内质网应激和NOD样受体蛋白3在重症中暑小鼠肠黏膜损伤中的作用

目的:研究内质网应激和NOD样受体蛋白3（NOD-like receptor protein 3，NLRP3）在重症中暑肠黏膜损伤中的作用及内质网应激抑制剂4-苯基丁酸（4-phenylbutyric acid，4-PBA）的保护效应。方法:30只雄性BALB /c小鼠随机（随机数字法）分成对照组（control）、重症中暑组（heat stroke，HS）和4-PBA预处理组（4-PBA+HS，4-PBA 120 mg/kg，腹腔注射）。Control组置于室温，HS组和4-PBA+HS组置于高温气候动物培养箱温度（35.5±0.5） ℃,湿度（60.0±5.0）%]，小鼠直肠温度达到42 ℃为重症中暑造模成功标准。中暑6 h后，采用比色法检测小肠匀浆丙二醛（MDA）及超氧化物歧化酶（SOD），ELISA法检测血清白介素-1β（IL-1β）及白介素-18 （IL-18），HE染色观察肠道组织病理，电镜下观察肠道超微结构，Western blot检测葡萄糖调节蛋白78（glucose regulated protein 78，GRP78）、CCAAT/增强子结合蛋白同源蛋白（CCAAT/enhancer-binding protein homologous protein，CHOP）、NLRP3和活化含半胱氨酸的天冬氨酸蛋白水解酶-1（cleaved caspase-1）蛋白表达。计量资料多组间比较如满足方差齐性采用单因素方差分析，组间两两比较采用LSD- t检验；如不满足方差齐性采用Welch分析，组间两两比较采用Dunnett's T3检验，以 P<0.05为差异有统计学意义。 结果:与control组相比，HS组小肠匀浆MDA升高（ t=14.243， P<0.01）而SOD下降（ t=7.781， P<0.01），血清IL-1β和IL-18显著升高（ t=12.664， P<0.01； t=16.240， P<0.01），小肠绒毛广泛破坏伴有炎症细胞浸润，内质网扩张及线粒体肿胀空泡化，小肠组织GRP78、CHOP、NLRP3和cleaved caspase-1蛋白表达增加（ t=14.824， P<0.01； t=12.667， P<0.01； t=9.298， P<0.01， t=6.588， P=0.001）。与HS组相比，4-PBA预处理降低MDA（ t=9.167， P<0.01）并升高SOD（ t=6.077， P<0.01），降低血清IL-1β和IL-18（ t=4.889， P=0.001； t=5.693， P<0.01），减轻小肠黏膜的病理改变及超微结构的损伤，降低了GRP78、CHOP、NLRP3和cleaved caspase-1蛋白表达（ t=9.080， P<0.01； t=7.152, P<0.01； t=4.249， P=0.005； t=3.650， P=0.011）。 结论:内质网应激和NLRP3参与重症中暑肠黏膜损伤，4-BPA通过抑制内质网应激及NLRP3炎症小体活化减轻重症中暑肠黏膜损伤。

Endoplasmic reticulum stress and NOD-like receptor protein 3 involves in intestinal mucosal injury induced by severe heat stroke

Objective:To observe whether endoplasmic reticulum stress and NOD-like receptor protein 3 (NLRP3) inflammasome activation were involved in severe heat stroke induced intestinal mucosal injury and to investigate the potential protective effect of the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA).Methods:Thirty male BALB/c mice were randomly (random number) assigned to 3 groups: the control group, heat stroke group (HS), and 4-PBA pretreatment group (4-PBA+HS, 4-PBA 120 mg/kg, intraperitoneal injection). Mice in the control group were placed at room temperature, while mice in the HS group and 4-PBA+HS group were placed in a prewarmed chamber temperature (35.5±0.5) °C, humidity (60.0±5.0)%]. A rectal temperature (Tc) that reached 42 °C was considered to indicate severe heat stroke. The concentrations of malondialdehyde (MDA) and superoxide dismutase (SOD) in intestinal homogenate were analyzed by a colorimetric method, serum interleukin-1β (IL-1β) and interleukin-18 (IL-18) were assessed by ELISA, intestinal histopathology was evaluated by hematoxylin and eosin (HE) staining, intestinal ultrastructure was observed by electron microscopy, and the protein expression of GRP78, CHOP, NLRP3 and cleaved caspase-1 were analyzed by Western blot. Data were statistically analyzed by ANOVA test and LSD- t multiple comparison test if homogeneous variance, or analyzed by Welch test and Dunnett's T3 multiple comparison test if heterogeneous variance. Results:The concentration of MDA in the HS group was increased ( t=14.243, P<0.01), while SOD was decreased compared with that in the control group ( t=7.781, P<0.01), and the concentrations of serum IL-1β and IL-18 were significantly elevated ( t=12.664, P<0.01; t=16.240, P<0.01). Under light microscopy, extensive destruction of small intestinal villi and inflammatory cell infiltration were observed in the intestines of mice with severe heat stroke. Transmission electron microscopy showed that endoplasmic reticulum structures were significantly expanded, and mitochondria were vacuolated in the intestines of mice with severe heat stroke. Compared with those in the control group, the protein expression levels of GRP78, CHOP, NLRP3 and cleaved caspase-1 in the small intestine were elevated in the HS group ( t=14.824, P <0.01; t=12.667, P<0.01; t=9.298, P<0.01; and t=6.588, P=0.001). Compared with those in the HS group, mice in the 4-PBA pretreatment group exhibited reduced concentrations of MDA ( t=9.167, P<0.01), increased SOD ( t=6.077, P<0.01) , and reduced serum IL-1β and IL-18 levels ( t=4.889, P= 0.001; t=5.693, P<0.01). In addition, 4-PBA pretreatment significantly alleviated the pathological disruption and ultrastructural damage to small intestine tissues. Moreover, 4-PBA pretreatment reduced GRP78, CHOP , NLRP3 and cleaved caspase-1 protein expression ( t=9.080, P<0.01; t=7.152, P<0.01; t=4.249, P=0.005; t=3.650, P=0.011). Conclusions:Endoplasmic reticulum stress and NLRP3 inflammasome are involved in intestinal mucosal injury induced by severe heat stroke. 4-PBA plays a protective role by alleviating endoplasmic reticulum stress and NLRP3 inflammasome activation.