Animals
Swiss albino mice (25±2.5g) of 7 weeks of age (procured from Central Drug Research Institute, Lucknow, India) were housed in polyvinyl chloride cages and maintained under laboratory conditions having 12:12 hrs light/dark cycle in a temperature (21o C ± 20C) and (55 ± 5%) humidity controlled room. The animals had free access of food (pellets) and water. The mice were subjected to experimentation after one week of acclimatization.
Experimental Design
Four groups of the mice (Group I, Group II, Group III & Group IV) were maintained (6 per group). Group II and Group III were exposed to LPS (1mg/kg bw) intraperitoneally for five days. After LPS exposure, multi strain probiotic VSL#3 (0.6gm/kg bw) was orally gavaged to Group III and Group IV (Only VSL#3) for 4 weeks. Mice of the Group I (Control) and Group II (LPS treated) were also maintained till 4 weeks. Food and water intake of mice were noted on daily basis. Mice were anaesthetized with pentobarbital (100mg/kg bw) on the termination of experiments and sacrificed.
Test Chemicals and Doses
The multi strain Probiotic (VSL#3) was purchased from Sun Pharmaceuticals, New Delhi, India. Lipopolysaccharide (LPS from Escherichia coli O26:B6) and the ELISA kits for Alanine Aminotransferases (ALT; Catalogue: MAK052), Aspartate Aminotransferase (AST; Catalogue: MAK055), Mouse TNF-α (Catalogue: RAB0477) and IL-6 (Catalogue: RAB0308) were procured from the Sigma Aldrich (Bengaluru, India). Rest of the chemicals was procured from Himedia, Mumbai, India. The dose of LPS (1mg/kg bw i.p.) was decided as it can cause increase expression of TLR-4 and its receptor as well as inflammatory mediators [33]. (Guo et al., 2013). The dose of VSL#3 (0.6gm/kg/ bw orally) decided on the basis of previous studies [34]. (Chang et al., 2013)
Cytokine Assay
For ELISA of the pro-inflammatory cytokines TNF-α and IL-6, blood was collected via cardiac puncture in micro centrifuge tubes and the plasma was separated by centrifugation at 3000 rpm for 15 minutes and was stored at -200C till cytokine assay. The level of TNF-α and IL-6 in plasma was determined using ELISA kits following the manufacturer’s protocols. Briefly, all reagents were brought to room temperature (RT) (220C) prior to use. The plasma samples were added in to the 96-well plate coated with specific antibody (Mouse TNF-α and IL-6 antibody) and the plate was incubated at RT for 2.30 hrs. The plate was washed 3 times with the wash buffer and firmly tapped on absorbent paper. The biotinylated detection antibody solution was added into each well and the plate was incubated at RT for 1hrs. After washing of the plate as mentioned above, HRP-Streptavidin solution was added to each well and was incubated with gentle shaking in RT for 45 minutes. The washing step was repeated and TMB (3, 3’, 5, 5’-tetramethyl benzidine) was added into each well and incubated with gentle shaking in dark for 30 minutes. Finally, the reaction was stopped by adding the stop solution and the concentrations of cytokines (TNF-α and IL-6) were calculated according to the absorbance measured at 450nm based on the standard curve.
Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) Assay
For assay of ALT and AST, blood was collected and was allowed to clot at room temperature. The clot was removed by centrifugation at 3000 rpm for 10 min in a refrigerated centrifuge and the supernatant (serum) was stored at -800 C till use for ALT and AST activity. Serum ALT and AST activities were estimated using the manufacturer’s protocols (Sigma Aldrich). In brief, all the required chemicals were brought to RT prior to use. Standard products (Pyruvate Standard for ALT & Glutamate standard for AST) and samples of each group added into a 96-well plate. Then 100 µl of the master reaction mixture was added to each of the wells, mixed properly using a horizontal shaker, incubated at 370 C for 2-3 minutes and initial reading (ALT A570nm & AST A450nm) was taken. Incubation was continued at 370 C taking measurement every 5 minutes until the value became greater than the value of the highest standard (10nmole/well). The activity of ALT and AST calculated according to manufacturer’s protocols.
