100 μl of the sample solution was added into each well and incubated for 90 min at 15–25 °C. After washing, 100 μl of conjugate solution (anti-DNA-peroxidase) was added to each well and the cells were further incubated for 90 min at 15–25 °C, followed by washing. 100 μl of ABTS (2-2′-azino-di-[3-ethylbenzthiazoline sulfonate]) was then added and incubated for 10 min and the absorbance measured at 405 nm. 2.6. Flow cytometry analysis All of the following assays were carried out on splenocytes treated with cadmium and piperine for different time intervals (60 min, 1.5, 3, 6 and 18 h) at 37 °C in a CO2 incubator. The flow cytometric analysis were done on BD-LSR flow cytometer. Cell debris, characterized by a low FSC/SSC was excluded from analysis. The data was analysed by Cell Quest software and mean fluorescence intensity was obtained by histogram statistics. 2.6.1. Apoptotic DNA analysis The cells with hypodiploid DNA were determined by cell cycle studies. After the treatment period, the harvested cells were washed with PBS and fixed by drop-by-drop addition of ice cold 70% ethanol and stored at 4 °C overnight. The fixed
cells were harvested, washed with PBS and suspended in 1 ml PBS. Phosphate citrate buffer (200 μl, pH 7.8) was added and the cells incubated for 60 min at room temp. After centrifugation, the cells were resuspended in 0.5 ml of propidium iodide stain (10 mg PI, 0.1 ml Triton X-100 and 3.7 mg EDTA in 100 ml PBS) and 0.5 ml of RNase A (50 μg/ml) and further incubated for 30 min in dark. The propidium iodide (PI) fluorescence was measured through a FL-2 filter (585 nm) and 10,000 events were acquired (Darzynkiewicz et al., 1992). 2.6.2. Assessment of apoptotic and necrotic cells The apoptotic and necrotic cell distribution was analysed by Annexin V binding and PI uptake. Positioning of quadrants on Annexin V/PI dot plots was performed and living cells (Annexin V−/PI−), early apoptotic/primary apoptotic cells (Annexin V+/ PI−), late apoptotic/secondary apoptotic cells (Annexin V+/PI+) and necrotic cells (Annexin V−/PI+) were distinguished (Vermes et al., 1995). Therefore, the total apoptotic proportion included the percentage of cells with fluorescence Annexin V+/PI− and Annexin V+/PI+. Briefly, after the treatment period (6 and 18 h), the harvested cells were suspended in 1 ml binding buffer (1X). An aliquot of 100 μl was incubated with 5 μl Annexin V-FITC and 10 μl PI for 15 min in dark at room temperature and 400 μl binding buffer (1X) was added to each sample. The FITC and PI fluorescence were measured through FL-1 filter (530 nm) and FL-2 filter (585 nm) respectively, and 10,000 events were acquired. 2.6.3. Assessment of T and B lymphocyte population The lymphocyte population of splenocytes was assessed by flow cytometry, based on CD3 and CD19 surface molecules. Briefly, after incubation time (6 and 18 h), the harvested cells were resuspended in 1 ml PBS. An aliquot of 100 μl was incubated with 5 μl FITC-conjugated anti-CD3 monoclonal antibody and 5 μl PE-conjugated anti-CD19 monoclonal antibody for 30 min in dark at room temperature, after which 400 μl PBS was added to each sample. The FITC and PE fluorescence were measured through FL-1 filter (530 nm) and FL-2 filter (585 nm) respectively, and 10,000 events were acquired. 2.6.4. Mitochondrial membrane potential and Bcl-2 protein expression For the detection of mitochondrial membrane potential, the splenocytes were incubated with rhodamine 123 (5 μg/ml final conc) for 60 min in dark at 37 °C, harvested and suspended in PBS. The mitochondrial membrane potential was measured by the fluorescence intensity (FL-1, 530 nm) of 10,000 cells (Bai et al., 1999). For Bcl-2 protein expression, cadmium and piperine (50 μg/ ml) treated cells were harvested and suspended in 1 ml RPMI buffer (containing 2% FBS and 0.1% sodium azide). An aliquot of 200 μl was incubated with 5 μl of 0.2 mg/ml stock solution of rabbit anti Bcl-2 polyclonal antibody for 20 min at 2–8 °C. The excess antibody was removed by washing cells twice with RPMI buffer. After washing, the cells were resuspended in 200 μl RPMI buffer and the binding of antibody was visualized
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Fig. 1. Effect of piperine on cadmium altered viability in murine splenocytes. Freshly isolated splenocytes (1.5 × 104) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 18 h. Absorbance was measured at 530 nm. Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
by adding 5 μl of 50 μg/ml stock solution of FITC-conjugated anti-rabbit Ig G antibody for 20 min at 2–8 °C. After incubation, cells were washed with RPMI buffer, then with PBS and resuspended in 400 μl PBS. The FITC fluorescence was measured through FL-1 filter (530 nm) and 10,000 events were acquired.
