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Objective:To explore the molecules mechanism of Pin1 in severe heat stroke induced acute lung injury by observing Pin1 regulate oxidative stress and apoptosis formation in pulmonary microvascular endothelial cells (PMVECs) and lung tissue in heat stressed mice.Methods:In vitro, a PMVECs heat stress (HS) model was established. In the control group, PMVECs were placed in a standard 37 °C, 5% CO 2 cell incubator; in the HS group, PMVECs were placed in a 43 °C cell incubator for 2 h, then the cells were further incubated at 37 °C for 1, 3, 6 or 12 h. PMVECs were pretreated with Pin1 inhibitor Juglone (1 μmol/L) 1 h before 43 °C of HS. In vivo, a severe heat stroke mouse model was established. In the HS group, the mice were kept at the simulation of climate chamber with temperature (35.5±0.5) °C, humidity (60±5)%, the rectum temperature in mice was measured by the anal rectal temperature table, when the temperature reached 42 °C, the heat exposure was stopped, and the mice were sacrificed at 1, 3, 6 or 12 h after HS. In the control group, the mice were kept at room temperature (25±0.5) °C. Mice received daily intraperitoneal administration of Pin1 inhibitor Juglone (1 mg/kg) for 3 d before HS. The protein level of Pin1, cleaved caspase-9 and cleaved caspase-3 were analyzed by Western blot, the level of O 2-˙ in cells was observed by DHE staining and fluorescence microscopy, the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in lung tissue were measured by ELISA, the pathological changes of mice in different group were detected by HE staining, and the expression of Pin1 in the lung tissue of different groups was detected by immunohistochemical staining, the apoptosis rate of the lung tissue in different groups was tested by TUNEL staining. Results:At 1 h after HS, the protein expression of Pin1 in PMVECs and lung tissue began to increase in a rewarming time-dependent manner ( F=771.6, P<0.05; F=1 035, P<0.05). Cleaved caspase-9 protein in PMVECs and lung tissue started to increase at 3 h post-HS, then increased with a rewarming time-dependent manner ( F=729.8, P<0.05; F=1 773, P<0.05). The protein expression of cleaved caspase-3 in PMVECs and lung tissue also started to increase at 3 h after HS and the expression continued to be increased with prolonged rewarming time, and the trend was consistent with cleaved caspase-9 ( F=1 084, P<0.05; F=1 252, P<0.05). In addition, HS induced the increased release of O 2-˙ from PMVECs, HS induced the imbalance of oxidation-antioxidant system in lung tissue of mice after HS which verified by the continuous release of MDA ( F=114.2, P<0.05) and the continuous inhibition of SOD activity ( F=99.15, P<0.05). Compared with the HS group, pretreatment with Pin1 inhibitor Juglone in PMVECs and mice before HS significantly inhibited the protein expression of Pin1, cleaved caspase-9 and cleaved caspase-3 (all P<0.05), pretreatment with Pin1 inhibitor greatly reduced the release of O 2-˙ in PMVECs after HS, and promoted the restore of the oxidation-antioxidant system balance of lung tissue in mice with severe heat stroke. In addition, compared with the HS group, inhibiting the expression of Pin1 significantly decreased HS induced MDA release [(11.53±0.84) nmol/mL vs (9.65±0.69) nmol/mL, t=12.52, P<0.05], promoted the restore of SOD activity [(41.18±3.45) U/mL vs (57.52±4.83) U/mL, t=5.57, P<0.05] and improved the pathological damage of lung tissue as well as decreased the occurrence of apoptosis in post-HS mice. Conclusion:It was confirmed that Pin1 is involved in heat stress induced acute lung injury mainly through mediating oxidative stress response and apoptosis.
