RvD1 improves resident alveolar macrophage self-renewal via the ALX/MAPK14/S100A8/A9 pathway in acute respiratory distress syndrome.

INTRODUCTION: Acute respiratory distress syndrome (ARDS) is a pulmonary inflammatory process primarily caused by sepsis. The resolution of inflammation is an active process involving the endogenous biosynthesis of specialized pro-resolving mediators, including resolvin D1 (RvD1). Resident alveolar macrophages (RAMs) maintain pulmonary homeostasis and play a key role in the resolution phase. However, the role of RAMs in promoting the resolution of inflammation by RvD1 is unclear. OBJECTIVES: Here, we investigated the mechanisms of RvD1 on regulating RAMs to promote the resolution of ARDS. METHODS: Mice were administered lipopolysaccharide and/or Escherichia coli via aerosol inhalation to establish a self-limited ARDS model. Then, RvD1 was administered at the peak inflammatory response. RAMs self-renewal was measured by flow cytometry, RAM phagocytosis was measured by two-photon fluorescence imaging. In addition, plasma was collected from intensive care unit patients on days 0-2, 3-5, and 6-9 to measure RvD1 and S100A8/A9 levels using triple quadrupole/linear ion trap mass spectrometry. RESULTS: RAMs were found to play a pivotal role in resolving inflammation during ARDS, and RvD1 enhanced RAM proliferation and phagocytosis, which was abrogated by a lipoxin A4 receptor (ALX, RvD1 receptor) inhibitor. Both primary RAMs transfected with rS100A8/A9 and/or S100A8/A9 siRNA and S100A9-/- mice (also deficient in S100A8 function) showed higher turnover and phagocytic function, indicating that RvD1 exerted its effects on RAMs by inhibiting S100A8/A9 production in the resolution phase. RvD1 reduced S100A8/A9 and its upstream MAPK14 levels in vivo and in vitro. Finally, in the patients, RvD1 levels were lower, but S100A8/A9 levels were higher. CONCLUSIONS: We propose that RvD1 improved RAM self-renewal and phagocytosis via the ALX/MAPK14/S100A8/A9 signaling pathway. Plasma RvD1 and S100A8/A9 levels were negatively correlated, and associated with the outcome of sepsis-induced ARDS.

Neutrophil reverse migration.

The behavior of neutrophils is very important for the resolution of inflammation and tissue repair. People have used advanced imaging techniques to observe the phenomenon of neutrophils leaving the injured or inflammatory site and migrating back into blood vessels in transgenic zebrafish and mice, which is called neutrophil reverse migration. Numerous studies have shown that neutrophil reverse migration is a double-edged sword. On the one hand, neutrophil reverse migration can promote the resolution of local inflammation by accelerating the clearance of neutrophils from local wounds. On the other hand, neutrophils re-enter the circulatory system may lead to the spread of systemic inflammation. Therefore, accurate regulation of neutrophil reverse migration is of great significance for the treatment of various neutrophil- mediated diseases. However, the mechanism of neutrophil reverse migration and its relationship with inflammation resolution is unknown. In this review, we reviewed the relevant knowledge of neutrophil reverse migration to elucidate the potential mechanisms and factors influencing reverse migration and its impact on inflammation in different disease processes.

The Effect and Mechanism of Lipoxin A4 on Neutrophil Function in LPS-Induced Lung Injury.

Excessive inflammatory response caused by infiltration of a large number of neutrophils is one of the important features of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Lipoxin A4 (LXA4) is an important endogenous mediator in the process of inflammation resolution, which has a strong role in promoting inflammation resolution. In this study, we examined the impact of LXA4 on the pulmonary inflammatory response and the neutrophil function in ARDS rats. Our results indicated that exogenous administration of LXA4 could reduce the degree of lung injury in ARDS rats and inhibit the release of pro-inflammatory factors TNF-α and IL-1ß in lung tissue homogenate. However, LXA4 has no lung protective effect on ARDS rats of neutropenia, nor can it inhibit the levels of pro-inflammatory factors TNF-α and IL-1ß in lung tissue homogenate. LXA4 can inhibit the production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in peripheral blood neutrophils of ARDS rats. At the same time, LXA4 can promote the phagocytosis of neutrophils in ARDS rats in vitro and can also promote the apoptosis of neutrophils in ARDS rats. In addition, the effect of LXA4 on the function of neutrophils in ARDS rats is mediated by its receptor ALX. LXA4 can inhibit the release of NE and MPO from neutrophils, thereby reducing the production of NETs. In summary, these findings indicate that LXA4 has a protective effect on LPS-induced ARDS rats by affecting the function of neutrophils.

