泛素—蛋白酶体途径对拟除虫菊酯神经毒性的影响及机制初探
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摘要
拟除虫菊酯(pyrethroids, Py)是继有机氯、有机磷和氨基甲酸酯之后迅速发展起来的新型农药,根据化学结构可分为不含氰基的Ⅰ型如联苯菊酯(bif enthrin, BF)和含有氰基的Ⅱ型如溴氰菊酯(deltamethrin, DM)。它们具有选择性毒性、哺乳动物体内代谢和排泄迅速、环境残留较少等特点。自上世纪70年代以来,拟除虫菊酯已经被广泛应用,而作为一种神经毒物,其神经毒性日益引起普遍关注。本室对DM的神经毒性的研究已有近20年的历史,发现DM染毒可使大鼠脑组织氧化损伤,神经细胞Ca2+内流增加,线粒体膜电位下降,细胞色素C释放增加以及caspase-3活力改变,对DM神经毒性的了解已有长足的进步,但目前确切的神经毒作用机制仍未十分明了。
自上世纪初始人们关注的热点主要集中在蛋白质的化学结构和生物学功能,以及蛋白质的合成。而对于蛋白质的降解过程关注较少。2004年诺贝尔化学奖授予科学家以表彰他们在蛋白质降解调节机制的重大发现,这个蛋白质降解机制即泛素-蛋白酶体途径(ubiquitin-proteasome pathway, UPP)。它是近年来受到重视的调节蛋白质降解与功能的重要系统,其主要功能在于降解细胞内半衰期较短的调节蛋白和结构异常、错构或受损伤的蛋白。UPP参与许多细胞生命过程,包括细胞周期调控、信号转导、DNA损伤修复、抗原提呈以及细胞凋亡等,并与神经系统许多疾病的发生发展密切相关,如帕金森病(Parkinson's disease,PD)、阿尔茨海默病(Alzheimer's disease, AD)等,因此对该途经的研究已成为目前医学研究的热点之一,而对该途径参与毒作用机制的报道并不多见。
本研究以大鼠为研究对象,在以往DM神经毒性的作用机制研究的基础上,探索蛋白质的降解是否参与Py的神经毒作用机制中,为深入了解神经毒性提供新的思路。从整体方面,给予DM和蛋白酶体抑制剂MG-132,利用现代生物化学和分子生物学方法和技术,观察泛素-蛋白酶体途径在DM致大鼠海马细胞凋亡的中的作用,进一步的探讨DM致大鼠神经毒性的新途径。由于泛素-蛋白酶体途径是非特异性的,以BF为Ⅰ型拟除虫菊酯类代表,验证泛素-蛋白酶体途径致大鼠的神经损伤中的作用,不仅观察细胞凋亡,而且增加了有关细胞氧化应激。在体外条件下,以SH-SY5Y细胞为模型,探讨泛素-蛋白酶体途径在DM和BF所致细胞毒性中的作用,试图为拟除虫菊酯类农药中毒的预防和治疗提供科学依据。
目的：观察DM对大鼠海马细胞泛素化蛋白表达的影响。
方法：采用DM 12.5mg/kg体重(溶剂色拉油)对雄性Wistar大鼠一次腹腔注射,分别于5h、24h、48h后处死,免疫组织化学法观察大鼠海马CA1、CA2、CA3、CA4区泛素化蛋白的表达；免疫蛋白印迹法(western blot)检测海马细胞泛素化蛋白的表达。
结果：经DM染毒后,不同时间点的泛素化蛋白表达用免疫组织化学方法测定。其结果：在海马CA1区,各DM处理组泛素化蛋白表达为5h(0.30±0.04)、24h(0.27±0.03)、48h(0.23±0.02),与对照组(0.18±0.02)相比,差异有统计学意义(P<0.05)。海马CA2区,各DM处理组泛素化蛋白表达为5h(0.29±0.04)、24h(0.26±0.03)、48h(0.22±0.02),与对照组(0.19±0.02)相比,差异有统计学意义(P<0.05)。海马CA3区,各DM处理组泛素化蛋白表达为5h(0.25±0.04)、24h(0.21±0.03)、48h(0.19±0.02),与对照组(0.15±0.01)相比,差异有统计学意义(P<0.05)。海马CA4区,各DM处理组泛素化蛋白表达为5h(0.28±0.03)、24h(0.23±0.02)、48h(0.19±0.03)组,与对照组(0.17±0.01)相比,差异均有统计学意义(P 小结：在本实验条件下,DM染毒可使大鼠海马细胞泛素化蛋白表达增加,提示泛素-蛋白酶体系统被大量激活。在接触DM时海马各区域泛素化蛋白表达最为明显为5小时,且随时间推移,逐渐减少,表明机体可能对接触DM有应激反应,将DM诱导的异常蛋白泛素化并降解,以维持细胞稳态。
目的：研究DM对大鼠海马细胞凋亡的影响。
方法：动物模型建立同第一节。将单侧新鲜海马制成单细胞悬液,流式细胞术(FCM) AV/PI法检测大鼠海马细胞凋亡率,并用单细胞凝胶电泳法(彗星试验)检测大鼠海马细胞DNA损伤。另一侧海马用裂解液匀浆取上清,western blot检测bcl-2蛋白及procaspase-3的表达。
结果：(1)流式细胞术检测发现,大鼠经DM分别染毒5h、24h、48h,其海马细胞早期凋亡率分别为(13.45±2.62)%,(23.81±2.97)%,(15.34±1.27)%,其中,5h、48h组与对照组(4.55±0.86)%相比,差异有统计学意义(P<0.05),24h组与对照组相比,差异有统计学意义(P<0.01)。
(2)彗星试验发现,DM染毒5h、24h、48h后,其海马细胞尾部DNA百分含量分别为(10.12±1.26),(11.91±1.28),(9.89±±1.07),与对照组(2.85±0.32)相比,差异有统计学意义(P<0.01)。DM染毒5h、24h、48h后,其海马细胞Olive尾矩(OTM)分别为(1.98±±0.23),(2.56±±0.31),(1.84±±0.23),与对照组(0.38±±0.041)相比,差异有统计学意义(P (3)经DM染毒后,bcl-2蛋白表达与对照组(0.42±±0.03)相比,24h组(0.12±0.02)、48h组(0.13±±0.02)分别下降了71%和69%,差异有统计学意义(P<0.01)。5h组(0.41±±0.04)与对照组相比,差异无统计学意义(P>0.05)。
(4)经DM染毒后,不同时间点procaspase-3蛋白表达均减少。与对照组(1.01±0.13)相比,5h组(0.72±±0.08)下降了28%,差异有统计学意义(P<0.05)；24h组(0.27±±0.04)、48h组(0.20±±0.04)分别下降了73%和80%,差异有统计学意义(P<0.01)。
小结：本实验利用流式细胞术AV/PI双标染色法和彗星试验,发现DM以12.5mg/kg体重分别处理大鼠5h、24h和48h后,其海马细胞早期凋亡率和DNA损伤均显著增加。其可能的机制是DM可抑制大鼠脑bc1-2蛋白表达；caspase-3前体的表达逐步减少,意味着已被水解成有活性的caspase-3的量逐渐增多,最终参与到细胞凋亡的进程中。
目的：观察UPP对DM致大鼠海马细胞凋亡的影响。
方法：雄性Wistar大鼠24只,随机分为4组：对照组(腹腔注射lml/kg的色拉油后24h处死)；DM组(以12.5 mg/kg的DM腹腔注射染毒,24h后处死)；MG-132+DM组(蛋白酶体抑制剂MG-132 0.5mg/kg体重腹腔注射2h后,在腹腔另一侧注射12.5 mg/kg DM,DM作用24h后处死)；MG-132组(MG-132以0.5mg/kg体重腹腔注射2h,再腹腔另一侧注射等容量的色拉油,色拉油作用24h后处死)。大鼠断头处死,冰上分离海马。将单侧新鲜海马制成单细胞悬液,流式细胞术(FCM)检测大鼠海马细胞凋亡率,并用单细胞凝胶电泳(彗星试验)检测大鼠海马细胞DNA损伤。另一侧海马用裂解液匀浆取上清,western blot检测bc1-2蛋白的表达以及用全波段多功能酶标仪测定大鼠海马caspase-3酶活力。
结果：(1)与DM组(27.29±2.41)%相比,MG-132+DM组其大鼠海马细胞早期凋亡率(19.94±2.07)%下降了27%,差异有统计学意义(P<0.05)。MG-132组的海马细胞早期凋亡率与对照组相比,差异无统计学意义(P>0.05)。
(2)彗星试验发现,MG-132+DM组其海马细胞尾部DNA百分含量为(8.47±0.89),与DM组(11.30±1.36)相比,差异有统计学意义(P<0.05)。MG-132+DM组其海马细胞Olive尾矩(OTM)为(1.51±0.23),与DM组(2.17±0.35)相比,差异有统计学意义(P<0.05)。MG-132组其海马细胞尾部DNA百分含量及0live尾矩(OTM)与对照组相比,差异无统计学意义(P>0.05)。