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During the onset of heat stroke, heat is the most fundamental cause of injury. It has been demonstrated in a number of animal and cell experiments that hyperthermia can directly induce tissue damage and cell death, and cells can activate apoptotic signals or direct necrosis depending on the extent of heat stress. In general, high heat stress activates apoptotic signals and induce apoptosis. Therefore, the form of damage of tissue cells during the onset of heat stroke is currently considered to be mainly apoptosis. In recent years, it has been found that the heat stress molecular biology research regulates the physiological activities of cells in a wide range and participates in the intracellular signal transduction process. Melatonin and its metabolites are broad-spectrum antioxidants and free radical scavengers that regulate a variety of molecular pathways, such as inflammation, proliferation, apoptosis, and metastasis, under different pathophysiological conditions. This article summarized the research on the effects of melatonin and heat shock on apoptosis, and evaluated the possible protective effects of melatonin on the pathogenesis of heat stroke, and provided new therapeutic ideas for the clinic.
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Objective To observe the oxidative stress, integrity of lysosome and apoptosis of intestinal epithelial cells 6 (IEC-6) after heat stress, and explore the pathogenesis of intestinal damage caused by heat stress.Methods In the heat stress groups,the cells were incubated at 43℃ for 1 hour, then, further incubated at 37℃ and 5% CO2 for 0, 1, 3, 6 and 12 hours respectively; in the medicine intervention group, the cells were pretreated with the medicine 1h before heat stress; while in control group, the cells were incubated at 37℃ and 5% CO2. The amount of reactive oxygen species (ROS) was assayed with 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) staining. The stability of lysosome membrane was checked by AO staining. Apoptosis was analyzed by flow cytometry using annexinⅤ-FITC/PI staining, CCK-8 assay was used to assess cellular viability.Results Compared with control group, cell viability decreased and apoptosis increased at 1 h after heat stress, which was the most obvious at 12h after rewarming (P<0.05). While ROS and pale cells increased immediately after heat stress and the increase become the most obvious (P<0.05). The cell viability in E-64 pretreatment group was significantly improved such as apoptosis reduction, compared with heat stress group (P<0.05).Conclusion Heat stress could induce robust increase of ROS, which mediates lysosome damage and results in cell apoptosis, thus suggesting that ROS-lysosome pathway may play an important role in intestinal injury in heat stress.
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Objective To observe the effect of different temperatures on endoplasmic reticulum stress, calcium overload, mitochondria and cell damage in pulmonary microvascular endothelial cells (PMVEC) induced by heat stress, and clarify the mechanism of endothelial cell injury in the process of heat stress to provide experimental basis for clinical prevention and treatment of heat stree. Methods Heat stress model of PMVEC cell was set up. Control group cells were incubated at 37℃, 5%CO2, while heat stress group cells were incubated at 39℃, 41℃, 43℃ for 2h, respectively, then further incubated at 37℃, 5%CO2 for 6h. Pretreatment of cells with 20μmol/L BAPTA-AM or 50μmol/L CsA before heat stress at 43℃. The protein levels of p-PERK, PERK p-eIF2a, eIF2a, ATF4 and GRP78 were analyzed by Western blotting. Intracellular Ca2+, mitochondrial membrane potential and the changes in mitochondrial permeability transition pore were investigated by flow cytometry. The change of caspase-3 was detected by Caspase Assay Kit. Millicell-ERS Volt-Ohm Meter and Accessories was used for determining the changes of transepithelium electrical resistance (TER). Results Compared with the control group, with the increase of heat stress temperature (41-43℃), the phosphorylation of p-PERK and p-eIF2a protein and the expressions of ATF4 and GRP78 proteins were gradually activated, intracellular Ca2+ increased, MPTP pore was opened, mitochondrial membrane potential decreased, cell permeability increased and apoptosis occurred, and it was the most obvious in the 43℃ heat stress group, and the difference was statistically significant (P<0.05). Pretreatment with Ca2+ inhibitors promoted the recovery of the MPTP hole, mitochondrial membrane potential and cell permeability, and reduced the occurrence of apoptosis. While pretreatment with the mitochondrial protective agent did not reduce the release of Ca2+, but it could promote the recovery of cell permeability and reduce the occurrence of apoptosis. Conclusion Heat stress activates endoplasmic reticulum stress response, induces intracellular Ca2+ overload mediated cell and mitochondrial damages in PMVEC cells, which may be one of the important mechanisms of endothelial cell injury induced by heat stress.
