| BACKGROUND AND AIMSHeat stress is a common stressor that affects many biological systems.Heat stroke can be fatal if not urgently and appropriately managed.When an individual is exposed to high ambient temperatures,heat stroke can affect vital organs;one of the primary organs affected by heatstroke is the gastrointestinal trac.Multiple studies have pathologically demonstrated damage to the small intestine following heat stress.Recently,studies in multiple animal models have detected heat stress-induced apoptosis in small intestinal tissue and intestinal epithelial cells.However,the precise molecular mechanism by which heat stress induces ROS and subsequent apoptosis in small intestinal tissue and small intestinal epithelial cells is still poorly understood.METHODS AND RESULTS Heat stress-induced increasing of ROS in SW480 cellsWe observed a time-dependent increase in intracellular ROS levels in SW480 cells that were treated with heat stress(43?).There was a noticeable increase immediately at Oh that peaked at 6h after heat stress.Lysosomal membrane permeabilization induced by heat stress in SW480 cellsFluorescent probes-AO are metachromatic fluorophores that yield red fluorescence when they accumulate within the lysosomes.Exposure of SW480 cells to heat stress(43?)led to a time-dependent decrease in red fluorescence detected by confocal laser scanning microscopy,which sharply decreased at 1h after heat stress.Furthermore,we used flow cytometry analysis to count cells with a reduced number of intact lysosomes('pale cells');the increase in the percentage of 'pale cells' was also time-dependent and increased drastically at 1h after heat stress.Heat stress activated cathepsin B release to cytosol in SW480 CellsIt has been demonstrated that lysosomal destabilization is followed by the release of lysosomal cathepsin proteases into the cytosol;cathepsin B,a cysteine protease,has been indicated as a necessary mediatorinthe activation of downstream events leading to cell death.Cathepsin B fluorescence was punctate and mainly perinuclear in untreated cells.However,following heat stress(43?)for 2h,cathepsin B became progressively diffuse as it was released from lysosomes into the cytosol when incubated at 37? for different times as indicated(1h,6h and 24h).Western blot analysis also indicated that heat stress caused cathepsin B release into the cytosol at 1h,peaked at 6h,and continued up to 24h after heat stress.Heat stress activates apoptosis of SW480 cells through the mitochondrial pathwayTo investigate mitochondrial function,mitochondrial depolarization(low ??m)was assayed and expressed as a change in JC-1 fluorescence from red to green.We found that the proportion of SW480 cells with low ??m increased from 8.4%in untreated cells to 36.8%in cells heat stressed for 3h,and further increased to 51.3%in cells heat stressed for 6h.A subsequent decrease in ??m to 42.7%and 37.9%was seen at 12h and 24h after heat stress,respectively.Then,we examined the influence of heat stress on the release of cytochrome C.The Western blots showed a significant increase in the release of cytochrome C into the cytosol induced by heat stress,which increased in a time-dependent manner beginning in 3h and lasting up to 24h after heat stress.Moreover,the abundance of Bax protein increased in a time-dependent fashion and with a similar increased expression trend as pro-apoptotic cytochrome C.Conversely,the expression of anti-apoptotic Bcl-2 decreased in the same time frame,suggesting an increase in the Bax/Bcl-2 ratio,which might be involved in apoptosis induced by heat stress.Cytochrome C interacts with apoptotic protease-activating factor-1(Apaf-1)in the cytosol resulting in the downstream recruitment,procession,and activation of pro-caspase-9 in the presence of dATP or ATP.Caspase-9 cleaves and activates pro-caspase-3 and pro-caspase-7,and serves as an activating protein in the mitochondrial apoptosis pathway.Caspase-9 activity significantly increased at 3h and peaked at 6h after heat stress.Caspase-3 expression was closely correlated with increased expression of caspase-9 activity.Cathepsin B activation of the mitochondrial apoptosis pathway in SW480 cellsLysosomal cysteine protease cathepsin B plays an important role in apoptosis.Cathepsin B can initiate apoptosis through activating caspases,a process related to the release of Bcl-2 and cytochrome C proteins and the activation of caspase-3.To investigate