Yong Wang1, Daizhi Peng, Wenhua Huang, Xin Zhou, Jin Liu, Yongfei Fang. 1. Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Abstract
AIM: To explore the mechanism of altered tumor necrosis factor-alpha (TNF-alpha) expression by peritoneal macrophages (PMPhi) and Panax notoginseng saponins (PNS) modulation in light of NF-kappaB signal transduction in severely scalded mice. METHODS: Eighteen percent total body surface area (TBSA) full-thickness scalded mice were used. PMPhi was collected at different time intervals (0, 2, 6, 12, 24 and 48 post-burn hour (PBH)) separately. The following parameters were measured: TNF-alpha mRNA and IL-10 mRNA expression (reverse transcription-polymerase chain reaction, RT-PCR), protein kinase C (PKC) activity (isotope incorporation analysis), NF-kappaB activity (electrophoretic mobility shift assay, EMSA), IkappaB-alpha expression (Western blot). RESULTS: After scald, increased expression of TNF-alpha mRNA of PMPhi peaked at 12 PBH. Meanwhile, expression of IL-10 mRNA dropped to the lowest level at 12 PBH. NF-kappaB activity was markedly activated and reached its peak at 2 PBH. Membrane PKC activity was up-regulated after scald and showed a positive correlation with the change of TNF-alpha mRNA. Expression of IkappaB-alpha first decreased at 2 PBH and then increased to high level at 24 PBH. When 12 PBH was chosen as the time point for in vitro intervention with the application of specific NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC), PKC inhibitor H-7 and PNS, both TNF-alpha mRNA expression and NF-kappaB activity decreased significantly. CONCLUSIONS: These results indicate that abnormal expression of TNF-alpha mRNA of macrophages might be regulated by PKC-NF-kappaB signaling following severe burn. PNS might play an anti-inflammatory effect by inhibiting NF-kappaB activity and TNF-alpha mRNA expression.
AIM: To explore the mechanism of altered tumor necrosis factor-alpha (TNF-alpha) expression by peritoneal macrophages (PMPhi) and Panax notoginsengsaponins (PNS) modulation in light of NF-kappaB signal transduction in severely scalded mice. METHODS: Eighteen percent total body surface area (TBSA) full-thickness scalded mice were used. PMPhi was collected at different time intervals (0, 2, 6, 12, 24 and 48 post-burn hour (PBH)) separately. The following parameters were measured: TNF-alpha mRNA and IL-10 mRNA expression (reverse transcription-polymerase chain reaction, RT-PCR), protein kinase C (PKC) activity (isotope incorporation analysis), NF-kappaB activity (electrophoretic mobility shift assay, EMSA), IkappaB-alpha expression (Western blot). RESULTS: After scald, increased expression of TNF-alpha mRNA of PMPhi peaked at 12 PBH. Meanwhile, expression of IL-10 mRNA dropped to the lowest level at 12 PBH. NF-kappaB activity was markedly activated and reached its peak at 2 PBH. Membrane PKC activity was up-regulated after scald and showed a positive correlation with the change of TNF-alpha mRNA. Expression of IkappaB-alpha first decreased at 2 PBH and then increased to high level at 24 PBH. When 12 PBH was chosen as the time point for in vitro intervention with the application of specific NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC), PKC inhibitor H-7 and PNS, both TNF-alpha mRNA expression and NF-kappaB activity decreased significantly. CONCLUSIONS: These results indicate that abnormal expression of TNF-alpha mRNA of macrophages might be regulated by PKC-NF-kappaB signaling following severe burn. PNS might play an anti-inflammatory effect by inhibiting NF-kappaB activity and TNF-alpha mRNA expression.