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Objective To explore the protective effect of propofol on endothelial cells during heat stress and its protective effect to mitochondra. Methods Heat stress model of human umbilical vein endothelial cell was established when cells were incubated at 43℃ for 2h, then further incubted at 37℃, 5%CO2 for 6h. The experimental group was subdivided into six groups, including 37℃ group, 37℃ plus intralipid group (negative control group), 37℃ plus propofol group, 43℃ plus propofol group, 43℃ plus intralipid group, H2O2 plus propofol group (positive control group); Pretreated with 50μmol/L propofol, 0.2ml intralipid or 25μmol/L H2O2 before heat stress at 43℃, while the cells in the control group were incubated at 37℃. Cell viability was tested by CCK-8. ROS, mitochondrial membrane potential and the changes in mitochondrial permeability transition pore were determined by flow cytometry. The level of ATP was detected by fluorescein-luciferase. The changes of caspase-9 and caspase-3 were analyzed by Caspase Activity Assay Kit. Results HUVESs cell viability and damage of mitochondra were significantly decreased after heat stress. Compared with 43℃ heat stress group, pretreatment with propofol induced the recovery of cell viability and the ROS levels were significantly decreased in HUVEC cells (P<0.05). Meanwhile, the number of cells representing the decrease of mitochondrial membrane potential (the proportion of JC-1 monomer) was significantly decreased (P<0.05) by propofol. The average fluorescence intensity of calcein which representing the MPTP changes and intracellular ATP content was significantly increased (P<0.05). In addition, the activation of mitochondrial apoptotic pathway mediated by caspase-9/3 was also inhibited. Conclusions Propofol have anti-oxidative, anti-apoptosis and mitochondria protective effect against endothelial cell injury during heat stress.
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As an important organ of the human body,vascular endothelial cells (VECs) play a vital role in heat stressinduced tissue damage.Its integrity not only serves as a barrier for maintaining vascular permeability but also has major impact on cellular structure and function during acute phase response to heat stress.In heat stroke,a series of acute and complicated pathophysiological changes,including microcirculation change,damage VECs and thereby induce or aggravate multiple organ dysfunction syndrome (MODS).Meanwhile,studies have shown that,during heat stroke,VECs are the major responding cells and one of the most common cells that experience morphological and functional changes.Therefore,VECs damage might be an important mechanism involved in heat stroke.This article reviews the mechanism of heat-induced damage of VECs and its effect on vital organs.
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Objective To evaluate the effect of propofol (PPF) on stress response and lung injury in rats with traumatic brain injury (TBI).Methods A total of 36 SD rats were randomly (random number)divided into sham group,intralipid group,TBI group,PPF1 h group,PPF 2 h group,PPF 6 h group (n =6 in each group).Fluid percussion brain injury models were used.By intraperitoneal injection,intralipid was administered in intralipid group after sham operations,while propofol 100 mg · kg-1 was given to rats in PPF1 h group,PPF 2 h group and PPF 6 h group 1,2,6 hours following injury,respectively.Nerve motor function were scored at different intervals,serum concentrations of adrenocorticotropic hormone (ACTH),cortisol (COR) and norepinephrine (NE) were measured 12 h after injury.Seventy-two hours later,all rats were sacrificed and brains were harvested for TTC staining,and lungs taken were stained with HE staining for observation under light microscope and electron microscopy.Results Compared with sham and intralipid group,nerve motor function scores were significantly decreased,and serum concentrations of ACTH,COR and NE were increased significantly in rats after injury.Compared with TBI group,the above biomarkers were improved significantly after propofol injection.There were no significant differences in above biomarkersbetweenPPF 1 hand PPF 2 h group (t=-0.816,t=-0.208,t=0.582,P＞0.05).The differences in COR and NE concentrations between PPF 2 h and PPF 6 h group were statistically significant (t =3.018,P =0.013;t =3.662,P =0.004).Light microscopy demonstrated abundant inflammatory cell infiltration and massive thickening of the alveolar walls,Electron microscopy showed Type Ⅱ lung epithelial cell swelling,vacuolar degeneration,osmiophilic lamellar corpuscle emptying in cytoplasm,microvilli protrusions decreases in some cytoplasm in TBI group,and pathological damage was improved significantly in PPF 2 h group.Conclusions Propofol may inhibit stress and protect the lung tissue from damage in TBI rats.
