强筋壮骨祛风合剂对髓核致炎大鼠背根神经节中3型酸敏感离子通道的影响
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摘要
目的:观察强筋壮骨祛风合剂对髓核致炎大鼠背根神经节中3型酸敏感离子通道(acid-sensing ion channel 3,ASIC3)的影响。方法:选取40只清洁级成年雄性SD大鼠,随机分为空白组、假手术组、模型组、阿米洛利组和中药组,每组8只。除空白组外,其余各组大鼠均手术暴露L5背根神经节。假手术组暴露L5背根神经节后直接缝合切口;模型组和中药组取出大鼠尾部的髓核组织置于暴露的L5背根神经节上,缝合切口;阿米洛利组将取自大鼠尾部的髓核组织置于暴露的L5背根神经节上,并在暴露的L5背根神经节周围滴入含0.1 mg盐酸的阿米洛利药液1 m L,缝合切口。自造模后第1天开始,中药组按5 m L·kg-1以强筋壮骨祛风合剂灌胃,每天2次,连续30 d;空白组、假手术组、模型组不进行药物干预。分别于造模前1 d及造模后3、5、9、15、23、29 d,采用Up-Down法测定大鼠左后足50%机械刺激缩足阈值(50%paw withdrawal threshold,50%PWT)。中药组药物干预结束后,处死所有大鼠,取出L5背根神经节分成2份,一份采用免疫组织化学法计算ASIC3阳性面积,另一份采用Western Blot技术检测ASIC3蛋白表达量。结果:造模后3 d内,模型组、阿米洛利组和中药组均有部分大鼠出现左后足外翻以及足趾背伸现象。造模前后不同时间50%PWT的差异有统计学意义,即存在时间效应(F=50.132,P=0.000)。5组50%PWT总体上比较,组间差异有统计学意义,即存在分组效应(F=288.219,P=0.000)。除造模前1 d时外(F=0.332,P=0.854),造模后各时点5组大鼠50%PWT比较,组间差异均有统计学意义(F=288.015,P=0.000;F=308.588,P=0.000;F=374.945,P=0.000;F=560.713,P=0.000;F=343.043,P=0.000;F=459.914,P=0.000);造模后各时点模型组的50%PWT均低于空白组、假手术组、阿米洛利组和中药组(P=0.010,P=0.011,P=0.001,P=0.001;P=0.023,P=0.022,P=0.000,P=0.004;P=0.013,P=0.013,P=0.003,P=0.001;P=0.012,P=0.002,P=0.002,P=0.002;P=0.004,P=0.000,P=0.000,P=0.000;P=0.003,P=0.005,P=0.003,P=0.013);造模后各时点空白组和假手术组50%PWT比较,差异均无统计学意义(P=0.640,P=0.710,P=0.590,P=0.480,P=0.330,P=0.390);造模后各时点阿米洛利组50%PWT均高于中药组(P=0.021,P=0.011,P=0.013,P=0.002,P=0.001,P=0.005)。时间因素与分组因素存在交互效应(F=2.358,P=0.002)。5组大鼠背根神经节中ASIC3阳性面积和ASIC3蛋白表达量比较,组间差异均有统计学意义(F=205.890,P=0.030;F=101.740,P=0.001);空白组、假手术组、阿米洛利组和中药组背根神经节中ASIC3阳性面积和ASIC3蛋白表达量均小于模型组(P=0.004,P=0.003,P=0.001,P=0.007;P=0.000,P=0.000,P=0.000,P=0.000);阿米洛利组背根神经节中ASIC3阳性面积和ASIC3蛋白表达量均大于中药组(P=0.001;P=0.000)。结论:强筋壮骨祛风合剂可以降低髓核致炎大鼠病变的背根神经节中ASIC3的水平,提高大鼠的疼痛阈值,但其作用效果不及盐酸阿米洛利。
Objective: To observe the effect of Qiangjin Zhuanggu Qufeng Heji( 强筋壮骨祛风合剂,QJZGQFHJ) on acid- sensing ion channel 3( ASIC3) of dorsal root ganglia( DRG) in rats with inflammation caused by nucleus pulposus. Methods: Forty clean adult male SD rats were selected and randomly divided into blank group,sham- operated group,model group,Amiloride group and TCD group,8 cases in each group. The surgeries were performed on rats in sham- operated group,model group,Amiloride group and TCD group to expose L5 DRG. The surgical incisions were sutured in sham- operated group right after the L5 DRG were exposed. The nucleus pulposuses in tails were fetched out and were placed on the denudate L5 DRG in model group and TCD group,and then the surgical incisions were sutured. The nucleus pulposuses in tails were fetched out and were placed on the denudate L5 DRG in Amiloride group,and Amiloride solution( 1 ml),containing hydrochloric acid( 0. 