| Systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS) are the major pathologic processes of infectious or traumatic diseases complicated with multiple organ dysfunction syndrome (MODS). The overproduction of the inflammatory mediators and the less or overproduction of the anti-inflammatory mediators in MODS can result in the uncontrolled inflammatory response, multiple systemic organ failure (MSOF) and ultimate death. The lung is easy to become a dysfunctional organ in the pathological process of uncontrolled inflammatory response and MODS. The acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common complicated with MODS. As important kinds of inflammatory mediators, cytokines play key roles in the pathological process of inflammatory response. The uncontrolled release of cytokines is the major pathophysiological processes of ARDS and MODS. According to the different function in host defending response, cytokines are subdivided into two kinds: proinflammatory cytokines and anti-inflammatory cytokines. In the inflammatory response, the proinflammatory cytokines such as TNF-α, IL-1β, IL-6 are secreted. At the same time, the anti-inflammatory cytokines such as IL-4, IL-10 are secreted too in order to resist the proinflammatory response and maintain the intra-homeostasis. The activated monocytes/macrophages play critical roles in the inflammatory response and release lots of various proinflammatory cytokines. Lipololysaccharide (LPS), the main outer membrane component of Gram-negative bacteria, is the most potent activator of monocytes/macrophages and plays a key role during severe Gram-negative infection, sepsis and septic shock. Cholecyetokinin (CCK), a typical braingut peptide, is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuromodulator to play a pivotal role. Recent years, a series of studies showed that CCK-8 had the effect of anti-inflammation. Pretreating endotoxin shock (ES) rats with CCK-8 led to a rise in mean arterial pressure and a reduction in pulmonary arterial hypertension (PAH), and the inflammatory lesions in the lung, liver and spleen tissues were reduced significantly. More recently, some studies of our laboratory proved the presence of CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR) mRNA expression in rat spleen cells, lung and heart tissues and pulmonary interstitial macrophages (PIMs), and the expression could be up-regulated by LPS obviously. A lot of data demonstrate that CCK-8 could cause an in vitro inhibition of LPS-induced NF-κB activity, sCD14 release, mCD14 and Toll-like receptor 4 (TLR4) expressions in rat PIMs via its receptors. Consistently, the production of proinflammatory cytokines including TNF-α, IL-1βand IL-6 in ES rat were also inhibited by CCK-8 in vivo and in vitro. Series studies showed that CCK-8 had conclusive anti-inflammatory effects on inflammation. The signal transduction mechanisms through which LPS activate macrophages are anfractuous. However it is undoubtful that the LPS—CD14—TLR4—IRAK—MAPKs—AP-1—cytokines pathway play pivotal role in the macrophage activation induced by LPS. The promoter in IL-1β, IL-6, IL-10 gene contain binding motifs for activate protein-1 (AP-1), suggesting AP-1 play a critical role in the expressions of IL-1β, IL-6 and IL-10. AP-1 is mainly regulated by mitogen-activated protein kinases (MAPKs). Some studies of our laboratory proved that CCK-8 could inhibit the expressions of TNF-α, IL-1βin rat PIMs induced by LPS via inhibiting the expressions of CD14 and TLR4. However, there are no reports about the effects of CCK-8 on activity of p38 MAPK, AP-1 and the expressions of anti-inflammatory cytokines such as IL-4, IL-10 in macrophages. To elucidate the anti-inflammatory mechanism of CCK-8 on balancing pro-and anti-inflammatory response, we performed a series of studies inendotoxemia mice and RAW264.7, a murine macrophage cell line. These studies included the dynamic changes of proinflammatory cytokines IL-1β, IL-6 and anti-inflammatory cytokines IL-10, IL-4 in inflammatory response, the effects of CCK-8 on IL-1β, IL-6, IL-4, IL-10 expressions induced by LPS and the related signal transduction mechanism. 