Effects And Mechanisms Of CTRP3 Attenuates Secondary Injury After Intracerebral Hemorrhage Via Regulating AMPK Pathway

Background:The deterioration of the nervous system in patients with cerebral hemorrhage is accompanied by a large number of neurons and vascular damage.The repair of vascular injury can prevent secondary neuronal injury after cerebral hemorrhage,and angiogenesis is an effective target for patients with cerebral hemorrhage.C1q/tumor necrosis factor(TNF)-related protein-3(CTRP3)is a newly discovered fat factor,which is a homologue of adiponectin.CTRP3 has many biological functions,such as promoting the development of new tube,anti inflammation,anti apoptosis,anti oxidative stress and so on.Animal experimental study on mice showed that CTRP3 can promote myocardial infarction blood vessels around the ischemic area of freshmen.However,whether CTRP3 can promote the formation of new blood vessels in the ischemic area around the hematoma has not been reported,and the specific picture of the relevant signal transduction mechanism has not been fully elucidated.Objective:In this paper,the model of intracerebral hemorrhage:(1)to investigate the neuroprotective effect of CTRP3 on secondary injury after intracerebral hemorrhage.(2)to investigate whether CTRP3 can promote the regeneration of new blood vessels around the hematoma after intracerebral hemorrhage,and to further explore its regulatory mechanism.Methods:1.Establishing a stable and reliable rat model of cerebral hemorrhage: whole blood(50μL),which was drawn from the femoral artery,was infused manually over 10 min via a 26 G needle inserted into the striatum at a depth of 5.8 mm below the surface of the skull.2.To observe the cereprotective effect of exogenous recombinant CTRP3(rCTRP3)protein after intracerebral hemorrhage:(1)Adult SD rats were randomly divided into the following four groups: sham operation(sham)group,intracerebral hemorrhage model(ICH)group,ICH + vehicle group and ICH + recombinant CTRP3(rCTRP3)group.(2)rCTRP3 was injected into the lateral ventricles of the brain(80 μg/kg)after 30 minutes of intracerebral hemorrhage,and then three times a week until the animals were killed.(3)The neurological function score assessed by the modified Garcia test,the balance beam test and the wire hanging test,hematoma volume,BBB integrity and brain edema were measured 7 days after intracerebral hemorrhage.(4)CD31 immunohistochemistry was used to detect the content of vascular markers.(5)Western blot was used to observe the changes of CTRP3,AMPK,HIF-1 and VEGF protein expression.(6)Real time fluorescent quantitative PCR was used to analyze the changes of CTRP3,HIF-1 and VEGF mRNA expression.3.To observe the cereprotective effect of lentivirus over expression of CTRP3(Lenti-CTRP3)on after intracerebral hemorrhage:(1)Adult SD rats were randomly divided into the following four groups: sham operation(sham)group,intracerebral hemorrhage model(ICH)group,ICH + null vector control(Lenti.null)group,ICH + lentivirus overexpression of CTRP3(Lenti.CTRP3)group.(2)Fourteen days after Lenti.CTRP3 intracerebroventricular injection,the rats model of intracerebral hemorrhage was established.(3)The neurological function score assessed by the modified Garcia test,the balance beam test and the wire hanging test,hematoma volume,BBB integrity and brain edema were measured 7 days after intracerebral hemorrhage.(4)CD31 immunohistochemistry was used to detect the content of vascular markers.(5)Western blot was used to observe the changes of CTRP3,AMPK,HIF-1 and VEGF protein expression.(6)Real time fluorescent quantitative PCR was used to analyze the changes of CTRP3,HIF-1 and VEGF mRNA expression.4.To investigate the AMPK/HIF-1α/VEGF signaling pathway in CTRP3 against intracerebral hemorrhage injury,we used antibodies specific to the phosphorylated and active forms of AMPK,HIF-1α and VEGF.To further elucidate the signaling pathways involved in CTRP3 mediated neuroprotection against the intracerebral hemorrhage,Compound C(AMPK axis inhibitor)or CBO-P11(VEGF inhibitor)was given together with rCTRP3 via the right lateral cerebral ventricle was used to study the AMPK signaling pathway.Results:1.In the present study,exogenous recombinant CTRP3 or lentivirus over expression of CTRP3 in ICH rat model demonstrated the same tendency to attenuate ICH-induced brain injury.CTRP3 promotes focal angiogenesis and attenuates ICH-induced brain edema and breakdown of the blood-brain barrier.There was significant statistical difference compared with ICH model group(P < 0.01).2.CTRP3 greatly intensified phosphorylation of AMP-activated protein kinase(AMPK)in addition to expression of hypoxia inducing factor-1a(HIF-1a)and vascular endothelial growth factor(VEGF).There was significant statistical difference compared with ICH model group(P < 0.01).3.In vivo studies,Compound C(AMPK axis inhibitor)or CBO-P11(VEGF inhibitor)was given together with rCTRP3 via the right lateral.Blocking AMPK activation abolished rCTRP3-induced pAMPK,HIF-1a and VEGF upregulation,and blocking VEGF activation only abolished rCTRP3-induced VEGF upregulation.In addition,CTRP3-induced capillary formation was entirely terminated when either AMPK or VEGF was inhibited.However,the neurological protective aspect of CTRP3 was completely blocked by Compound C and partially blocked by CBO-P11.Conclusion:1.CTRP3 can reduce the nerve function defect,up regulate the expression of angiogenic factors,induce angiogenesis,protect blood brain barrier,so as to resist the secondary injury of cerebral hemorrhage.2.CTRP3 may exert its angiogenic effect through AMPK/HIF-1α/VEGF signaling.