The Protective Effect Of Dexmedetomidine On Heatstroke Mice And Its Mechanism Based On Intestinal Barrier

BackgroundHeatstroke is a life-threatening illness characterized by severe hyperthermia(Tc ? 40 oC)associated with central nervous system(CNS)disturbances,such as confusion,ataxia or coma.Endotoxin released from the “leaky” intestinal barrier induced by heatstroke played a key role for aggravating systemic inflammatory response to multi-organ dysfunction,which pathogenesis showed a high similarity with sepsis.Therefore,the intestine has been considered as the motor of multiple organ injury,and maintaining the intestinal integrity has been a potential target for heatstroke treatment.Dexmedetomidine(DEX),a highly selective ?2 adrenoceptor agonist(?2-AR),is widely used in ICU and surgical room as a sedative and analgesic.As we know,endotoximia and sepsis are the common complications associated with high mortality in ICU patients.It has been reported that DEX,used as a sedative in critically ill patients,could substantially decrease serum levels of TNF-?,IL-6 and IL-1?.Meanwhile,its potential anti-inflammatory and anti-apoptosis effects also have been verified in kinds of animal models,including endotoxemia,sepsis and so on.Therefore,it can be inferred that DEX may have a protective effect on heatstroke.According to the data,?2A-AR was high-expressed in the gut.Specifically,growing studies have found that DEX could decrease inflammation and apotosis,and protect against organ injury via inhibiting TLR4/NF-?B pathway through ?2A-AR in various models.Therefore,it can be inferred that DEX can attenuate intestinal injury,inhibit systemic inflammatory response,decrease apoptosis and further protecting against multi-organ dysfunction under heatstroke.ObjectiveIn this study,we focused on the protective effect of DEX on inflammation and organ injury induced by heatstroke in mice.We also exploring the mechanism underlying the effects of DEX on heatstroke through dectecting the permeability,tight junctions,ultrastructure,inflammation and apoptosis of the intestine.The ?2A-AR and TLR4 expression in the intestine were detected,and its downstream NF-?B,NLRP3 inflammsome signal pathway and apoptosis were also researched.Finally,?2A-AR agoniost BRL-44408 was pre-treated to identify whather the effects of DEX on heatstroke were rely on ?2A-AR,therefore providing a potential new therapeutic drug for heatstroke in clinic.Methods Part One: heatstroke mice modelMethod to induce heatstroke: The mice judged suitable for experiment were placed in the pre-warmed artificial climate chamber(provided by Department of Enviromental Hygiene of the Second Military Medical University)maintained at 40 ± 1 oC with a relative humidity of 55 ± 5 %,until the Tco increased to 42.7 oC,the received criterion of heatstroke,mice were moved out of the chamber,and recovered passively at room temperature(22 ± 2 oC)in a new cage with food and water ad libitum.The levels of inflammatory cytokines: The blood samples were harvested at 2 hours,6 hours and 24 hours after the onset of heatstroke,respectively.Serum levles of TNF-?,IL-6 and IL-1? were detected to choose the best time point for the subsequent research.Multi-organ injury induced by heatstroke on mice: Based on the best time point above,the mice were killed,blood and tissues were collected for analysis.First,the biomarker of liver function(ALT,AST and ALP)and kidney function(BU and CREA)were detected.Then,the levels of plasma endotoxin as a biomarker of the intestinal function were also detected.Finally,the samples of liver,kidney,lung,spleen and ileal segment 5 cm away from the ileocecal valve were harvested immediately following blood withdrawal,and then were paraffin embedded,sectioned,stained with hematoxylin and eosin(H&E)and examined microscopically in a blind way.Part Two: the protective effect of DEX on heatstroke miceThe dose choosen for DEX: The mice were intraperitoneally injected with DEX at the dose of 5 ?g/kg,10 ?g/kg and 25 ?g/kg,respectively,immediately after the onset of heatstroke.The blood samples were harvested 6 hours after heatstroke,based on the results above.Inflammatory cytokines were detected to identify the effective dose of DEX for the subsequent research.The protective effect of DEX against multi-organ injury: DEX were injected intraperitoneally,at the best effective dose,immediately after the onset of heatstroke.All