[1]张博宇,骆鹏任,秦晓宽,等.益肾养髓方对大鼠脊髓缺血再灌注损伤的影响及机制研究[J].中医正骨,2025,37(04):1-10.
　ZHANG Boyu,LUO Pengren,QIN Xiaokuan,et al.Effects and mechanism of Yishen Yangsui Fang(益肾养髓方)on spinal cord ischemia-reperfusion injury in rats:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(04):1-10.
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益肾养髓方对大鼠脊髓缺血再灌注损伤的影响及机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年04期
页码:
1-10
栏目:
基础研究
出版日期:
2025-04-20

文章信息/Info

Title:
Effects and mechanism of Yishen Yangsui Fang(益肾养髓方)on spinal cord ischemia-reperfusion injury in rats:an experimental study
作者:
张博宇1; 骆鹏任1; 秦晓宽1; 张典2; 许博1; 马国梁1; 杨博文1; 银河1; 陈忻1; 金哲峰1; 朱立国1
1.中国中医科学院望京医院,北京 100102; 2.北京市平谷区中医医院,北京 101200
Author(s):
ZHANG Boyu1; LUO Pengren1; QIN Xiaokuan1; ZHANG Dian2; XU Bo1; MA Guoliang1; YANG Bowen1; YIN He1; CHEN Xin1; JIN Zhefeng1; ZHU Liguo1
1.Wangjing Hospital of CACMS,Beijing 100102,China 2.Pinggu District Hospital of Traditional Chinese Medicine,Beijing 101200,China
关键词:
脊髓型颈椎病;  再灌注损伤;  益肾养髓方;  氧化性应激;  大鼠;  Sprague-Dawley;  动物实验
Keywords:
cervical spondylotic myelopathy;  reperfusion injury;  Yishen Yangsui Fang;  oxidative stress;  rats; Sprague-Dawley;  animal experimentation
摘要:
目的:观察益肾养髓方对大鼠脊髓缺血再灌注损伤(spinal cord ischemia-reperfusion injury,SCII)的影响,并分析其作用机制。方法:将56只SPF级雌性SD大鼠随机分为7组,每组8只。压迫组、生理盐水组、甲强龙组及益肾养髓方各浓度组,通过在C6～7椎间隙植入吸水膨胀材料进行脊髓压迫造模,假手术组将压迫材料植入30 s后取出。压迫手术结束4周后,生理盐水组、甲强龙组及益肾养髓方各浓度组取出压迫材料进行减压,压迫组在移除压迫材料30 s后再次放入,假手术组仅咬除相应位置相同大小的椎板。减压手术前1周,益肾养髓方高、中、低浓度组分别按16.74 g·kg-1、8.37 g·kg-1和4.19 g·kg-1以益肾养髓方药液灌胃(上述药物剂量为生药量),每天1次,连续2周; 假手术组、压迫组和生理盐水组均按0.01 mL·g-1灌服生理盐水。甲强龙组在减压手术前30 min和术后1周,分别进行1次甲泼尼龙琥珀酸钠尾静脉注射,给药量为30 mg·kg-1。减压手术中,采用激光散斑血流成像系统检测生理盐水组大鼠的脊髓局部血流量; 压迫后1周和干预结束后,采用纤维丝机械刺激法测定大鼠机械痛阈值,采用推拉力计测定大鼠前爪抓力; 干预结束后,采用ELISA技术测定大鼠血清超氧化物歧化酶(superoxide dismutase,SOD)抑制率及丙二醛(malondialdehyde,MDA)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量,取大鼠C5～C7节段脊髓组织采用HE染色进行病理学观察,并采用免疫荧光技术检测脊髓组织中8-羟基鸟嘌呤(8-oxoguanine,8-oxoG)阳性率。结果:①脊髓局部血流量测定结果。与减压前相比,减压后10 min生理盐水组大鼠的脊髓局部血流量增大(t=6.562,P=0.005)。②机械痛阈值测定结果。压迫后1周,压迫组、生理盐水组、益肾养髓方各浓度组及甲强龙组的机械痛阈值均低于假手术组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。干预结束后,益肾养髓方各浓度组及甲强龙组的机械痛阈值均高于压迫组(P=0.019,P=0.032,P=0.011,P=0.011),生理盐水组与压迫组机械痛阈值的差异无统计学意义; 益肾养髓方各浓度组及甲强龙组与生理盐水组机械痛阈值的组间差异均无统计学意义; 益肾养髓方各浓度组及甲强龙组机械痛阈值的组间差异均无统计学意义。③前爪抓力测定结果。压迫后1周,压迫组、生理盐水组、益肾养髓方各浓度组及甲强龙组的前爪抓力均低于假手术组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。干预结束后,生理盐水组、益肾养髓方各浓度组及甲强龙组的前爪抓力均高于压迫组(P=0.000,P=0.004,P=0.000,P=0.000,P=0.000); 益肾养髓方各浓度组及甲强龙组的前爪抓力均高于生理盐水组(P=0.033,P=0.005,P=0.003,P=0.000); 益肾养髓方各浓度组及甲强龙组前爪抓力的组间差异均无统计学意义。④血清SOD抑制率测定结果。假手术组的血清SOD抑制率高于压迫组(P=0.000); 假手术组、益肾养髓方中浓度组、益肾养髓方低浓度组、甲强龙组的血清SOD抑制率均高于生理盐水组(P=0.000,P=0.000,P=0.000,P=0.008),压迫组、益肾养髓方高浓度组与生理盐水组血清SOD抑制率的组间差异均无统计学意义; 益肾养髓方各浓度组与甲强龙组血清SOD抑制率的组间差异均无统计学意义; 益肾养髓方高浓度的血清SOD抑制率高于中浓度组(P=0.048),益肾养髓方高、中浓度组与低浓度组血清SOD抑制率的组间差异均无统计学意义。⑤血清MDA含量。假手术组的血清MDA含量低于压迫组(P=0.037); 假手术组和益肾养髓方中浓度组的血清MDA含量均低于生理盐水组(P=0.000,P=0.011),压迫组、益肾养髓方高浓度组、益肾养髓方低浓度组、甲强龙组与生理盐水组血清MDA含量的组间差异均无统计学意义; 益肾养髓方各浓度组及甲强龙组血清MDA含量的组间差异均无统计学意义。⑥血清IL-6含量。假手术组与压迫组血清IL-6含量的差异无统计学意义; 假手术组、压迫组、益肾养髓方各浓度组及甲强龙组的血清IL-6含量均低于生理盐水组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 益肾养髓方高、低浓度组的血清IL-6含量均高于甲强龙组(P=0.029,P=0.033),益肾养髓方中浓度组与甲强龙组血清IL-6含量的差异无统计学意义; 益肾养髓方各浓度组血清IL-6含量的组间差异均无统计学意义。⑦血清TNF-α含量。