1 Effects of CCK-8 on the expressions of related pro-and anti-inflammatory cytokines of endotoxemia mice induced by LPS Objective: To observe the effects of CCK-8 on the mortality rate, the expressions of proinflammatory cytokines IL-1β,IL-6 and anti-inflammatory cytokines IL-4,IL-10 in endotoxemia mice induced by LPS. Methods: To observe the effect of CCK-8 on mortality rate, several Kunming (KM) mice were randomly assigned to four groups injected different agents via intraperitoneal injection. For group receiving LPS, a bolus dose of LPS was injected into abdominal cavity. For group of LPS+CCK-8, a bolus dose of CCK-8 was pre-injected 30 min before the injection of LPS. Saline or CCK-8 was injected separately in control or CCK-8 group. Meanwhile, the productions of IL-1β, IL-6, IL-4 and IL-10 in the serum and lung tissues and the expressions of IL-1β, IL-6, IL-4 and IL-10 mRNA in the lung tissues were observed injected with LPS alone or plus CCK-8 in different groups at different time (The bolus dose of CCK-8 was 60μg/kg. When observing mortality rate, the bolus dose of LPS was 25mg/kg; When observing the expressions of cytokines, the bolus dose of LPS was 10mg/kg). The contents of cytokines in the serum and lung tissues was measured with the method of Enzyme linked immunoabsorbant assay (ELISA); The expressions of cytokines mRNA in the lung tissues were assayed by means of reverse transcription polymerase chain reaction (RT-PCR). Data were presented as x ±s and analyzed with ANOVA and least significant difference (LSD) using SPSS statistical program. A level of P<0.05 was considered statistically significant. Using Chi-square test (X2) to analyse the mortality rate. A level of P<0.007 was considered statistically significant according to the emendation equation.Results: (1) After 48 h of injection, the mortality rate of control group and CCK-8 group mice was 0 (0/20). The mortality rate of LPS group mice was 70% (14/20, P<0.007 vs control group). The mortality rate of LPS+CCK-8 group mice was 10% (2/20, P<0.007 vs LPS group). (2) The concentrations of IL-6 and IL-4 were 54.03±6.57 ng/L, 37.76±7.17 ng/L respectively and the contents of IL-1β, IL-10 were under detectable limit in the serum of control group mice. The concentrations of cytokines in the serum of different groups rose by injection of LPS, the concentrations of IL-1β, IL-6, IL-4 and IL-10 in the serum reached the peak at the time of 2 h, 4 h, 4 h, 6 h after injection of LPS and the concentrations were 342.14±14.89 ng/L, 4022.76±39.72 ng/L, 193.02±10.95 ng/L and 104.33±11.95 ng/L respectively. The different peak times of cytokines expressions were selected to investigate the effects of CCK-8 on different cytokines. The study showed that pre-injection of CCK-8 (LPS+CCK-8 group) reduced the contents of IL-1β(2 h), IL-6 (4 h) by 86.9% and 62.8% compared with LPS group (44.76±10.19 ng/L vs 342.14±14.89 ng/L, 1497.59±55.43 ng/L vs 4022.76±39.72 ng/L, P<0.01). But the IL-1βand IL-6 concentrations of LPS+CCK-8 group were still higher than that of control group (P<0.01 vs control group). However the IL-4 concentrations of LPS+CCK-8 group (4 h and 12 h) rose respectively by 59.7% and 35.1% compared with LPS group (308.15±11.24 ng/L vs 193.02±10.95 ng/L, 170.33±9.48 ng/L vs 126.14±10.95 ng/L, P<0.01). The IL-10 concentrations of LPS+CCK-8 group (6 h and 12 h) rose respectively by 73.6% and 56.2% compared with LPS group (181.11±9.03 ng/L vs 104.33±11.95 ng/L, 108.84±16.28 ng/L vs 69.70±10.43 ng/L, P<0.01). (3) The concentrations of IL-6, IL-4 were 56.14±15.78 ng/L, 44.92±7.17 ng/L respectively in the lung tissues of control group and the contents of IL-1β, IL-10 were under detectable limit. The concentrations of cytokines in the lung tissues of different groups rose by injection of LPS. The concentrations of IL-1β, IL-6, IL-4 and IL-10 in the lung tissues reached the peak at the time of 2 h, 4 h, 6 h, 6 h after injection of LPS and the concentrations were 1167.67±35.16 ng/L, 4033.28±72.32 ng/L, 289.76±20.99 ng/L and 187.13±11.37 