华蟾毒精对骨癌痛大鼠镇痛作用及机制研究

Study on analgesic effect and mechanism of cinobufagin on rats with bone cancer pain

目的研究华蟾毒精(CBG)对骨癌痛大鼠的镇痛作用及毒蕈碱受体M4亚型(M4 mAChR)在其中的作用与机制。方法100只雌性SD大鼠采用随机数字表法分为5组(n=20):假手术组(S组)、胫骨癌痛组(A组)、生理盐水+ CBG溶剂组(ANS组)、生理盐水+ 1 mg/kg CBG组(ANC组)、Tropicamide + 1 mg/kg CBG组(ATC组)。S组左胫骨上端骨髓腔各注入Hank′s液10 μl,A组、ANS组、ANC组、ATC组左胫骨上端骨髓腔各注入Walker256癌细胞悬液10μl。模型制备成功后第9天,ANS组、ANC组、ATC组鞘内分别注射生理盐水15μl、生理盐水15μl、10nmol M4 mAChR阻断剂Tropicamide,10min后,ANS组、ANC组、ATC组腹腔分别注射CBG溶剂、1mg/kg CBG、1mg/kg CBG。造模前(T0)每组大鼠测3次取平均值作为其基础痛阈,腹腔给药前20min(T1),给药后10min(T2)、30min(T3)、60min(T4)、90min(T5)、120min(T6)分别测定其痛阈值。并且给药后60min取L4~L6左侧脊髓背角和背根神经节(DRG),采用蛋白质印迹法(Western blot)检测钙离子/钙调素依赖性蛋白激酶Ⅱa(CaMKⅡa)、磷酸化钙离子/钙调素依赖性蛋白激酶Ⅱa(pCaMKⅡa)表达情况。结果在T1~T6各时间点,S组机械痛阈值分别为(8.69±0.45)、(8.63±0.44)、(8.65±0.39)、(8.84±0.23)、(8.80±0.14)、(8.75±0.14)g,A组分别为(6.37±0.30)、(6.42±0.13)、(6.29±0.17)、(6.25±0.22)、(6.34±0.33)、(6.36±0.34)g,差异均有统计学意义(t=-16.41、-23.47、-30.25、-17.35、-19.52、-22.56,均P<0.01);在T3~T5各时间点,ANS组机械痛阈值分别为(6.42±0.32)、(6.39±0.34)、(6.26±0.32)g,ANC组分别为(7.29±0.34)、(7.81±0.15)、(7.54±0.19)g,差异均有统计学意义(t=13.52、14.22、17.33,均P<0.01);在T3~T5各时间点,与ANC组比较,ATC组机械痛阈值下降,分别为(6.55±0.23)、(6.84±0.46)、(6.80±0.43)g,差异均有统计学意义(t=-12.69、-11.26、-10.33,均P<0.01)。T4时间点,各组大鼠脊髓背角pCaMKⅡa表达水平分别为(0.67±0.05)、(1.64±0.12)、(1.57±0.14)、(0.78±0.09)、(1.39±0.11),DRG表达水平分别为(1.65±0.39)、(3.59±0.17)、(3.43±0.32)、(2.17±0.34)、(2.95±0.23),差异均有统计学意义(F=179.89、198.76,均P<0.01);与S组比较,A组pCaMⅡa表达上调;与ANS组比较,ANC组pCaMKⅡa表达下调;与ANC组比较,ATC组pCaMKⅡa表达上调;CaMKⅡa在脊髓背角及DRG内各组表达差异均无统计学意义(F=1.25、2.79,均P>0.05)。结论M4 mAChR阻断剂参与介导华蟾毒精缓解骨癌痛大鼠的痛觉过敏,其机制可能与pCaMKⅡa/CaMKⅡa信号通路有关。

Objective To evaluate the analgesic effects of cinobufagin (CBG) on cancer-induced bone pain in rat and study the role of the muscarinic receptor M4 subtype (M4 mAChR) in its involvement.Methods A total of 100 Female Sprague-Dawley rats were randomly divided into 5 groups (n=20): Sham group (group S),Cancer group (group A),Normal saline + CBG vehicle solution group (group ANS),Normal saline + 1 mg/kg CBG group (group ANC) and Tropicamide + 1 mg/kg CBG group (group ATC).Rats in group S were injected 10 μl Hank′s solution into the left tibia medullar cavity,while rats in group A,ANS,ANC,and ATC were injected Walker 256 mammary cancer cells (10 μl,2×107 cells/ml) into the same place.On day 9 post-inoculation rats in group ANS,ANC,and ATC were respectively received Saline (0.9%,15 μl,i.t.),Saline (0.9%,15 μl,i.t.)and 10 nmol of M4 mAChR blocker Tropicamide.After 10 min,ANS group,ANC group and ATC group were intraperitoneally injected with CBG vehicle solution,1 mg/kg CBG and 1 mg/kg CBG.Model rats in each group were tested three times average as its basis pain threshold before injection cancer cells (T0).Mechanical withdrawal thresholds were measured on left hind paws,before 20 min (T1) and after 10 min (T2),30 min (T3),60 min (T4),90 min (T5) and 120 min (T6) intrathecal injection.Left L4-L6 spinal dorsal horn and DRG were removed for determination of the expression of CaM-dependent kinaseⅡa (CaMKⅡa) and pCaMKⅡa by Western Blot after 60 min drug delivery.Results At each time point from T1 to T6,the mechanical pain thresholds of group S were (8.69±0.45),(8.63±0.44),(8.65±0.39),(8.84±0.23),(8.80±0.14),(8.75±0.14) g,respectively,and the mechanical pain thresholds of group A were (6.37±0.30),(6.42±0.13),(6.29±0.17),(6.25±0.22),(6.34±0.33),(6.36±0.34) g,the difference was statistically significant (t=-16.41,-23.47,-30.25,-17.35,-19.52,-22.56,all P<0.01).At each time point from T3 to T5,the mechanical pain thresholds of the ANS group were (6.42±0.32),(6.39±0.34),(6.26±0.32) g,respectively,and the mechanical pain thresholds of the ANC group were (7.29±0.34),(7.81±0.15),(7.54±0.19) g,the difference was statistically significant (t=13.52,14.22,17.33,all P<0.01).At each time point from T3 to T5,compared with the ANC group,the mechanical pain threshold of the ATC group decreased (6.55±0.23),(6.84±0.46),(6.80±0.43) g,and the difference was statistically significant (t=-12.69,-11.26,-10.33,all P<0.01).At the time of T4,the expressions of pCaMKⅡa in the spinal dorsal horn of each group were (0.67±0.05),(1.64±0.12),(1.57±0.14),(0.78±0.09),(1.39±0.11),respectively,and the expressions of pCaMKⅡa in DRG of each group were (1.65±0.39),(3.59±0.17),(3.43±0.32),(2.17±0.34),(2.95±0.23).The differences were statistically significant (F=179.89,198.76,both P<0.01).Compared with the S group,the expression of pCaMⅡa was up-regulated in group A.Compared with ANS group,the expression of pCaMKⅡ a was down-regulated in ANC group.Compared with ANC group,the expression of pCaMK Ⅱ a was up-regulated in ATC group.The expression of CaMKⅡa in spinal dorsal horn and DRG was not statistically significant (F=1.25,2.79,both P>0.05).Conclusions These results demonstrated that M4mAChR participated in mediating the alleviation of hyperalgesia by cinobufagin in rats with bone cancer pain,and its mechanism may be related to pCaMKⅡa/CaMKⅡa signaling pathway.