Protectin DX promotes the inflammatory resolution via activating COX-2/L-PGDS-PGD2 and DP1 receptor in acute respiratory distress syndrome.

PURPOSE: Acute respiratory distress syndrome (ARDS) is characterized by uncontrollable inflammation. Cyclooxygenase-2(COX-2) and its metabolite prostaglandins are known to promote the inflammatory resolution of ARDS. Recently, a newly discovered endogenous lipid mediator, Protectin DX (PDX), was also shown to mediate the resolution of inflammation. However, the regulatory of PDX on the pro-resolving COX-2 in ARDS remains unknown. MATERIAL AND METHODS: PDX (5 µg/kg) was injected into rats intravenously 12 h after the lipopolysaccharide (LPS, 3 mg/kg) challenge. Primary rat lung fibroblasts were incubated with LPS (1 µg/ml) and/or PDX (100 nM). Lung pathological changes examined using H&E staining. Protein levels of COX-2, PGDS and PGES were evaluated using western blot. Inflammatory cytokines were tested by qPCR, and the concentration of prostaglandins measured by using ELISA. RESULTS: Our study revealed that, COX-2 and L-PGDS has biphasic activation characteristics that LPS could induce induced by LPS both in vivo and in vitro.. The secondary peak of COX-2, L-PGDS-PGD2 promoted the inflammatory resolution in ARDS model with the DP1 receptor being activated and PDX up-regulated the inflammatory resolutionvia enhancing the secondary peak of COX-2/L-PGDS-PGD2 and activating the DP1 receptor. CONCLUSION: PDX promoted the resolution of inflammation of ARDS model via enhancing the expression of secondary peak of COX-2/L-PGDS-PGD2 and activating the DP1 receptor. PDX shows promising therapeutic potential in the clinical management of ARDS.

RvD1 accelerates the resolution of inflammation by promoting apoptosis of the recruited macrophages via the ALX/FasL-FasR/caspase-3 signaling pathway.

The uncontrolled inflammatory response caused by a disorder in inflammation resolution is one of the reasons for acute respiratory distress syndrome (ARDS). The macrophage pool markedly expands when inflammatory monocytes, known as recruited macrophages, migrate from the circulation to the lung. The persistent presence of recruited macrophages leads to chronic inflammation in the resolution phase of inflammation. On the contrary, elimination of the recruited macrophages at the injury site leads to the rapid resolution of inflammation. Resolvin D1 (RvD1) is an endogenous lipid mediator derived from docosahexaenoic acid. Mice were administered RvD1 via the tail vein 3 and 4 days after stimulation with lipopolysaccharide. RvD1 reduced the levels of the inflammatory factors in the lung tissue, promoted the anti-inflammatory M2 phenotype, and enhanced the phagocytic function of recruited macrophages to alleviate acute lung injury. We also found that the number of macrophages was decreased in BAL fluid after treatment with RvD1. RvD1 increased the apoptosis of recruited macrophages partly via the FasL-FasR/caspase-3 signaling pathway, and this effect could be blocked by Boc-2, an ALX/PRP2 inhibitor. Taken together, our findings reinforce the concept of therapeutic targeting leading to the apoptosis of recruited macrophages. Thus, RvD1 may provide a new therapy for the resolution of ARDS.

