雪莲注射液防治骨关节炎的作用及其机制探讨
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摘要
目的:鉴于目前中成药物防治膝OA,尤其是在“延缓关节软骨退变和促进透明软骨再生”的层面上,研究较少的情况,故而,本课题将结合现代医学试验的理论与方法,运用Hulth造模法制作兔OA动物模型,运用雪莲注射液对膝骨关节炎动物模型进行干预,通过对软骨和滑膜标本大体观察、对软骨细胞成分、基质的光镜和电镜下观测,以及从细胞因子、透明质酸、氧自由基代谢等多方面指标的改变,来探索中医药——雪莲注射液防治OA的作用机理,为临床使用提供理论支持。
方法:1.体内实验:动物随机编为4组,A组、B组、C组和D组,A、B、C组建立骨关节炎动物模型,D组正常喂养。A、B组分别注射雪莲注射液和生理盐水,C组不用药。按设计术后六周,C组、D组随机抽取实验动物两只,拍摄右侧后肢膝关节侧位X线片。术后6、12、24周各组动物采血检测血清一氧化氮(NO,Nitric oxide)含量后,处死动物,右膝关节注入无菌生理盐水1.0ml,混匀后抽取关节液,检测白介素-1β(IL-1β)和透明质酸水平,然后取关节软骨标本,常规石蜡切片,HE/SOFC双染色,Mankin评分比较各组关节软骨损伤程度。免疫组化法(IHC)检测各组关节软骨基质Ⅰ、Ⅱ型胶原的表达情况。透射电镜(TEM)观察各组关节软骨超微结构变化。测定12周滑膜组织中超氧化物歧化酶(SOD,Superoxide dismutase)活性及丙二醛(MDA,Malondialdehyde)含量。2.体外试验:分离培养软骨细胞,传代细胞分Ⅰ、Ⅱ组,Ⅰ组给予不同浓度雪莲注射液刺激,Ⅱa组予不同浓度雪莲注射液干预24小时再予脂多糖(LPS,Lipopolysaccharide)刺激,Ⅱb组先予脂多糖(LPS)刺激24小时再予不同浓度雪莲注射液干预,72小时后收集上清液检测IL-1β水平。
结果:1.X片观察:C组与D组相比,关节内侧间隙明显变窄,关节面粗糙变形,关节边缘有明显骨赘,软骨下骨骨密度明显增高,说明造模成功。2.光镜、电镜观察发现,A组动物在各个时间点的软骨结构、细胞排列和形态、软骨基质对Safranin-0异染性均优于B、C组。3.雪莲注射液能够降低骨关节炎模型动物关节液与体外培养软骨细胞分泌IL-1β的水平。4A组在各时点Ⅰ型胶原阳染面积低于B组和C组,Ⅱ型胶原阳染面积高于B组和C组。D组在各时点关节软骨仅在表层存在少量Ⅰ型胶原阳性染色表达。4.A组在各时段的HA水平与D组之间没有显著性差异(P＞0.05),A组与B、C组各时段的HA水平有显著(P＜0.05),B、C组和D组的HA水平有显著性差异(P＜0.05),B、C组间没有显著性差异。5.除D组以外,各组12周时血清中NO含量均高于6周时;24周时血清中NO含量均高于12周时。NO的表达均逐渐升高,说明NO是参与膝OA发病进程的重要介质,病变的程度越重则含量越高;A组在各时点血清中NO含量均低于B组和C组(P＜0.05),说明雪莲注射液能有效抑制骨性关节炎发病病理过程中的一氧化氮的产生;试验显示A组动物滑膜组织中SOD活性显著高于B、C组(P＜0.05),而MDA含量显著低于B、C组(P＜0.05),B组与C组无显著性差异(P＞0.05)。
结论:雪莲注射液能对软骨细胞蛋白多糖PG表型的表达具有维持和保护作用,使破坏的胶原纤维与弹力纤维得到修复,使软骨细胞的代谢恢复正常,进而维持其结构的完整性,廷缓关节软骨退变;有效抑制IL-1、NO的生物活性,增强SOD的生物活性,改善微循环,降低骨内压,从而达到保护关节软骨的目的;促进滑膜细胞产生透明质酸和调节关节液中微量元素,恢复关节滑液和关节组织基质的流变学特性,缓解滑膜炎症,减轻软骨破坏,改善关节功能;其主要成分黄酮总甙具有抗炎、镇痛、消肿的作用,直接对抗炎性介质,改善关节的内环境,改善微血管状态,降低毛细血管通透性,减少渗出,消除滑膜及周围组织炎症,降低细胞因子、金属蛋白酶水平,使组织代谢恢复正常,延缓关节软骨的退变过程。
目的:探讨雪莲注射液对膝骨关节炎模型动物软骨组织形态学的影响,试图从形态学角度阐明雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板,逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。术后六周,C组、D组随机抽取实验动物两只,拍摄患膝侧位X线片。术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。切取右膝负重关节面全层软骨,石蜡包埋切片,苏木紫-伊红(HE)染色、番红O/固绿(Safranin'O/FastGreen)组织化学染色,中性树脂封片,光镜观察,按Mankin评分标准评分。透射电镜观察各组关节软骨超微结构变化。
结果:(1)X片观察:C组与D组相比,关节内侧间隙明显变窄,关节面粗糙变形,关节边缘有明显骨赘,软骨下骨骨密度明显增高,说明造模成功。(2)光镜、电镜观察发现,A组动物在各个时间点的软骨结构、细胞排列和形态、软骨基质对Safranin-O异染性均优于B、C组。
结论:(1)从CR平片和光镜、电镜观察比较可以判断,兔膝骨性关节炎模型建立成功。(2)雪莲注射液能够促进软骨细胞合成代谢,促进软骨基质蛋白多糖(PG)的合成,对关节软骨具有保护作用。