1 mg),was dropped arround the denudate L5 DRG,then the surgical incisions were sutured. Since the 1st day after modeling,the rats in TCD group were intragastric administrated with QJZGQFHJ( 5ml / kg),twice a day for 30 consecutive days. No drug interventions were performed on rats in blank group,sham- operated group and model group. The 50% paw withdrawal threshold( 50% PWT) of left posterior feet were measured by using Up- Down methods at 1 day before modeling and at 3,5,9,15,23 and 29 days after modeing respectively. After the end of drug interventions,all rats in TCD group were executed and the L5 DRGs were fetched out and were divide into 2 parts. One of them was used for calculating the ASIC3 positive areas by using immunohistochemical method,and the other potion was used for detecting the ASIC3 protein expression by using Western Blot assay. Results: Left posterior foot eversion and toe dorsal extension were found in some rats of model group,Amiloride group and TCD group within 3 days after modeling. There was statistical difference in 50% PWT between different timepoints before and after modeling,in other words,there was time effect( F = 50. 132,P = 0. 000).There was statistical difference in 50% PWT between the 5 groups in general,in other words,there was group effect( F = 288. 219,P =0. 000). Except at 1 day before modeling( F = 0. 332,P = 0. 854),there was statistical difference in 50% PWT between the 5 groups( F =288. 015,P = 0. 000; F = 308. 588,P = 0. 000; F = 374. 945,P = 0. 000; F = 560. 713,P = 0. 000; F = 343. 043,P = 0. 000; F = 459. 914,P = 0. 000). At different timepoints after modeling,the 50% PWT were lower in model group compared to blank group,sham- operated group,Amiloride group and TCD group( P = 0. 010,P = 0. 011,P = 0. 001,P = 0. 001; P = 0. 023,P = 0. 022,P = 0. 000,P = 0. 004; P =0. 013,P = 0. 013,P = 0. 003,P = 0. 001; P = 0. 012,P = 0. 002,P = 0. 002,P = 0. 002; P = 0. 004,P = 0. 000,P = 0. 000,P = 0. 000; P =0. 003,P = 0. 005,P = 0. 003,P = 0. 013). There was no statistical difference in 50% PWT between blank group and sham- operated group( P = 0. 640,P = 0. 710,P = 0. 590,P = 0. 480,P = 0. 330,P = 0. 390) and the 50% PWT were higher in Amiloride group compared to TCD group( P = 0. 021,P = 0. 011,P = 0. 013,P = 0. 002,P = 0. 001,P = 0. 005) at different timepoints after modeling. There was interaction between time factor and group factor( F = 2. 358,P = 0. 002). There was statistical difference in ASIC3 positive areas and ASIC3 protein expression in DRG between the 5 groups( F = 205. 890,P = 0. 030; F = 101. 740,P = 0. 001). ASIC3 positive areas and ASIC3 protein expression were less in blank group,sham- operated group,Amiloride group and TCD group compared to model group( P = 0. 004,P =0. 003,P = 0. 001,P = 0. 007; P = 0. 000,P = 0. 000,P = 0. 000,P = 0. 000),and were greater in Amiloride group compared to TCD group( P = 0. 001; P = 0. 000). Conclusion: QJZGQFHJ can reduce the ASIC3 level in diseased DRG and increase the pain threshold in rats with inflammation caused by nucleus pulposus,while it is inferior to Amiloride in effectiveness.