Resolvin Conjugates in Tissue Regeneration 1 Promote Alveolar Fluid Clearance by Activating Alveolar Epithelial Sodium Channels and Na, K-ATPase in Lipopolysaccharide-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS), a common and fatal clinical condition, is characterized by the destruction of epithelium and augmented permeability of the alveolar-capillary barrier. Resolvin conjugates in tissue regeneration 1 (RCTR1) is an endogenous lipid mediator derived from docosahexaenoic acid , exerting proresolution effects in the process of inflammation. In our research, we evaluated the role of RCTR1 in alveolar fluid clearance (AFC) in lipopolysaccharide-induced ARDS/acute lung injury (ALI) rat model. Rats were injected with RCTR1 (5 µg/kg) via caudal veins 8 hours after lipopolysaccharide (LPS) (14 mg/kg) treatment, and then AFC was estimated after 1 hour of ventilation. Primary type II alveolar epithelial cells were incubated with LPS (1 ug/ml) with or without RCTR1 (10 nM) for 8 hours. Our results showed that RCTR1 significantly enhanced the survival rate, promoted the AFC, and alleviated LPS-induced ARDS/ALI in vivo. Furthermore, RCTR1 remarkably elevated the protein expression of sodium channels and Na, K-ATPase and the activity of Na, K-ATPase in vivo and in vitro. Additionally, RCTR1 also decreased neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) level via upregulating Ser473-phosphorylated-Akt expression. Besides this, inhibitors of receptor for lipoxin A4 (ALX), cAMP, and phosphatidylinositol 3-kinase (PI3K) (BOC-2, KH-7, and LY294002) notably inhibited the effects of RCTR1 on AFC. In summary, RCTR1 enhances the protein levels of sodium channels and Na, K-ATPase and the Na, K-ATPase activity to improve AFC in ALI through ALX/cAMP/PI3K/Nedd4-2 pathway, suggesting that RCTR1 may become a therapeutic drug for ARDS/ALI. SIGNIFICANCE STATEMENT: RCTR1, an endogenous lipid mediator, enhanced the rate of AFC to accelerate the resolution of inflammation in the LPS-induced murine lung injury model. RCTR1 upregulates the expression of epithelial sodium channels (ENaCs) and Na, K-ATPase in vivo and in vitro to accelerate the AFC. The efficacy of RCTR1 on the ENaC and Na, K-ATPase level was in an ALX/cAMP/PI3K/Nedd4-2-dependent manner.

Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis.

BACKGROUND: Endothelial glycocalyx loss is integral to increased pulmonary vascular permeability in sepsis-related acute lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel macrophage-derived lipid mediator exhibiting potential anti-inflammatory and pro-resolving benefits. METHODS: PCTR1 was administrated intraperitoneally with 100 ng/mouse after lipopolysaccharide (LPS) challenged. Survival rate and lung function were used to evaluate the protective effects of PCTR1. Lung inflammation response was observed by morphology and inflammatory cytokines level. Endothelial glycocalyx and its related key enzymes were measured by immunofluorescence, ELISA, and Western blot. Afterward, related-pathways inhibitors were used to identify the mechanism of endothelial glycocalyx response to PCTR1 in mice and human umbilical vein endothelial cells (HUVECs) after LPS administration. RESULTS: In vivo, we show that PCTR1 protects mice against lipopolysaccharide (LPS)-induced sepsis, as shown by enhanced the survival and pulmonary function, decreased the inflammatory response in lungs and peripheral levels of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Moreover, PCTR1 restored lung vascular glycocalyx and reduced serum heparin sulphate (HS), syndecan-1 (SDC-1), and hyaluronic acid (HA) levels. Furthermore, we found that PCTR1 downregulated heparanase (HPA) expression to inhibit glycocalyx degradation and upregulated exostosin-1 (EXT-1) protein expression to promote glycocalyx reconstitution. Besides, we observed that BAY11-7082 blocked glycocalyx loss induced by LPS in vivo and in vitro, and BOC-2 (ALX antagonist) or EX527 (SIRT1 inhibitor) abolished the restoration of HS in response to PCTR1. CONCLUSION: PCTR1 protects endothelial glycocalyx via ALX receptor by regulating SIRT1/NF-κB pathway, suggesting PCTR1 may be a significant therapeutic target for sepsis-related acute lung injury.