目的:探讨雪莲注射液对膝骨关节炎模型动物关节液中白介素1水平的影响,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:(1)体内试验:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。分别于术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。右膝关节注入无菌生理盐水1.0ml,混匀后抽取关节液,检测IL-Iβ。
(2)体外试验:4周龄新西兰白兔4只,无菌条件下切取股骨髁和胫骨平台关节软骨,经漂洗、消化、过滤、离心、接种和培养等,分离培养出软骨细胞,传代细胞分Ⅰ、Ⅱ组,Ⅰ组给予不同浓度雪莲注射液刺激,Ⅱa组予不同浓度雪莲注射液干预24小时再予脂多糖(LPS,Lipopolysaccharide)刺激,Ⅱb组先予脂多糖(LPS)刺激24小时再予不同浓度雪莲注射液干预,72小时后收集上清液检测IL-1β水平。采用放射免疫分析法(RIA,Radio Immunity Analyse),结果以pg/ml表示。各组数据均采用x±s表示,结果由SPSS统计软件分析处理。
结果:雪莲注射液能够降低骨关节炎模型动物关节液与体外培养软骨细胞分泌IL-1β的水平。
结论:雪莲注射液可以通过抑制白介素-1β的水平对骨关节炎起防治作用。
目的:探讨雪莲注射液对膝骨关节炎模型动物软骨组织胶原表达的影响,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。切取右膝负重关节面全层软骨,石蜡包埋切片,脱蜡至水后,3%过氧化氢室温下10min,抗原修复液37℃2min,山羊血清封闭液10min。依次加入一抗(oncogene)37℃22h;二抗(生物素标记IgG)37℃20min;链霉素抗生物素蛋白—过氧化酶溶液37℃30min;DAB显色,甲基绿复染,中性树脂封片。Olympus BH—2光镜观察,LeicaQw 550图像分析软件定量分析,结果以阳性染色面积比例(PAR)表示,PAR=(阳性染色面积/总面积)×100%。
结果:A组在各时点Ⅰ型胶原阳染面积低于B组和C组,Ⅱ型胶原阳染面积高于B组和C组。D组在各时点关节软骨仅在表层存在少量Ⅰ型胶原阳性染色表达。
结论:应用雪莲注射液早期干预在翻译水平能有效缓解基质胶原降解破坏的程度,延缓OA的发生发展。
目的:探讨雪莲注射液对膝骨关节炎模型动物关节液中透明质酸含量的影响,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。右膝关节抽取关节液,检测透明质酸含量。采用放射免疫分析法(RIA),结果以mg/ml表示。各组数据均采用x±s表示,结果由SPSS统计软件分析处理。
结果:雪莲注射液能够促进骨关节炎模型动物关节液中透明质酸含量的提高。
结论:雪莲注射液可以通过提高关节液中透明质酸的水平对骨关节炎起防治作用。
目的:探讨中药雪莲注射液对膝骨性关节炎模型动物氧自由基代谢的影响,,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。分别于术后6、12、24周每组随机取5只兔,采血检测血清一氧化氮(NO,Nitric oxide)含量(硝酸还原酶法),然后用过量麻醉法处死,打开右膝关节腔,取出滑膜,测定滑膜组织中超氧化物歧化酶(SOD,Superoxide dismutase)活性及丙二醛(MDA,Malondialdehyde)含量。各组数据均采用x±s表示,结果由SPSS统计软件分析处理。
结果:(1)除D组以外,各组12周时血清中NO含量均高于6周时;24周时血清中NO含量均高于12周时。NO的表达均逐渐升高,说明NO是参与膝OA发病进程的重要介质,病变的程度越重则含量越高。(2)A组在各时点血清中NO含量均低于B组和C组(P＜0.05),说明雪莲注射液能有效抑制骨性关节炎发病病理过程中的一氧化氮的产生。(3)试验显示A组动物滑膜组织中SOD活性显著高于B、C组(P＜0.05),而MDA含量显著低于B、C组(P＜0.05),B组与C组无显著性差异(P＞0.05)。
结论:雪莲注射液可有效抑制骨性关节炎发病病理过程中的一氧化氮的产生,显著提高SOD的活性,降低MDA含量,从而起到延缓关节软骨的退变,促进软骨修复的作用。
[Objective]OA(osteoarthritis)of knee joint is a kind of common disease.It effects seriously on work and live of people.It is thought that the pathogenesis of osteoarthritis of knee joint results from such factors as stress,cytokine(CK), apoptosis,auto-immune,heredity,inflammation,matrixmetallo proteinase(MMP)and tissue-inhibitor mentallo proteinase(TIMP).However,the pathogenesis of OA is still not dearly determined.At present,the authors with an attempt to further explore the mechanism of OA.
