Mechanism of altered TNF-α expression by macrophage and the modulatory effect of Panax notoginseng saponins in scald mice

Burns 32 (2006) 846–852 www.elsevier.com/locate/burns

Mechanism of altered TNF-a expression by macrophage and the modulatory effect of Panax notoginseng saponins in scald mice Yong Wang a,b, Daizhi Peng a,*, Wenhua Huang a, Xin Zhou a, Jin Liu a, Yongfei Fang b a

Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing 400038, China b Department of Traditional Chinese Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China Accepted 31 January 2006

Abstract Aim: To explore the mechanism of altered tumor necrosis factor-alpha (TNF-a) expression by peritoneal macrophages (PMF) and Panax notoginseng saponins (PNS) modulation in light of NF-kB signal transduction in severely scalded mice. Methods: Eighteen percent total body surface area (TBSA) full-thickness scalded mice were used. PMF was collected at different time intervals (0, 2, 6, 12, 24 and 48 post-burn hour (PBH)) separately. The following parameters were measured: TNF-a mRNA and IL-10 mRNA expression (reverse transcription-polymerase chain reaction, RT-PCR), protein kinase C (PKC) activity (isotope incorporation analysis), NFkB activity (electrophoretic mobility shift assay, EMSA), IkB-a expression (Western blot). Results: After scald, increased expression of TNF-a mRNA of PMF peaked at 12 PBH. Meanwhile, expression of IL-10 mRNA dropped to the lowest level at 12 PBH. NF-kB 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-a mRNA. Expression of IkB-a 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-kB inhibitor pyrrolidine dithiocarbamate (PDTC), PKC inhibitor H-7 and PNS, both TNF-a mRNA expression and NF-kB activity decreased significantly. Conclusions: These results indicate that abnormal expression of TNF-a mRNA of macrophages might be regulated by PKC-NF-kB signaling following severe burn. PNS might play an anti-inflammatory effect by inhibiting NF-kB activity and TNF-a mRNA expression. # 2006 Elsevier Ltd and ISBI. All rights reserved. Keywords: Burns; Tumor necrosis factor; Macrophage; Protein kinase C; Nuclear factor-kB

1. Introduction Severe burn induces hyper-inflammation and immunosuppression that predispose patients to sepsis and multiple organ failure. These are major complications associated with burn trauma and recent evidence suggests that activation of a pro-inflammatory cascade plays an important role in their development. After severe burn, macrophages (MF) are postulated to play a vital role in this response and activated by various stimuli such as stress, necrotic tissue components, ischemia, infected bacteria and some cytokines derived from * Corresponding author. Tel.: +86 23 68754174; fax: +86 23 65460398. E-mail address: [email protected] (D. Peng). 0305-4179/$30.00 # 2006 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2006.02.001

other inflammatory cells or MF itself [1–5]. While several groups have addressed the relationship between immune dysfunction and burn injury, the mechanism of tumor necrosis factor-a (TNF-a) secretion in this response has yet to be elucidated. With regard to this, MF is major producer of proinflammatory mediators (i.e. TNF-a, interleukin (IL)-6), prostaglandin E2 (PGE2), nitric oxide (NO). Moreover, burn increases the productive capacity of MF for these mediators [6–9]. Dysregulation of MF activity, leading to increased release of pro-inflammatory factors (i.e. MF hyperactivity), appears to be of fundamental importance in the development of immune dysfunction after burn [4,10]. TNF-a is believed to be the initiating cytokine that induces