ng/L respectively. The different peaktimes of cytokines expressions were selected to investigate the effects of CCK-8 on different cytokines. The study showed that pre-injection of CCK-8 (LPS+CCK-8 group) reduced the contents of IL-1β(2 h), IL-6 (4 h) respectively by 51.2% and 67.2% compared with LPS group (569.65±66.37 ng/L vs 1167.6±35.16 ng/L, 1323.99±65.71 ng/L vs 4033.28±72.32 ng/L, P<0.01). But the IL-1βand IL-6 concentrations of LPS+CCK-8 group were still higher than that of control group (P<0.01 vs control group). However the IL-4 concentrations in the lung tissues of LPS+CCK-8 group (6 h and 12 h) rose respectively by 47.6% and 24.0% compared with LPS group (388.89±21.50 ng/L vs 263.48±16.42 ng/L, 252.74±16.42 ng/L vs 203.77±14.48 ng/L, P<0.01). The IL-10 concentrations in the lung tissues of LPS+CCK-8 group (6 h and 12 h) rose respectively by 45.9% and 27.0% compared with the LPS group (272.94±13.80 ng/L vs 187.13±11.37 ng/L, 184.12±9.40 ng/L vs 144.97±16.28 ng/L, P<0.01). (4) After 2 h of injection, the expression of IL-1βmRNA was unable to be detected in the lung tissues of the control group and CCK-8 group. The expression of IL-1βmRNA rose significantly in the lung tissues of the LPS group (P<0.01 vs control group). The pre-injection of CCK-8 (LPS+CCK-8 group) inhibited the expression of IL-1βmRNA significantly and the inhibiting rate was 59.9% (P<0.01 vs LPS group). After 4 h of injection, weak IL-6 mRNA expressions were detected in the lung tissues of the control group and CCK-8 group. The expression of IL-6 mRNA rose significantly in the lung tissues of the LPS group (P<0.01 vs control group). The pre-injection of CCK-8 (LPS+CCK-8 group) inhibited the expression of IL-6 mRNA significantly and the inhibiting rate was 68.1% (P<0.01 vs LPS group). After 6 h of injection, weak IL-4, IL-10 mRNA expressions were detected in the lung tissues of the control group and CCK-8 group. The expressions of IL-4, IL-10 mRNA rose significantly in the lung tissues of the LPS group (P<0.01 vs control group). The pre-injection of CCK-8 further improved the expressions of IL-4, IL-10 mRNA and the up-regulation rates were 50.4% and 51.9% respectively (P<0.01 vs LPS group).Conclusions: The above results demonstrated that the anti-inflammatory cytokines IL-4, IL-10 produced after the proinflammatory cytokines IL-1β, IL-6 in endotoxemia mice. In addition, the peak times of cytokines expressions, quantity and the effects of CCK-8 on cytokines expressions were not consistent in the serum and lung tissues of endotoxemia mice. CCK-8 reduced the mortality rate of endotoxemia mice significantly, and participated in the anti-inflammatory response by inhibiting the expressions of proinflammatory cytokines IL-1β, IL-6 and increasing the expressions of anti-inflammatory cytokines IL-4, IL-10 in the serum and lung tissues of endotoxemia mice, which would be of important significance to alleviate the inflammatory response in the lung tissues induced by LPS and reduce the mortality rate of endotoxemia mice. 2 Effects of CCK-8 on the expressions of pro-and anti-inflammatory cytokines in LPS-stimulated RAW264.7 cells Objective: Macrophages are the critical effector cells and the major target cells in the inflammatory response. The activated macrophages are the important sources of many cytokines such as IL-1β, IL-6 and IL-10. So in the present study, effects of CCK-8 on the expressions of proinflammatory cytokines IL-1β, IL-6 and anti-inflammatory cytokine IL-10 in LPS-stimulated RAW264.7 cells were observed. Methods: RAW264.7 cells were stimulated with LPS in the presence or absence of CCK-8. The contents of IL-1β, IL-6, IL-10 in RAW264.7 cells culture supernatant were measured with the method of ELISA. The expressions of IL-1β, IL-6 and IL-10 mRNA were assayed by means of RT-PCR. Data were presented as x ±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) The concentrations of IL-1β, IL-6 and IL-10 were under detectable limit in RAW264.7 cells culture supernatant of control groups. The concentrations of IL-1β, IL-6, IL-10 in RAW264.7 cells culture supernatant