Study On The Mechanism Of Heparin Regulating PTN/syndecan-3 In The Repair Of Brain Injury After Cardiopulmonary Resuscitation

Background:With the continuous innovation of cardiopulmonary resuscitation technology and widespread social popularity,the recovery rate of autonomic circulation after cardiopulmonary resuscitation can reach 50%,but in the long term,only less than 5%of patients have a good prognosis of neurological function.The damage of synaptic function of brain nerve cells after resuscitation has been the main cause of the poor long-term prognosis of cardiopulmonary resuscitation.Repairing synaptic function of resuscitated brain nerve cells plays an important role in improving the prognosis of neural function.Studies have shown that Syndecan3（SDC3）is an important factor in synaptic growth and repair after hypoxic injury of neurons,and Pleiotrophin（PTN）signals are transmitted to nerve cells to participate in synaptic migration,neuron growth and development,etc.At the same time,SDC3 is a proteoglycan specific to the surface of endogenous nerve cell membrane in the body,and the extracellular region of heparan sulfate has a high structural similarity with commonly used clinical exogenous heparin.Therefore,it is of great significance to study whether heparin improves brain function and synaptic growth in rats after resuscitation by affecting PTN/Syndecan3 pathway,and to propose new therapeutic strategies for protecting brain function after cardiopulmonary resuscitation.ObjectiveClinical results showed that PTN and SDC3 were correlated with neurological recovery in CA-CPR patients.Animal level verified that heparin improved the cerebral nerve function and the expression of PTN and SDC3 after cardiopulmonary resuscitation.To investigate the target of heparin on PTN/SDC3 pathway in hippocampal neuron OGD/R model at cellular level.Method:1.In clinical study,we selected patients who had a cardiac arrest and had received CPR and had achieved recovery of autonomic circulation.GCS,TICS-m score,CPC score and survival were recorded within 7 days after resuscitation.Blood samples were collected from 2 hours to 7 days after resuscitation,and serum expressions of PTN,SDC3 and neurospecific enolase（NSE）were detected.The correlation between neurological function score and serum PTN,SDC3 and NSE after resuscitation was analyzed,and the ability of serological indicators to predict neurological function prognosis and death was examined by area under ROC curve（AUC）.2.A cardiopulmonary resuscitation model of asphyxiated cardiac arrest was established in SD rats,which were randomly divided into Sham group（S）,Heparin group（H）and normal saline group（C）to evaluate the changes of neurological function within 7 days after resuscitation.The expression of PTN,SDC3 and NF200in brain tissue and serum of rats after resuscitation was detected.The change of water content in brain tissue after resuscitation was detected by dry-wet ratio.The expression of PTN,SDC3,NF200 and Tau in hippocampus of brain tissue was analyzed by immunofluorescence staining.3.Mouse hippocampal neuronal cells（HT22）were used to prepare OGD/R models to simulate the ischemia hypoxia reperfusion injury of hippocampal neuronal cells.The expression of PTN,SDC3 and NF200 after the intervention of OGD/R by heparin was detected.The expression of PTN,SDC3 and NF200 in hippocampal neurons was mapped by immunofluorescence staining,and the HT22 cell lines with continuous down-expression of PTN and SDC3 were screened by lentivirus infection.The expression of PTN,SDC3,NF200 and PSD95 in HT22 with OGD/R intervention of PTN and SDC3 persistently down-regulated expression was detected to determine the target of heparin in PTN/SDC3 pathway.Results:1.There was a complete linear correlation between GCS and TICS m scores within 7 days after resuscitation in 23 patients with cardiac arrest and cardiopulmonary resuscitation,with correlation R=0.9970,P<0.05.Serum PTN and SDC3 changed within 7 days after resuscission.The ability of serum NSE,PTN and SDC3 indexes to predict 7-day death was