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a cascade of secondary cytokines and humoral factors that can lead to local and systemic sequelae [11]. Moreover, TNF-a is a potent mediator of the shock-like state associated with severe burn and sepsis [12]. On the other hand, recent findings demonstrated that blockade of endogenous interleukin (IL)-10 enhanced IL-6 and TNFa release in response to lipopolysaccharide (LPS), and addition of exogenous IL-10 to the MF cultures suppressed inflammatory mediator release [13]. It is well known that nuclear transcription factors such as nuclear factor-kB (NF-kB), cAMP response element-binding protein (CREB), activating protein-1 (AP-1) regulate the expression of pro-inflammatory factors such as TNF-a, inducible nitric oxide synthase (iNOS) and Cycloxygenase-2 (COX-2) [14–15]. With regards to NF-kB dimers, under resting conditions it is in the cytosol in complex with its inhibitory kB (IkB) whose promoter region also contains a NF-kB-binding site [16]. Upon stimulation, protein kinase C (PKC) family members phosphorylate IkB kinases, and IkB is phosphorylated and proteolyzed, which lead to NF-kB translocation and binding to specific promoter regions inducing gene transcription [17]. Alterations in NF-kB, important in the regulation of MF gene expression after burn, has not been investigated but may be important in understanding the mechanisms by which MF hyperactivity occurs. Panax notoginseng saponins (PNS), which is the principal ingredient extracted from the traditional Chinese herb medicinal P. notoginseng (Burk.), has obvious anti-inflammatory effect and its mechanisms are related to the inhibition of intracellular free calcium concentration level and phospholipase A2 activity in neutrophils, and reduction of dinoprostone content [18]. Up to now little is known about whether PNS has taken its anti-inflammatory effects through regulating NF-kB activity and TNF-a production following severe burn. Therefore, our laboratory sought to explore the mechanism of altered TNF-a expression by peritoneal macrophages (PMF) and PNS modulation in light of NF-kB signal transduction following severe burn. In this study, changes of TNF-a expression, PKC activity, NF-kB activity, expressions of IkB-a in PMF at different time points in scalded mice were measured, then the effects of the specific NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC), PKC inhibitor H-7 and Chinese herb PNS on regulation of TNF-a mRNA and NF-kB activity were determined.

2. Materials and methods 2.1. Experimental burn Either sex of Kunming mice (6–8 weeks of age, 20–24 g) were obtained from the Experimental Animal Center of Third Military Medical University (Chongqing, China). The research was conducted in accordance with the institutional

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accepted principles for laboratory animal use and care. All the animals were maintained in cages at 20
2 8C with free access to pellet food and water and were kept. This study complies with current ethical regulations on animal research of this institute and all animals used in the experiment receive humane care. Mice received a full-thickness scald burn as previously described [4], which was confirmed by pathological slices. Briefly, the entire dorsal surface of anesthetized mice was shaved and fixed under a thick rubber board with a hole in order to let 18% total body surface area (TBSA) expose on the shaved back. Then, the exposed area was subjected to high pressure vapor (1.0–1.2 kg/cm2) with a distance of 4 cm away from its outlet for 10 s to produce 18% TBSA skin-full-thickness scalds, then mice were resuscitated with 2 ml sterile normal saline intraperitoneally. On waking, the mice were returned to the animal facility in different cages. The control group was treated as same as above, except the hot vapor was replaced by air. The injured mice were randomly assigned into different postburn hour (PBH) groups, the control group, or in vitro different reagents treated groups. There were five mice in each group. 2.2. Isolation and culture of mouse peritoneal macrophages (PMF) [4,8] The injured mice were killed by bleeding at the designated time points after injury (i.e. 2, 6, 12, 24 and 48 PBH) and thoroughly cleansed with 70% ethyl alcohol. A small incision was made into the abdomen, and the peritoneal cavity was rinsed with 5 ml pre-cooled phosphate-buffered saline (PBS) (pH 7.2) twice aseptically. The rinsed PBS was collected and centrifuged at 1000  g for 5 min. The cell pellet was re-suspended with complete RPMI 1640 medium containing 10% fetal bovine serum (Gibco-BRL, USA) before counting and were incubated in plastic cell culture plates for 90 min at 37 8C, 5% CO2 to allow the macrophages to adhere. The non-adherent peritoneal cells were removed by washing the plate twice with warm serum-free medium, and more than 90% of the adherent cell population determined by morphology and non-specific esterase staining was MF. The medium was removed from macrophage monolayers (3.0  106 cell per well) in six-well tissue culture plate. PMF of each well at different PBH intervals were incubated in 3 ml complete RPMI 1640 medium containing 10 mg/ml LPS (Escherichia coli, serotype O111:B4, Sigma). When the 12 PBH was chosen as the time point for intervention of the PMF, specific NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC), PKC inhibitor H-7 (Sigma, USA) and PNS were used with the final concentration of 100 mmol/l, 50 mmol/l and 0.8 mg/ml, respectively. After being cultured for 2 h, the supernatants were discarded from each well and the cells were prepared for RNA isolation, PKC activity measurement and nuclear protein extraction as follows.

