Nf κb p65

To induce cutaneous inflammation CFA (Sigma–Aldrich, Germany), was injected into the plantar surface of one hind paw (20 μl/paw). Carrageenan (Sigma–Aldrich, Germany) (100 μl/paw) was administered in separate cohorts of mice as an additional model of cutaneous tissue injury and inflammation, griess reagent, gabapentin (5 mg/kg), tramadol (50 mg/kg), piroxicam (5 mg/kg) (Merck, Darmstadt, Germany), dexamethasone (5 mg/kg), chloroform, distilled water, 2% DMSO were of research grade. Honokiol was isolated as previously described (Park et al., 2009 (link)) (10 mg/kg i.p.) dissolve in 2% DMSO and normal saline was administered before CFA or Carrageenan induced inflammation. Elisa kits of TNF-α and IL-1β (Thermo Fisher Scientific, United States), NF-κB (p65) (Cayman Chemical, Ann Arbor, MI, United States).

The tissue samples from all the groups (vehicle, negative, Dexa, and honokiol (10 mg/kg i.p.) were determined using a commercially available ELISA kits. TNF-α and IL-1β (eBioscience, Inc., San Diego, CA, United States) and NF-κB (p65) (Cayman Chemical, Ann Arbor, MI, United States) kits were used as per manufacturer instructions. For sample preparation the paw skin tissue was removed, tissue protein extraction was done by using Phosphate buffer saline (PBS) in concentration of 100 mg tissue/ml PBS, to which 0.4 M NaCl, 0.05%, Tween 20, and protease inhibitors were added. The prepared sample was then centrifuged at 3000 g for 10 min as method described previously (Khan et al., 2013b (link)).