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cells in 200 μl of complete medium were seeded in 96-well plate with or without one or more reagents (2.5 μg/ml concanavalin A, cadmium and piperine) and incubated for 72 h at 37 °C in a CO2 incubator. The plate was centrifuged and supernatant was used for measuring cytokine release by ELISA. The ELISA plates were incubated overnight with 100 μl/well of capture antibody in coating buffer under cold conditions. After aspirating the coating solution, the wells were washed 3 times with 300 μl wash buffer and after removing the buffer carefully, 200 μl of blocking solution was added for 1.0 h at 15–25 °C. The plates were washed and the standard/sample solution (100 μl) were added into each well and incubated for 2.0 h at 15–25 °C. After washing the plates 5 times, 100 μl of detection antibody solution was added to each well and the plates were further incubated for 1.0 h at 15–25 °C, followed by washing. 100 μl of Avidin-HRP was then added and incubated for 30 min and the plates were again washed 7 times. The plates were further incubated with 100 μl of substrate solution for 15 min,
2.6.5. Reactive oxygen species measurement The generation of reactive oxygen species was detected by DCF fluorescence. Splenocytes were incubated with cadmium and piperine and DCFH-DA (100 μM final concentration) was added simultaneously to the medium. The cells were harvested, suspended in PBS and reactive oxygen species generation was measured by DCF fluorescence intensity (FL-1, 530 nm) of 10,000 cells (Wang et al., 1996). 2.6.6. Glutathione (GSH) measurement Intracellular GSH in splenocytes was monitored by CMFDA. After treatment, the cells were incubated with CMF-DA (1 μM final concentration) for 30 min in dark at 37 °C. After harvesting, the cells were suspended in PBS and GSH was measured by CMF fluorescence intensity (FL-1, 530 nm) of 10,000 cells (Okada et al., 1996). 2.7. [3H] Thymidine incorporation measurement To measure splenic cell proliferation, 1.0 × 104 cells were seeded in 96-well plates in 200 μl of complete medium with or without one or more reagents {2.5 μg/ml concanavalin A, 5 μg/ml lipopolysaccharide (LPS), cadmium and piperine} and incubated for 24 and 72 h at 37 °C in a CO2 incubator. [3H] Thymidine (2 μCi) was added to the wells, 18 h prior to the completion of incubation time. The cells were collected with cell harvester and incorporated radioactivity was measured in a liquid scintillation counter (Hewett Packard). 2.8. Release of cytokines The release of Interleukin-2 and gamma-Interferon in splenocytes was measured by using Mouse Interleukin-2 and gamma-Interferon ELISA kits respectively. Briefly, 1.0 × 104
Fig. 2. Effect of piperine on cadmium induced reactive oxygen species generation. Freshly isolated splenocytes (1.5 × 106) were incubated with DCFHDA (100 μM), cadmium (25 μM) and piperine (1–50 μg/ml) for 1.5, 3, 6 and 18 h at 37 °C. DCF fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A, 6 h) and mean fluorescence was obtained from the histogram statistics (B, C). Each bar represents mean ± S.D. (n = 3).⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
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piperine (50 μg/ml) restored the viability to near normal (Fig. 1). The 50% loss in cell viability by cadmium was checked even with 1 μg/ml piperine (P b 0.05). Piperine alone did not elicit any cytotoxicity. Following this observation, we next investigated the antioxidative potential of piperine in cadmium induced oxidatively stressed splenocytes. 3.2. Effect of piperine on reactive oxygen species generation and intracellular GSH Reactive oxygen species and GSH are oxidative stress markers and act as early intracellular signals for splenocytes to undergo programmed cell death (Pathak and Khandelwal, 2006b). Cadmium at all time points (1.5–18 h) caused significant reactive oxygen species generation which peaked at 6 h. DCFH-DA, the fluorescent probe is mainly used for the determination of wide range of reactive oxygen species and DCF fluorescence is directly proportional to intracellular
