Chapter-II. Materials and Methods

Transcription

1 Materials and Methods

2 1. Materials 1.1 Reagents 12-O-tetradecanoyl-phorbol-13-acetate(TPA),7,12-dimethylbenz(a)anthracene(DMBA), oxidized glutathione (GSSG), reduced glutathione (GSH), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), dithiothreitol, bovine serum albumin (BSA), flavin adenine dinucleotide (FAD), horseradish peroxidase (HRP), xanthine, agrose, pyrogallol, poly-l-lysine, dichlorophenolindophenol (DCPIP),tris base, tris-hcl, o-dianisidine dihydrochloride, Griess reagent and Mayer's hematoxylin were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India. Proinflammatory cytokine TNF-α, IL-6 and IL-1β were purchased from e-bioscience,usa. Anti-phospho-p38 (dilution 1:200, Santa Cruz), anti-vegf (dilution 1:200, Thermo scientific), anti-inos (dilution 1:200, Thermo scientific), anti-ki-67 (dilution 1:200, Thermo scientific), anti- COX-2 (dilution 1:200, Santa Cruz), anti-nf-kb (p65) (1:300, Biolegend) and anti-pcna (dilution 1:200, Thermo scientific) primary antibodies were used. Poly- HRP plus one detection system (Thermo scientific). Hydrogen peroxide, xylene, ethanol, magnesium chloride, sulphosalicylic acid, perchloric acid, thiobarbituric acid (TBA), trichloroacetic acid (TCA), tween-20, ferric chloride, folin ciocalteau reagent (FCR), copper sulphate, sodium potassium tartrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide, sodium citrate, hexadecyl trimethyl ammonium bromide, sodium chloride, potassium chloride, acetone, methanol, sodium azide, formaldehyde, acetic acid, hydrochloric acid (HCl) and sulfuric acid were purchased from E. Merck Limited, India. All the other reagents used were of highest purity and commercially available. 49

3 1.2 Natural chemopreventive agents Caffeic Acid, Geraniol, Silibinin and Quercetin were purchased from Sigma Aldrich Chemicals Pvt. Ltd., India. Soy Isoflavones from Novasoy, USA. 1.3 Animals Six to eight week old female Swiss albino mice (25 30 g), free from pathogens, were obtained from the Central Animal House facility of Hamdard University, New Delhi, India. Animals were housed in a well-ventilated room at 25 o C under a 12 h light dark cycle in polypropylene cages and have free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India) and water ad libitum. Study protocols were approved by the Institutional Animal Ethics Committee (IAEC) of the university and animals were undergone to experiment with the approved ethical guidelines. The dorsal portions of the mice skin were shaved with an electric clipper (Oster A2) followed by the application of hair removing cream (Anne French, Geoffery Manners, Bombay, India) at least two days before treatment. Excess cream was washed off with lukewarm water dipped in cotton sorbs. Only mice that showed no signs of hair regrowth were included in the experiments. 2. Methodology 2.1 Tissue processing After the desired time period, control and treated animals were sacrificed by cervical dislocation. Skin tissues from the sacrificed animals of each group were excised and washed with ice cold sodium chloride (0.85 %). A piece of skin was preserved in 10% neutral buffered formalin for histological observation Homogenization Skins tissues were homogenised in appropriate buffer (usually, phosphate buffer, 0.1 M, ph 7.4) using polytron homogenizer (Kinematica-AGPT 3000). 50

4 2.1.2 Subcellular fractionation The tissue homogenates were filtered through a muslin cloth and was centrifuged in a Remi Cooling Centrifuge (C-24 DL) at 3000 rpm for 10 min at 4 C to separate the nuclear debris. The aliquot so obtained was centrifuged at 10,500 x g for 30 min at 4 C to obtain post mitochondrial supernatant (PMS), which was used as a source of various enzymes Parameters Edema measurement Skin edema was assessed by the method of Afaq et al. (2005). Weight of skin punch (1cm diameter, free from extraneous materials) was measured. After drying for 24 h at 50 C, the skin punch was reweighed and loss of water content was determined. An increase in the mass of skin punch is directly proportional to the degree of inflammation. The extent of skin edema was determined by using the difference in the water gain between control and treated groups Measurement of nitric oxide (NO) Production of NO was evaluated by measuring the level of nitrite (an indicator of NO) in the skin tissue supernatants using Griess reagent as described by Green et al. (1982). Briefly, 100 μl of tissue supernatants were mixed with 100 μl Griess reagent [0.1% N- (1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H 3 PO 4 ]. After incubation at room temperature for 10 min, the absorbance at 540 nm was measured by Microplate ELISA reader (Bio RAD, U.S.A.).The concentration of nitrite in the sample was determined from a sodium nitrite (NaNO 2 ) standard curve and was expressed as μmol nitrite/mg protein Assay for myeloperoxidase (MPO) activity MPO activity was assayed by the method of Bradley et al. (1982). Skin tissue homogenate was prepared in 50 mm K 2 HPO 4 buffer (ph 6.0) containing 0.5% hexadecyl trimethyl 51