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2.3. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the detection of cytokines [19] Two micrograms of total RNA was reversibly transcribed using M-MuLV Reverse Transcriptase (40 units) at 37 8C for 1 h. Five microliters of 20 ml total RT reaction was used as template DNA for PCR. PCR was performed by 60 s denaturation at 94 8C, 60 s renaturation with specific primers at 60 8C, and 60 s extension at 72 8C for 35 cycles. Primers were based on published nucleotide sequence for mouse TNF-a (50 -GGC AGG TCT ACT TTG GAG TCA TTG C-30 , 50 -ACATTC GAG GCT CCA GTG AAT TCG G30 , with an amplified product of 307 bp), mouse IL-10 (50 ACC TGG TAG AAG TGA TGC CCC AGG CA-30 ; 50 -CTA TGC AGT TGA TGA AGA TGT CAA A-30 , with an amplified product of 237 bp), and mouse b-actin (50 -TGG AAT CCT GTG GCA TCC ATG AAA C-30 ; 50 -TAA AAA CGC AGC TCA GTA ACA GTC CG-30 , with an amplified product of 348 bp). After they were electrophoresed in 1.5% agarose gel, the PCR products were scanned and relative intensity of the signals was determined by LabImage 2.6 software. The polymerase chain reaction experiments were performed three times with similar results. The ratio of arbitrary unit of target genes over b-actin was used for expressing the relative level of mRNA expression. 2.4. Preparation and assays of PKC All manipulations were performed at 4 8C except as otherwise noted. PMF were rinsed three times with cold PBS. After routine centrifugation, pelleted cells were re-suspended in 2 ml cold buffer A (15 mmol/l Tris–HCl (pH 7.4), 1 mmol/l EGTA, 2.5 mmol/l MgCl2, 0.34 mol/l sucrose, 50 mmol/l 2-mercaptoethanol, 1 mmol/l PMSF, 1 mg /ml leupeptin, 1 mg/ml pepstinin) and sonicated immediately with three 10-s bursts of 250 mA each. The broken cells preparations were centrifuged at 27,500  g for 60 min. The supernatant containing plasma soluble PKC component were collected. The sediments were re-suspended in 2 ml cold buffer B containing 0.1% (w/v) Triton-X 100 (the rest components were as same as that of buffer A), sonicated with three 10-s bursts of 300 mA each and centrifuged at 27,500  g for 30 min by turns. The supernatant containing trans-membrane soluble PKC component were collected [20]. Protein concentrations were determined after the technique by modified Bradford method [21,22]. PKC activity was determined by measuring the incorporation of g-phosphorus 32-labeled adenosine triphosphate (g-[32P]-ATP) into histone type III-s according to the modified method described by Helfman et al. [23]. Twenty microliters of sample solution containing PKC was added into 250 ml of reaction solution (20 mmol/l Tris–HCl (pH 7.5), 8 mmol/l MgCl2, 160 mg/ml histone, 2 mCi g-[32P]ATP (50 mCi at 5000 Ci/mmol, Yahui Corp., China), 10 mg/ ml phosphatidyl serine, 0.5 mmol/l CaCl2) and incubated at