(3)bc1-2蛋白的表达：MG-132+DM组海马细胞bc1-2蛋白的表达为(2.01±0.23),与DM组(0.43±0.06)相比,提高了367%,差异有统计学意义(P<0.01)。MG-132组,其海马细胞bc1-2蛋白的表达与对照组相比,差异无统计学意义(P>0.05)。
(4)caspase-3酶活力：MG-132+DM组海马细胞caspase-3的活力为(4.55±0.46),与DM组相比降低了14%,差异有统计学意义(P0.05)。
小结：本实验中,MG-132以0.5mg/kg体重提前处理大鼠2h再给予DM染毒,整体凋亡率、DNA损伤、caspase-3酶活力三个细胞凋亡指标与DM组相比均明显下降,bc1-2蛋白的表达显著上升。提示蛋白酶体抑制剂MG-132以0.5mg/kg提前处理大鼠2h能一定程度抑制DM致大鼠海马细胞的凋亡,说明UPP途径参与DM致大鼠海马细胞凋亡,阻断UPP途径能一定程度抑制DM对海马细胞的损伤。
目的：探讨UPP在DM致大鼠海马细胞凋亡的机制。
方法：动物分组同第三节。大鼠断头处死,冰上取海马,采用免疫共沉淀方法检测bc1-2与泛素的相互作用,并观察MG-132对此相互作用的影响。
结果：大鼠经DM染毒24h后,其海马细胞bcl-2蛋白泛素化水平增高了约90%,与对照组相比,差异有统计学意义(P<0.01)。MG-132+DM组其bc1-2蛋白泛素化水平下降了约46%,与DM组相比,差异有统计学意义(P<0.01)；MG-132组其bc1-2蛋白泛素化水平与对照组相比,差异无统计学意义(P>0.05)。
小结：DM致大鼠海马细胞凋亡的机制可能是提高bc1-2蛋白的泛素化,MG-132能抑制DM诱导的bc1-2蛋白的泛素化,减少bc1-2蛋白的降解,以保护细胞免受毒物的损害作用。
目的：研究UPP对BF致大鼠海马细胞凋亡的影响。
方法：21只健康成年Wistar雄性大鼠,体重180-200g,按体重随机分为7组(每组3只)：对照组(腹腔注射色拉油后24h处死)；BF 24h组(以7mg／kg的BF腹腔注射染毒,24h后处死)；BF5d组(以7mg/kg的BF腹腔注射染毒,连续5d,每天一次,最后一次给药24h后处死)；MG-132+BF 24h组(MG-132以0.5mg/kg体重腹腔注射1h后,BF以7mg/kg体重在另一侧腹腔注射,BF作用24h后处死)；MG-132+BF 5d组：MG-132腹腔注射1h后,在另一侧腹腔注射BF,连续5d,最后一次BF染毒24h后处死；MG-132 24h组(MG-132以0.5mg/kg体重腹腔注射1h后,在另一侧腹腔注射色拉油,色拉油作用24h后处死)；MG-132 5d组(MG-132以0.5mg/kg体重腹腔注射1h后,在另一侧腹腔注射色拉油,连续5d,最后一次给予色拉油24h后处死)。大鼠断头处死,冰上分离海马。将单侧新鲜海马制成单细胞悬液,流式细胞术(FCM)检测大鼠海马细胞凋亡率。
结果：BF 24h组和5d组,其海马细胞早期凋亡率分别(51.57±5.22)%,(50.23±6.86)%,与对照组(33.43±7.06)%相比,分别升高了约54.26%和50.25%,差异有统计学意义(P<0.05)。MG-132+BF 24h组,其海马细胞早期凋亡率为(40.23±7.69)%,与BF 24h组相比,下降了约21.99%,差异有统计学意义(P<0.05)。MG-132+BF 5d组海马细胞晚期凋亡率为(39.33±9.31)%,与BF 5d组相比,差异无统计学意义(P>0.05)。MG-132 24h组其海马细胞早期凋亡率为(51.03±5.08)%,与对照组相比,升高了约52.65%,差异有统计学意义(P<0.05)。MG-132 5d组其海马细胞早期凋亡率为(51.77±3.59)%,与对照组相比,升高了约54.86%,差异有统计学意义(P<0.05)。
小结：BF染毒后,能诱导大鼠海马细胞发生凋亡。预先给予MG-132,能在一定程度抑制BF诱导细胞凋亡的发生,提示UPP参与BF致大鼠海马细胞凋亡的过程。
目的：探讨UPP对BF致大鼠海马细胞氧化应激的影响。
方法：动物分组同第一节。大鼠断头处死取单侧海马组织制成10%的匀浆液,试剂盒测定其MDA、SOD、GSH的含量变化。
结果：(1)丙二醛(MDA)含量：BF 24h和5d组,海马细胞MDA含量分别为(4.07±0.53)和(4.21±0.45),与对照组(2.53±0.21)相比,分别升高约61%和66%,差异有统计学意义(P<0.05)。MG-132+BF 24h组,其海马细胞MDA含量为(1.94±0.38),与BF 24h组相比,降低约52%,差异有统计学意义(P<0.01)。MG-132+BF 5d组与BF 5d组相比,差异无统计学意义(P>0.05)。MG-132 24h和5d组,其海马细胞MDA含量与对照组相比,差异无统计学意义(P>0.05)。
(2)超氧化物歧化酶(SOD)活力单位：BF 5d组其海马细胞SOD活力单位为(19.51±2.15),与对照组(30.41±8.27)相比下降约36%,差异有统计学意义(P<0.05)。BF 24h组与对照组相比差异无统计学意义(P>0.05)。MG-132+BF 5d组(28.01±3.49)与BF 5d组(19.52±2.15)相比升高约43%,差异有统计学意义(P<0.05)。MG-132+BF 24h组其海马细胞SOD活力单位与BF 24h组相比,差异无统计学意义(P>0.05)。MG-132 24h和5d组与对照组相比,差异无统计学意义(P>0.05)。
(3)谷胱甘肽(GSH)含量：BF 5d组为(2.13±0.56),与对照组(3.16±0.36)相比,降低约33%,差异有统计学意义(P<0.05)。BF 24h组与对照组相比,差异无统计学意义(P>0.05)。MG-132+BF 24h组,其海马细胞GSH含量为
(4.33±0.53),与BF 24h组(2.47±0.55)相比,显著增高,升幅约75%,差异有统计学意义(P<0.01)。MG-132+BF 5d组与BF 5d组相比,差异无统计学意义(P>0.05)。MG-132 24h和5d组,其海马细胞GSH含量与对照组相比,差异无统计学意义(P>0.05)。
小结：BF染毒后能诱导大鼠海马细胞氧化损伤,MDA含量增高,SOD、GSH含量下降。MG-132能在一定程度的抑制BF诱导的大鼠海马细胞氧化损伤,提示UPP参与BF致大鼠海马细胞氧化应激的过程。
目的：探讨UPP对Py致SH-SY5Y细胞存活率的影响。
方法：设7个组,每组8个平行样。分别为空白对照组,溶剂对照(1‰DMSO)组,DM组、MG-132+DM组、BF组、MG-132+BF组,MG-132组,其中DM、BF各设7个剂量组,均为1×10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、1×10-3mol／L。MG-132终浓度为1×10-6μmol/L,分别提前2h和1h处理细胞后与DM、BF联合处理。DM、BF处理24h后以MTT法测定细胞存活率。
结果：(1)DM组1×10-9-1×10-3mol／L的细胞存活率分别为105.48%,95.32%,98.15%,82.09%,74.79%,72.06%和80.45%。其中1×10-6、1×10-5、1×10-4、1×10-3mol／L四个DM剂量组细胞的存活率与空白对照组相比明显下降,差异有统计学意义(P<0.01)。经过浓度为1×10-6μmol／L的MG-132预处理2h后,DM各剂量组的细胞存活率分别为109.35%,97.14%,95.67%,91.86%,82.53%,75.49%,78.74%。其中1×10-6、1×10--mol／L DM染毒剂量的细胞存活率与相应DM染毒的细胞存活率相比,显著升高,差异有统计学意义(P<0.05)。
(2)BF组1×10-9-1×10-2mol／L的细胞存活率分别为101.12%,94.24%,92.52%,92.61%,87.67%,76.22%和66.95%。其中,1×10-5、1×10-4、1×10-3mol／L三个BF剂量组细胞的存活率与空白对照组相比明显下降,差异有统计学意义(P<0.01)。经过浓度为1×10-6μmol／L的MG-132预处理1h后,BF各剂量组的细胞存活率分别为105.64%,98.32%,94.75%,90.68%,95.24%,86.24%,70.84%,其中1×10-51×10-4mol／L BF染毒剂量的细胞存活率与相应BF染毒的细胞存活率相比,显著升高,差异有统计学意义(P<0.05)。