The pathology of OA includes mainly the histology and metabolic change of the chondrocyte and the biochemical and configuration change of extra cellular matrix (ECM)and secondary synovitis.The well-balanced chondrocyte can spontaneously excrete microcrystalline interlukin-1β,but the chondrocyte of knee joint osteoarthritis can excrete interlukin-1βexceptionally manifold,and consequently bring on apoptosis and decreasing level of collagenⅡand PG(proteoglycan).Finally extra cellular matrix(ECM)suffer degradation.
The mail remedy of knee joint osteoarthritis is to stave off the cataplasia of cartilage and to promote the regenesis of hyaline cartilage.As an important dispelling rheumatism medicine,XueLian injection has been widely used in curing RA (rheumatoid arthritis)and OA(osteoarthritis)and has gained well curative effect.
This topic tries to find out the mechanism and the preventive method of XueLian injection.We infused XueLian injection into osteoarthritis rabbit model by intramuscular injection to study the effect on histology,collagen phenotype,ultramicrostructure,interlukin-1βlevel,HA,NO and OFR in OA model.
[Method]OA model animals were randomly divided into 4 groups:group A,group B,group C and group D.Animals in group A received XueLian injection and animals in group B received sterilized normal saline intramuscularly,group C was control and received nothing and another group(group D)was used for normal control.After the animals were killed,cartilage specimen were obtained and sections were made and stained With HE/SOFG for Mankin score;change of ultramicrostructure of cartilage was observed with transmission electron microscope. After the animals were killed,the IL-1βlevel in joint fluid of A group was determined and compared with that of group B,C,D.in-vitro:cartilage of rabbit were obtained, isolated and cultured.The secondary passes cells were divided into groupⅠandⅡ, different concentrations of XueLian injection were added to groupⅠfor 72 hours; groupⅡa received different concentrations of XueLian injection for 24 hours and then received LPS for 72 hours,groupⅡb received LPS first,after 24 hours different concentrations of XueLian injection solution were added.72 hours later,the supernatant of each group were collected and the IL-1βlevel was determined by RIA. After the animals were killed,cartilage specimen were obtained and sections were tested by immunohistochemistry.The hyaluronic acid level in joint fluid of A group was determined and compared with that of group B,C,D.The nitric oxide level in osteoarthritis serum and superoxide dismutase and malondialdehyde level in osteoarthritis synovialis of A group was determined and compared with that of group B,C,D.
[Result]Mankin score in group A was better than that in group B and C at each test period;cartilage impairment degree in group A was lower than in group B and C,and impairment manifestation in group B and C was similar on the whole. XueLian injection can significantly decrease the interlukin-1βlevel in the joint fluid of OA model animal and the in-vitro cultured chondrocyte supernatant.Rresult of IHC shows that positive stain area of collagenⅠWas lower in group A than that in group B and C.and positive stain area of collagenⅡWas higher than that in group B and C at each test period;cartilage impairm ent degree in group A Was lower than in group B and C,and impairment manifestation in group D and K was similar on the whole.XueLian injection can significantly increase the hyaluronic acid level in the joint fluid of OA model animal.XueLian injection can significantly decrease the nitric oxide level in osteoarthritis serum of OA model animal.
[Conclusion]XueLian injection may protect articular cartilage by maintaining and promoting the chondrocyte synthesizing proteoglycan,by decreasing the IL-1βlevel in joint fluid and inhibiting the secretion of IL-1βfrom chondrocytes,by maintaining and promoting the typeⅡcollagen,by increasing the hyaluronic acid level in joint fluid,by decreasing the nitric oxide level in osteoarthritis serum of OA model animal.