Determination of Oxidative Status (Gut & Liver)
Lipid Peroxidation (LPx)
Endogenous Lipid Peroxidation (LPx) levels in samples were assessed by monitoring the formation of thiobarbituric acid reactive substances (TBARS) according to the protocol of Ohkawa, 1979 [35]. In brief, LPx cocktail was prepared by mixing following solutions: 162 µl of 10% (w/v) SDS, 375 µl of 20% (v/v) acetic acid (pH 3.5), 300 µl of 1% (w/v) TBA solution, 20 µl of 1% (w/v) BHT solution and 13 µl of distilled water. Supernatant 130µl (10% homogenate of the Gut/Liver in phosphate buffer followed by centrifugation) was added in 870 µl of the LPx cocktail. The mixture was vortexed and heated in water bath at 950 C for 60 minutes. Tubes were cooled down to room temperature and centrifuged at 1,000Xg for 10 minutes. Absorbance of the supernatant was recorded at 532nm against an appropriate blank. The concentration of TBARS was calculated from the extinction coefficient of 1.56X105M-1cm-1. Result was expressed as nmol/MDA/TBARS formed/mg protein.
Superoxide Dismutase (SOD) Activity
The activity of superoxide dismutase (SOD) enzyme was measured by the method of Beauchamp and Fridovich [36]. In brief, 10% homogenate of Gut/Liver tissue was prepared in 0.4 M PBS (pH= 7.8), followed by centrifugation at 10,000 rpm for 15 min. 100 μl supernatant mixed with 900μl reaction mixtures [50 mM PBS (45 μl SS), 100 mM Methionine (180 μl SS), 100 mM EDTA (90 μl SS), 450 μM NBT (40.5 μl SS), 1 mM Riboflavin (9 μl SS) and 535.5 μl dH2O]. The mixture was kept in foil-lined box and illuminated with a 25 W light for 15 min. Absorbance of purple-colored complex was measured at 560 nm against blank. The SOD activity was directly proportional to percent inhibition of NBT reduction expressed per ml (50 % of NBT reduction corresponds to one unit of enzyme). The enzyme activity was calculated per mg protein. Protein content was determined by the Bradford method [37].
Catalase (CAT) Activity
CAT activity was assessed on the basis of decomposition of H2O2 according to the method of Aebi [38]. In brief, 10% homogenate of Gut/Liver tissue was prepared in 0.4 M phosphate buffer (pH = 7.8), followed by centrifugation at 10,000 rpm for 15 min. 500 μl supernatant mixed with 50 μl ethanol and kept in ice for 30 min. After this, 450 μl mixtures mixed with 50μl Tritan-X100, vortexed and from this sample, 100 μl taken in a cuvette and mixed 1.4ml of 13 mM H2O2. Absorbance was measured at 240 nm for 1 min, with the help of UV–VIS spectrophotometer (Shimadzu, UV-1800 pharma spec), using extinction coefficient of H2O2 (0.041/μM/cm2 at 240 nm) and is expressed as Units/min/mg of protein.
Glutathione Reductase (GR) Activity
GR activity was assayed following the method of [39]. In brief, to 867µl of 50 mM phosphate buffer pH7.4, 33.3 µl each of 120 mM GSSG and 4.5 mM NADPH were added. After one minute, reaction was initiated by adding 66.4 µl sample to it. The absorbance was recorded at one minute interval at 5 minutes at 340 nm with running a parallel blank sample. Enzyme activity was expressed as Unit NADPH oxidized/min/mg protein considering NADPH molar extinction coefficient as 6.22 mM-1 cm-1.
Histopathology
Tissues (Gut & Liver) were dissected out and fixed in 4% paraformaldehyde solution. After 24 hrs of fixation, the tissues were processed for paraffin (m.p. 58-600C) embedding. Sections of 6 µm thickness were cut and stretched on sterilized glass slides. After deparaffinization, the sections were processed for Hematoxylin-Eosin staining [40] and observed under light microscope (Leica DFC450 C, Germany).
Morphometric Analysis
Morphometric analysis was done by the image-J software NIH, Bethesda, USA. For quantitative evaluation of villous height and crypt depth of small intestine, 10 randomly selected sections of different groups (100 villi & 100 crypts/each group) were measured at X40. For counting of the goblet cells 10 sections of colon of each group were randomly selected. From each section goblet cell were counted on 10 different sites of mucosal epithelium in a counting frame (100X100 µm2). The size of the goblet cells was determined measuring 100 cells (10 sections/group) from each group.
Statistical Analysis
All the data were presented as mean ± SEM. The statistical significance of the differences between the means was analyzed by one-way ANOVA with Tukey’s post hoc test. Statistical significance was assigned when the value was p <0.05.