Abdominal paracentesis drainage ameliorates severe acute pancreatitis in rats by regulating the polarization of peritoneal macrophages

AIM To investigate the role of peritoneal macrophage(PM) polarization in the therapeutic effect of abdominal paracentesis drainage(APD) on severe acute pancreatitis(SAP).METHODS SAP was induced by 5% Na-taurocholate retrograde injection in Sprague-Dawley rats. APD was performed by inserting a drainage tube with a vacuum ball into the lower right abdomen of the rats immediately after the induction of SAP. To verify the effect of APD on macrophages, PMs were isolated and cultured in an environment, with the peritoneal inflammatory environment simulated by the addition of peritoneal lavage in complete RPMI 1640 medium. Hematoxylin and eosin staining was performed. The levels of pancreatitis biomarkers amylase and lipase as well as the levels of inflammatory mediators in the blood and peritoneal lavage were determined. The polarization phenotypes of the PMs were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry, qPCR and immunohistochemical staining. The protein expression in macrophages that had infiltrated the pancreas was determined by Western blot.RESULTS APD treatment significantly reduced the histopathological scores and levels of amylase, lipase, tumor necrosis factor-α and interleukin(IL)-1β, indicating that APD ameliorates the severity of SAP. Importantly, we found that APD treatment polarized PMs towards the M2 phenotype, as evidenced by the reduced number of M1 macrophages and the reduced levels of proinflammatory mediators, such as IL-1β and L-selectin, as well as the increased number of M2 macrophages and increased levels of anti-inflammatory mediators, such as IL-4 and IL-10. Furthermore, in an in vitro study wherein peritoneal lavage from the APD group was added to the cultured PMs to simulate the peritoneal inflammatory environment, PMs also exhibited a dominant M2 phenotype, resulting in a significantly lower level of inflammation. Finally, APD treatment increased the proportion of M2 macrophages and upregulated the expression of the anti-inflammatory protein Arg-1 in the pancreas of SAP model rats.CONCLUSION These findings suggest that APD treatment exerts antiinflammatory effects by regulating the M2 polarization of PMs, providing novel insights into the mechanism underlying its therapeutic effect.

Hsp70 confines tumor progression of rat histiocytoma and impedes the cytotoxicity induced by natural killer cells and peritoneal macrophages

Objective:To study the role of inducible form of heat shock protein 70(Hsp70) in the host tumor regression of rat tumor model.Methods:We examined the role of Hsp70 in host tumorigenicity and in vitro cellular cytotoxicity using a rat histocytoma.The differential tumor growth and regression kinetics were studied and correlated with the expression of Hsp70,activation of macrophages and natural killer(NK) cells,and circulating or tumor infiltrating immune molecules in the host system.Results:The sub cuteaneous(s.c.) tumor regression was correlated with increased serum cytokines such as IL-12,TNF P,IFNγand Hsp70.Despite of similar increase of Hsp70 in intraperitoneal(i.p.) tumor implanted animals,animals succumb to tumor growth,further,evidently,no immune molecule activation was observed.The viral promoter driven Hsp70 over expression in these tumor cells restrained solid tumor growth,however,failed to inhibit ascites growth.The NK cells from s.c.immunized animals induces cytotoxicity in the presence of anti-tumor antibody,which necessitated CD40-L expression,conversely,NK cells from i.p.immunized animals failed to induce cytotoxicity.The NK cells from s.c.or i.p.implanted animals with Hsp70 positive tumor cells failed to induce such cytotoxicity.The peritoneal macrophages isolated from s.c.tumor implanted animals when co-cultured with parental BC-8 cells lyses tumor cells,nevertheless entail macrophage specific TNFαexpression.On the contrary,Hsp70 expressing BC-8 tumor cells were resistant to peritoneal macrophage induced cytolysis.Conclusions:This study brings out that Hsp70 possibly involved in regulating the host tumor response and cellular cytotoxicity.