假手术组的血清TNF-α含量低于压迫组(P=0.001); 假手术组、压迫组、益肾养髓方中浓度组、益肾养髓方低浓度组、甲强龙组的血清TNF-α含量均低于生理盐水组(P=0.043,P=0.033,P=0.006,P=0.001,P=0.022),益肾养髓方高浓度组与生理盐水组血清TNF-α含量的差异无统计学意义; 益肾养髓方各浓度组及甲强龙组血清TNF-α含量的组间差异均无统计学意义。⑧脊髓组织病理学观察结果。假手术组脊髓组织中细胞形态基本无改变,未见神经元胞体肿胀、细胞核固缩等病理表现; 压迫组脊髓组织中可见大量瘢痕组织及空泡样改变,并可见部分神经元损伤; 生理盐水组脊髓组织中部分区域可见瘢痕组织及空泡样改变,并可见部分神经元形态改变; 益肾养髓方各浓度组空泡样改变及瘢痕较压迫组和生理盐水组明显减少,神经元细胞核局限性固缩、散在或游离分布,细胞形态逐渐恢复; 甲强龙组脊髓组织中可见神经元胞体膨大,细胞核固缩。⑨脊髓组织中8-oxoG阳性率检测结果。假手术组与压迫组8-oxoG阳性率的差异无统计学意义; 假手术组、压迫组、益肾养髓方各浓度组及甲强龙组的8-oxoG阳性率均低于生理盐水组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 益肾养髓方各浓度组的8-oxoG阳性率均低于甲强龙组(P=0.000,P=0.000,P=0.000); 益肾养髓方各浓度组8-oxoG阳性率的组间差异均无统计学意义。结论:益肾养髓方对大鼠SCII具有治疗作用,其机制可能与其能够减少神经元氧化损伤有关。
Abstract:
Objective:To observe the effects of Yishen Yangsui Fang(益肾养髓方,YSYSF)on spinal cord ischemia-reperfusion injury(SCII)in rats,and to analyze its underlying mechanism.Methods:Fifty-six SPF-grade female SD rats were randomized into sham-operated group,compression group,normal saline(NS)group,methylprednisolone(MP)group,YSYSF low-concentration group,YSYSF medium-concentration group,and YSYSF high-concentration group,with 8 cases in each group.All rats but the ones in sham-operated group were mo-deled by implanting water-absorbing swelling materials into the C6-7 intervertebral space to compress the spinal cord for inducing cervical spondylotic myelopathy(CSM),while the ones in sham-operated group was implanted with the same compression materials at the corresponding sites for 30 seconds and then removed.Four weeks after the end of the compression surgery,the rats in the NS group,MP group,and YSYSF low-,medium-,and high-concentration groups were further modeled by decompression via removal the implanted compression materials for inducing SCII,while,the ones in compression group underwent removal the compression materials for 30 seconds,followed by re-insertion,and the ones in sham-operated group were only bited lamina of the same size at the corresponding sites.One week before the decompression surgery,the rats in YSYSF high-,medium- and low-concentration groups were intervened by intragastric administration with YSYSF solution at the crude doses of 16.74,8.37,and 4.19 g/kg,respectively,once a day for consecutive 2 weeks; the ones in sham-ope-rated group,compression group and NS group by oral application of NS at a dose of 0.01 mL/g; while,the ones in MP group by injection of MP sodium succinate at a dose of 30 mg/kg via the tail vein at 30 minutes before the decompression surgery and at 1 week after the decompression surgery,respectively.During the decompression surgery,the local spinal cord blood flow(SCBF)in rats of NS group was detected using a laser speckle blood flow imaging system.One week after the compression and after the end of the intervention,the mechanical pain threshold(MPT)and forepaw grip strength were measured by the Von Frey filament test and a push-pull dynamometer,respectively.After the end of the intervention,the inhibition rate of superoxide dismutase(SOD)and the levels of malondialdehyde(MDA),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in serum of rats were measured by using ELISA.After that,the spinal cord tissues were harvested from C5–C7 segments,and were stained with HE for observing the pathological changes,and the positive rate of 8-oxoguanine(8-oxoG)in spinal cord tissues was detected by using immunofluorescence staining.Results:①The local SCBF.The local SCBF increased in rats of NS group at 10 minutes after decompression compared to pre-decompression(t=6.562,P=0.005).