reached the peak at the time of 3 h, 24 h, 24 h after LPS stimulation and theconcentrations were 209.41±10.04 ng/L, 2071.64±9.08 ng/L and 207.65±16.27 ng/L respectively. The different peak times of cytokines expressions were selected to investigate the effects of CCK-8 on different cytokines. It was found that pre-treating RAW264.7 cells with CCK-8 led to a dose-dependent inhibition of IL-1β, IL-6 productions and a dose-dependent augmentation of IL-10 production. Low doses of CCK-8 (10-10 mol/L and 10-9 mol/L) had no obvious effects on the LPS-induced productions of IL-1β, IL-6 and IL-10. While 10-8 mol/L, 10-7 mol/L, 10-6 mol/L CCK-8 could inhibit LPS-induced IL-1β, IL-6 productions and augment the LPS-induced IL-10 production significantly. When incubating cells for 24 h, the extents of CCK-8 augmentation LPS-induced IL-10 expression were higher than incubating cells for 48 h. The IL-1βconcentrations of LPS+CCK-8 (10-8, 10-7, 10-6 mol/L) group were 103.26±10.04 ng/L, 64.66±7.37 ng/L, 37.32±7.37 ng/L respectively. The inhibiting rates were 50.7%, 69.1%, 82.2% compared with LPS group. The IL-6 concentrations were 1274.19±13.87 ng/L, 982.11±13.62 ng/L, 665.85±13.87 ng/L respectively. The inhibiting rates were 38.5%, 52.6%, 67.9% respectively. The IL-10 concentrations of LPS+CCK-8 (10-8, 10-7, 10-6 mol/L) group were 379.04±18.72 ng/L, 439.26±7.08 ng/L, 456.25±19.29 ng/L respectively when incubating cells for 24 h and the productions of IL-10 were increased 0.83 times, 1.12 times and 1.20 times respectively compared with LPS group. When incubating cells for 48 h, the IL-10 concentrations were 195.30±8.02 ng/L, 220.00±7.08 ng/L, 241.62±12.26 ng/L and the productions of IL-10 were increased 0.51 times, 0.70 times, 0.87 times respectively (every group P<0.01 vs LPS group). 10-8 mol/L CCK-8 incubation alone did not affect IL-1β, IL-6 and IL-10 productions (P>0.05 vs control group). (2) The weak expressions of IL-10 and IL-6 mRNA were detected in control group, while the expression of IL-1βmRNA was unable to be detected in control group RAW264.7 cells. The expressions of IL-1β, IL-6 and IL-10 mRNA were increased significantly by LPS stimulation, which reached the peak at the time of 3 h, 24 h and 24 h respectively after LPS stimulation. The different peak times of cytokines expressions were selected toinvestigate the effects of CCK-8 on different cytokines. It was found that pre-treating RAW264.7 cells with CCK-8 led to a dose-dependent inhibition of IL-1β, IL-6 mRNA expressions and a dose-dependent augmentation of IL-10 mRNA expression. Low doses of CCK-8 (10-10 mol/L) had no obvious effects on LPS-induced IL-1β, IL-6 and IL-10 mRNA expressions. While 10-8 mol/L and 10-6 mol/L CCK-8 could inhibit the LPS-induced IL-1β, IL-6 mRNA expressions and augment the LPS-induced IL-10 mRNA expression significantly. Compared with LPS group, 10-8 mol/L and 10-6 mol/L CCK-8 could inhibit the LPS-induced IL-1βmRNA expressions by 50.5%, 72.5% and inhibit the LPS-induced IL-6 mRNA expressions by 43.6%, 65.6% respectively. The IL-10 expression in LPS+CCK-8 (10-8, 10-6 mol/L) group was increased 0.76 times, 1.18 times respectively compared with LPS group (every group P<0.01 vs LPS group). 10-8 mol/L CCK-8 incubation alone did not affect IL-1β, IL-6 and IL-10 mRNA expressions (P>0.05 vs control group). Conclusions: The results demonstrated that LPS induced IL-1β, IL-6 and IL-10 expressions in a time-dependent manner in RAW264.7 cells and the anti-inflammatory cytokines IL-10 produced after the proinflammatory cytokines IL-1βand IL-6. CCK-8 participated in the anti-inflammatory response by inhibiting the expressions of proinflammatory cytokines IL-1β, IL-6, meanwhile augmenting the expression of anti-inflammatory cytokine IL-10 induced by LPS, which was important to regulate the pro-and anti-inflammatory cytokines, balance pro-and anti-inflammatory mediators and inhibit the inflammatory response. 