[1]杜江,黄远鹏,李奕修,等.龟鹿二仙胶及其拆方对去势骨关节炎大鼠血清雌二醇浓度及膝关节软骨细胞Ⅱ型胶原表达的影响[J].中医正骨,2013,25(03):11-20.
　DU Jiang*,HUANG Yuan-peng,LI Yi-xiu,et al.Effect of GUILU ERXIAN gelatin and its formula components on the concentrations of serum estradiol and expressions of Collagen-Ⅱ in the chondrocytes of knee joint for ovariectomized rats with osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(03):11-20.
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龟鹿二仙胶及其拆方对去势骨关节炎大鼠血清雌二醇浓度 及膝关节软骨细胞Ⅱ型胶原表达的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期数:
2013年03期
页码:
11-20
栏目:
基础研究
出版日期:
2013-03-20

文章信息/Info

Title:
Effect of GUILU ERXIAN gelatin and its formula components on the concentrations of serum estradiol and expressions of Collagen-Ⅱ in the chondrocytes of knee joint for ovariectomized rats with osteoarthritis
作者:
杜江; 黄远鹏; 李奕修; 余添赐; 魏迎辰; 王和鸣; 李楠
福建中医药大学,福建 福州 350122
Author(s):
DU Jiang*; HUANG Yuan-peng; LI Yi-xiu; YU Tian-ci; WEI Ying-chen; WANG He-ming; LI Nan.
*Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China
关键词:
骨关节炎 龟鹿二仙胶 拆方研究 雌二醇 胶原Ⅱ型 大鼠
Keywords:
Osteoarthritis;  GUILU ERXIAN gelatin;  Formula components analysis;  Estradiol;  Collagen type Ⅱ;  Rats
摘要:
目的:观察龟鹿二仙胶及其拆方对去势骨关节炎大鼠血清雌二醇浓度及膝关节软骨细胞Ⅱ型胶原表达的影响。方法:采用抽签法将168只4月龄雌性SD清洁级大鼠随机分为2组,手术造模组141只,假手术组27只。切除手术造模组大鼠双侧的卵巢和双侧膝关节的内侧副韧带和前交叉韧带,制造肝肾亏虚型骨关节炎大鼠模型; 切除假手术组大鼠双侧卵巢附近的脂肪组织。每天驱赶实验大鼠1 h,连续驱赶4周。4周后分别从手术造模组与假手术组各取5只大鼠,采用HE染色和甲苯胺蓝染色法观察子宫与膝关节软骨的组织形态学变化,采用甲苯胺蓝染色法观察膝关节软骨基质中糖胺聚糖的表达情况,采用放射免疫法测定血清雌二醇浓度,鉴定大鼠模型。造模成功后将假手术组作为空白对照组(空白组),并将其分为干预2、4、8周的3小组,每组7只,留1只备用; 采用抽签法将手术造模组随机分为6组,即模型对照组(模型组)、龟鹿二仙胶组(全方组)、龟甲鹿角组(龟鹿组)、龟甲鹿角人参组(龟鹿参组)、龟甲鹿角枸杞组(龟鹿杞组)、盐酸氨基葡萄糖对照组(盐酸氨基葡萄糖组),再分别将每组分为干预2、4、8周的3小组,每组7只,留10只备用。参照人与大鼠药物等效剂量换算公式,将龟甲胶、鹿角胶、人参、枸杞、盐酸氨基葡萄糖分别配成浓度为81 mg·mL-1、54 mg·mL-1、10.8 mg·mL-1、27 mg·mL-1、13.5 mg·mL-1的药液。各组大鼠均以10 mL·kg-1体质量以各组药液灌胃,空白组与模型组以生理盐水灌胃; 全方组以配成的龟鹿二仙胶混合液灌胃; 龟鹿组以配成的龟甲胶和鹿角胶混合液灌胃; 龟鹿参组以配成的龟甲胶、鹿角胶与人参混合液灌胃; 龟鹿杞组以配成的龟甲胶、鹿角胶、枸杞子混合液灌胃; 盐酸氨基葡萄糖组以配成的盐酸氨基葡萄糖溶液灌胃。每天灌胃1次,各组分别连续灌胃2、4、8周。药物干预结束后,从腹主动脉抽血后处死大鼠,取出的血经静置、离心后吸取上层血清,保存待检; 取出大鼠右侧中下1/3段股骨,制备石蜡标本。采用放射免疫法检测大鼠血清雌二醇浓度,采用免疫组化染色法观察关节软骨中Ⅱ型胶原的表达。结果:①形态观察。手术造模组与假手术组大鼠的子宫与膝关节软骨的组织形态学变化显示手术造模组造模成功。②药物干预前血清雌二醇浓度和膝关节软骨基质中糖胺聚糖含量。药物干预前,手术造模组的血清雌二醇浓度明显低于假手术组[(87.130±39.720)ng·L-1,(195.080±42.449)ng·L-1; t=4.152,P=0.003]; 手术造模组的软骨基质中糖胺聚糖含量低于假手术组[(0.239±0.066),(0.597±0.053); t=9.457,P=0.000]。显示手术造模组造模成功。③药物干预后血清雌二醇浓度。用药2周后,各组间血清雌二醇浓度比较,差异有统计学意义[(204.082±50.995)ng·L-1,(80.191±29.921)ng·L-1,(129.583±48.763)ng·L-1,(121.223±51.514)ng·L-1,(117.582±50.131)ng·L-1,(128.181±45.987)ng·L-1,(114.479±39.346)ng·L-1; F=4.520,P=0.001]。组间两两比较,空白组高于模型组、全方组、龟鹿组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.000,P=0.002,P=0.001,P=0.001,P=0.003,P=0.001); 模型组低于全方组(P=0.039); 其余各组间血清E2浓度比较,差异均无统计学意义(P>0.05)。用药4周后,各组间血清雌二醇浓度比较,差异有统计学意义[(204.819±53.802)ng·L-1,(91.613±32.654)ng·L-1,(148.727±51.860)ng·L-1,(146.576±32.976)ng·L-1,(145.225±49.217)ng·L-1,(146.365±45.800)ng·L-1,(132.197±46.032)ng·L-1; F=4.278,P=0.002]。