among the patients with heatstroke at the mecca pilgrimage , most of them had diabetes with hyperglycemia . indeed , compared with nondiabetic rats , diabetic rats were more susceptible to heat stroke occurrence [ 2 , 3 ] . most of the heatstroke manifestations ( e.g. , excessive hyperthermia associated with a systemic inflammatory response that led to multiple organ dysfunction in which the brain dysfunctions predominated ) could be reproduced by exposing the streptozotocin - induced diabetic rats to hot environments . the heatstroke - diabetic rats ( hsdr ) displayed excessive hyperthermia , brain inflammation , ischemia and oxidative damage , and multiple organ dysfunction or failure . honokiol was one of main constituents of the chinese herb , magnolia officinalis , and had a variety of pharmacological actions , including anti - inflammatory effects , antimicrobial activity [ 6 , 7 ] , and antioxidative effects . however , it was not known whether hypothalamic ischemia and oxidative stress with systemic inflammation and multiple organ dysfunction in hsdr could be affected by honokiol . heatstroke was a form of hyperthermia associated with a systemic inflammatory response that led to multiple organ dysfunction , in which central nervous system ( cns ) disorders predominated . based on triad of factors ( hyperthermia , cns disorders , and a history of heat stress ) , anesthetized rodents all displayed a uniform response which was similar to responses of humans with heatstroke . the present study was to assess the effects of heat stress on body core temperature ( tco ) , mean arterial pressure ( map ) , hypothalamic values of cerebral blood flow ( cbf ) , partial pressure of o2 ( po2 ) , cellular ischemia marker ( e.g. , glutamate ) , organ damage markers ( e.g. , glycerol and lactate dehydrogenase ) , toxic oxidizing radicals ( e.g. , nitric oxide and dihydroxybenzoic acid ) , and serum levels of inflammatory markers ( e.g. , interleukin-1 , interleukin-6 , tumor necrosis factor- , and myeloperoxidase ) in hsdr with or without honokiol pretreatment . adult male sprague - dawley rats ( weighing 223 to 256  g ) were obtained from the animal resource center of the national science council of the republic of china ( taipei , taiwan ) . the animals which were allowed to become acclimated for at least one week were housed four per cage at an ambient temperature ( ta ) of 22  1c with a 12-h light / dark cycle and were supplied with rat chow and tap water ad libitum . the experimental protocol was approved by the animal ethic committee of the chi mei medical center ( tainan , taiwan ) under guidelines of the national science council . animal care and experiments were according to the guide for the care and use of laboratory animals published by the usa national institutes of health ( nih publication number 85 - 23 revised 1996 ) . adequate anesthesia was maintained to abolish the corneal reflex and pain reflexes induced by tail pinching throughout all experiments ( approximately 480 minutes in duration ) by a single intraperitoneal dose of urethane ( 1.4  g / kg body weight ) . at the end of the experiments , control rats and all rats that had survived heat stroke the right femoral artery and vein of rats under urethane anesthesia were cannulated with polyethylene tubing ( pe50 ) , for blood pressure monitoring and drug administration . the tco temperature ( tco ) was monitored continuously by means of a thermocouple , while map was monitored continuously with a pressure transducer . louis , mo , usa ) at 30  mg / ml / kg of body weight in the tail veins of unanesthetized rats . the animals were maintained for 4 - 5 weeks before heat stress was applied . at the day of thermal experiments , the right femoral artery and vein of rats under general anesthesia were cannulated with polyethylene tubing ( pe50 ) for blood pressure monitoring and drug administration or biochemical determination , respectively . in the present study , the core temperature ( at about 37c ) of the anesthetized animals was maintained with an infrared light lamp except in the heat stress experiments . heatstroke was induced by putting the animals in a bold heating pad of 43c controlled by circulating hot water . the instant in which map dropped to ~50  mmhg was found to be about 60 minutes after the start of heat stress ( figure 1 ) . at 60 minutes , the heating was removed and the animals were allowed to recover at room temperature ( 26c ) . survival time values ( interval between the start of heat stress and animal death ) were determined . it was seen in figure 1 that the heated rats displayed both hyperthermia ( ~42.5c ) and hypotension ( ~50  mmhg ) at 60 minutes , suggesting the occurrence of heatstroke . animals were assigned randomly to one of the following three major groups : normothermic diabetic rats treated with vehicle solution ( ntdr + v ) , heatstroke - diabetic rats treated with vehicle solution ( hsdr + v ) , and heatstroke - diabetic rate treated with honokiol ( hsdr + h ) . sixty minutes before the start of heat stress , honokiol ( h8c2o2 ) ( 0.5  mg , 1.5  mg , or 5.0  mg per ml per kg of body weight ; sigma - aldrich , saint louis , mo , usa ) or vehicle ( dmso ) solution ( 1  ml per kg body weight ) was administered via the veins . a 100  m diameter thermocouple and two 230  m fibers oxylite 2000 ( oxford optronix ltd . , oxford , uk ) was a 2-channel device ( measuring po2 and temperature at two sites simultaneously ) , whereas oxyflo 2000 was a 2-channel laser doppler perfusion monitoring instrument . this combined probe was implanted stereotaxically into the right hypothalamus according to the stereotaxic coordinates of paxinos and watson   to measure both cbf and po2 in the hypothalamus . for determination of nox and dhba , the nox concentrations in the dialysates were measured with the eicomo-20 nox analysis system ( eicom , kyoto ) . the concentrations of hydroxyl radicals were measured by a modified procedure based on the hydroxylation of sodium salicylates by hydroxyl radicals , leading to the production of 2,3-dhba and 2,5-dhba . for determination of creatinine , blood urea nitrogen ( bun ) , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , alkaline phosphatase ( alp ) , and lactate dehydrogenase ( ldh ) were estimated in blood samples collected at 0 and 60 minutes after the start of heat stress . the serum levels of creatinine , bun , alt , ast , and alp were determined by spectrophotometry ( hitachi7600 , tokyo , japan ) . in addition , ldh was measured to evaluate the extent of organ injury by fuji dri - chem 3030 ( fuji photo film co. , ltd . , blood samples were collected 0 and 60 minutes after the start of heat stress and stored at 80c until they could be assayed . we used commercially available elisa kits for the determination of serum il-1 , il-6 , tnf- , il-10 , and icam-1 levels ( quantikine , r&d systems inc . , minneapolis , mn , usa ) according to the manufacturer 's instruction . peripheral polymorphonuclear ( pmn ) cells were isolated from the whole blood of rats and treated with heparin ( 100  units / ml ) . erythrocytes were allowed to sediment for 30 minutes after the addition of 3  ml of 6% dextran ( weight / volume in pbs ) to 10  ml blood . after sedimentation , the plasma containing leukocytes was centrifuged twice at 300  g for 5  min each . the precipitates were mixed with 70% osmolality - adjusted percoll and centrifuged at 30,000  g for 30  min at 26c . each fraction was washed twice with hanks ' balanced salt solution , and the cell number was counted . the purity of the pmns was determined to exceed 95% by giemsa staining cells ( 1  10 cells / tube ) which were incubated with a rabbit polyclonal antibody to ( cd62e ( ab18981 ; abcam pic332 , cambridge , uk ) or control . after washing , the cells were stained with a secondary antibody ( goat polyclonal to rabbit igg - h & l [ fitc ]  [ ab6717 ] ; abcam pic ) . they were mixed with oligasaccharides and incubated for 20 minutes , and then coincubated with km93 for 60 minutes . a 100  l aliquot of serum was mixed with 900  l of 50  mmol / l phosphate buffer ( ph-6.0 ) containing 0.167  mg / ml of o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide . one unit of peroxidase activity equaled the amount of enzyme decomposing 1  mol of hydrogen peroxide which was calculated from the oxidation of o - dianisidine using an absorption coefficient of 11.3/mm / cm at 460  nm . plasma corticosterone and acth were assayed using corticosterone double antibody ria kit ( mp biomedicals , solon , oh , usa ) and acth ( rat , mouse)-ria kit ( phoenix pharmaceuticals , burlingame , ca , usa ) , respectively . one - way analysis of variance with tukey 's multiple comparisons test was used for serum markers and physiological parameters . diabetic rats had smaller body weight , higher plasma glucose levels , and lower plasma insulin when compared to healthy rats ( table 1 ) . the survival time values for ( hsdr + v ) rats were decreased from the ( ntdr + v ) control values of 480    3  min to new values of 79  3  min after the start of heat stress ( table 2 ) . heatstroke - diabetic rats ( hsdr ) treated with h ( 0.5 , 1.5 , and 5.0  mg / ml / kg of honokiol ) 60 minutes before heat stress had significantly and dose - dependently higher values of survival time ( 114273  min ) than those treated with vehicle . however , in the following experiments , only the effects of one dose of 1.5  mg / ml / kg of honokiol were on determining other parameters during heatstroke . figure 1 showed the effects of heat exposure ( 43c for 60 minutes ) on both tco and map in ( ntdr + v ) rats , ( hsdr + v ) rats , and ( hsdr + h ) rats . as shown in this figure , sixty minutes after the start of heat exposure , the values of map in the ( hsdr + v ) group were significantly lower than those in the ( ntdr + v ) group ( ~51  mmhg versus ~92  mmhg ; p < 0.05 ) . on the other hand , the values of tco in the ( hsdr + v ) group were significantly higher than those in the ( ntdr + v ) group ( ~42.2c versus ~37.3c ; p < 0.01 ) . heat - induced hypotension and hyperthermia were all significantly reduced by honokiol pretreatment as shown in ( hsdr + h ) group . figure 2 showed that the hypothalamic levels of po2 and cerebral blood flow in the ( hsdr + v ) rats were significantly lower at 60 minutes after the start of heat exposure than in the ( ntdr + v ) group ( ~100  bpu versus ~400  bpu for cbf ; ~5  mmhg versus ~20  mmhg for po2 ; p < 0.05 ) . in contrast , the hypothalamic levels of glutamate and glycerol in the ( hsdr + v ) rats were significantly higher at 60 minutes after the start of heat exposure ( ~82  mmol / l versus ~3  mmhg for glutamate ; ~22  mol versus ~5  mol for glycerol ; p < 0.05 ) . heat - induced hypothalamic hypoxia , ischemia , and damage at 60 minutes were all significantly reduced by pretreatment with honokiol . figure 3 showed that the hypothalamic levels of 2,3-dhba and nox in the ( hsdr + v ) rats were significantly higher at 60 minutes after the start of heat exposure than in the ( ntdr + v ) rats ( ~180% versus ~100  mmhg for dhba ; ~9.6  m versus ~2.6  m for nox ; p < 0.05 ) . as shown in this figure , heat - induced increased hypothalamic levels of all these parameters were significantly reduced by honokiol pretreatment in the ( hsdr + h ) rats . figure 4 showed that the hypothalamic values of tunel - positive cells in the ( hsdr + v ) were significantly higher at 60 minutes after the start of heat stress than that in the ( ntdr + v ) rats ( ~148 cells versus ~5 cells ; p < 0.01 ) . as shown in this figure , heat - induced increased hypothalamic values of tunel - positive cells were significantly reduced by honokiol pretreatment in the ( hsdr + h ) rats . figure 5 showed that the serum values of il-1 , il-6 , tnf- , e - selectin , icam-1 , lactate dehydrogenase , and mpo for ( hsdr + v ) were significantly higher at 60 minutes after the start of heat stress than that in the ( ntdr + v ) rats . heat - induced increased values of these parameters were all significantly reduced by honokiol pretreatment in the ( hsdr + h ) rats . in addition , serum levels of il-10 in the ( hsdr + h ) rats were significantly higher than that in the ( hsdr + v ) rats at 60 minutes after the start of heat stress . table 3 showed that the plasma levels of both acth and corticosterone in the ( hsdr + v ) rats were significantly higher at 60 minutes after the start of heat stress than that in the ( ntdr + v ) rats . additionally , ( hsdr + h ) rats had significantly ( p < 0.05 ) higher plasma levels of both acth and corticosterone than ( hsdr + v ) rats . table 4 showed that the serum level of bun , creatinine alt , ast , and ap in the ( hsdr + v ) rats were significantly higher at 60  min after the start of heat stress than that in the ( ntdr + v ) rats . heat - increased serum levels of bun , creatinine , alt , ast , and ap in the ( hsdr + v ) rats were significantly reduced by honokiol pretreatment in the ( hsdr + h ) rats . severe heat stress induced excessive hyperthermia , splanchnic vasoconstriction , and multiple organ dysfunction or failure . during heatstroke , excessive hyperthermia facilitates the leakage of endotoxin from the intestine to the circulating blood and resulted in leukocytes infiltration and excessive activation of endothelial cells [ 2 , 13 ] . tissue ischemia , hypoxia , and damage secreted proinflammatory cytokines which led to excessive production or release of the toxic oxidizing radicals like nox and dhba and resulted in multiple organ damage . amplification of the resultant activated inflammatory state induced accumulation of free radicals and resulted in augmentation of local tissue injury . this systemic inflammation response led to polymorphonuclear cells sequestration in both the lung and liver and resulted in multiple organ dysfunction or failure . in particular , heat - induced multiple organ dysfunction might be associated with hypothalamo - pituitary - adrenocortical ( hpa ) axis impairment . their data showed that intolerance to heat exposure was associated with an hpa axis impairment , possibly related to changes occurring in the inflammatory mediators levels . as mentioned in section 1 , the patients with diabetes were susceptible to heat stroke at mecca pilgrimage . our present study further showed that heat - induced multiple organ dysfunction or failure in streptozotocin - induced diabetic rats was related to tissue inflammation and oxidative stress . especially , pretreatment with honokiol significantly ( a ) attenuated hyperthermia , hypotension and hypothalamic ischemia , hypoxia , and neuronal apoptosis , ( b ) reduced the plasma index of the toxic oxidizing radicals including nox and 2,3-dhba , ( c ) diminished the indices of hepatic and renal dysfunction including creatinine , blood urea nitrogen , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , and lactate hydrogenase , ( d ) attenuated the plasma systemic inflammatory response molecules like soluble intercellular adhesion