the mice were killed after 6 houres,blood and tissues were collected for analysis.First,we checked the serum levles of TNF-?,IL-6 and IL-1?.Then,the biomarker of liver function(ALT,AST and ALP),kidney function(BU and CREA)and intestinal function(plasma endotoxin levels)were detected.Finaly,Samples of liver,kidney,lung,spleen and ileal segment 5 cm away from the ileocecal valve were harvested immediately following blood withdrawal,and then were examined microscopically in a blind way.Permeability of the intestine: An FD4 solution was gently gavaged to mice,the plasma FITC concentration was measured at 6 hours after the onset of heatstroke.Plasma D-lactate levels were measured by a D-lactate Colorimetric Assay Kit according to the manufacturer's instructions.Expression of tight junction proteins: Both WB and immunohistochemisty staining were used to check the expression of tight junction proteins occludin and ZO-1 of the intestine.Ultrastructure of the intestine: Tansmission electron microscopy was employed to check the ultrastructure of the intestine under heatstroke.Part Three: the mechanism of DEX on protecting intestine of the heatstroke miceThe inflammatory pathway related to TLR4 in the intestine: Western blot was used to detect the expression of ?2A-AR and TLR4.WB was used to detect the expression of p65,phospho-p65 and phospho-I?B? as the key proteins of NF-?B pathway.The level of nuclear localization of NF-?B p65 was detected by immunofluorescent staining.WB was used to detect the expression of NLRP3,ASC and caspase-1 p10,and the level of NLRP3 and IL-1? was detected by immunofluorescent staining.Finaly,m RNA levels of TNF-?,IL-6 and IL-1? in the intestine were detected through q PCR.Apoptosis level in the intestine: The expression of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were mearsured by western blot,and the numbers of apoptotic cells were mearsured by TUNEL immunofluorescent staining.The mice were pretreated with BRL-44408 to verfy whether the protective effect of dexmedetomidine on heatstroke was depending on the ?2A-AR.The expression of ?2A-AR and TLR4 in the intestine,the intestinal m RNA and the serum levles of TNF-?,IL-6 and IL-1?,and the pathological examination of liver,kidney,lung,spleen and ileal were both detected.Results 1.Heatstroke mice modelThe core temperature: Ten male C57BL/6 mice were placed in a controlled climate chamber(40 ± 1 oC,55 ± 5 % humidity)for 218.0 ± 19.9 min,they reached to the maximumcore temperature(Tc,Max)of 42.7oC,the received criterion of heatstroke.During heating,the mice showed moving restlessly,delirium,dehydration and even coma.The temperature of heatstroke mice showed a lipid fall without heating,fall to(30.5 ± 0.9)oC between 80 min – 180 min.After the heatstroke mice recoved for approximately 6 hours,the temperature increased to(36.9±0.8)oC,with a similar signs with control mice(n=5).Levels of serum inflammatory cytokines: The concentration of serum TNF-?,IL-6 and IL-1? were increased significantly at 6 h after the onset of heatstroke compared with control mice(n=8).Multi-organ dysfunction: The biomarker of liver function(ALT,AST and ALP),kidney function(BU and CREA)and the plasma levels of endotoxin as the biomarker of the intestine in HS group were increased significantly at 6 h after heatstroke.The hepatocytes from heatstroke mice showed visible edema,hyaline and hydropic degenerations,and typical renal histopathology injuries were observed primarily in cortex,as characterized by hyaline cast.Furthermore,in the lung sections,visible interstitial congestion,thrombi and incidental pulmonary alveoli congestion were observed in heatstroke mice.Spleen injury was also detected in white pulp,characterized by cellular debris within macrophages.Meanwhile,intestinal lesions in heatstroke group were characterized by markedly dilated central lacteals of the villi and disordered structure of the lamina propria(n=8).Accroding the literature,the model of heatstroke in mice was established successfully.2.The protective effect of DEX on heatstroke miceAnti-inflammatroy effects of DEX: The concentration of serum TNF-?,IL-6 and IL-1? were decreased significantly in DEX group(25 ?g/kg)at 6 h after the onset of heatstroke compared with heatstroke mice(n=8).Multi-organ protective effect of DEX: The biomarker of liver function(ALT,AST and ALP),kidney function(BU and CREA)and plasma levels of endotoxin