小结：DM、BF分别以一定剂量(DM:1×10-6-1×10-3mol／L;BF:1×10-5-1×10-3mol／L)能抑制SH-SY5Y细胞的生长,MG-132对Py的神经细胞毒作用具有较好的抑制作用,提示UPP途径参与Py的神经细胞毒性过程。
总之,本课题“泛素-蛋白酶体途径对拟除虫菊酯农药致大鼠神经毒性的影响及机制初探”的研究仅是个开端,在毒作用机制研究中,考虑蛋白降解途径。初步得出的结论是：在本研究中,Ⅱ型拟除虫菊酯农药DM可引起神经细胞凋亡,泛素化蛋白增加。利用蛋白酶体抑制剂MG-132,证实泛素-蛋白酶体途径参与DM诱导的细胞凋亡,增加细胞DNA损伤,提高bcl-2蛋白泛素化。并且发现bcl-2蛋白的泛素化降解很可能是DM引起神经细胞凋亡的机制之一。同时以Ⅰ型拟除虫菊酯农药联苯菊酯(bifenthrin,BF),验证了UPP参与BF所致神经细胞毒性,包括细胞凋亡和氧化损伤。在体外试验也初步发现阻断UPP能减低Py的细胞毒性,提高存活率。综上所述,在神经系统或神经细胞中UPP通路是参与农药中毒的机制,可以抑制该通路发挥其对机体和细胞的神经保护作用。
Pyrethroid (Py) pesticides, the major class of insecticides, are commonly used in agriculture and urban settings due to their high potency and selectivity as nerve poisons and low persistent residues compared with other classes of insecticides. However, the pyrethroids can elicit neurotoxicity, characterized by hyperexcitation, incoordination, tremor and paralysis. A large amount of researches demonstrate that typeⅡpyrethroids such as deltamethrin (DM) can induce the activity of caspase-3 and apoptosis in nerve cells, but the exact mechanism of apoptosis induced by DM is not known yet.
The ubiquitin-proteasome pathway (UPP) is the major extra-lysosomal pathway responsible for intracellular protein degradation in eukaryotes. It is composed of the ubiquitin-conjugating system and the 26S proteasome. Degradation of proteins through this pathway involves two distinct and successive steps. Firstly, target proteins, usually abnormal proteins, are conjugated to polyubiquitin, called ubiquitinated proteins. Secondly, they are recognized and recruited to the 26S proteasome where they are rapidly degraded. The UPP is important in controlling intracellular levels of a variety of short-lived proteins and in maintaining cellular growth and metabolism. The UPP plays a role in many cellular processes, including cell cycle controlling, signal transduction, DNA damage and repair, apoptosis, but there are few reports about how the UPP play a role in toxic mechanism.
Based on departed researches on Py neurotoxic mechanism, the aims of this study were to determine how the Py poisoning was associated with the ubiquitin-proteasome pathway. Taking advantage of proteasome inhibitor MG-132, to test the hypothesis that if the UPP induces neurocyte toxicity. The present study will provide new insights to molecular mechanisms of the UPP in the toxic mechanism of pesticide.
Objective:To explore the effects of DM on the expression of ubiquitinated proteins in hippocampus of rats.
Methods:Male Wistar rats weighing 180-220g were divided into 4 groups:control group, 12.5 mg/kg body weight DM were administered by intraperitoneal injection. The animals were decapitated after treatment of DM for 5 h,24 h and 48 h. Immunohistochemical method was applied to measure the expression of ubiquitinated proteins in CA1, CA2, CA3, CA4 area of hippocampus of rat brain. The expression of ubiquitinated proteins in hippocampus were detected by western blot.
Results:The results of immunohistochemistry showed that after treatment of DM, the expression of ubiquitinated proteins enhanced in all areas of hippocampus in 5 h,24 h and 48 h groups. Compared to control group, there was significant difference of optical density (P<0.05). Western blot experiment indicated that after treatment of DM on different time points, the expression of ubiquitinated proteins increased in hippocampus. Compared to control group, there was significant difference of relative band intensity (P<0.05).