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Objective To observe the effect of severe heatstroke on intestinal mucosal barrier function,and explore its correlation with systemic inflammatory reaction.Methods The SPF male BALB/c mice were randomly divided into normal control group,40 ℃ heat stress group and 42 ℃ heat stress group,with 6 mice in each group.The mice in normal control group were observed at normal temperature with (25.0 ± 0.5)℃,and the mice in heat stress groups were challenged with a temperature of (35.5 ± 0.5) ℃ and a humidity of (60 ± 5)％ until body temperature increase up to 40 ℃ or 42 ℃ followed by recovering the surroundings temperature to normal temperature for 12 hours.The blood of medial angle of eye of mice in each group was collected for determination of plasma lipopolysaccharide (LPS),tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) levels,and diamine oxidase (DAO) activity with enzyme linked immunosorbent assay (ELISA).The level of D-lactic acid was determined with ultraviolet spectrophotometer.Then the mice in each group were sacrificed,and mesenteric lymph node (MLN),liver,spleen,lung,kidney tissues,and the blood from portal vein and caval vein were collected for colony count to observe the intestinal bacterial translocation.The ileum tissue was collected for observation of changes in histomorphology and ultrastructure of small intestine mucous membrane with microscope.Pearson linear regression analysis was used to explore the correlation between intestinal mucosal barrier dysfunction and systemic inflammatory response.Results Compared with normal control group,plasma LPS,inflammatory parameters such as TNF-α and IL-6,and gut barrier function parameters such as DAO and D-lactic acid levels as well as the rate of bacterial translocation after heat stress were significantly increased,and the differences were more obvious in 42 ℃ heat stress group [LPS (EU/L):740±50 vs.340±40,TNF-α (ng/L):148.06±36.61 vs.12.89 ± 1.67,IL-6 (ng/L):110.91 ± 9.97 vs.18.02 ± 2.20,DAO (U/L):1 760 ± 400 vs.670± 50,D-lactic acid (mg/L):9.60 ± 1.48 vs.5.08 ± 0.28,rate of bacterial translocation:78.6％ (33/42) vs.9.5％ (4/42),all P ＜ 0.01].It was shown by Pearson linear regression analysis that plasma LPS,TNF-α,IL-6 were positively correlated with DAO activity (r values were 0.834,0.808,0.836,respectively) and D-lactic acid (r values were 0.811,0.811,0.800,respectively) in 42 ℃ heat stress group (all P =0.000).It was showed by microscope that the changes in histomorphology and ultrastructure changes in intestinal mucosa were found after heat stress,and was obvious in 42 ℃ heat stress group as following:villus atrophy and falling off,infiltration of neutrophils and lymphocytes,the microvillus on the surface of mucosa cells were short and small,arranged in disorder,the tight junction between epithelial cells became widen,the mitochondrion and endoplasmic reticulum swelled obviously.Conclusion During the early stage of severe heatstroke,the damage of intestinal mucosal was obvious,and it has close correlation with systemic inflammatory response.