in DEX group were significantly decreased in DEX group compared with HS group.The histological changes in the liver,kidney,lung,spleen and intestine were attenuated by DEX treatment,obviously(n=6~8,P<0.05 or 0.01).Effect of DEX on intestinal permeability: The increased plasma levels of FD4 and D-lactate in heatstroke mice were significantly restored by DEX treatment(n=8,P<0.05).Effect of DEX on intestinal TJ proteins: The intestinal expression of occludin and ZO-1 were markedly reduced in the heatstroke mice compared with control mice.However,the expression of tight junction protein was restored partly by DEX treatment(n=6~8).Effect of DEX on intestinal ultrastructure: The intestinal ultrastructure in heatstroke mice showed:(1)dehisced or loosened tight junction and irregularly widened gap between adjoining cells;(2)destroyed or shorter microvilli;(3)damaged mitochondria characterized as swelling,vacuolar and vague cristae with slight dilated outer membrane.However,the ultrastructural changes of the intestine in the DEX group were attenuated distinctly,which showed basically normal tight junction and regularly aligned microvilli,in addition to slight mitochondria swelling and occasional vacuolar.3.The mechanism of DEX on protecting intestine of the heatstroke miceEffect of DEX on intestinal ?2A-AR and TLR4: DEX treatment increased the expression of ?2A-AR and decreased the expression of TLR4 compared with HS mice(n=6~8,P<0.05 or P<0.001).Effect of DEX on NF-?B signal pathway: Heatstroke induced phosphorylation of the intestinal I?B? and NF-?B p65 in mice,while the levels of phosphorylated I?B? and phosphorylated p65 were attenuated significantly by DEX.And NF-?B p65 in heatstroke mice were seen in nuclear localization,colocalised with DAPI,while the nuclear levels of NF-?B p65 were decreased significantly in DEX treated mice compared with HS(n=6~8).Effect of DEX on NLRP3 inflammasome signal pathway: The increased expression of intestinal NLRP3,ASC and caspase-1 p10 in heatstroke mice were siginificantly attenuated by DEX treatment.By immunofluorescent staining,colocalised NLRP3 and IL-1? in HS mice were seen,while the level of NLRP3 and IL-1? were decreased significantly in DEX treatment mice compared with the heatstroke mice(n=6~8).Effect of DEX on intestinal inflammatory cytokines: DEX decreased the m RNA levels of TNF-?,IL-6 and IL-1? compared with HS mice,indicated that DEX inhibited the intestinal inflammation(n=6~8).Effect of DEX on intestinal apoptosis: Heatstroke increased the expression of Bax and decreased the expression of Bcl-2,while DEX treatment decreased the expression of Bax and increased the expression of Bcl-2.Moreover,TUNEL immunofluorescent staining showed that there were much more TUNEL-positive cells in the intestine from heatstroke mice compared with control mice,whereas only a few TUNEL-positive cells were observed in the intestine from DEX treatment mice(n=6~8).Effect of DEX was related to ?2A-AR: BRL-44408 administration decreased the expression of ?2A-AR,increased the expression of TLR4 compared with DEX treated mice.The decreased intestinal and serum levels of TNF-?,IL-6 and IL-1? by DEX were restored partly by BRL-44408 treatment.BRL-44408 treatment partly restored the protective effect of DEX against heatstroke induced multi-organ injury,such as liver,kidney,lung,spleen and intestine(n=6~8).Conclusions1.Adult male C57BL/6 mice placed in a chamber(40 ± 1 oC,55 ± 5 % humidity)for approximately 4 hours,could reached to 42.7 oC,the received criterion of heatstroke.Six hours after the onset of heatstroke,obvious systemic inflammation was detected in mice,accompanied by multiple organ damage,including liver,kidney,lung,spleen and intestine.2.DEX could protect against inflammation and multi-organ injury,including liver,kidney,lung,spleen and intestine of mice under heatstroke.3.DEX could exert the protective effects through protecting against the intestinal injury,maintaining the ultrastructure,therefore inhibiting endotoxin leaking via maintaining the intestinal integrity.4.DEX exerted the protective effects on intestinal injury under heatstroke in this research may be attributed to:(1)DEX decreased the activation of NF-?B and NLRP3 inflammasome signal pathway,through increased the expression of ?2A-AR and decreased the expression of TLR4;(2)DEX decreased the levels of apoptosis of the intestine in a Bax/Bcl-2 dependent manner.