HSP70 Alleviates Spinal Cord Injury Through Activation Of Wnt/β-catenin Signaling Pathway

Research background:Spinal cord injury(SCI),a class of transverse injury to spinal cord,caused by traumatic and non traumatic factors temporarily or permanently.The damage of spinal cord results in decline in the function,such as muscle disorder,loss of sensory,dysfunction of autonomic nerve.Most of the time,SCI traumatic factors include serious traffic accident,violent shooting,falling from tall altitude and physical damage directly.For the non traumatic causes,bacterial infections,Lack of blood supply,injury by tumor indirectly and other diseases are involved in the lesion.Besides,spinal cord vascular disease and pathogenesis of disc disease may also lead to SCI.SCI is a serious worldwide clinical problem;however,the mechanism of SCI is complex and still considerable ambiguity.The pathgenesis of SCI include both direct mechanical injury and succedent degeneration of peripheric tissue.Succedent degeneration often causes ischemia,swelling of cells,electrolyte disturbances and other pathological responses,which plays a pivotal role in the phase of the secondary injury.Ischemia,hypoxia and inflammation could promote the release of reactive oxygen species(ROS),resulting in oxidative damage of motor neuron in spinal cord,nerve cells apoptosis and necrosis occurre subsequently.Thus,finding effective drugs to decrease the release of ROS and protect neurons is a research hotspot in the treatment of spinal cord injury.Heat shock protein-70 kDa(HSP70),an important member of heat shock factor(HSFs)family,is expressed in the cytoplasm,endoplasmic reticulum and mitochondria widely.HSP70 is a highly conserved molecular chaperone and found in different species,tissue,as wells as many kinds of cells,which maintains intracellular homeostasis and prevents protein degeneration caused by the high temperature and other stressful stimuli.It has been reported that upregulation of HSP70 protein in keloid fibroblasts and keloid tissue might be tightly bound up with the hypernomic collagen production by keloid fibroblast.In addition,the fact that HSP70 involved in the inflammatory reaction has been illustrated by animal experiments and HSP70 maybe a pharmacological target of the anti-inflammatory for enteritis.Previous experiments have pay more attention to analyze the function of HSP70,However,there is rare study about the relationship between HSP70 and SCI.Therefore,our study aims to test this relationship and verify whether HSP70 alleviates SCI through activation of Wnt/β-catenin signaling pathway.In order to verify this hypothesis,three parts of experiments were carried out.The first part aims to observe whether there is differential expression of HSP70 and Wnt/β-catenin signaling pathway in SCI rats,and positive correlation between HSP70 and Wnt/β-catenin signaling pathway.On the basis of the results of Part one second part experiment was performed to observe the effect of HSP70 on the movement of rats and Wnt/β-catenin signaling pathway,and to further explore whether this effect affected the neuronal apoptosis through up-or down-regulating HSP70.The third part verified the conclusion of the second part at the cellular level.Based on the results of the appeal experiment,the corresponding conclusions are drawn.In this study,the pathogenesis of SCI was explored from the perspective of classic molecular and signal pathways,and the relationship between HSP70 and Wnt/β-catenin signaling pathway was revealed and verified for the first time in the SCI model.Section Ⅰ The expression of HSP70,Wnt3a,Wnt5a and β-catenin in SCI rat modelObjective:The expression of HSP70,Wnt3a,Wnt5a andp-catenin in SCI rat model were detected and analyzed,which could provide the theoretical basis for the follow-up study on mechanisms.9Methods:1.The spinal cord was compressed by a microvascular clip at the T9～T10 level to induce SCI and those successfully established models were used as SCI group(n=15).The adult male SD rats underwent the same disposition with SCI group except that spinal cord was compressed at the T9～T10 level and those rats were used as control group(n=15).The spinal cord of SCI group and control group were collected for following study.2.Co-immunoprecipitation(Co-IP)was used to test the interaction between HSP70 and Wnt3a,HSP70 and Wnt5a,and HSP70 and p-catenin.3.The mRNA expression of HSP70,Wnt3a,Wnt5a and β-catenin in SCI rat model were detected by RT-qPCR.4.The protein expression of HSP70,Wnt3a,Wnt5a and β-catenin in SCI rat model were detected by western blot.Results:1.Positive interaction between HSP70 and Wnt3a,HSP70 and