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Results With the prolonged exposure to heat , the mice exhibited swollen and disorderly arranged neurons , shrunken cells , and contracted and deeply stained nuclei , with significantly higher scores on nerve pathological injury evaluation at 6, 12, and 24 h (2.78 ± 0.71, 3.21 ±0.56, and 3.36 ±0.63) than the control mice (0.43 ±0.10) (P<0.05).ELISA showed remarkably elevated levels of UCH-L1 in the serum (F=147.7, P=0.05) and brain tissue (F=145.7, P=0.05) in the heat stress group as compared with the con-trol, and Western blot also revealed a markedly higher expression of UCH-L1 in the brain tissue in the former group than in the latterObjective The abnormal expression of ubiquitin C-terminal hydrolase-L1 ( UCH-L1 ) has an important role in the diagnosis and prognosis of brain damage .This study was to investigate the changes of UCH-L1 in the serum and brain tissue in the mouse model of heat stress . Methods Twelve BALB/c mice were randomly divided into a control and a heat stress group of equal number, the former placed at a temperature of (25.0 ±0.5)℃and a relative humidity of (35 ±5)%and the latter in a simulated in-cubator at (35.5 ±0.5)℃and a relative humidity of (60 ±5)%.When the rectal temperature reached 42℃, the animals were re-moved from the incubator and cooled at an ambient temperature of (25.0 ±0.5)℃and a humidity of (35 ±5)%for 0, 6, 12, and 24 h.Then the brain tissues of all the animals were harvested for HE staining , evaluation of neuronal injury under the light microscope , measurement of the UCH-L1 levels in the serum and brain tissue by ELISA , Western blot, and immunohistochemistry , respectively. (F=261.2, P=0.01).Immunohistochemistry manifested that , with the prolonged exposure to heat , the UCH-L1 expression in the brain tissue was characterized by gradually increased light brown of the neurons at staining . Conclusion Severe heatstroke causes brain injury in a time-dependent manner , and the abnormally elevated levels of UCH-L1 in the serum and brain tissue can be a marker of heatstroke-induced brain injury .
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Objective To observe the expressions of cytochrome C,Caspase-9,Caspase-3 and their relationships,and investigate apoptosis signal transduction mechanism after heat stress-induction in human umbilical vein endothelial cell (HUVEC),and explore pathogenesis of vascular endothelial damage in the wake of severe heat stroke.Methods Human umbilical vein endothelial cell heat stress model was set up.Control group were incubated at 37℃,while heat stress group of cells were incubated at 39℃,41℃,and 43℃ for 2h,then all the cells were further incubated at 37℃ for 24 h.Mitochondria of human umbilical vein endothelial cell were examined with electron microscopy.Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining,and protein levels of cytochrome C,caspase-9,caspase-3 were analyzed by western blot.Results In the control group (37℃),the structure of mitochondrial was intact in HUVEC under transmission electron microscope.In contrast,mitochondrial swelling was found in the group of 43℃ heat stress.Compared with control group,as increasing in heat stress temperature,the rates of induced apoptosis were 17.8％ at 41℃ and 25.6％ at 43℃,and the levels of cytochrome C,Caspase-9,and caspase-3 were significantly increased (P ＜0.05).There was no obvious change at 39℃ heat stress (P ＞ 0.05).Conclusions Mitochondrial apoptosis pathway is involved in apoptosis of human umbilical vein endothelial cells in the wake of heat stress.The vascular endothelial cells apoptosis may be associated with the occurrence of severe heat stroke.
