| Chapter ?The pathological characteristics of peripheral inflammatory cells from patients and rat after intracerebral hemorrhageBackground and purposeIntracerebral hemorrhage(ICH),which has a high rate of mortality and disability,is one of the main causes of death and disability besides cardiovascular diseases in China.Although surgical treatment shows slightly superior than drug administration in cases of superficial lobe hemorrhage,neither therapies is effective in deep lobe hemorrhage.Several clinical studies have shown that higher neutrophils and NLR ratio were associated with poor ICH outcome,suggesting that inflammatory response played an important role in ICH-induced secondary brain injury.Therefore,we retrospectively analyzed the epidemiology and blood routine examination of ICH-patients hospitalized,and explored the pathological characteristics of peripheral blood and infiltrated PMNs in in ICH rats.Materials and methodsThe study was divided into 2 parts.First,retrospective analysis of clinical cases:We retrospectively review the medical records of acute spontaneous ICH patients hospitalized at the Neurosurgery department from January 1 2016 to January 30 2019,who underwent admission routine blood sampling and cranial computed tomographic neuroimaging within24 hours from symptom onset.Demographics,medical history,admission blood pressure and were considered.Total leukocyte(Leu),absolute Polymorphonuclear neutrophils(PMNs)count,and absolute lymphocyte(Lym)count were collected from admission blood work.ICH patients were divided into subgroup PMNs elevated group(ICH-PMNs-H)and PMNs normal group(ICH-PMNs-N)according to whether the PMNs count in peripheral blood routine was greater than 6.3*10~9/L.Inflammatory cells changes were further analyzed.The control group were meningiomas patients at Same time period.Second,Rat ICH model was established by injecting autologous non-anticoagulant whole blood into the right basal ganglia.The infiltration of lesions and inflammatory cells in peripheral blood were analyzed after ICH.Result1.Ninety-six ICH patients were included in this retrospective analysis.Leu and PMNs counts were(10.02±3.63)*10~9/L and(8.31±3.53)*10~9/L,respectively.In control group,75patients were included.Leu and PMNs counts were(5.83±1.62)*10~9/L and(3.73±1.44)*10~9/L,respectively.It indicated that Leu and PMNs counts in ICH group were significantly higher than control group(p<0.01).The Lym counts is lower in ICH group than control group,there have significant statistical difference in both groups((1.26±0.81)*10~9/L vs(1.63±0.50)*10~9/L,p<0.01).NLR ratio in ICH group is about three times higher than control group(7.97±4.90 vs 2.61±1.68,p<0.01)2.ICH subgroup analysis showed that the proportion of ICH-PMNs-H group was64.58%(62/96),and ICH-PMNs-N group was 35.41%(34/96).The percentage of PMNs in Leu in ICH group,ICH-PMNs-H group,ICH-PMNs-N group and Control group were(81.62±0.08)%,(84.54±0.06)%,(76.30±0.08)%,(62.61±0.10)%;(62.61±0.10)%,respectively.There were statistical differences between ICH group with Control group(p<0.01)and ICH subgroup with Control group(p<0.01)There were also statistical differences there have significant statistical difference in PMNs,Lym count,NLR ratio between ICH-PMNs-N and Control group(p<0.01).3.The ICH model in SD rats showed that the Leu count and PMNs count is higher than Sham group,have significant statistical difference in both group(p<0.01).NLR increased significantly in ICH group than Sham group(p<0.01).In addition,there are a large number of PMNs infiltrating in the brain parenchyma around the intracranial hematoma after ICH model.Conclusion1.The Leu counts,PMNs counts and NLR increased,and the Lym counts decreased after ICH in patients.2.In the ICH model of SD rats,the Leu counts,PMNs counts and NLR also increased,and PMNs infiltrated around the hematoma,suggesting that the ICH model of autologous blood was suitable to study the role of PMNs and the relevant intervention research after ICH.Chapter ? The role and mechanism of simvastatin in regulating polymorphonuclear neutrophils Mcl-1 in reducing intracerebral hemorrhage injuryBackground and purposeStatin is widely used to regulate blood lipid and cardiovascular diseases,and has been proved to have good clinical efficacy.A study has shown that simvastatin treatment can increase PMNs apoptotic rate and reduce inflammation in patients after cardiac bypass surgery.Increasing evidence suggests that statin has potential neuroprotective effects after ICH