| Intracerebral hemorrhage is a kind of common injury diseases in the central nervous system,which frequently occurs in the basal ganglia area and is associated with a high mortality and disability.After onset of intracerebral hemorrhage,in addition to the initial anatomical damage,the secondary injury caused by inflammatory reaction is often accompanied.Cerebral edema after stroke is a typical outcome of inflammatory injury,which is mainly related to the breakdown of the blood-brain barrier.The factors that cause damage to the blood-brain barrier mainly include two aspects: firstly,physical damage such as squeezing and fracturing caused by hematoma;secondly,biochemical damage caused by local inflammatory factor cascade caused by a large number of pro-inflammatory substances released after the rupture of blood cells.The action of multiple blood components and their metabolites can overactivate local immune cells,resulting in excessive inflammatory responses that disrupt the tight connection of the blood-brain barrier.Early surgical intervention can only relieve the physical damage,not stop the inflammatory storm that has already started,and provides non-significant improvement on long-term prognosis for intracerebral hemorrhage patients.Recent studies have also shown that the inflammatory responses after intracerebral hemorrhage are not confined to the central nervous system,but exsits close signal crosstalk to the peripheral environment.In the meanwhile,after intracerebral hemorrhage,patients are prone to getting infection due to the metabolic disorders and immune suppression,which consequently results in strengthening peripheral inflammatory signals and mobilizing monocytes from bone marrow to infiltrate into the lesion.As a receptor widely expressed on a variety of inflammation-related cells,C-C chemokine receptor 5(CCR5)plays an important role in the proliferation,activation and cytokine production of immune cells,especially in controlling immunocyte migration under physiological and pathological conditions.It has been confirmed that it is involved in the regulation of neuronal pyrosis after intracerebral hemorrhage.However,the role of CCR5 in peripheral-central inflammatory interactions after intracerebral hemorrhage is still lacking.In this study,we focus on the essence of post-hemorrhage cerebral edema,namely blood-brain barrier disruption.Aiming to the central-peripheral inflammation crosstalk,we started from the CCR5,and used clinical demonstration,experimental verification and mechanism exploration to elaborate the role and mechanism of peri-central inflammation mediated by CCR5 in the pathological process of cerebral edema after intracerebral hemorrhage.It mainly includes the following three aspects:Part 1 Intense peripheral inflammation induced by sepsis worsened the cerebral edema and neurological functional prognosis in patients with intracerebral hemorrhageObjectives:To explore the effects of intense peripheral inflammation induced by sepsis on the occurrence of cerebral edema and prognosis of neurological function in patients with spontaneous intracerebral hemorrhage.Methods:Patients with spontaneous intracerebral hemorrhage admitted to our center from January2018 to June 2020 were retrospectively collected and divided into two groups according to whether sepsis occurred after intracerebral hemorrhage.Clinical data and prognostic scores between the two groups were compared and analyzed.The peak of inflammation in patients with intracerebral hemorrhage combined with sepsis was recorded and compared with the peak of cerebral edema to find the relationship between them.Risk factors for poor prognosis were identified by stepwise regression analysis.Results:1.The peak of inflammation in patients with sepsis after intracerebral hemorrhage was consistent with that of cerebral edema;2.The occurrence of sepsis is an independent risk factor for poor prognosis in patients with intracerebral hemorrhage;3.Sepsis increased the 28-day mortality rate and decreased the neurological function score in patients with intracerebral hemorrhage;4.Intracerebral hemorrhage patients with sepsis had the worsen GCS score at admission,and higher incidence of hematoma rupture into ventricle.Conclusions:The incidence of sepsis in patients with intracerebral hemorrhage is high and is associated with poor prognosis.As an important indicator to evaluate the degree of inflammation in sepsis,PCT level usually peaks at 3-5 days after intracerebral hemorrhage complicated with sepsis,which is consistent with the peak of cerebral edema and closely correlated with poor prognosis,thus confirming that the aggravation of peripheral inflammation is involved in the deterioration of central inflammatory injury.Part 2 CCR5 effects for blood-brain barrier disruption under the condition of increased peripheral inflammation after intracerebral hemorrhageObjectives:To explore whether peripheral inflammation promotes the occurrence of cerebral edema and blood-brain barrier injury after cerebral hemorrhage,and to demonstrate whether CCR5 signal participates in this process.Methods:Based on the constructed mouse model of increased peripheral inflammation after intracerebral hemorrhage,the motor function,cerebral edema and blood-brain barrier disruption of the mice were evaluated in various ways,and then the expression of CCR5 signal was verified by WB,IF and other methods.Results:1.The occurrence of peripheral inflammation after intracerebral hemorrhage aggravated motor dysfunction and brain edema and blood-brain barrier disruption in mice,especially at day 3;2.The expression of CCR5 and CCL5 in brain tissue and CCL5 in peripheral blood increased due to the aggravation of peripheral inflammation after intracerebral hemorrhage,and reached a peak at day 3;3.The expression of CCL5 and CCR5 was related to the movement disorder of mice,cerebral edema and blood-brain barrier injury;4.CCR5 was co-localized with GFAP,IBA-1 and MPO via immunofluorescence.Conclusions:CCR5 is expressed in astrocytes,microglia and monocytes,and activated by CCL5 ligand,participates in the occurrence of peripheral inflammation,which aggravates the cerebral edema,blood-brain barrier disruption and motor dysfunction after intracerebral hemorrhage in mice.Part 3 Maraviroc inhibited the CCR5 expression and subsequently reduce blood-brain barrier damage induced by increased peripheral inflammation after intracerebral hemorrhageObjectives:To explore the specific molecular mechanism of CCR5-mediated peripheral inflammation on the blood-brain barrier injury after intracerebral hemorrhage,and to demostrate the clinical transformation possibility for maraviroc,CCR5 tageted antagonist,to be regarded as the drug for alleviating the secondary brain injury after intracerebral hemorrhage.Methods:Based on the constructed mouse model of increased peripheral inflammation after intracerebral hemorrhage,the downstream molecules of CCR5 were identified by WB.After the expression of CCR5 was specifically interfered with,the motor function,the degree of cerebral edema and the integrity of the blood-brain barrier in mice as well as the changes of downstream molecular expression were evaluated.Then CRISPR plasmid was used to specifically knockdown the downstream molecule of CCR5 namely JAK2 to further demonstrate the scientific rationality of this molecular pathway.Results:1.Increased peripheral inflammation after intracerebral hemorrhage can promote the expression of CCR5/JAK2/STAT3/MMP9 pathway;2.Maraviroc specifically block the CCR5 expression,which can down-regulate the expression of JAK2,STAT3 and MMP9 protein,and alleviate the blood-brain barrier damage and neurological dysfunction caused by increased peripheral inflammation after intracerebral hemorrhage;3.The specific activation of CCR5 by r CCL5 can up-regulate the expression of JAK2,STAT3 and MMP9,and worsen the blood-brain barrier disruption and neurological dysfunction caused by the aggravation of peripheral inflammation after intracerebral hemorrhage;4.CRISPR plasmid specific knockdown of JAK2 partially reversed blood-brain barrier disruption and neurological dysfunction caused by up-regulation of CCR5 expression.Conclusions:CCR5 is partially involved in blood-brain barrier injury and motor dysfunction induced by increased peripheral inflammation after intracerebral hemorrhage through upregulating JAK2/STAT3/MMP9 pathway expression.Maraviroc can alleviate these impairments by specifically inhibiting CCR5 expression. |
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