3 Effect of CCK-8 on the DNA binding activity of AP-1 in LPS-stimulated RAW264.7 cells Objective: Some studies have showed that the promoter in IL-1β, IL-6 and IL-10 gene contains binding motifs for activate protein-1 (AP-1), which appear to be important in those cytokines expressions. Some experiments have demonstrated that p38 MAPK might be one of the upstream regulation molecules of AP-1. Recently our laboratory reported that CCK-8 inhibitedNF-κB activity in ES rat lung tissues and rat PIMs induced by LPS. While there is no report about the effect of CCK-8 on AP-1 activity in LPS-stimulated RAW264.7 cells. So in the present study, effects of CCK-8 on the DNA binding activity of AP-1 in LPS-stimulated RAW264.7 cells were observed. Methods: RAW264.7 cells were cultured and stimulated with LPS in the absence or presence of CCK-8 and/or SB203580, a specific inhibitor of p38 MAPK. The DNA binding activity of AP-1 was analyzed by means of electrophoretic mobility shift assay (EMSA). Data were presented as x ±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) The DNA binding activity of AP-1 was significantly higher in RAW264.7 cells stimulated with 1mg/L LPS in comparison with unstimulated cells (P<0.01 vs control group). 10-10 mol/L CCK-8 had no obvious effect on the LPS-induced AP-1 activity. 10-8 and 10-6 mol/L CCK-8 could inhibit the LPS-induced AP-1 activity by 43.9% and 67.2% respectively in a dose-dependent manner (P<0.01 vs LPS group). CCK-8 alone had no effect on the DNA binding activity of AP-1 (P>0.05 vs control group). (2) p38 MAPK specific inhibitor SB203580 (10μmol/L) inhibited the LPS-induced AP-1 activity by 51.7% (P<0.01 vs LPS group); 10μmol/L SB203580, 10-8 mol/L CCK-8 co-incubation (LPS+SB203580+CCK-8 group) further inhibited the DNA binding activity of AP-1 in LPS-induced RAW264.7 cells. The inhibiting rates were 69.8%, 46.1% and 37.4% respectively compared with LPS group, LPS+CCK-8 10-8 mol/L group and LPS+SB203580 group. 10μmol/L SB203580 alone had no effect on the AP-1 activity (P>0.05 vs control group). The binding specificity was confirmed by using homologous (AP-1) and nonhomologous (AP-2) oligonucleotides as competitors. Conclusion: These results showed that CCK-8 inhibited AP-1 activity in LPS-induced RAW264.7 cells, which might be the upstream regulatory mechanism of the inhibitory effects of CCK-8 on LPS-induced IL-1βand IL-6 expressions. p38 MAPK might be one of the upstream regulation molecules ofAP-1 and besides p38 MAPK, other signal pathways might participated in the regulation of CCK-8 on LPS-induced AP-1 activity. 4 Effects of CCK-8 on p38 MAPK activity in LPS-stimulated RAW264.7 cells and the relations between IL-1β, IL-6, IL-10 mRNA expressions and p38 MAPK activity 4.1 Effect of CCK-8 on p38 MAPK activity in LPS-stimulated RAW264.7 cells Objective: It is well known that the MAPK pathway is the important signal system which mediates cell response. The p38 MAPK is one of the important members of MAPKs which mainly regulates the inflammatory response and produces cytokines. This pathway is activated in many clinical conditions such as sepsis, ARDS, severe burns and acute pancretitis. Many studies have demonstrated that blocking p38 MAPK cascades can alleviate the inflammatory response. So in the present study, effect of CCK-8 on p38 MAPK activity in LPS-stimulated RAW264.7 cells were observed in order to elucidate the anti-inflammatory mechanism of CCK-8. Methods: RAW264.7 cells were cultured and p38 MAPK activity was analyzed by Western blot 30 min after stimulation. Data were presented as x ±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) In RAW264.7 cells, the activity of p38 MAPK (the phosphorylated p38 MAPK) was induced by LPS in a time-dependent manner, and reached the peak after being stimulated with LPS for 30 min, then reduced gradually. The p38 MAPK activity in RAW264.7 cells stimulated by LPS for 5 min, 15 min, 30 min, 45min, 60 min and 120 min increased 2.86 times, 6.37 times, 8.96 times, 4.56 times, 3.63 times and 1.73 times in comparison with LPS-stimulation for 0 min (P<0.01). The amount of non-phosphorylated forms of p38 MAPK had not obvious changed. (2) CCK-8 inhibited the activity of p38 MAPK in a dose-dependent manner in LPS-stimulated RAW264.7 cells. 