Adiponectin Alleviated Mouse Neuronal Apoptosis After Return Of Spontaneous Circulation Through Regulating The Biogenesis Of Small Extracellular Vesicles Derived From Cerebral Microvascular Endothelial Cells

BackgroundCardiac arrest(CA)patients have a poor prognosis.About 60%of the surviving patients have neurological dysfunction of varying degrees.A number of studies have shown that neurological dysfunction after ROSC is closely related to cerebral ischemia/reperfusion(I/R)injury caused by CA-CPR,which is represented by significant brain tissue edema and neuronal injury.Actively looking for treatment measures to improve the neurological dysfunction of CA survivors has important research value.Adiponectin(APN)is an important cytokine secreted by adipocytes in vivo,which can significantly reduce brain I/R damage.Previous studies of our group confirmed that after CA-CPR-ROSC,the expressions of B-cell lymphoma-2(Bcl-2)and caspase-3 in mouse brain tissues and anti-apoptotic proteins were increased.In this study,preliminary experimental results showed that in vitro conditioned medium of BMECs administrated by APN could significantly alleviate the apoptosis of neurons after OGD/R,suggesting that APN could affect the function of neurons by regulating the secretion of BMECs.But the specific molecular mechanism of action is still unclear.Small extracellular vesicles(sEVs),which exist in most biological fluids,are recently discovered as important mediators of signal transduction.A number of studies have confirmed that sEVs derived from vascular Endothelial cells(ECs)can promote neuronal regeneration and reduce apoptosis after brain I/R injury,thus playing a protective role in brain tissue.Previous studies have confirmed that APN can promote the biogenesis of sEVs in endothelial cells through T-cadherin.However,does APN reduce neuronal apoptosis by regulating the releasing of sEVs from BMSCs after CA-CPR-ROSC?If so,what are the specific molecules which regulate sEVs releasing?Therefore,this study intends to explore the protective effect on neuronal apoptosis of adiponectin through regulation on the number of BMEC sEVs in vivo and in vitro and the specific molecular mechanism.This study provides a new therapeutic strategy for alleviating brain injury and neurological dysfunction caused by brain F/R after return of spontaneous circulation.Materials and MethodChapter 1 Adiponectin alleviated neuronal apoptosis in WT mice after return of spontaneous circulation through regulating plasma sEVs1.The effect of adiponectin on neuronal apoptosis after ROSC in WT mice(1)The effect of APN on neuronal apoptosis in WT mice after ROSCUsing the random number table method,15 wild-type C57BL/6 mice were divided into 3 groups(n=5 in each group):①Sham group,②CPR group,③CPR+APN group.In the CPR+APN group,APN 10 μg was injected intravenously 10 min after ROSC.Three hours after ROSC,frozen sections of the cerebral cortex tissues of the three groups of mice were obtained,and the double fluorescent staining of Tunel combined with Neun was performed.(2)Apoptosis of neurons in different types of mice after ROSCUsing the random number table method,10 wild-type C57BL/6 mice and 10 Adipoq-/-C57 BL/6 mice were randomly divided into 2 groups(n=5 for each group):①WT Sham group;②WT CPR group ③Adipoq-/-Sham group;④Adipoq-/-CPR group.The same detection method as(1)was used to compare the neuronal apoptosis of mice in each group at 3h after ROSC.2.Preparation and fluorescent labeling of plasma sEVs in Adipoq-/-mice and WT mice(1)Extraction of plasma sEVsCollect blood from 10 wild-type C57BL/6 and Adipoq-/-C57 BL/6 mice,separate the plasma,and extract plasma sEVs by ultracentrifugation.(2)Identification of plasma sEVsThe transmission electron microscope(TEM)was used to identify the morphology of plasma sEVs.Nanoparticle tracking analysis(NTA)was used to identify the particle size of plasma sEVs.Western blotting was used to detect the expression levels of sEVs marker proteins CD63 and CD81.