Drugs and chemicals
Nerolidol (Sigma-Aldrich, USA), CFA (Sigma-Aldrich, USA), trizole solution, cDNA synthesis kit (Gene Direx, USA), forward / reverse primers (Gene Direx, USA), Cybergreen, deionized water, ethanol, chloroform.
Sprague Dawley rats (150-250g) of either sex were used for investigational procedure. Animals were housed at animal house of Department of Pharmacology, College of Pharmacy, University of Sargodha with recommended housing conditions. All animals fed on water and standard diet and they were controlled by following the guidance in accordance with National Research Council. All tests were approved by animal ethics and review committee at University of Sargodha (Approval NO. SU/Pharm/Animal Ethics Approval/2019/215).
Complete Freund’s adjuvant induced rheumatoid arthritis model
Animals were separated randomly into six different groups (n=5). The 1st group designated as normal control group and 2nd group (arthritic control group) received 2% tween 80 (3mL/kg). The 3rd group (standard group) was given naproxen 20mg/kg while 4th, 5th and 6th group served as treatment groups and received 200, 400 and 800mg/kg of nerolidol respectively. Arthritis was persuaded by inoculation of 0.1mL of CFA injection (containing 1mg/mL of heat killed M. tuberculosis in 0.15mL mono-oleate and 0.85mL paraffin oil) into left hind footpad of each rats except normal control group. The day of CFA shot was served as 0 day. The oral administration of different doses to treatment groups was continued for 28 days consecutively. Arthritis in all groups were evaluated by recording various factors (Mahdi et al., 2018).
Evaluations of arthritis from body weights and paw volume of rats
During the period of treatments, body weight of each rat was observed at every 7th day. Paw size/oedema was measured using digital plathysmometer. Percent inhibition in paw oedema /size was calculated by following this equation.
“VC” and “VT” are the paw volume of arthritic control and treatment group.
Assessment of arthritis from serum and blood
At 28th day, all rats were sacrificed and through cardiac puncture blood was collected for the assessment of biochemical and haematological markers comprising WBCs, RBCs, Hb, c-Reactive Proteins, RF, ESR, Platelets, ALP, SGOT, SGPT, Creatinine and urea. These tests were performed at diagnostic center, University of Sargodha (Hassan et al., 2019).
Estimation of mRNA expression levels of TNF-α, IL-1β, IL-6, COX-2, IL-4, NF-Kβ and IL-10.
Collected blood samples were used for appraisal of expression of mRNA TNF-α, IL-1β, IL-6, COX-2, NF-Kβ, IL-4, and IL-10. TRIzol method was used for the extraction of total RNA from blood. According to this method, into 500uL of blood, 700uL of trizole solution was added. It was mixed gently and incubated it for 10 minutes. Then 200uL of chloroform was added into it. Shake thoroughly and placed it in the centrifuge machine for 15 minutes at 12000 rpm and 4oC. The aqueous layer was taken and then added 500uL of isopropanol into it and again mixed vigorously. Samples were incubated for 10 minutes and retained it in the centrifuge machine at 12000 rpm and 4 ºC for 15 minutes. Discarded the supernatant solution and the RNA pellet was washed with absolute ethanol. Air dried the RNA pellet and added 30uL of purified water into it and then quantified the RNA from Nano drop reader. After this, cDNA was synthesized by following the kit manufacturer procedure (Gene- Direx). In brief, 1uL of RNA solution, 1uL of oligo (dT) 20, 1uL of dNTP Mix and then RNA free water was added into it. The reaction mixture was heated at 65°C for 3-5 minutes, spin it and placed promptly on frost. Then added 4uL of 1st strand buffer, 1uL DTT, 1uL of Script RTase and finally made volume up to 20uL. Incubated it for 30-60 minutes at 50°C and the enzyme was inactivated at 70°C for 15 minutes.
Real time quantitative PCR was used to intensify and quantify the reaction by using Bio-Rad scheme in Pharmacology department, University of Health Sciences, Lahore. Afterwards, templates of cDNA were mixed with qPCR master mix and added the specific primers of genes, nuclease free water, and then placed it in a thermal cycler for 45 cycles with denaturation temperature at 95°C, annealing at 56°C, extension at 72°C and then terminated the reaction at 72°C. Various markers of genes were nominated from Ensemble Genome Browser for determination of specific gene primers physically by using Input primer 3 (v. 0.4.0.) which is available online software. The sequences of primers are provided in Table1 (Shabbir et al., 2016, Lim et al., 2017).
ELISA (Enzyme linked immuno-sorbent assay) for Prostaglandin E2
ELISA test was performed for quantitative identification of rat Prostaglandin E2 in serum samples according to kit manufacturer procedures (rat Prostaglandin E2 ELISA kit, Bio-assay technology laboratory having Cat No. E0504Ra, standard curve range = 0.05ng/ml-15ng/ml, size = 96 wells, sensitivity= 0.026ng/ml). By adding acidic solution, reaction was terminated and absorbance was measured at 450nm (micro-plate reader with 450 ± 10nm).
Estimation of peroxidase antioxidant enzyme activity
Peroxidase activity was measured by determining its capability to decrease hydrogen peroxide at wavelength of 470nm (Zia et al., 2011). The 0.06mL of enzyme extract was added in 3mL of buffer substrate solution that comprised of 47mL of phosphate buffer (0.2M), 0.7mL of guaiacol and 0.32mL of H2O2. After three minutes of enzyme reaction, optical density was measured at 470 nm spectrophotometer against blank (phosphate buffer guaiacol). Peroxidase activity was measured by using the undermentioned formula.
A = Absorbance at 470nm, 26.6= extinction coefficient of guaiacol (Mm-1cm-1, 0.06= volume of enzyme extract (mL), 3.0 = volume of phosphate buffer (mL)
Estimation of catalase antioxidant enzyme activity
Assay was performed for determining the catalase activity of antioxidant enzymes and its ability was checked to reduce H2O2 at 240nm. Reaction mixture was comprised of 3.0mL of K2PO4 buffer (50Mm, pH 7), 0.1mL of hydrogen peroxide (30Mm) and 0.1mL of enzyme extract. Absorbance was observed after 3 minutes of reaction time at 240nm (Chance and Maehly, 1955).Catalase activity was determined by given formula
A3= Absorbance at 240nm, 0.04= Extinction coefficient for H2O2 (M-1CM-1)
Evaluation of superoxide dismutase
The SOD bustle was performed to check its ability to inhibit the photo reduction of nitro-blue tetrazolium. This test was performed by adopting the protocol with slight modification as discussed by Worthington 1988. Assay mixture was contained 1mL of 0.0067M potassium phosphate buffer (7.8pH), 0.05mL extract of enzyme and 0.016mL of 0.012mM solution of riboflavin. The reaction mixture was incubated in a light box for 12 minutes. After that, 0.067mL of EDTA/NaCN solution and 0.033mL of nitroblue tetrazolium solution was added into the reaction mix. After 30 second of reaction time, the absorbance was observed against blank through spectrophotometer at wavelength of 560nm. The activity of SOD was calculated by under mentioned formula:
Histopathological assessment of ankle joints
At the end of the treatment, ankle joints of arthritic control and treated rat paws were collected and static in 10% solution of formalin for the assessment of histopathology of joints (Shabbir et al., 2014).
Radio graphical assessment of joints
The legs were removed at knee joints and were subjected for radio graphical assessment with computerized radio graphical system (Toshiba 630 M) (Uttra and Hasan, 2017).