5 ammonium bromide. Samples were centrifuged at 2500 x g for 30 min at 4 C immediately after three cycles of sonication and freezing thawing. In 0.1 ml of supernatant MPO activity was measured, using 0.3 ml phosphate buffer (50 mm, ph 6.0) containing mg/ml o-dianisidine dihydrochloride and % hydrogen peroxide as substrates, at 460 nm over 5 min. MPO activity was expressed in unit which is defined as quantity of the enzyme degrading the 1µ mol of H 2 O 2 / min at 25 c. The result was expressed as Units/min/mg protein Measurement of lipid peroxidation (LPO) The assay for membrane lipid peroxidation was done by the method of Wright et al. (1981) with slight modifications. Reaction mixture in a total of 3 ml containing 1 ml of (10% tissue homogenate), 1.0 ml of TCA (10%), and 1.0ml TBA (0.67%). The reaction mixture was incubated in boiling water for 45 min. After cooling the tubes were centrifuged at 2500 g for 10 min. Absorbance of the supernatant was taken at 532 nm. Rate of LPO was assessed by measuring MDA content as nmol MDA formed / g tissue at 37 o C by using a molar extinction coefficient of 1.56 x 10 5 M 1 cm Measurement of reduced glutathione (GSH) GSH content of the skin tissue was determined by the method of Jollow et al. (1974). Briefly 1 ml of PMS (10%) was mixed with 1.0 ml of sulphosalicylic acid (4%). The samples were incubated at 4 o C for 1 h and then centrifuge at 1200 g for 15 min at 4 o C. The assay mixture (3ml) consists of 0.4 ml supernatant, 2.2 ml phosphate buffer (0.1 M, ph 7.4) and 0.4 ml DTNB (4 mg/ml). The yellow colour developed was read immediately at 412 nm. The GSH concentration was calculated as nmol DTNB conjugate formed/g tissue Assay for glutathione-s-transferase (GST) activity Glutathione-S-transferase activity was estimated by the method of Habig et al. (1974). The reaction mixture consists of ml sodium phosphate buffer (0.1 M, ph 7.4),

6 ml reduced glutathione (1 mm), ml CDNB (1 mm) and 0.2 ml PMS in a total volume of 2.0 ml. The changes in the absorbance was recorded at 340 nm and enzyme activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of M 1 cm Measurement of glutathione peroxidase (GPx) activity The glutathione peroxidase activity was measured by the method of Mohandas et al. (1984). The reaction mixture consists of 1.44 ml phosphate buffer (0.1 M, ph 7.4), 0.1 ml of EDTA (1 mm), 0.1 ml of sodium azide (1.0 mm), 0.05 ml of GR (1 eu / ml), 0.05 ml of GSH (1.0 mm), 0.1 ml of NADPH (0.2 mm), and 0.01 ml of H 2 O 2 (0.25 mm) and 0.1 ml PMS (10%) in a total volume of 2.0 ml. Disappearance of NADPH at 340 nm was recorded. The enzyme activity was calculated as nmol NADPH oxidized / min / mg protein using a molar extinction coefficient of M 1 cm Measurement of glutathione reductase (GR) activity The GR activity was measured by the method of Carlberg and Mannervik, (1975). The assay mixture consists of 1.65 ml phosphate buffer (0.1 M, ph 7.6), 0.1 ml EDTA (0.5 mm), 0.05 ml oxidized glutathione (1.0 mm), 0.1 ml NADPH (0.1 mm) and 0.1 ml PMS (10%) in a total volume of 2.0 ml. The enzyme activity was recorded at 25 C by measuring the disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized / min / mg protein using molar extinction coefficient of M 1 cm Measurement of glucose-6-phosphate dehydrogenase (G6PD) activity The activity of glucose-6-phosphate dehydrogenase was determined by the method of Zaheer et al. (1965). The reaction mixture consisted of 0.3 ml Tris HCl buffer (0.05 M, ph 7.6), 0.1 ml NADP (0.1 mm), 0.1 ml glucose-6-phosphate (0.8 mm), 0.1 ml MgCl2 (8 mm), 0.3 ml PMS (10%) and 2.1 ml distilled water in a total volume of 3 ml. The changes 53

