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[1]李記天,馬言,羅漫麗.淫羊藿苷干預(yù)大鼠椎間盤源性腰痛的實(shí)驗(yàn)研究[J].中醫(yī)正骨,2020,32(03):7-13.
　LI Jitian,MA Yan,LUO Manli.An experimental study of icariin for intervention of discogenic low back pain in rats[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2020,32(03):7-13.
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淫羊藿苷干預(yù)大鼠椎間盤源性腰痛的實(shí)驗(yàn)研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第32卷
期數(shù):
2020年03期
頁碼:
7-13
欄目:
基礎(chǔ)研究
出版日期:
2020-03-20

文章信息/Info

Title:
An experimental study of icariin for intervention of discogenic low back pain in rats
作者:
李記天; 馬言; 羅漫麗
(河南省洛陽正骨醫(yī)院/河南省骨科醫(yī)院,河南 鄭州 450016)
Author(s):
LI Jitian; MA Yan; LUO Manli
Luoyang Orthopedic-Traumatological Hospital,Zhengzhou 450016,Henan,China
關(guān)鍵詞:
腰痛;  椎間盤退行性變;  淫羊藿甙;  大鼠; Sprague-Dawley;  動(dòng)物實(shí)驗(yàn)
Keywords:
low back pain;  intervertebral disc degeneration;  icariin;  rats; Sprague-Dawley;  animal experimentation
摘要:
目的:探討淫羊藿苷(icariin,ICA)干預(yù)大鼠椎間盤源性腰痛的效果及可能的作用機(jī)制。方法:將45只8周齡SPF級(jí)雄性SD大鼠隨機(jī)分為5組,假手術(shù)組、生理鹽水組、ICA 50 mg·kg-1組、ICA 100 mg·kg-1組各10只,塞來昔布組5只。生理鹽水組、ICA 50 mg·kg-1組、ICA 100 mg·kg-1及塞來昔布組大鼠通過L4～5和L5～6椎間盤穿刺建立大鼠腰痛模型; 假手術(shù)組僅顯露椎間盤,不進(jìn)行穿刺。自造模后第7天開始進(jìn)行藥物干預(yù),至造模后第21天結(jié)束。ICA 50 mg·kg-1組和ICA 100 mg·kg-1組以ICA溶液進(jìn)行灌胃(ICA溶于去離子水中),每天劑量分別為50 mg·kg-1和100 mg·kg-1; 塞來昔布組以塞來昔布膠囊進(jìn)行灌胃(塞來昔布膠囊粉劑溶于去離子水中),每天100 mg·kg-1; 生理鹽水組以生理鹽水灌胃,每天100 mg·kg-1; 假手術(shù)組常規(guī)飼養(yǎng),不進(jìn)行干預(yù)。從各組隨機(jī)選取5只大鼠,分別于造模前、造模后第7天、第14天、第21天、第28天、第35天進(jìn)行足底機(jī)械性疼痛閾值檢測。造模后第35天,足底疼痛閾值測定結(jié)束后處死假手術(shù)組、生理鹽水組、ICA 50 mg·kg-1組和ICA 100 mg·kg-1組的5只大鼠,以qRT-PCR法測定椎間盤組織中P物質(zhì)(substance P,SP)mRNA和降鈣素基因相關(guān)肽(calcitonin gene related peptide,CGRP)mRNA的含量。造模后第21天時(shí),處死假手術(shù)組、生理鹽水組、ICA 50 mg·kg-1組和ICA 100 mg·kg-1組其余的5只大鼠,以ELISA法測定大鼠椎間盤組織中細(xì)胞因子誘導(dǎo)的中性粒細(xì)胞趨化劑-1(cytokine-induced neutrophil chemoattractant-1,CINC-1)含量。結(jié)果:①足底機(jī)械性疼痛閾值。時(shí)間因素和分組因素存在交互效應(yīng)(F=17.971,P=0.001)。5組大鼠的足底機(jī)械性疼痛閾值總體比較,差異有統(tǒng)計(jì)學(xué)意義,即存在分組效應(yīng)(F=146.660,P=0.000)。造模前后不同時(shí)點(diǎn)間足底機(jī)械性疼痛閾值的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=118.057,P=0.000)。假手術(shù)組足底機(jī)械性疼痛閾值隨時(shí)間未見明顯變化,一直處于較高水平[(14.60±0.89)g,(14.79±0.47)g,(15.00±0.00)g,(15.00±0.00)g,(15.00±0.00)g,(15.00±0.00)g,F=0.836,P=0.537]。生理鹽水組足底機(jī)械性疼痛閾值造模后明顯降低,后期一直維持在較低水平[(14.67±0.74)g,(3.44±2.22)g,(3.19±1.37)g,(2.84±0.96)g,(3.92±1.36)g,(3.02±0.98)g,F=58.882,P=0.000]。ICA 50 mg·kg-1組、ICA 100 mg·kg-1組和塞來昔布組造模前后的足底機(jī)械性疼痛閾值變化趨勢基本一致,造模后均先降低,藥物干預(yù)開始后逐漸回升,藥物干預(yù)結(jié)束后又逐漸降低[(15.00±0.00)g,(2.78±0.81)g,(4.64±1.67)g,(5.59±1.25)g,(8.52±1.63)g,(6.11±1.49)g,F=56.088,P=0.000;(14.66±0.76)g,(2.53±1.37)g,(10.65±2.69)g,(9.67±2.41)g,(10.67±2.04)g,(8.59±2.95)g,F=16.684,P=0.000;(15.00±0.00)g,(2.28±1.03)g,(12.09±1.28)g,(12.37±1.34)g,(6.71±2.89)g,(5.18±1.88)g,F=44.668,P=0.000]。