Effect And Mechanism Of Curcumin Against Hippocampal Injury Induced By Pseudorabies Virus In Rats

Pseudorabies（PR）is an acute and highly contagious disease caused by Pseudorabies virus（PRV）in pigs of different ages.Among them,nervous system disorder and organ injury are the main pathological features.The disease is one of the major diseases that endanger the healthy development of the pig industry,and has caused huge economic losses to the pig industry.Previous studies have shown that PRV infection can disrupt the mitochondrial function of cells,and the destruction of mitochondrial function is closely related to the pathological changes of the nervous system.PRV has been reported to induce apoptosis in cultured porcine neurons.In addition,viral infection often causes a wide range of oxidative stress in the body,thus accelerating the occurrence and development of the disease.It is well known that oxidative stress and apoptosis are closely related to cellular mitochondrial dysfunction.Therefore,the above research information suggests that neuronal oxidative stress,mitochondrial dysfunction and apoptosis are the key factors of nervous system pathological changes caused by PRV infection,which provides a potential target for the use of antiviral drugs to control PRV infection.Curcumin（CUR）is the pharmacological active ingredient of turmeric.It has antioxidant,anti-inflammatory,anti-cancer and other biological activities.In recent years,the potential of CUR as an antiviral drug has attracted much attention.Studies have found that CUR has anti-virus activity,such as herpes simplex virus and human immunodeficiency virus and so on.However,whether CUR can resist the damage of neurons caused by PRV infection has not been reported.Because CUR has the ability to cross the blood-brain barrier,it has a broad application prospect for the prevention and treatment of nervous system diseases.Therefore,we hypothesized that CUR can reduce neuronal damage caused by PRV infection,and oxidative stress and apoptosis pathways,which are closely related to mitochondrial dysfunction,may be involved in this process.Brain-derived neurotrophic factor（BDNF）is a member of the neurotrophin family,which is most widely expressed in the brain of mammals.Previous studies have shown that BDNF can play the role of anti-oxidative injury and anti-apoptosis by binding to the transmembrane tyrosine kinase B（Trk B）receptor located in the mitochondrial membrane.The increased BDNF can resist the mitochondrial dysfunction in the primary rat cortical culture induced by3-nitropropionic acid.However,there are no studies to prove whether PRV infection can affect the expression of BDNF in hippocampal neurons.If the answer is yes,then we speculate that BDNF/Trk B pathway may mediate the neuroprotective effect of CUR during PRV infection.In order to verify the above hypotheses and speculations,to explore the neuroprotective effect of CUR on PRV infection and its potential mechanism.The following experiments were carried out in this study.In vitro experiment:the hippocampal neurons of rats were cultured in primary culture within 24 hours of birth.After purity detection and identification,the best infection time of PRV infected neurons was determined to establish oxidative stress model.After that,different concentrations of CUR were added to explore the best concentration of CUR to protect neurons against virus infection,and to further explore whether the antioxidant,anti-mitochondrial disorder and apoptosis pathways initiated by BDNF mediate the neuroprotective effect of CUR during PRV infection.It was verified by adding ANA-12,a specific inhibitor of BDNF receptor Trk B,to block BDNF/Trk B signal pathway.The results are as follows.1.The primary cultured hippocampal neurons in vitro were identified with a purity of more than 95%.Compared with the control group,the content of MDA in hippocampal neurons infected with PRV with virus titer3.06×10⁵TCID₅₀increased in a time-dependent manner at 6 and 8 hours after infection,but increased significantly at 2 hours（P<0.05）,while the activity of SOD enzyme decreased in a time-dependent manner and reached a very significant decrease at 2 hours（P<0.01）.At the same time,the content of NO also increased significantly at 2h（P<0.01）.These results showed that oxidative damage of hippocampal neurons could be caused by PRV infection for 2h.2.Hippocampal neurons were treated with different concentrations of CUR for24 hours.The results showed that curcumin at concentrations above 20μmol/L could affect the activity of hippocampal neurons.Then curcumin was pretreated with curcumin less than 20μmol/L for 24 hours,and PRV was added to infect neurons for 2hours.It was found that 2.5-10μM curcumin could protect hippocampal neurons from infection,and the activity of neurons was the highest when the concentration of curcumin was 10μmol/L.therefore,the best concentration of curcumin against PRV infection was 10μmol/L.3.The changes of BDNF protein level in hippocampal neurons were detected by Western-Blot.Compared with the control group,the BDNF protein level in the PRV infection group was significantly decreased（P<0.05）,and the BDNF protein level in the curcumin treatment group was significantly higher than that in the PRV infection group（P<0.05）.The results showed that BDNF mediated the protective effect of curcumin on hippocampal neurons against PRV infection.4.In order to further verify whether BDNF/Trk B mediates the protective effect of curcumin on hippocampal neurons against PRV infection,this study investigated the effect of Trk B inhibitor ANA-12 on neuronal viability by detecting the activity of lactate dehydrogenase（LDH）.The