Fig. 3. Effect of piperine on cadmium inhibited GSH levels. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (1– 50 μg/ml) for 1.5, 3, 6 and 18 h at 37 °C. CMF-DA was added and incubated for 30 min. CMF fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A, 6 h) and mean fluorescence obtained from the histogram statistics (B, C). Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
50 μl of stop solution was added to each well and the absorbance measured at 450 nm. 2.9. Statistical analysis Significance of mean of different parameters between the treatment groups was analysed using one-way analysis of variance (ANOVA) after ascertaining the homogeneity of variance between the treatments. Pair wise comparisons were done by calculating the least significant difference. 3. Results 3.1. Effect of piperine on Cd induced cell viability Piperine when added to cadmium treated splenocytes could enhance the cell viability to an extend that the highest dose of
Fig. 4. Effect of piperine on cadmium altered apoptotic and necrotic cell distribution. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) and the cell distribution was analysed using Annexin V binding and PI uptake. The FITC and PI fluorescence were measured using flow cytometer with FL-1 and FL-2 filters, respectively. Results were expressed as dot plot representing as one of the three independent experiments. LL — living cells (Annexin V−/PI−), LR — early/primary apoptotic cells (Annexin V+/PI−), UR — late/secondary apoptotic cells (Annexin V+/PI+) and UL — necrotic cells (Annexin V−/PI+).
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Fig. 5. Effect of piperine on cadmium induced apoptotic DNA. Freshly isolated splenocytes (1.5×106) were treated with cadmium (25 μM) and piperine (1–50 μg/ ml) for 18 h. The PI fluorescence was measured using a flow cytometer with FL-2 filter. Results were expressed as histogram representing the sub-G1 population (18 h).
reactive oxygen species. The latter was gradually suppressed with time and dose of piperine (Fig. 2). At initial time point i.e. 1.5 h, piperine (at all doses) exhibited significant inhibition of reactive oxygen species and the maximum effect being exhibited by 50 μg/ml piperine, which could almost completely abolish reactive oxygen species generation at 3 h and beyond. Even, ∼50% lowering of reactive oxygen species was observed with 1 μg/ml piperine at 3, 6 and 18 h. Regarding GSH in cadmium treated splenocytes, the levels remained diminished at all times with maximum depletion at 6 h. A dose dependent increase in GSH was seen when piperine was added to the cadmium treated cells (Fig. 3). Best efficacy was monitored by the highest dose of piperine which raised the GSH levels close to controls from 3 h onwards. Concurrent to reactive oxygen species inhibition, the lowest dose of piperine was equally effective in partially preventing the cells to undergo GSH deprivation even in the presence of cadmium. The results on reactive oxygen species and GSH suggest that the free radical scavenging and antioxidant characteristics (Mittal and Gupta, 2000; Vijayakumar et al., 2004) of piperine appear to play a vital role in maneuvering the oxidative stress scenario. After establishing its anti-oxidative potential, we subsequently explored the influence of piperine on various apoptotic markers. 3.3. Effect of piperine on cadmium induced apoptosis The underlying nature of cadmium toxicity via apoptotic cell death is established in various cell types (Pulido and Parrish,
