热休克蛋白在大鼠慢性吗啡镇痛耐受中的作用及机制  

Heat shock protein 60 mediates chronic morphine antinociceptive tolerance through TLR4-p38MAPK in rats

作　　者：赵彦芬 赵峰 刘鹏森 毛轲 Zhao Yan-Fen;Zhao Feng;Liu Peng-Sen;Mao Ke(Department of Anesthesiology,Henan Hospital of Traditional Chinese Medicine/the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450002,China)

机构地区：[1]河南省中医院/河南中医药大学第二附属医院麻醉科,郑州450002

出　　处：《解放军医学杂志》2020年第12期1242-1247,共6页Medical Journal of Chinese People's Liberation Army

摘　　要：目的探讨热休克蛋白60(HSP60)在慢性吗啡镇痛耐受中的作用及机制。方法50只雄性SD大鼠随机分为对照组(n=10)、吗啡组(n=10)、siRNA-阴性对照(siRNA-NC)+吗啡组(n=10)、siRNA-HSP60+吗啡组(n=10)与脂多糖(LPS)+siRNA-HSP60+吗啡组(n=10)。对照组和吗啡组分别于鞘内注射10μl生理盐水和吗啡;siRNA-NC+吗啡组和siRNA-HSP60+吗啡组分别给予10μl siRNA-NC和siRNA-HSP60,然后鞘内注射吗啡;LPS+siRNA-HSP60+吗啡组给予10μl siRNA-HSP60和LPS,然后鞘内注射吗啡。各组均连续处理7 d。采用RT-PCR和Western blotting检测大鼠脊髓组织中HSP60的mRNA和蛋白表达水平;采用水浴甩尾法观察沉默HSP60对疼痛耐受的影响;免疫荧光染色法检测脊髓小胶质细胞标志物OX-42的表达;RT-PCR法检测大鼠脊髓组织中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)mRNA的表达;Western blotting检测大鼠脊髓组织中Toll样受体4(TLR4)和磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)的表达水平。结果鞘内注射siRNA-HSP60可显著下调吗啡诱导的HSP60的表达(P<0.05)。与对照组相比,吗啡组大鼠对疼痛的耐受度降低(P<0.05),HSP60缺失可增强大鼠对疼痛的耐受(P<0.05)。与siRNA-NC+吗啡组比较,siRNA-HSP60+吗啡组大鼠脊髓组织中OX-42阳性细胞数以及IL-1β、TNF-αmRNA的表达显著下调(P<0.05),TLR4和p-p38MAPK的表达水平降低(P<0.05),而LPS处理可上调TLR4和p-p38MAPK的表达(P<0.05),并能逆转HSP60缺失对吗啡耐受的作用(P<0.05)。结论HSP60缺失通过抑制小胶质细胞的活化及炎症反应改善吗啡耐受的形成,其机制可能与抑制TLR4-p38MAPK信号通路有关。Objective To investigate the function and potential mechanisms of heat shock protein 60(HSP60)in chronic morphine antinociceptive tolerance.Methods Fifty male rats were randomly divided into five groups(n=10):control group,morphine group,siRNA-negative control(siRNA-NC)+morphine group,siRNA-HSP60+morphine group,lipopolysaccharide(LPS)+siRNA-HSP60+morphine group.The control group and the morphine group were injected intrathecally with 10μl of saline or morphine;the siRNA-NC+morphine group and the siRNA-HSP60+morphine group were given 10μl of siRNA-NC and siRNA-HSP60 respectively,and then intrathecal morphine;LPS+siRNA-HSP60+morphine group was given 10μl siRNA-HSP60 and LPS,and then intrathecal injection of morphine.All groups were treated continuously for 7 days.Real-time polymerase chain reaction(RT-PCR)and Western blotting were used to evaluate the expression of HSP60 in the spinal cord tissues of rats.The effect of siRNA-HSP60 on pain tolerance was explored by hot-water tail-flick test.Immunohistochemistry assay was applied to detect the expression of OX-42 in the spinal cord tissues of rats.RT-PCR was used to detect the mRNA levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the spinal cord tissues of rats.Western blotting was used to measure the protein levels of TLR4,p-p38MAPK in the spinal cord tissues of rats.Result Intrathecal injection of siRNA-HSP60 significantly decreased morphineinduced HSP60 expression(P<0.05).Compared with the control group,morphine group rats were less tolerant to pain(P<0.05),and HSP60 deficiency enhanced analgesic effect(P<0.05).Compared with the siRNA-NC+morphine group,the expression levels of OX-42,IL-1βand TNF-αin siRNA-HSP60+morphine group decreased significantly(P<0.05).In addition,the protein levels of TLR4 and p-p38MAPK in siRNA-HSP60+morphine group were lower than those in the siRNA-NC+morphine group(P<0.05).LPS treatment increased the expression of TLR4 and p-p38MAPK(P<0.05);LPS reversed the effect of HSP60 deficiency on morphine tolerance(P<0.0

关 键 词：热休克蛋白60 TLR4-p38MAPK 吗啡镇痛耐受 

分 类 号：R614[医药卫生—麻醉学]