The first section:Effect of XueLian injection on histomorphology in cartilage of osteoarthritis animal model
[Objective]The object of this research is to study the effect of XueLian injection on histology and ultramicrostructurc in cartilage of osteoarthritis animal model[Method]OA model animals were randomly divided into 4 groups:group A,group B,group C and group D.Animals in group A received XueLian injection and animals in group B received sterilized normal saline intramuscularly,group C was control and received nothing and another group(group D)was used for normal control.After the animals were killed,cartilage specimen were obtained and sections were made and stained with HE/SOFG for Mankin score;change of ultramicrostructure of cartilage was observed with transmission electron microscope.[Result]Mankin score in group A was better than that in group B and C at each test period;cartilage impairment degree in group A was lower than in group B and C,and impairment manifestation in group B and C was similar on the whole. [Conclusion]XueLian injection may protect articular cartilage by maintaining and promoting the chondrocyte synthesizing proteoglycan.
The second section:Effect of XueLiau injection on interlukin-1 level in osteoarthritis joint fluid and chondrocyte culture supernatant
[Objective]The object of this research is to study the effect of XueLian injection on interlukin-1βlevel in OA model and in-vitro culture chondrocyte supematant.[Methods]in-vivo:OA model animals were randomly divided into 4 groups:A XueLian injection group,B Saline group,C Control group. Group A B received XueLian injection im and sterilized normal saline im respectively, group C received nothing,and anothergroup(group D)was used for normal control. After the animals were killed,the IL-1βlevel in joint fluid of A group was determined and compared with that of group B,C,D.in-vitro:cartilage of rabbit were obtained, isolated and cultured.The secondary passes cells were divided into groupⅠandⅡ, different concentrations of XueLian injection were added to groupⅠfor 72 hours; groupⅡa received different concentrations of XueLian injection for 24 hours and then received LPS for 72 hours,groupⅡb received LPS first,after 24 hours different concentrations of XueLian injection solution were added.72 hours later,the supernatant of each group were collected and the IL-1βlevel was determined by RIA. [Results]XueLian injection can significantly decrease the interlukin-1βlevel in the joint fluid of OA model animal and the in-vitro cultured chondrocyte supernatant. [Conclusion]The therapeutic effect of XueLian injection on OA can be achieved by decreasing the IL-1βlevel in joint fluid and inhibiting the secretion of IL-1βfrom chondrocytes.
The third section:Effect of XueLian injection on histomorphology in cartilage of osteoarthritis animal model
[Objective]The object of this research is to study the efect of XueLian injection on collagen phenotype in cartilage of osteoarthritis animal model.[Method]OA model animals were randomly divided into the expression level of collagenⅠandⅡin mamx was 4 groups:group A,group B,group C and group D.Animals in group A received XueLian injection and animals in group B received sterilized normal saline intramuscularly,group C was control and received nothing and another group(group D)was used for normal control.After the animals were killed,cartilage specimen were obtained and sections were tested by immunohistochemistry.[Result]Rresult of IHC shows that positive stain area of collagenⅠWas lower in group A than that in group B and C.and positive stain area of collagenⅡWas higher than that in group B and C at each test period;cartilage impairment degree in group A Was lower than in group B and C,and impairment manifestation in group D and K was similar on the whole.[Conclusion]XueLian injection may protect articular cartilage by maintaining and promoting the typeⅡcollagen.
The fourth section:Effect of XueLian injection on Hyaluronic acid level in osteoarthritis joint fluid
[Objective]The object of this research is to study the effect of XueLian injection on hyaluronic acid level in OA model.[Methods]OA model animals were randomly divided into 4 groups:A XueLian injection group,B Saline group,C Control group.Group A B received XueLian injection im and sterilized normal saline im respectively,group C received nothing,and anothergroup(group D) was used for normal control.After the animals were killed,the hyaluronic acid level in joint fluid of A group was determined and compared with that of group B,C,D. [Results]XueLian injection can significantly increase the hyaluronic acid level in the joint fluid of OA model animal.[Conclusion]The therapeutic effect of XueLian injection on OA can be achieved by increasing the hyaluronic acid level in joint fluid.