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Objective To observe the effect of heat stress-induced reactive oxygen species (ROS) burst on the regulation of expression of Bcl-2 and Bax in human umbilical vein endothelial cell (HUVEC) apoptosis induced by heat stress,and explore the pathogenesis of vascular endothelial damage caused by severe heat stroke.Methods HUVEC heat stress model was reproduced.Cells of heat stress group were incubated at either 39,41,or 43 ℃ for 2 hours,then all the cells were further incubated at 37 ℃ and 5％ CO2 for 24 hours.Before heat stress,cells of 43 ℃ heat stress group were pretreated with 10 μmol/L MnTMPyP,which was a specific scavenger of ROS,for 1 hour.Cells of control group were incubated at 37 ℃ and 5％ CO2.The amount of ROS was assayed with 2',7'-dichlorofluorescin diacetate (DCFH-DA) and dihydroethidium (DHE) staining.Apoptosis was determined by using staining with Hoechst33258.The mRNA expressions of Bcl-2 and Bax were determined by reverse transcription-polymerase chain reaction (RT-PCR).The protein levels of Bcl-2,Bax,caspase-3 were analyzed by Western Blot.In addition,the effect of MnTMPyP on heat stress-induced apoptosis was also studied.Results Compared with control group,there was no obvious change in cells after 39 ℃ heat stress.With the increase in heat stress temperature up to 41 ℃ and 43 ℃,viability of cells showed a lowering trend,with a burst of ROS,and an increase of mRNA and protein of Bax,and the protein of caspase-3 was significantly increased,the mRNA and protein of Bcl-2 were significantly decreased in a temperature-dependent manner.These changes were marked in 43 ℃ heat stress group as compared with those of the control group [cell viability:(46.00 ±4.00)％ vs.(96.33 ± 1.53)％,t=20.164,P=0.001; ROS (fluorescence relative value):400.67 ± 12.10 vs.99.33 ±4.04,t=32.909,P=0.001; Bax mRNA (A value):3.03 ±0.15 vs.1.00 ± 0.00,t=23.056,P=0.001; Bax protein (gray value):3.97 ±0.21 vs.1.00 ± 0.00,t=24.684,P=0.001; caspase-3 protein (gray value):4.80 ± 0.20 vs.1.00 ± 0.00,t=32.909,P=0.001 ; Bcl-2 mRNA(A value):0.42 ± 0.30 vs.1.00 ± 0.00,t=33.072,P=0.001 ; Bcl-2 protein (gray value):0.39 ± 0.25 vs.1.00 ± 0.00,t=42.212,P=0.001].It was shown that pre-condition with the antioxidant MnTMPyP significantly decreased the heat stress-induced expression of Bax,caspase-3,and apoptosis,and the expression of Bcl-2 was elevated [Bax mRNA (A value):2.00 ± 0.20 vs.3.33 ±0.25,t=7.184,P=0.002; Bax protein (gray value):2.03 ±0.25 vs.3.23 ±0.25,t=5.840,P=0.004; caspase-3 protein (gray value):2.07 ± 0.21 vs.5.00 ± 0.20,t=17.600,P=0.001 ; Bcl-2 mRNA(A value):0.71 ± 0.40 vs.0.42 ± 0.26,t=8.126,P=0.002; Bcl-2 protein (gray value):0.57 ± 0.31 vs.0.40 ± 0.06,t=5.091,P=0.007].Conclusions A burst in an increase of ROS plays an important role on heat stress-induced HUVEC apoptosis,and the mechanism is probably related to the expressions of Bcl-2 and Bax.The vascular endothelial cells apoptosis may be one of the pathogenetic factor in severe heat stroke.
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Objective To explore the effect of activation of caspase-3 on apoptosis of human umbilical vein endothelial cells induced by heat stress. Methods A model of human umbilical vein endothelial cell heat stress was established. The endothelial cells in the control group were incubated at 37 ℃, while the cells in the heat stress group were incubated at 39 ℃, 41 ℃, or 43 ℃ for 2 h , then all the cells were further incubated at 37 ℃ for 24 h. Apoptosis was detected using staining with Hoechst33258; protein levels of caspase-3 were analyzed by Western blot; and the effect of caspase-3 inhibitor Z-DEVD-FMK on heat stress-induced apoptosis was determined. Results As compared with the control group, with an elevation in heat stress temperature (41℃ to 43 ℃), apoptosis was markedly increased and level of caspase-3 was significantly increased. In addition , caspase-3 inhibitor Z-DEVD-FMK significantly decreased the heat stress-induced apoptosis and levels of caspase-3. Conclusions Caspase-3 mediates apoptosis of human umbilical vein endothelial cells can be induced by heat stress.