Macrophage Inflammatory Protein-2 mRNA Research Articles

Diabetes-Mediated Toxicity Resulted in the Expression of CD80 and CD86 on Neutrophils after Delayed Wound Healing in Male Rats.

Background Polymorphonuclear neutrophils (PMNs) play an essential role in the innate immune response, and their number increases after prolonged inflammatory diabetic wounds and prolonged wounds in older rats. The expression of CD80 and CD86 on PMNs confirms their participation in acquired immunity, wherein these molecules are involved in antigen presentation. Materials and Methods We investigated CD80 and CD86 expression on PMNs by flow cytometry and analyzed the mRNA expression of neutrophil chemoattractants macrophage inflammatory protein-2 (MIP-2) and MIP-1α by real-time polymerase chain reaction (PCR) in diabetic wound, which was healed by a camel milk peptide (CMP). The animals were allocated to the following wounded groups: control, diabetic (DM), and diabetic treated with CMP (DM-CMP). Results Alkaline phosphatase, gamma-glutamyl transpeptidase, and lactate dehydrogenase levels were elevated in DM rats but decreased in peptide-treated rats. The expression of CD80 and CD86 was significantly higher in DM rats with prolonged wounds than in control rats. The expression of both markers was restored to normal levels in diabetic rats treated with CMP. RT-PCR analysis revealed the upregulation in MIP-2 mRNA expression in DM rats. However, neutrophil number at wounded sites of DM rats declined at day 1 after wounding as compared to that in control rats. MIP-2 mRNA expression and neutrophil number were restored in CMP-treated diabetic rats. Conclusion Prolonged wound stress induced toxicity in DM rats and significantly increased the expression of CD80 and CD86 on PMNs. CMP peptide ameliorated the levels of toxicity markers, CD80 and CD86, and chemoattractant molecules in diabetic rats.

Protective effect and mechanism of alpha-lipoic acid on partial hepatic ischemia-reperfusion injury in adult male rats.

In order to reduce tissue damage caused by ischemia-reperfusion injury, this study aims to investigate the protective effect and mechanism of ?-lipoic acid on hepatic ischemia-reperfusion injury in rats. The bloodstream of rats was blocked in the left middle and left lateral liver lobes of the liver. Forty rats were randomly divided into two groups: treatment group and injury group. Rats were injected with either 25 mg/1 ml of alpha-lipoic acid (treatment group) or 1 ml of saline (injury group) into the caudal vein 15 min before hepatic ischemia-reperfusion. Rat serum alanine aminotransferase (GPT), glutathione (GSH) and superoxide dismutase (SOD) levels were examined at various time points (1, 3, 6 and 12 h) in both groups. Changes in nuclear factor kappa B P65 (NF-kappaB P65) expression in ischemia-reperfusion liver at various time points after reperfusion (1, 3, 6 and 12 h) were evaluated through immunohistochemistry assay. Changes in macrophage inflammatory protein-2 (MIP-2) mRNA and inducible nitric oxide synthase (iNOS) mRNA expression in ischemic reperfused rat livers were detected by RT-PCR. Serum GPT level was significantly higher in the injury group than in the treatment group (P<0.01). NF-kappaB P65, MIP-2 mRNA and iNOS mRNA expression in ischemic reperfused rat livers were significantly higher in the injury group than in the treatment group (P<0.01). Serum GSH and SOD levels were higher in the treatment group than in the injury group (P<0.01). Alpha-lipoic acid significantly reduced ischemia-reperfusion injury in rat livers. This may be associated to the direct scavenging of oxygen-free radicals, increased GSH production, and the activation of downstream media due to decreased NF-kappaB and GSH consumption.

Effect of resveratrol on the expression of MIP-2 in P.e-LPS-induced mouse osteoblasts

To investigate the effect of lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) on the expression of macrophage inflammatory protein-2 (MIP-2) mRNA and protein levels in MC3T3-E1 cells and the influence of resveratrol on the expression of MIP-2 protein in P.e-LPS induced cells. MC3T3-E1 cells were treated with different concentrations of P.e-LPS(0-50 mg/L) and 20 mg/L P.e-LPS for different time (0-48 h). The expression of MIP-2 mRNA and protein was detected by real-time RT-PCR and enzyme linked immunosorbent assay (ELISA). MC3T3-E1 cells were pretreated with resveratrol for 1 h in the presence of 20 mg/L P.e-LPS for 24 h，which was detected by ELISA. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. Treatment of MC3T3-El cells with different concentrations of P.e-LPS(0-50 mg/L) caused a significantly increase in MIP-2 mRNA and protein expression in dose-dependent manners.The expression of MIP-2 protein increased from (41.86±2.49) ng/L to (3126.74±158.30) ng/L, and the difference was significant(P<0.05). In the observation time (0-48 h), the impact of 20 mg/L P.e-LPS on induction of MIP-2 in MC3T3-El cells exhibited a time-dependent manner. At 48 h, the maximal induction of MIP-2 protein expression was (2102.55±123.27) ng/L(P<0.01). Incubation of cells with 10 μmol/L resveratrol for 1h significantly decreased the expression of MIP-2 protein from (1805.33±67.54) ng/L to（813.82±47.21) ng/L, and the difference was significant(P<0.05). The results suggest that P.e-LPS may mediate MIP-2 expression in MC3T3-E1 cells, and resveratrol has a significant inhibitory effect on this process.

