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[1]張博宇,駱鵬任,秦曉寬,等.益腎養(yǎng)髓方對大鼠脊髓缺血再灌注損傷的影響及機(jī)制研究[J].中醫(yī)正骨,2025,37(04):1-10.
　ZHANG Boyu,LUO Pengren,QIN Xiaokuan,et al.Effects and mechanism of Yishen Yangsui Fang(益腎養(yǎng)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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益腎養(yǎng)髓方對大鼠脊髓缺血再灌注損傷的影響及機(jī)制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期數(shù):
2025年04期
頁碼:
1-10
欄目:
基礎(chǔ)研究
出版日期:
2025-04-20

文章信息/Info

Title:
Effects and mechanism of Yishen Yangsui Fang(益腎養(yǎng)髓方)on spinal cord ischemia-reperfusion injury in rats:an experimental study
作者:
張博宇1; 駱鵬任1; 秦曉寬1; 張典2; 許博1; 馬國梁1; 楊博文1; 銀河1; 陳忻1; 金哲峰1; 朱立國1
1.中國中醫(yī)科學(xué)院望京醫(yī)院,北京 100102; 2.北京市平谷區(qū)中醫(yī)醫(yī)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
關(guān)鍵詞:
脊髓型頸椎病;  再灌注損傷;  益腎養(yǎng)髓方;  氧化性應(yīng)激;  大鼠;  Sprague-Dawley;  動物實(shí)驗
Keywords:
cervical spondylotic myelopathy;  reperfusion injury;  Yishen Yangsui Fang;  oxidative stress;  rats; Sprague-Dawley;  animal experimentation
摘要:
目的:觀察益腎養(yǎng)髓方對大鼠脊髓缺血再灌注損傷(spinal cord ischemia-reperfusion injury,SCII)的影響,并分析其作用機(jī)制。方法:將56只SPF級雌性SD大鼠隨機(jī)分為7組,每組8只。壓迫組、生理鹽水組、甲強(qiáng)龍組及益腎養(yǎng)髓方各濃度組,通過在C6～7椎間隙植入吸水膨脹材料進(jìn)行脊髓壓迫造模,假手術(shù)組將壓迫材料植入30 s后取出。壓迫手術(shù)結(jié)束4周后,生理鹽水組、甲強(qiáng)龍組及益腎養(yǎng)髓方各濃度組取出壓迫材料進(jìn)行減壓,壓迫組在移除壓迫材料30 s后再次放入,假手術(shù)組僅咬除相應(yīng)位置相同大小的椎板。減壓手術(shù)前1周,益腎養(yǎng)髓方高、中、低濃度組分別按16.74 g·kg-1、8.37 g·kg-1和4.19 g·kg-1以益腎養(yǎng)髓方藥液灌胃(上述藥物劑量為生藥量),每天1次,連續(xù)2周; 假手術(shù)組、壓迫組和生理鹽水組均按0.01 mL·g-1灌服生理鹽水。甲強(qiáng)龍組在減壓手術(shù)前30 min和術(shù)后1周,分別進(jìn)行1次甲潑尼龍琥珀酸鈉尾靜脈注射,給藥量為30 mg·kg-1。減壓手術(shù)中,采用激光散斑血流成像系統(tǒng)檢測生理鹽水組大鼠的脊髓局部血流量; 壓迫后1周和干預(yù)結(jié)束后,采用纖維絲機(jī)械刺激法測定大鼠機(jī)械痛閾值,采用推拉力計測定大鼠前爪抓力; 干預(yù)結(jié)束后,采用ELISA技術(shù)測定大鼠血清超氧化物歧化酶(superoxide dismutase,SOD)抑制率及丙二醛(malondialdehyde,MDA)、白細(xì)胞介素-6(interleukin-6,IL-6)、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)含量,取大鼠C5～C7節(jié)段脊髓組織采用HE染色進(jìn)行病理學(xué)觀察,并采用免疫熒光技術(shù)檢測脊髓組織中8-羥基鳥嘌呤(8-oxoguanine,8-oxoG)陽性率。結(jié)果:①脊髓局部血流量測定結(jié)果。與減壓前相比,減壓后10 min生理鹽水組大鼠的脊髓局部血流量增大(t=6.562,P=0.005)。②機(jī)械痛閾值測定結(jié)果。壓迫后1周,壓迫組、生理鹽水組、益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的機(jī)械痛閾值均低于假手術(shù)組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。干預(yù)結(jié)束后,益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的機(jī)械痛閾值均高于壓迫組(P=0.019,P=0.032,P=0.011,P=0.011),生理鹽水組與壓迫組機(jī)械痛閾值的差異無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組與生理鹽水組機(jī)械痛閾值的組間差異均無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組機(jī)械痛閾值的組間差異均無統(tǒng)計學(xué)意義。③前爪抓力測定結(jié)果。壓迫后1周,壓迫組、生理鹽水組、益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的前爪抓力均低于假手術(shù)組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000)。