Conclusions:In this condition, DM could enhance the expression of ubiquitinated proteins, which indicated that UPP was activated.
Objective:To observe the effect of DM on apoptosis in hippocampus cell of rat.
Methods:The animal model was the same as sectionⅠ. The utilization of flow cytometry (FCM), the comet assay and western blot was aim to analyze apoptosis of hippocampus of rat from many aspects.
Results:FCM analysis indicated that in all DM treated group, the early apoptotic rate were increased (P<0.05). The results of the comet assay showed that in all DM treated group, DNA damage was worse compared to the control group (P＜0.05). Western blot indicated that bcl-2 was decreased in 24h and 48h of DM-treated group (P<0.01), but didn't show significant change in 5h group. Procaspase-3 was decreased in 5h of DM-treated group (P<0.05), there was significant difference (P<0.01) in 24h and 48h group.
Conclusions:The apoptosis of hippocampus cell could be induced after treatment of DM.
Objective:To study the effect of UPP on DM-induced apoptosis in hippocampus cell of rat.
Methods:Male wistar rats were divided into 4 groups:(1) control group (salad oil); (2) DM 12.5mg/kg BW; (3) MG-132 (0.5mg/kg BW, dissolved in DMSO) administered 2 h before DM (12.5mg/kg BW), both chemicals were intraperitoneal injected; (4) MG-132 (0.5mg/kg BW) then 2 h later coin oil. After treatment rats were sacrificed by decapitation and the hippocampus were immediately removed for later use. FCM, the comet assay, western blot and the caspase-3 activity kit were utilized to measure the potential protetive effect of MG-132.
Results:FCM showed that compared to DM group, the early apoptotic rate of hippocampus cells were decreased in MG-132+DM group (P＜0.05). The comet assay indicated that compared to DM group, the DNA damage were decreased in MG-132+DM group (P<0.05). The bcl-2 western blot showed that compared to DM group, the expression of bcl-2 was increased in MG-132+DM group (P＜0.01). The activity of caspase-3 in MG-132+DM group was lower than DM group (P<0.05). Compared to control group, MG-132 treated alone group didn't show any apoptotic change (P>0.05).
Conclusions:In this experimental condition, using MG-132 at the dose of 0.5mg/kg and pre-treated for 2h could attenuate the apoptotic effect induced by DM.
Objective:To observe the mechanism of UPP on DM-induced apoptosis in hippocampus cell of rat.
Methods:The animal model was the same as sectionⅢ. Co-immunoprecipitation was utilized to detect the interaction between bcl-2 and ubiquitin.
Results:The co-immunoprecipitation showed that compared to control group, the bcl-2 ubiquitination was significantly increased (P＜0.01). Pre-treatment with MG-132 almost completely blocked the increase of bcl-2 ubiquitination (P＜0.01). MG-132 treated alone group didn't show any change compared to control group (P>0.05).
Conclusions:In this experimental condition, DM could induce bcl-2 ubiquitination which could be inhibited by MG-132.
Objective:To study the effect of UPP on BF-induced apoptosis in hippocampus cell of rat.
Methods:Male wistar rats were divided into 7 groups:(1) control group (salad oil); (2) BF 7mg/kg BW for 24h; (3) rats were treated daily with BF in the dose of 7mg/kg BW for 5d; (4) MG-132 (0.5mg/kg BW, dissolved in DMSO) administered 1 h, then treated with BF (12.5mg/kg BW) for 24h, both chemicals were intraperitoneal injected; (5) MG-132 administered 1 h, then treated with BF, five successive days (6) MG-132+salad oil for 24h; (7) MG-132+salad oil for five successive days. After treatment, rats were sacrificed by decapitation and the hippocampus were immediately removed to detect apoptotic rate through FCM.
Results:Compared to control group, the early apoptosis rate was increased in BF 24h and 5d group (P<0.05). Compared to BF 24h group, the early apoptosis rate was decreased in MG-132+BF for 24h group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group(P>0.05). Compared to control group, the early apoptosis rate was increased in MG-132 treated alone for 24h and 5d group (P<0.05).
Conclusions:In this experimental condition, BF could induce apoptosis of hippocampus cell, and this induction could be blocked by MG-132, which indicated that UPP may play a role in BF-induced apoptosis.
Objective:To study the effect of UPP on BF-induced oxidative stress in hippocampus of rat.
Methods:The animal model was the same as sectionⅠ. SOD kit, MDA kit and GSH kit were utilized to detect oxidative stress.
Results:(1) MDA level:compared to control group, the level of MDA was increased in BF 24h and 5d group (P<0.05). Compared to BF 24h group, the level of MDA was decreased in MG-132+BF for 24d group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group (P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05). (2) SOD activity unit:compared to control group, the SOD activity unit was decreased in BF 5d group (P<0.05) but not in 24h group (P>0.05). Compared to BF 5d group, the SOD activity unit was increased in MG-132+BF for 5d group (P<0.05). But there wasn't significant change between BF 24h group and MG-132+BF for 24h group(P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05). (3) GSH level:compared to control group, the level of GSH was decreased in BF 5d group (P<0.05) but not in 24h group (P>0.05). Compared to BF 24h group, the level of GSH was increased in MG-132+BF for 24h group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group(P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05).
Conclusions:In this experimental condition, BF could induce oxidative stress of hippocampus cell, and this induction could be inhibited by MG-132, which indicated that UPP may take part in BF-induced oxidative damage.
Objective:To study the effect of UPP on cytotoxicity induced by Py in SH-SY5Y cell.
Methods:Cells were divided into 7 groups:blank control group, vehiche control group (1‰DMSO), DM group, MG-132+DM group, BF group, MG-132+BF group, MG-132 group. The dose of DM and BF was the same:1×10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、1×10-3 mol/L. The final concentration of MG-132 was 1×10-6μmol/L (DM, BF, MG-132 were all dissolved in DMSO). MG-132 was treated 2h before DM and 1h before BF. After DM or BF treated for 24h, the cell survival rate was measured by the method of MTT.
Results:(1) The cell survival rate of DM group was 105.48%,95.32%,98.15%,82.09%, 74.79%,72.06%,80.45%, there were significantly lower than blank control group in the dose of 1 10-6、1×10-5、1×10-4、1×10-3 mol/L (P<0.01). Pretreated with 1×10-6μmol/L MG-132, the cell survival rate of DM all groups were 109.35%,97.14%,95.67%,91.86%,82.53%, 75.49%,78.74%, there were significantly higher than DM group in the dose of 1×10-6、1×10-5 mol/L (P＜0.05). (2) The cell survival rate of BF group was 101.12%,94.24%,92.52%,92.61 %,87.67%,76.22%,66.95%, there were significantly lower than blank control group in the dose of 1×10-5、1×10-4、1×10-3 mol/L (P＜0.01). After 1×10-6μmol/L MG-132 intervention, the cell survival rate of BF all groups were 105.64%,98.32%,94.75%,90.68%,95.24%, 86.24%,70.84%, there were significantly higher than BF group in the dose of 1×10-5、1×10-4 mol/L (P<0.05).
Conclusions:In this research, DM and BF under certain concentration could induce cytotoxicity of SH-SY5Y cell. MG-132 can inhibit the adverse effect induced by DM and BF, which implied that UPP may play a role in the induction of cytotoxicity.