②The MPT.One week after the compression,the MPT was lower in compression group,NS group,MP group,and YSYSF low-,medium- and high-concentration groups compared to sham-operated group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).After the end of the intervention,the MPT was higher in YSYSF high-,medium- and low-concentration groups and MP group compared to compression group(P=0.019,P=0.032,P=0.011,P=0.011),with no significant difference between NS group and compression group.Furthermore,the MPT was not significantly different between YSYSF low-concentration group and NS group,between YSYSF medium-concentration group and NS group,between YSYSF high-concentration group and NS group,as well as between MP group and NS group,additionally,it was not significantly different among YSYSF low-concentration group,YSYSF medium-concentration group,YSYSF high-concentration group,and MP group.③The forepaw grip strength.One week after the compression,the forepaw grip strength was smaller in compression group,NS group,MP group,and YSYSF low-,medium- and high-concentration groups compared to sham-operated group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).After the end of the intervention,the forepaw grip strength was greater in NS group,MP group,and YSYSF low-,medium- and high-concentration groups compared to compression group(P=0.000,P=0.000,P=0.004,P=0.000,P=0.000),and was greater in MP group,and YSYSF low-,medium- and high-concentration groups compared to NS group(P=0.000,P=0.033,P=0.005,P=0.003),with no significant difference among YSYSF low-concentration group,YSYSF medium-concentration group,YSYSF high-concentration group,and MP group.④The serum SOD inhibition rate.The inhibition rate of SOD in serum was higher in sham-operated group compared to compression group(P=0.000),and was higher in sham-operated group,YSYSF medium-concentration group,YSYSF low-concentration group,and MP group compared to NS group(P=0.000,P=0.000,P=0.000,P=0.008),while,it was not significantly different between compression group and NS group,as well as between YSYSF high-concentration group and NS group.Furthermore,the inhibition rate of SOD was not significantly different between YSYSF low-concentration group and MP group,between YSYSF medium-concentration group and MP group,and between YSYSF high-concentration group and MP group,but it was higher in YSYSF high-concentration group compared to YSYSF medium-concentration group(P=0.048),with no significant difference between YSYSF high-concentration group and YSYSF low-concentration group,as well as between YSYSF medium-concentration group and YSYSF low-concentration group.⑤The serum MDA level.The serum level of MDA was lower in sham-operated group compared to compression group(P=0.037),and was lower in sham-operated group and YSYSF medium-concentration group compared to NS group(P=0.000,P=0.011),with no significant difference between compression group and NS group,between YSYSF high-concentration group and NS group,between YSYSF low-concentration group and NS group,as well as between MP group and NS group.Furthermore,it was not significantly different among YSYSF low-concentration group,YSYSF medium-concentration group,YSYSF high-concentration group,and MP group.