Protective Effect And Mechanism Of Curcumin Against TNF-α-induced Neuronal Damage In Rat Hippocampus

Since the first acquired immunodeficiency syndrome(AIDS) patient was found in 1981,scientists and doctors have done a lot of researches on AIDS and human immunodeficiency virus(HIV).The application of highly active antiretroviral therapy (HAART) has prolonged the lives of HIV-1 infected patients,and lowered the death rate.But after the death rate went down,AIDS patients become suffering from systematic complications.One of the most common and severe complications is central nervous system(CNS) complications,HIV-1 associated dementia(HAD).HAD is impairments of cognition,movement and behavior,and happens in over 60%of AIDS patients.It has become one of the most important causes of death in AIDS patients.It is reported that HAD has become the most common cause of dementia for people under 60 years old.However HAART is not very effective for HAD.It did not lessen the pathological changes in HAD patients' brains,and improve the clinical symptoms.It confused researchers that there was low virus load in HAD patients' brains which could not explain the severe pathological changes.Then,what damaged neurons so badly? According to the newest reports,HIV-1 and its viral proteins(gp120,gp41, Tat) can directly damage neurons.But the immune response and the inflammation induced by cytokines,such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β) and interleukin-6(IL-6),which are secreted from HIV-1 activated macrophages and microglias are thought to play a more important role in the process of HAD.Among the cytokines,TNF-αis more neurotoxic.It can activate astrocytes and microglias to secrete a large mount of excitatory amino acids-glutamate(Glu),and inhibit uptake of Glu by astrocytes.The concentration of Glu becomes higher and higher.And neurons are overexcited and damaged.Curcumin has many multiple pharmacal activities,such as anti-inflammation, anti-HIV-1 and anti-tumor.It can directly or indirectly inhibit the long terminal repeat (LTR),protease and integrase of HIV-1 so as to inhibit the viral duplication.Curcumin also can inhibit inflammatory factors,such as TNF-α,IL-1βand IL-8,and lessen inflammatory symptoms.But the application of curcumin on HAD treatment has not been reported at home or overseas.Our previous study has proved curcumin could improve learning and memory ability in memory disorder rats induced by gp120.Based on that,the present study aims on protective effect and mechanism of curcumin against TNF-α-induced neuronal damage in rat hippocampus,contributing a new experimental support to the prevention and treatment of cognitive dysfunction patients.ObjectiveTo observe the structural and functional protective effect of curcumin against TNF-α-induced neuronal damage in rat hippocampus,and explore the mechanism.To provide experimental support for applying curcumin on treating HAD.Methods1.30 Spraugue-Dawley(SD) fetal rats which were born in 1 day were used in hippocampal neuron culture.Cultivate rat hippocampal neurons for 7 days.Mark Tau protein of neurons by fluorescent staining.Identify the neurons by observing their morphologic characteristics under inverted fluorescent microscope and laser confocal microscope.2.Incubate the neurons with low,medium and high doses of curcumin and TNF-αrespectively.Determine safe dose(low dose,1μmol/L)of curcumin and damage dose(high dose,100ng/mL) of TNF-αby observing morphological changes of neurons.3.After Cultivating rat hippocampal neurons for 5 days,neurons were divided into 4 gropus:control group(no drug in medium),TNF-αgroup(100ng/mL TNF-αin medium),curcumin group(1μmol/L curcumin in medium),and curcumin plus TNF-αgroup.After drug treatment,take dynamic photo series of neurons for 1 hour and static photos 48 hours later under inverted microscope to observe morphological changes of neurons.Another same grouped rat hippocampal neurons were cultivated for 7 days.Observe the concentration changes of fluorescently-labeled Ca²⁺ in neurons immediately after TNF-αand curcumin treatment under laser confocal microscope.4.50 SD rats were divided into 10 groups randomly:control group,TNF-αbaseline group,NMDA baseline group,curcumin baseline group,TNF-αgroup,heated TNF-αgroup,NMDA group,curcumin group,curcumin