10-10 mol/L CCK-8 had no effect on the activity of p38 MAPK (P>0.05 vs LPS group, P<0.01 vs control group). 10-8, 10-6 mol/L CCK-8 inhibited theLPS-induced p38 MAPK activity significantly by 44.1% (P<0.01 vs LPS group, P<0.01 vs control group) and 71.9% (P<0.01 vs LPS group, P<0.05 vs control group), but the activity of p38 MAPK was still higher than that of the control group. 10-8 mol/L CCK-8 alone had no effect on the activity of p38 MAPK (P>0.05 vs control group). The amount of non-phosphorylated forms of p38 MAPK had not obvious changed. Conclusion: These results indicated that CCK-8 inhibited the activity of p38 MAPK in LPS-stimulated RAW264.7 cells in a dose-dependent manner, which might be one of the signal transduction mechanisms of CCK-8 on anti-inflammatory response. 4.2 Effects of p38 MAPK on CCK-8 regulation the IL-1β, IL-6 and IL-10 mRNA expressions in LPS-stimulated RAW264.7 cells Objective: It is well known that p38 MAPK cascade is one of the important signal pathways that mediates the producing of inflammatory mediators. p38 MAPK is phosphorylated and activated, leading downstream transcription factors AP-1 to translocate into the nucleus, and initiating the related gene transcription, such as the expressions of cytokines IL-1β, IL-6 and IL-10. We have demonstrated that CCK-8 inhibited the activity of p38 MAPK in LPS-stimulated RAW264.7 cells in a dose-dependent manner. So in the present study, effects of p38 MAPK on CCK-8 regulation the IL-1β, IL-6 and IL-10 mRNA expressions in LPS-stimulated RAW264.7 cells were observed in order to elucidate the anti-inflammatory mechanism of CCK-8. Methods: RAW264.7 cells were cultured and stimulated with LPS in the absence or presence of CCK-8 and/or SB203580, a specific inhibitor of p38 MAPK for different times and the mRNA expressions of IL-1β, IL-6 and IL-10 was analyzed by RT-PCR. Data were presented as x ±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) SB203580 inhibited the activity of p38 MAPK in LPS-stimulated RAW264.7 cells in a dose-dependent manner. Compared with the LPS group, 1μmol, 5μmol/L, 10μmol/L and 20μmol/L SB203580 couldinhibit the LPS-induced p38 MAPK activity by 7.8% (P>0.05 vs LPS group), 61.7%, 85.4% and 87.2%,respectively (P<0.01 vs LPS group). However, increasing concentration of SB203580 to 20μmol/L could not further improve the inhibiting effect of SB203580 compared with the LPS+SB 10μmol/L group ( P>0.05). The amount of non-phosphorylated forms of p38 MAPK had not obvious changed. (2) 10μmol/L SB203580 significantly inhibited IL-1β, IL-6 mRNA expressions in LPS-induced RAW264.7 cells by 56.7% and 34.3% respectively(P<0.01 vs LPS group); 10μmol/L SB203580 and CCK-8 co-incubition further inhibted LPS-induced IL-1β, IL-6 mRNA expressions by 78.4% and 67.2%, but the mRNA expressions were still higher than those of the control group (P<0.01 vs LPS, LPS+SB, LPS+CCK-8 and control group). 10μmol/L SB203580 significantly inhibited the IL-10 mRNA expression in LPS-induced RAW264.7 cells by 53.7%, while partially inhibited the effect of CCK-8 on augmenting the LPS-induced IL-10 mRNA expression with the inhibiting rate just about 19.1% (P<0.01 vs LPS+CCK-8, LPS, LPS+SB and control group). Conclusion: These results indicated that CCK-8 inhibited IL-1β, IL-6 mRNA expressions via inhibiting the activity of p38 MAPK and other pathways outside of p38 MAPK, and CCK-8 augmented the LPS-induced IL-10 mRNA expression mainly via other pathways outside of p38 MAPK to participate in the anti-inflammatory response. 