Recent insights into the characteristics and role of peritoneal macrophages from ascites of cirrhotic patients

Macrophages are a diverse myeloid cell population involved in innate and adaptive immune responses,embryonic development,wound repair,and regulation of tissue homeostasis.These cells link the innate and adaptive immunities and are crucial in the development and sustainment of various inflammatory diseases.Macrophages are tissue-resident cells in steady-state conditions;however,they are also recruited from blood monocytes after local pathogen invasion or tissue injury.Peritoneal macrophages vary based on their cell complexity,phenotype,and functional capabilities.These cells regulate inflammation and control bacterial infections in the ascites of decompensated cirrhotic patients.Our recent work reported several phenotypic and functional characteristics of these cells under both healthy and pathological conditions.A direct association between cell size,CD14/CD16 expression,intracellular level of GATA-6,and expression of CD206 and HLA-DR activation/maturation markers,indicate that the large peritoneal macrophage CD14^(high)CD16^(high)subset constitutes the mature phenotype of human resident peritoneal macrophages during homeostasis.Moreover,elevated expression of CD14/CD16 is related to the phagocytic capacity.The novel large CD14^(high)CD16^(high)peritoneal subpopulation is increased in the ascites of cirrhotic patients and is highly sensitive to lipopolysaccharide(LPS)-induced activation,thereby exhibiting features of inflammatory priming.Thus,phosphorylation of ERK1/2,PKB/Akt,and c-Jun is remarkably increased in response to LPS in vitro,whereas that of p38 MAPK is reduced compared with the monocyte-derived macrophages from the blood of healthy controls.Furthermore,in vitro activated monocyte-derived macrophages from ascites of cirrhotic patients secreted significantly higher levels of IL-6,IL-10,and TNF-αand lower amounts of IL-1βand IL-12 than the corresponding cells from healthy donor’s blood.Based on these results,other authors have recently reported that the surface expression level of CD206 can be used to identify mature,resident,inflammatory peritoneal macrophages in patients with cirrhosis.Soluble CD206 is released from activated large peritoneal macrophages,and increased concentrations in patients with cirrhosis and spontaneous bacterial peritonitis(SBP)indicate reduced odds of survival for 90 d.Hence,the level of soluble CD206 in ascites might be used to identify patients with SBP at risk of death.In conclusion,peritoneal macrophages present in ascites of cirrhotic patients display multiple phenotypic modifications characterized by reduced ratio of cells expressing several membrane markers,together with an increase in the ratios of complex and intermediate subpopulations and a decrease in the classiclike subset.These modifications may lead to the identification of novel pharmaceutical targets for prevention and treatment of hepatic damage.

Influence of whole peptidoglycan of bifidobacterium on cytotoxic effectors produced by mouse peritoneal macrophages

INTRODUCTIONBifidobacteria are physiologically beneficial bacteria which are perdominant in human intestine ,and possess the most important functions .They play an important role in maintaining microbial balance of the intestine .Furthermore , their presence is thought to be an important indication of health of the body [1-4].Whole peptidoglycan ( WPG) is the major component in the cell wall of bifidobacterium ,which is also a biological responsemodifier with nontoxic side dffcets.

THE EFFECTS OF RADIX SALVIAE MILTIORRHIZAE ON LIPID ACCUMULATION OF PEROXIDIZED LOW DENSITY LIPOPROTEIN IN MOUSE PERITONEAL MACROPHAGES ?LIPID ANALYSIS AND MORPHOLOGICAL STUDIES

Mouse peritoneal macrophages were incubated in DMEM with pox-LDL and Rradlx Salviae Miltiorrhizae (RSM) to investigate the effects of RSM on the internalization of peroxidized low density lipoprotein (pox-LDL) by using lipid analysis and electron microscopy. Lipid peroxide (LPO) concentrations were increased slightly in the medium after incubation of macrophages with normal LDL (n-LDL), while decreased significantly in the media after incubation of macrophages with pox-LDL. In the three groups with pox-LDL, it could be found that there was a dose-dependent decrease of concentrations of LPO and total cholesterol (TCH) in the two RSM groups, and the decrease in the two RSM groups was much greater than in the group without RSM. RSM accelerated a more decrease of LPO than cholesterol contents in the media containing pox-LDL. The ultrastructural studies also showed that RSM induced the accumulation of lipid droplets in the cytoplasm of mouse peritoneal macrophages. The results suggested that RSM could accelerate the phagocytosis and degradation of pox-LDL by macrophages.