molecule-1 , e - selectin , tumor necrosis factor - alpha , interleukin-1 , and interleukin-6 , ( e ) promoted plasma levels of an anti - inflammatory cytokine , interleukin-10 , ( f ) reduced an index of infiltration of polymorphonuclear neutrophils in the serum as measured by myeloperoxidase activity , ( g ) enhanced the plasma levels of corticosterone and adrenocorticotrophic hormone , and ( h ) promoted the survival time fourfold in hsdr . hence , we suggested that isolating honokiol from magnolia officinalis might be a potential adjunct in the prevention of heatstroke . in fact , the biological activities of honokiol include potent inhibition of lipid peroxidation [ 8 , 18 ] , scavengers of hydroxyl radicals , inhibition of leukotriene synthesis in leukaemia cells , and inhibition of uv - induced mutations . serum molecules like il-1 , il-6 , tnf- , icam-1 , and e - selectin were involved in the pathophysiology of systemic inflammatory response syndromes [ 2225 ] . activation of both neutrophils and endothelial cells is associated with overproduction of these systemic inflammatory response syndrome molecules in patients   or rats   with heatstroke . in the present study , both hyperthermia and overproduction of these systemic inflammatory response syndrome molecules in rats could be significantly attenuated by honokiol . in addition , honokiol pretreatment facilitated the production of serum il-10 , an anti - inflammatory cytokine ,   that occurred during heatstroke . thus , honokiol might downregulate the extent of activated inflammation via attenuating the heatstroke - induced excessive hyperthermia . the severity of heat illness was believed to depend on the degree of hyperthermia and its duration . it was promoted that decreased heat tolerance was associated with hpa axis impairment . as shown in the present study , hypothalamic ischemia , hypoxia , and neuronal apoptosis were associated with hpa impairment ( evidenced by insufficient release of both acth and corticosterone ) in hsdr which could be significantly reversed by honokiol . it was likely that honokiol might increase heat tolerance by reducing hpa axis impairment ( as reflected by producing more acth and corticosterone in plasma in response to heat stress ) in rats . the hypothesis is supported by our previous finding showing that glucocorticoids reduced interleukin-1 concentration and resulted in neuroprotective effects in rat heatstroke . it should be mentioned that the normal rats should release more acth and corticosterone in response to heat stress than the normothermic controls ( nonetheless , the data are not reported here ) . however , the plasma levels of both acth and corticosterone in heated diabetic rats should be lower than that of heated nondiabetic rats . the reduced release of both acth and corticosterone displayed by heated diabetic rats could be reversed by honokiol supplement . in summary , we demonstrated that honokiol protected against hyperthermia , hypotension , and multiorgan injury in hsdr . the beneficial effects of honokiol might be on the reduction of ( a ) systemic inflammatory response syndrome molecules like tnf- , il-1il-6 , icam-1 , mpo , and e - selectin in the plasma , ( b ) the toxic oxidizing radicals like nox and 2,3-dhba , in the plasma , ( c ) the organ injury indicators like ldh , creatinine , blood urea nitrogen , alanine aminotransferase , aspartate aminotransferase , and alkaline phosphatase in the plasma , and ( d ) an indicator for leukocytes accumulation like mpo in the serum , and thus leading to maintenance of hpa axis function and increased survival in rats with heatstroke - associated multiple organ dysfunction . our data suggested that honokiol was useful in reducing both inflammation and oxidative stress ; lowering heat - induced multiple organ dysfunction .
we aimed at investigating the effect of honokiol on heatstroke in an experimental rat model . 
 sprogue - dawley rats were divided into 3 groups : normothermic diabetic rats treated with vehicle solution ( ntdr+v ) , heatstroke - diabetic rats treated with vehicle ( hsdr+v ) , and heatstroke rats treated with konokiol ( 0.55  mg / ml / kg ) ( hsdr+h ) . 
 sixty minutes before the start of heat stress , honokiol or vehicle solution was administered . 
 ( hsdr+h ) 
 significantly ( a ) attenuated hyperthermia , hypotension and hypothalamic ischemia , hypoxia , and neuronal apoptosis ; ( b ) reduced the plasma index of the toxic oxidizing radicals ; ( c ) diminished the indices of hepatic and renal dysfunction ; ( d ) attenuated the plasma systemic inflammatory response molecules ; ( e ) promoted plasma levels of an anti - inflammatory cytokine ; ( f ) reduced the index of infiltration of polymorphonuclear neutrophils in the serum ; and ( g ) promoted the survival time fourfold compared with the ( hsdr+v ) group . in conclusion , honokiol protected against the outcome of heatstroke by reducing inflammation and oxidative stress - mediated multiple organ dysfunction in diabetic rats .