Chemicals
β-sitosterol was purchased from Himedia Laboratories (India). Indomethacin, Substance P, L-arginine and L-NAME were purchased from Sigma-Aldrich (USA). The interleukin-6 ELISA kit was purchased from Krishgen Biotech (India). All the other reagents and chemicals of analytical grade were purchased from authorized dealers. β-sitosterol was suspended in carboxymethyl cellulose by triturating thoroughly followed by micronizing in an ultrasonicator. The suspension was freshly prepared before experiments.
Animals
Swiss albino mice of either sex with body weight in a range of 20-25g (10 weeks age) were used. Animals were procured from the National Institute of Pharmaceutical Education and Research, Mohali, India and kept in the central animal house of Guru Nanak Dev University, maintained under standard environmental conditions 25 ± 5ºC with 12-hour light/dark cycle and water and feed were provided ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Guru Nanak Dev University, Amritsar (226/CPCSEA/2018/41). All experimental procedures were undertaken as per the guidelines for the use and care of laboratory animals.
Experimental Protocol
Formalin-induced nociception and acetic acid-induced writhing tests were used.β-sitosterol was administered at low (10 mg/kg), medium (20 mg/kg) and high dose (40 mg/kg) prior to acetic acid and formalin in acetic acid induced writhing and formalin induced hyperalgesia respectively. For mechanistic studies, the role of nitric oxide was studied by using NO precursor L-arginine (40 mg/kg; ip) and nitric oxide synthase blocker, L-NAME (10 mg/kg, ip). Whereas, the role of COX was studied by using substance P (10µg/kg; ip), a neurokinin known to stimulate COX-2 (Koon et al., 2006). L-NAME, L-arginine and substance were administered 30 min prior to the β-sitosterol (40 mg/kg, ip). After β-sitosterol treatment, 2% formalin was injected into the hind paw and the licking and tapping behaviour was recorded for 60 min. After behavioural evaluation in the formalin test, the animals were anaesthetized (ketamine 50 mg/kg) and blood samples were taken for serum IL-6 estimation. Thereafter, animals were sacrificed and the brain samples were extracted for oxidative stress analysis. The formalin-injected paw was excised for the histological examination. The detailed experimental design of the current study is depicted in Figure 1.
Assessment of analgesic activity
Acetic Acid induced writhing test
The acetic acid-induced writhing test was used to evaluate the peripheral antinociceptive activity (Nirmal et al., 2011). Acetic acid-induced writhings were induced by injecting acetic acid (0.6% v/v) intraperitoneally at a dose of 10 ml/kg. The number of writhings was counted for a period of 30 min post acetic acid as an indicator of hyperalgesia. The test and standard drugs were administered 30 min before the acetic acid treatment [17].
Formalin-induced licking and taping test
In this method, 2% formalin at a dose of 50 µL was injected subcutaneously into the intraplantar region of the right hind paw and the number of licking and tapings was counted for a period of 60 as a measure of hyperalgesia. The first phase of 0-5 min is the neurogenic phase and the second phase of 25-30 min is the inflammatory phase. The test and standard drugs were administered 30 min before formalin injection [18].
Biochemical estimations
Serum IL-6 estimation
The animals were anaesthetized with ketamine (50 mg/kg) after respective acetic acid and formalin tests. The blood samples were collected by the retro-orbital route. Serum was separated from the blood by centrifuging the blood samples at 3000 rpm for 10 min. Serum IL-6 estimation was done by using the commercially available Elisa kits (Krishgen Biotech).
Estimation of oxidative stress
Preparation of brain homogenate
Brain homogenate was prepared by adding the dithiothreitol (15.4 mg) and triton X (500 mg) to the phosphate buffered saline solution of pH 7.4. Homogenate was centrifuged at 2000 g for 10 min to obtain a clear supernatant. The pellets were discarded and the supernatant was used for estimation of reduced glutathione and lipid peroxidation.
Thiobarbituric acid reactive substances (TBARS) estimation
An increased level of TBARS in tissue is considered an index of increased lipid peroxidation in tissue. 0.4 ml of TCA-TBA-HCl reagent was taken and added to 0.2 ml of supernatant. The solution was kept in a boiling water bath for 5 min, brought to room temperature and centrifuged at 10,000 g for 10 min. The coloured supernatant was collected and measured spectrophotometrically at 535 nm [19].
Reduced Glutathione estimation
Ellman method was used for measuring the glutathione content in brain tissue. 1 ml of 0.3 M disodium hydrogen phosphate was added to the 0.25 ml of supernatant followed by addition of 0.125 ml of 0.001M freshly prepared DTNB. The resultant yellow-colored solution was measured spectrophotometrically at 412 nm [19].
Histological studies
Hematoxylin and eosin staining of the treated right hind paw
The histological analysis of paw tissue was carried out to investigate the effect of β-sitosterol on formalin-induced paw inflammation. Skin from the paw was isolated and preserved in 10% formalin solution for 24 h [20]. The tissue was dehydrated by exposure to alcohol, immersed in xylene and then embedded in paraffin [21]. Sections of 3-4 µm thickness were cut and placed on a slide using the mounting medium. After mounting the section paraffin wax was removed by gently warming the slide followed by washing with xylene. This was followed by alternate washings with varying concentrations of alcohol (70%, 80%, 90% and 100%) and water to prevent dehydration. Thereafter, the sections were stained with hematoxylin for 15 min. The washing of stained sections was done with water. Then the sections were treated with 1% acid-alcohol mixture for 20 s. The acid alcohol mixture was washed off with water and counterstained with a 1% aqueous solution of eosin for 2 min. The excess eosin was washed with water and sections were dehydrated with alcohol. The dehydrated sections were mounted with Canada balsam as a mounting agent under a coverslip carefully to avoid bubbles. The slides were observed under the microscope at 40x [22].
Statistical analysis
The data were expressed as mean ± standard error mean (SEM). Statistical analysis was done by one-way analysis of variance (ANOVA) followed by post-hoc analysis by Tukey’s multiple comparison test using Graph pad Prism software version 7.0.