Development of a mouse model of eosinophilic chronic rhinosinusitis with nasal polyp by nasal instillation of an Aspergillus protease and ovalbumin.

One subtype of chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by the development of a T-helper type 2 (Th2) response and eosinophilic infiltration. Here, we aimed to establish an eosinophilic CRSwNP murine model, which would be essential to understand the underlying pathogenesis and establish a treatment strategy. C57BL/6 mice were challenged intranasally with a mixture of an Aspergillus oryzae-derived protease (AP) and ovalbumin (OVA) for 6, 8, or 12 consecutive weeks (12 mice/group); control mice received the same volume of phosphate-buffered saline for 12weeks (n=12). Sinonasal samples were evaluated histologically, and interleukin (IL)-4, IL-5, IL-13, eotaxin, keratinocyte chemoattractant, and macrophage inflammatory protein-2 mRNA levels in sinonasal mucosa were measured by real-time PCR. Protein levels of Th2 cytokines, INF-γ, IL-17A, and chemokines in nasal lavage fluid, and total serum IgE were measured by ELISA. Greater eosinophil infiltration in the subepithelial layer was observed in the challenged groups, compared with the control group. Polypoid mucosal lesions were predominantly observed in the 12-week group, which also exhibited mucosal thickening on micro-CT scans. The IL-4, IL-5, and IL-13 mRNA and protein levels were elevated in the sinonasal mucosa and nasal lavage fluid. INF-γ and IL-17A were undetectable or not elevated relative to the control group levels. In contrast, eotaxin levels were particularly elevated in the sinonasal mucosa and nasal lavage fluid in the 12-week group. In conclusion, intranasal AP and OVA exposure successfully induced Th2-specific CRSwNP in a murine model.

Induced histamine regulates Ni elution from an implanted Ni wire in mice by downregulating neutrophil migration

Histamine regulates various inflammatory reactions. We have reported that the expression of histidine decarboxylase (HDC) was induced by subcutaneous implantation of nickel (Ni) wire. However, the source and functions of histamine in Ni elution and Ni wire-induced inflammation have not been completely studied. We aimed to elucidate the effects of de novo synthesized histamine on leucocyte infiltration and Ni elution. Implantation of Ni wire induced an increase in the Ni ion content of the surrounding tissues and serum and in the mRNA levels of HDC, a histamine-producing enzyme, macrophage inflammatory protein-2 (MIP-2), a chemoattractant for neutrophils, and monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes. The Ni wire induced HDC expression even in mast cell-deficient WBB6F1-W/WV mice. In HDC knockout (HDC KO) mice, the Ni wire-induced increase in MIP-2 mRNA expression was significantly higher than that in wild-type mice but not MCP-1. MIP-2 expression was enhanced in histamine H2 receptor knockout (H2R KO) mice but not in WBB6F1-W/WV mice. Histamine inhibited NiCl2 -induced MIP-2 mRNA expression in mouse bone marrow-derived macrophages (BMDMs) obtained from wild-type mice; this inhibition was not observed in BMDMs from H2R KO mice. Ni elution increased in HDC KO mice, in which leucocyte infiltration also increased, and was suppressed in mice treated with neutrophil-specific antibody. These results suggest that the Ni wire induced HDC expression in non-mast cells and that, in the chronic phase of inflammation, endogenous histamine reduced Ni elution, probably through regulation of MIP-2 expression and neutrophil migration.