⑥The serum IL-6 level.The serum level of IL-6 was not significantly different between sham-operated group and compression group,while,it was lower in sham-operated group,compression group,MP group and YSYSF low-,medium-,and high-concentration groups compared to NS group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),and was higher in YSYSF high- and low-concentration groups compared to MP group(P=0.029,P=0.033),with no significant difference between YSYSF medium-concentration group and MP group.Furthermore,it was not significantly different among YSYSF low-,medium-,and high-concentration groups.⑦The serum TNF-α level.The serum level of TNF-α was lower in sham-operated group compared to compression group(P=0.001),and was lower in sham-operated group,compression group,YSYSF me-dium-concentration group,YSYSF low-concentration group and MP group compared to NS group(P=0.043,P=0.033,P=0.006,P=0.001,P=0.022),with no significant difference between YSYSF high-concentration group and NS group.Furthermore,it was not significantly different among YSYSF low-concentration group,YSYSF medium-concentration group,YSYSF high-concentration group,and MP group.⑧The pathological changes in spinal cord tissues.In the sham-operated group,the cell morphology in the spinal cord tissue was essentially unchanged,with no pathological manifestations such as swollen neuronal cell bodies or nucleus pyknosis observed.The changes,manifesting as extensive scar tissues,vacuolar changes,and partially damaged neurons were observed in the spinal cord tissues in rats of compression group.While,in rats of NS group,the scar tissues and vacuolar changes were observed in partial areas of the spinal cord tissues,with neuronal morphological changes.Compared with that of compression group and NS group,a significant improvement was observed in vacuolar changes and scar tissue in rats of YSYSF low-,medium-,and high-concentration groups,with locally pyknotic,scatteredly or free distributed neuronal nuclei,and gradually recovered cell morphology.In the MP group,the enlarged neuronal cell bodies and pyknotic nuclei were observed in the spinal cord tissues.⑨The positive rate of 8-oxoG in spinal cord tissues.The positive rate of 8-oxoG in spinal cord tissues was not significantly different between sham-operated group and compression group,while,it was lower in sham-operated group,compression group,MP group and YSYSF low-,medium-,and high-concentration groups compared to NS group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000),and was lower in YSYSF low-,medium-,and high-concentration groups compared to MP group(P=0.000,P=0.000,P=0.000).Furthermore,it was not significantly different among YSYSF low-,medium-,and high-concentration groups.Conclusion:YSYSF exerts therapeutic effects on SCII in rats,it may work by mitigating the oxidative damage to neurons.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(82205151); 中国中医科学院望京医院基础研究苗圃培育计划课题(WJYY-YJKT-2022-13); 中国中医科学院基本科研业务费优秀青年科技人才培养专项课题(ZZ16-YQ-025)
通讯作者:金哲峰 E-mail:jzfchmjxt@163.com
更新日期/Last Update: 1900-01-01