Taken together, these findings demonstrate that DM can induce neuron apoptosis through stimulating ubiquitin-proteasome pathway (UPP). DM induces an activation of ubiquitinated proteins and caspase-3, as well as the DNA damage. Meanwhile DM lowers the expression of bcl-2. The inhibitor of UPP, MG-132, may block the apoptosis in a certain degree induced by DM. Furthermore, bcl-2 ubiquitination was inhibited after MG-132 treated, which suggested that bcl-2 ubiquitination may be a mechanism of DM neurotoxicity. Besides, UPP could play a role in BF-induced neuron cytotoxicity, including apoptosis and oxidative stress. At the same time, MG-132 had a certain protection against cytotoxicity induced by DM and BF in vitro. These findings imply that UPP play a role in the toxic mechanism of pesticide.
自上世纪初始人们关注的热点主要集中在蛋白质的化学结构和生物学功能,以及蛋白质的合成。而对于蛋白质的降解过程关注较少。2004年诺贝尔化学奖授予科学家以表彰他们在蛋白质降解调节机制的重大发现,这个蛋白质降解机制即泛素-蛋白酶体途径(ubiquitin-proteasome pathway, UPP)。它是近年来受到重视的调节蛋白质降解与功能的重要系统,其主要功能在于降解细胞内半衰期较短的调节蛋白和结构异常、错构或受损伤的蛋白。UPP参与许多细胞生命过程,包括细胞周期调控、信号转导、DNA损伤修复、抗原提呈以及细胞凋亡等,并与神经系统许多疾病的发生发展密切相关,如帕金森病(Parkinson's disease,PD)、阿尔茨海默病(Alzheimer's disease, AD)等,因此对该途经的研究已成为目前医学研究的热点之一,而对该途径参与毒作用机制的报道并不多见。
本研究以大鼠为研究对象,在以往DM神经毒性的作用机制研究的基础上,探索蛋白质的降解是否参与Py的神经毒作用机制中,为深入了解神经毒性提供新的思路。从整体方面,给予DM和蛋白酶体抑制剂MG-132,利用现代生物化学和分子生物学方法和技术,观察泛素-蛋白酶体途径在DM致大鼠海马细胞凋亡的中的作用,进一步的探讨DM致大鼠神经毒性的新途径。由于泛素-蛋白酶体途径是非特异性的,以BF为Ⅰ型拟除虫菊酯类代表,验证泛素-蛋白酶体途径致大鼠的神经损伤中的作用,不仅观察细胞凋亡,而且增加了有关细胞氧化应激。在体外条件下,以SH-SY5Y细胞为模型,探讨泛素-蛋白酶体途径在DM和BF所致细胞毒性中的作用,试图为拟除虫菊酯类农药中毒的预防和治疗提供科学依据。
目的：观察DM对大鼠海马细胞泛素化蛋白表达的影响。
方法：采用DM 12.5mg/kg体重(溶剂色拉油)对雄性Wistar大鼠一次腹腔注射,分别于5h、24h、48h后处死,免疫组织化学法观察大鼠海马CA1、CA2、CA3、CA4区泛素化蛋白的表达；免疫蛋白印迹法(western blot)检测海马细胞泛素化蛋白的表达。
结果：经DM染毒后,不同时间点的泛素化蛋白表达用免疫组织化学方法测定。其结果：在海马CA1区,各DM处理组泛素化蛋白表达为5h(0.30±0.04)、24h(0.27±0.03)、48h(0.23±0.02),与对照组(0.18±0.02)相比,差异有统计学意义(P<0.05)。海马CA2区,各DM处理组泛素化蛋白表达为5h(0.29±0.04)、24h(0.26±0.03)、48h(0.22±0.02),与对照组(0.19±0.02)相比,差异有统计学意义(P<0.05)。海马CA3区,各DM处理组泛素化蛋白表达为5h(0.25±0.04)、24h(0.21±0.03)、48h(0.19±0.02),与对照组(0.15±0.01)相比,差异有统计学意义(P<0.05)。海马CA4区,各DM处理组泛素化蛋白表达为5h(0.28±0.03)、24h(0.23±0.02)、48h(0.19±0.03)组,与对照组(0.17±0.01)相比,差异均有统计学意义(P
目的：研究DM对大鼠海马细胞凋亡的影响。
方法：动物模型建立同第一节。将单侧新鲜海马制成单细胞悬液,流式细胞术(FCM) AV/PI法检测大鼠海马细胞凋亡率,并用单细胞凝胶电泳法(彗星试验)检测大鼠海马细胞DNA损伤。另一侧海马用裂解液匀浆取上清,western blot检测bcl-2蛋白及procaspase-3的表达。
结果：(1)流式细胞术检测发现,大鼠经DM分别染毒5h、24h、48h,其海马细胞早期凋亡率分别为(13.45±2.62)%,(23.81±2.97)%,(15.34±1.27)%,其中,5h、48h组与对照组(4.55±0.86)%相比,差异有统计学意义(P<0.05),24h组与对照组相比,差异有统计学意义(P<0.01)。
(2)彗星试验发现,DM染毒5h、24h、48h后,其海马细胞尾部DNA百分含量分别为(10.12±1.26),(11.91±1.28),(9.89±±1.07),与对照组(2.85±0.32)相比,差异有统计学意义(P<0.01)。DM染毒5h、24h、48h后,其海马细胞Olive尾矩(OTM)分别为(1.98±±0.23),(2.56±±0.31),(1.84±±0.23),与对照组(0.38±±0.041)相比,差异有统计学意义(P
(4)经DM染毒后,不同时间点procaspase-3蛋白表达均减少。与对照组(1.01±0.13)相比,5h组(0.72±±0.08)下降了28%,差异有统计学意义(P<0.05)；24h组(0.27±±0.04)、48h组(0.20±±0.04)分别下降了73%和80%,差异有统计学意义(P<0.01)。
小结：本实验利用流式细胞术AV/PI双标染色法和彗星试验,发现DM以12.5mg/kg体重分别处理大鼠5h、24h和48h后,其海马细胞早期凋亡率和DNA损伤均显著增加。其可能的机制是DM可抑制大鼠脑bc1-2蛋白表达；caspase-3前体的表达逐步减少,意味着已被水解成有活性的caspase-3的量逐渐增多,最终参与到细胞凋亡的进程中。
目的：观察UPP对DM致大鼠海马细胞凋亡的影响。
方法：雄性Wistar大鼠24只,随机分为4组：对照组(腹腔注射lml/kg的色拉油后24h处死)；DM组(以12.5 mg/kg的DM腹腔注射染毒,24h后处死)；MG-132+DM组(蛋白酶体抑制剂MG-132 0.5mg/kg体重腹腔注射2h后,在腹腔另一侧注射12.5 mg/kg DM,DM作用24h后处死)；MG-132组(MG-132以0.5mg/kg体重腹腔注射2h,再腹腔另一侧注射等容量的色拉油,色拉油作用24h后处死)。大鼠断头处死,冰上分离海马。将单侧新鲜海马制成单细胞悬液,流式细胞术(FCM)检测大鼠海马细胞凋亡率,并用单细胞凝胶电泳(彗星试验)检测大鼠海马细胞DNA损伤。另一侧海马用裂解液匀浆取上清,western blot检测bc1-2蛋白的表达以及用全波段多功能酶标仪测定大鼠海马caspase-3酶活力。
结果：(1)与DM组(27.29±2.41)%相比,MG-132+DM组其大鼠海马细胞早期凋亡率(19.94±2.07)%下降了27%,差异有统计学意义(P<0.05)。MG-132组的海马细胞早期凋亡率与对照组相比,差异无统计学意义(P>0.05)。
(2)彗星试验发现,MG-132+DM组其海马细胞尾部DNA百分含量为(8.47±0.89),与DM组(11.30±1.36)相比,差异有统计学意义(P<0.05)。MG-132+DM组其海马细胞Olive尾矩(OTM)为(1.51±0.23),与DM组(2.17±0.35)相比,差异有统计学意义(P<0.05)。MG-132组其海马细胞尾部DNA百分含量及0live尾矩(OTM)与对照组相比,差异无统计学意义(P>0.05)。
(3)bc1-2蛋白的表达：MG-132+DM组海马细胞bc1-2蛋白的表达为(2.01±0.23),与DM组(0.43±0.06)相比,提高了367%,差异有统计学意义(P<0.01)。MG-132组,其海马细胞bc1-2蛋白的表达与对照组相比,差异无统计学意义(P>0.05)。
(4)caspase-3酶活力：MG-132+DM组海马细胞caspase-3的活力为(4.55±0.46),与DM组相比降低了14%,差异有统计学意义(P