Effects of propofol on the proliferation and migration of liver cancer cells.

Liver cancer is a malignant cancer with worldwide prevalence. It has been reported that cancer cells are usually exposed to a hypoxic microenvironment, which is associated with a poor prognosis in patients with cancer. Propofol is an intravenous anesthetic that is widely used in cancer surgery. The present study aimed to determine the effects of propofol stimulation on the viability, proliferation and migration of liver cancer cells under normoxia and cobalt chloride (CoCl2)-induced hypoxia. Under normoxia, HepG2 and HCCLM3 cells were randomly divided into six groups as follows: i) Control group; ii) 10 µM propofol group; iii) 25 µM propofol group; iv) 50 µM propofol group; v) 100 µM propofol group; and vi) DMSO group. Cell viability and proliferation were analyzed using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, respectively, following 24 or 48 h of propofol treatment. In addition, wound healing and Transwell migration assays were used to determine the changes in cell migration. Under CoCl2-induced hypoxia, the protein levels of hypoxia inducible factor-1α (HIF-1α) of HepG2 cells were analyzed using western blotting. Subsequently, CCK-8 and wound healing assays were used to determine the effect of propofol on cell viability and migration. The results of the present study revealed that propofol stimulation had no significant effect on the viability, proliferation and migration of HepG2 and HCCLM3 cells under normoxia. The protein levels of HIF-1α were significantly upregulated following the treatment with 200 µM CoCl2 for 12 h. However, no significant differences were found in the viability and migration of HepG2 cells following the stimulation with propofol in the presence of CoCl2. In conclusion, the findings of the present study revealed that propofol exerted no effect on the viability, proliferation and migration of HepG2 and HCCLM3 cells under normoxic and hypoxic conditions.

Rosuvastatin Enhances Alveolar Fluid Clearance in Lipopolysaccharide-Induced Acute Lung Injury by Activating the Expression of Sodium Channel and Na,K-ATPase via the PI3K/AKT/Nedd4-2 Pathway.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are devastating clinical conditions characterized by pulmonary epithelial damage and protein-rich fluid accumulation in the alveolar spaces. Statins are a class of HMG-CoA reductase inhibitors, which exert cholesterol-lowering and anti-inflammatory effects. METHODS: Rosuvastatin (1 mg/kg) was injected intravenously in rats 12 h before lipopolysaccharide (LPS, 10 mg/kg) administration. Eight hours later after LPS challenge, alveolar fluid clearance (AFC) was detected in rats (n = 6-8). Rosuvastatin (0.3 µmol/mL) and LPS were cultured with primary rat alveolar type II epithelial cells for 8 h. RESULTS: Rosuvastatin obviously improved AFC and attenuated lung-tissue damage in ALI model. Moreover, it enhanced AFC by increasing sodium channel and Na,K-ATPase protein expression. It also up-regulated P-Akt via reducing Nedd4-2 in vivo and in vitro. Furthermore, LY294002 blocked the increase in AFC in response to rosuvastatin. Rosuvastatin-induced AFC was found to be partly rely on sodium channel and Na,K-ATPase expression via the PI3K/AKT/Nedd4-2 pathway. CONCLUSION: In summary, the findings of our study revealed the potential role of rosuvastatin in the management of ALI/ARDS.

LXA4 Inhibits Lipopolysaccharide-Induced Inflammatory Cell Accumulation by Resident Macrophages in Mice.