and can improve the prognosis of ICH patients.Mcl-1 has been identified as a key gene regulating apoptosis of PMNs.Therefore,we hypothesize that simvastatin can promote PMNs apoptosis by down-regulating Mcl-1 gene-related protein expression,reduce NLR ratio and reduce excessive aggregation of PMNs cerebral hemorrhage area,which may play a beneficial role in the prognosis of ICH patients.Therefore,on the basis of the first part of the experimental study,we focus on the role of simvastatin in the occurrence and development of ICH and further explain whether statin plays a neuroprotective role by down-regulating the expression of Mcl-1.Materials and methodsThis research was divided into 3 parts.First,to examine whether simvastatin could decrease peripheral PMNs count and PMNs brain-infiltrating,ninety rats had an intracaudate injection of 100 ml of blood.The sham control received only needle injection.The animals were randomly assigned to three groups.Group 1 received simvastatin(2 mg/kg/d,i.p.)from 5 days before ICH until sacrificed,and the control group received the same volume of vehicle.Some rats(n= 4 per group,each time point)were sacrificed for RT-PCR analysis of TNF-a,IL-6,CCL2,CXCL10 and ICAM-1 at 6,12,and 24 h after ICH.Other animals(n=5 for sham group,n =8 for ICH+Simva.group and ICH+Veh.Group each time point)were employed for blood cell count and MPO staining at 1,3,and 7 days after ICH.In the second part,to evaluate the neuroprotective effect of simvastatin treatment on brain injury following ICH,rats were randomly divided into three groups and treated as part I.Neurological function scoring was conducted in some rats one day before ICH and 1,3,and 7 days following ICH(n=6 per group).Other animals(n=6 per group,each time point)were sacrificed for brain water content measurement at 24 and 72 h after ICH.In the third part,to explore the mechanism underlying simvastatin-mediated against peripheral PMNs brain-invading after ICH,rats were randomly divided into three groups and treated as part I.Flow cytometric analysis of peripheral PMNs apoptosis(n=6 per group,each time point)and lymphocytes apoptosis(n=6 per group,each time point)were conducted at days 1,3,and 7 after ICH.Then,the level of apoptotic related proteins was detected using Western blotting analysis at 24 and 72 h after ICH(n=4 per group for each time point).Result 1.Blood routine examinationCompared with the sham group,the ICH rats presented higher PMNs count and higher NLR at 24 h after blood injection.Then,to test the potential effect of simvastatin on modulating Leu change following ICH,we conducted the dynamic blood routine analysis.The total Leu count in Veh-group has no difference among the Simva-,Veh-and shamgroups on day1,but markedly increased on day3,and reached a peak on day7 after ICH.However,the total Leu count in the Simva-group remains stable on the 3 days and 7 days post-ICH.The PMNs count in the Simva-group was significantly lower than the control group on day1 and day7 after ICH.Of note,24 h after ICH,the Simva-group displayed dramatically decreased NLR compared to Vehicle controls.These results suggest that elevated PMNs count and NLR also presented in the experimental ICH model in a rat,which was effectively reversed after simvastatin treatment.Furthermore,we also analyzed the count of peripheral blood mononuclear cells(PBMCs)and monocyte in this rat model of ICH.PBMCs elevates on 72 h after ICH and stay high for up to 7 days.Simvastatin effectively reduced the PBMCs count on day 7 post-ICH.Notably,simvastatin significantly decreased the monocyte count from 24 h to day 7 after ICH.2.MPO immunofluorescence staining Compared with the Sham group,both Simva-group and Veh-group showed a lot of MPO positive cells in the area around the hematoma,in which the MPO positive cells count in the Simva-group was less than the control one on the days 1,3,and 7 post-ICH.This data indicate that simvastatin effectively prevented peripheral PMNs infiltrating into brain parenchyma,which may be attributed by cutting down the number of PMNs in the circulation after ICH.3.Expressions of inflammatory factors,chemokines and adhesion factorsThe expressions of IL-6 and TNF-a,CCL2,CXCL10 and ICAM-1 in the brain tissues around the hematoma in Sham group were at very low levels.TNF-a and CXCL10 reached peak at 6 hours after ICH,then decreased.IL-6,CCL2 and ICAM-1 reached peak at 12 hours after ICH,and decreased at 24 hours.RT-PCR analysis showed a lower expression level of all above chemokines