SiRNA干扰慢性炎性痛大鼠患侧背根神经节MrgC表达及PKCε磷酸化的研究

SiRNA interference on expression of MrgC and phosphorylation of PKCε in ipsilateral dorsal root ganglion of rats with chronic inflammatory pain

目的观察鞘内注射小干扰RNA(small interference RNA,siRNA)对完全弗氏佐剂(complete freund’s adjuvant,CFA)诱导的慢性炎性痛大鼠患侧背根神经节(dorsal root ganglion,DRG)中Mrg C(Mas-related G protein-coupled receptor C,Mrg C)mRNA与蛋白表达的干扰作用,并观察该作用对大鼠患足痛阈及患侧DRG PKCε丝氨酸729点位磷酸化(phosphorylation of PKCεSer729,p-PKCεSer729)水平的影响。方法健康雄性SD(Sprague-Dawley,SD)大鼠16只,随机分为对照siRNA组,Mrg C siRNA组,每组8只。大鼠脊髓鞘内插管成功后,两组大鼠给予相应药物鞘内注射4 d,1次/d,5μg/d/只。给药第4 d,大鼠右后足底注射CFA 0.1 m L建立慢性炎性痛模型,此后隔日注射药物,直至给药第11 d处死。分别于鞘内置管前、给药前、给药4 d(CFA造模0 h)、给药5 d(CFA造模24 h)、给药11 d(CFA造模7 d)5个时点检测大鼠患足机械缩腿阈(Paw withdrawal thresholds,PWTs)的变化。荧光定量PCR法检测患侧DRG Mrg C的mRNA的表达,免疫荧光法检测患侧DRG Mrg C表达量及p-PKCεSer729的含量。结果与给药4 d比较,给药5 d两组大鼠的PWTs均有显著的下降(P<0.01);给药前后各时点,两组大鼠之间PWTs没有明显差异。观察给药11d时大鼠患侧L4-L6 DRG Mrg C mRNA的表达,与对照siRNA组比较,Mrg C siRNA组各神经节Mrg C mRNA的表达均明显下降(P<0.01);观察大鼠给药11d时大鼠患侧L4-L6 DRG Mrg C与p-PKCεSer729的表达,与对照siRNA组比较,Mrg C siRNA组患侧DRG的Mrg C阳性细胞率明显减少(P<0.01),而p-PKCεSer729的阳性细胞率显著上升(P<0.05)。结论 Mrg C siRNA片段可有效干扰CFA慢性炎性痛大鼠患侧L4-L6 DRG Mrg C mRNA与Mrg C的表达,对Mrg C的干扰作用能显著上调PKCεSer729磷酸化的水平,但不影响大鼠患足机械缩腿阈。

Objective To observe the Interference effects of siRNA（ small interference RNA） intrathecal injection on the expression of mRNA and protein of MrgC, on PWTs （ paw withdrawal thresholds） and the phosphorylation of PKCεSer729 in ipsilateral DRG（ dorsal root ganglion） of rats with chronic inflammatory pain induced by CFA（ complete freund’ s adjuvant）.Methods 16 health adult male SD （Sprague-Dawley） rats were randomly divided into 2 groups： control siRNA group and MrgC siRNA group, 8 rats in each group.After success of intrathecal catheterization, corresponding siRNA was injected in rats for 4d, once a day, 5μg/d per rat.The model of chronic inflammatory pain was established by CFA （0.1ml per rat） subcutaneously injected into the right hind paw at 4th day post-administration, then two groups were administrated corresponding siRNA on alternate day and executed at the 11th day post-administration.The PWTs were measured at 5 time points of pre-intrathecal catheterization, pre-administration, 4th day post-administration（0h post-CFA injection）, 5th day post-administration（24h post-CFA injection）, 11th day post-administration（7 d post-CFA injection）. The expression of MrgC mRNA in ipsilateral DRG was detected by fluorogenic quantitative PCR, and the expression of MrgC and the phosphorylation of PKCε Ser729 in ipsilateral DRG was detected by immumofluorescence method.Result Compared with 4th day post-administration, PWTs of both two groups at 5th day post-administration decreased significantly （ P〈0.01 ） .While there was no significant difference of PWTs between two groups at every detective time point. Compared with control siRNA group, the expression of MrgC mRNA and the rate of MrgC positive cells in MrgC siRNA group both decreased significantly （ P〈0.01,P 〈0.05）, whereas the rate of p-PKCε Ser729 positive cells increased obviously （ P〈0.05） at 11th day post-administration.Conclusion MrgC siRNA can effectively interfere the expression of mRNA and protein of MrgC in L4-L6 ipsilateral DRGs of rats with chronic inflammatory pain induced by CFA, and the siRNA interference on MrgC can obviously up-regulate the phosphorylation of PKCε Ser729, while it has no significant effect on PWTs of rats.