5 Effects of cAMP-PKA and PKC signal pathway on CCK-8 augmenting the LPS-induced IL-10 mRNA expression Objective: The regulation mechanism of IL-10 was complicated, involving many signal pathways, transcription factors and regulatory mechanisms after transcription. We have demonstrated that CCK-8 inhibited IL-1βand IL-6 expressions in LPS-induced RAW264.7 cells via the p38 MAPK-AP-1 signal pathway and LPS induced IL-10 expression via this pathway, but this pathway was not mainly mediated the effect of CCK-8 on augmenting the LPS-induced IL-10 mRNA expression. Some studies indicated that under nonpathological conditions or stimulation by LPS, p38 MAPKactivation was critical for IL-10 production, while the signal transduction cascade for the induction of IL-10 expression was altered following the conditions such as thermal injury and other stimulation. The promoter in IL-10 gene contains binding motifs for activate protein-1 (AP-1) and cAMP response element binding protein (CREB), the former one is regulated by PKC signal pathway while the later one is regulated by the PKA signal pathway. So in the present study, effects of cAMP-PKA and PKC signal pathway on CCK-8 augmenting the LPS-induced IL-10 mRNA expression were observed in order to elucidate the signal mechanism of CCK-8 on augmenting the LPS-induced IL-10 mRNA expression. Methods: RAW264.7 cells were cultured and stimulated with LPS in the absence or presence of CCK-8 and/or cAMP activator Forskolin (FsK), PKA selective inhibitor H89 and PKC inhibitor GF109203X and the mRNA expression of IL-10 was analyzed by RT-PCR. Data were presented as x ±s and analyzed with ANOVA and LSD using SPSS statistical program. A level of P<0.05 was considered statistically significant. Results: (1) PKA selective inhibitor H-89, partially inhibited the LPS-induced IL-10 mRNA expression in RAW264.7 cells (The inhibiting rate was 18.6%, P<0.01 vs LPS group) and completely conversed the effect of CCK-8 on augmenting the LPS-induced IL-10 mRNA expression (P>0.05 vs LPS group, P<0.01 vs LPS+CCK-8 group). H-89 alone had no effect on IL-10 mRNA expression (P>0.05 vs control group). FsK increased the expression of IL-10 mRNA in RAW264.7 cells, FsK and LPS co-incubation further increased LPS-induced IL-10 mRNA expression by 1 times (P<0.01 vs LPS group). FsK, CCK-8 and LPS co-incubition further increased LPS-induced IL-10 mRNA expression, increasing 1.56 times, 0.21 times and 0.26 times compared with LPS group, LPS+CCK-8 group and LPS+FsK group (P<0.01). (2) PKC inhibitor GF109203X, partially inhibited the LPS-induced IL-10 mRNA expression in RAW264.7 cells (The inhibiting rate was 14.1%, P<0.05 vs LPS group) and had no effect on CCK-8 augmenting the LPS-induced IL-10 mRNA expression (P>0.05 vs LPS+CCK-8 group, P<0.01 vs LPSgroup). GF109203X alone had no effect on IL-10 mRNA expression (P>0.05 vs control group). Conclusion: These results indicated that the effect of CCK-8 on augmenting the LPS-induced IL-10 mRNA expression in RAW264.7 cells was mediated by cAMP-PKA-dependent and PKC-independent pathways. CONCLUSIONS In the present study, we first systematically investigated the modulatory effects of CCK-8 on LPS-activated RAW264.7 cells and endotoxemia mice at the levels from protein kinases and transcription factors to the gene expressions of pro-and anti-inflammatory cytokines. We found that CCK-8 had direct anti-inflammatory effects via inhibiting the expressions of proinflammatory cytokines and at the same time, increasing the expressions of anti-inflammatory cytokines in inflammatory response. 1 CCK-8 could inhibit the expressions of proinflammatory cytokines IL-1β, IL-6 and increase the expressions of anti-inflammatory cytokines IL-4, IL-10 in the serum and lung tissues of endotoxemia mice and reduce the mortality rate of endotoxemia mice significantly. 2 CCK-8 could inhibit the expressions of proinflammatory cytokines IL-1β, IL-6 and augment the expression of anti-inflammatory cytokine IL-10 in LPS-stimulated RAW264.7 cells in a dose-dependent manner. 3 CCK-8 could inhibit DNA binding activity of AP-1 in LPS-induced RAW264.7 cells, which might be one of the upstream mechanisms of the inhibitory effects of CCK-8 on LPS-induced proinflammatory cytokines IL-1βand IL-6 expressions. 4 CCK-8 could inhibit the activity of p38 MAPK in LPS-induced RAW264.7 cells in a dose-dependent manner. CCK-8 inhibited IL-1β, IL-6 mRNA expressions via inhibiting the activity of p38 MAPK and other pathways outside of p38 MAPK, and CCK-8 might augment the LPS-induced IL-10 mRNA expression mainly via other pathways outside of p38 MAPK.
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