in the ICH+Simva group than in the ICH+Veh group at 6,12,and 24 h after ICH.4.Evaluation of brain edema The brain water content of ipsilateral basal ganglia and cortex in ICH+Veh group was significantly higher than that in Sham group at 24 hours and 72 hours after blood injection,moreover the brain water content at 3days was higher than 24 h after ICH.while the water content of ipsilateral basal ganglia and cortex in ICH+Simva group was significantly lower at 24 hours and 72 hours than that in ICH+Veh group.The brain water content of ipsilateral basal ganglia and cortex was dramatic decline when use simvastatin therapy.There was no significant difference in contralateral basal ganglia,cortex and cerebellum in every group.5.Neurobehavioral Assessment The animals scores of m NSS was increased significantly,the number of forelimbs placed and extended to the left forelimb decreased significantly,and the ability to turn to the left decreased significantly in the corner test after ICH,there was a significant difference between ICH+Veh group and Sham group(p<0.05).The results of m NSS score,forelimb placement and corner test showed improvement at 24 h,3days and 7days when use simvastatin therapy,but there only has significant difference at 7days in ICH+Simva group and ICH+Veh group(p<0.05).6.Apoptosis of PMNs and Lym A: Apoptosis of PMNs cells.The apoptotic ratio of PMNs in peripheral blood was(13.04±1.65)%,(13.74±1.90)% and(12.73±1.47)% at 24 h,3days and 7days,respectively.The apoptotic rate of PMNs in peripheral blood increased(15.34±1.50)% at 24 hours,and maintained a low level(5.36±1.56)% and(4.88±2.09)%)at 3 days and 7 days after blood injection,there was significantly difference compare with Sham group(p<0.05).The apoptotic rate of PMNs in ICH+Simva group increased more significantly at 24 hours(17.68±1.86)%,and decreased to(8.02±2.27)% at 3 days,both all higher than ICH+Veh group(p<0.05).At 7 days,the apoptotic rate of PMNs has no significantly difference in ICH+Simva group and ICH+Veh group(p>0.05).B: Apoptosis of Lym cells.The apoptotic ratio of Lym cells were(13.93±1.23)%,(13.23±2.11)% and(12.49±1.76)% in Sham group at 24 h,3days and 7days,respectively.In ICH group,the apoptotic ratio of Lym cells has a short dip at 24 hours(11.79±1.55)%,but rapidly rising at 3days(27.70±3.63)% and then decreased at 7days(17.01±3.13)%.Simvastatin administration can effectively increased the apoptotic ratio of lymphocytes on days 1 and 7 post-ICH(16.49±1.30)% and(22.96±2.63)%,there was significantly difference than ICH +Veh group(p<0.05),suggesting simvastatin has potential proapoptotic effect on various subtypes of leukocyte not just on PMNs.7.Western blot assay At 24 hours after ICH,the expression of anti-apoptotic protein Mcl-1 and Bcl-2 in PMNs decreased,while the expression of pro-apoptotic protein Bax and Cleaved caspase-3 increased,there was significantly different compared with Sham group(p<0.05).The expression of anti-apoptotic protein Mcl-1 and Bcl-2 was significantly reduce and the pro-apoptotic protein Bax and Cleaved caspase-3 was significantly higher than ICH group after simvastatin treatment,there was significantly different compared to ICH group(p<0.05).Conclusion 1.PMNs and NLR increased after ICH,PMNs infiltrated around hematoma,and the expression levels of chemokines,adhesion factors and inflammatory factors increased,which together led to the aggravation of brain edema,BBB injury and neurological dysfunction after ICH.2.Simvastatin pretreatment can reduce Leu and PMNs counts,increase Lym counts and decrease NLR ratio after ICH,reduce PMNs infiltration and the expression of chemokines,adhesion factors and inflammatory factors,alleviate brain edema,protect BBB and improve neurological function.3.Preliminarily explain the protective mechanism of simvastatin on ICH:down-regulate the expression of anti-apoptosis gene-related proteins Mcl-1 and Bcl-2 in PMNs,up-regulate the expression of apoptosis-related proteins Bax and Cleaved caspase-3,and promote the apoptosis of PMNs.Chapter ?: The mechanism of simvastatin on alleviating brain injury after intracerebral hemorrhage through mediating PMNs apoptosis via FPR2-MAPK-Mcl-1 signaling pathwayBackground and purposeOur previous studies showed that HMG-Co A reductase inhibitor-simvastatin could effectively reduce PMNs count and NLR ratio in peripheral blood,inhibit PMNs infiltration,alleviate brain edema and improve neurological dysfunction.Further studi es showed that simvastatin-mediated neuroprotective