Studies on the Electron Microscopic Localization of Con A and WGA Receptors in Human Peritoneal Macrophages

The electron microscopic topology of Con A and WGA receptors was investi-gated in human peritoneal macrophagcs by utilizing avidin-gold colloids.Our observa-tions confirmed that both the receptors were distributed on the cell membranes,yet thereceptor expression showed variations among the individual cells.Some cells had abun-dant receptors,while others had only few receptors.The data obtained in the presentstudy indicate that human peritoneal macrophages may be a functionally andbiochemically heterogenous population,and the study may also serve as a theorcticalreference for future clinical exploitation of human peritoneal macrophages.

Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophages

Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40μmol/ L) inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0. 5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.

Lipopolysaccharide enhances the inhibition of NF-κB expression in NNK-mediated peritoneal macrophages

Objective： The aim of the study was to investigate the effect of lipopolysaccharide （LPS） on the expression of nuclear factor kappa B （NF-κB） in 4-（methylitrosamino）-1-（3-pyridyl）-1-butanone （NNK）-mediated primary mouse peritoneal macrophages in vitro. Methods： The activity of peritoneal rnacrophages treated with different concentrations of LPS was detected by MTT assay in rider to find the optimal concentration. Peritoneal macrophages were also treated with NNK （100-500 μM）, with or without LPS for 9 h. The expression of NF-κB was demonstrated via immunocytochemistry （ICC） and Western- blot, respectively. Results： The concentration of LPS at 25 μg/mL was found to be the optimal concentration to improve the activity of peritoneal macrophages （P 〈 0.01）. Simultaneously, LPS （25 μg/mL） increased the expression of NF-κB in both the nucleus and cytoplasm and facilitated transfer of NF-κB to the nucleus. NNK treatment significantly inhibited the expression of NF-κB in a concentration-dependent manner, among the LPS-stimulated or unstimulated peritoneal macrophages, especially when cotreated with LPS （25 μg/mL, P 〈 0.01 ）. Furthermore, NNK treatment （500 μM） with LPS yielded a significant decrease in NF-κB translocation to nucleus and inhibited the expression of NF-κB （P 〈 0.005）. Conclusion： LPS enhances the suppression of NF-κB expression in NNK-mediated mouse peritoneal macrophages, which may provide a theoretical basis for the inhibition of cancer.

Aqueous extract of Ocimum gratissimum Linn and ascorbic acid ameliorate nicotine-induced cellular damage in murine peritoneal macrophage

Objective:To test the in vitro protective role of aqueous extract of Ocimum gratissimum Linn. (0.gratissimum) and ascorbic acid against nicotine-induced murine peritoneal macrophage. Methods:Peritoneal macrophages from mice were treated with nicotine(10 mM),nicotine (10 mM) with aqueous extract of O.gratissimum(1 to 25μg/mL),and nicotine(10 mM) with ascorbic acid(0.01 mM) for 12 h in cell culture media,while the control group was treated with culture media.Levels of free radical generation,lipid peroxidation,protein carbonyls,oxidized glutathione levels and DNA damage were observed and compared.Results:Phytochemical analysis of aqueous extract has shown high amount of phenolics and flavonoids compound present in it.The significantly increased free radical generation,lipid peroxidation,protein carbonyls,oxidized glutathione levels and DNA damage were observed in nicotine-treated group as compared to the control group:those were significantly reduced in aqueous extract of O. gratissimum and ascorbic acid supplemented groups.Moreover,significantly reduced antioxidant status in nicotine exposed murine peritoneal macrophage was effectively ameliorated by these two products.Among the different concentration of aqueous extract of O.gratissimum,the maximum protective effect was observed at 10μg/mL which does not produce any significant change in the normal cell.Conclusions:These findings suggest the potential use and beneficial role of O.gratissimum as a modulator of nicotine-induced cellular damage in murine peritoneal macrophage.

