Objective To investigate the effect of hemin on neuronal necroptosis and its mechanism in mice after intracerebral hemorrhage.Methods (1) Experiment one: 24 C57BL/6 mice were divided into sham-operated group, saline control group, 5 mmol/L hemin group, and 10 mmol/L hemin group (n=6); mice in the latter two groups were injected 20μL 5 mmol/L or 10 mmol/L heme solution into the corpus striatum to prepare cerebral hemorrhage models, and the normal saline group was injected 20μL normal saline; Western blotting was used to detect the protein expressions of heme oxygenase-1 (HO-1), receptor-interacting protein (RIP)1, RIP3, and mixed lineage kinase domain like protein (MLKL). Three C57BL/6 mice were injected 20μL 10 mmol/L heme solution into the corpus striatum to prepare cerebral hemorrhage models; 24 h after that, double-labelling immunofluorescence was used to detect the expressions of Caspases-3 and neuron-specific nuclear protein (NeuN) in the hippocampus tissues. (2) Experiment two: the primary cultured hippocampal neurons were stimulated with 0, 10, 20, 40 and 80μmol/L heme for 6 h and 40μmol/L heme for 0, 1, 2, 4 and 6 h, respectively; lactic dehydrogenase (LDH) method was used to determine the neuron damage. After the primary cultured hippocampal neurons being stimulated with 40μmol/L heme for 0, 1, 2, 4 and 6 h, respectively, the protein expressions of interleukin-1 receptor (IL-1R)1, RIP1, RIP3, MLKL and caspase-3 in the neurons were detected by Western blotting, and the level of inflammatory factor interleukin-1β(IL-1β) was detected by ELISA. After primary cultured hippocampal neurons being stimulated with 0, 40μmol/L heme for 6 h, the expressions of NeuN and Caspase-3 in the neurons were detected by double-labelling immunofluorescence. (3) Experiment three: the primary cultured hippocampal neurons were pre-incubated with 0, 0.5, 1.0, and 1.5μg/mL IL-1R antagonist for one h, and then, stimulated with 40μmol/L heme for 6 h; the toxic injury of neurons was detected by LDH method, and the protein expressions of IL-1R1, RIP1, RIP3 and MLKL in neurons were detected by Western blotting.Results (1) As compared with those in the sham-operated group and saline control group, protein expressions of HO-1, RIP1, RIP3 and MLKL in the 5 mmol/L heme group and 10 mmol/L heme group were significantly increased (P<0.05); at 24 h after 10 mmol/L heme injection, Caspase-3 expression was found in frozen sections of hippocampal tissues of mice, but no co-localization of Caspase-3 and NeuN was found. (2) As compared with those in the 0μmol/L heme group, LDH release rates in the heme groups of different concentrations were significantly increased and concentration-dependent (P<0.05); as compared with the heme group of 0 h stimulation, the heme groups of different times of stimulation had significantly increased release rates of LDH in time dependent manner (P<0.05). As compared with the heme group of 0 h stimulation, the heme groups of different times of stimulation had significantly increased IL-1R1, RIP1, RIP3, and MLKL protein expressions (P<0.05); Caspase-3 showed no obvious changes. As compared with the heme group of 0 h stimulation, the heme groups of different times of stimulation had significantly increased IL-1βlevels in time dependent manner (P<0.05). No obvious Caspase-3 expression was noted in the heme group of 6 h stimulation. (3) As compared with the 40μmol/L heme group, pre-incubation groups of 0.5, 1.0, and 1.5μg/mL IL-1R antagonist had significantly reduced LDH release rates and protein expressions of IL-1R1, RIP1, RIP3 and MLKL (P<0.05).Conclusion sHemin could induce neuron cell death directly via a necroptosis pathway rather than Caspase3-mediated apoptosis. The cytotoxicity of hemin could be alleviated by blocking IL-1R.

Background and Purpose- Accumulated evidence suggests that hemin-a breakdown product of hemoglobin-plays a pivotal role in the inflammatory injuries that result after hemorrhagic stroke through the Toll Like Receptor 2-Toll Like Receptor 4 signal pathway. However, the mechanism of how hemin triggers neuronal necroptosis directly after intracranial hemorrhage (ICH) is still an area of active research. As animal model and preclinical studies have shown, the recombinant interleukin-1 receptor antagonist (IL-1RA) improves clinical outcomes after stroke. As such, we have chosen to investigate the mechanism of how IL-1RA exerts protective effect in hemin-induced neuronal necroptosis after ICH. Methods- Our ICH model was induced by hemin injection in C57BL/6 mice and IL-1R1-/- mice. In addition, we used primary cultured neurons to assess hemin-induced cell death. Co-immunoprecipitation, immunoblot, immunofluorescent staining, neurological deficit scores, and brain water content were used to study the mechanisms of IL-1R1 modulation in neuronal necroptosis both in vitro and in vivo. Results- Free hemin could mediate neuronal necroptosis directly by assembling necrosome complex and then to trigger cell death. This phenomenon was driven by IL-1R1 as IL-1R1 can form a complex with necrosome. After treatment with IL-1RA, both the expression and translocation of the necrosome decreased while disruption of the interaction between IL-1R1 and RIP1/RIP3 (receptor interacting protein 1/3) increased neuron survival. In addition, the IL-1R1-deficient mice demonstrated lower levels of necrosome components, including RIP1, RIP3, and MLKL (mixed lineage kinase domain-like protein), compared with control groups after hemin treatment. In addition, the neurological deficit scores, brain water content, and inflammatory response were all also reduced in the IL-1R1-deficient mice. Conclusions- Functional inhibition of the interaction between IL-1R1 and the necrosome complex improves neuron survival and promotes the recovery of neurological function in experimental ICH. Targeting IL-1R1/RIP1/RIP3 assembly could be a promising therapeutic strategy for patients with ICH.

