Effects Of TRPV4 On LPS-induced Fever, The Content Of CAMP And The Concentration Of [Ca²⁺]_i In Rat Hypothalamus

INTRODUCTIONRecently many research have found that cAMP is a principal central medium in EP induced febrile response.In addition,people are paying more attention on the change of calcium concentration in hypothalamus tissuses,and want to know how it does in febrile response.But the mechanisum is not clear.TRPV4 is a member of the transient receptor potential vanilloid family.It is a nonselective cation channel which can be activated by warm temperature(above 27 degree).And it can cause a large calcium influx.Reserrchers find that there are much TRPV4 expression in hypothalamus,and this region ia a primary thermalregulation center.So TRPV4 may play a role in thermalregulation.In this experiment,we use lipoolysaccharide to make rats fever and inhibit the activity of TRPV4 with ruthenium red to observe the relationship of TRPV4 content, calcium concentration and cAMP content.To explore whether TRPV4 participate the thermalregulation or not.MATERIALS AND METHODS一,Animals and SurgeryHealthy male Sprague-Dawley rats,weighing between 200 and 250g,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(Ketamine hydrochloride and xylazine),a 1mm of diameter cannula was implanted into the lateral cerebral ventricle by stereotaxic apparatus according to the rat brain altas,and the cannyla was anchored with dental acrylic,sealing off upper end.Rats were individually housed a week before experiments.Examine body temperature:the digital thermometer probe 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-handling.After administration,rectal temperature was recorded respectively during experiments(to measure once every other 15 min in 0-1h,every other 30 min in other time).when the experiment was over,cannula's position was checked to discard incorrect ones.二,cAMP level assayRats were put to death immediately at peak of LPS-induced fever,then the rat brain tissues were put into liquid nitrogen.50mg hypothalamus tissue was weighted, homogenate,centurfuged and assay concentration of cAMP with radio-immunoassay.三,Calcium concentration assayRats were put to death immediately at peak of LPS-induced fever,1/4 hypothalamus tissue was weighted,digested with pancreatin for 30min in 37 degree water bath.Stop the digestion with calf serum,loaded with Fura-2/AM for 45min, centurfuged,added D-Hanks into the precipitation and assay concentration of cellular calcium.å››,The expression of TRPV4 in rat hypothalamus tissue assayHypothalamus tissue was homogenated,centrifuged and the supernatants were used to measure the expression of TRPV4 by western blot assay.五,Statistical analysisResults were presented as mesn±SD((?)±s).Change in body temperature were presented and analyzed withâ–³T.Data were analyzed using t-test. RESULTSGiven RR solely decreased the temperature Compared with LPS group,the increase amplitude ofâ–³T,camp,[Ca²⁺]_i all degrade in RR+LPS group And the content of TRPV4 are less than control groupDISCUSSIONIn this experiment,we use lipoolysaccharide to make rats fever and detect the the expression of cAMP in hypothalamus.The result suggested there is obvious correlation between the change of cAMP content and the change of temperature.It confirmed that cAMP is a important central fever medium.To explore the role of TRPV4 in thermalregulation,we use LPS to make rats fever,and inhibit TRPV4 with blocker ruthenium red.We observed that the temperature of RR+LPS group increased obviously compared with control group.But the fever amplitude decreased and the latency prolonged compared with LPS group.And the temperature of RR group decreased obviously compared with control group.This suggested TRPV4 participate the maintenance of normothermia and play a role in LPS-induced fever.Further research,we tested the expression of TRPV4 in different groups.And we found that the expression of TRPV4 in LPS group was increased obviously compared with control group on peak time.This illustrate that the rising temperature can make TRPV4 express more.And we inject RR into ventricle in advance,the expression of TRPV4 in RR group and R+L group is lower than control group.This suggested that RR can inhibit the expression of TRPV4.So how did TRPV4 play in LPS-induced fever? Accordingly,TRPV4 is a nonselective cation channel and it can cause a large calcium influx when it was activated.So we speculated that because of the opening of TRPV4 channel there was a larger calcium influx,and then the cAMP concentration increased.For this reason,we tested the viscosity of calcium and concentration of cAMP.We found that the concentration of calcium in LPS group increased obviously than control group and we speculate the increasing was a result of TRPV4 opening.And the concentration of cAMP increased more than control group at the same time.Correlation analysis suggested hypothalamus calcium concentration have positive correlation with cAMP content.In RR+LPS group,calcium concentration was higher than control group but lower than LPS group.The cAMP content was lower than LPS group.This experiment suggested the expression and opening of TRPV4 can influence the level of calcium and this is an important way to maintain the normothermia and LPS-induced fever.The further evidence need more research such as electric physiology.CONCLUSIONâ‘ TRPV4 may participate the maintenance of normothermia;â‘¡The ferverscence was implemented by activating TRPV4 channel to induce a large calcium influx and then increasing the content of cAMP...