组间两两比较,空白组高于模型组、全方组、龟鹿组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.000,P=0.022,P=0.013,P=0.015,P=0.013,P=0.002); 模型组低于全方组、龟鹿组、龟鹿参组、龟鹿杞组(P=0.027,P=0.027,P=0.037,P=0.027); 其余各组间血清E2浓度比较,差异均无统计学意义(P>0.05)。用药8周后,各组间血清雌二醇浓度比较,差异有统计学意义[(192.060±57.040)ng·L-1,(97.504±40.006)ng·L-1,(183.140±50.887)ng·L-1,(130.757±39.285)ng·L-1,(167.763±32.878)ng·L-1,(158.112±48.309)ng·L-1,(112.494±46.687)ng·L-1; F=3.296,P=0.009]。组间两两比较,空白组高于模型组、龟鹿组、盐酸氨基葡萄糖组(P=0.000,P=0.011,P=0.001); 模型组低于全方组、龟鹿参组、龟鹿杞组(P=0.002,P=0.005,P=0.012); 全方组高于盐酸氨基葡萄糖组(P=0.010); 其余各组间比较,差异均无统计学意义(P>0.05)。④药物干预后膝关节软骨基质中Ⅱ型胶原的表达量。用药2周后,各组间膝关节软骨基质中Ⅱ型胶原的表达量比较,差异有统计学意义[(0.598±0.096),(0.226±0.113),(0.310±0.070),(0.310±0.072),(0.311±0.075),(0.296±0.081),(0.389±0.068); F=33.533,P=0.000)]。组间两两比较,空白组高于模型组、全方组、龟鹿组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 模型组低于全方组、龟鹿组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.007,P=0.006,P=0.006,P=0.021,P=0.000); 全方组低于盐酸氨基葡萄糖组(P=0.008)。其余各组间比较,差异均无统计学意义(P>0.05)。用药4周后,各组间膝关节软骨基质中Ⅱ型胶原的表达量比较,差异有统计学意义[(0.603±0.083),(0.223±0.113),(0.387±0.090),(0.270±0.083),(0.371±0.059),(0.300±0.081),(0.442±0.067); F=37.064,P=0.000]。组间两两比较,空白组高于模型组、全方组、龟鹿组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 模型组低于全方组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.000,P=0.000,P=0.011,P=0.000); 全方组高于龟鹿组、龟鹿杞组(P=0.000,P=0.003)。其余各组间Collagen-Ⅱ表达量比较,差异均无统计学意义(P>0.05)。用药8周后,各组间软骨基质中Ⅱ型胶原的表达量比较,差异有统计学意义[(0.608±0.067),(0.217±0.106),(0.437±0.065),(0.263±0.110),(0.380±0.073),(0.273±0.081),(0.443±0.068); F=42.071,P=0.000]。组间两两比较,空白组高于模型组、全方组、龟鹿组、龟鹿参组、龟鹿杞组、盐酸氨基葡萄糖组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 模型组低于全方组、龟鹿参组、盐酸氨基葡萄糖组(P=0.000,P=0.000,P=0.000); 全方组高于龟鹿组、龟鹿参组、龟鹿杞组(P=0.000,P=0.049,P=0.000); 其余各组间Collagen-Ⅱ表达量比较,差异均无统计学意义(P>0.05)。结论:在提高血清雌二醇浓度方面,龟甲胶与鹿角胶配伍人参或枸杞均是有效配伍,且用药2周时配伍人参和枸杞的效果要优于单独配伍人参或单独配伍枸杞。但随着用药时间的延长,龟甲胶与鹿角胶配伍人参提高雌二醇浓度的效果好还是配伍枸杞的效果好,还是龟鹿二仙胶全方效果好,仍需进一步研究证实。在促进膝关节软骨基质中Ⅱ型胶原表达方面,人参和枸杞在龟鹿二仙胶方中发挥了不可缺少的作用。
Abstract:
Objective:To observe the effect of GUILU ERXIAN gelatin and its formula components on the concentrations of serum estradiol(E2)and expressions of Collagen-Ⅱ in the chondrocytes of knee joint for ovariectomized rats with osteoarthritis(OA).Methods:One hundred and sixty-eight clean female SD rats of 4 months old were randomly divided into 2 groups,141 cases in operation modeling group,while the others in sham operation group.Rats in operation modeling group were administrated with ovariectomy and removal of medial collateral ligament(MCL)and anterior cruciate ligament(ACL)of bilateral knee joints to build OA models of LIVER-KIDNEY DEFICIENCY type,while the cases in sham operation group were administrated with fat removal around the ovary.The experimental rats were forced to run 1 hour every day for 4 weeks,and then 5 rats were selected from operation modeling group and sham operation group respectively.The histological and morphological changes of uteruses and knee articular cartilages were observed though HE staining and toluidine