The fifth section:Effect of XueLian injection on Nitric oxide level in osteoarthritis serum and Superoxide dismutase and Malondiaidehyde level in osteoarthritis synovialis
[Objective]The object of this research is to study the effect of XueLian injection on nitric oxide level in osteoarthritis serum and superoxide dismutase and malondialdehyde level in osteoarthritis synovialis[Methods]OA model animals were randomly divided into 4 groups:A XueLian injection group,B Saline group,C Control group.Group A B received XueLian injection im and sterilized normal saline im respectively,group C received nothing,and anothergroup(group D)was used for normal control.After the animals were killed, the nitric oxide level in osteoarthritis serum and superoxide dismutase and malondialdehyde level in osteoarthritis synovialis of A group was determined and compared with that of group B,C,D.[Results]XueLian injection can significantly decrease the nitric oxide level in osteoarthritis serum of OA model animal [Conclusion]The therapeutic effect of XueLian injection on OA can be achieved by decreasing the nitric oxide level in osteoarthritis serum of OA model animal
方法:1.体内实验:动物随机编为4组,A组、B组、C组和D组,A、B、C组建立骨关节炎动物模型,D组正常喂养。A、B组分别注射雪莲注射液和生理盐水,C组不用药。按设计术后六周,C组、D组随机抽取实验动物两只,拍摄右侧后肢膝关节侧位X线片。术后6、12、24周各组动物采血检测血清一氧化氮(NO,Nitric oxide)含量后,处死动物,右膝关节注入无菌生理盐水1.0ml,混匀后抽取关节液,检测白介素-1β(IL-1β)和透明质酸水平,然后取关节软骨标本,常规石蜡切片,HE/SOFC双染色,Mankin评分比较各组关节软骨损伤程度。免疫组化法(IHC)检测各组关节软骨基质Ⅰ、Ⅱ型胶原的表达情况。透射电镜(TEM)观察各组关节软骨超微结构变化。测定12周滑膜组织中超氧化物歧化酶(SOD,Superoxide dismutase)活性及丙二醛(MDA,Malondialdehyde)含量。2.体外试验:分离培养软骨细胞,传代细胞分Ⅰ、Ⅱ组,Ⅰ组给予不同浓度雪莲注射液刺激,Ⅱa组予不同浓度雪莲注射液干预24小时再予脂多糖(LPS,Lipopolysaccharide)刺激,Ⅱb组先予脂多糖(LPS)刺激24小时再予不同浓度雪莲注射液干预,72小时后收集上清液检测IL-1β水平。
结果:1.X片观察:C组与D组相比,关节内侧间隙明显变窄,关节面粗糙变形,关节边缘有明显骨赘,软骨下骨骨密度明显增高,说明造模成功。2.光镜、电镜观察发现,A组动物在各个时间点的软骨结构、细胞排列和形态、软骨基质对Safranin-0异染性均优于B、C组。3.雪莲注射液能够降低骨关节炎模型动物关节液与体外培养软骨细胞分泌IL-1β的水平。4A组在各时点Ⅰ型胶原阳染面积低于B组和C组,Ⅱ型胶原阳染面积高于B组和C组。D组在各时点关节软骨仅在表层存在少量Ⅰ型胶原阳性染色表达。4.A组在各时段的HA水平与D组之间没有显著性差异(P＞0.05),A组与B、C组各时段的HA水平有显著(P＜0.05),B、C组和D组的HA水平有显著性差异(P＜0.05),B、C组间没有显著性差异。5.除D组以外,各组12周时血清中NO含量均高于6周时;24周时血清中NO含量均高于12周时。NO的表达均逐渐升高,说明NO是参与膝OA发病进程的重要介质,病变的程度越重则含量越高;A组在各时点血清中NO含量均低于B组和C组(P＜0.05),说明雪莲注射液能有效抑制骨性关节炎发病病理过程中的一氧化氮的产生;试验显示A组动物滑膜组织中SOD活性显著高于B、C组(P＜0.05),而MDA含量显著低于B、C组(P＜0.05),B组与C组无显著性差异(P＞0.05)。
结论:雪莲注射液能对软骨细胞蛋白多糖PG表型的表达具有维持和保护作用,使破坏的胶原纤维与弹力纤维得到修复,使软骨细胞的代谢恢复正常,进而维持其结构的完整性,廷缓关节软骨退变;有效抑制IL-1、NO的生物活性,增强SOD的生物活性,改善微循环,降低骨内压,从而达到保护关节软骨的目的;促进滑膜细胞产生透明质酸和调节关节液中微量元素,恢复关节滑液和关节组织基质的流变学特性,缓解滑膜炎症,减轻软骨破坏,改善关节功能;其主要成分黄酮总甙具有抗炎、镇痛、消肿的作用,直接对抗炎性介质,改善关节的内环境,改善微血管状态,降低毛细血管通透性,减少渗出,消除滑膜及周围组织炎症,降低细胞因子、金属蛋白酶水平,使组织代谢恢复正常,延缓关节软骨的退变过程。
目的:探讨雪莲注射液对膝骨关节炎模型动物软骨组织形态学的影响,试图从形态学角度阐明雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板,逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。术后六周,C组、D组随机抽取实验动物两只,拍摄患膝侧位X线片。术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。切取右膝负重关节面全层软骨,石蜡包埋切片,苏木紫-伊红(HE)染色、番红O/固绿(Safranin'O/FastGreen)组织化学染色,中性树脂封片,光镜观察,按Mankin评分标准评分。透射电镜观察各组关节软骨超微结构变化。
