Effects Of Tetrandrine On Glial Cells By Modulating Neuroinflammatory Respons In A Rat Model Of Vascular Dementia

Objective:Vascular dementia (VaD) is defined as a clinical syndrome of acquiredimpairment of intellectual function resulting from brain injury due tocerebrovascular disorder. The exact mechanisms of VaD are not known, butactivation of glial cells and release of inflammatory cytokines has emergedas a critical component involved in the pathophysiology of VaD. S100B isan acidic calcium binding protein. It derived from astrocytes and has beenregarded as an important factor which functions in the brain-damagingfeedback loop leading to neurotoxicity. Tetrandrine (Tet), abis-benzylisoquinoline alkaloid, is isolated from the root of StephaniaTetrandrae S. Moore. It is a natural calcium channel blocker andtraditionally used as a herb with multiple biological activities, includinganti-inflammatory, antitumor and immunosuppressive activities.Recently, it was reported that Tet have protective effects on rats withAlzheimer’s disease by modulating inflammatory agent. Enhanced andprolonged neuroinflammatory response is a common pathogenic mechanism shared by VD and AD, especially in the hippocampus and cerebral cortex.The aims of this study were, therefore, to determine the effects of Tet onlearning and memory dysfunction and pathological changes inhippocampual CA1zone of rats with VaD induced by permanent bilateralcommon carotid artery ligation (2-VO) through modulating S100B andpro-inflammatory IL-1β、iNOS.Methods:1. VaD rats were induced by ligation of bilateral common carotid arteries(2-VO) and maintained for8weeks until behavioral testing. Escape latencywas abtained from Morris water maze test to choose subjects in accordancewith the literature. Rats were classified as dementia group if the proportionwas greater than20%and chosen to further experiment.2. Rats were treated with Tet (10mg/kg or30mg/kg) intraperitoneallythrice weekly for4weeks. Morris water maze test was then adopted to testability of learning and memory ability in all rats12weeks after surgery.H&E staining,Nissl staining and TdT mediated dUTP nick end labeling(TUNEL) were used to detect the pathological changes and neuronalapoptosis．3. Aanimals (n=10in each group) were decapitated after anesthesia.Total RNA and protein in the hippocampus was isolated to analyze thecontents of IL-1β, S100B and iNOS. At the same time, brains wereembedded with paraffin, and then the paraffin-embedded tissues were cut into4-mm sections and mounted at poly-L-lysine-coated slides. Morriswater maze test was then adopted to test ability of learning and memoryability in all rats12weeks after surgery. H&E staining,Nissl staining andTdT mediated dUTP nick end labeling (TUNEL) were used to detect thepathological changes and neuronal apoptosis．Fluorescence histochemistrywas used to observe the expression of S100B protein in the hippocampalCA1zone. Also,the change of [Ca2+] in the hippocampi were detected bythe Ca2+indicator Fluo-3AM under a confocal laser scanning microscope.Results：1. Morris water maze test showed that Escape latency was significantlyshortened, the times of entries and swimming distance in the target zone wassignificantly increased in VaD rats administrated with Tet when comparedwith those in model group(P<0.05). At the same time, rats had improvedpathological changes such as nuclear shrink or disappearance, cellularedema and the loss of Nissl bodies in pyramidal neuron, and numbers of theTUNEL positive cells was significantly decreased in the CA1zone ofhippocampus in Tet-treated rats(P<0.05)．2. FQ-PCR, Western blot, immunofluorescence analysis showed that2-VO surgery can induce astrocyte-derived S100B activation ofshippocampal CA1zone in situ, and S100B mRNA and S100B proteinexpression increased in the hippocampal cells (P <0.05)， proinflammatorycytokine IL-1β and proinflammatory enzyme iNOS expression was also significantly increased (P <0.05). However,in the Tet-treated group,levels of S100B,IL-1β and iNOS were significantly reduced (P <0.05).[Ca2+] i concentration of hippocampus in Tet-treated group was decreasedsignificantly compared with model group (p <0.05).Conclusion:1. Ischemic VaD rat model is successfully established induced byligation of bilateral common carotid arteries (2-VO)2. Tet could relieved the learning and memory dysfunction andpathological lesions, reduced the quantity of the TUNEL positive cells in theCA1zone of hippocampus in VaD rats．3. Tet ameliorates learning and memory dysfunction in rats with VaDprobably through inhibiting glial activation and hippocampal S100Bactivities and release of proinflammatory IL-1β and iNOS.