小结：本实验中,MG-132以0.5mg/kg体重提前处理大鼠2h再给予DM染毒,整体凋亡率、DNA损伤、caspase-3酶活力三个细胞凋亡指标与DM组相比均明显下降,bc1-2蛋白的表达显著上升。提示蛋白酶体抑制剂MG-132以0.5mg/kg提前处理大鼠2h能一定程度抑制DM致大鼠海马细胞的凋亡,说明UPP途径参与DM致大鼠海马细胞凋亡,阻断UPP途径能一定程度抑制DM对海马细胞的损伤。
目的：探讨UPP在DM致大鼠海马细胞凋亡的机制。
方法：动物分组同第三节。大鼠断头处死,冰上取海马,采用免疫共沉淀方法检测bc1-2与泛素的相互作用,并观察MG-132对此相互作用的影响。
结果：大鼠经DM染毒24h后,其海马细胞bcl-2蛋白泛素化水平增高了约90%,与对照组相比,差异有统计学意义(P<0.01)。MG-132+DM组其bc1-2蛋白泛素化水平下降了约46%,与DM组相比,差异有统计学意义(P<0.01)；MG-132组其bc1-2蛋白泛素化水平与对照组相比,差异无统计学意义(P>0.05)。
小结：DM致大鼠海马细胞凋亡的机制可能是提高bc1-2蛋白的泛素化,MG-132能抑制DM诱导的bc1-2蛋白的泛素化,减少bc1-2蛋白的降解,以保护细胞免受毒物的损害作用。
目的：研究UPP对BF致大鼠海马细胞凋亡的影响。
方法：21只健康成年Wistar雄性大鼠,体重180-200g,按体重随机分为7组(每组3只)：对照组(腹腔注射色拉油后24h处死)；BF 24h组(以7mg／kg的BF腹腔注射染毒,24h后处死)；BF5d组(以7mg/kg的BF腹腔注射染毒,连续5d,每天一次,最后一次给药24h后处死)；MG-132+BF 24h组(MG-132以0.5mg/kg体重腹腔注射1h后,BF以7mg/kg体重在另一侧腹腔注射,BF作用24h后处死)；MG-132+BF 5d组：MG-132腹腔注射1h后,在另一侧腹腔注射BF,连续5d,最后一次BF染毒24h后处死；MG-132 24h组(MG-132以0.5mg/kg体重腹腔注射1h后,在另一侧腹腔注射色拉油,色拉油作用24h后处死)；MG-132 5d组(MG-132以0.5mg/kg体重腹腔注射1h后,在另一侧腹腔注射色拉油,连续5d,最后一次给予色拉油24h后处死)。大鼠断头处死,冰上分离海马。将单侧新鲜海马制成单细胞悬液,流式细胞术(FCM)检测大鼠海马细胞凋亡率。
结果：BF 24h组和5d组,其海马细胞早期凋亡率分别(51.57±5.22)%,(50.23±6.86)%,与对照组(33.43±7.06)%相比,分别升高了约54.26%和50.25%,差异有统计学意义(P<0.05)。MG-132+BF 24h组,其海马细胞早期凋亡率为(40.23±7.69)%,与BF 24h组相比,下降了约21.99%,差异有统计学意义(P<0.05)。MG-132+BF 5d组海马细胞晚期凋亡率为(39.33±9.31)%,与BF 5d组相比,差异无统计学意义(P>0.05)。MG-132 24h组其海马细胞早期凋亡率为(51.03±5.08)%,与对照组相比,升高了约52.65%,差异有统计学意义(P<0.05)。MG-132 5d组其海马细胞早期凋亡率为(51.77±3.59)%,与对照组相比,升高了约54.86%,差异有统计学意义(P<0.05)。
小结：BF染毒后,能诱导大鼠海马细胞发生凋亡。预先给予MG-132,能在一定程度抑制BF诱导细胞凋亡的发生,提示UPP参与BF致大鼠海马细胞凋亡的过程。
目的：探讨UPP对BF致大鼠海马细胞氧化应激的影响。
方法：动物分组同第一节。大鼠断头处死取单侧海马组织制成10%的匀浆液,试剂盒测定其MDA、SOD、GSH的含量变化。
结果：(1)丙二醛(MDA)含量：BF 24h和5d组,海马细胞MDA含量分别为(4.07±0.53)和(4.21±0.45),与对照组(2.53±0.21)相比,分别升高约61%和66%,差异有统计学意义(P<0.05)。MG-132+BF 24h组,其海马细胞MDA含量为(1.94±0.38),与BF 24h组相比,降低约52%,差异有统计学意义(P<0.01)。MG-132+BF 5d组与BF 5d组相比,差异无统计学意义(P>0.05)。MG-132 24h和5d组,其海马细胞MDA含量与对照组相比,差异无统计学意义(P>0.05)。
(2)超氧化物歧化酶(SOD)活力单位：BF 5d组其海马细胞SOD活力单位为(19.51±2.15),与对照组(30.41±8.27)相比下降约36%,差异有统计学意义(P<0.05)。BF 24h组与对照组相比差异无统计学意义(P>0.05)。MG-132+BF 5d组(28.01±3.49)与BF 5d组(19.52±2.15)相比升高约43%,差异有统计学意义(P<0.05)。MG-132+BF 24h组其海马细胞SOD活力单位与BF 24h组相比,差异无统计学意义(P>0.05)。MG-132 24h和5d组与对照组相比,差异无统计学意义(P>0.05)。
(3)谷胱甘肽(GSH)含量：BF 5d组为(2.13±0.56),与对照组(3.16±0.36)相比,降低约33%,差异有统计学意义(P<0.05)。BF 24h组与对照组相比,差异无统计学意义(P>0.05)。MG-132+BF 24h组,其海马细胞GSH含量为
(4.33±0.53),与BF 24h组(2.47±0.55)相比,显著增高,升幅约75%,差异有统计学意义(P<0.01)。MG-132+BF 5d组与BF 5d组相比,差异无统计学意义(P>0.05)。MG-132 24h和5d组,其海马细胞GSH含量与对照组相比,差异无统计学意义(P>0.05)。
小结：BF染毒后能诱导大鼠海马细胞氧化损伤,MDA含量增高,SOD、GSH含量下降。MG-132能在一定程度的抑制BF诱导的大鼠海马细胞氧化损伤,提示UPP参与BF致大鼠海马细胞氧化应激的过程。
目的：探讨UPP对Py致SH-SY5Y细胞存活率的影响。
方法：设7个组,每组8个平行样。分别为空白对照组,溶剂对照(1‰DMSO)组,DM组、MG-132+DM组、BF组、MG-132+BF组,MG-132组,其中DM、BF各设7个剂量组,均为1×10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、1×10-3mol／L。MG-132终浓度为1×10-6μmol/L,分别提前2h和1h处理细胞后与DM、BF联合处理。DM、BF处理24h后以MTT法测定细胞存活率。
结果：(1)DM组1×10-9-1×10-3mol／L的细胞存活率分别为105.48%,95.32%,98.15%,82.09%,74.79%,72.06%和80.45%。其中1×10-6、1×10-5、1×10-4、1×10-3mol／L四个DM剂量组细胞的存活率与空白对照组相比明显下降,差异有统计学意义(P<0.01)。经过浓度为1×10-6μmol／L的MG-132预处理2h后,DM各剂量组的细胞存活率分别为109.35%,97.14%,95.67%,91.86%,82.53%,75.49%,78.74%。其中1×10-6、1×10--mol／L DM染毒剂量的细胞存活率与相应DM染毒的细胞存活率相比,显著升高,差异有统计学意义(P<0.05)。
(2)BF组1×10-9-1×10-2mol／L的细胞存活率分别为101.12%,94.24%,92.52%,92.61%,87.67%,76.22%和66.95%。其中,1×10-5、1×10-4、1×10-3mol／L三个BF剂量组细胞的存活率与空白对照组相比明显下降,差异有统计学意义(P<0.01)。经过浓度为1×10-6μmol／L的MG-132预处理1h后,BF各剂量组的细胞存活率分别为105.64%,98.32%,94.75%,90.68%,95.24%,86.24%,70.84%,其中1×10-51×10-4mol／L BF染毒剂量的细胞存活率与相应BF染毒的细胞存活率相比,显著升高,差异有统计学意义(P<0.05)。
小结：DM、BF分别以一定剂量(DM:1×10-6-1×10-3mol／L;BF:1×10-5-1×10-3mol／L)能抑制SH-SY5Y细胞的生长,MG-132对Py的神经细胞毒作用具有较好的抑制作用,提示UPP途径参与Py的神经细胞毒性过程。
总之,本课题“泛素-蛋白酶体途径对拟除虫菊酯农药致大鼠神经毒性的影响及机制初探”的研究仅是个开端,在毒作用机制研究中,考虑蛋白降解途径。初步得出的结论是：在本研究中,Ⅱ型拟除虫菊酯农药DM可引起神经细胞凋亡,泛素化蛋白增加。利用蛋白酶体抑制剂MG-132,证实泛素-蛋白酶体途径参与DM诱导的细胞凋亡,增加细胞DNA损伤,提高bcl-2蛋白泛素化。并且发现bcl-2蛋白的泛素化降解很可能是DM引起神经细胞凋亡的机制之一。同时以Ⅰ型拟除虫菊酯农药联苯菊酯(bifenthrin,BF),验证了UPP参与BF所致神经细胞毒性,包括细胞凋亡和氧化损伤。在体外试验也初步发现阻断UPP能减低Py的细胞毒性,提高存活率。综上所述,在神经系统或神经细胞中UPP通路是参与农药中毒的机制,可以抑制该通路发挥其对机体和细胞的神经保护作用。