结果:(1)X片观察:C组与D组相比,关节内侧间隙明显变窄,关节面粗糙变形,关节边缘有明显骨赘,软骨下骨骨密度明显增高,说明造模成功。(2)光镜、电镜观察发现,A组动物在各个时间点的软骨结构、细胞排列和形态、软骨基质对Safranin-O异染性均优于B、C组。
结论:(1)从CR平片和光镜、电镜观察比较可以判断,兔膝骨性关节炎模型建立成功。(2)雪莲注射液能够促进软骨细胞合成代谢,促进软骨基质蛋白多糖(PG)的合成,对关节软骨具有保护作用。
目的:探讨雪莲注射液对膝骨关节炎模型动物关节液中白介素1水平的影响,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:(1)体内试验:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。分别于术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。右膝关节注入无菌生理盐水1.0ml,混匀后抽取关节液,检测IL-Iβ。
(2)体外试验:4周龄新西兰白兔4只,无菌条件下切取股骨髁和胫骨平台关节软骨,经漂洗、消化、过滤、离心、接种和培养等,分离培养出软骨细胞,传代细胞分Ⅰ、Ⅱ组,Ⅰ组给予不同浓度雪莲注射液刺激,Ⅱa组予不同浓度雪莲注射液干预24小时再予脂多糖(LPS,Lipopolysaccharide)刺激,Ⅱb组先予脂多糖(LPS)刺激24小时再予不同浓度雪莲注射液干预,72小时后收集上清液检测IL-1β水平。采用放射免疫分析法(RIA,Radio Immunity Analyse),结果以pg/ml表示。各组数据均采用x±s表示,结果由SPSS统计软件分析处理。
结果:雪莲注射液能够降低骨关节炎模型动物关节液与体外培养软骨细胞分泌IL-1β的水平。
结论:雪莲注射液可以通过抑制白介素-1β的水平对骨关节炎起防治作用。
目的:探讨雪莲注射液对膝骨关节炎模型动物软骨组织胶原表达的影响,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。切取右膝负重关节面全层软骨,石蜡包埋切片,脱蜡至水后,3%过氧化氢室温下10min,抗原修复液37℃2min,山羊血清封闭液10min。依次加入一抗(oncogene)37℃22h;二抗(生物素标记IgG)37℃20min;链霉素抗生物素蛋白—过氧化酶溶液37℃30min;DAB显色,甲基绿复染,中性树脂封片。Olympus BH—2光镜观察,LeicaQw 550图像分析软件定量分析,结果以阳性染色面积比例(PAR)表示,PAR=(阳性染色面积/总面积)×100%。
结果:A组在各时点Ⅰ型胶原阳染面积低于B组和C组,Ⅱ型胶原阳染面积高于B组和C组。D组在各时点关节软骨仅在表层存在少量Ⅰ型胶原阳性染色表达。
结论:应用雪莲注射液早期干预在翻译水平能有效缓解基质胶原降解破坏的程度,延缓OA的发生发展。
目的:探讨雪莲注射液对膝骨关节炎模型动物关节液中透明质酸含量的影响,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。术后6、12、24周每组随机取5只兔,过量麻醉法处死,以A06、A12、A24代表A组6、12和24周;B组、C组、D组类推。右膝关节抽取关节液,检测透明质酸含量。采用放射免疫分析法(RIA),结果以mg/ml表示。各组数据均采用x±s表示,结果由SPSS统计软件分析处理。
结果:雪莲注射液能够促进骨关节炎模型动物关节液中透明质酸含量的提高。
结论:雪莲注射液可以通过提高关节液中透明质酸的水平对骨关节炎起防治作用。
目的:探讨中药雪莲注射液对膝骨性关节炎模型动物氧自由基代谢的影响,,从而探讨雪莲注射液治疗骨关节炎的作用机理。
方法:健康日本大耳兔60只,随机编为4组即雪莲注射液实验组(A组)、生理盐水对照组(B组)、空白对照组(C组)和正常组(D组),每组15只。参考Hulth造模方法,将A、B、C三组动物用3%戊巴比妥钠40mg/kg的剂量进行腹腔注射麻醉。仰卧绑于手术台上固定,取右侧后肢膝关节内侧纵切口长约4cm,探查关节腔无原发病变后仔细切断前后交叉韧带,切断内侧副韧带,完整切除内侧半月板(勿损伤关节软骨面),逐层缝合伤口,无菌敷料包扎,术侧肢体不固定,分笼饲养。术后第二天起A组动物每日肌肉注射雪莲注射液0.4ml,B组动物每日肌肉注射生理盐水0.4ml,连续4周。C组动物不用药。分别于术后6、12、24周每组随机取5只兔,采血检测血清一氧化氮(NO,Nitric oxide)含量(硝酸还原酶法),然后用过量麻醉法处死,打开右膝关节腔,取出滑膜,测定滑膜组织中超氧化物歧化酶(SOD,Superoxide dismutase)活性及丙二醛(MDA,Malondialdehyde)含量。各组数据均采用x±s表示,结果由SPSS统计软件分析处理。
结果:(1)除D组以外,各组12周时血清中NO含量均高于6周时;24周时血清中NO含量均高于12周时。NO的表达均逐渐升高,说明NO是参与膝OA发病进程的重要介质,病变的程度越重则含量越高。(2)A组在各时点血清中NO含量均低于B组和C组(P＜0.05),说明雪莲注射液能有效抑制骨性关节炎发病病理过程中的一氧化氮的产生。(3)试验显示A组动物滑膜组织中SOD活性显著高于B、C组(P＜0.05),而MDA含量显著低于B、C组(P＜0.05),B组与C组无显著性差异(P＞0.05)。
结论:雪莲注射液可有效抑制骨性关节炎发病病理过程中的一氧化氮的产生,显著提高SOD的活性,降低MDA含量,从而起到延缓关节软骨的退变,促进软骨修复的作用。
[Objective]OA(osteoarthritis)of knee joint is a kind of common disease.It effects seriously on work and live of people.It is thought that the pathogenesis of osteoarthritis of knee joint results from such factors as stress,cytokine(CK), apoptosis,auto-immune,heredity,inflammation,matrixmetallo proteinase(MMP)and tissue-inhibitor mentallo proteinase(TIMP).However,the pathogenesis of OA is still not dearly determined.At present,the authors with an attempt to further explore the mechanism of OA.