In this study, we explored the potential mechanisms of how PTEN regulating LPS induced TLR4 signaling pathway. The initial findings from ELISA demonstrate that PTEN influences TNF-α secretion by its lipid phosphatase activity. Subsequently, western blot, immunoprecipitation assay, and immunofluorescence were performed to explore the activation process of PTEN by stimulation with LPS. As early as 20 minutes after LPS stimulation, reduced phosphorylation of PTEN was found obviously. Accordingly, the whole cell-scattered PTEN translocated towards the cell membrane 20 minutes after stimulating with LPS. Moreover, the weak physical association between PTEN and TLR4 in resting RAW264.7 cells increased gradually after the stimulation of LPS. Furthermore, our study showed PTEN decreased LPS-induced Akt activity and upregulated NF-κB-dependent gene transcription, identifying indirectly that the PTEN could regulate the activation of NF-κB by its downstream Akt kinase. In summary, our study illustrates the potential signal transduction process of PTEN while stimulated by LPS: by increasing the association of TLR4, PTEN recruits to its phosphoinositide substrate PI(3,4,5)P3 located on the cell membrane and exerts its dephosphorylated function and subsequently depresses the activity of downstream molecule Akt and results in activation of NF-κB, followed by the secretion of inflammatory mediators TNF-α.

Objective To investigate the effect of dopamine on lipopolysaccharide (LPS)-induced inflammatory response in mouse peritoneal macrophages (MPMs).Methods MPMs were isolated after injection of thioglycolate broth into the peritoneal cavity.MPMs from wild type C57 mice were distributed into control group,LPS group,dopamine pretreatment group,dopamine D1-like and and D2-like receptor antagonist groups (D1 and D2 antagonist groups).In the latter two groups,MPMs were pretreated with Dl-like and D2-like receptor antagonist respectively for 30 min,and then stimulated with dopamine and LPS.MPMs from wild type (TLR4 +/+) and TLR4 knock-out (TLR4-/-) mice were only divided into LPS group and dopamine pretreatment group.In LPS group,MPMs were stimulated with 1 μg/ml LPS for 6 h.In dopamine pretreatment group,MPMs were pretreated with 10-4 mol/L dopamine for 2 h,and then stimulated with 1μg/ml LPS for 6 h.Expressions of TLR4 and pro-IL-1βwere detected by Western blot and tumor necrosis factor-α (TNF-α) in cell culture supernatant by ELISA method.Results (1) Expressions of TLR4,pro-IL-1β and TNF-α in control group were (0.56 ± 0.07),(0.65 ± 0.11) and (1,770.6 ±448.8) pg/ml;in LPS group were (1.12 ± 0.15),(1.24 ± 0.20) and (15,569.5 ± 822.7) pg/ml;in dopamine pretreatment group were (0.28 ± 0.11),(0.22 ± 0.08) and (7,800.7 ±862.6)pg/ml;in D1 antagonist group were (0.25 ±0.12),(0.18 ±0.09) and (7,065.0 ± 1016.8)pg/ml;in D2 antagonist group were (0.80 ±0.09),(0.44 ±0.08) and (14,299.6 ± 1430.9)pg/ml.The three indicators in LPS group were increased compared to control group and dopamine pretreatment group (P ＜ 0.05),and in D2 antagonist group were increased compared to dopamine pretreatment group (P ＜ 0.05).There were no obvious differences between D1 antagonist group and dopamine pretreatment group(P ＞0.05).(2) After LPS stimulation,expressions of pro-IL-1 β and TNF-α in TLR4+/+ mice were (0.94 ±0.17) and (15,109.0 ± 1,903.4)pg/ml,and were (0.08 ±0.04) and (5,063.6 ± 512.8) pg/ml in TLR4-/-mice (P ＜0.05).After dopamine pretreatment,expressions of proIL-1β and TNF-α were (0.45 ±0.12) and (6,383.9 ± 1,287.3) pg/ml in TLR4+/+ mice,and were (0.05 ± 0.02) and (4,863.0 ± 824.7) pg/ml in TLR4-/-mice.Expressions of pro-IL-1β and TNF-o were higher in TLR4 +/+ mice than in TLR4-/-mice after LPS stimulation (P ＜ 0.05).Expressions of pro-IL-1β and TNF-αin TLR4 +/+ mice were reduced after dopamine pretreatment compared to LPS stimulation (P ＜ 0.05),while were similar in TLR4-/-mice (P ＞ 0.05).Conclusion Dopamine can inhibit LPS-induced inflammatory response in MPMs,and the inhibition is strongly related to dopamine D2-1ike receptor and TLR4.