(3)Comparison of the number of plasma sEVs.The NTA method was used to detect the number of plasma sEVs in Adipoq-/-mice and wild-type mice.(4)Fluorescent labeling of plasma sEVs.PKH67 fluorescent dye was used to label wild-type mouse and Adipoq-/mouse plasma sEVs respectively.Reserved for use in Part 3 of this chapter.3.Observation on neuronal uptaken of plasma sEVs from different types of mice(In vitro)Divide HT22 cells into 4 groups:①Control+(WT Plasma sEVs)group②Control+(Adipoq-/-Plasma sEVs)group③OGD/R+(WT Plasma sEVs)group④OGD/R+(Adipoq-/-plasma sEVs)group.③④group established an in vitro neuronal OGD/R model.At the corresponding time point the four groups were added WT Plasma sEVs and Adipoq-/-Plasma sEVs as planned.After 6 hours,immunofluorescence staining was performed to detect the uptake of plasma sEVs by neurons.4.The effect of different types of mouse plasma sEVs on neuronal apoptosis after ROSC in wild-type mice(1)The effect of different types of mouse plasma sEVs on neuronal apoptosis after ROSC in WT miceUsing the random number table method,20 wild-type C57BL/6 mice were randomly divided into 4 groups(n=5 in each group):①Sham group②CPR group③WT Plasma sEVs group ④Adipoq-/-Plasma sEVs group.The different types of plasma sEVs were injected into the③④mice through the tail vein.After 48 hours,the CA-CPR-ROSC model was established in the②③④group.After ROSC and 3 hours after the sham operation,frozen sections of the cerebral cortex tissue of 4 groups of mice were obtained,and the double fluorescence staining of Tunel and Neun was performed.(2)The effect of different types of mouse plasma sEVs on neuronal apoptosis challenged with OGD/R(in vitro)Divide HT22 cells into 4 groups:①Control group,②OGD/R group,③WT Plasma sEVs group,④Adipoq-/-Plasma sEVs group.The②③④group established an OGD/R model.After the end of glucose and oxygen deprivation,the medium was changed to complete medium,but the corresponding plasma sEVs was added to the③④group.After 24 hours,the survival rate of neurons、LDH releasing and Caspase3 activity were measured.Chapter 2 Adiponectin alleviated mouse neuronal apoptosis after return of spontaneous circulation through regulating the number of BMEC sEVs1.Extraction and identification of sEVs from BMECsBrain microvascular endothelial cells(BMECs)were divided into 4 groups:①(Control+Vehicle)BMEC sEVs group②(Control+APN)BMEC sEVs group③(OGD/R+Vehicle)BMEC sEVs group④(OGD/R+APN)BMEC sEVs group.The ②④group was administrated with APN10 μg/ml to the culture medium;the③④group established a neuronal OGD/R model in vitro.Ultracentrifugation was used to extract sEVs,and transmission electron microscopy,nanoparticle tracking analysis,and western blotting were used to identify sEVs.2.The effect of APN on regulating the number of sEVs BMECs(1)The number of CD63-GFP BMEC sEVs gegulated by APN in vivoUsing the random number table method,15 wild-type C57BL/6 mice and 15 Adipoq-/-mice were divided into 6 groups(n=5 for each group):①WT Sham group ②Adipoq-/-Sham group③AAV-BR1-Cd63-gfp WT Sham④AAV-BR1-Cd63-gfp Adipoq-/-Sham⑤AAV-BR1-Cd63-gfp WT CPR⑥AAV-BR1-Cd63-gfp Adipoq-/-CPR.A.Labeling of sEVs derived from BMECsIn groups③④⑤⑥,AAV-BR1-Cd63-gfp was injected into the jugular vein of mice,so that BMECs sEVs contained the specific marker CD63-GFP green fluorescent protein.B.The number of BMEC CD63-GFP-sEVs regulated by APNAfter 4 weeks,the CA-CPR-ROSC model was established in group ⑤⑥.For all groups,after ROSC and 3 hours after sham operation,blood was collected to extract sEVs for WB detection of CD63-GFP protein expression level,and sEVs containing specific marker CD63-GFP green fluorescent protein was retained for use in Part 3 of this chapter.