造模后第7天時(shí),ICA 50 mg·kg-1組、ICA 100 mg·kg-1組和塞來昔布組的足底機(jī)械性疼痛閾值兩兩比較,組間差異均無統(tǒng)計(jì)學(xué)意義。造模后第14天時(shí),ICA 100 mg·kg-1組和塞來昔布組的足底機(jī)械性疼痛閾值均高于ICA 50 mg·kg-1組(P=0.001; P=0.000); ICA 100 mg·kg-1組和塞來昔布組的足底機(jī)械性疼痛閾值比較,差異無統(tǒng)計(jì)學(xué)意義。造模后第21天時(shí),ICA 100 mg·kg-1組和塞來昔布組的足底機(jī)械性疼痛閾值均高于ICA 50 mg·kg-1組(P=0.009; P=0.000); ICA 100 mg·kg-1組和塞來昔布組的足底機(jī)械性疼痛閾值比較,差異無統(tǒng)計(jì)學(xué)意義。造模后第28天時(shí),ICA 100 mg·kg-1組的足底機(jī)械性疼痛閾值高于塞來昔布組(P=0.049); ICA 50 mg·kg-1組與ICA 100 mg·kg-1組、塞來昔布組比較,組間差異均無統(tǒng)計(jì)學(xué)意義。造模后第35天時(shí),ICA 50 mg·kg-1組、ICA 100 mg·kg-1組和塞來昔布組的足底機(jī)械性疼痛閾值兩兩比較,組間差異均無統(tǒng)計(jì)學(xué)意義。②椎間盤組織CINC-1含量。造模后第21天,假手術(shù)組、生理鹽水組、ICA 50 mg·kg-1組及ICA 100 mg·kg-1組大鼠椎間盤組織中CINC-1含量比較,差異有統(tǒng)計(jì)學(xué)意義[(534.2±142.6)pg·mL-1,(28 376.0±976.7)pg·mL-1,(21 866.0±1 536.0)pg·mL-1,(9 956.0±1 010.0)pg·mL-1,F=423.000,P=0.000]。生理鹽水組的CINC-1含量高于假手術(shù)組、ICA 50 mg·kg-1組和ICA 100 mg·kg-1組(P=0.000; P=0.003; P=0.000); ICA 50 mg·kg-1組的CINC-1含量高于ICA 100 mg·kg-1組(P=0.000)。③椎間盤組織SP mRNA和CGRP mRNA含量。造模后第35天,假手術(shù)組、生理鹽水組、ICA 50 mg·kg-1組及ICA 100 mg·kg-1組大鼠椎間盤組織中SP mRNA和CGRP mRNA含量比較,組間差異均有統(tǒng)計(jì)學(xué)意義(1.04±0.49,183.50±118.60,55.50±10.26,19.53±8.05,F=9.499,P=0.000; 0.74±0.21,6.29±2.06,1.55±0.69,1.19±0.37,F=27.180,P=0.000)。生理鹽水組的SP mRNA含量高于假手術(shù)組、ICA 50 mg·kg-1組和ICA 100 mg·kg-1組(P=0.008; P=0.042; P=0.015); ICA 50 mg·kg-1組的SP mRNA含量高于ICA 100 mg·kg-1組(P=0.000)。生理鹽水組的CGRP mRNA含量高于假手術(shù)組、ICA 50 mg·kg-1組和ICA 100 mg·kg-1組(P=0.000; P=0.001; P=0.000); ICA 50 mg·kg-1組和ICA 100 mg·kg-1組的CGRP mRNA含量比較,差異無統(tǒng)計(jì)學(xué)意義。結(jié)論:ICA可有效緩解大鼠椎間盤源性腰痛,其鎮(zhèn)痛效果與劑量有關(guān),與同劑量的塞來昔布相比其鎮(zhèn)痛效果持續(xù)時(shí)間更長,降低大鼠椎間盤組織中CINC-1的水平可能是其鎮(zhèn)痛的作用機(jī)制之一。
Abstract:
Objective:To explore the effects of icariin(ICA)on discogenic low back pain(DLBP)in rats and its possible mechanism of action.Methods:Forty-five 8-week-old SPF-grade male SD rats were selected and were randomly divided into sham-operated group(10),normal saline group(10),ICA 50 mg/kg group(10),ICA 100 mg/kg group(10)and celecoxib group(5).The lumbago models were built in rats of normal saline group,ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group by puncturing L4/L5 and L5/L6 intervertebral disc,while the surgeries were performed on rats in sham-operated group to expose their intervertebral discs and no intervertebral disc puncture was performed.The drug intervention were performed on rats from day 7 to day 21 after modeling.The rats in ICA 50 mg/kg group were intragastric administrated with ICA solution(ICA was dissolved into deionized water)in daily dosages of 50 mg/kg.The rats in ICA 100 mg/kg group,celecoxib group and normal saline group were intragastric administrated with ICA solution,celecoxib solution(celecoxib capsule powders were dissolved into deionized water)and normal saline respectively in daily dosage of 100 mg/kg,while the rats in sham-operated group