Wnt5a,and HSP70 and(3-catenin were detected by Co-IP.2.On the mRNA level,the expression of HSP70,Wnt3a,Wnt5a and β-catenin in SCI group were observably decreased compared to those in control group(P<0.05)by RT-qPCR.3.In the protein level,the expression of HSP70,Wnt3a,Wnt5a and β-catenin in SCI group were observably decreased compared to those in control group(P<0.05)by western blot.Conclusion:1.HSP70 could interact Wnt/β-catenin signaling pathway.2.HSP70 and Wnt/β-catenin were downregulated in SCI rats.Section Ⅱ The upregulation of HSP70 alleviates spinal cord injury in rat through activating Wnt/β-catenin signaling pathway and suppressing caspase3-induced apoptosisObjective:To investigated whether HSP70 alleviates spinal cord injury in rat through activating Wnt/β-catenin signaling pathway and suppressing caspase3-induced apoptosis,adenoviruses were used to upregulate HSP70 expression and XAV-939 was used to inhibit Wnt/β-catenin signaling pathway.Methods:Verification of adenovirus replication:20 rats were randomly divided into Ad-con group and Ad-HSP70 group.Adenoviruses were injected into spinal cord at the T9～T10 level.After 7 days of injection,spinal cord at the T9～T10 level was collected.A portion of the spinal cord was used to make frozen section and detect fluorescence of adenoviruses through microscope.Another part of the spinal cord was used to detect the expression of HSP70 by western blot.Inhibitory effect of XAV-939:20 rats were randomly divided into NS group and XAV-939 group.The rats of XAV-939 group received 100 μM XAV-939 by intraperitoneal injection everyday for 28 days and the rats of NS group received equal saline by intraperitoneal injection everyday for 28 days.After 28 days of injection,spinal cord at the T9～T10 level was collected.The expression of Wnt3a,Wnt5a and p-catenin were detected by western blot.The effects of HSP70 upregulation on SCI and Wnt/β-catenin signaling pathway:60 rats were randomly divided into SCI group,SCI+Ad-con group and SCI+Ad-HSP70 group.All rats underwent SCI surgery.The rats of SCI+Ad-con group and SCI+Ad-HSP70 group received 3 μl Ad-con and 3 μl Ad-HSP70 respectively before 7 days of SCI surgery.After 1 day,3 days,7 days,14 days,21 days and 28 days of SCI surgery,behavioral recordings were detected according to BBB scale.After 28 days of SCI surgery,spinal cord at the T9～T10 level was collected.The expression of HSP70,Wnt3a,Wnt5a,β-catenin,Bcl-2,Bax,pro-caspase3 and cleaved-caspase3 were detected by western blot.A portion of the spinal cord was used to make frozen section and detect the apoptosis by TUNEL staining.The effects of XAV-939 on SCI and Wnt/β-catenin signaling pathway:40 rats were randomly divided into SCI+Ad-HSP70 group and SCI+Ad-HSP70+XAV-939 group.All rats underwent SCI surgery and received 3 μl Ad-HSP70 before 7 days of SCI surgery.The rats of SCI+Ad-HSP70 group and SCI+Ad-HSP70+XAV-939 group received saline and XAV-939 respectively by intraperitoneal injection everyday for 28 days after SCI surgery.After 1 day,3 days,7 days,14 days,21 days and 28 days of SCI surgery,behavioral recordings were detected according to BBB scale.After 28 days of SCI surgery,spinal cord at the T9～T10 level was collected.The expression of HSP70,Wnt3a,Wnt5a,β-catenin,Bcl-2,Bax,pro-caspase3 and cleaved-caspase3 were detected by western blot.A portion of the spinal cord was used to make frozen section and detect the apoptosis by TUN EL staining.Results:Verification of adenovirus replication:After 7 days of adenoviruses injection,abundant green fluorescence was detected in the frozen section of spinal cord.The expression of HSP70 detected by western blot in Ad-HSP70 group was observably increased compared to Ad-con group(P<0.05).Inhibitory effect of XAV-939:The expression of Wnt3a,Wnt5a and β-catenin detected by western blot in XAV-939 group were observably decreased compared to NS group(P<0.05).The effects of HSP70 upregulation on SCI and Wnt/β-catenin signaling pathway:The BBB scores in SCI+Ad-HSP70 group were significantly higher than that in SCI+Ad-con group(P<0.05).The expression of HSP70,Wnt3a,Wnt5a,β-catenin and Bcl-2 detected by western blot in SCI+Ad-HSP70 group were observably increased compared to SCI+Ad-con group(P<0.05);the expression of Bax and cleaved-caspase3 detected by western blot in SCI+Ad-HSP70 group were observably decreased compared to SCI+Ad-con group(P<0.05);The expression of pro-caspase3 detected by western blot in SCI+Ad-HSP70 group were undifferentiated compared to SCI+Ad-con group(P>0.05).TUNEL staining indicated that the apoptosis rate of spinal neurons of SCI+Ad-HSP70 was observably decreased compared to SCI+Ad-con