The pathology of OA includes mainly the histology and metabolic change of the chondrocyte and the biochemical and configuration change of extra cellular matrix (ECM)and secondary synovitis.The well-balanced chondrocyte can spontaneously excrete microcrystalline interlukin-1β,but the chondrocyte of knee joint osteoarthritis can excrete interlukin-1βexceptionally manifold,and consequently bring on apoptosis and decreasing level of collagenⅡand PG(proteoglycan).Finally extra cellular matrix(ECM)suffer degradation.
The mail remedy of knee joint osteoarthritis is to stave off the cataplasia of cartilage and to promote the regenesis of hyaline cartilage.As an important dispelling rheumatism medicine,XueLian injection has been widely used in curing RA (rheumatoid arthritis)and OA(osteoarthritis)and has gained well curative effect.
This topic tries to find out the mechanism and the preventive method of XueLian injection.We infused XueLian injection into osteoarthritis rabbit model by intramuscular injection to study the effect on histology,collagen phenotype,ultramicrostructure,interlukin-1βlevel,HA,NO and OFR in OA model.
[Method]OA model animals were randomly divided into 4 groups:group A,group B,group C and group D.Animals in group A received XueLian injection and animals in group B received sterilized normal saline intramuscularly,group C was control and received nothing and another group(group D)was used for normal control.After the animals were killed,cartilage specimen were obtained and sections were made and stained With HE/SOFG for Mankin score;change of ultramicrostructure of cartilage was observed with transmission electron microscope. After the animals were killed,the IL-1βlevel in joint fluid of A group was determined and compared with that of group B,C,D.in-vitro:cartilage of rabbit were obtained, isolated and cultured.The secondary passes cells were divided into groupⅠandⅡ, different concentrations of XueLian injection were added to groupⅠfor 72 hours; groupⅡa received different concentrations of XueLian injection for 24 hours and then received LPS for 72 hours,groupⅡb received LPS first,after 24 hours different concentrations of XueLian injection solution were added.72 hours later,the supernatant of each group were collected and the IL-1βlevel was determined by RIA. After the animals were killed,cartilage specimen were obtained and sections were tested by immunohistochemistry.The hyaluronic acid level in joint fluid of A group was determined and compared with that of group B,C,D.The nitric oxide level in osteoarthritis serum and superoxide dismutase and malondialdehyde level in osteoarthritis synovialis of A group was determined and compared with that of group B,C,D.
[Result]Mankin score in group A was better than that in group B and C at each test period;cartilage impairment degree in group A was lower than in group B and C,and impairment manifestation in group B and C was similar on the whole. XueLian injection can significantly decrease the interlukin-1βlevel in the joint fluid of OA model animal and the in-vitro cultured chondrocyte supernatant.Rresult of IHC shows that positive stain area of collagenⅠWas lower in group A than that in group B and C.and positive stain area of collagenⅡWas higher than that in group B and C at each test period;cartilage impairm ent degree in group A Was lower than in group B and C,and impairment manifestation in group D and K was similar on the whole.XueLian injection can significantly increase the hyaluronic acid level in the joint fluid of OA model animal.XueLian injection can significantly decrease the nitric oxide level in osteoarthritis serum of OA model animal.
[Conclusion]XueLian injection may protect articular cartilage by maintaining and promoting the chondrocyte synthesizing proteoglycan,by decreasing the IL-1βlevel in joint fluid and inhibiting the secretion of IL-1βfrom chondrocytes,by maintaining and promoting the typeⅡcollagen,by increasing the hyaluronic acid level in joint fluid,by decreasing the nitric oxide level in osteoarthritis serum of OA model animal.
