鞘内注射siRNA-P300抑制CaMKⅡ磷酸化缓解佐剂性关节炎大鼠疼痛与炎症的作用机制  

Intrathecal injection of P300 siRNA to relieve pain and inflammation in rats with adjuvant arthritis by inhibiting the phosphorylation of CaMKⅡ

作　　者：谈俊[1] 高巍巍 滕志鹏 王广[2] 代震宇[3] 李炜 TAN Jun;GAO Weiwei;TENG Zhipeng(Department of Pain,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China)

机构地区：[1]重庆市中医院疼痛科,400021 [2]重庆市中医院神经外科,400021 [3]重庆市中医院骨伤科,400021

出　　处：《河北医药》2023年第15期2271-2276,共6页Hebei Medical Journal

基　　金：国家自然科学基金项目(编号:81702202);重庆市自然科学基金面上项目(编号:cstc2020jcyj-msxmX0116)。

摘　　要：目的研究敲低转录辅因子P300对佐剂性关节炎大鼠的痛觉和炎症的影响并探讨钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)介导的机制。方法60只SPF级别健康成年SD大鼠,随机分为A、B、C、D、E、F共6组(n=10)。A组:对照组;B组:关节模型组;C组:siRNA-P300的阴性对照(siRNA-NC)+关节炎模型组;D组:siRNA-P300+关节炎模型组;E组:siRNA-P300+二甲基亚砜(DMSO)+关节炎模型组;F组:siRNA-P300+油酸+关节炎模型组。各组大鼠造模前1 d及造模后1 d、7 d、14 d的5个时间点分别使用Von Frey纤维丝刺激针以及热辐射痛检测仪分别检测大鼠左后足的机械痛阈值(MWT)和热缩足潜伏期(PWTL),使用电子千分卡尺测左后足跖厚度;于造模后14 d处死动物取脊髓L4~L5节段,采用免疫组织化学法检测脊髓P物质(SP)、c-fos、p-CaMKⅡ的表达水平;蛋白免疫印迹检测CaMKⅡ的磷酸化(p-CaMKⅡ)水平。ELISA法检测血清TNF-α、L-1β、IL-6的水平。结果与A组比较,B组造模后1 d、7 d、14 d的MWT和PWTL均下调,差异均有统计学意义(P<0.05),B组左后足跖厚度增加且TNF-α、L-1β、IL-6上调,差异均有统计学意义(P<0.05),脊髓的SP、c-fos、p-CaMKⅡ的表达水平均上调,差异均有统计学意义(P<0.05)。与C组比较,D组在造模后1 d、7 d、14 d的MWT和PWTL均上调,差异均有统计学意义(P<0.05),D组的足跖厚度减少且TNF-α、IL-1β、IL-6下调,差异均有统计学意义(P<0.05),脊髓的SP、c-fos、p-CaMKⅡ阳性率和表达水平均下调,差异均有统计学意义(P<0.05)。与E组比较,F组在造模后1 d、7 d、14 d的MWT和PWTL均下调,差异均有统计学意义(P<0.05),F组TNF-α、L-1β、IL-6有小幅度上调,差异均有统计学意义(P<0.05),脊髓的SP、c-fos、p-CaMKⅡ的表达水平均上调,差异均有统计学意义(P<0.05)。结论鞘内注射siRNA-P300可通过抑制CaMKⅡ磷酸化缓解佐剂性关节炎大鼠疼痛与炎症作用。Objective To study the effect of knockdown of transcriptional cofactoRP300 on pain and inflammation in rats with adjuvant arthritis and the underlying mechanism by mediating calmodulin-dependent protein kinaseⅡ(CaMKⅡ).Methods A total of 60 healthy adult Sprague-Dawley(SD)rats at the specific-pathogen free(SPF)level were randomly divided into group A(blank control),B(modeling of adjuvant arthritis),C(modeling of adjuvant arthritis plus intrathecal injection of siRNA-NC),D(modeling of adjuvant arthritis plus intrathecal injection of P300 siRNA),E(modeling of adjuvant arthritis plus intrathecal injection of dimethyl sulfoxide[DMSO]and P300 siRNA)and F(modeling of adjuvant arthritis plus intrathecal injection of oleic acid and P300 siRNA),with 10 in each group.Mechanical withdrawal threshold(MWT)and paw withdrawal thermal latency(PWTL)of the rat left hind paw were assessed 1 day before modeling and 1,7 and 14 days afteRmodeling using Von Frey fibers and a thermal analgesiometer,respectively.The plantaRthickness was measured using an electronic micrometeRcaliper.Rats were sacrificed 14 days afteRmodeling,and the L4-L5 spinal segments were collected.Positive expressions of substance P(SP),c-fos and phosphorylated CaMKII(p-CaMKⅡ)in the spinal cord were detected by immunohistochemical staining.The protein level of p-CaMKII was detected by Western blot.Serum levels of tumoRnecrosis factoRalpha(TNF-α),interleukin-1 beta(IL-1β),interleukin-6(IL-6)were measured by Enzyme-linked immunosorbent assay(ELISA).Results Compared with those of group A,rats in group B presented the significantly loweRMWT and PWTL at 1d,7d,and 14d afteRmodeling,largeRleft planteRthickness,higheRserum levels of TNF-α,IL-1βand IL-6,and higheRpositive expressions of SP,c-fos and p-CaMKⅡin the spinal cord(P<0.05).Compared with those of group C,rats in group D presented the significantly higheRMWT and PWTL at 1 d,7 d,and 14 d afteRmodeling,less left planteRthickness,loweRserum levels of TNF-α,IL-1βand IL-6,loweRpositive expressions of SP,c-fos

关 键 词：P300 CaMKⅡ 佐剂性关节炎 疼痛 炎症 

分 类 号：R684.3[医药卫生—骨科学]