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2003; Shih et al., 2003; Lemarie et al., 2004), including thymocytes (Pathak and Khandelwal, 2006c) and splenocytes (Pathak and Khandelwal, 2006b). Cadmium causes almost 27– 30% splenic cell death by apoptosis at 18 h. The necrotic cell death being negligible. In our earlier studies, cadmium induced both necrosis and apoptosis in murine thymocytes in vitro as well as in vivo, but only apoptotic cell death of splenocytes in vivo. Our present results on splenic apoptosis corroborated with our previous findings. Piperine could substantially suppress apoptosis both at 6 and 18 h. With the Annexin binding assay, it is possible to identify cells in early and late apoptotic phase. At 18 h, out of the total cells undergoing apoptosis, ∼ 90% appeared normal with the highest dose of piperine and the apoptotic index levelled at 6% compared to 3.8% in controls (Fig. 4). The anti-apoptotic efficacy of piperine was observed even with the minimal dose as early as 6 h. Another assay for determining apoptosis i.e. the hypodiploid DNA (sub G1 population) (Fig. 5, Table 1) correlated with the Annexin binding data. In addition to the flow cytometric analysis of apoptosis, the intranucleosomal fragmentation (DNA ladder) induced by cadmium was observed to be diminished by piperine in a dose related fashion (Fig. 6). The Sandwich ELISA method which measures mono- and oligonucleosomes as shown in Table 2 also demonstrated the dose dependent decrease in absorbance at 405 nm by piperine. The anti-apoptotic nature of piperine was clear from the results of Apoptosis and DNA fragmentation. Piperine, itself did not elicit any adverse influence on the splenic cells as apparent by oxidative stress and apoptotic data. 3.4. Mitochondrial membrane potential, Bcl-2 protein and caspase-3 activity To further evaluate the effect of piperine on mitochondrial apoptosis signaling pathway, we examined whether piperine affects apoptosis by modulating the expression of Bcl-2 and caspase-3. The mitochondrial membrane depolarization being the early marker of apoptosis was monitored at 1.5, 3 and 6 h. Mitochondrial membrane potential substantially lowered by Table 1 Effect of piperine on Cd induced apoptotic DNA % of apoptotic cells Groups
3h
6h
18 h
Control pip 1 pip 10 pip 50 Cd 25 μM Cd + pip 1 Cd + pip 10 Cd + pip 50
1.8 ± 0.5 1.6 ± 0.3 1.9 ± 0.3 2.1 ± 0.7 1.4 ± 0.8 1.9 ± 0.6 1.6 ± 0.4 2.0 ± 0.5
2.6 ± 0.6 2.2 ± 0.8 2.4 ± 0.4 2.8 ± 1.2 16.8 ± 2.6 15.4 ± 2.3 2.6 ± 2.2c 10.5 ± 2.4b
3.7 ± 1.2 4.1 ± 1.9 3.8 ± 1.5 3.7 ± 1.4 26.1 ± 48 18.9 ± 3.5b 11.2 ± 2.8a 6.1 ± 1.8a
Freshly isolated splenocytes (1.5 × 106) were treated with Cd (25 μM) and piperine (1–50 μg/ml) for 3, 6 and 18 h. The propidium iodide fluorescence was measured using a flow cytometer with FL-2 filter. Results were expressed as the percentage of apoptotic cells obtained from the histogram statistics. Each value represents mean ± S.D. (n = 3). aP b 0.001, bP b 0.01,cP b 0.05 as compared to Cd group, using one-way ANOVA.
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3.5. Effect of piperine on cadmium altered splenic cell population
Fig. 6. Effect of piperine on cadmium induced DNA fragmentation by (0.7%) agarose gel electrophoresis. Freshly isolated splenocytes (3.0 Effect of piperine on cadmium induced DNA fragmentation by (0.7%) agarose gel electrophoresis. Freshly isolated splenocytes (3.0 × 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 18 h.
