Preliminary Study Of Curcumin Inhibits Encephalitis Induced By Pseudorabies Virus And The Underlying Mechanism

In recent years,the variation of porcine pseudorabies virus strain in China has led to the decline of the protective ability of the original pseudorabies virus vaccine,which resulted in an outbreak trend in pig farms and caused a great economic losses to the pig industry.Porcine pseudorabies is mainly characterized by nervous system diseases and reproductive system diseases,such as encephalitis,ataxia,abortion and so on.Microglia is the immune cells in the central nervous system,which is widely distributed throughout the central nervous system.It can constantly remove damaged nerves,plaques and infectious substances,and plays a very important role in the injury of the central nervous system and the prognosis of diseases.According to related studies,pseudorabies virus infection can cause the activation and proliferation of microglia in brain tissue,which suggests that microglia and their mediated brain inflammation play an important role in the prognosis of PRV-damaged brain tissue and neurological diseases.Curcumin is a yellow pigment extracted from the rhizome of Zingiberaceae,and has many pharmacological effects such as anti-inflammation,anti-oxidation,antibacterial,improving immunity and so on.Scholars have found that CUR can pass through the blood-brain barrier and has the effect of anti-virus in recent years.Therefore,we speculate that,pseudorabies virus may cause encephalitis and activation of microglia after rats were infected and curcumin can improve pseudorabies virus-induced encephalitis and may have a certain inhibitory effect on virus replication.In order to confirm the above hypothesis and investigate the underlying molecular mechanism,we carried out the following experimental studies.In vivo experiment:After the optimal infection dose was determined in 40200±20g SD rats（half male and half female）,60 200±20g SD rats（half male and half female）were randomly divided into 6 groups:including control group（Control）,virus infection group（PRV）,resveratrol（50mg/kg BW）positive control group（RES）,low dose（25mg/kg BW）curcumin group（CUR L）,medium dose（50mg/kg BW）curcumin group（CUR M）and high dose（100mg/kg BW）curcumin group（CUR H）.The positive control group and curcumin groups were injected intraperitoneally with corresponding concentrations of drugs every day for 14 days,the control group and virus infection group were injected intraperitoneally with the same dose of carrier solvent（0.5%carboxymethyl cellulose）every day for 14 days.On the 8th day of the experiment,except for the control group,the rats in other groups were inoculated with 100μL(2.85×10³TCID₅₀/0.1m L)PRV solution.The excited state of central nervous system was tested by open field test,and the daily changes of body weight,temperature and death were recorded.At the end of the experiment,the rats under anesthesia were killed by decapitation.The cortex of rats were taken to observe the pathological changes of the cortex by making pathological sections.Our study aimed at comprehensively analyzing the anti-inflammatory and antiviral effect of curcumin.The results in this experiment showed that,CUR improved the clinical symptoms such as elevated body temperature,growth inhibition and central excitement caused by PRV infection in SD rats.Furthermore,CUR also alleviated the pathological symptoms such as cerebral hyperemia,cortical vascular entrapment and pathological changes caused by PRV infection in SD rats.In vitro experiment:BV2 microglial cell lines were subcultured and the cytotoxicity of different concentrations of curcumin was determined by CCK-8 method.After that,BV2 cells were pretreated with safe concentrations of curcumin（2.5-20μM）for 2 hours and then infected with PRV（MOI=0.5）for 24 hours.The progeny virus titer was detected by TCID₅₀method,and the changes of inflammatory cytokines TNF-α,IL-6 and MCP-1 were detected by ELISA.Western Blot analysis was applied to investigate the expression levels of HO-1 protein.The aim of our study is to comprehensively analyze the anti-inflammatory and antiviral effects of CUR and the underlying mechanisms.The results in this experiment showed that,the concentrations of CUR below 20μM had no obvious cytotoxicity to BV2 cells,and CUR reduced the titer of PRV progeny virus,inhibitd the increase of inflammatory factors such as TNF-α,IL-6 and MCP-1 caused by PRV infection in BV2 cells,and activated the expression of HO-1 to play an anti-inflammatory effect.Conclusion:（1）In vivo test,CUR alleviated the central excitement,high body temperature,reduced growth and low survival of rate caused by PRV infection.（2）In vivo test,CUR relieved congestion and cortical lesions of rats brain tissue caused by PRV infection.（3）In vitro,CUR prevented the decrease of cell viability caused by PRV infection,reduced the titer of offspring virus,and inhibited the increase of TNF-α,IL-6 and MCP-1 expression caused by PRV infection,indicating that CUR has antiviral and anti-inflammatory effects.（4）In vitro,HO-1 may mediate the response of CUR against microglial inflammation.