7 in absorbance were recorded at 340 nm and enzyme activity was calculated as µmol NADP reduced/min/mg protein using a molar extinction coefficient of M-1 cm Assay for hydrogen peroxide Hydrogen peroxide (H 2 O 2 ) was assayed by H 2 O 2 -mediated horseradish peroxidasedependent oxidation of phenol red by the method of Pick and Keisari (1980). 2.0 ml of supernatant, suspended in 1.0 ml of solution containing phenol red (0.28 nm), horse radish peroxidase (8.5 units), dextrose (5.5 nm) and phosphate buffer (0.05 M, ph 7.0) was incubated at 37 C for 60 min. The reaction was stopped by the addition of 0.01 ml of NaOH (10N) and then centrifuged at 800 g for 5 min. The absorbance of supernatant was recorded at 610 nm against a reagent blank. The quantity of H 2 O 2 produced was expressed as nmol H 2 O 2 /h/gm tissue based on the standard curve of H 2 O 2 mediated oxidation of phenol red Measurement of catalase activity Catalase activity was assayed by the method of Claiborne, (1985). Briefly the reaction mixture consists of 1.95 ml phosphate buffer (0.1M, ph 7.4), 1.0 ml hydrogen peroxide (0.019M) and 0.05 ml PMS in a final volume of 3 ml. The changes in absorbance were recorded at 240 nm. Catalase activity was calculated as nmol H 2 O 2 consumed per min per mg protein Measurement of superoxide dismutase (SOD) activity The SOD activity was measured by the method of Marklund and Marklund, (1974). The reaction mixture consisted of ml Tris HCl buffer (50 mm, ph 8.5), pyrogallol (24mM in 10mM HCl) and 100 μl PMS in a total volume of 3 ml. The enzyme activity was measured at 420 nm and was expressed as units/mg protein. One unit of enzyme is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%. 54

8 Measurement of xanthine oxidase (XO) activity Xanthine oxidase catalyzes the conversion of xanthine to uric acid, which has a characteristic absorption at 290 nm. It was assayed as described by Stirpe and Della Corte, (1969). The reaction mixture, containing 0.2 ml PMS diluted to 1ml with phosphate buffer (0.1 M, ph 7.4) and incubated for 5 min at 37 o C. Reaction was started by adding 0.1 ml xanthine and mixture was kept at 37 C for 20 min. The reaction was terminated by the addition of 0.5 ml ice-cold perchloric acid (10%). After 10 min, 2.5 ml distilled water was added and the mixture was centrifuged at 4000 rpm for 10 min. The absorbance of the clear supernatant was read at 290 nm. The result was expressed as µg uric acid formed/mg protein Measurement of quinone reductase (QR) activity The QR activity was determined by the method of Benson et al. (1980). The 3ml reaction mixture consists of 2.13 ml Tris HCl buffer (25 mm, ph 7.4), 0.7 ml BSA, 0.1 ml FAD, 0.02 ml NADPH (0.1 mm), and 50 μl PMS (10%). The reduction of dichlorophenolindophenol (DCPIP) was recorded calorimetrically at 600 nm and the enzyme activity was calculated as μmol of DCPIP reduced/min/mg protein using molar extinction coefficient of M -1 cm Histological examination Fixation, dehydration, infiltration and block preparation. Skin tissue were excised out and fixed in Bovin s fluid for hrs. The tissues were then rehydrated by passing through graded series of ethyl alcohol (50%, 70%, 90% and 100%) for one hour in each giving two changes. These were then cleared in xylene (two changes of one hour each). The cleared tissue were placed for five minutes in xylene containing molten paraffin wax at C for infiltration. Sections were deparaffinised by dipping in xylene and given two changes. These slides were then passed through graded concentrations of ethyl alcohol (30%, 50%, 70%, 90% and 100%) with two changes of two minutes each. Then keep 55