blue staining,the expressions of glycosaminoglycan(GAG)in knee articular cartilages matrix were observed though toluidine blue staining,and the concentrations of serum E2 were detected through radioimmunoassay to identify the rat models.After successful modeling,sham operation group was referred as blank control group(blank group),then it was further divided into 3 groups according to intervention times of 2,4 and 8 weeks,7 rats in each group,and 1 spared rat for resurance.The operation modeling group was randomly divided into 6 groups,including model control group(model group),GUILU ERXIAN gelatin group(complete formula group),tortoiseshell-deerhorn group(TD group),tortoiseshell-deerhorn-ginseng group(TDG group),tortoiseshell-deerhorn-wolfberry group(TDW group)and glucosamine hydrochloride group(GHC group),and then they were respectively divided into 3 groups according to intervention times of 2,4 and 8 weeks,7 rats in each group,and 10 spared rat for resurance.According to the conversion equation of drug equivalent dose for human and rats,the components as tortoiseshell glue,deerhorn glue,ginseng,wolfberry and GHC were respectively prepared into solution with concentrations of 81 mg·mL-1,54 mg·mL-1,10.8 mg·mL-1,27 mg·mL-1 and 13.5 mg·mL-1.Rats in blank group and in model group were intragastric administrated with normal saline(10 mL·kg-1); rats in complete formula group were intragastric administrated with mixed liquor(10 mL·kg-1)of GUILU ERXIAN gelatin; rats in TD group were intragastric administrated with mixed liquor(10 mL·kg-1)of tortoiseshell glue and deerhorn glue; rats in TDG group were intragastric administrated with mixed liquor(10 mL·kg-1)of tortoiseshell glue,deerhorn glue and ginseng; rats in TDW group were intragastric administrated with mixed liquor(10 mL·kg-1)of tortoiseshell glue,deerhorn glue and wolfberry; while rats in GHC group were intragastric administrated with glucosamine hydrochloride solution(10 mL·kg-1).Rats in each group were intragastric administrated once per day for 2,4 and 8 weeks respectively.After medicine intervention,the rats were executed by drawing blood from abdominal aorta.After standing and centrifuging,the upper serum was sucked from the blood preparation for further inspection.After that,the middle and bottom thirds of right femur of rats were fetched out for paraffin specimen preparation.Concentrations of serum E2 of rats were detected through radioimmunoassay,and expressions of Collagen-Ⅱ in the articular cartilage were detected through immunohistochemical staining.Results:Successful modeling of operation modeling group was shown from the histological and morphological changes of uteruses and knee articular cartilages of rats in operation modeling group and sham operation group respectively.Concentrations of serum E2 of operation modeling group were significantly lower than those of sham operation group[(87.130±39.720)ng·L-1,(195.080±42.449)ng·L-1; t=4.152,P=0.003]before