To address whether the cell protective efficacy of piperine is predominant with B or T cells, we determined their population with the help of cell surface markers: CD3 and CD19 for T and B cells, respectively. Alterations in T and B cell percentage was seen not earlier than 18 h. The CD3 population fell from 39.8 to 24.2% by cadmium and in the presence of piperine, the CD3 cells were restored in a dose dependent manner. With 50 μg/ml piperine, the number of T cells became very close to control cells i.e. 41.5%. Similarly, the suppressed B cell population (from 54.8 to 31.5%) was also observed coming close to the control with piperine. The highest dose of piperine, completely restored B cells to 55.2% (Fig. 9).
cadmium at 3 and 6 h was significantly reverted by piperine (Fig. 7) and with the highest concentration, the depolarization effect of cadmium was completely nullified. Similar was the case with caspase-3 activation by cadmium and its amelioration by piperine (Table 3). The higher the dose of piperine, the greater was the inhibition of caspase-3 activity, being maximum at 3 h. The Bcl-2 family proteins control the rate of apoptosis. Members of the Bcl-2 family have been demonstrated to be associated with the mitochondrial membrane and regulate its integrity (Adams and Corey, 1998; Hengartner, 2000). In the mitochondrial death pathway, the ratio of expression of the proapoptotic Bax protein and the anti-apoptotic Bcl-2 or Bcl-xl protein ultimately determine the fate of the cell. The reduced expression of Bcl-2 by cadmium was substantially upregulated by piperine (50 μg/ml) and the alkaloid alone had no influence on the protein (Fig. 8). The outcome of above results is a clear indication of chemopreventive efficacy of piperine (anti-oxidative and antiapoptotic). As strongly suggested for the anti-apoptotic potential of most herbal antioxidants, modulation of reactive oxygen species and GSH at early time points may lead to the amelioration of cadmium induced lymphocyte apoptosis.
Table 2 Effect of piperine on Cd induced DNA fragmentation (mono- and oligonucleosomes) Groups
O.D. 405 (6 h)
O.D. 405 (18 h)
Control Cd 25 μM Pip 1 Pip 10 Pip 50 Cd + Pip 1 Cd + Pip 10 Cd + Pip 50
100 ± 8.6 130 ± 8.6 106 ± 4.8 98 ± 6.5 105 ± 10.5 122 ± 4.6 115 ± 5.7b 106 ± 12.6a
100 ± 6.5 183 ± 18.8 105 ± 16.5 102 ± 11.2 96 ± 10.5 159 ± 8.6b 126 ± 12.4a 109 ± 7.8a
Freshly isolated splenocytes (1.5 × 106) were treated with Cd (25 μM) and piperine (1–50 μg/ml) for 6 and 18 h at 37 °C. DNA fragments were determined by Cell Death Detection ELISA. Each value represents mean ± S.D (n = 3). a P b 0.001, bP b 0.01 as compared to Cd group, using one-way ANOVA.
Fig. 7. Effect of piperine on cadmium induced mitochondrial membrane depolarization. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 1.5, 3 and 6 h at 37 °C. Rh 123 was added and incubated for 60 min. The fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A, 3 h) and mean fluorescence obtained from the histogram statistics (B, C). Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
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Table 3 Effect of piperine on Cd induced caspase-3 activity Groups
1.5 h
3.0 h
6.0 h
Control Cd 25 μM Pip 1 Pip 10 Pip 50 Cd + Pip 1 Cd + Pip 10 Cd + Pip 50
0.06 ± 0.01 0.11 ± 0.02 0.06 ± 0.01 0.07 ± 0.01 0.06 ± 0.02 0.12 ± 0.01 0.10 ± 0.03 0.09 ± 0.01
0.08 ± 0.02 0.34 ± 0.06 0.07 ± 0.01 0.09 ± 0.03 0.10 ± 0.02 0.29 ± 0.04c 0.27 ± 0.01c 0.12 ± 0.01a
0.09 ± 0.01 0.42 ± 0.01 0.12 ± 0.01 0.11 ± 0.01 0.12 ± 0.01 0.32 ± 0.01b 0.20 ± 0.01a 0.13 ± 0.01a
Freshly isolated splenocytes (3.0 × 106) were treated with Cd (25 μM) and piperine (1–50 μg/ml) for 1.5, 3 and 6 h at 37 °C. The enzyme activity was determined by Fluorometric Assay using AFC as standard and the activity was expressed as nmol AFC/60 min. The fluorescence was measured at Ex: 400 nm and Em: 505 nm. aP b 0.001, bP b 0.01, cP b 0.05 as compared to cadmium group, using one-way ANOVA.
