不同剂量氟对大鼠肾皮质IRE1α-ASK1-JNK蛋白表达的影响

Effects of different doses of fluoride on expression of IRE1α-ASK1-JNK proteins in rat renal cortex

目的探讨内质网应激凋亡通路相关蛋白在慢性氟中毒大鼠肾皮质中的表达变化及意义。方法24只健康SD大鼠,根据体质量(100~120 g)采用随机数字表法分为4组(6只/组,雌雄各半);对照组大鼠饮用自来水(含氟量<0.5 mg/L),低、中、高氟组分别饮用含氟量为10、50、100 mg/L(氟化钠)的自来水。各组大鼠饲养180 d后,观察氟斑牙发生情况;收集24 h尿样,氟离子选择电极法检测尿氟含量;大鼠麻醉后处死取肾脏组织,苏木精-伊红(HE)染色观察大鼠肾皮质病理学改变;免疫组织化学染色法观察大鼠肾皮质内质网应激凋亡通路相关蛋白[肌醇依赖性激酶1α(inositol-requiring enzyme 1α,IRE1α)、凋亡信号调节激酶1(apoptosis signal-regulating kinase 1,ASK1)、C-Jun氨基酸末端激酶(C-Jun N-terminal kinase,JNK)及磷酸化JNK(phosphorylated-JNK,P-JNK)]的表达水平。结果对照组大鼠未检出氟斑牙,低氟组大鼠氟斑牙发生率为2/6,中氟组为5/6,高氟组为6/6。与对照组[(5.707±1.190)mg/L]比较,低、中、高氟组大鼠尿氟[(17.028±3.006)、(34.378±12.045)、(94.759±31.773)mg/L]均较高(P均<0.05),且高氟组明显高于低、中氟组(P均<0.05)。HE染色可见,与对照组比较,中、高氟组大鼠肾皮质区肾小管及肾小球细胞体积增大,细胞排列紧密,细胞质嗜酸性增强。免疫组织化学染色结果显示,对照组和低、中、高氟组大鼠肾皮质JNK蛋白表达水平比较差异无统计学意义(F=0.07,P>0.05);高氟组大鼠肾皮质IRE1α、ASK1、P-JNK蛋白表达水平与对照组和低、中氟组比较均较高(P均<0.05),且中氟组IRE1α、ASK1蛋白表达水平均明显高于对照组及低氟组(P均<0.05)。结论长期过量氟摄入可导致大鼠肾皮质损伤,其损伤机制可能与内质网应激凋亡通路IRE1α-ASK1-JNK的活化有关。

Objective To investigate the expression and significance of endoplasmic reticulum stress apoptosis pathway related proteins in renal cortex of rats with chronic fluorosis.Methods Twenty four healthy SD rats were divided into 4 groups(6 rats/group,half male and half female)according to their body mass(100-120 g)by random number table method,rats in control group drank tap water(fluoride content<0.5 mg/L),and in low,medium and high fluoride groups drank tap water with fluoride content(sodium fluoride)of 10,50 and 100 mg/L,respectively.After 180 days of feeding,dental fluorosis was examined,24-hour urine sample was collected and the content of fluoride in urine was detected by fluoride ion selective electrode method.Renal tissue was taken after anesthesia,and the pathological changes of renal cortex were observed by hematoxylin-eosin(HE)staining.The expressions of endoplasmic reticulum stress apoptosis pathway related proteins[inositol-requiring enzyme 1α(IRE1α),apoptosis signal-regulating kinase 1(ASK1),C-Jun N-terminal kinase(JNK)and phosphorylated JNK(P-JNK)]were determined by immunohistochemical staining in rat renal cortex.Results No dental fluorosis was found in control group.The incidence of dental fluorosis in low,medium and high fluoride groups were 2/6,5/6 and 6/6,respectively.Compared with control group[(5.707±1.190)mg/L],the urinary fluoride in low,medium and high fluoride groups[(17.028±3.006),(34.378±12.045),(94.759±31.773)mg/L]was significantly higher(P<0.05),and the urinary fluoride in high fluoride group was higher than that in low and medium fluoride groups(P<0.05).HE staining showed that,compared with control group,the cell volume of renal tubules and glomeruli in medium and high fluoride groups increased,the cells arranged closely,and the eosinophilia of the cytoplasm increased.The immunohistochemical staining results showed that there was no significant difference in the expression of JNK protein in rat renal cortex between control group and low,medium and high fluoride groups(F=0.07,P>0.05).The expressions of IRE1α,ASK1 and P-JNK proteins in rat renal cortex in high fluoride group were higher than those of control,low and medium fluoride groups(P<0.05),and the expressions of IRE1αand ASK1 proteins in medium fluoride group were significantly higher than those in control and low fluoride groups(P<0.05).Conclusion Long-term excessive fluoride intake can lead to renal cortex injury in rats,and the mechanism of injury may be related to the activation of IRE1α-ASK1-JNK endoplasmic reticulum stress apoptosis pathway.