medicine intervention; also the GAG content in knee articular cartilages matrix of operation modeling group was lower than that of sham operation group[(0.239±0.066),(0.597±0.053); t=9.457,P=0.000].Successful modeling of operation modeling group was shown from above results.There was statistical difference in concentrations of serum E2 among all the groups after 2 weeks medication[(204.082±50.995)ng·L-1,(80.191±29.921)ng·L-1,(129.583±48.763)ng·L-1,(121.223±51.514)ng·L-1,(117.582±50.131)ng·L-1,(128.181±45.987)ng·L-1,(114.479±39.346)ng·L-1; F=4.520,P=0.001].Further pairwise comparison showed that the serum E2 concentrations of blank group were higher than those of other groups respectively(P=0.000,P=0.002,P=0.001,P=0.001,P=0.003,P=0.001),and the serum E2 concentrations of model group were lower than those of complete formula group(P=0.039),while there was no statistical difference in serum E2 concentrations between any other rest groups(P>0.05).There was statistical difference in serum E2 concentrations among all the groups after 4 weeks medication[(204.819±53.802)ng·L-1,(91.613±32.654)ng·L-1,(148.727±51.860)ng·L-1,(146.576±32.976)ng·L-1,(145.225±49.217)ng·L-1,(146.365±45.800)ng·L-1,(132.197±46.032)ng·L-1; F=4.278,P=0.002].Further pairwise comparison showed that the serum E2 concentrations of blank group were higher than those of other groups respectively(P=0.000,P=0.022,P=0.013,P=0.015,P=0.013,P=0.002); and the serum E2 concentrations of model group were lower than those of complete formula group,TD group,TDG group and TDW group respectively(P=0.027,P=0.027,P=0.037,P=0.027); while there was no statistical difference in serum E2 concentrations between any other rest groups(P>0.05).There was statistical difference in serum E2 concentrations among all the groups after 8 weeks medication[(192.060±57.040)ng·L-1,(97.504±40.006)ng·L-1,(183.140±50.887)ng·L-1,(130.757±39.285)ng·L-1,(167.763±32.878)ng·L-1,(158.112±48.309)ng·L-1,(112.494±46.687)ng·L-1; F=3.296,P=0.009].Further pairwise comparison showed that the serum E2 concentrations of blank group were higher than those of model group,TD group and GHC group respectively(P=0.000,P=0.011,P=0.001); the serum E2 concentrations of model group were lower than those of complete formula group,TDG group and TDW group respectively(P=0.002,P=0.005,P=0.012); the serum E2 concentrations of complete formula group were higher than those of GHC group(P=0.010); while there were no statistical difference between any other rest groups(P>0.05).There were statistical difference in Collagen-Ⅱexpressions in knee articular cartilages matrix among all the groups after 2 weeks medication[(0.598±0.096),(0.226±0.113),(0.310±0.070),(0.310±0.072),(0.311±0.075),(0.296±0.081),(0.389±0.068); F=33.533,P=0.000]].Further pairwise comparison showed that Collagen-Ⅱexpressions of blank group were higher than those of other groups