30 8C for 10 min. The reaction was terminated by the addition of 1 ml cold 20% trichloroacetic acid (TCA). Precipitated protein was collected on Millipore 0.45 mm type HA filters, washed thoroughly with 5% TCA, dried, and counted by liquid scintillation spectrometry. PKC activity was expressed as pmol/min/mg protein. 2.5. Electrophoretic mobility shift assay (EMSA) for NF-kB activity [19,24] After nuclear protein was extracted by a modification of methods [25], a double-stranded oligonucleotide containing mouse nuclear factor-kB (NF-kB) site (P1: 50 -AGT TGA GGG GAC TTT CCC AGG C-30 ; P2: 50 -GCC TGG GAA AGT CCC CTC AAC T-30 ) was end-labeled with g-[32P]ATP (50 mCi at 5000 Ci/mmol, Yahui Corp., China) with T4 polynucleotide kinase. Binding assays were performed in 21 ml of binding reaction mixture containing 3 mg nuclear extract protein, DNA binding buffer and 1 ng 32P-labeled DNA probe. Reactions were incubated at room temperature for 30 min and analyzed by electrophoresis on a 5% nondenaturing polyacrylamide gel at 110 V for 2 h using the high ionic strength conditions. The specificity of binding was confirmed by addition of a 100-fold excess of unlabeled double-stranded oligonucleotides containing mouse NF-kB site to separate reaction mixtures. As an additional control, a 100-fold excess of cold oligonucleotide bearing the AP-1 binding site was added to separate reaction mixtures. Competition reactions were incubated for 10 min before addition of labeled oligonucleotide. After electrophoresis, gels containing DNA–protein complexes were exposed to Kodak X film for 18 h at 70 8C. The autoradiograms were quantified by scanning densitometry with LabImage 2.6 software and measured value was expressed as relative density units (RDU). 2.6. Western blot analysis for IkB-a protein level The IkB-a protein level was determined by Western blot analysis as previously described [26]. Cytosolic extracts from PMF were electrophoresed on 10% SDS polyacrylamide gels and transferred to polyvinylidene fluoride membrane (PVDF) in 25 mM Tris, 192 mM glycine, 20% methanol at 135 mA for 6 h. The filters were blocked to reduce non-specific binding by incubation in blocking buffer (3% non-fat milk, 0.01 M PBS, 0.02% Tween-20) overnight. The blots were then incubated with rabbit polyclonal antiIkB-a antibody at 1:2000 dilution for 1 h. After being washed with PBS–Tween-20, the blots were incubated with secondary antibody conjugated to horseradish peroxidase blocking buffer for 1 h. The blots were washed extensively with PBS–Tween-20 and developed by the enhanced diaminobenzidine methods. The intensity of the bands representing IkB-a protein was scanned using BioRad image analysis and quantified by LabImage 2.6 software, measured in relative density units (RDU).

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2.7. Statistical analysis The data were expressed as mean
S.D. Statistical analysis was evaluated by one-way analysis of variance, followed by Dunnett’s t-test for between-group comparisons. Correlations between variables were tested by Spearman’s correlation coefficients. These statistical analyses were done with SPSS statistical program. The level of P < 0.05 was considered statistically significant.

3. Results 3.1. TNF-a and IL-10 expression in PMF following burn During the early phase after burn in this model, analysis of expression with RT-PCR demonstrated that the expression of TNF-a in PMF was increased, reaching the peak level at 12 PBH, and then decreased gradually, but at 48 PBH, the expression was still remarkably higher than that of the control group (P < 0.01) (Fig. 1). On the contrary, expression of IL-10 mRNA by murine PMF decreased continuously after scald and dropped to the lowest level at 12 PBH before returning to the normal level at 24 PBH. 3.2. Changes of PKC activity following burn It was found that the pattern of change of both plasma and membrane PKC were different at the early stage after burn injury (Fig. 2). Compared with the control group, the activity of plasma PKC following burn was slightly increased, reaching the value of 82.82
27.09 pmol/min/mg protein at 12 PBH, which no significance was found between the control group and 12 PBH. On the other hand, membrane PKC activity ascended in wave shape. It rapidly elevated to 512.10
33.42 pmol/min/mg protein (P < 0.01) at 12 PBH

Fig. 1. Semiquantitive reverse transcription polymerase chain reaction (RTPCR) analysis of TNF-a and IL-10 mRNA expression in PMF following burn. Data are reported as the ratio of TNF-a or IL-10 to b-actin signals and are mean
S.D. of three mice and the experiments in each animal included triplicate sets. *P < 0.05 and #P < 0.01 as compared with the control group values.

Fig. 2. Isotope incorporation analysis of membrane and plasma PKC activities in PMF after burn. Data are reported as pmol/min/mg protein and are mean
S.D. of five mice. *P < 0.05 and #P < 0.01 as compared with the control group values.

and hitting its peak of 530.49
28.54 pmol/min/mg protein (P < 0.01) at 48 PBH, respectively. 3.3. Changes of NF-kB activity and IkB-a protein expression following burn The specificity of the shift bands in EMSA was verified by competition assays: all the shift bands were suppressed by incubation with a 100-fold excess of unlabeled NF-kB probe and unchanged by competition with a similar amount of another irrelevant oligonucleotide such as AP-1 (data not shown). Compared with the control group, NF-kB activity increased significantly 2 h after the scald and reached the peak level. At 6 PBH, the activity was decreased slightly, but followed by increase again till 48 PBH. Seen from the overall pattern of the changes, it could be concluded that NFkB had been highly activated in the early post-burn stage (Fig. 3). IkB-a content in the PMF was lowered at 2 PBH followed by gradual increasing till 24 PBH about twice as much as the normal level (Fig. 4).

