α-硫辛酸经氧化-内质网应激信号通路对氟中毒大鼠生殖损伤的保护作用

The protective effects ofα-lipoic acid on fluoride-induced reproductive lesion in rats via oxidative stress-mediated endoplasmic reticulum stress

目的:明确氟中毒大鼠生殖损伤状态下的氧化应激和内质网应激水平,并观察α-硫辛酸(α-LA)干预后各相关指标的变化,探讨α-LA在氟中毒大鼠生殖损伤中的作用及可能机制。方法:将40只雄性Sprague-Dawley(SD)大鼠采用随机数字法分为四组,分别为对照组(0.9%氯化钠);α-LA组(100 mg/kg α-LA);氟化钠(NaF)组(25 mg/kg NaF);NaF和α-LA联合作用组(25 mg/kg NaF+100 mg/kg α-LA)。采用灌胃的方式染毒,染毒8周后,各组分别进行精子质量分析、睾丸氟含量测定及HE染色;TUNEL法检测睾丸组织细胞凋亡;生化法测定氧化应激相关指标;Western印迹检测睾丸组织中GRP78、PERK及CHOP的蛋白表达水平。结果:与对照组相比,NaF组的精子密度及精子存活率较低[(5.99±1.45)×10^6/ml比(10.96±1.83)×10^6/ml, P<0.01;(33.40±2.71)%比(66.41±3.33)%, P<0.01],精子畸形率较高[(26.43±2.43)%比(11.44±1.55)%, P<0.01)];与NaF组相比,NaF+α-LA组的精子密度及精子存活率较高[(8.47±0.82)×10^6/ml比(5.99±1.45)×10^6/ml, P<0.05;(49.97±3.51)%比(33.40±2.71)%, P<0.05],而精子畸形率较低[(22.69±2.39)%比(26.43±2.43)%, P<0.05]。与对照组相比,NaF组的睾丸氟含量较高[(11.14±0.77)μg/g比(5.78±0.28)μg/g, P<0.01]。光学显微镜下可见NaF组的生精细胞间结构较为疏松,生精细胞和成熟精子数量减少,与α-LA联合作用后,生精细胞结构损伤有所改善,管腔脱落细胞减少。TUNEL检测发现,NaF组的凋亡指数较高[(61.32±7.14)%比(6.99±2.17)%, P<0.01];与NaF组相比,NaF+α-LA组细胞凋亡指数较低[(45.96±5.31)%比(61.32±7.14)%, P<0.01]。氧化应激水平检测发现,与对照组相比,NaF组的丙二醛(MDA)较高[(5.46±0.30)nmol/mgprot比(3. 24±0.58)nmol/mgprot, P<0.01],超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)较低[(6.04±0.71)U/mgprot比(7.19±0.52)U/mgprot, P<0.01;(23.67±0.99)U/mgprot比(26.91±1.67)U/mgprot, P<0.01];与NaF组相比,NaF+α-LA组MDA较低[(4.66±0.70)nmol/mgprot比(5.46±0.30)nmol/mgprot, P<0.05],GSH-Px较高[(25.90±1.93)U/mgprot比(23.67±0.99)U/mgprot, P<0.05]。内质网应激水平检测发现,与对照组相比,NaF组的GRP78、PERK和CHOP蛋白表达水平明显上调(0.79±0.05比0.45±0.09, P<0.01;0.71±0.04比0.40±0.05, P<0.01;0.79±0.09比0.19±0.08, P<0.01),与NaF组相比,α-LA抑制了NaF介导的GRP78和CHOP蛋白表达上调(0.46±0.06比0.79±0.05, P<0.01;0.52±0.09比0.79±0.09, P<0.01)。 结论:α-LA可通过抑制氧化-内质网应激信号通路,对氟中毒大鼠生殖损伤起到一定的保护作用。