Pyrethroid (Py) pesticides, the major class of insecticides, are commonly used in agriculture and urban settings due to their high potency and selectivity as nerve poisons and low persistent residues compared with other classes of insecticides. However, the pyrethroids can elicit neurotoxicity, characterized by hyperexcitation, incoordination, tremor and paralysis. A large amount of researches demonstrate that typeⅡpyrethroids such as deltamethrin (DM) can induce the activity of caspase-3 and apoptosis in nerve cells, but the exact mechanism of apoptosis induced by DM is not known yet.
The ubiquitin-proteasome pathway (UPP) is the major extra-lysosomal pathway responsible for intracellular protein degradation in eukaryotes. It is composed of the ubiquitin-conjugating system and the 26S proteasome. Degradation of proteins through this pathway involves two distinct and successive steps. Firstly, target proteins, usually abnormal proteins, are conjugated to polyubiquitin, called ubiquitinated proteins. Secondly, they are recognized and recruited to the 26S proteasome where they are rapidly degraded. The UPP is important in controlling intracellular levels of a variety of short-lived proteins and in maintaining cellular growth and metabolism. The UPP plays a role in many cellular processes, including cell cycle controlling, signal transduction, DNA damage and repair, apoptosis, but there are few reports about how the UPP play a role in toxic mechanism.
Based on departed researches on Py neurotoxic mechanism, the aims of this study were to determine how the Py poisoning was associated with the ubiquitin-proteasome pathway. Taking advantage of proteasome inhibitor MG-132, to test the hypothesis that if the UPP induces neurocyte toxicity. The present study will provide new insights to molecular mechanisms of the UPP in the toxic mechanism of pesticide.
Objective:To explore the effects of DM on the expression of ubiquitinated proteins in hippocampus of rats.
Methods:Male Wistar rats weighing 180-220g were divided into 4 groups:control group, 12.5 mg/kg body weight DM were administered by intraperitoneal injection. The animals were decapitated after treatment of DM for 5 h,24 h and 48 h. Immunohistochemical method was applied to measure the expression of ubiquitinated proteins in CA1, CA2, CA3, CA4 area of hippocampus of rat brain. The expression of ubiquitinated proteins in hippocampus were detected by western blot.
Results:The results of immunohistochemistry showed that after treatment of DM, the expression of ubiquitinated proteins enhanced in all areas of hippocampus in 5 h,24 h and 48 h groups. Compared to control group, there was significant difference of optical density (P<0.05). Western blot experiment indicated that after treatment of DM on different time points, the expression of ubiquitinated proteins increased in hippocampus. Compared to control group, there was significant difference of relative band intensity (P<0.05).
Conclusions:In this condition, DM could enhance the expression of ubiquitinated proteins, which indicated that UPP was activated.
Objective:To observe the effect of DM on apoptosis in hippocampus cell of rat.
Methods:The animal model was the same as sectionⅠ. The utilization of flow cytometry (FCM), the comet assay and western blot was aim to analyze apoptosis of hippocampus of rat from many aspects.
Results:FCM analysis indicated that in all DM treated group, the early apoptotic rate were increased (P<0.05). The results of the comet assay showed that in all DM treated group, DNA damage was worse compared to the control group (P＜0.05). Western blot indicated that bcl-2 was decreased in 24h and 48h of DM-treated group (P<0.01), but didn't show significant change in 5h group. Procaspase-3 was decreased in 5h of DM-treated group (P<0.05), there was significant difference (P<0.01) in 24h and 48h group.