3.6. Effect of piperine on cadmium altered splenocyte proliferation Another aspect of piperine studied in the present study is its influence on mitogenic stimulated splenic cell proliferation. Piperine effectively mitigated the inhibitory effect of cadmium studied by thymidine uptake in concanavalin A and lipo polysachharide stimulated T and B cells, respectively. The mitogenically stimulated and non-stimulated splenic cells, incorporated 50% less thymidine, on cadmium treatment. Piperine, alone did not influence blastogenesis. As seen in
Fig. 9. Effect of piperine on cadmium altered splenic cell population. Freshly isolated splenocytes (1.5× 106) were treated with cadmium (25 μM) and piperine (1–50 μg/ml) for 18 h and the cells were stained with FITC-conjugated anti-CD3 monoclonal antibody for T cells and PE-conjugated anti-CD19 monoclonal antibody for B cells. The FITC and PE fluorescence were measured independently, using flow cytometer with FL-1 and FL-2 filters, respectively. Results were expressed as dot plot representing as one of the three independent experiments.
Fig. 10, with the highest dose of piperine, the cadmium induced inhibited mitogenic proliferative response was almost abolished and the thymidine uptake both in concanavalin A and lipo polysachharide stimulated cells reached to near normal values. A similar activity of piperine was also observed in nonstimulated splenic cells. After establishing significant protection by piperine against cadmium induced apoptosis, oxidative stress, splenic phenotypes and blastogenesis, we further explored the role of piperine in modulation of immune function i.e. the release of cytokines (Interleukin-2 and gamma-Interferon). 3.7. Effect of piperine on the release of cytokines Fig. 8. Effect of piperine on cadmium lowered Bcl-2 protein. Freshly isolated splenocytes (1.5 × 106) were treated with cadmium (25 μM) and piperine (50 μg/ ml) for 18 h at 37 °C. Bcl-2 protein was measured using rabbit anti Bcl-2 polyclonal antibody and FITC-conjugated anti-rabbit Ig G antibody. FITC fluorescence was measured using a flow cytometer with FL-1 filter. Results were expressed as a representative histogram (A) and mean fluorescence obtained from the histogram statistics (B). Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001 as compared to cadmium group, using one-way ANOVA.
Cadmium caused ∼ 2 fold suppression of Interleukin-2 and gamma-Interferon in concanavalin A stimulated splenic cells (Fig. 11). With piperine addition, the level of Interleukin-2 and gamma-Interferon continued to rise with increase in dose and with the highest dose of piperine, the cytokines were close to normal. In non-stimulated cells, there was no influence of
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piperine. The above results suggest the enhanced efficacy of piperine in the release of cytokines by splenic cells. 4. Discussion
Fig. 10. Effect of piperine on cadmium altered blastogenesis. Freshly isolated splenocytes (1.0 × 104) were treated with cadmium (25 μM), piperine (1– 50 μg/ml), concanavalin A (2.5 μg/ml) and LPS (5 μg/ml) for 72 h at 37 °C. [3H] Thymidine (2 μCi) was added, 18 h prior to completion of incubation time. The radioactivity was measured in a liquid scintillation counter and the results were expressed as cpm (A) concanavalin A, lipopolysachharide and piperine, (B) concanavalin A, lipopolysachharide, cadmium and piperine. Each bar represents mean ± S.D. (n = 3). aP b 0.001, bP b 0.01, cP b 0.05 as compared to Cd, ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01 as compared to cadmium + concanavalin A, x P b 0.001, yP b 0.01 as compared to cadmium + lipopolysachharide group, using one-way ANOVA.