were bred normally and were not given any drug interventions.Five rats were randomly selected out from each group,and plantar mechanical pain threshold values were measured before modeling and at the 7th,14th,21st,28th and 35th day after the modeling respectively.At the 35th day after the modeling,the five rats in sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group were executed after the last measurement of pelma pain threshold value and the contents of substance P(SP)mRNA and calcitonin gene related peptide(CGRP)mRNA in intervertebral disc tissues were measured by using qRT-PCR assays.At the 21st day after the modeling,the other 5 rats in sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group were executed,and the content of cytokine-induced neutrophil chemoattractant-1(CINC-1)in intervertebral disc tissues of rats were measured by using ELISA.Results:There was interaction between time factor and group factor in plantar mechanical pain threshold values(F=17.971,P=0.001).There was statistical difference in plantar mechanical pain threshold values between the 5 groups in general,in other words,there was group effect(F=146.660,P=0.000).There was statistical difference in plantar mechanical pain threshold values between different timepoints before and after the modeling,in other words,there was time effect(F=118.057,P=0.000).No significant time-dependent change of plantar mechanical pain threshold values were found in sham-operated group,and the pain threshold values remained at a high level(14.60+/-0.89,14.79+/-0.47,15.00+/-0.00,15.00+/-0.00,15.00+/-0.00,15.00+/-0.00 g,F=0.836,P=0.537).The plantar mechanical pain threshold values decreased significantly in normal saline group after modeling,and then remained at a low level(14.67+/-0.74,3.44+/-2.22,3.19+/-1.37,2.84+/-0.96,3.92+/-1.36,3.02+/-0.98 g,F=58.882,P=0.000).The plantar mechanical pain threshold values were basically consistent with each other in the variation tendency in ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group before and after the modeling,and they all decreased firstly after the modeling and increased gradually after the beginning of drug intervention and then decreased gradually after the end of drug intervention(15.00+/-0.00,2.78+/-0.81,4.64+/-1.67,5.59+/-1.25,8.52+/-1.63,6.11+/-1.49 g,F=56.088,P=0.000; 14.66+/-0.76,2.53+/-1.37,10.65+/-2.69,9.67+/-2.41,10.67+/-2.04,8.59+/-2.95 g,F=16.684,P=0.000; 15.00+/-0.00,2.28+/-1.03,12.09+/-1.28,12.37+/-1.34,6.71+/-2.89,5.18+/-1.88 g,F=44.668,P=0.000).At the 7th day after the modeling,further pairwise comparison showed that there was no statistical difference in the plantar mechanical pain threshold values between ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group.At the 14th day after the modeling,the plantar mechanical pain threshold values were higher in ICA 100 mg/kg group and celecoxib