Objective: To observe the effect of Qiangjin Zhuanggu Qufeng Heji( 强筋壮骨祛风合剂,QJZGQFHJ) on acid- sensing ion channel 3( ASIC3) of dorsal root ganglia( DRG) in rats with inflammation caused by nucleus pulposus. Methods: Forty clean adult male SD rats were selected and randomly divided into blank group,sham- operated group,model group,Amiloride group and TCD group,8 cases in each group. The surgeries were performed on rats in sham- operated group,model group,Amiloride group and TCD group to expose L5 DRG. The surgical incisions were sutured in sham- operated group right after the L5 DRG were exposed. The nucleus pulposuses in tails were fetched out and were placed on the denudate L5 DRG in model group and TCD group,and then the surgical incisions were sutured. The nucleus pulposuses in tails were fetched out and were placed on the denudate L5 DRG in Amiloride group,and Amiloride solution( 1 ml),containing hydrochloric acid( 0. 1 mg),was dropped arround the denudate L5 DRG,then the surgical incisions were sutured. Since the 1st day after modeling,the rats in TCD group were intragastric administrated with QJZGQFHJ( 5ml / kg),twice a day for 30 consecutive days. No drug interventions were performed on rats in blank group,sham- operated group and model group. The 50% paw withdrawal threshold( 50% PWT) of left posterior feet were measured by using Up- Down methods at 1 day before modeling and at 3,5,9,15,23 and 29 days after modeing respectively. After the end of drug interventions,all rats in TCD group were executed and the L5 DRGs were fetched out and were divide into 2 parts. One of them was used for calculating the ASIC3 positive areas by using immunohistochemical method,and the other potion was used for detecting the ASIC3 protein expression by using Western Blot assay. Results: Left posterior foot eversion and toe dorsal extension were found in some rats of model group,Amiloride group and TCD group within 3 days after modeling. There was statistical difference in 50% PWT between different timepoints before and after modeling,in other words,there was time effect( F = 50. 132,P = 0. 000).There was statistical difference in 50% PWT between the 5 groups in general,in other words,there was group effect( F = 288. 219,P =0. 000). Except at 1 day before modeling( F = 0. 332,P = 0. 854),there was statistical difference in 50% PWT between the 5 groups( F =288. 015,P = 0. 000; F = 308. 588,P = 0. 000; F = 374. 945,P = 0. 000; F = 560. 713,P = 0. 000; F = 343. 043,P = 0. 000; F = 459. 914,P = 0. 000). At different timepoints after modeling,the 50% PWT were lower in model group compared to blank group,sham- operated group,Amiloride group and TCD group( P = 0. 010,P = 0. 011,P = 0. 001,P = 0. 001; P = 0. 023,P = 0. 022,P = 0. 000,P = 0. 004; P =0. 013,P = 0. 013,P = 0. 003,P = 0. 001; P = 0. 012,P = 0. 002,P = 0. 002,P = 0. 002; P = 0. 004,P = 0. 000,P = 0. 000,P = 0. 000; P =0. 003,P = 0. 005,P = 0. 003,P = 0. 013). There was no statistical difference in 50% PWT between blank group and sham- operated group( P = 0. 640,P = 0. 710,P = 0. 590,P = 0. 480,P = 0. 330,P = 0. 390) and the 50% PWT were higher in Amiloride group compared to TCD group( P = 0. 021,P = 0. 011,P = 0. 013,P = 0. 002,P = 0. 001,P = 0. 005) at different timepoints after modeling. There was interaction between time factor and group factor( F = 2. 358,P = 0. 002). There was statistical difference in ASIC3 positive areas and ASIC3 protein expression in DRG between the 5 groups( F = 205. 890,P = 0. 030; F = 101. 740,P = 0. 001). ASIC3 positive areas and ASIC3 protein expression were less in blank group,sham- operated group,Amiloride group and TCD group compared to model group( P = 0. 004,P =0. 003,P = 0. 001,P = 0. 007; P = 0. 000,P = 0. 000,P = 0. 000,P = 0. 000),and were greater in Amiloride group compared to TCD group( P = 0. 001; P = 0. 000). Conclusion: QJZGQFHJ can reduce the ASIC3 level in diseased DRG and increase the pain threshold in rats with inflammation caused by nucleus pulposus,while it is inferior to Amiloride in effectiveness.
引文
[1]舒剑臣,唐小穗,赵京元.腰椎间盘退变引起腰腿痛的相关化学因素研究进展[J].中华临床医师杂志:电子版,2012,6(6):1534-1537.
[2]HUMZAH MD,SOAMES RW.Human intervertebral disc:structure and function[J].Anat Rec,1988,220(4):337-356.
[3]SLUKA KA,RASMUSSEN LA,EDGAR MM,et al.AcidSensing ion Channel 3 deficiency increases inflammation but decreases pain behavior in murine arthritis[J].Arthritis Rheum,2013,65(5):1194-1202.
[4]WEMMIE JA,TAUGHER RJ,KREPLE CJ.Acid-sensing ion channels in pain and disease[J].Nat Rev Neurosci,2013,14(7):461-471.