干預(yù)結(jié)束后,生理鹽水組、益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的前爪抓力均高于壓迫組(P=0.000,P=0.004,P=0.000,P=0.000,P=0.000); 益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的前爪抓力均高于生理鹽水組(P=0.033,P=0.005,P=0.003,P=0.000); 益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組前爪抓力的組間差異均無統(tǒng)計學(xué)意義。④血清SOD抑制率測定結(jié)果。假手術(shù)組的血清SOD抑制率高于壓迫組(P=0.000); 假手術(shù)組、益腎養(yǎng)髓方中濃度組、益腎養(yǎng)髓方低濃度組、甲強(qiáng)龍組的血清SOD抑制率均高于生理鹽水組(P=0.000,P=0.000,P=0.000,P=0.008),壓迫組、益腎養(yǎng)髓方高濃度組與生理鹽水組血清SOD抑制率的組間差異均無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方各濃度組與甲強(qiáng)龍組血清SOD抑制率的組間差異均無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方高濃度的血清SOD抑制率高于中濃度組(P=0.048),益腎養(yǎng)髓方高、中濃度組與低濃度組血清SOD抑制率的組間差異均無統(tǒng)計學(xué)意義。⑤血清MDA含量。假手術(shù)組的血清MDA含量低于壓迫組(P=0.037); 假手術(shù)組和益腎養(yǎng)髓方中濃度組的血清MDA含量均低于生理鹽水組(P=0.000,P=0.011),壓迫組、益腎養(yǎng)髓方高濃度組、益腎養(yǎng)髓方低濃度組、甲強(qiáng)龍組與生理鹽水組血清MDA含量的組間差異均無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組血清MDA含量的組間差異均無統(tǒng)計學(xué)意義。⑥血清IL-6含量。假手術(shù)組與壓迫組血清IL-6含量的差異無統(tǒng)計學(xué)意義; 假手術(shù)組、壓迫組、益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的血清IL-6含量均低于生理鹽水組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 益腎養(yǎng)髓方高、低濃度組的血清IL-6含量均高于甲強(qiáng)龍組(P=0.029,P=0.033),益腎養(yǎng)髓方中濃度組與甲強(qiáng)龍組血清IL-6含量的差異無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方各濃度組血清IL-6含量的組間差異均無統(tǒng)計學(xué)意義。⑦血清TNF-α含量。假手術(shù)組的血清TNF-α含量低于壓迫組(P=0.001); 假手術(shù)組、壓迫組、益腎養(yǎng)髓方中濃度組、益腎養(yǎng)髓方低濃度組、甲強(qiáng)龍組的血清TNF-α含量均低于生理鹽水組(P=0.043,P=0.033,P=0.006,P=0.001,P=0.022),益腎養(yǎng)髓方高濃度組與生理鹽水組血清TNF-α含量的差異無統(tǒng)計學(xué)意義; 益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組血清TNF-α含量的組間差異均無統(tǒng)計學(xué)意義。⑧脊髓組織病理學(xué)觀察結(jié)果。假手術(shù)組脊髓組織中細(xì)胞形態(tài)基本無改變,未見神經(jīng)元胞體腫脹、細(xì)胞核固縮等病理表現(xiàn); 壓迫組脊髓組織中可見大量瘢痕組織及空泡樣改變,并可見部分神經(jīng)元損傷; 生理鹽水組脊髓組織中部分區(qū)域可見瘢痕組織及空泡樣改變,并可見部分神經(jīng)元形態(tài)改變; 益腎養(yǎng)髓方各濃度組空泡樣改變及瘢痕較壓迫組和生理鹽水組明顯減少,神經(jīng)元細(xì)胞核局限性固縮、散在或游離分布,細(xì)胞形態(tài)逐漸恢復(fù); 甲強(qiáng)龍組脊髓組織中可見神經(jīng)元胞體膨大,細(xì)胞核固縮。⑨脊髓組織中8-oxoG陽性率檢測結(jié)果。假手術(shù)組與壓迫組8-oxoG陽性率的差異無統(tǒng)計學(xué)意義; 假手術(shù)組、壓迫組、益腎養(yǎng)髓方各濃度組及甲強(qiáng)龍組的8-oxoG陽性率均低于生理鹽水組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 益腎養(yǎng)髓方各濃度組的8-oxoG陽性率均低于甲強(qiáng)龍組(P=0.000,P=0.000,P=0.000); 益腎養(yǎng)髓方各濃度組8-oxoG陽性率的組間差異均無統(tǒng)計學(xué)意義。結(jié)論:益腎養(yǎng)髓方對大鼠SCII具有治療作用,其機(jī)制可能與其能夠減少神經(jīng)元氧化損傷有關(guān)。
Abstract:
Objective:To observe the effects of Yishen Yangsui Fang(益腎養(yǎng)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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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金項目(82205151); 中國中醫(yī)科學(xué)院望京醫(yī)院基礎(chǔ)研究苗圃培育計劃課題(WJYY-YJKT-2022-13); 中國中醫(yī)科學(xué)院基本科研業(yè)務(wù)費(fèi)優(yōu)秀青年科技人才培養(yǎng)專項課題(ZZ16-YQ-025)
通訊作者:金哲峰 E-mail:[email protected]
更新日期/Last Update: 1900-01-01