| Background: Endotoximia(ETM)is a pathological disease caused by large amounts of endotoxin either from bacterial infection or endotoxin polluted injection.Mortality rate can reach 70%~90% with cardiac dysfunction during severe ETM.Nevertheless,there are no efficient approaches to prevent and treat ETM induced cardiac dysfunction,decrease high mortality rate or prolong survival time.Therefore,it is very significant to study the pathogenesis of cardiac dysfunction induced by ETM and seek the effective therapeutic targets.TRPC,which are short for transient receptor potential canonical channels,are the nonselective cation channels.Recent studies have showed that TRPC play crucial roles on many physiological and pathological processes including nervous system development,neuronal survival,cardiac pacing,cardiac conduction,and cardiac contraction,hypoxic pulmonary vasoconstriction,pulmonary hypertension and pathological cardiac hypertrophy.Our study demonstrated that that the protein expression levels of TRPC1 and TRPC6 were markedly increased in cardiac tissues of ETM mice,suggesting that TRPC may participate in the development of cardiac dysfunction induced by ETM.Objective: The objective of this research were to study the probable role of TRPC on cardiac dysfunction induced by ETM from different perspectives of molecules,organs and animals using relevant experimental techniques combined pharmacology and gene intervention,then explore the probable mechanism underlie the effects of TRPC on cardiac dysfunction induced by ETM,and thus provide experimental evidence for finding novel therapeutic targets in ETM prevention.Method:(1)The models of ETM mice were established by 40 mg/kg LPS injection.After treating LPS for different time,the protein expression levels of all subtypes of TRPC were detected by western blot experiment in cardiac tissues acquired from the anima l models.The genotypes of mice were identified by PCR.The expression and location of TRPC1 and TRPC6 in myocardial cells was detected by immunofluorescence.After injecting LPS into WT mice and knockout mice,the alterations of cardiac inflammatory pathological damage,cardiac function and survival time of mice were observed,respectively.(2)The mRNA level of relevant genes,including Tnf-?,Il-1?,Il-6,Tlr2,Tlr3,Tlr4,Traf6,Gapdh,Myd88,Trif,Tirap,Tab2,I?b?,Ap1,Nox2,Nox4,Gsh-px1,Ncf1,Ncf2,Nrf2,Caspase3,Calmodulin and Calcineurin etc,were measured by real-time PCR technique.After stimulating with LPS,the levels of TNF-?,IL-1?,IL-10 and IL-6 were measured by enzyme-linked immunosorbent assays.The phosphorylation and protein expression levels of inflammatory pathway relevant molecules(TLR4?MyD88?TRAF6?IRAK1?IRAK4?TRIF?TIRAP and TRAM),MAPK molecules(ERK1/2?JNK?p38 and ERK5),NF-?B molecules(p65,I?B? and IKK)and CaM were measured by western blotting.The relationships between TRPC1/6 and relevant proteins were forward investigated by immunoprecipitation.(3)Mice were pretreated with different doses(5 mg/kg,10 mg/kg and 20 mg/kg)of TRPC inhibitor SKF96365 or vehicle before injection with 40 mg/kg LPS and the alteration of cardiac inflammatory pathological damage,cardiac function and survival time of mice were observed,respectively.Results:(1)The models of ETM mice were successfully established by administrating LPS.The protein expressions of TRPC1 and TRPC6 were higher than the control group(1.6±0.2 folds and 1.9±0.2 folds).What's more,the changes of TRPC1 and TRPC6 expression levels were time-dependent from WB and immunofluorescence experiments.The result of WB experiments showed that the alteration of TRPC1 protein expression increased before 3 h and descreased after 3 h.In 3 h,the protein expression of TRPC1 was 1.5±0.2 folds that of 0 h.Similarly,the alteration of TRPC6 protein expression increased before 2 h and descreased after 2 h.In 2 h,the protein expression of TRPC6 was 2.6±0.2 folds that of 0 h.Besides,the locations of TRPC1 and TRPC6 were detected in myocardial cells.After administrating LPS,the LVEF was reduced from 69.5±2.74% to 35.2±1.26% in WT mice.The LVFS was reduced