Aminophylline Effect on Renal Ischemia-Reperfusion Injury in Mice

BackgroundAminophylline increases the intracellular concentration of cAMP and exerts an anti-inflammatory effect. The aim of this study was to investigate the effect of aminophylline on renal ischemia-reperfusion (I/R) injury in mice. MethodsThirty C57BL/6 mice were divided into 3 groups. In the sham group (group S, n = 10), only right nephrectomy was performed. In the control group (group C, n = 10), after right nephrectomy, the mice were subjected to 30 minutes of left renal ischemia. In the aminophylline group (group A, n = 10), an intraperitoneal injection of aminophylline (5 mg/kg) was performed before renal ischemia. Twenty-four hours after reperfusion, the mice were euthanized, and plasma and kidney samples were obtained to analyze the serum creatinine, renal histology, and expression levels of nuclear factor-kappa B (NF-kB) and pro-inflammatory cytokines. ResultsThe serum creatinine concentration in group C was markedly elevated at 24 hours after reperfusion. Aminophylline treatment significantly reduced serum creatinine, compared with group C. Aminophylline also reduced the histological evidence of renal damage. The expression levels of NF-kB, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and intercellular adhesion molecule-1 (ICAM-1) mRNA were significantly increased in group C (P < .001). Group A showed lower expression of NF-kB, TNF-α, MCP-1, MIP-2, and ICAM-1 mRNA than group C (P < .01). ConclusionsAminophylline treatment improved the renal function and indexes of renal inflammation, which suggests that it provided reno-protection against renal I/R injury.

Role of chemokine CXC-ligand 16 in renal ischemia-reperfusion injury in mice

Objective To investigate the role of chemokine CXC-ligand 16 (CXCL16) in renal ischemia-reperfusion (I/R) injury in the mice. Methods Twelve healthy male C57BL/6 mice and 12 CXCL16-knockout (CXCL16-KO) mice, aged 8-10 weeks, weighing 20-30 g, were studied.The 12 C57BL/6 mice were randomly divided into 2 groups (n=6 each) using a random number table: C57BL/6 sham operation group (group C-S) and C57BL/6 I/R group (group C-I/R). The 12 CXCL16-KO mice were randomly divided into 2 groups (n=6 each) using a random number table: CXCL16-KO sham operation group (group KO-S) and CXCL16-KO I/R group (group KO-I/R). The right kidney was removed, and the left kidney was exposed, and the renal artery was then clamped for 45 min with atraumatic microclips followed by 24 h reperfusion to establish the model of renal I/R in anesthetized mice.Venous blood samples were taken at 24 h of reperfusion for determination of the serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations.The renal specimens were obtained at 24 h of reperfusion for microscopic examination of the pathological changes, and the damage to the renal tubules was scored.The number of myeloperoxidase (MPO) positive cells (MPO+ cells), F4/80+ cells and CD3+ cells in renal tissues was counted by immunohistochemistry.The expression of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-6, and macrophage inflammatory protein-2 (MIP-2) mRNA in renal tissues was determined by real-time reverse transcriptase polymerase chain reaction. Results Compared with group C-S, the serum BUN and Cr concentrations, renal tubular damage score, and the number of MPO+ , F4/80+ , and CD3+ cells were significantly increased, and the expression of TNF-α, IL-1β, IL-6 and MIP-2 mRNA was significantly up-regulated in group C-I/R (P<0.05). Compared with group KO-S, the serum BUN and Cr concentrations, renal tubular damage score, and the number of MPO+ , F4/80+ , and CD3+ cells were significantly increased, and the expression of TNF-α, IL-1β, IL-6 and MIP-2 mRNA was significantly up-regulated in group KO-I/R (P<0.05). Compared with group C-I/R, the serum BUN and Cr concentrations, renal tubular damage score, and the number of MPO+ , F4/80+ , and CD3+ cells were significantly decreased, and the expression of TNF-α, IL-1β, IL-6 and MIP-2 mRNA was significantly down-regulated in group KO-I/R (P<0.05). Conclusion CXCL16 is involved in the pathophysiological process of renal I/R injury in the mice. Key words: Chemokines, CXC; Kidney; Reperfusion injury

Protective effect of quercetin on acute lung injury in rats with sepsis and its influence on ICAM-1 and MIP-2 expression.

This study aimed to explore the protective effect of quercetin on acute lung injury (ALI) in rats with sepsis and the related mechanism. Rats were administered different doses of quercetin intraperitoneally, and blood samples and lung tissue were collected at 24 h after treatment. Arterial blood gases, lung water content, protein content, and cell counts in bronchoalveolar lavage fluid (BALF) were measured. Morphological changes in lung tissue pathology were observed under a light microscope. Serum intercellular adhesion molecule (ICAM)-1 and macrophage inflammatory protein 2 (MIP-2) levels were detected and ICAM-1 and MIP-2 mRNA expression in lung tissue was determined. Compared with that in the control model group, arterial blood gases, lung water content, protein content, and cell counts in BALF improved in the high- and low-dose quercetin groups (P < 0.05), with maximal improvement observed for the high-dose quercetin (P < 0.05). Lesions on the lungs improved in the high- and low-dose quercetin groups than those in the control model group, and the high-dose quercetin group showed better improvement than the low-dose group (P < 0.05). Compared with that in the sham-operated group, both serum and lung tissue ICAM-1 and MIP-2 expression increased significantly in the model group (P < 0.05). The quercetin groups presented lower ICAM-1 and MIP-2 expression than the control model group, with the lowest expression observed in the high-dose group (P < 0.05). Quercetin may protect against ALI in rats with sepsis by inhibiting ICAM-1 and MIP-2 expression.