(2)APN regulate the number of BMEC sEVs in vitroBMECs cultured under normal condition were divided into 2 groups:①Control group;②APN group.Add APN10μg/ml to the medium of group ②BMECs challenged with OGD/R were divided into 3 groups:①Control group;②Vehicle+OGD/R group;③APN+OGD/R group.The②③group established an in vitro neuronal OGD/R model,and the third group was added APN10μg/ml to the culture medium.The NTA method was used to obtain the number of BMEC sEVs.3.BMEC sEVs was uptaken by neuron in vivo and in vitro(1)BMEC sEVs was uptaken by neuron in vivo.Using the random number table method,10 WT and 10 Adipoq-/C57BL/6 mice were randomly divided into 4 groups(n=5 in each group),①WT Sham group;②WT CPR group;③Adipoq-/-Sham group ④Adipoq-/CPR group.Mice in each group were injected with BMEC-specific ally infected AAV-BR1-Cd63-gfp through the jugular vein.After 4w,②④ group established CA-CPR-ROSC model.After ROSC and 3h after sham operation,frozen sections of mouse cerebral cortex were obtained in 4 groups and stained with Neun combined with CD63-GFP.The uptake of neurons was observed by immunofluorescence.(2)BMEC sEVs was uptaken by neuron in vitro.A.Labeling BMEC sEVsPKH67 fluorescent dye was used to label each group of BMEC sEVs extracted and identified in Part 1 of this chapter.B.Neurons uptake BMEC sEVsDivide HT22 cells into 8 groups:①Control+(Control+Vehicle)BMEC sEVs group;②Control+(Control+APN)BMEC sEVs group;③Control+(OGD/R+Vehicle)BMEC sEVs group;④Control+(OGD/R+APN)BMEC sEVs Group;⑤OGD/R+(Control+Vehicle)BMEC sEVs group;⑥OGD/R+(Control+APN)BMEC sEVs group;⑦OGD/R+(OGD/R+Vehicle)BMEC sEVs group;⑧OGD/R+(OGD/R+APN)BMEC sEVs group.⑤⑥⑦⑧group established OGD/R model.After the end of glucose and oxygen deprivation,the corresponding BMEC sEVs were added to the 8 groups.After 6 hours,the uptake of BMEC sEVs by neurons was detected.4.The effect of BMEC sEVs with APN administration on neuronal apoptosis in WT mice after ROSC(1)The effect of BMEC sEVs with APN administration on neuronal apoptosis in WT mice after ROSC.A.The effect of sEVs derived from BMECs under normal condition on apoptosis of neurons after ROSC in WT miceUsing the random number table method,20 WT C57BL/6 mice were randomly divided into the following groups(n=5 in each group):①Sham group ②CPR group ③(Control+Vehicle)BMEC sEVs group ④(Control+APN)BMEC sEVs groupEach group of BMEC sEVs extracted in the first part of this chapter was injected into the jugular vein of each group of mice.The CA-CPR-ROSC model was established 48h later,and the cerebral cortex tissue was taken for frozen section 3h after ROSC.Tunel combined with Neun double fluorescent staining was detected.B.The effect of sEVs derived from BMECs with OGD/R challenged on apoptosis of neurons after ROSC in WT miceUsing the random number table method,20 WT C57BL/6 mice were randomly divided into the following groups(n=5 in each group):①Sham group ②CPR group ③(OGD/R+Vehicle)BMEC sEVs group ④(OGD/R+APN)BMEC sEVs group.The following processing is the same as above.(2)The effect of BMEC sEVs on neuronal apoptosis induced by OGD/R with APN administration.A.The effect of sEVs derived from BMECs under normal condition on neurons challenged with OGD/RHT22 cells were divided into 4 groups:①Control group ②OGD/R group③(Control+Vehicle)BMEC sEVs group;④(Control+APN)sEVs BMEC groupAfter the end of glucose and oxygen deprivation,the anaerobic and sugar-free medium was replaced with a complete medium containing the corresponding BMEC sEVs.After 24 hours,the survival rate of neurons、LDH releasing and caspase3 activity were measured.B.The effect of sEVs derived from BMECs challenged with OGD/R on neurons challenged with OGD/RHT22 cells were divided into 4 groups:①Control group ②OGD/R group ③(OGD/R+Vehicle)BMEC sEVs group;④(OGD/R+APN)BMEC sEVs group.The following processing is the same as above.Chapter 