Fig. 3. Dynamic changes of NF-kB activity in PMF after thermal injury. Representative pictures of three experiments of Electrophoretic mobility shift assay analysis. Lane 1: negative control; lane 2: control; lane 3: 2PBH; lane 4: 6PBH; lane 5: 12PBH; lane 6: 24PBH; lane 7: 48PBH.

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Fig. 4. IkB-a protein levels in PMF after thermal injury. Representative pictures of three experiments of Western blot analysis. Lane 1: control; lane 2: 2PBH; lane 3: 6PBH; lane 4: 12PBH; lane 5: 24PBH; lane 6: 48PBH.

Fig. 5. (A) TNF-a mRNA expression increased significantly at 12 PBH compared with the control group, while treatment with PDTC, H-7 and PNS at corresponding concentration in vitro induced marked attenuation of TNF-a mRNA expression respectively. Lane 1: control; lane 2: burn; lane 3: burn + PDTC; lane 4: burn + H-7; lane 5: burn + PNS. (B) NF-kB binding activity increased significantly at 12 PBH compared with the control group, whereas addition of PDTC, H-7 and PNS resulted in significant decrease of NF-kB binding activity respectively. Lane 1: negative control; lane 2: control; lane 3: burn; lane 4: burn + PDTC; lane 5: burn + H-7; lane 6: burn + PNS.

3.4. Effects of PDTC, H-7 and PNS on TNF-a expression and NF-kB activity at 12 PBH In this study, 12 PBH was chosen as the time point for intervention with the application of specific NF-kB inhibitor PDTC and PKC inhibitor H-7. It was showed that both TNFa mRNA expression and NF-kB activity increased significantly 12 h after the injury compared with the control group, while the addition of PDTC at a final concentration of 100 mmol/l for 90 min or H-7 at 50 mmol/l concentration for 90 min induced marked attenuation of TNF-a mRNA expression and NF-kB activity, respectively. When antiinflammatory herb PNS was added to the culture medium to a final concentration of 0.8 mg/ml with the treatment duration of 90 min, not only the expression of TNF-a mRNA, but also the activity of NF-kB were significantly decreased when measured by RT-PCR and EMSA. Similar results were obtained from repeated experiments for three times (Fig. 5A and B).

4. Discussion The release of pro-inflammatory cytokines by MF is an important mechanism by which MF regulate the inflam-

matory response. TNF-a is one of the mononuclear factors created earliest by macrophages after burn or trauma, which is involved in immune defense against infection. Appropriate quantity of TNF-a has protective effects, but the marked increased TNF-a production in macrophages after burn is harmful, which would cause organ injury and negative nitrogen balance [13,27]. On the other hand, macrophages, as well as T-cells, produce the antiinflammatory cytokine IL-10, which can block inducible nitric oxide synthase expression, TNF-a production and other aspects of cell-mediated immunity [28,29]. Recent findings have demonstrated that blockade of endogenous IL10 enhanced IL-6 and TNF-a release in response to LPS, and addition of exogenous IL-10 to the MF cultures suppressed inflammatory mediator release [14]. Our results showed that the increased expression of TNF-a mRNA arrived to its peak at 12 PBH and lasted till 48 PBH. On the contrary, expression of IL-10 mRNA by murine PMF decreased after scald and dropped to the lowest level at 12 PBH before returning to the normal level at 48 PBH. It suggested that there is an imbalance between TNF-a and IL10 expression, which may be one of the initiating factors for the development of SIRS and multiple organ failure in the early after burn period. PKC constitutes an expanding multigene family of serine/ threonine kinases that is involved in the transduction of extracellular signals conveyed by growth factors, neurotransmitters, hormones and other biological molecules [30]. To date, 12 known isoforms have been classified into three subfamilies on the basis of their structure and ability to bind cofactors [31]. In inactivated cells, PKC generally exists in cytoplasm in inactivated mode. Extracellular stimulation causes diacylglycerol or phorbol ester to combine with PKC, which activates PKC due to allosteric effect, and subsequently translocates from cytoplasm to various subcellular sites presumably containing appropriate substrates followed by phosphorylating the elements down stream to exert its bioactivity. For example, PKC isoenzyme PKC-z directly or indirectly phosphorylates substrate protein IkB kinase-b to induce LPS-induced NF-kB p65 subunit nuclear translocation in human myometrial cells [32,33]. Some research indicates that membrane and plasma PKC in T-lymphocytes is activated in mice after burn to generate nuclear translocation and thereby induces IL-2 and IL-10 secretion [34]. Our experimental results indicated that NF-kB activity was markedly activated and reached its peak at 2 PBH after serious scalding. Membrane PKC ascended in wavy shape and rapidly elevated to 512.10
33.42 pmol/min/mg protein at 12 PBH while cytoplasm PKC activity reached the value of 82.82
27.09 pmol/min/mg protein at 12 PBH, suggesting much of PKC was activated and shifted from the cytoplasm to the membrane. The correlation analysis among changes of TNF-a expression, membrane PKC and NF-kB activity indicated a significant positive correlation (r = 0.859, P < 0.01), which means there might be an internal relationship among these items. When 12 PBH was chosen as the time