Objective To determine the oxidative stress and endoplasmic reticulum stress and their changes afterα-lipoic acid(α-LA)intervention,and to explore the effect and mechanism offluoride-induced reproductive lesion.Methods A total of 40 male Sprague-Dawley(SD)rats were randomly divided into four groups,control group(0.9%sodium chloride),α-LA group(100 mg/kgα-LA),NaF group(25 mg/kg NaF),and NaF+α-LA group(25 mg/kg NaF+100 mg/kgα-LA).Each group was treated in the way of intragastric administration for eight weeks.Sperm quality and the content of NaF in testis were analyzed.The morphologic changes of the testis were observed with the use of HE staining and the apoptosis was detected by the TUNEL assay.Biochemical method was used to measure oxidative stress.Western blot was used to detect the expression of endoplasmic reticulum stress markers,such as GRP78,PERK,and CHOP.Results Compared with the control group,the NaF group had a low level in sperm density[(5.99±1.45)×10^6/ml to(10.96±1.83)×10^6/ml,P<0.01]and sperm vitality[(33.40±2.71)%vs(66.41±3.33)%,P<0.01],but a high level in sperm abnormality rate[(26.43±2.43)%vs(11.44±1.55)%,P<0.01].Compared with the NaF group,the NaF+α-LA group had a high level in both sperm density[(8.47±0.82)×10^6/ml vs(5.99±1.45)×10^6/ml,P<0.05]and sperm vitality[(49.97±3.51)%vs(33.40±2.71)%,P<0.05],but a low level in sperm abnormality rate[(22.69±2.39)%vs(26.43±2.43)%,P<0.05].There was a significantly higher content of NaF in testis in the NaF group[(11.14±0.77)μg/g vs(5.78±0.28)μg/g,P<0.01]than the control group.Optical microscope was used to observe the morphologic changes of the testis,and it was showed that loose structure appeared both in spermatogenic cells and mature sperm cells while the amount of them decreased.However,after the administration ofα-LA,there were complete organelles structure and exfoliated cells in the lumen ameliorated.TUNEL assay found that the apoptotic cells were in a high level in the NaF group[(61.32±7.14)%vs(6.99±2.17)%,P<0.01],whileα-LA significantly suppressed the percentage of apoptotic cells in the NaF+α-LA group compared with the Naf group[(45.96±5.31)%vs(61.32±7.14)%,P<0.01].Oxidative stress assays showed that there were higher express of Malondialdehyde(MDA)content[(5.46±0.30)nmol/mgprot vs(3.24±0.58)nmol/mgprot,P<0.01],the activity of Superoxide Dismutase(SOD)[(6.04±0.71)U/mgprot vs(7.19±0.52)U/mgprot,P<0.01]and Glutathione peroxidase(GSH-Px)[(23.67±0.99)U/mgprot vs(26.91±1.67)U/mgprot,P<0.01]in the NaF group than the control group.To compared with the NaF group,the counterpart in the NaF+a-LA group of MDA content was less[(4.66±0.70)nmol/mgprot vs(5.46±0.30)nmol/mgprot,P<0.05]and the GSH-Px activity was high[(25.90±1.93)U/mgprot vs(23.67±0.99)U/mgprot,P<0.05].Towards the detection of endoplasmic reticulum stress,we found that there were all in higher level in the NaF group that the expression of GRP78[(0.79±0.05)vs(0.45±0.09),P<0.01],PERK[(0.71±0.04)vs(0.40±0.05),P<0.01],and CHOP[(0.79±0.09)vs(0.19±0.08),P<0.01]than the control group,and to compared with the NaF group,α-LA significantly supressed the expression of GRP78[(0.46±0.06)vs(0.79±0.05),P<0.01]and CHOP[(0.52±0.09)vs(0.79±0.09),P<0.01].Conclusionα-lipoic acid plays a protective role in fluoride-induced reproductive lesion in rats by oxidative stress-mediated endoplasmic reticulum stress.