3 Under different conditions,APN regulated the biogenesis and releasing of BMEC sEVs through regulating CHMP4B or RAB27A,respectively.1.Screen and determine the role molecules of APN to promote BMECs production and releasing BMEC sEVs(1)Screen and determine the role molecules of APN in promoting BMECs production and releasing of BMEC sEVs under normal conditionbEnd.3 cells were divided into 3 groups:①Control group②Control+APN5μg/mlgroup③Control+APN10μg/ml group.RT-qPCR screens the transcription levels of molecules that promote the biogenesis of BMEC sEVs under normal condition administrated with APN.bEnd.3 cells were divided into 2 groups:①Control group②Control+APN group.WB method detects the protein expression level of the molecules screened out by RT-qPCR.(2)Screen and determine the role molecules of APN in promoting BMECs in production and releasing of BMEC sEVs challenged with OGD/RThe bEnd.3 cells were divided into 2 groups:①OGD/R group ②OGD/R+APN group.After RT-qPCR screening for OGD/R,APN promotes the production of BMEC sEVs and the transcription level of the molecules.The target protein expression level was detected with WB.2.Overexpression plasmid preparation and transfection efficiency verificationConstruction of Chmp4b and Rab27a overexpression plasmids(Jikai Biotechnology Company).The BMECs were divided into 2 groups:②Control+NC group②Control+Chmp4b OE group.After BMECs were transfected with empty plasmid and Chmp4b overexpression plasmid,WB method was used to detect the expression level of CHMP4B protein in BMECs;the same method was used to verify the transfection efficiency of Rab27a overexpression plasmid.3.Under normal condition,APN regulates the biogenesis of BMEC sEVs through CHMP4BbEnd.3 cells were divide into 3 groups ①Control group:②APN+NC group;③APN+Chmp4b OE group.①group was transfected with pCS2 HRS-RFP(red fluorescent)tracer plasmid,②group was transfected with pCS2 HRS-RFP tracer plasmid and empty plasmid at the same time,③group was transfected with pCS2 HRS-RFP and Chmp4b overexpression plasmid.②③Group APN(10μμg/ml)was added 24h after plasmid transfection.After BMECs plasmid was transfected for 48 hours,the particle size of red fluorescence(pCS2 HRS-RFP)was detected by immunofluorescence.The particle diameter of green fluorescence(Rab5-Q79L-GFP)was also detected by immunofluorescence as the same way.4.Challenged with OGD/R,APN regulates the releasing of BMEC sEVs through RAN27AThe bEnd.3 cells were divided into 4 groups:①Control group:②OGD/R group;③OGD/R+NC+APN group④OGD/R+NC+Rab27a OE group;Group ②does not transfect plasmid,group③transfects empty plasmid,group ④Rab27a overexpression plasmid transfects BMECs.In the②③④group,OGD/R was performed 18h after transfection,and BMEC sEVs was extracted 24h later.NTA was used to detect the number of BMEC sEVs particles in each group,and WB was used to detect the expression levels of CD63 and CD81 proteins.ResultsChapter 1 Adiponectin alleviated mouse neuronal apoptosis after return of spontaneous circulation through regulating plasma sEVs1.The effect of APN on neuronal apoptosis after ROSC in mice(1)APN reduces neuronal apoptosis in wild-type mice after ROSCThe apoptosis of cerebral cortex neurons in WT mice increased significantly after ROSC(Tunel+Neun+,18.10%,P<0.01).APN intervention significantly reduced neuronal apoptosis in the cerebral cortex(Tunel+Neun+,7.24%,P<0.01).