[5]LI WG,YU Y,ZHANG ZD,et al.ASIC3 channels integrate agmatine and multiple inflammatory signals through the nonproton ligand sensing domain[J].Mol Pain,2010,6:88.
[6]贺秋兰,魏明.酸敏感离子通道ASIC3与炎性痛觉敏感关系[J].实用医学杂志,2010,26(15):2843-2845.
[7]CHAPLAN SR,BACH FW,POGREL JW,et al.Quantitative assessment of tactile allodynia in the rat paw[J].J Neurosci Methods,1994,53(1):55-63.
[8]FREBURGER JK,HOLMES GM,AGANS RP,et al.The rising prevalence of chronic low back pain[J].Arch Intern Med,2009,169(3):251-258.
[9]OHTORI S,INOUE G,KOSHI T,et al.Up-regulation of acid-sensing ion Channel 3 in dorsal root ganglion neurons following application of nucleus pulposus on nerve root in rats[J].Spine(Phila Pa 1976),2006,31(18):2048-2052.
[10]杨宇,何睿,张宇宁,等.酸敏感离子通道与炎性痛中枢敏化机制的研究[J].中国现代药物应用,2014,09(9):228-228.
[11]郭婕,张前德.补肾方骨青颗粒对佐剂性关节炎大鼠的干预和影响软骨表达酸敏感离子通道3的实验研究[J].南京医科大学学报(自然科学版),2012,32(6):773-778.
[12]杨廷智,罗兵,杨明庭.加味独活寄生汤配合手法治疗腰椎间盘突出症100例[J].陕西中医,2012,33(12):1606-1607.
[13]李国强.身痛逐瘀汤加味合电针、牵引治疗腰椎间盘突出症93例临床观察[J].四川中医,2009,26(12):117-118.
[14]王久瑞.补肾化瘀法治疗腰椎间盘突出症临床观察[J].中医学报,2013,28(8):1239-1240.
[2]HUMZAH MD,SOAMES RW.Human intervertebral disc:structure and function[J].Anat Rec,1988,220(4):337-356.
[3]SLUKA KA,RASMUSSEN LA,EDGAR MM,et al.AcidSensing ion Channel 3 deficiency increases inflammation but decreases pain behavior in murine arthritis[J].Arthritis Rheum,2013,65(5):1194-1202.
[4]WEMMIE JA,TAUGHER RJ,KREPLE CJ.Acid-sensing ion channels in pain and disease[J].Nat Rev Neurosci,2013,14(7):461-471.
[5]LI WG,YU Y,ZHANG ZD,et al.ASIC3 channels integrate agmatine and multiple inflammatory signals through the nonproton ligand sensing domain[J].Mol Pain,2010,6:88.
[6]贺秋兰,魏明.酸敏感离子通道ASIC3与炎性痛觉敏感关系[J].实用医学杂志,2010,26(15):2843-2845.
[7]CHAPLAN SR,BACH FW,POGREL JW,et al.Quantitative assessment of tactile allodynia in the rat paw[J].J Neurosci Methods,1994,53(1):55-63.
[8]FREBURGER JK,HOLMES GM,AGANS RP,et al.The rising prevalence of chronic low back pain[J].Arch Intern Med,2009,169(3):251-258.
[9]OHTORI S,INOUE G,KOSHI T,et al.Up-regulation of acid-sensing ion Channel 3 in dorsal root ganglion neurons following application of nucleus pulposus on nerve root in rats[J].Spine(Phila Pa 1976),2006,31(18):2048-2052.
[10]杨宇,何睿,张宇宁,等.酸敏感离子通道与炎性痛中枢敏化机制的研究[J].中国现代药物应用,2014,09(9):228-228.
[11]郭婕,张前德.补肾方骨青颗粒对佐剂性关节炎大鼠的干预和影响软骨表达酸敏感离子通道3的实验研究[J].南京医科大学学报(自然科学版),2012,32(6):773-778.
[12]杨廷智,罗兵,杨明庭.加味独活寄生汤配合手法治疗腰椎间盘突出症100例[J].陕西中医,2012,33(12):1606-1607.
[13]李国强.身痛逐瘀汤加味合电针、牵引治疗腰椎间盘突出症93例临床观察[J].四川中医,2009,26(12):117-118.
[14]王久瑞.补肾化瘀法治疗腰椎间盘突出症临床观察[J].中医学报,2013,28(8):1239-1240.