THE EFFECT OF DIALYSIS ON THE RELEASE OF TNF-α FROM PERITONEAL MACROPHAGE IN VIVO

Objective To investigate the consistent inhibitory effect olperitoneal dialysate on the capacity ofperitoneal macrophage release of TNF- α and the inhibition associated with different glucose concentration invivo. Methods We compared the peritoneal macrophage activity obtained from the irrigation solution of theuremia patients (n=2) immediately after inserting the catheter and with those from the long - dwell time dialysate(≥10h) of peritoneal dialysis patients (n= 7). The capacity of peritoneal macrophage to release TNF - x was assayedby L929 cytotoxicity. At the same time, we compared TNF- 7 activity among different glucose concentration (1.5%,2.5% and 4.25% dextrose) of dialysate. Results The levels of TNF- a in CAPD and control group were 170 and517 pg/106 respectively (P<0.01). The patients who were using dialysate containing higher glucose concentrationhad lower TNF- x level (P<0.05, P<0.01). Conclusion It is suggested that the inhibitory effect of peritonealdialysate on peritoneal macrophages might continue at least 10h in vivo. The higher glucose concentration ofdialysate may have worse effect on peritoneal macrophage activity.

Inhibiting Effect of Lobenzarit Disodium on the Inter-leukin-1 Activity in Normal and in Adjuvant ArthritisKats and on the H_2O_2 Release from Rat Peritoneal Macro-phages

The effects of Lobenzarit disodium (CCA) upon the activity of interleukin-1(IL-1) of peritoneal macrophages(PMΦ)in normal and in adjuvant arthritis AA) rats and on the release of hydrogen peroxide(H_2O_2)of rat peritoneal macrophages were studied. CCA 10～200 μg/ml could inhibit IL-1activity in normal rat in vitro;CCA 10 and 50 mg/kg could depress the level of increased IL-1 in AA rats in vivo;CCA 5～100μg/ml could inhibit the release of H_2O_2 from rat PMΦin vitro.The results suggest that these effects could be one of the mechanisms of anti-inflammatory action of CCA.

Essential role of monocytes and macrophages in the progression of acute pancreatitis

Acute pancreatitis(AP) is an inflammatory condition of the pancreas caused by an imbalance in factors involved in maintaining cellular homeostasis.Earliest events in AP occur within acinar cells accompanied by other principal contributors to the inflammatory response i.e.the endothelial cells,immunocytes(granulocytes,monocytes/macrophages,lymphocytes) and neutrophils.Monocytes/macrophages are important inflammatory mediators,involved in the pathophysiology of AP,known to reside in the peritoneal cavity(in the vicinity of the pancreas) and in peripancreatic tissue.Recent studies suggested that impaired clearance of injured acini by macrophages is associated with an altered cytokine reaction which may constitute a basis for progression of AP.This review focuses on the role of monocytes/macrophages in progression of AP and discusses f indings on the inflammatory process involved.

Alteration of some cellular function in amikacin resistant Pseudomonas aeruginosa transfected macrophages:a time dependent approach

Objective:To evaluate the free radical generation and antioxidant enzymes status in murine peritoneal macrophage during in vitro amikacin resistant Pseudomonas aeruginosa(ARPA) treatment with different time interval.Methods:Peritoneal macrophages were treated with 1× 10~2 CFU/mL ARPA cell suspension in vitro for different time interval(1,2,3,6,12,and 24 h) and super oxide anion generation,NO generation,reduced glutathione level and antioxidant enzymes status were analyzed.Results:Super oxide anion generation and NO generation got peak at 12 h,indicating maximal free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during ARPA transfection.Reduced glutathione level and antioxidant enzymes status were decreased significantly(P【0.05) with increasing time of ARPA transfection. All the changes in peritoneal macrophages after 12 h in vitro ARPA transfection had significant difference(P【0.05).Conclusions:From this study,it may be summarized that in vitro ARPA infection not only generates excess free radical but also affects the antioxidant system and glutathione cycle in murine peritoneal macrophage.

