Study For The Mechanisms Of FGF21 Alleviates Endothelial Mitochondrial Damage And Prevents Bbb From Disruption After Intracranial Hemorrhage

In recent years,Intracranial hemorrhage(ICH)has increasingly become a major cause of death in modern society.Its morbidity is approximately account for 10～20%in stroke diseases[1,2],which always leads to severe neural dysfunction even death.Its incidence is higher in male and Asian people[3],especially for patient inhibited in low-and middleincome countries[5].Compared to the ischemia stroke,ICH caused more death events and held a higher possibility of contributing to severe neural dysfunction.According to the published literatures,the mortality of ICH could be 40%per month and 54%per year.Only 12%-39%patients can return long-term norm life after ICH[5].Multiple risk factors would lead to ICH,such as hypertension,smoking,alcohol abuse and drug[6-10].Due to different location of the hemorrhage and hematoma volume,individual present different symptoms[11].The disruption of the brain-blood barrier(BBB)is a vital event for the secondary damage caused by ICH.Under normal conditions,the BBB is responsible for preventing harmful materials from entering the central nervous system(CNS)and maintaining routine exchange with the peripheral circulatory system[12,13].Damage to the BBB aggravates brain swelling and permits inflammatory cell infiltration.Such events form a vicious cycle and cause secondary injury after ICH[14].As an essential component of the BBB,the neurovascular unit consists of cerebral microvascular endothelial cells(ECs),astrocytes,pericytes,neurons and an extracellular matrix.Among these structures,ECs and their membrane proteins,such as tight junction proteins(TJ-ps)and adherens junction proteins(AJ-ps),execute a series of important functions.Due to detrimental stresses caused by ICH,the energy supply for ECs is gradually exhausted.Thus,under conditions of ICH injury,maintaining the metabolic homeostasis of ECs by preserving normal mitochondrial function could be a rational treatment strategy.Fibroblast growth factor 21(FGF21),which is an endocrine molecule,belongs to the fibroblast growth factor(FGF)family.According to the literature,FGF s widely take part in maintaining the metabolic homeostasis of different cells and exerting beneficial effects in a variety of diseases[15-17].Mouse FGF21 and human FGF21 are highly homologous(～75%identity.Due to its unique characteristic of a low binding affinity for heparin,FGF21 is capable of crossing the BBB and binding to fibroblast growth factor receptor 1(FGFR1)to accomplish different biological functions.According to accumulating evidence,administration of FGF21 to rodents alleviates neural damage in multiple diseases.For example,it protects endothelial cells in ischemia/reperfusion(I/R)or traumatic brain injury,alleviates hippocampal damage in obesity-induced cognitive dysfunction,prevents neuronal ferroptosis after spinal cord injury and attenuates neuronal inflammation and oxidant stress in aging and diabetes.However,few studies have evaluated the protective effects of FGF21 against ICH.Therefore,in this study,we aimed to explore whether FGF21 can alleviate BBB damage and promote brain recovery after ICH.First,the clinical data of 77 ICH patients were collected and analyzed to evaluate the correlation between the serum level of FGF21 and ΔGCS%.Next,given the energy-exhausted states caused by ICH and the vital role of mitochondria in preserving metabolic homeostasis,we hypothesized that FGF21 may maintain the stability of ECs and the BBB,at least partly,by alleviating mitochondrial damage.Part 1 Study on the different patients’ serum level of FGF21 after ICH attackObjectiveTo determine the difference of the serum level regarding to FGF21 among different ICH patients,analyze the relevance between this difference and the coma severity after ICH.Methods77 emergency room patients aged 56.7 ± 10.9 years who suffered ICH with hematomas of 15.13 ± 2.7 mL were enrolled in this study.Patients with severe neurological or hepatic diseases or other severe systemic diseases were excluded.The Glasgow Coma Scale(GCS)was used to evaluate the severity of coma.The serum level of FGF21 at 24 h from the onset of ICH was measured.Patients’GCS score at the time of admission and discharge was recorded.Δ GCS%indicates the following ratio:which is(GCSdischarge_GCSadmission)/GCSadmission.Positive values indicate neurological improvement,while negative values indicate deterioration during hospitalization.According to the ΔGCS%,patients were assigned to 2 groups:which were the ΔGCS%>0 group and the ΔGCS%<0 group.The relationship between the serum level of FGF21 and the ΔGCS%was determined.ResultsThe serum level of FGF21 in ΔGCS%>0 group was 434.15±99.6 pg/mL,and the serum level of FGF21 in ΔGCS%<0 group was 250.84±125.8 pg/mL.There was obvious difference between these 2 groups regarding to the serum level of FGF21 after ICH(p<0.01).Receiver operating characteristic(ROC)curve analysis was employed to assess the ability of plasma FGF21 levels to discriminate patients’ coma severity(AUC:83.9%).In addition,a positive correlation was found between the serum level of FGF21 and the ΔGCS%.ConclusionThere was obvious difference regarding to the serum level of FGF21 among different ICH patients,and this difference was positively correlated with the ΔGCS%.Part 2 Study on the protective effects of FGF21 for the neural function and the blood brain barrier