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[1]杜江,黃遠鵬,李奕修,等.龜鹿二仙膠及其拆方對去勢骨關節(jié)炎大鼠血清雌二醇濃度及膝關節(jié)軟骨細胞Ⅱ型膠原表達的影響[J].中醫(yī)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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龜鹿二仙膠及其拆方對去勢骨關節(jié)炎大鼠血清雌二醇濃度 及膝關節(jié)軟骨細胞Ⅱ型膠原表達的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期數(shù):
2013年03期
頁碼:
11-20
欄目:
基礎研究
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; 黃遠鵬; 李奕修; 余添賜; 魏迎辰; 王和鳴; 李楠
福建中醫(yī)藥大學,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
關鍵詞:
骨關節(jié)炎 龜鹿二仙膠 拆方研究 雌二醇 膠原Ⅱ型 大鼠
Keywords:
Osteoarthritis;  GUILU ERXIAN gelatin;  Formula components analysis;  Estradiol;  Collagen type Ⅱ;  Rats
摘要:
目的:觀察龜鹿二仙膠及其拆方對去勢骨關節(jié)炎大鼠血清雌二醇濃度及膝關節(jié)軟骨細胞Ⅱ型膠原表達的影響。方法:采用抽簽法將168只4月齡雌性SD清潔級大鼠隨機分為2組,手術造模組141只,假手術組27只。切除手術造模組大鼠雙側(cè)的卵巢和雙側(cè)膝關節(jié)的內(nèi)側(cè)副韌帶和前交叉韌帶,制造肝腎虧虛型骨關節(jié)炎大鼠模型; 切除假手術組大鼠雙側(cè)卵巢附近的脂肪組織。每天驅(qū)趕實驗大鼠1 h,連續(xù)驅(qū)趕4周。4周后分別從手術造模組與假手術組各取5只大鼠,采用HE染色和甲苯胺藍染色法觀察子宮與膝關節(jié)軟骨的組織形態(tài)學變化,采用甲苯胺藍染色法觀察膝關節(jié)軟骨基質(zhì)中糖胺聚糖的表達情況,采用放射免疫法測定血清雌二醇濃度,鑒定大鼠模型。造模成功后將假手術組作為空白對照組(空白組),并將其分為干預2、4、8周的3小組,每組7只,留1只備用; 采用抽簽法將手術造模組隨機分為6組,即模型對照組(模型組)、龜鹿二仙膠組(全方組)、龜甲鹿角組(龜鹿組)、龜甲鹿角人參組(龜鹿參組)、龜甲鹿角枸杞組(龜鹿杞組)、鹽酸氨基葡萄糖對照組(鹽酸氨基葡萄糖組),再分別將每組分為干預2、4、8周的3小組,每組7只,留10只備用。參照人與大鼠藥物等效劑量換算公式,將龜甲膠、鹿角膠、人參、枸杞、鹽酸氨基葡萄糖分別配成濃度為81 mg·mL-1、54 mg·mL-1、10.8 mg·mL-1、27 mg·mL-1、13.5 mg·mL-1的藥液。各組大鼠均以10 mL·kg-1體質(zhì)量以各組藥液灌胃,空白組與模型組以生理鹽水灌胃; 全方組以配成的龜鹿二仙膠混合液灌胃; 龜鹿組以配成的龜甲膠和鹿角膠混合液灌胃; 龜鹿參組以配成的龜甲膠、鹿角膠與人參混合液灌胃; 龜鹿杞組以配成的龜甲膠、鹿角膠、枸杞子混合液灌胃; 鹽酸氨基葡萄糖組以配成的鹽酸氨基葡萄糖溶液灌胃。每天灌胃1次,各組分別連續(xù)灌胃2、4、8周。藥物干預結(jié)束后,從腹主動脈抽血后處死大鼠,取出的血經(jīng)靜置、離心后吸取上層血清,保存待檢; 取出大鼠右側(cè)中下1/3段股骨,制備石蠟標本。采用放射免疫法檢測大鼠血清雌二醇濃度,采用免疫組化染色法觀察關節(jié)軟骨中Ⅱ型膠原的表達。結(jié)果:①形態(tài)觀察。手術造模組與假手術組大鼠的子宮與膝關節(jié)軟骨的組織形態(tài)學變化顯示手術造模組造模成功。②藥物干預前血清雌二醇濃度和膝關節(jié)軟骨基質(zhì)中糖胺聚糖含量。藥物干預前,手術造模組的血清雌二醇濃度明顯低于假手術組[(87.130±39.720)ng·L-1,(195.080±42.449)ng·L-1; t=4.152,P=0.003]; 手術造模組的軟骨基質(zhì)中糖胺聚糖含量低于假手術組[(0.239±0.066),(0.597±0.053); t=9.457,P=0.000]。顯示手術造模組造模成功。③藥物干預后血清雌二醇濃度。用藥2周后,各組間血清雌二醇濃度比較,差異有統(tǒng)計學意義[(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]。組間兩兩比較,空白組高于模型組、全方組、龜鹿組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.000,P=0.002,P=0.001,P=0.001,P=0.003,P=0.001); 模型組低于全方組(P=0.039); 其余各組間血清E2濃度比較,差異均無統(tǒng)計學意義(P>0.05)。