The first section:Effect of XueLian injection on histomorphology in cartilage of osteoarthritis animal model
[Objective]The object of this research is to study the effect of XueLian injection on histology and ultramicrostructurc in cartilage of osteoarthritis animal model[Method]OA model animals were randomly divided into 4 groups:group A,group B,group C and group D.Animals in group A received XueLian injection and animals in group B received sterilized normal saline intramuscularly,group C was control and received nothing and another group(group D)was used for normal control.After the animals were killed,cartilage specimen were obtained and sections were made and stained with HE/SOFG for Mankin score;change of ultramicrostructure of cartilage was observed with transmission electron microscope.[Result]Mankin score in group A was better than that in group B and C at each test period;cartilage impairment degree in group A was lower than in group B and C,and impairment manifestation in group B and C was similar on the whole. [Conclusion]XueLian injection may protect articular cartilage by maintaining and promoting the chondrocyte synthesizing proteoglycan.
The second section:Effect of XueLiau injection on interlukin-1 level in osteoarthritis joint fluid and chondrocyte culture supernatant
[Objective]The object of this research is to study the effect of XueLian injection on interlukin-1βlevel in OA model and in-vitro culture chondrocyte supematant.[Methods]in-vivo:OA model animals were randomly divided into 4 groups:A XueLian injection group,B Saline group,C Control group. Group A B received XueLian injection im and sterilized normal saline im respectively, group C received nothing,and anothergroup(group D)was used for normal control. After the animals were killed,the IL-1βlevel in joint fluid of A group was determined and compared with that of group B,C,D.in-vitro:cartilage of rabbit were obtained, isolated and cultured.The secondary passes cells were divided into groupⅠandⅡ, different concentrations of XueLian injection were added to groupⅠfor 72 hours; groupⅡa received different concentrations of XueLian injection for 24 hours and then received LPS for 72 hours,groupⅡb received LPS first,after 24 hours different concentrations of XueLian injection solution were added.72 hours later,the supernatant of each group were collected and the IL-1βlevel was determined by RIA. [Results]XueLian injection can significantly decrease the interlukin-1βlevel in the joint fluid of OA model animal and the in-vitro cultured chondrocyte supernatant. [Conclusion]The therapeutic effect of XueLian injection on OA can be achieved by decreasing the IL-1βlevel in joint fluid and inhibiting the secretion of IL-1βfrom chondrocytes.
The third section:Effect of XueLian injection on histomorphology in cartilage of osteoarthritis animal model
[Objective]The object of this research is to study the efect of XueLian injection on collagen phenotype in cartilage of osteoarthritis animal model.[Method]OA model animals were randomly divided into the expression level of collagenⅠandⅡin mamx was 4 groups:group A,group B,group C and group D.Animals in group A received XueLian injection and animals in group B received sterilized normal saline intramuscularly,group C was control and received nothing and another group(group D)was used for normal control.After the animals were killed,cartilage specimen were obtained and sections were tested by immunohistochemistry.[Result]Rresult of IHC shows that positive stain area of collagenⅠWas lower in group A than that in group B and C.and positive stain area of collagenⅡWas higher than that in group B and C at each test period;cartilage impairment degree in group A Was lower than in group B and C,and impairment manifestation in group D and K was similar on the whole.[Conclusion]XueLian injection may protect articular cartilage by maintaining and promoting the typeⅡcollagen.
The fourth section:Effect of XueLian injection on Hyaluronic acid level in osteoarthritis joint fluid
[Objective]The object of this research is to study the effect of XueLian injection on hyaluronic acid level in OA model.[Methods]OA model animals were randomly divided into 4 groups:A XueLian injection group,B Saline group,C Control group.Group A B received XueLian injection im and sterilized normal saline im respectively,group C received nothing,and anothergroup(group D) was used for normal control.After the animals were killed,the hyaluronic acid level in joint fluid of A group was determined and compared with that of group B,C,D. [Results]XueLian injection can significantly increase the hyaluronic acid level in the joint fluid of OA model animal.[Conclusion]The therapeutic effect of XueLian injection on OA can be achieved by increasing the hyaluronic acid level in joint fluid.
The fifth section:Effect of XueLian injection on Nitric oxide level in osteoarthritis serum and Superoxide dismutase and Malondiaidehyde level in osteoarthritis synovialis
[Objective]The object of this research is to study the effect of XueLian injection on nitric oxide level in osteoarthritis serum and superoxide dismutase and malondialdehyde level in osteoarthritis synovialis[Methods]OA model animals were randomly divided into 4 groups:A XueLian injection group,B Saline group,C Control group.Group A B received XueLian injection im and sterilized normal saline im respectively,group C received nothing,and anothergroup(group D)was used for normal control.After the animals were killed, the nitric oxide level in osteoarthritis serum and superoxide dismutase and malondialdehyde level in osteoarthritis synovialis of A group was determined and compared with that of group B,C,D.[Results]XueLian injection can significantly decrease the nitric oxide level in osteoarthritis serum of OA model animal [Conclusion]The therapeutic effect of XueLian injection on OA can be achieved by decreasing the nitric oxide level in osteoarthritis serum of OA model animal
引文
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