from 32.9±2.07% to 13.5±0.57% in WT mice.However,after administrating LPS,the LVEF was reduced from 71.3±3.23% to 45.0±1.55% in Trpc1-/-mice.The LVFS was reduced from 34.6±2.23% to 18.1±0.76% in Trpc1-/-mice.Similarly,,after administrating LPS in Trpc6-/-mice,the LVEF was reduced from 64.7±1.81% to 50.8±0.53%.The LVFS was reduced from 29.4±1.24% to 21.1±0.28%.Compared with LVEF and LVFS of WT+LPS group,LVEF and LVFS of knockout mice were increased in response to LPS,suggesting that TRPC1 and TRPC6 may participate in the process of cardiac dysfunction induced by LPS.Histomorphology and survival rates evaluation results suggested that survival rates of ETM mice were increased from 0% to 70% and inflammatory damage of myocardium were decreased in Trpc1-/-and Trpc6-/-mice.(2)The results of ELISA experiments showed that the level of TNF-? increased from 50.5±4.3 pg/?l to 176.0±4.5 pg/?l,the level of IL-1? increased from 13.2±1.6 pg/?l to 34.1±1.5 pg/?l,the level of IL-6 increased from 0.5±0.8 ng/?l to 1.8±0.1 ng/?l in LPS+WT mice.In Trpc1-/-+LPS mice,the level of TNF-? increased from 56.6±2.1 pg/?l to 72.3±3.1 pg/?l,the level of IL-1? increased from 16.2±0.7 pg/?l to 24.6±0.8 pg/?l,the level of IL-6 increased from 0.6±0.1 ng/?l to 1.16±0.1 ng/?l.In Trpc6-/-+LPS mice,the level of TNF-? increased from 65.5±4.2 pg/?l to 80.0±7.1 pg/?l,the level of IL-1? increased from(17)(16)(13)(15)±1.4 pg/?l to 27.2±1.7 pg/?l,the level of IL-6 increased from 0.5±0.1 ng/?l to 0.9±0.1 ng/?l.Those results suggested that the levels of pro-inflammation cytokines in Trpc1-/-and Trpc6-/-mice were lower than those in WT mice after administrating LPS.In addition,in WT+LPS mice,the level of IL-10 increased from 10.4±1.7 pg/?l to 16.4±3.0 pg/?l.In Trpc1-/-+LPS mice,the level of IL-10 increased from 24.2±1.1 pg/?l to 36.0±4.7 pg/?l.In Trpc6-/-+LPS mice,the level of IL-10 increased from 21.6±1.6 pg/?l to 40.7±3.6 pg/?l.Those results suggested that the levels of IL-10 in Trpc1-/-and Trpc6-/-mice were higher than those in WT mice after administrating LPS.Furthermore,in WB and PCR experiments,the activation of TLR4-MyD88-TRAF6,phosphorylation of MAPK and NF-?B signaling pathway related protiens induced by LPS were attenuated in Trpc1-/-and Trpc6-/-mice.In addition,TRPC1 and TRPC6 could interact with CaM which can further interact with TLR4,MyD88 and TRAF6 by Co-IP experiments.(3)The cardiac function detected by M-Echo showed that the LVEF and LVFS were 73.0+2.06(4)and 35.4+1.65(4)in Vehicle group,68.6±1.30% and 32.0±0.94% in 10 mg/kg SKF group,35.4+2.39(4)and 13.6±1.08(4)in LPS group,(4)and 6±0.81(4)in 5 mg/kg SKF+LPS group,49.3±1.40(4)and 20.3±0.73(4)in 10 mg/kg SKF+LPS group,56.3±2.08(4)and 24.2±1.24(4)in 20 mg/kg SKF+LPS group,respectively.The results suggested that SKF96365 could improve cardiac dysfunction induced by LPS in a dose-dependent manner.In addition,SKF96365 also could alleviate damages of myocardium induced by LPS in a dose-dependent manner.The experiments of urvival analysis showed that survival rates of mice were 10% in LPS group within 60 h.After injection with low,medium and high doses of SKF96365,the survival rates of ETM mice were 10%,20% and 30%,respectively.Further,the survival rates of ETM mice can reach about 70% when ETM mice were injected with medium dose of SKF96365 each 8 h for three times.Conclusions:(1)The study proved that TRPC1 and TRPC6 were involved in the process of cardiac dysfunction induced by LPS.The inhibition of TRPC1 or TRPC6 could alleviate damages of myocardium induced by LPS,improve cardiac function of ETM mice and enhance its survival rates.(2)TRPC1 and TRPC6 could amplify the inflammatory signaling pathway cascade by inhibiting the protein expression of CaM,reducing the binding of Ca M to TLR4,MyD88 and TRAF6 proteins.(3)SKF96365 could significantly alleviate damages of myocardium induced by LPS,improve cardiac function of ETM mice and enhance survival rates of ETM mice.Therefore,TRPC were potential targets for drug therapy of ETM-induced cardiac dysfunction. |
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