Mechanism of Elevated Vascular Endothelial Growth Factor Levels in Peritoneal Fluids from Patients with Endometriosis

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.

JNK/CCl2通路诱导巨噬细胞募集并促进PM(2.5)颗粒物暴露诱导的幼年大鼠过敏性气道炎症

目的:基于JNK/CCl2信号通路诱导的巨噬细胞募集探讨PM(2.5)暴露对幼年大鼠气道炎症的作用及其机制。方法:幼年SD大鼠50只,随机分为5组(n=10),其中对照组不进行任何处理,PM(2.5)组幼年大鼠接受PM(2.5)颗粒物暴露,PM(2.5)+茴香霉素组幼年大鼠接受PM(2.5)暴露以及静脉注射JNK的激活剂茴香霉素,PM(2.5)+SP600125组幼年大鼠接受PM(2.5)暴露以及静脉注射JNK拮抗剂SP600125,PM(2.5)+吡非尼酮组幼年大鼠接受PM(2.5)暴露以及静脉注射CCl2拮抗剂吡非尼酮。安乐死幼年大鼠取肺组织,Western blot法检测JNK、磷酸化的JNK(p-JNK)和CCl2的蛋白表达水平变化,用苏木精-伊红(HE)染色检测肺部气道组织的病理变化并进行肺支气管炎症评分。流式细胞术分析肺泡灌洗液中巨噬细胞含量的变化。ELISA检测各组幼年大鼠的肺泡灌洗液中促炎因子IL-6、IL-1β、TNF-α水平。结果:各组间JNK、p-JNK、CCl2的表达水平(F=205.296、950.408、260.019;均P<0.001),巨噬细胞含量(F=48.414;P<0.001),肺支气管炎症评分(F=101.703;P<0.001)以及IL-6(H=44.890;P<0.001)、IL-1β(H=42.071;P<0.001)、TNF-α(F=297.154;P<0.001)的水平差异均有统计学意义。与对照组相比,PM(2.5)组JNK/CCl2通路蛋白JNK、p-JNK、CCl2的表达上调(均P<0.05),同时巨噬细胞含量增加(P<0.05),肺支气管炎症评分升高(P<0.05),IL-6、IL-1β、TNF-α水平上调(均P<0.05)。与PM(2.5)组相比,PM(2.5)+茴香霉素组中巨噬细胞含量均上调(P<0.05),肺支气管炎症评分升高(P<0.05),另外IL-6、IL-1β、TNF-α的水平升高(均P<0.05),且JNK、p-JNK、CCl2的表达水平升高(均P<0.05)。与PM(2.5)组相比,PM(2.5)+SP600125组、PM(2.5)+吡非尼酮组的巨噬细胞含量均下调(P<0.05),且肺支气管炎症评分降低(P<0.05),另外IL-6、IL-1β、TNF-α的水平均下调(均P<0.05)。与PM(2.5)组相比,PM(2.5)+SP600125组的JNK、p-JNK、CCl2的表达水平下调(均P<0.05),PM(2.5)+吡非尼酮组的CCl2的表达水平下调(均P<0.05)。结论:JNK/CCl2通路诱导巨噬细胞募集促进PM(2.5)颗粒物暴露诱导的幼年大鼠过敏性气道炎症。

PM2.5暴露对鸦胆子油乳治疗荷瘤肺癌小鼠腹腔巨噬细胞的作用

目的探讨PM2.5暴露对鸦胆子油乳治疗荷瘤肺癌小鼠腹腔巨噬细胞的活性调节作用。方法建立肺癌荷瘤A549细胞小鼠模型32只,随机分为4组:治疗组1(鸦胆子油乳治疗组)和对照组1,治疗组2(PM2.5暴露联合鸦胆子油乳治疗组)和对照组2,每组8只。收集腹腔巨噬细胞,检测520 nm吸光度值。结果腹腔巨噬细胞吞噬能力显示,治疗组1高于对照组1,差异有统计学意义(P<0.05);治疗组2低于对照组2及治疗组1高于治疗组2,差异有统计学意义(P<0.01)。结论 PM2.5暴露对鸦胆子油乳治疗荷瘤肺癌小鼠腹腔巨噬细胞的活性有抑制作用。

