Furthermore, inhibition of p38 MAPK with its specific inhibittors SB239063 and SB203580 counteracted diabetes-associated GSH depletion in the peripheral nerve and COX-2, inducible nitric oxide synthase, and tumor necrosis factor- overexpression in dorsal root ganglion neurons [28,52]. into two subgroups that were managed with or without treatment with cinnamyl-3,4-dihydroxy- em /em -cyanocinnamate (CDC), 8 mg kg/d, s.c., for another 4 weeks. CDC, at the K114 aforementioned dose, counteracted multiple manifestations of diabetic neuroapthy and oxidative-nitrosative stress in peripheral nerve and spinal cord in our previous study [24]. Non-fasting blood glucose measurements were performed after induction of diabetes and at the end of the study period. 2.3. Anesthesia, Euthanasia and Tissue Sampling The animals were sedated by CO2, and immediately sacrificed by Rabbit Polyclonal to STK24 cervical dislocation. Sciatic nerves and spinal cords were rapidly dissected and frozen in liquid nitrogen for subsequent assessment of LO as well as total and phosphorylated p38 MAPK, ERK, and SAPK/JNK levels, and 12(S)-HETE concentrations. 2.4. Human Schwann Cell Culture Schwann cells play a key role in the pathology of various inflammatory, metabolic, and hereditary polyneuropathies, including diabetic neuropathy [34,35]. Previous studies exhibited that cultured human Schwann cells (cell collection K114 cat. #1700, ScienCell, Carlsbad, CA) manifest increased superoxide production, accumulation of nitrated and poly(ADP-ribosyl)ated proteins and K114 4-hydroxynonenal adducts, inducible nitric oxide synthase overexpression, 12/15-Lipoxygenase overexpression and activation, increased p38 MAPK phosphorylation, downregulation of taurine transporter, as well as impaired insulin signaling early (1 – 7 d) after exposure to high glucose [24,36-38]. They therefore represent a good model for studying interactions among individual pathobiochemical mechanisms in the peripheral nerve. In the present study, human Schwann cells (passages 7 – 10) were cultured in 6-well plates in media made up of 5.5 mM D-glucose. At 70% confluence, the media were replaced with those made up of either 5.5 mM D-glucose or 30 mM D-glucose with or without CDC, 10 M (6 – 8 plates per condition). After 24 hr, the cells were utilized for assessment of total and phosphorylated p38 MAPK, ERK, and SAPK/JNK. 2.5. Specific Methods 2.5.1. Western Blot Analyses of LO and Total and Phosphorylated p38 MAPK, ERK, and SAPK/JNK Sciatic nerve and spinal cord materials (3 – 10 mg) or scraped human Schwann cells were placed on ice in 100 L of buffer made up of 50 mmol/l Tris-HCl, pH 7.2; 150 mmol/l NaCl; 0.1% sodium dodecyl sulfate; 1% NP-40; 5 mmol/l EDTA; 1 mmol/l EGTA; 1% sodium deoxycholate and the protease/ phosphatase inhibitors leupeptin (10 g/ml), pepstatin (1 g/ml), aprotinin (20 g/ ml), benzamidine (10 mM), phenylmethylsulfonyl fluoride (1 mM), sodium orthovanadate (1 mmol/l), and homogenized on ice. The homogenates were sonicated and centrifuged at 14,000 g for 20 min. All the afore-mentioned steps were performed at 4C. The lysates (20 g protein for sciatic nerve and 40 g for spinal cord and human Schwann cells) were mixed with equivalent volumes of 2 sample-loading buffer made up of 62.5 mmol/l Tris-HCl, pH 6.8; 2% sodium dodecyl sulfate; 5% -mercaptoethanol; 10% glycerol, and 0.025% bromophenol blue, and fractionated in 10 %10 % (total and phosphorylated MAPKs) or 7.5% (lipoxygenase) SDS-PAGE in an electrophoresis cell (Mini-Protean III; Bio-Rad Laboratories, Richmond, CA). Electrophoresis was conducted at 15 mA constant current for stacking, and at 25 mA for protein separation. Gel contents were electrotransferred (80 V, 2 hr) to nitrocellulose membranes using Mini Trans-Blot cell (Bio-Rad Laboratories, Richmond, CA) and Western transfer buffer (10 Tris/Glycine buffer, Bio-Rad Laboratories, Richmond, CA) diluted with 20% (v/v) methanol. Free binding sites were blocked in 5% (w/v) BSA in 20 mmol/l Tris-HCl buffer, pH 7.5, containing 150 mmol/l NaCl and 0.05% Tween.