(2)Apoptosis of neurons in Adipoq-/-mice increases after ROSCCompared with WT mice,the apoptosis of cerebral cortex neurons in Adipoq-/-mice increased significantly after ROSC(Tunel+Neun+,12.32%,P<0.01).2.Preparation and fluorescent labeling of plasma sEVs in Adipoq-/-mice and wild-type miceThe results of TEM detection suggested that the collected Plasma sEVs of Adipoq-/-and WT mice had a "cup-plate" morphology.NTA results indicate that the peak and average particle size are in the range of 30-150 nm.CD63 and CD81 were highly expressed in Adipoq-/-and WT mouse plasma and Plasma sEVs.NTA test showed that compared with WT mice,the number of Plasma sEVs in Adipoq-/-mice was significantly reduced(40%,P<0.01).3.Adipoq-/-and WT mouse plasma sEVs were taken up by neuronsUnder normal condition,Adipoq-/-and WT mouse Plasma sEVs labeled with PKH green fluorescent dye can be observed in neurons after OGD/R.4.Adipoq-/-plasma sEVs promotes neuronal apoptosis(1)Adipoq-/-plasma sEVs promotes neuronal apoptosis after ROSC in WT miceCompared with mice in the CPR group,the apoptosis of cerebral cortex neurons in the WT Plasma sEVs group was significantly reduced(Tunel+Neun+,13.29%,P<0.01),while neuron apoptosis in Adipoq-/-Plasma sEVs group increased(Tunel+Neun+,8.90%,P<0.01).(2)Adipoq-/-plasma sEVs promotes apoptosis of neuron challenged with OGD/RAdipoq-/-Plasma sEVs significantly reduced the survival rate of neurons challenged with OGD/R(33.19%,P<0.01),promoted the releasing of cell death marker LDH(3.88-fold,P<0.01),and increased the activity of caspase 3(37%,P<0.01).Chapter 2 Adiponectin alleviated mouse neuronal apoptosis after return of spontaneous circulation through regulating the number of BMEC sEVs1.Extraction and identification of BMEC sEVsBMEC sEVs had a "cup-disk-like" morphology detected by TEM.NTA results indicate that the peak and average particle size are in the range of 30-150 nm.BMECsEVs highly express CD63 and Flotillin-1 detected by WB.2.APN regulates the number of BMEC sEVs(1)APN regulates the number of CD63-GFP-sEVs derived from BMECs in vivoCD63-GFP protein was detected in sEVs derived from WT and Adipoq-/plasma.Compared with WT mice,the expression of CD63-GFP protein in Plasma sEVs of Adipoq-/-mice was significantly reduced(0.75-fold,P<0.01).After ROSC,the expression of CD63-GFP protein in plasma sEVs of WT mice increased significantly(0.55-fold,P<0.01;the expression of CD63-GFP protein in plasma sEVs of Adipoq-/-mice increased significantly(0.87-fold,P<0.01)).(2)The number of BMEC sEVs regulated by APN in vitroCompared with the Control group,the number of BMEC sEVs in the Control+APN group was significantly increased(2.85-fold,P<0.01).Compared with the Control group,the number of BMEC sEVs in the OGD/R group increased significantly(3.63-fold,P<0.01).Compared with the OGD/R group,the number of BMEC sEVs in OGD/R+APN group was significantly reduced(2.77-fold,P<0.01).3.Neurons uptake of BMEC sEVs(1)Neurons uptake of BMEC sEVs in vivoCD63-GFP-labeled BMEC sEVs could be observed in WT and Adipoq-/mouse neurons no matter normal or after ROSC detected by immunofluorescence staining.(2)Neuron uptake of BMEC sEVs in vitroUnder normal condition and challenged with OGD/R,BMEC sEVs labeled with PKH67 green fluorescent dye can be observed in neurons.4.APN alleviated neuronal apoptosis in WT mice after ROSC through regulating the number of BMEC sEVs(1)APN alleviated neuronal apoptosis in WT mice after ROSC through regulating the number of BMEC sEVsA.Under normal condition,sEVs released by BMECs with APN administration alleviated neuronal apoptosis after ROSC in WT miceCompared with the(Control+Vehicle)BMEC sEVs group,(Control+APN)BMEC sEVs group significantly increased neuron apoptosis(Tunel+Neun+,5.40%,P<0.01).B.sEVs derived from BMECs challenged with OGD/R promoted neuronal apoptosis in mice after ROSC,while sEVs derived from BMECs challenged with OGD/R plus APN alleviated neuronal apoptosisCompared with(OGD/R+Vehicle)BMEC sEVs group,the(OGD/R+APN)BMEC sEVs group significantly reduced neuronal apoptosis(Tunel+Neun+,8.58%,P<0.01).