respectively(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); Collagen-Ⅱexpressions of model group were lower than those of complete formula group,TD group,TDG group,TDW group and GHC group respectively(P=0.007,P=0.006,P=0.006,P=0.021,P=0.000); Collagen-Ⅱexpressions of complete formula group were lower than those of GHC group(P=0.008); while there was no statistical difference between any other rest groups(P>0.05).There were statistical difference in Collagen-Ⅱexpressions in knee articular cartilages matrix among all the groups after 4 weeks medication[(0.603±0.083),(0.223±0.113),(0.387±0.090),(0.270±0.083),(0.371±0.059),(0.300±0.081),(0.442±0.067); F=37.064,P=0.000].Further pairwise comparison showed that Collagen-Ⅱexpressions of blank group were higher than those of other groups respectively(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); Collagen-Ⅱexpressions of model group were lower than those of complete formula group,TDG group,TDW group and GHC group respectively(P=0.000,P=0.000,P=0.011,P=0.000); Collagen-Ⅱexpressions of complete formula group were higher than those of TD group and TDG group respectively(P=0.000,P=0.003); while there was no statistical difference in Collagen-Ⅱexpressions between any other rest groups(P>0.05).There was statistical difference in Collagen-Ⅱexpressions in knee articular cartilages matrix among all the groups after 8 weeks medication[(0.608±0.067),(0.217±0.106),(0.437±0.065),(0.263±0.110),(0.380±0.073),(0.273±0.081),(0.443±0.068); F=42.071,P=0.000].Further pairwise comparison showed that Collagen-Ⅱexpressions of blank group were higher than those of other groups respectively(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); Collagen-Ⅱexpressions of model group were lower than those of complete formula group,TDG group and GHC group respectively(P=0.000,P=0.000,P=0.000); Collagen-Ⅱexpressions of complete formula group were higher than those of TD group,TDG group and TDW group respectively(P=0.000,P=0.049,P=0.000); while there was no statistical difference in Collagen-Ⅱexpressions between any other rest groups(P>0.05).Conclusion:Tortoiseshell glue and deerhorn glue combined with either ginseng or wolfberry can effectively increase serum E2 concentrations.Moreover,after 2 weeks medication,the effect of GUILU ERXIAN gelatin,which including tortoiseshell glue,deerhorn glue,ginseng and wolfberry,is better than that of tortoiseshell glue and deerhorn glue combined with either ginseng or wolfberry.Howerer,further research is needed to find out the most effective combination for increasing serum E2 concentrations as the time of medication is prolonged.Both ginseng and wolfberry play an important role in the formula of GUILU ERXIAN gelatin in the improvement of Collagen-Ⅱexpressions in knee articular cartilages matrix.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金(30772814),福建省自然科学基金(C0710028),福建省高等学校新世纪优秀人才支持计划(闽教科2007年20号)
通讯作者:李楠 E-mail:mr.linan@126.com
更新日期/Last Update: 2013-03-20