Effect of milrinone on the inflammatory response and NF-kB activation in renal ischemia-reperfusion injury in mice

BackgroundMilrinone increases intracellular adenosine 3',5'-cyclic monophosphate concentration and enhances vascular relaxation. Nuclear factor-kappa B (NF-kB) plays a key role in inflammatory responses during ischemia-reperfusion (I/R) injury. We aimed to investigate the effect of milrinone on the inflammatory responses and NF-kB activation in renal I/R injury in mice.MethodsThirty C57BL/6 mice were allocated into 3 groups. In group S (n = 10), only right nephrectomy was done. In group C (n = 10), the left kidney was subjected to 30 min of ischemia after right nephrectomy. In group M (n = 10), milrinone (5 µg/kg) was administered before ischemia. After 24 hours of reperfusion, the serum creatinine was measured, kidney samples were obtained for histology, and expressions of NF-kB and proinflammatory cytokines were analyzed.ResultsIn group C, the serum creatinine concentration was markedly elevated, compared with group S. Creatinine concentration in group M was also elevated, but it was significantly lower than that in group C. Histologic evidence of renal damage was severe in group C, but it was improved in group M. In groups C and M, expression of NF-kB, tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) mRNA increased significantly compared with group S (P < 0.05). But group M showed a lower expression of NF-kB, TNF-α, ICAM-1, MCP-1 and MIP-2 mRNA than group C (P < 0.05).ConclusionsMilrinone treatment attenuates the renal inflammatory response and activation of NF-kB, resulting in improvement of renal function and tissue injury.

Myeloperoxidase deficiency in mice exacerbates lung inflammation induced by nonviable Candida albicans

This study aimed to evaluate the effect of myeloperoxidase (MPO) deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice were inoculated intranasally with nCA, and accumulation of neutrophils and macrophages in the bronchoalveolar lavage fluid was analyzed by flow cytometry. The levels of macrophage inflammatory protein 2 (MIP-2), keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in the lung were measured by ELISA. Production of MIP-2 and KC from neutrophils and macrophages was quantified in vitro. MIP-2 mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the extent of phosphorylation of ERK1/2 and Syk in the neutrophils was analyzed by Western blotting. The MPO(-/-) mice that received nCA showed more severe pneumonia than wild-type mice. Within 12 h of nCA administration, MPO(-/-) mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-2 and KC relative to the responses seen in wild-type mice. Neutralization of MIP-2 and KC in vivo significantly reduced neutrophil infiltration. In vitro, production of MIP-2, but not that of KC, was enhanced in the nCA-stimulated neutrophils from MPO(-/-) mice, concomitant with up-regulation of Syk and ERK1/2. At 1 and 3 days after nCA administration, MPO(-/-) mice had significantly higher lung concentrations of TNF-α and IL-1β than wild-type mice. Pulmonary administration of nCA produced an altered inflammatory response in MPO(-/-) mice relative to wild-type mice. Enhanced MIP-2 production by MPO(-/-) neutrophils may at least partly contribute to exacerbated inflammation in mutant mice.

Effect of B7-H3 protein on the expression of macrophage-inflammatory protein 2 mRNA in Streptococcus pneumococcal meningitis mouse models

Objective To investigate the effect of B7-H3 protein, a collaborative signal molecule, on macrophage-inflammatory protein 2(MIP-2) mRNA level in Streptococcus pneumococcal meningitis mouse models. Methods Forty-eight healthy male BALB/C mice at the age of 1.5-2.0 months were randomly divided into 4 groups: 9 g/L saline group(NS group), B7-H3 protein group(B7-H3 group), Streptococcus neumonia group(SP group), and Streptococcus neumonia plus B7-H3 protein group(combination group), 12 mice in each group.Mouse models were established by intracerebral ventricular injection with 9 g/L saline, B7-H3 protein, Streptococcus neumonia type 3 or Streptococcus neumonia type 3 plus B7-H3 protein.Neurobehavior of different groups was evaluated according to loeffler rule after injection for 6 h and 24 h, then the mice were sacrificed and MIP-2 mRNA levels were tested by Real-time PCR.The results were analyzed by SPSS 18.0 software. Results Neurobehavior scoring results showed that there were no significant differences between B7-H3 group and NS group(P>0.05) after infection for 6 h and 24 h, while the score of SP group was decreased compared with that of NS group(P 0.05), while at 24 h post infection, the relative MIP-2 mRNA expressions in SP group were significantly increased compared with that of NS group(12.880±7.792 vs 1.000±0.091), the difference was significant (P 0.05); while at 24 h post infection, the difference was significant(38.760±6.601 vs 12.880±7.792)(P<0.05). Conclusion Collaborative signal molecule B7-H3 protein may increase MIP-2 mRNA level in Streptococcus pneumococcal meningitis mouse model, and exaggerate the clinical disease condition. Key words: Bacterial meningitis; Streptococcus pneumoniae; B7-H3 protein; Macrophage-inflammatory protein 2