results showed that the level of LDH in the PRV infection group was significantly higher than that in the control group（P<0.01）,and the LDH level in the curcumin treatment group was significantly lower than that in the PRV infection group（P<0.01）.Compared with the curcumin treatment group,the level of LDH in the inhibitor group was significantly higher than that in the curcumin group（P<0.01）,and the inhibitor alone did not affect the neuronal activity,indicating that curcumin protected hippocampal neurons from PRV infection by regulating BDNF/Trk B pathway.5.In order to investigate whether the oxidative stress and apoptosis molecular pathway regulated by BDNF/Trk B pathway mediate the protective effect of curcumin against PRV infection,Western-Blot detected the protein levels or activities of Trk B,n NOS and apoptosis/anti-apoptosis related molecules.The results showed that compared with the control group,the protein levels of n NOS（P<0.01）and Bax（P<0.05）were significantly increased,while the protein levels of Bcl-2（P<0.01）and Trk B（P<0.05）were significantly decreased,and the activity of Caspase-3 was significantly increased in PRV infection group（P<0.01）.Compared with PRV infection group,the protein levels of n NOS（P<0.05）and Bax（P<0.01）in curcumin group were significantly decreased,while the protein levels of Bcl-2 and Trk B were significantly increased（P<0.01）,and the activity of Caspase-3 was significantly decreased in curcumin treatment group（P<0.01）.Compared with curcumin treatment group,the protein levels of n NOS and Bax in inhibitor group increased significantly（P<0.01）,while the protein levels of Bcl-2 and Trk B decreased significantly（P<0.01）,whiletheactivityofCaspase-3increased significantly（P<0.05）.These results showed that curcumin decreased the up-regulation of n NOS and apoptosis-related protein level or activity induced by PRV,and increased the down-regulation of anti-apoptosis-related protein induced by PRV.Blocking the BDNF/Trk B pathway eliminated this effect.6.In order to investigate whether the mitochondrial function regulated by BDNF/Trk B pathway mediates the protective effect of curcumin against PRV infection,the levels of mitochondrial membrane potentialΔψm and ATP were measured.The results showed that the levels of ATP andΔψm in PRV infection group were significantly lower than those in control group（P<0 01）,and the levels of ATP andΔψm in curcumin treatment group were significantly higher than those in PRV infection group（P<0 01）.Compared with curcumin treatment group,the levels of ATP andΔψm in inhibitor group decreased significantly（P<0 01）.However,the addition of Trk B inhibitor blocked the protective effect of curcumin,indicating that BDNF/Trk B pathway mediates the protective effect of curcumin against neuronal mitochondrial dysfunction induced by PRV infection.In order to further confirm the neuroprotective effect of curcumin against PRV infection,we carried out in vivo experiments.In vivo experiment:60 220g SD rats were randomly divided into 6 groups:control group,PRV infection group,PRV+CUR low（25mg/kg）group,PRV+CUR medium（50mg/kg）,PRV+CUR high dose（100mg/kg）group and resveratrol group（positive control group）.The curcumin anti-infection group and the positive control group were injected intraperitoneally with the corresponding dose for 7 days,while the control group and PRV infection group were injected with the same dose of 5%sodium carboxymethyl cellulose solution.On the 14th day,except the control group,the other groups were injected with PRV 100μL with a titer of 2.85×10³.On the 7th day after PRV infection,the rats in each group were anesthetized and the corresponding indexes were detected.The results are as follows:1.As of the 14th day of the test,the 7th day of challenge,the survival rate of rats in the control group was 100%,the survival rate of the PRV group was 70%,the survival rate of the PRV+curcumin low-dose group was 70%,and the survival rate of the PRV+curcumin medium-dose group The rate is100%,the survival rate of the PRV+curcumin high-dose group is 90%,and the survival rate of the positive control group is 90%.The results show that the middle dose of curcumin,that is,50mg/kg,has the best antiviral effect,so the medium-dose group was selected conduct follow-up experiments.2.Detection of oxidation/antioxidation indicators found that compared with the control group,the MDA content in the brain tissue,liver and lungs of the PRV group was significantly increased（P<0.05）,and the GSH content and SOD activity were significantly reduced（P<0.05）,indicating that In vivo PRV infection caused oxidative damage to body tissues;compared with the PRV group,the MDA content in the brain tissue,liver and lungs of the curcumin middle-dose group was significantly reduced（P<0.05）,and the GSH content and SOD activity were significantly increased（P<0.05）,indicating that CUR can relieve oxidative stress in rats caused by PRV infection.3.The results of pathological sections showed that there were different degrees of pathological changes in the hippocampus of the PRV group compared with the control group,and curcumin significantly improved these lesions compared with the PRV group.In summary,this study demonstrated that CUR can protect rat hippocampal neurons from oxidative stress,apoptosis and mitochondrial dysfunction induced by PRV,which is mediated by up-regulated BDNF/Trk B pathway.In addition,through in vivo experiments,it was further confirmed that curcumin could reduce the damage of rat tissue,especially hippocampal tissue caused by PRV infection,which laid a foundation for the research and development of curcumin as an anti-pseudorabies virus drug and neuroprotective agent.