Conclusions:The apoptosis of hippocampus cell could be induced after treatment of DM.
Objective:To study the effect of UPP on DM-induced apoptosis in hippocampus cell of rat.
Methods:Male wistar rats were divided into 4 groups:(1) control group (salad oil); (2) DM 12.5mg/kg BW; (3) MG-132 (0.5mg/kg BW, dissolved in DMSO) administered 2 h before DM (12.5mg/kg BW), both chemicals were intraperitoneal injected; (4) MG-132 (0.5mg/kg BW) then 2 h later coin oil. After treatment rats were sacrificed by decapitation and the hippocampus were immediately removed for later use. FCM, the comet assay, western blot and the caspase-3 activity kit were utilized to measure the potential protetive effect of MG-132.
Results:FCM showed that compared to DM group, the early apoptotic rate of hippocampus cells were decreased in MG-132+DM group (P＜0.05). The comet assay indicated that compared to DM group, the DNA damage were decreased in MG-132+DM group (P<0.05). The bcl-2 western blot showed that compared to DM group, the expression of bcl-2 was increased in MG-132+DM group (P＜0.01). The activity of caspase-3 in MG-132+DM group was lower than DM group (P<0.05). Compared to control group, MG-132 treated alone group didn't show any apoptotic change (P>0.05).
Conclusions:In this experimental condition, using MG-132 at the dose of 0.5mg/kg and pre-treated for 2h could attenuate the apoptotic effect induced by DM.
Objective:To observe the mechanism of UPP on DM-induced apoptosis in hippocampus cell of rat.
Methods:The animal model was the same as sectionⅢ. Co-immunoprecipitation was utilized to detect the interaction between bcl-2 and ubiquitin.
Results:The co-immunoprecipitation showed that compared to control group, the bcl-2 ubiquitination was significantly increased (P＜0.01). Pre-treatment with MG-132 almost completely blocked the increase of bcl-2 ubiquitination (P＜0.01). MG-132 treated alone group didn't show any change compared to control group (P>0.05).
Conclusions:In this experimental condition, DM could induce bcl-2 ubiquitination which could be inhibited by MG-132.
Objective:To study the effect of UPP on BF-induced apoptosis in hippocampus cell of rat.
Methods:Male wistar rats were divided into 7 groups:(1) control group (salad oil); (2) BF 7mg/kg BW for 24h; (3) rats were treated daily with BF in the dose of 7mg/kg BW for 5d; (4) MG-132 (0.5mg/kg BW, dissolved in DMSO) administered 1 h, then treated with BF (12.5mg/kg BW) for 24h, both chemicals were intraperitoneal injected; (5) MG-132 administered 1 h, then treated with BF, five successive days (6) MG-132+salad oil for 24h; (7) MG-132+salad oil for five successive days. After treatment, rats were sacrificed by decapitation and the hippocampus were immediately removed to detect apoptotic rate through FCM.
Results:Compared to control group, the early apoptosis rate was increased in BF 24h and 5d group (P<0.05). Compared to BF 24h group, the early apoptosis rate was decreased in MG-132+BF for 24h group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group(P>0.05). Compared to control group, the early apoptosis rate was increased in MG-132 treated alone for 24h and 5d group (P<0.05).
Conclusions:In this experimental condition, BF could induce apoptosis of hippocampus cell, and this induction could be blocked by MG-132, which indicated that UPP may play a role in BF-induced apoptosis.
Objective:To study the effect of UPP on BF-induced oxidative stress in hippocampus of rat.
Methods:The animal model was the same as sectionⅠ. SOD kit, MDA kit and GSH kit were utilized to detect oxidative stress.
Results:(1) MDA level:compared to control group, the level of MDA was increased in BF 24h and 5d group (P<0.05). Compared to BF 24h group, the level of MDA was decreased in MG-132+BF for 24d group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group (P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05). (2) SOD activity unit:compared to control group, the SOD activity unit was decreased in BF 5d group (P<0.05) but not in 24h group (P>0.05). Compared to BF 5d group, the SOD activity unit was increased in MG-132+BF for 5d group (P<0.05). But there wasn't significant change between BF 24h group and MG-132+BF for 24h group(P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05). (3) GSH level:compared to control group, the level of GSH was decreased in BF 5d group (P<0.05) but not in 24h group (P>0.05). Compared to BF 24h group, the level of GSH was increased in MG-132+BF for 24h group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group(P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05).
Conclusions:In this experimental condition, BF could induce oxidative stress of hippocampus cell, and this induction could be inhibited by MG-132, which indicated that UPP may take part in BF-induced oxidative damage.
Objective:To study the effect of UPP on cytotoxicity induced by Py in SH-SY5Y cell.
Methods:Cells were divided into 7 groups:blank control group, vehiche control group (1‰DMSO), DM group, MG-132+DM group, BF group, MG-132+BF group, MG-132 group. The dose of DM and BF was the same:1×10-9、1×10-8、1×10-7、1×10-6、1×10-5、1×10-4、1×10-3 mol/L. The final concentration of MG-132 was 1×10-6μmol/L (DM, BF, MG-132 were all dissolved in DMSO). MG-132 was treated 2h before DM and 1h before BF. After DM or BF treated for 24h, the cell survival rate was measured by the method of MTT.
Results:(1) The cell survival rate of DM group was 105.48%,95.32%,98.15%,82.09%, 74.79%,72.06%,80.45%, there were significantly lower than blank control group in the dose of 1 10-6、1×10-5、1×10-4、1×10-3 mol/L (P<0.01). Pretreated with 1×10-6μmol/L MG-132, the cell survival rate of DM all groups were 109.35%,97.14%,95.67%,91.86%,82.53%, 75.49%,78.74%, there were significantly higher than DM group in the dose of 1×10-6、1×10-5 mol/L (P＜0.05). (2) The cell survival rate of BF group was 101.12%,94.24%,92.52%,92.61 %,87.67%,76.22%,66.95%, there were significantly lower than blank control group in the dose of 1×10-5、1×10-4、1×10-3 mol/L (P＜0.01). After 1×10-6μmol/L MG-132 intervention, the cell survival rate of BF all groups were 105.64%,98.32%,94.75%,90.68%,95.24%, 86.24%,70.84%, there were significantly higher than BF group in the dose of 1×10-5、1×10-4 mol/L (P<0.05).
Conclusions:In this research, DM and BF under certain concentration could induce cytotoxicity of SH-SY5Y cell. MG-132 can inhibit the adverse effect induced by DM and BF, which implied that UPP may play a role in the induction of cytotoxicity.
Taken together, these findings demonstrate that DM can induce neuron apoptosis through stimulating ubiquitin-proteasome pathway (UPP). DM induces an activation of ubiquitinated proteins and caspase-3, as well as the DNA damage. Meanwhile DM lowers the expression of bcl-2. The inhibitor of UPP, MG-132, may block the apoptosis in a certain degree induced by DM. Furthermore, bcl-2 ubiquitination was inhibited after MG-132 treated, which suggested that bcl-2 ubiquitination may be a mechanism of DM neurotoxicity. Besides, UPP could play a role in BF-induced neuron cytotoxicity, including apoptosis and oxidative stress. At the same time, MG-132 had a certain protection against cytotoxicity induced by DM and BF in vitro. These findings imply that UPP play a role in the toxic mechanism of pesticide.
引文
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