甘草酸治疗对骨癌痛小鼠痛行为及脊髓炎症反应的影响

Effect of glycyrrhizin on bone cancer-induced pain behavior and spinal inflammation in mice

目的:探讨甘草酸对骨癌痛小鼠痛行为及脊髓炎症反应的影响。方法:雄性C3H/HeJ小鼠共40只,4~6周龄,体重20~25 g,采用随机数字法,将其分为4组(n=10):假手术组(Sham组)、肿瘤组(Tumor组)、低剂量甘草酸给药组(G1组)和高剂量甘草酸给药组(G2组)。小鼠右侧股骨远端骨髓腔接种NCTC纤维肉瘤细胞,建立骨癌痛模型。Sham组小鼠右侧股骨远端骨髓腔仅注射不含肿瘤细胞的α-MEM培养基,Tumor组小鼠右侧股骨远端骨髓腔接种NCTC纤维肉瘤细胞,G1组和G2组股骨远端接种肿瘤细胞后2、24和48小时分别腹腔注射甘草酸100和200 mg/kg。于肿瘤细胞接种前两小时、肿瘤细胞接种后4、7、10、14、21和28天测量机械缩足阈值和自发抬足次数;肿瘤细胞接种后28天,采用Western blot法检测右侧脊髓L3~5节段HMGB1、IL-1β、IL-10、p-Akt、p-mTOR蛋白表达,免疫荧光法检测右侧脊髓小胶质细胞标记物脊髓背角Iba-1阳性细胞密度和Iba-1免疫荧光阳性面积。结果:与Sham组比较,Tumor组小鼠肿瘤接种后10、14、21和28天机械缩足阈值显著下降,7、10、14、21和28天自发抬足次数明显增多(P <0.05)。与Tumor组比较,G1和G2组小鼠肿瘤接种后14、21和28天机械缩足阈值升高,且自发抬足次数减少(P <0.05);与Sham组比较,Tumor组小鼠肿瘤接种后28天损伤侧脊髓HMGB1、IL-1β、p-Akt、p-mTOR蛋白表达水平显著升高(P <0.05),脊髓背角Iba-1阳性小胶质细胞密度和Iba-1免疫荧光阳性面积明显增加(P <0.05)。与Tumor组比较,G1和G2组小鼠肿瘤接种后28天损伤侧脊髓HMGB1、IL-1β、p-Akt、p-mTOR蛋白表达水平明显下调(P <0.05),IL-10蛋白表达水平显著上调(P <0.05),脊髓背角Iba-1阳性小胶质细胞密度和Iba-1免疫荧光阳性面积下调(P <0.05)。结论:甘草酸可缓解骨癌痛小鼠痛行为的形成并减轻脊髓炎症反应。

Objective:To investigate the effect of glycyrrhizin on bone cancer-induced pain behavior and spinal inflammation in mice.Methods:A total of 40 male C3H/HeJ mice,4 to 6 weeks old,weighting 20 to 25 g,were divided into 4 groups(n=10):Sham operation group(Sham group),tumor group(Tumor group),low-dose glycyrrhizin treatment group(G1 group)and high-dose glycyrrhizin treatment group(G2 group).To establish the bone cancer pain model,we inoculated the right femur with NCTC fibrosarcoma cells.For the mice in the Sham group,the right distal femur was merely injected with theα-MEM medium void of any tumor cell.The mice in the Tumor group were injected with NCTC fibrosarcoma cells in the distal right femur.The mice in the G1 and G2 groups received the inoculation of NCTC fibrosarcoma followed by intraperitoneal treatment with 100 and 200 mg/kg of glycyrrhizin,respectively,at 2,24,and 48 hours after tumor cell seeding.At two hours before tumor cell inoculation and 4,7,10,14,21,and 28 days after tumor cell inoculation,the mechanical paw withdrawal threshold(MPWT)and the number of spontaneous lifts(NSL)were measured.At twenty-eight days after tumor cell inoculation,the protein expression of HMGB1,IL-1β,IL-10,p-Akt,p-mTOR in the spinal cord was detected by Western blot L3~5 segment protein expression,and immunofluorescence analysis was used to detect the density of Iba-1 positive cell density and the immunoreactive area.Results:Compared with the Sham group,the MPWT of mice in the Tumor group decreased on 10,14,21,and 28 days after tumor inoculation,and the NSL increased significantly on 7,10,14,21,and 28 days after tumor inoculation(P<0.05).Compared with Tumor group,the MPWT in G1 and G2 groups increased on 14,21,and 28 days after tumor inoculation,whereas the NSL decreased(P<0.05).Compared with Sham group,the protein expression of HMGB1,IL-1β,p-Akt,and p-mTOR in the spinal cord elevated significantly in the Tumor group(P<0.05),and the density of Iba-1 positive microglia and the area of Iba-1 immunofluorescence in the spinal dorsal horn increased significantly(P<0.05).Compared with Tumor group,the protein expression of HMGB1,IL-1β,p-Akt and p-mTOR protein in the spinal cord of the G1 and G2 groups were significantly down-regulated(P<0.05),and IL-10 protein expression level was up-regulated(P<0.05),the density of Iba-1 positive microglia and the Iba-1 immunoreactive area were down-regulated(P<0.05).Conclusion:Treatment with glycyrrhizin can alleviate pain behavior-induced by bone cancer pain and reduce the spinal inflammation in mice.