Plant alkaloids like berberine, tetrandrine, vinblastin, taxol, piperine etc. are recognized as chemopreventive agents and are believed to be pharmacologically harmless (Henderson et al., 2003; Ho et al., 2004; Sikorska et al., 2004; Hwang et al., 2006). Piperine with a long history of medicinal use, has been experimentally evaluated for its multiple biological activities, such as anti-metastatic, anti-inflammatory, hepatoprotective, antithyroid, antitumor and immunomodulatory (Koul and Kapil, 1993; Pradeep and Kuttan, 2002; Panda and Kar, 2003; Sunila and Kuttan, 2004). The mechanism of immunomodulation by piperine, has been studied by us in the present investigation. Oxidative stress and apoptosis, the two closely interlinked phenomena (Pulido and Parrish, 2003; Lemarie et al., 2004; Pathak and Khandelwal, 2006b) have been successfully proven in cadmium exposed rodent models as well as various cell types (Hart et al., 1999; Fujimaki et al., 2000; Pathak and Khandelwal, 2007). There are reports that thiol antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate reduce apoptosis by modulation of oxidative stress markers (GSH and reactive oxygen species) (Aruoma et al., 1989; Schreck et al., 1991; Shih et al., 2003). Recently, Pathak and Khandelwal (2006c) have also demonstrated oxidative stress followed by apoptosis leading to altered immune function and cell proliferative mitogenic response in murine thymocytes. The same group further reported that piperine supplementation effectively restored immune function
Fig. 11. Effect of piperine on cadmium altered cytokine levels. Freshly isolated splenocytes (1.0 × 104) were treated with cadmium (25 μM), and concanavalin A (2.5 μg/ml) for 72 h at 37 °C. The cytokines (Interleukin-2 and gamma-Interferon) were measured by ELISA kits. Each bar represents mean ± S.D. (n = 3). ⁎⁎⁎P b 0.001, ⁎⁎P b 0.01, ⁎P b 0.05 as compared to cadmium group, using one-way ANOVA.
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and proliferation notably by curbing oxidative stress damage to murine thymocytes (Pathak and Khandelwal, 2006a). Similarly, splenocytes comprising of both B and T cells respond favourably to piperine, resulting in complete abrogation of the influence of cadmium and leading to restoration of the B and T cell population in this study. Piperine by itself remains non cytotoxic (MTT assay). As reported by Vijayakumar et al. (2004), the antioxidant efficacy of black pepper and piperine in rats with high fat diet induced oxidative stress, was shown by lowering TBARS and Conjugated dienes. The SOD, CAT, GPX, GST and GSH levels were maintained close to controls. The anti-oxidative efficacy of piperine observed in our study could be attributed to its free radical scavenging property (Mittal and Gupta, 2000). Piperine (50 μg/ml) could effectively enhance the GSH levels at 3 h and lower the reactive oxygen species production as early as 1.5 h and may account for corrective signalling by these critical cellular biomarkers. These in turn could lead to apoptotic attenuation and at 18 h almost all the splenic cells undergoing apoptosis behaved as normal. Similar to the T cell phenotypic alterations (Pathak and Khandelwal, 2006a), cadmium also alters the splenic cell population to a significant extend. The anti-apoptotic nature of piperine appears quite evident in the normalization of B and T splenic population. Considering that viable cells are responsible for any immune response and function, we observed that piperine appears to mitigate the adverse effects of cadmium on cytokines (Interleukin-2 and gamma-Interferon) and cell proliferative mitogenic response. The data on cadmium exposed splenic cells and piperine revealed normal immune responsive behaviour of viable cells and could explain the chemopreventive action of piperine. Changes in reactive oxygen species and GSH by cadmium may be associated with alterations in the expression of several antioxidant and apoptotic genes and interaction of piperine through redox sensitive pathways is quite plausible, since Li et al. (2007) observed that piperine could upregulate the mRNA level of brain-derived neurotrophic factor in hippocampus of chronic mild stressed mice. It is unclear at present whether the anti-oxidative and free radical scavenging property of piperine is wholly responsible for its immuno-compromised restorative ability or it is the additive influence of its multiple cellular activities which remain undetermined so far. We can therefore, conclude that immunomodulation by piperine may be clearly attributed to its multi faceted activities such as anti-oxidative, anti-apoptotic and restorative ability against cell proliferative mitogenic response, splenic B and T cell population and cytokine release. These cytoprotective properties of piperine are suggestive of its therapeutic usefulness in immuno-compromised situations. Acknowledgements Authors are grateful to Director, ITRC for his keen interest in this work and to Dr. Y. Shukla for providing flow cytometer facility. The secretarial assistance of Mr. R.S. Verma is acknowledged.
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