Inhibitory Effects of Ethyl Acetate Extract of Andrographis paniculata on NF‐κB Trans‐Activation Activity and LPS‐Induced Acute Inflammation in Mice

This study was to investigate anti-inflammatory effect of Andrographis paniculata (Burm. f.) Nees (Acanthaceae) (AP). The effects of ethyl acetate (EtOAc) extract from AP on the level of inflammatory mediators were examined first using nuclear factor kappa B (NF-κB) driven luciferase assay. The results showed that AP significantly inhibited NF-κB luciferase activity and tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), macrophage inflammatory protein-2 (MIP-2) and nitric oxide (NO) secretions from lipopolysaccharide (LPS)/interferon-γ stimulated Raw264.7 cells. To further evaluate the anti-inflammatory effects of AP in vivo, BALB/c mice were tube-fed with 0.78 (AP1), 1.56 (AP2), 3.12 (AP3) and 6.25 (AP4) mg kg−1 body weight (BW)/day in soybean oil, while the control and PDTC (pyrrolidine dithiocarbamate, an anti-inflammatory agent) groups were tube-fed with soybean oil only. After 1 week of tube-feeding, the PDTC group was injected with 50 mg kg−1 BW PDTC and 1 h later, all of the mice were injected with 15 mg kg−1 BW LPS. The results showed that the AP1, AP2, AP3 and PDTC groups, but not AP4, had significantly higher survival rate than the control group. Thus, the control, AP1, AP2, AP3 and PDTC groups were repeated for in vivo parameters. The results showed that the AP and PDTC groups had significantly lower TNF-α, IL-12p40, MIP-2 or NO in serum or peritoneal macrophages and infiltration of inflammatory cells into the lung of mice. The AP1 group also had significantly lower MIP-2 mRNA expression in brain. This study suggests that AP can inhibit the production of inflammatory mediators and alleviate acute hazards at its optimal dosages.

Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic acini

Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-κB, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-κB silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-κB and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-κB inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-κB and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-κB and AP-1 in these cells. Thus dual inhibition of NF-κB and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-κB alone.

Mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of IL-1β, MIP-2 and LTB 4

In the present study, the role of macrophages and mast cells in mineral trioxide aggregate (MTA)-induced release of neutrophil chemotactic factor was investigated. MTA suspension (50 mg/mL) was plated over inserts on macrophages or mast cells for 90 minutes. Untreated cells served as controls. Cells were washed and cultured for 90 minutes in RPMI without the stimuli. Macrophages and mast cell supernatants were injected intraperitoneally (0.5 mL/cavity), and neutrophil migration was assessed 6 hours later. In some experiments, cells were incubated for 30 minutes with dexamethasone (DEX, 10 muM/well), BWA4C (BW, 100 muM/well) or U75302 (U75, 10 muM/well). The concentration of Leukotriene B(4) (LTB(4)) in the cell-free supernatant from mast cells and macrophage culture was measured by ELISA. Supernatants from MTA-stimulated macrophages and mast cells caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages and mast cells was significantly inhibited by DEX, BW, or U75. Macrophages and mast cells expressed mRNA for interleukin-1 (IL-1)beta and macrophage inflammatory protein-2 (MIP-2) and the pretreatment of macrophages and mast cells with DEX, BW, or U75 significantly altered IL-1beta and MIP-2 mRNA expression. LTB(4) was detected in the MTA-stimulated macrophage supernatant but not mast cells. MTA-induces the release of neutrophil chemotactic factor substances from macrophages and mast cells with participation of IL-1beta, MIP-2, and LTB(4).