用藥4周后,各組間血清雌二醇濃度比較,差異有統(tǒng)計學意義[(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]。組間兩兩比較,空白組高于模型組、全方組、龜鹿組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.000,P=0.022,P=0.013,P=0.015,P=0.013,P=0.002); 模型組低于全方組、龜鹿組、龜鹿參組、龜鹿杞組(P=0.027,P=0.027,P=0.037,P=0.027); 其余各組間血清E2濃度比較,差異均無統(tǒng)計學意義(P>0.05)。用藥8周后,各組間血清雌二醇濃度比較,差異有統(tǒng)計學意義[(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]。組間兩兩比較,空白組高于模型組、龜鹿組、鹽酸氨基葡萄糖組(P=0.000,P=0.011,P=0.001); 模型組低于全方組、龜鹿參組、龜鹿杞組(P=0.002,P=0.005,P=0.012); 全方組高于鹽酸氨基葡萄糖組(P=0.010); 其余各組間比較,差異均無統(tǒng)計學意義(P>0.05)。④藥物干預后膝關節(jié)軟骨基質(zhì)中Ⅱ型膠原的表達量。用藥2周后,各組間膝關節(jié)軟骨基質(zhì)中Ⅱ型膠原的表達量比較,差異有統(tǒng)計學意義[(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)]。組間兩兩比較,空白組高于模型組、全方組、龜鹿組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 模型組低于全方組、龜鹿組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.007,P=0.006,P=0.006,P=0.021,P=0.000); 全方組低于鹽酸氨基葡萄糖組(P=0.008)。其余各組間比較,差異均無統(tǒng)計學意義(P>0.05)。用藥4周后,各組間膝關節(jié)軟骨基質(zhì)中Ⅱ型膠原的表達量比較,差異有統(tǒng)計學意義[(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]。組間兩兩比較,空白組高于模型組、全方組、龜鹿組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 模型組低于全方組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.000,P=0.000,P=0.011,P=0.000); 全方組高于龜鹿組、龜鹿杞組(P=0.000,P=0.003)。其余各組間Collagen-Ⅱ表達量比較,差異均無統(tǒng)計學意義(P>0.05)。用藥8周后,各組間軟骨基質(zhì)中Ⅱ型膠原的表達量比較,差異有統(tǒng)計學意義[(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]。組間兩兩比較,空白組高于模型組、全方組、龜鹿組、龜鹿參組、龜鹿杞組、鹽酸氨基葡萄糖組(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); 模型組低于全方組、龜鹿參組、鹽酸氨基葡萄糖組(P=0.000,P=0.000,P=0.000); 全方組高于龜鹿組、龜鹿參組、龜鹿杞組(P=0.000,P=0.049,P=0.000); 其余各組間Collagen-Ⅱ表達量比較,差異均無統(tǒng)計學意義(P>0.05)。結(jié)論:在提高血清雌二醇濃度方面,龜甲膠與鹿角膠配伍人參或枸杞均是有效配伍,且用藥2周時配伍人參和枸杞的效果要優(yōu)于單獨配伍人參或單獨配伍枸杞。但隨著用藥時間的延長,龜甲膠與鹿角膠配伍人參提高雌二醇濃度的效果好還是配伍枸杞的效果好,還是龜鹿二仙膠全方效果好,仍需進一步研究證實。在促進膝關節(jié)軟骨基質(zhì)中Ⅱ型膠原表達方面,人參和枸杞在龜鹿二仙膠方中發(fā)揮了不可缺少的作用。
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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備注/Memo

備注/Memo:
基金項目:國家自然科學基金(30772814),福建省自然科學基金(C0710028),福建省高等學校新世紀優(yōu)秀人才支持計劃(閩教科2007年20號)
通訊作者:李楠 E-mail:[email protected]
更新日期/Last Update: 2013-03-20