the involvement of cathepsin B in heat stress-induced apoptosis,the effects of the transfection of cathepsin B siRNA on the cellular features of apoptosis were studied.The transfected cells were assayed for cathepsin B protein expression by Western blot assay.Of the several tested siRNA target sequences,one siRNA was effective in decreasing protein levels.Cathepsin B levels measured by RT-PCR were significantly decreased in cathepsin B-transfected cells compared to control cells(scrambled siRNA).The cathepsin B siRNA-transfected cells expressed reduced levels of cathepsin B protein,and were less susceptible to heat stress-induced mitochondrial depolarization compared with control cells.Compared with control cells,cathepsin B siRNA-transfected SW480 cells showed a drastic reduction in the release of cytochrome C,caspase-9 activityand caspase-3 activity.Meanwhile,compared to control cells,the expression of Bcl-2 was up-regulated,whereas the expression of Bax was down-regulated).Effect of antioxidant NAC on heat stress-induced lysosomal membrane permeabilization,cathepsin B release,and apoptosis in vitro and in vivoTo confirm the role of ROS in heat stress-induced lysosomal membrane permeabilization,cathepsin B release,and cell apoptosis,the SW480 cells were pretreated with a well-known antioxidant(NAC)that can inhibit ROS generation.NAC significantly inhibited heat stress-induced ROS formation.When cells were pretreated with NAC,it promoted the recovery of lysosome stability,indicated by a much brighter fluorescence and decreased percentage of 'pale cells'compared to no treatment with NAC.After NAC treatment,cathepsin B fluorescence in the cytosol was reduced and the expression levels of cathepsin B induced by heat stress were down-regulated.The depletion of ROS by the antioxidant NAC significantly decreased the heat stress-mediated mitochondrial depolarization,cytochrome C release from mitochondria),Bax/Bcl-2 ratio,caspase-9 activity,and caspase-3 activity.To further explore the mechanism of intestinal damage and the effect of ROS in vivo,mice were pretreated with NAC.Histopathologic examination was performed on the small intestine(ileum).In the control group that did not undergo heat stress,no marked damage was observed in the small intestinal mucosa.In the heat stroke group,marked villous stroma broadening,focal necrosis,and some epithelial cell detachment accompanied by marked edema and congestion were observed on histologic evaluation with H&E staining.The Chiu score was also increased.After NAC treatment,we observed reduced heat stroke-induced intestinal damage and decreased Chiu scores.NAC also reduced the expression of cathepsin B in the small intestine tissue analyzed by immunohistochemical stain and Western blot.In the mice pretreated with NAC,there were reduced levels of apoptosis,cytochrome C release from mitochondria,Bax/Bcl-2 ratios,caspase-9 activity and caspase-3 activity in the tissue from the small intestine.Effect of cathepsin B inhibitor on heat stress induced apoptosis in vitro and in vivoTo further verify that cytosol cathepsin B accumulation is associated with heat stress-induced cell apoptosis,we administered a highly selective cathepsin B inhibitor(CA-074 Me).After pretreatment with CA-074 Me there was a significant reduction in vivo of cytosolic heat stress-induced cathepsin B on immunohistochemical stain and Western blot.Meanwhile,pretreatment with CA-074 Me was shown to reduce intestinal tissue injury,decreased Chiu score,and decrease the degree of apoptosis caused by heat stroke.In addition,we found that a significant decrease in cytosolic cytochrome C,Bax/Bcl-2 ratio,caspase-9and caspase-3 activation were observed both in vitro and in vivo.Mitochondrial depolarization was also alleviated in cells SW480 pretreated with CA-074 Me compared with control cells.CONCLUSIONIn conclusion,our data suggest that both intestinal tissue and epithelial cells undergo apoptosis soon after heat stress.Heat stress-induced apoptosis is associated with the lysosomal-mitochondrial apoptosis pathway by causing lysosomal membrane permeabilization and cathepsin B release into the cytosol.This process appears to be mediated by ROS generation both in vitro and in vivo.These findings may offer potentially targets to combat heat stress and provide strategies to repair intestinal epithelial tissue injured by heat stroke. |
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