Regulation and gene expression of chemokines and adhesion molecules in rat liver after gamma-irradiation

Radiation Induced Liver Diseases (RILD) is a serious clinical complication. CXC and CC chemokines are known to attract inflammatory cells and adhesion molecules play a role in transmigration process to the site of inflammation. Aim of study was to investigate gene expression of CXC, CC-chemokines and their receptor in relation to two adhesion molecules in rat liver after single dose gamma-irradiation. Macrophage inflammatory protein-2 (MIP-2) concentration in serum was measured by ELISA after exposing the rat liver to gamma-irradiation (25Gy). Livers of irradiated animals and of controls (sham-irradiated) were investigated at 1, 3, 6, 12, 24, and 48h after liver irradiation. Cryostat sections were used to detect inflammatory cells by immunohistochemistry and total RNA was extracted. Hepatocytes and kupffer cells primary cultures were irradiated. Increased numbers of neutrophil granulocytes attached to wall of the portal vessels (max. at 3–6h) were observed after single-dose of gamma-irradiation. Number of ED-1 (mononuclear phagocytes) positive cells were not increased at any time points after irradiation. Moncyte chemoattractant protein -1 (MCP-1) was increasingly detected in the wall of the portal vessels.In CXC chemokines, MIP-2 serum concentration was significantly increased up to (774±80 pg/ml) 3h after irradiation as compared to sham-irradiated controls. A statistically significant induction of MIP-2, CXCL-1, LIX and MCP-3 (CC-chemokine) in RNA was also observed at 1h with peak at 3h and followed by a continuous decrease thereafter. CXCR-2 receptor gene expression was also early up-regulated (1–48h) and a quick (1–3h) induction of ICAM-1 but no significant change of PECAM-1 was observed after rat liver irradiation. Irradiation also up-regulated MIP-2 mRNA and CXCR-2 in isolated hepatocytes (8Gy) and kupffer cell (8Gy). Single dose (unfractionated) gamma- irradiation administrated to the liver induces a transient accumulation of granulocytes within the portal area. Although, gamma-irradiation is able to induce significant increase of gene expression of chemokines, known to be involved also in recruitment of mono-nuclear phagocytes, no increase of these cells at any time point was observed. This may influence repair process.

Effect and mechanism of emodin on cholestatic hepatitis

To explore the therapeutic effect and mechanism of emodin on cholestatic hepatitis. Rats were divided into 5 groups: 1 group was untreated, the other 4 groups were treated with alpha-naphthylisothiocyanate (ANIT), ANIT and emodin, ANIT and ursodeoxycholic acid, or ANIT and dexamethasone, respectively. At 24 h, 48 h and 72 h after the treatment, NF-kappa B, early growth response factor-1 (Egr-1), cytokine-induced neutrophil chemoattractant 1 (CINC-1), macrophage inflammatory protein 2 (MIP-2), intercellular adhesion molecule 1 (ICAM-1),tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) were assayed by immunohistochemistry, real-time PCR , western-blot and ELISA. The level of malondialdehyde (MDA), superoxide Dismutase(SOD) and myeloperoxidase (MPO) were assayed by thiobarbituric acid method, xanthine oxidase method and colorimetric method, respectively. (1) Compared to the controls, emodin had a notable effect on total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT) at all time points (all P less than 0.05). Compared to ursodeoxycholic acid, emodin had a notable effect on TB and DB at 24 h after the treatments, however, after 48 h, emodin had a notable effect only on TB (all P less than 0.05). Compared to Dexamethasone, emodin had a notable effect on TB at 48 h time point, and it had a notable effect on ALT at all time points (all P less than 0.05). (2) The nuclei NF-kappa B p65 staining was significantly increased at 24 h and 48 h after ANIT treatment (all P less than 0.05), and emodin treatment could block the increase (all P less than 0.05). (3) Egr-1 mRNA level was not affected by emodin treatment (P more than 0.05); levels of CINC-1, MIP-2 mRNA and ICAM-1 protein were significantly decreased after emodin treatment (all P less than 0.05). (4) The levels of TNF alpha and IL-6 were decreased after emodin treatment(all P less than 0.05). (5) The levels of MDA at all time points and MPO at 24 h, 48 h time points were notably down-regulated by emodin treatment, while the level of SOD was markedly elevated at all time points after emodin treatment (all P less than 0.05). Emodin treatment can reduce the levels of TB, DB and ALT in ANIT induced-cholestatic hepatitis. The effect may be due to inhibition of NF-kappa B signal pathway.