(2)APN alleviated neuronal apoptosis challenged with OGD/R through regulating the number of BMEC sEVs in vitroA.Under normal condition,sEVs released by BMECs with APN administration alleviated neuronal apoptosis challenged with OGD/RCompared with the(Control+Vehicle)BMEC sEVs group,the neuron survival rate of the(Control+APN)BMEC sEVs group increased(13.83%,P<0.01),the releasing of LDH decreased(1.25-fold,P<0.01),the activity of apoptosis marker protein caspase 3 decreased(33%,P<0.01).B.sEVs derived from BMECs challenged with OGD/R promoted neuronal apoptosis challenged with OGD/R,while sEVs derived from BMECs challenged with OGD/R plus APN alleviated neuronal apoptosisCompared with(OGD/R+Vehicle)BMEC sEVs group,the neuron survival rate of(OGD/R+APN)BMEC sEVs group increased(15.81%,P<0.05),the releasing of LDH decreased(1.55-fold,P<0.01),and the activity of apoptosis marker protein caspase 3 decreased(32%,P<0.01).Chapter 3 APN attenuated the biogenesis and releasing of BMEC sEVs by down-regulating CHMP4B and RAB27A,respectively1.Screen and determine the molecules role of APN to promote BMEC production and releasing BMEC sEVs(1)Screening and determining the molecules role of APN to promote BMEC production and releasing BMEC sEVs under normal conditionCompared with the Control group,in the Control+APN group the expression levels of Chmp4b,Stam1 and Vps4b in the ESCRT-dependent sEVs production pathway in BMECs were significantly increased(6.92-fold,6.77-fold and 2.77-fold,P<0.01,P<0.01 and P<0.05),among which Chmp4b has the most significant difference.Compared with the Control group,the CHMP4B and VPS4B protein expression levels of BMECs in the Control+APN group were significantly reduced(0.79-fold,0.63-fold;P<0.05),and the STAMI protein level was unchanged.It is confirmed that APN mainly promotes the biogenesis of BMEC sEVs through down-regulating CHMP4B under normal condition.(2)Screening and determining the molecules role of APN to promote BMEC production and releasing BMEC sEVs challenged with OGD/RCompared with the OGD/R group,the expression levels of rab27a and ykt-6 genes in BMECs in the OGD/R+APN group were significantly reduced(0.50-fold and 0.56-fold;P<0.05 and P<0.01).Compared with the Control group,the RAB27A protein level in the OGD/R group was significantly increased(1.37-fold,P<0.05).Compared with the OGD/R group,the expression level of RAB27A protein in the OGD/R+APN group was significantly reduced(1.32-fold,P<0.05).2.Overexpression plasmid preparation and transfection efficiency verificationCompared with the NC group,the CHMP4B protein expression level in the Chmp4b OE group was significantly higher(9.27-fold,P<0.01),and the RAB27A protein expression in the Rab27a OE group was significantly higher(37.73-fold,P<0.05).3.Under normal condition,APN alleviated the biogenesis of BMEC sEVs through down-regulating CHMP4BCompared with the Control+APN+NC group,the size of red fluorescent particles(pCS2 HRS-RFP)and green fluorescent particles(Rab5-Q79L-GFP)in the Control+APN+Chmp4b OE group reduced,which indicating that the formation of MVBs was reduced,and then the biogenesis of sEVs was reduced.4.After OGD/R,APN alleviated the releasing of sEVs from BMECs through down-regulating RAN27AThe number of sEVs particles in OGD/R+Rab27 OE+APN was significantly increased compared with OGD/R+APN group detected by NTA(2.84-fold,P<0.05).Compared with the OGD/R+APN group,the CD63 and CD81 protein levels in the BMEC sEVs of the OGD/R+Rab27 OE+APN group increased(0.63-fold,0.90-fold,P<0.01).Conclusion1.Adiponectin alleviated mouse neuronal apoptosis after return of spontan-eous circulation through regulating plasma sEVs;2.Adiponectin alleviated mouse neuronal apoptosis after return of spontan-eous circulation through regulating the number of BMEC sEVs;3.Under normal condition,APN down-regulated CHMP4B to promote the formation of MVBs,which in turn promoted the generation of BMEC sEVs and increased the number of BMEC sEVs.APN down-regulated RAB27A to inhibit the releasing of sEVs from damaged BMECs,and reduces the number of BMEC sEVs.