Phosphoinositide 3-kinase/Akt pathway is involved in mediating the anti-inflammation effects of magnesium sulfate

Phosphoinositide 3-kinase β, phosphoinositide 3-kinase δ, and phosphoinositide 3-kinase γ mediate the anti-inflammatory effects of magnesium sulfate

“…Culture media from each group were collected after endotoxin exposure for 6 h or comparable duration in groups that were not exposed to endotoxin. An incubation interval of 6 h was determined on the basis of our previously published protocol for endotoxininduced upregulation of inflammatory mediators in RAW264.7 cells [4,7]. The concentrations of inflammatory mediators, including macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor a (TNF-a), and interleukin 6 (IL-6) from the culture media were analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits for MIP-2, TNF-a, and IL-6 (Pierce Biotechnology, Inc, Rockfold, IL).…”

“…Cell culture of RAW264.7 cells from each group was harvested after endotoxin exposure for 30 min or comparable duration in groups without endotoxin exposure. The interval for cell harvesting was determined on the basis of our previously published data that exposure to endotoxin for 30 min induced significant NF-kB and PI3K activation in RAW264.7 cells [4,7]. Nuclear and cytosolic extracts of the cell cultures were prepared and immunoblotting assay was performed, according to our previous reports [12,18].…”

“…We recently demonstrated that in endotoxin-activated murine macrophages, MgSO 4 induced phosphoinositide 3-kinase (PI3K) activation and nonspecific inhibition of PI3K by either LY294002 or Wortmannin (Sigma-Aldrich, St. Louis, MO) blocked the anti-inflammatory effects of MgSO 4 [7]. To date, four different classes of PI3K (i.e., classes IeIV) have been identified in the PI3K family [8].…”

Anti-inflammation effects of naloxone involve phosphoinositide 3-kinase delta and gamma

“…In groups receiving LPS, naloxone, and the PI3K inhibitors, the PI3K inhibitors were added 30 min before naloxone. The dosages of naloxone [6] and LY294002 [20] were determined according to previous studies. Moreover, the dosages of IC87114 [21] and AS252424 [22] were determined according to the half maximal inhibitory concentration (IC 50 ) of IC87114 and AS252424 in inhibiting PI3Kd and PI3Kg, respectively.…”

“…Immunoblotting assays were performed to measure the level of cyclooxygenase-2 (COX-2, the enzyme that regulates PGE 2 production) expression, NF-kB activation, and Akt activation (i.e., the indicator of PI3K activity), as we have previously reported [20,23]. In brief, the nuclear and cytosolic extracts of the cell cultures were collected and then separated with electrophoresis followed by protein transfer from gel to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA).…”