Therapeutic effects of emodin on acute cholestatic hepatitis and mechanism thereof: experiment with rats

To investigate the therapeutic effects of emodin on acute cholestatic hepatitis and mechanism thereof. 96 SD rats were randomly divided into 4 groups to be treated with emodin, ursodeoxycholic acid, dexamethasone, or normal saline respectively for 4 days. On the 5th day gastric perfusion of alpha-naphthylisothiocyanate (ANIT) was performed to establish models of cholestatic hepatitis. 4 - 6 hours after the establishment of model the above mentioned agents were given continuously. 24, 48, and 72 hours after the model establishment blood samples were collected from abdominal aorta to examine the total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT), and total bile acid (TBA). Specimen of liver was collected to undergo pathological examination. PCR was used to detect the mRNA expression of cytokine-induced neutrophil chemoattractant 1 (CINC-1) and macrophage inflammatory protein 2(MIP-2), and Western blotting was used to detect the protein expression of intercellular adhesion molecule 1 (ICAM-1). Twenty-four rats treated with NS only were used as controls. The pathological changes of behaviors and liver of the emodin and ursodeoxycholic acid groups were all remarkably milder than those of other model groups. The levels of TB [(32.8 +/- 3.7) micromol/L, (61.0 +/- 16.4) micromol/L, (10.8 +/- 4.5) micromol/L], DB[ (26.03 +/- 3.10) micromol/L, (49.40 +/- 18.16) micromol/L, (8.04 +/- 3.03) micromol/L], and ALT [(314 +/- 50) U/L, (664 +/- 97) U/L, (200 +/- 60) U/L], at the time points 24, 48, and 72 hours, the 48 and 72 hours AST levels, the 48 hours ALP level, the 72 hours GGT level, and the 48 and 72 hours TBA levels of the emodin group were all significantly lower than those of the model group (all P < 0.05). The 24 and 48 hours TB levels, 24 hours DB and ALT, and 24, 72 hours TBA levels of the emodin group were all significantly lower than those of the ursodeoxycholic acid group (all P < 0.05). The 24, 48 hours TB and TBA levels, and the ALT, AST and GGT levels at all time points of the emodin group were all significantly lower than those of the dexamethasone group (all P < 0.05). The CINC-1 and MIP-2 mRNA expression levels and ICAM-1 protein expression levels at all time points of the emodin group were all significantly lower than those of the model group (all P < 0.05). Decreasing the levels of TB, DB and ALT in particular, emodin has a protective effect on cholestatic hepatitis. Its effects are quicker than ursodeoxycholic acid, and it has better effects on ALT, AST GGT, and TBA than dexamethasone. These effects may be due to inhibition of the activation of CINC-1, MIP-2, and ICAM-1.

Effects of N-acetylcysteine on mRNA expression of monocyte chemotactic protein and macrophage inflammatory protein 2 in acute necrotizing pancreatitis: experiment with rats

To explore the potential role of monocyte chemotactic protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) in the pathogenesis of acute necrotizing pancreatitis (ANP), and to study the effect of N-acetylcysteine (NAC) on the mRNA expression of MCP-1 and MIP-2. Thirty-five SD rats were randomly divided into 3 groups: sham-operation (SO) group (n = 5), acute necrotizing pancreatitis (ANP) group (n = 15), and NAC-pretreated group (n = 15), ANP were induced by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct. The NAC- groups underwent intraperitoneal injection of NAC 500 mg/kg 30 minutes before the induction of sodium taurocholate. The ANP and NAC groups were re-divided into 3 equal subgroups respectively: 3 h, 6 h, and 12 h subgroups. 3, 6, and 12 hours after the establishment of models heart blood samples were collected from 5 rats from each model group to detect the serum amylase. Then the rats were killed by blood letting with their pancreases taken out. HE staining and microscopy were used to observe the pathological changes of the pancreas. The wet/dry ratio of pancreas was determined. Enzyme histochemical method was used to detect the activity of myeloperoxidase (MPO) in the pancreas. RT-PCR was used to examine the mRNA expression of MCP-1 and MIP-2. The levels of serum amylase, wet/dry ratio of pancreas, pancreatic MPO activity, and histological score of pancreas of the ANP group increased time-dependently, all the levels at different time-points were significantly higher than those of the SO group (all P < 0.01). The expression levels of MCP-1 mRNA at the time points 3, 6, and 12 h of the ANP group were 0.3653 +/- 0.0213, 0.5890 +/- 0.0225, and 0.7164 +/- 0.0275 respectively, all significantly higher than that of the SO group (0.1492 +/- 0.0036, all P < 0.01). The expression levels of MIP-2 mRNA at different time points of the ANP group were 0.3871 +/- 0.0286, 0.6040 +/- 0.0448, and 0.7692 +/- 0.0620 respectively, all significantly higher than that of the SO group (0.1593 +/- 0.0117, all P < 0.01). The intrapancreatic MPO levels at the time points 3 h, 6 h, and 12 h of the NAC group were 0.63 +/- 0.03, 0.88 +/- 0.05, and 1.31 +/- 0.09 respectively, all significantly lower than those of the ANP groups (0.89 +/- 0.03, 1.42 +/- 0.14, and 1.94 +/- 0.07, all P < 0.05). The MCP-1 mRNA expression levels at different time points the NAC group were 0.2497 +/- 0.0168, 0.4457 +/- 0.0097, and 0.6306 +/- 0.0423 respectively, and the MIP-2 mRNA expression levels at the time points 3 h, 6 h, and 12 h of the NAC group were 0.2436 +/- 0.0099, 0.4312 +/- 0.0221, and 0.6302 +/- 0.0288 respectively, all significantly lower than those of the ANP group (all P < 0.05). The pancreatic histological scores at different time points of the NAC group were 3.50 +/- 0.61, 5.60 +/- 0.65, and 7.50 +/- 0.79, all significantly lower than those of the ANP group (5.10 +/- 0.42, 7.50 +/- 0.50, and 9.90 +/- 0.96, all P < 0.05). The MCP-1 mRNA expression and MIP-2 mRNA expression were both correlated with the severity of pancreatic injury (r = 0.76 and 0.82, both P < 0.05). The chemokines of MCP-1 and MIP-2 are overexpressed at the early stage of acute pancreatitis. Both of them may play an important role in the pathogenesis of AP. NAC may have beneficial effects on AP through downregulation of the expression of MCP-1 and MIP-2.

