Effects Of TRPV1 On LPS-induced Fever And The Content Of CAMP And [Ca²⁺]_i In Hypothalamus In Rats

INTRODUCTIONIt is generally considered that preoptic area of the anterior hypothalamus(POAH) is an important thermoregulatory control center in the brain and contains temperature sensitive neuron.This area integrates peripheral and central thermometric information. Capsaicin receptor,recently termed vanilloid receptor(transient receptor potential vanilloid,TRPV1),is a ligand-gated ion channel whose activation by capsaicin,protons (pH＜6.0),and noxious heat(43℃)increases intracellular Ca²⁺in vitro.Recently many researchers have found that TRPV1 mRNA are involved in hypothalamus,therefore we suppose that TRPV1 may play a important role in thermoregulation.Cyclic adenosine monophosphate(cAMP)is a crucial meditator regulating cell function synopsis transmition,and is also a major central neurogen in febrile response.TRPV1 receptor antagonist capsazepine has been extensively studied and shown to inhibit activated by noxious heat.In this study,we observed the variation of temperature and the content of cAMP and Ca²⁺in hypothalamus by replicating the models for LPS-induced fever in the rat after injected capsazepine through center,in order to explore the effect of TRPV1 receptor on LPS-induced fever and its mechanism.Materials and methods一,Group and surgeryHealthy male Sprague-Dawley rats,weighing between 250 g and 280 g,were provided by China Medical University Animal Center.They were maintained in a room at(22±2)℃and individually housed in wire-mesh cages with a 12-h light:12-h dark cycle and fed with dry powder chow,with tap water available ad libitum.Under intraperitoneal anesthesia(10%Chloral Hydrate),an 1 mm of diameter cannula was implanted into the lateral cerebral ventricle by stereotaxic apparatus according to the rat brain altas,and the cannula was anchored with dental acrylic,sealing off upper end. Rats were individually housed a week before experiments.Examine body temperature: the digital thermometer probes were inserted 6 centimeters in rats' rectum.Value was recorded after stabilization.Mean of three values 1 hour before administration was taken as baseline of pre-hangling.After administration,rectal temperature was recorded respectively during experiments(to measure once every other 15 min in 0-1 h,every other 30 min in 1-8h).When the experiment was over,cannula's position was checked to discard incorrect ones.二,cAMP level assayRats were put to death immediately at the time of 300 min after LPS-induced fever, and then the rat brain tissues were put into liquid nitrogen.50mg hypothalamus tissue was weighted,homogenate,3500r·min^-1-centurfuged 15min and assay concentration of cAMP with radioimmunoassay.三,Intracellular Ca²⁺concentration in hippocampusIntracellular Ca²⁺concentration in hypothalamus cells was measured by the technique of Fura-2/AM calcium ions fluorescence indicator in SPF.The excitation wave is 340nm and the emission wave is 510nm.å››,Statistical analysisResults were presented as mean+ SD.Change in body temperature were presented and analyzed with T.Interclass data were tested by ANOVA.Results1.In the febrile rats induced by LPS,cAMP level and[Ca²⁺]_i in hypothalamus were significantly increased.2.Pretreatment with capsazepine could obviously reinforced LPS-induced fever and meanwhile the content of cAMP and[Ca²⁺]_i were higher.DiscussionIn our study,the body temperature of the rats advances obviously after administration LPS,and peaked at 240 min.The fever lasted more than 6 hours.And the cAMP level in hypothalamus was significantly higher,which was consistent with the previous report.Our research observed that LPS-induced fever was obviously reinforced by pretreatment with i.c.v TRPV1 receptor antagonist capsazepine,and cAMP level in hypothalamus was significantly higher than LPS group,which suggested that TRPV1 receptor was blocked by capsazepine and can not induce effect.Meanwhile in intracellular Ca²⁺concentration in hypothalamus cells was significantly lower,which suggested that capsazepine blocked TRPV1 receptor through decreasing intracellular Ca²⁺or Ca²⁺external flowing,the level than even lower than the normal.It suggested that TRPV1 played an important role to limit febrile response.But intracellular Ca²⁺ how to restrict the content of cAMP is not clear.It must research further.These findings suggest that TRPV 1 is involved in LPS-induced febrile response especially play a critical role for limiting the extent of fervescence.Conclusion1.TRPV1 channel may have no influence on normal temperature.2.TRPV1 receptor plays a critical role for limiting the extent of fervescence.