BACKGROUND:Wound healing is impaired in diabetes mellitus. The underlying mechanism involved in this process is still unknown. The Akt/mTOR signaling pathway plays a crucial role in the pathogenesis of diabetes.OBJECTIVE:we investigated the role of the Akt/mTOR pathway in diabetic wounds and the mechanisms that growth factors activate this pathway to promote diabetic wound healing.METHODS:Full-thickness skin excisional wounds were created on the backs of normal and streptozotocin-induced diabetic rats. The expression of key proteins in the Akt/mTOR pathway was assayed using western blotting; topical effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on diabetic wounds and activation of the Akt/mTOR pathway were subsequently investigated. Activation of the Akt/mTOR pathway by GM-SCF in vitro was examined in rat primary fibroblasts.RESULTS:The results indicate that the Akt/mTOR pathway was activated in the wound tissue of both non-diabetic and diabetic rats, as indicated by a remarkable increase in expression of total and phosphorylated key proteins in this pathway. However, the expression level of these proteins was dramatically attenuated in diabetic wounds compared with non-diabetic wounds. Upon topical application of GM-CSF, the diabetic wound healing was remarkably improved concomitantly with increased expression and phosphorylation of key proteins in the Akt/mTOR pathway. In addition, rat fibroblast proliferation induced by GM-CSF depended on the Akt/mTOR pathway activation.CONCLUSION:Impaired wound healing results from the dysfunction of the Akt/mTOR pathway in diabetic rats. The pharmacologic elevation of this pathway may represent an attractive intervention strategy to improve prognosis of diabetic wounds.

Objective: Inflammation plays a critical role in secondary brain damage after intracerebral hemorrhage (ICH). However, the mechanisms of inflammatory injury following ICH are still unclear, particularly the involvement of NLRP3 inflammasome, which are crucial to sterile inflammatory responses. In this study, we aim to test the hypothesis that NLRP3 signaling pathway takes a vital position in ICH-induced secondary inflammatory damage and detect the role of N-methyl-D-aspartic acid receptor 1 (NMDAR1) in this progress.Methods: ICH was induced in mice by microinjection of hemin into the striatum. The protein levels of NMDAR1, NMDAR1 phosphorylation, NLRP3 and IL-1 beta were measured by Western blot. The binding of NMDAR1 to NLRP3 was detected by immunoprecipitation.Results: The expression of NMDAR1, NMDAR1 phosphorylation, NLRP3 and IL-1 beta were rapidly increased after ICH. Hemin treatment enhanced NMDAR1 expression and NMDAR1 phosphorylation, as well in cultured microglial cells treated by hemin. Hemin up-regulated NLRP3 and IL-1 beta level, which was reversed by MK801 (NMDAR antagonist) in vitro. Hemin also promoted the binding of NMDAR1 to NLRP3.Conclusion: Our findings suggest that NMDAR1 plays a pivotal role in hemin-induced NLRP3-mediated inflammatory damage through synergistic activation. (C) 2015 Daping Hospital and the Research Institute of Surgery of the Third Military Medical University. Production and hosting by Elsevier B.V.

Ischemic stroke induces microglial activation and release of proinflammatory cytokines, contributing to the expansion of brain injury and poor clinical outcome. Propofol has been shown to ameliorate neuronal injury in a number of experimental studies, but the precise mechanisms involved in its neuroprotective effects remain unclear. We tested the hypothesis that propofol confers neuroprotection against focal ischemia by inhibiting microglia-mediated inflammatory response in a rat model of ischemic stroke. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. Propofol (50 mg/kg/h) or vehicle was infused intravenously at the onset of reperfusion for 30 minutes. In vehicle-treated rats, MCAO resulted in significant cerebral infarction, higher neurological deficit scores and decreased time on the rotarod compared with sham-operated rats. Propofol treatment reduced infarct volume and improved the neurological functions. In addition, molecular studies demonstrated that mRNA expression of microglial marker Cd68 and Emr1 was significantly increased, and mRNA and protein expressions of proinflammatory cytokines tumor necrosis factor-α, interleukin-1β and interleukin-6 were augmented in the peri-infarct cortical regions of vehicle-treated rats 24 h after MCAO. Immunohistochemical study revealed that number of total microglia and proportion of activated microglia in the peri-infarct cortical regions were markedly elevated. All of these findings were ameliorated in propofol-treated rats. Furthermore, vehicle-treated rats had higher plasma levels of interleukin-6 and C-reactive protein 24 h after MCAO, which were decreased after treatment with propofol. These results suggest that propofol protects against focal cerebral ischemia via inhibition of microglia-mediated proinflammatory cytokines. Propofol may be a promising therapeutic agent for the treatment of ischemic stroke and other neurodegenerative diseases associated with microglial activation.