High-molecular-weight hyaluronan – a possible new treatment for sepsis-induced lung injury: a preclinical study in mechanically ventilated rats

IntroductionMechanical ventilation with even moderate-sized tidal volumes synergistically increases lung injury in sepsis and has been associated with proinflammatory low-molecular-weight hyaluronan production. High-molecular-weight hyaluronan (HMW HA), in contrast, has been found to be anti-inflammatory. We hypothesized that HMW HA would inhibit lung injury associated with sepsis and mechanical ventilation.MethodsSprague–Dawley rats were randomly divided into four groups: nonventilated control rats; mechanical ventilation plus lipopolysaccharide (LPS) infusion as a model of sepsis; mechanical ventilation plus LPS with HMW HA (1,600 kDa) pretreatment; and mechanical ventilation plus LPS with low-molecular-weight hyaluronan (35 kDa) pretreatment. Rats were mechanically ventilated with low (7 ml/kg) tidal volumes. LPS (1 or 3 mg/kg) or normal saline was infused 1 hour prior to mechanical ventilation. Animals received HMW HA or low-molecular-weight hyaluronan via the intraperitoneal route 18 hours prior to the study or received HMW HA (0.025%, 0.05% or 0.1%) intravenously 1 hour after injection of LPS. After 4 hours of ventilation, animals were sacrificed and the lung neutrophil and monocyte infiltration, the cytokine production, and the lung pathology score were measured.ResultsLPS induced lung neutrophil infiltration, macrophage inflammatory protein-2 and TNFα mRNA and protein, which were decreased in the presence of both 1,600 kDa and 35 kDa hyaluronan pretreatment. Only 1,600 kDa hyaluronan completely blocked both monocyte and neutrophil infiltration and decreased the lung injury. When infused intravenously 1 hour after LPS, 1,600 kDa hyaluronan inhibited lung neutrophil infiltration, macrophage inflammatory protein-2 mRNA expression and lung injury in a dose-dependent manner. The beneficial effects of hyaluronan were partially dependent on the positive charge of the compound.ConclusionsHMW HA may prove to be an effective treatment strategy for sepsis-induced lung injury with mechanical ventilation.

Exacerbation of wood smoke-induced acute lung injury by mechanical ventilation using moderately high tidal volume in mice

We investigated the effects of mechanical ventilation with a moderately high tidal volume (VT) on acute lung injury (ALI) induced by wood smoke inhalation in anesthetized mice. Animals received challenges of air, 30 breaths of smoke (30SM) or 60 breaths of smoke (60SM) and were then ventilated with a VT of 10 ml/kg (10VT) or 16 ml/kg (16VT). After 4-h mechanical ventilation, the bronchoalveolar-capillary permeability, pulmonary infiltration of inflammatory cells, total lung injury score and pulmonary expressions of interleukin-1beta and macrophage inflammatory protein-2 mRNA and proteins in the 30SM+16VT and 60SM+16VT groups were greater than those in the 30SM+10VT and 60SM+10VT groups, respectively. Additionally, the wet/dry weight ratio of lung tissues and lung epithelial cell apoptosis in the 60SM+16VT group were greater than those in the 60SM+10VT group. These differences between the 16VT and 10VT groups were not seen in animals with air challenge. Thus, mechanical ventilation with a moderately high VT in mice exacerbates ALI induced by wood smoke inhalation.