effects after ICH might be partially by accelerating apoptosis of PMNs,reducing the expression of chemokines,intercellular adhesion molecule and inflammatory factors.Simvastatin has potential neuroprot ective effects by acting on PMNs,which may become a novel therapeutic target.N-formyl peptide receptor 2(FPR2)is located on the surface of PMNs.Mitochondrial gene-encoded mitochondrial peptides(FP),cardiolipin,ATP,etc.can be used as damage-related molecular patterns(DAMPs)to activate FPR2 receptor,recruit periphery inflammatory cells including PMNs and promote inflammation cells infiltration in perihematomal and causes secondary brain injury.Studies have shown that FP binding to FPR2 receptors of PMNs membrane can causes intracellular calcium homeostasis imbalance and phosphorylation of MAPKs(P38,PERK),and induces secretion of IL-6 and TNF-?to produce inflammatory response.Based on the above,we speculate that simvastatin may play pro-apoptotic role in PMNs through FPR2-MAPK-Mcl-1 pathway.Therefore,this part of the experimental focusing on the Mcl-1 upstream pathway of regulate PMNs apoptotic,and further exploration base on our previous studies.Materials and methodsThis study was divided into 3 parts.First,to examine whether simvastatin and Boc-2 can cause LXA4 content changes in plasma at 24 h and 72 h by Elisa assays and to examine whether simvastatin,LXA4 and Boc-2 could influence peripheral PMNs count.Thirty-nine rats had an intracaudate injection of 100 ml of blood.The sham control received only needle injection.Some rats(n=5 for sham group,ICH+Veh.Group,ICH+Simva group and ICH+Simva+Boc-2 group)were sacrificed for Elisa assays analysis of LXA4 content in plasma after ICH.Other animals(n=6 for sham group,ICH+Veh.Group,ICH+LXA4 group,ICH+Simva group and ICH+Simva+Boc-2 group)were employed for blood cell count at 24 h.Simvastatin(2 mg/kg/d,i.p.)from 5 days before ICH until sacrificed,LXA4(10ug/kg,i.p.)and Boc-2(100ug/kg,i.p.)was used 30 minutes before surgery,and the control group received the same volume of vehicle.In the second part,to explore the Mcl-1 upstream pathway of simvastatin-mediated peripheral PMNs.The level of related proteins-FPR2,MAPK(P38/pp38,PERK/P-PERK),Mcl-1 and Bax,were detected using Western blotting analysis at 24 h after ICH(n=4 for ICH+Veh.Group,ICH+LXA4 group,ICH+Simva group and ICH+Simva+Boc-2 group),and treated as part I.In the third part,to verify whether Boc-2 blocked the neuroprotective effect of simvastatin.A.Extract brain tissue proteins around hematoma and detect the expression of inflammatory factors(TNF-a and IL-6),complement C3 and Myelin basic protein(MBP)expression(n=4 per group).B.To examine PMNs infiltration by MPO immunofluorescence staining(n=5 for per group);to determine brain water content by Wet-dry weighting method(n=5 for per group);Neurobehavioral assessment was performed at 1d,3d and 7d after ICH(n=6 for per group)to assay the neurological dysfunction.Result1.Simvastatin can increase the level of LXA4 in peripheral blood plasma of rats after ICH.2.Leu and PMNs counts in peripheral blood increased after ICH,and simvastatin or LXA4 treatment can reduce the increased Leu and PMNs counts;Boc-2 could block the therapeutic effect of simvastatin.3.Simvastatin and LXA4 can up-regulate the expression of FPR2 receptor of PMNs,decrease the phosphorylation level of MAPK-related protein(P38,PERK)of PMNs,down-regulate the expression of anti-apoptotic protein Mcl-1 and up-regulate the expression of pro-apoptotic protein Bax of PMNs,and Boc-2 can block these effects of simvastatin.4.Simvastatin and LXA4 can reduce the expression of inflammatory factors and complement C3 of brain tissue,up-regulate the expression of MBP of brain tissue;and Boc-2 can block these effects of simvastatin.5.Simvastatin and LXA4 can alleviate brain edema and promote the recovery of nerve function after ICH,while brain edema and neurobehavioral recovery is slowed down when use Boc-2.Conclusion1.Simvastatin can up-regulate the level of LXA4 in plasma after ICH,and then promote the PMNs apoptosis through FPR2-MAPK-Mcl-1 pathway;Boc-2 can block the pro-apoptotic effect of simvastatin on PMNs.2.Simvastatin and LXA4 can reduce Leu and PMNs counts in peripheral blood,decreases PMNs infiltration in perihematoma,decreases inflammatory factors and complement C3 levels,increases MBP content,alleviates brain edema and promotes neurological recovery,and Boc-2 can block these effects of Simvastatin. |
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