INTRODUCTION: Alveolar macrophages that regulate the inflammatory response in lungs are the main target cell for the treatment of inflammatory pulmonary pathologies, such as acute respiratory distress syndrome (ARDS). Yolk sac derived alveolar resident macrophages play an important role in the pulmonary inflammatory response. With regards to anti-inflammatory actions, lipoxin A4 (LXA4) has been identified as an inflammatory "braking signal". METHODS: In vivo, LXA4 (0.1 µg/mouse) was injected intraperitoneally after intratracheal (1 mg/kg) lipopolysaccharide (LPS) administration; flow cytometry was used to measure peripheral blood monocyte derived recruited macrophage and neutrophil numbers; resident alveolar macrophage was depleted by liposome clodronate; CXCL2, CCL2, MMP9 level was detected by RT-PCR and ELISA. In vitro, sorted resident macrophages (1×106) were cultured with LPS (1 µg/mL) and LXA4 (100 nmol/mL) with or without BOC-2 (10 µM) for 24 h to gain a better understanding of the mechanisms of LXA4. RESULTS: LXA4 inhibited tumor necrosis factor-a (TNF-a) and interleukin-1ß (IL-1ß) production induced by LPS. LXA4 also mediated LPS-induced macrophage recruitment and showed that this was dependent on CCL2 secretion and release by resident macrophages. LXA4 protects lung tissue by inhibiting neutrophil recruitment, partly through the CXCL2/MMP-9 signaling pathway. CXCL2 and MMP-9 are mainly expressed by resident macrophages and neutrophils, respectively. Finally, LXA4's beneficial effects were abrogated by BOC-2, an LXA4 receptor inhibitor. CONCLUSION: These results suggest that LXA4 may be a promising therapy for preventing and treating ARDS.

Emodin alleviates LPS-induced inflammatory response in lung injury rat by affecting the function of granulocytes.

BACKGROUND: Acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) are critical life-threatening syndromes characterized by the infiltration of a large number of granulocytes (mainly neutrophils) that lead to an excessive inflammatory response. Emodin (Emo) is a naturally occurring anthraquinone derivative and an active ingredient of Chinese medicine. It is believed to have anti-inflammatory effects. In this study, we examined the impact of Emo on the pulmonary inflammatory response and the granulocytes function in a rat model of lipopolysaccharide (LPS)-induced ALI. RESULTS: Treatment with Emo protected rat against LPS-induced ALI. Compared to untreated rat, Emo-treated rat exhibited significantly ameliorated lung pathological changes and decreased tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). However, Emo has no protective effect on the rat model of acute lung injury with granulocyte deficiency. In addition, treatment with Emo enhanced the bactericidal capacity of LPS-induced granulocytes via the up-regulation of the ability of granulocytes to phagocytize bacteria and generate neutrophil extracellular traps (NETs). Emo also downregulated the respiratory burst and the expression of reactive oxygen species (ROS) in LPS-stimulated granulocytes, alleviating the damage of granulocytes to surrounding tissues. Finally, Emo can accelerate the resolution of inflammation by promoting apoptosis of granulocytes. CONCLUSION: Our results provide the evidence that Emo could ameliorates LPS-induced ALI via its anti-inflammatory action by modulating the function of granulocytes. Emo may be a promising preventive and therapeutic agent in the treatment of ALI.

PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury.

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

MCTR1 enhances the resolution of lipopolysaccharide-induced lung injury through STAT6-mediated resident M2 alveolar macrophage polarization in mice.

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro-resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post-treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS-induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin-1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS-induced lung injury.

RvD1 ameliorates LPS-induced acute lung injury via the suppression of neutrophil infiltration by reducing CXCL2 expression and release from resident alveolar macrophages.

Acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) are life-threatening critical syndromes characterized by the infiltration of a large number of inflammatory cells that lead to an excessive inflammatory response. Resolvin D1 (RvD1), an endogenous lipid mediator, is believed to have anti-inflammatory and proresolving effects. In the present study, we examined the impact of RvD1 on the pulmonary inflammatory response, neutrophil influx, and lung damage in a murine model of lipopolysaccharide (LPS)-induced ALI. Treatment with RvD1 protected mice against LPS-induced ALI, and compared to untreated mice, RvD1-treated mice exhibited significantly ameliorated lung pathological changes, decreased tumor necrosis factor-α (TNF-α) concentrations and attenuated neutrophil infiltration. In addition, treatment with RvD1 attenuated LPS-induced neutrophil infiltration via the downregulation of CXCL2 expression on resident alveolar macrophages. Finally, BOC-2, which inhibits the RvD1 receptor lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2), reversed the protective effects of RvD1. These data demonstrate that RvD1 ameliorates LPS-induced ALI via the suppression of neutrophil infiltration by an ALX/FPR2-dependent reduction in CXCL2 expression on resident alveolar macrophages.