of mice after ICHObjectiveTo determine the protective effects of FGF21 regarding to neural function and BBB integrity of C57BL/6 mice after ICH.MethodsMimicked the intracranial hemorrhage in C57BL/6 mice by injected the collagenaseⅣ into striatum,then injected the recombinant FGF21 intraperitoneally for 7 consecutive days.Distributed the C57BL/6 mice into 4 groups,which were sham group,ICH group,ICH+vehicle group and ICH+FGF21 group.Occupied the MNSS test,corner turn test and wire hanging test to detect the sensorimotor alterations of mice among different groups after ICH.Employed the MRI to detect the lesion volume after ICH.Harvested the mice brains,through detect the brain water content to assess the brain edema.Collected lesion samples,through TUNEL stain to analyze the neural apoptosis.Assisted with Evan blue test and western blot,we analyzed the difference regarding to the integrity of the BBB among different groups after ICH.ResultsFGF21 could restrict the lesion volume after ICH and alleviate brain edema in vivo.According to multiple sensorimotor tests,FGF21 could improve the mice neural dysfunction and reduce the neural apoptosis after ICH.Moreover,FGF21 hold the ability of maintaining the integrity of the BBB and improving the expression of proteins related to the BBB after ICH.ConclusionFGF21 could improve the neural dysfunction of mice and maintain the integrity of the BBB after ICH in vivo.Part 3 Study on the protective effects of FGF21 for the human brain microvascular endothelial cells and the relative mechanismObjectiveTo determine the protective effects of FGF21 for HCMECs and its mitochondrial homeostasis,and explore relative mechanism in vitro.MethodsCultivated the HCMECs,and employed the oxyhemoglobin to mimic the hemorrhage in vitro.Distributed these HCMECs into 4 groups,which were control group,oxyhemoglobin group,oxyhemoglobin+vehicle group and oxyhemoglobin+FGF21 group.Through multiple experimental methods,such as permeability test of monolayer endothelial cells,CCK-8 test,western blot,qPCR,MitoTracker and MitoSOX test,to detect difference among different groups.Furthermore,we employed small interfering RNA to downregulate the expression of SIRT6,to determine the involving of SIRT6 in vitro.ResultsAfter hemoglobin injury,FGF21 held the ability of alleviating endothelial cells apoptosis,maintaining the integrity of monolayer HCMECs and improving the level of proteins regarding to the BBB.Meanwhile,the mitochondrial morphology and function of the HCMECs both alternated after hemoglobin injury.However,FGF21 treatment could partly reverse such detrimental alteration.Combined with literatures and our experiments,we found the level of SIRT6 was dramatically decreased after oxyhemoglobin injury,and the FGF21 could partly reverse this decline.Furthermore,when downregulated the SIRT6 by small interfering RNA,the protective effects of FGF21 for HCMECS and its mitochondria were all diminished.ConclusionFGF21 was capable of protecting the HCMECS and maintaining its mitochondrial homeostasis after the oxyhemoglobin injury in vitro,and these benefit effects were partly involving the participation of SIRT6.Part 4 Study on the relevance between SIRT6 and the protective effects of FGF21 assisted with the conditioned knockout miceObjectiveAssisted by the endothelial specific SIRT6 knockout mice to determine the relevance between SIRT6 and the benefit effects of FGF21 after ICH in vivo.MethodsConstructed the endothelial specific SIRT6 knockout mice(eSIRT6-/-)as experimental group,and employed the SIRT6flox/flox as control group.Occupied the Collagnase Ⅳ to induce intracranial hemorrhage in vivo.Through MNSS,corner turn test and wire hanging test to determine the sensorimotor alterations after ICH.Collected the lesion samples from mice brains,and through the TUNEL stain to determine the neural apoptosis.Harvested the mice brain and weighted to determine the alteration of brain water content.Meanwhile,through the Evans blue test to determine the permeability of the BBB.Analyzed the difference between these 2 groups to reveal the relevance between SIRT6 and the protective effects of FGF21 for the neural function and the BBB.ResultsThrough multiple sensorimotor tests,such as MNSS,corner turn test and wire hanging test,FGF21 could still improve the performance of SIRT6flox/flox mice after the ICH attack.Meanwhile,through Evans blue test,TUNEL stain and brain water content test,we found FGF21 was capable of protecting the neural system and the BBB in SIRT6flox/flox mice after ICH.In contrast,such benefit effect all diminished in eSIRT6-/-mice.ConclusionThe protective effects of FGF21 for the neural function and the BBB were partly rely on the cooperation of SIRT6Part 5 Study on the mechanism of FGF21 upregulated the expression of SIRT6ObjectiveTo determine the mechanism of FGF21 upregulated the expression of SIRT6MethodsCultivated the HCMECs and employed the oxyhemoglobin to mimic the ICH damage in vitro.Through the qPCR and western blot to determine the difference between different groups.ResultsIn vitro,the level of SIRT6 mRNA was decreased after the transfection of siFoxO3a.Meanwhile,after treated with FGF21,the alteration of SIRT6 level was accordance with the phosphorylation of AMPK-FOXO3a pathway.Furthermore,FGF21 failed to upregulate the SIRT6 level after the utilization of Compound C which is a specific inhibitor of AMPK.ConclusionFGF21 might upregulate the expression of SIRT6 by AMPK-FoxO3a pathway.