AUC_PTN=0.8917,AUC_SDC3=0.8333,AUC_NSE=0.6964 respectively.The patients with mild and severe neurological dysfunction after resuscitation were distinguished according to the CPC grading.It was found that there was a difference in NSE between the two groups 1 day after resuscitation（P<0.05）,while there was no statistically significant difference in PTN and SDC3 between the two groups（P>0.05）.2.The incidence of ROSC was 79.65%in group H,84.31%in group H,and75.81%in group C.There was no significant difference in the success rate of resuscitation（P>0.05）.The body weight of groups C and H was lower than that of group S（P<0.05）.There were no significant differences in the time from asphyxia to cardiac arrest（TA-C）,the time from cardiopulmonary resuscitation to spontaneous circulation recovery（TC-R）,the time from spontaneous circulation recovery to extubation（TR-E）and 7-day survival rate between group C and group H（P>0.05）.After resuscitation,the neurological function of group H was significantly different from that of group C（P<0.05）.Within 7 days after resuscitation,there was significant difference in water content of brain tissue among the three groups（P<0.05）.The expressions of PTN,SDC3 and NF200 in brain tissue of C and H groups were statistically significant at 1,3 and 7 days after resuscitation（P<0.05）.There were statistically significant differences in serum PTN at 3 and 7 days and NF200 at 1day between group H and group C（P<0.05）,but no statistically significant differences in PTN,SDC3 and NF200 at other time points（P>0.05）.Immunofluorescence results showed that there was no significant difference between group C and group H in SDC3,NF200 and Tau at 1 day after resuscitation and Tau at3 days after resuscitation（P>0.05）.At other time points,the expressions of PTN,SDC3,NF200 and Tau in hippocampal neurons were statistically different between the two groups（P<0.05）.3.HT22 cells of mouse hippocampal neurons cultured in vitro showed the best activity at heparin concentration of 4u/100ul.The optimum MOI of HT22 lentivirus was 120,and the infection enhancement fluid was A.After lentivirus infection with sh RNA-PTN and sh RNA-SDC3,stable cell lines with PTN and SDC3 deletion were obtained by single cell cloning.The mrna expressions of PTN and SDCwere significantly different from those of normal HT22（P<0.05）.The mrna and protein of PTN and NF200were significantly different in HT22,HT22-OGd/R and HT22-Ogd/R-Hep groups（P<0.05）,but there was no significant difference in SDC3m RNA and protein expression among the three groups（P>0.05）.There were no significant differences in mrna and protein expressions of PTN and SDC3 in HT22-PTN-,HT22-PTN--OGD/R and HT22-PTN--OGD/r-Hep groups（P>0.05）.The mrna and protein of NF200 and PSD95were significantly different between HT22-p TN-vs HT22-PTN--OGD/R and HT22-PTN--OGD/R vs HT22-PTN--OGD/r-Hep（P<0.05）.There were significant differences in PTNm RNA and protein expression in HT22-SDC3-,HT22-SDC3--OGD/R and HT22-SDC3--OGD/r-Hep groups（P<0.05）,but no significant differences in SDC3m RNA and protein expression in HT22-SDC3--Ogd/r-Hep groups（P>0.05）.The mrna and protein levels of NF200and PSD95were significantly different between HT22-sd C3-vs HT22-SDC3--OGD/R and HT22-sdc3-vs HT22-SDC3--OGD/r-Hep（P<0.05）.Conclusion:1.Serum PTN and SDC3 in patients with cardiopulmonary resuscitation after cardiac arrest changed with the recovery of neurological function,and they had a certain ability to predict death within 7 days after resuscitation.2.The 5-minute cardiopulmonary resuscitation model of rats with asphyxiating cardiac arrest can cause severe brain injury after resuscitation,and the injury is most significant 3 days after resuscitation;3.Heparin promoted the recovery of cerebral nerve function within 7 days after resuscitation,and improved the expression levels of PTN and NF200 in the brain tissue after resuscitation,but did not affect the expression of SDC3 in the early stage after resuscitation.4.Heparin can up-regulate the synaptic growth of hippocampal neurons after hypoxia injury by affecting the function of SDC3.