Effects of spermatogenic cycle on Stem Leydig cell proliferation and differentiation.

We reported previously that stem Leydig cells (SLC) on the surfaces of rat testicular seminiferous tubules are able to differentiate into Leydig cells. The proliferation and differentiation of SLCs seem likely to be regulated by niche cells, including nearby germ and Sertoli cells. Due to the cyclical nature of spermatogenesis, we hypothesized that the changes in the germ cell composition of the seminiferous tubules as spermatogenesis proceeds may affect tubule-associated SLC functions. To test this hypothesis, we compared the ability of SLCs associated with tubules at different stages of the cycle to differentiate into Leydig cells in vitro. SLCs associated with stages IX-XI were more active in proliferation and differentiation than SLCs associated with stages VII-VIII. However, when the SLCs were isolated from each of the two groups of tubules and cultured in vitro, no differences were seen in their ability to proliferate or differentiate. These results suggested that the stage-dependent local factors, not the SLCs themselves, explain the stage-dependent differences in SLC function. TGFB, produced in stage-specific fashion by Sertoli cells, is among the factors shown in previous studies to affect SLC function in vitro. When TGFB inhibitors were included in the cultures of stages IX-XI and VII-VIII tubules, stage-dependent differences in SLC development were reduced, suggesting that TGFB may be among the paracrine factors involved in the stage-dependent differences in SLC function. Taken together, the findings suggest that there is dynamic interaction between SLCs and germ/Sertoli cells within the seminiferous tubules that may affect SLC proliferation and differentiation.

Maresin 1 attenuates mitochondrial dysfunction through the ALX/cAMP/ROS pathway in the cecal ligation and puncture mouse model and sepsis patients.

Inflammation always accompanies infection during sepsis. Mitochondrial dysfunction and the role of reactive oxygen species (ROS) produced by mitochondria have been proposed in the pathogenesis of sepsis. Maresins have protective and resolving effects in experimental models of infection. In the present study, we investigated the effects of maresin 1 (MaR1) on mitochondrial function in cecal ligation and puncture (CLP)-induced sepsis and sepsis patients to identify mechanisms underlying maresin 1-mediated stimulation of ROS in mitochondria. We found that treatment with MaR1 significantly inhibited production of cytokines, decreased bacterial load in the peritoneal lavage fluid, reduced the number of neutrophils, decreased lactic acid level and upregulated cyclic AMP (cAMP) concentration, with the outcome of decreased lung injury in CLP-induced sepsis in mice. The effects of MaR1 on downregulation nitric oxide (NOX) activity, improvement CAT and SOD activity to inhibit ROS production in mitochondria was dependent on lipoxin receptor (ALX) and cAMP. Survival rates were significantly increased after the treatment of mice with MaR1. In BMDM stimulated with LPS, MaR1 inhibited the ROS production, downregulated enzyme activity, reduced mtO2 production, increased mitochondrial membrane potential, improved adenosine triphosphate (ATP) content and mitochondrial DNA (mtDNA) copy number. Finally, the effects of MaR1 on ROS production in the blood of healthy volunteers stimulated with LPS or sepsis patients were associated with ALX and cAMP. Taken together, these data suggest that treatment with MaR1 could attenuate mitochondrial dysfunction during sepsis through regulating ROS production.

Effects of miR-338 on morphine tolerance by targeting CXCR4 in a rat model of bone cancer pain.