group(P<0.05).All above indicators were undifferentiated between SCI+Ad-con group and SCI+Ad-HSP70 group(P>0.05).The effects of XAV-939 on SCI and Wnt/β-catenin signaling pathway:The BBB scores in SCI+Ad-HSP70+XAV-93 group were significantly lower than that in SCI+Ad-HSP70 group(P<0.05).The expression of HSP70,Wnt3a,Wnt5a,β-catenin and Bcl-2 detected by western blot in SCI+Ad-HSP70+XAV-93 group were observably decreased compared to SCI+HSP70 group(P<0.05);the expression of Bax and cleaved-caspase3 detected by western blot in SCI+Ad-HSP70+XAV-93 group were observably increased compared to SCI+HSP70 group(P<0.05);The expression of pro-caspase3 detected by western blot in SCI+Ad-HSP70+XAV-93 group were undifferentiated compared to SCI+Ad-HSP70 group(P>0.05).TUNEL staining indicated that the apoptosis rate of spinal neurons of SCI+Ad-HSP70+XAV-93 was observably increased compared to SCI+HSP70 group(P<0.05).Conclusion:Upregulation of HSP70 could alleviate movement disorder of SCI rats.Upregulation of HSP70 could activate Wnt/β-catenin signaling pathway in SCI rats.Upregulation of HSP70 could suppress caspase3-induced apoptosis.Section Ⅲ The upregulation of HSP70 protects PC-12 cells through activating Wnt/β-catenin signaling pathway and suppressing caspase3-induced apoptosisObjective:To investigated whether HSP70 alleviates H202-induced in PC-12 cells through activating Wnt/β-catenin signaling pathway and suppressing caspase3-induced apoptosis,adenoviruses were used to upregulate HSP70 expression.Methods:Verification of adenovirus replication:PC-12 cells were randomly divided into Ad-con group and Ad-HSP70 group.2.5 MOI Adenoviruses were transfected into PC-12 cells.After 24 h of transfection,RT-qPCR and western blot were used to detect the expression of HSP70.The effects of HSP70 upregulation on PC-12 cells and Wnt/β-catenin signaling pathway:PC-12 cells were randomly divided into Ad-congroup,Ad-HSP70 group,H2O2+Ad-con group and H2O2+Ad-HSP70 group.After 24 h of transfection,500μM H2O2 was added into culture medium of H202+Ad-con group and H2O2+Ad-HSP70 group.ROS was detected by intracellular reactive oxygen detection kit.cell viability was detected by MTT.Cell apoptosis was detected by flow cytometry.The expression of HSP70,Wnt3a,Wnt5a,[3-catenin,Bcl-2,pro-caspase3 and cleaved-caspase3 were detected by western blot.Results:Verification of adenovirus replication:The expression of HSP70 detected by RT-qPCR in Ad-HSP70 group was observably increased compared to Ad-con group(P<0.05).The expression of HSP70 detected by western blot in Ad-HSP70 group was observably increased compared to Ad-con group(P<0.05).The effects of HSP70 upregulation on PC-12 cells and Wnt/β-catenin signaling pathway:The ROS activity in PC-12 cells of H2O2+Ad-con group was significantly higher than that in PC-12 cells of Ad-con group(P<0.05)and the ROS activity in PC-12 cells of H2O2+Ad-HSP70 group was significantly lower than that in PC-12 cells of H2O2+Ad-con group(P<0.05).The cell viability in PC-12 cells of H2O2+Ad-con group was significantly decreased compared to that in PC-12 cells of Ad-con group(P<0.05)and the cell viability in PC-12 cells of H2O2+Ad-HSP70 group was significantly increased compared to that in PC-12 cells of H202+Ad-con group(P<0.05).The cell apoptosis in PC-12 cells of H202+Ad-con group was significantly increased compared to that in PC-12 cells of Ad-con group(P<0.05)and the cell apoptosis in PC-12 cells of H2O2+Ad-HSP70 group was significantly decreased compared to that in PC-12 cells of H202+Ad-con group(P<0.05).The expression of Wnt3a、Wnt5a、β-catenin and Bcl-2 in PC-12 cells of H202+Ad-con group was significantly decreased compared to that in PC-12 cells of Ad-con group(P<0.05).The expression of Wnt3a、Wnt5a、β-catenin and Bcl-2 in PC-12 cells of H2O2+Ad-HSP70 group was significantly increased compared to that in PC-12 cells of H2O2+Ad-con group(P<0.05).The expression of cleaved-caspase3 in PC-12 cells of H2O2+Ad-con group was significantly increased compared to that in PC-12 cells of Ad-con group(P<0.05).The expression of cleaved-caspase3 in PC-12 cells of H2O2+Ad-HSP70 group was significantly decreased compared to that in PC-12 cells of H202+Ad-con group(P<0.05).The expression of pro-caspase3 was undifferentiated among Ad-congroup,Ad-HSP70 group,H202+Ad-con group and H2O2+Ad-HSP70 group.Conclusion:H2O2 induced the decrease of Wnt/β-catenin signaling pathway and the increase of caspase3-induced apoptosis pathway in PC-12 cells.Upregulation of HSP70 could alleviate H202-induced injury.Upregulation of HSP70 could activate Wnt/β-catenin signaling pathway and suppress caspase3-induced apoptosis in PC-12 cells.