| Research background and purpose:Cerebral ischemia is one of the main causes of death and disability in humans,recirculation of blood in a short period of time is the key to improve patients’ recovery.However,relevant studies have shown that after reperfusion,pathophysiological phenomena such as inflammatory factors appear in brain tissue,thus brain is faced with the risk of aggravated injury,which greatly reduces the clinical benefits of reperfusion therapy.CO as a product of heme,has shown neuroprotective effects in previous studies,but its effective release is an unsolved problem in research.HBOC can carry and release oxygen and play a protective role against various organ injuries.Interestingly,hemoglobin is much more capable to carbon monoxide than oxygen,so it may be a potential carrier of CO.Therefore,a new agent,CO-HBOC(also known as CO-Poly PHb),has been prepared in this study.And we explored its influence on cerebral ischemia reperfusion injury,this experiment aims to provide a new idea for the effective utilization of CO,and provide a new direction for reducing this injury.Materials and Methods:The first part: 45 SPF male C57BL/6 mice,were divided into: sham+ NS(group C),t-MCAO+ NS(group M),t-MCAO+ CO-HBOC(group MC).The mice in the group M and group MC were performed occlusion of blood flow for 90 mins.The group MC was given CO-HBOC 0.3g/Kg/day through tail vein at the time of reperfusion for 3consecutive days,and the other two groups were given the same amount of normal saline.After 72 hours of reperfusion,we evaluated the neurological function,measured the cerebral infarction area of mice in each group,and observed the damage of brain cells by HE staining.And we detected the release of inflammatory factors in plasma by ELISA,the relative expression of IL-1β,IL-6,TNF-α,and TREM-1,ERK and NF-κB was detected by RT-q PCR,and the concentration of TREM-1 and P-ERK/P-NF-κB were detected by WB.The second part: 30 mice were randomly divided into: t-MCAO + CO-HBOC(group MC),t-MCAO + CO-HBOC +LP17(group MCL).The group MC was given CO-HBOC only as the first part,and the group MCL was also given the LP17 1mg/ Kg/day via nose,consecutive 3 days.After 72 hours,we evaluated the the same metrics as in section 1.Graphpad Prism 6.0 was used for data analysis.The normal distribution data was expressed as mean ± standard error(SEM).The ANOVA was used to test the data of samples of multiple groups,and the T test was used for samples of two groups.Data that did not conform to homogeneity of variance were tested by Kruskal-Wallis H.P <0.05 indicated statistically significant difference.Results:The first part: 1.The neurological function of mice in group M was significantly impaired,but this in group MC was recovered,and the neurological function score was decreased(all P < 0.01).2.The brain tissue in group C was normal by TTC,but in group M showed obvious infarction,while the infarct size in group MC was smaller(P< 0.01).3.HE staining of brain tissue showed severe brain injury in mice of group M,while CO-HBOC could reverse neuronal injury in group MC.4.Compared to group C,the levels of IL-1β,IL-6 and TNF-α in plasma of group M had increased(all P < 0.01),and the relative RNA levels were also rised(all P < 0.01).While the plasma expression of inflammatory factors in group MC was decreased(IL-1β: P < 0.05,IL-6: P < 0.01,TNF-α: P < 0.01);and the relative RNA levels were also decreased(all P < 0.01).5.WB results indicated the levels of LC3 II was risd meanwhile P62 was reduced in group M(all P < 0.05).however,autophagy of group MC was weakened comparatively(all P< 0.05).6.Compared with group C,the relative RNA expression of TREM-1 and ERK/NF-κB was increased in group M(all P < 0.01);and the protein content of TREM-1,P-ERK and P-NF-κB increased(TREM-1: P < 0.01,P-ERK: P < 0.05,P-NF-κB: P < 0.05).Compared with group M,the relative RNA expression of this factors in group MC were decreased(all P < 0.01);and the concentrates of proteins were also declined(all P < 0.05).The second part: 1.When CO-HBOC was combined with LP17,the protein expression in group MCL was reduced more significantly compared with group MC(TREM-1: P < 0.05,P-ERK: P < 0.05,P-NF-κB: P < 0.01).2.The group MCL inhibited the inflammatory cytokines more significantly than group MC(IL-1β: P<0.05,TNF-α:P<0.05).3.It was also found that compared with group MC,the LC3 II expression was suppressed more significantly,and P62 was upped in group MCL(all P< 0.05).4.In the animal models,we further confirmed that the recovery of neurological dysfunction in group MCL was more obviously than that in group MC(general: P < 0.01,focal: P <0.05);and infarction size also declined more evidently(P < 0.05).Conclusion:1.CO-HBOC can reduce inflammatory response,inhibit autophagy,and inhibit TREM-1/ERK/NF-κB signaling pathway,protect mice from cerebral ischemia reperfusion injury;2.CO-HBOC can reduce the inflammatory response and autophagy by inhibiting TREM-1. |
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