The present study aimed to investigate the effects of miR-338 on morphine tolerance through the targeting of CXC chemokine receptor-4 (CXCR4) in a rat model of bone cancer pain (BCP). Sprague-Dawley (SD) rats were obtained and divided into model saline (n=10), model morphine (n=50), normal saline (n=10) and normal morphine (healthy rats, n=10) groups. After BCP rat model establishment, the remaining SD rats (n=40) in the model saline group were assigned into pLV-THM-miR-338, pLV-THM-anti-miR-338, CXCR4 shRNA, blank and PBS groups. Luciferase reporter gene assay was used for luciferase activity. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to detect the miR-338 and CXCR4 mRNA and protein expression. The model saline group showed increased mRNA and protein expressions of CXCR4 but decreased miR-338 compared with the model saline group, and the model morphine group had increased mRNA and protein expressions of CXCR4 but decreased miR-338 compared with the model saline group. The mRNA and protein expressions of miR-338 in the pLV-THM-miR-338 group increased remarkably while those of the pLV-THM-anti-miR-338 group decreased significantly compared with the CXCR4 shRNA, blank and PBS groups. The pLV-THM-miR-338, pLV-THM-anti-miR-338, CXCR4 shRNA and CXCR4 mRNA groups all had lower mRNA and protein expressions of CXCR4 than those in the blank and PBS groups. miR-338 exerts significant influence in the inhibition of morphine tolerance by suppressing CXCR4 in BCP.

The effect of propofol on intrathecal morphine-induced pruritus and its mechanism.

BACKGROUND: Previous studies have shown that a low dose of propofol IV bolus had a beneficial effect on intrathecal morphine-induced pruritus in humans. However, its exact mechanism has not been fully understood. In this study, we hypothesized that propofol relieved intrathecal morphine-induced pruritus in rats by upregulating the expression of cannabinoid-1 (CB[1]) receptors in anterior cingulate cortex (ACC). METHODS: Twenty-four Sprague-Dawley rats were divided into a control group and 20, 40, 80 µg/kg morphine groups to create an intrathecal morphine-induced scratching model. The effects of propofol on intrathecal 40 µg/kg morphine-induced scratching responses were then evaluated. Sixty rats were randomly assigned to control, normal saline, intralipid, and propofol groups, with pruritus behavior observation or killed 8 minutes after venous injection of normal saline, intralipid, or propofol, and brain tissues were then collected for assay. Immunohistochemistry was then performed to identify the expression of CB (1) receptor in ACC, and the concentration of CB(1) receptor in ACC was determined by Western blot analysis. RESULTS: Compared with the control group, rats in the 20, 40, 80 µg/kg morphine groups had higher mean scratching response rates after intrathecal morphine injection (P =0.020, 0.005, and 0.002, respectively). There was a statistical difference between 20 and 40 µg/kg morphine groups at 10 to 15 and 15 to 20 timepoints after intrathecal morphine injection (P = 0.049 and 0.017, respectively). Propofol almost abolished the scratching response that was induced by 40 µg/kg intrathecal morphine injection (F[2, 15] = 46.87, P < 0.001; F[22, 165] = 2.37, P = 0.001). Compared with the intralipid and normal saline groups, the scratching behavior was significantly attenuated in the propofol group (P < 0.001). Compared with control, normal saline, and intralipid groups, the protein expression of CB(1) receptor in ACC (Western blot) in the propofol group increased (0.86 ± 0.21, 0.94 ± 0.18, 0.86 ± 0.13, and 1.34 ± 0.32, respectively, P < 0.001). There was no significant difference among control, normal saline, and intralipid groups. Compared with the control, normal saline, and intralipid groups, the average number of neurons of CB(1) receptor in the ACC area were higher in the propofol group (21.0 ± 1.4, 19.3 ± 1.8, 24.8 ± 7.7, and 37.2 ± 3.3, respectively, P < 0.001). CONCLUSIONS: Morphine elicits dose-independent scratching responses after intrathecal injection in rats. Morphine 40 µg/kg intrathecal injection-induced scratching responses can be prevented by propofol. Increased protein expression of CB(1) receptors in ACC may contribute to the reversal of intrathecal morphine-induced scratching.