Effects of shenmai injection on expression of TNF-α mRNA in peritoneal macrophages of scald mice

Objective To explore the effect of shenmai injection (SI) on expression of TNF-α mRNA in peritoneal macrophages (pMΦs) of scald mice.Methods BALB/c mice were inflicted with 11% of body surface area Ⅲ degree scald and injected intraperitoneally (ip) with SI daily for 5 days, and expression of TNF-α mRNA in pMΦs was determined by semi-quantitative RT-PCR.Results In scald mice, the expression of TNF-α mRNA in pMΦs increased significantly, but it was reduced obviously (P<0.01) after SI administration, while the livability was increased markedly (P<0.05).Conclusions For scald mice, the cause of death at early stage might be related to the high expression of TNF-α mRNA in pMΦs and the use of SI can decrease the death rate.

PM2.5暴露对荷瘤肺癌小鼠免疫功能影响研究

目的:通过对肺癌小鼠腹腔巨噬细胞的活性和5种细胞因子表达量研究,探讨细颗粒物(Particulate Matter 2.5,PM2.5)的作用机制。方法:尾静脉注射法建立肺癌荷瘤小鼠模型,30只随机分为3组:荷瘤对照组、PM2.5暴露组(气管滴注法暴露PM2.5)、NS暴露组(滴注等量生理盐水)。中性红比色法检测PM2.5对肺癌小鼠腹腔巨噬细胞活性的影响;ELISA法检测血清和腹腔巨噬细胞培养液上清中IL-2、IL-10、TNF-α、IFN-γ和MIF 5种细胞因子的表达情况。结果:腹腔巨噬细胞吞噬能力实验结果显示,PM2.5暴露组和NS暴露组分别与荷瘤对照组比较,吞噬活性显著提高(P<0.05),PM2.5暴露组明显高于NS暴露组(P<0.05)。以上3组吞噬率间两两比较均具有统计学差异(P<0.05)。ELISA结果显示,血清和巨噬细胞细胞培养液上清中IL-2和MIF的表达量显著降低,IL-10表达量显著升高(P<0.05)。而TNF-α在血清中的表达量显著降低,但在细胞培养液上清中的表达量显著增高(P<0.05)。结论:空气细颗粒物暴露对荷瘤肺癌小鼠腹腔巨噬细胞的活性虽然具有上调作用,但同时损伤了巨噬细胞和机体的非特异性免疫和特异性免疫功能。

Inhibition of Emodin on LPS-induced Nitric Oxide Generation by Suppressing PLC-γ Phosphorylation in Rat Peritoneal Macrophages

Objective To investigate the inhibitory mechanism of emodin on lipopolysaccharide (LPS)-induced nitric oxide (NO) generation in rat peritoneal macrophages. Methods NO production and iNOS expression were measured through nitrite assay and Western blotting assay, respectively. NF-κB activity and nuclei P65 expression were estimated by dual-luciferase and Western blotting assay, respectively. Intracellular free Ca2+ ([Ca2+]i) was detected using the ratiometric fluorescent calcium indicator dye, Fura-2, and a microspectrofluorometer. PLC-γ phosporylation was analyzed by Western blotting assay. Results First, emodin was found playing active roles in suppressing LPS-induced NF-κB activation in rat peritoneal macrophages. Second, emodin down-regulated transient [Ca2+]i and could increase in NF-κB upstream signal. Finally, emodin suppressed phosphorylation of PLC-γ by LPS stimulation in the upstream of [Ca2+]i. Conclusion Suppression of PLC-γ phosphorylation is involved in emodin inhibiting NO generation by LPS stimulation in rat peritoneal macrophages.

Comparison of the characteristics of macrophages derived from murine spleen, peritoneal cavity, and bone marrow

Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity（BMs, SPMs, and PCMs, respectively） were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex（MHC） Ⅱ and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, mannose receptor（MR）, and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction（PCR） analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.