group compared to ICA 50 mg/kg group(P=0.001; P=0.000),and there was no statistical difference between ICA 100 mg/kg group and celecoxib group.At the 21st day after the modeling,the plantar mechanical pain threshold values were higher in ICA 100 mg/kg group and celecoxib group compared to ICA 50 mg/kg group(P=0.009; P=0.000),and there was no statistical difference between ICA 100 mg/kg group and celecoxib group.At the 28th day after the modeling,the plantar mechanical pain threshold values were higher in ICA 100 mg/kg group compared to celecoxib group(P=0.049),and there was no statistical difference between ICA 50 mg/kg group and ICA 100 mg/kg group and between ICA 50 mg/kg group and celecoxib group.At the 35th day after the modeling,further pairwise comparison showed that there was no statistical difference in the plantar mechanical pain threshold values between ICA 50 mg/kg group,ICA 100 mg/kg group and celecoxib group.At the 21st day after the modeling,there was statistical difference in the content of CINC-1 in intervertebral disc tissues of rats between sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group(534.2+/-142.6,28 376.0+/-976.7,21 866.0+/-1 536.0,9 956.0+/-1 010.0 pg/mL,F=423.000,P=0.000).The content of CINC-1 was higher in normal saline group compared to sham-operated group,ICA 50 mg/kg group and ICA 100 mg/kg group(P=0.000; P=0.003; P=0.000),and was higher in ICA 50 mg/kg group compared to ICA 100 mg/kg group(P=0.000).At the 35th day after the modeling,there was statistical difference in the contents of SP mRNA and CGRP mRNA in intervertebral disc tissues of rats between sham-operated group,normal saline group,ICA 50 mg/kg group and ICA 100 mg/kg group(1.04+/-0.49,183.50+/-118.60,55.50+/-10.26,19.53+/-8.05,F=9.499,P=0.000; 0.74+/-0.21,6.29+/-2.06,1.55+/-0.69,1.19+/-0.37,F=27.180,P=0.000).The content of SP mRNA in intervertebral disc tissues was higher in normal saline group compared to sham-operated group,ICA 50 mg/kg group and ICA 100 mg/kg group(P=0.008; P=0.042; P=0.015),and was higher in ICA 50 mg/kg group compared to ICA 100 mg/kg group(P=0.000).The content of CGRP mRNA in intervertebral disc tissues was higher in normal saline group compared to sham-operated group,ICA 50 mg/kg group and ICA 100 mg/kg group(P=0.000; P=0.001; P=0.000),and there was no statistical difference between ICA 50 mg/kg group and ICA 100 mg/kg group.Conclusion:ICA can effectively relieve DLBP in rats,and its analgesic effect is associated with the dose and lasted longer than that of same dose of celecoxib.It can decrease the level of CINC-1 in intervertebral disc tissues,which may be one of its analgesic mechanisms.

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備注/Memo

備注/Memo:
(收稿日期:2020-02-06 本文編輯:李曉樂) 基金項(xiàng)目:河南省中醫(yī)藥科學(xué)研究專項(xiàng)課題(2019ZYZD02) 通訊作者:李記天 E-mail:[email protected]
更新日期/Last Update: 2020-03-10