9 stained with hematoxylin for one minute and again washed in running water thoroughly. Slides were then passed through 50% and 70% ethyl alcohol and subsequently put into eosin stain {prepared in 70%, 90%, 100%, 100% + xylene (1:1)} and finally they were given two changes of xylene. All slides were mounted in DPX. They were covered with glass cover slips and kept at room temperature for drying. Histological changes/ analysis were observed with microscope (fluorescent microscope, Olympus) at least in six different regions Measurement of proinflammatory cytokines TNF-α, IL-6 and IL-1β proteins level was measured by enzyme-linked immunosorbent assay (ELISA) kit (ebioscience, Inc., San Diego., USA). Samples were prepared in phosphate buffered saline (PBS) containing protease inhibitor cocktail. Analysis was performed according to the manufacturer s instruction Protein estimation The protein concentration in all samples was determined by the method of Lowry et al. (1951). Peptide bonds form a complex with alkaline copper sulphate reagent, which gives a blue colour with Folins reagent. Briefly, 0.1ml (10% w/v) was diluted to 1ml water and protein precipitated with equal volume of TCA (10%), samples were kept overnight 4 0 C and centrifuged at 800 X g for 5 minutes. The supernatant was decanted and discarded. The pellet was dissolved in 5ml of NaOH (1N). Finally 0.1ml of diluted aliquot was taken for colour development. 0.1ml of aliquot was further diluted to 1ml with water and then 2.5ml of alkaline copper sulphate reagent containing sodium carbonate (2%), copper sulphate (1%), and sodium potassium tartarate (2%) was added. Following 10 minutes after addition of alkaline copper sulphate reagent to allow complex formation 0.25ml of Folin s reagent was added. After 30 minutes blue colour developed that was read at 660 nm for standard Bovine serum albumin (BSA 0.1mg/ml) was used. 56

10 Immunohistochemical analysis The dorsal skin tissues were fixed in formalin and paraffinized sections of 5 μm thickness were cut on to poly-lysine coated glass slides. Sections were deparaffinized three times (5 min each) in xylene fallowed by dehydration in graded ethanol and finally rehydrated in running tap water. For antigen retrieval sections were boiled in 10 mm citrate buffer (Ph 6.0) for 5-7 min. Sections were incubated with hydrogen peroxide for 15 min to minimize nonspecific staining and then washed three times (5 min each) with 1X TBST (0.05% Tween-20). Sections were incubated first with power block for 10 min and then for overnight with the primary antibodies at 4 C. Further processing was done by using poly- HRP plus one detection system from Thermo scientific. The peroxides complex was visualized with 3, 3- diaminobenzidine. Lastly the slides were counterstained with haematoxylin. Slides were then cleaned in xylene, gradually dehydrated with ethanol, after DPX mounting microscopic (Fluorescent Microscope, Olympus) analysis was done Genomic DNA extraction and gel electrophoresis Genomic DNA was extracted according to the method of Maniatis et al. (1982) with some modifications. Briefly, tissue samples ( g) were homogenized in 5 ml of a DNA extraction buffer (1M Tris HCL, 0.5M EDTA, RNase 5 mg, 5% SDS, ph 8.0) and incubated for 1h. After adding 100 µl proteinase K (10 mg/ml stock) the samples were incubated over night in a shaking water bath at C. DNA was extracted twice with a mixture of equal volume of saturated phenol (ph 8.0) and centrifuged at 3,000 x g for 10 min. Then 1 ml of ammonium acetate (10M) was added to each sample and mixed gently for 5 min. DNA was precipitated with 10 ml of chilled EtOH (100 %). After extensive washing with EtOH (70 %), DNA strands were hook out with pipette. DNA was then dried and resuspended in 100µl of Tris-EDTA buffer or distilled water. Thereafter, DNA (10 μl) from each sample was loaded onto 1.2 % agarose gel, containing 0.5 μg/ml ethidium bromide and electrophoresed at 25V for 12 h in TBE buffer for the analysis of 57

11 DNA fragmentation which was detected by using the gel documentation system (Alpha Innotech). 3. Statistical analysis The data from individual groups are presented as the means ± standard error of the mean (SEM). Differences between groups were analyzed by using one way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test and minimum criterion for statistical significance was set at p<0.05 for all comparisons. 58