plus TNF-αgroup, curcumin plus NMDA group.Make rat brain slices,and incubate brain slices in artificial cerebrospinal fluid(ACSF) which was saturated by mixed oxygen(95% O₂,5%CO₂) for at least 1 hour.Then incubate brain slices for another 30 min after putting drug into ACSF.Record the excitatory postsynaptic potential(EPSP) of CAl pyramidal layer of rat hippocampal brain slices with extracellular electrophysiological techniques.Measure and calculate the initial slope of EPSP of 10 groups.The drug treatment of 10 groups was as follows:control group had no drug in ACSF;TNF-αbaseline group and TNF-αgroup had 4.5ng/mL TNF-αin ACSF;heated TNF-αgroup had 4.5ng/mL TNF-αin ACSF which was bathed at 70℃for 30min;NMDA baseline group and NMDA group had 0.5mmol/L NMDA in ACSF;curcumin baseline group and curcumin group had 1μmol/L curcumin in ACSF;curcumin plus TNF-αgroup had 1μmol/L curcumin and 4.5ng/mL TNF-αin ACSF;curcumin plus NMDA group had 1μmol/L curcumin and 0.5mmol/L NMDA in ACSF.Results1.Tau protein fluorescently-labeled by Cy3 showed the shape of neurons limpidly. The neuron cell body was pyramidal or elliptic.There were lots of axons and secondary branches stretching out from cell body and forming network.They could be distinguished from glia cells easily.2.Low dose of curcumin(1μmol/L) did not influence the shape and growth of neurons.But medium(10μmol/L) and high doses(20μmol/L) of curcumin would inhibit the shape and growth of neurons.When the concentration was higher than 20μmol/L,neurons died.When the concentration of TNF-αwas over 100ng/mL,it obviously inhibited the growth of neurons.Neuron cell body was small and spindle-shaped.Rare axons and secondary branches stretched out from cell body which could not form network structure.3.1 hour after drug treatment,cell bodies of neurons of control group and curcumin group were normal.Axons and secondary branches grew well.But cell bodies of neurons of TNF-αgroup swelled.And axons and secondary branches gradually shrunk,and finally vanished.However,cell bodies of neurons of curcumin plus TNF-αgroup were normal.Axons and secondary branches became normal too. 48h after treatment,it could be seen under microscope that neurons of control group and curcumin group grew closely.Their cell bodies were comparatively large and plump.There were a lot of axons and secondary branches stretching out from cell bodies which formed network structure.However,neurons of TNF-αgroup grew sparsely.And neuron cell body was small.There were rare axons and secondary branches stretching out from cell body which could not form network structure.But neurons of curcumin plus TNF-αgroup grew well. Axons and secondary branches recovered back to mormal.4.Laser confocal scanning indicated that TNF-αinduced rapid influx of Ca²⁺ in neuron endochylema.Fluorescence intensity greatly increased.Curcumin could partly inhibit the Ca²⁺ increase induced by TNF-α(P＜0.05 vs control group),and maintain the Ca²⁺ homeostasis of neuron endochylema.5.After high frequency stimulation(HFS,100Hz,1000ms×2,20s interval) which was gave on Schaffer branches,record long term-potentiation(LTP) in CA1 region of hippocampus.TNF-αand NMDA could obviously inhibit LTP of hippocampal brain slices of rat.Initial slope of EPSP both went down(TNF-αgroup:P＜0.05 vs control group;NMDA group:P＜0.05 vs control group).But TNF-α,NMDA and curcumin did not influence basal EPSR Furthermore, curcumin did not influence LTP itself(P＞0.05 vs control group).Curcumin could recover the LTP inhibited by TNF-αor NMDA.Initial slope of EPSP became normal(P＜0.05 vs TNF-αgroup;P＜0.05 vs NMDA group).Conclusion1.Low dose of curcumin can protect neuron from TNF-α.Pretreating curcumin makes neuron grow healthily and maintain normal shape.Curcumin has structural protective effect on hippocampal neuron of rat.2.Curcumin also can prevent the electrophysiological disfunction of rat hippocampal neuron induced by TNF-αand NMDA,and stabilize LTP.Thus, curcumin has functional protective effect on hippocampal neuron of rat.3.The mechanism of structural and functional protective effect of curcumin against TNF-α-induced neuronal damage in rat hippocampus is that curcumin can prevent the rapid Ca²⁺ influx induced by TNF-αand maintain the Ca²⁺ homeostasis in neuron endochylema through NMDA receptor.