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point for intervention with the application of PDTC or H-7, both TNF-a mRNA expression and NF-kB activity were inhibited at the same time. Either human or mice, there are several NF-kB combination sites on TNF-a promoters [35,36]. Thereby, the participation of PKC-NF-kB signal pathway after scalding could be considered as one of the reasons that cause increased TNF-a expression. In normal mice, cytoplasmic IkB-a expression is maintained at a relatively high level. But in this study, it was decreased significantly at 2 h post-burn, and then increased gradually till reaching the peak level at 24 h about twice as much as the normal level. The course of variation of IkB-a coincided with NF-kB activation, indicating that IkBa in the macrophages is activated rapidly after burn, immediately followed by ubiquitination and degradation through phosphorylation by PKC or IkB kinase, leading to decrease of its cytoplasmic content [19,30]. At 2 PBH, because of significant decrease of IkB-a, NF-kB activity reached its peak rapidly. At 12 PBH, the burn itself and development of endotoxemia cause the activity of NF-kB to increase again to the second peak, but IkB-a expression increased to almost normal level without descent, possibly because IkB-a gene promotor contains multiple NF-kBbinding sequences to allow activated NF-kB to initiate IkBa gene transcription. The high-level expression of IkB-a thus inhibits the binding between DNA and NF-kB through a negative feedback mechanism, and induces the NF-kB bound to DNA to dissociate and terminates NF-kB-mediated transcription process. This also results in anchorage of NFkB dimers in the cytoplasm for its inactivation [37,38]. At 24 and 48 PBH, as IkB-a synthesis is accelerated, large amount of activated NF-kB is inhibited, so that the activity of NF-kB decreases gradually, and both of them maintain high levels with inhibitory interactions. PNS, also known as sanchi ginseng, contains 24 dammarene type saponins (ginsenosides) including sanchinoside or notoginsenoside which is unique to P. notoginseng [39]. It has been reported to be effective in improving blood circulation and regulating the immune system after burn, such as inhibiting platelet aggregation, improving blood flow in the coronary arteries, reducing myocardial oxygen consumption, increasing myocardial Gsa mRNA expression and elevating (Na+–K+)-ATPase and (Ca2+–Mg2+)-ATPase activities of cardiomyocyte membrane [40,41]. In this study, it was found that cell viability of PMF exceeded 95% by trypan blue staining followed by PNS was added to the culture medium to a final concentration of 0.8 mg/ml for 90 min, suggesting the drug has no cytotoxicity effect on PMF at this concentration in vitro. Both the expression of TNF-a mRNA and the activity of NF-kB were significantly inhibited by PNS using RT-PCR and EMSA detection. In conclusion, severe burn provokes the syndrome of excessive inflammatory responses and immune dysfunction. There exists an imbalance between increased TNF-a and decreased IL-10 mRNA expression following burn. Abnormal expression of TNF-a mRNA of macrophages may be

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regulated by PKC-NF-kB signaling following severe burn. PNS may play an anti-inflammatory effect by inhibiting NFkB activity and TNF-a mRNA expression, it might be a beneficial guidance for us to correct the imbalance between TNF-a and IL-10 and prevent the progress of SIRS and multiple organ failure after severe burns.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 39290700-01) and the Military Ninth Five-year Instructive Topic (No. 96L042).

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