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[1]梁文娜,李西海,胡柳,等.二至丸抑制絕經(jīng)后骨質(zhì)疏松大鼠骨代謝紊亂的作用機(jī)制研究[J].中醫(yī)正骨,2017,29(11):1-7,14.
　LIANG Wenna,LI Xihai,HU Liu,et al.Study on mechanism of action of Erzhi Wan(二至丸)in inhibiting bone metabolism disorder in rats with postmenopausal osteoporosis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(11):1-7,14.
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二至丸抑制絕經(jīng)后骨質(zhì)疏松大鼠骨代謝紊亂的作用機(jī)制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期數(shù):
2017年11期
頁(yè)碼:
1-7,14
欄目:
基礎(chǔ)研究
出版日期:
2017-11-20

文章信息/Info

Title:
Study on mechanism of action of Erzhi Wan(二至丸)in inhibiting bone metabolism disorder in rats with postmenopausal osteoporosis
作者:
梁文娜; 李西海; 胡柳; 丁珊珊; 康潔; 王洋; 沈建英; 李燦東
福建中醫(yī)藥大學(xué),福建 福州 350122
Author(s):
LIANG Wenna; LI Xihai; HU Liu; DING Shanshan; KANG Jie; WANG Yang; SHEN Jianying; LI Candong
Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China
關(guān)鍵詞:
骨質(zhì)疏松; 絕經(jīng)后;  大鼠;  二至丸;  骨密度;  骨特異性堿性磷酸酶;  抗酒石酸酸性磷酸酶5b;  基質(zhì)金屬蛋白酶9;  組織蛋白酶K
Keywords:
Key words osteoporosis; postmenopausal;  rats;  Erzhi Pill;  bone density;  bone alkaline phosphotase;  tartrate-resistant acid phosphatase 5b;  matrix metalloproteinase 9;  cathepsin K
摘要:
目的:探討二至丸抑制絕經(jīng)后骨質(zhì)疏松大鼠骨代謝紊亂的作用機(jī)制。方法:2月齡雌性清潔級(jí)SD大鼠60只,采用隨機(jī)數(shù)字表隨機(jī)分為假手術(shù)組15只與手術(shù)組45只。假手術(shù)組在雙側(cè)卵巢周?chē)谐僭S脂肪組織,手術(shù)組摘除雙側(cè)卵巢建立絕經(jīng)后骨質(zhì)疏松癥大鼠模型。造模后2周,再將手術(shù)組大鼠隨機(jī)分為模型組、二至丸組與雌二醇組,每組15只。二至丸組和雌二醇組大鼠,分別用二至丸混懸液和戊酸雌二醇片混懸液灌胃; 假手術(shù)組和模型組大鼠,用生理鹽水灌胃; 每日1次,連續(xù)灌胃12周。藥物干預(yù)結(jié)束后,采用ELISA法檢測(cè)大鼠血清中骨代謝標(biāo)記物骨特異性堿性磷酸酶(bone alkaline phosphotase,BALP)、抗酒石酸酸性磷酸酶-5b(tartrate-resistant acid phosphatase-5b,TRACP-5b)、基質(zhì)金屬蛋白酶-9(matrix metalloproteinase-9,MMP-9)、組織蛋白酶K(cathepsin K,Cath-K)含量; 并每組隨機(jī)取3只大鼠的L5椎體,以4%多聚甲醛固定,行骨密度(bone mineral density,BMD)及組織學(xué)檢測(cè); 每組其余12只大鼠L5腰椎以液氮保存,行骨代謝標(biāo)記物mRNA表達(dá)的檢測(cè)。并對(duì)各組檢測(cè)結(jié)果進(jìn)行比較。結(jié)果:①BMD檢測(cè)結(jié)果。藥物干預(yù)12周后,4組間腰椎BMD檢測(cè)結(jié)果比較,差異有統(tǒng)計(jì)學(xué)意義[(0.16±0.02)g·cm-2,(0.10±0.03)g·cm-2,(0.13±0.02)g·cm-2,(0.14±0.02)g·cm-2; F=5.800,P=0.005]; 假手術(shù)組、二至丸組與雌二醇組高于模型組(P=0.001,P=0.024,P=0.010); 假手術(shù)組與二至丸組、雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.123,P=0.236); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.701)。②組織學(xué)檢測(cè)結(jié)果。藥物干預(yù)12周后,假手術(shù)組腰椎骨小梁致密,分布均勻,排列規(guī)則,表面光滑,交織成網(wǎng)狀,骨小梁間隙均勻。模型組腰椎骨小梁變細(xì)、分布稀疏、散亂、不連續(xù),表面粗糙,完整性差,出現(xiàn)碎片、斷裂等。二至丸組與雌二醇組腰椎骨小梁變細(xì),數(shù)量減少,分布稀疏,排列較規(guī)則。4組間骨小梁面積百分比比較,差異有統(tǒng)計(jì)學(xué)意義[(23.25±5.32)%,(11.82±3.87)%,(17.10±3.83)%,(18.08±2.59)%; F=8.144,P=0.001]; 模型組、二至丸組與雌二醇組低于假手術(shù)組(P=0.000,P=0.015,P=0.038); 二至丸組與雌二醇組高于模型組(P=0.034,P=0.014); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.677)。③骨代謝標(biāo)記物血清含量檢測(cè)結(jié)果。藥物干預(yù)12周后,4組間血清BALP含量比較,差異有統(tǒng)計(jì)學(xué)意義[(58.14±9.04)單位·L-1,(40.91±6.47)單位·L-1,(51.13±7.88)單位·L-1,(52.23±7.41)單位·L-1; F=5.239,P=0.008]; 假手術(shù)組、二至丸組與雌二醇組高于模型組(P=0.001,P=0.034,P=0.012); 假手術(shù)組與二至丸組、雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.133,P=0.286); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.645)。4組間血清TRACP-5b含量比較,差異有統(tǒng)計(jì)學(xué)意義[(0.71±0.13)pg·mL-1,(0.98±0.22)pg·mL-1,(0.79±0.09)pg·mL-1,(0.77±0.07)pg·mL-1; F=3.874,P=0.025]; 假手術(shù)組、二至丸組與雌二醇組低于模型組(P=0.004,P=0.038,P=0.022); 假手術(shù)組與二至丸組、雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.320,P=0.459); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.795)。4組間血清MMP-9含量比較,差異有統(tǒng)計(jì)學(xué)意義[(1.63±0.23)pg·mL-1,(2.01±0.35)pg·mL-1,(1.64±0.25)pg·mL-1,(1.58±0.18)pg·mL-1; F=3.507,P=0.034]; 假手術(shù)組、二至丸組與雌二醇組低于模型組(P=0.021,P=0.023,P=0.009); 假手術(shù)組與二至丸組、雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.972,P=0.699); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.673)。4組間血清Cath-K含量比較,差異有統(tǒng)計(jì)學(xué)意義[(2.55±0.40)pg·mL-1,(4.06±0.87)pg·mL-1,(3.07±0.77)pg·mL-1,(2.73±0.69)pg·mL-1; F=5.539,P=0.006]; 假手術(shù)組、二至丸組與雌二醇組低于模型組(P=0.001,P=0.024,P=0.004); 假手術(shù)組與二至丸組、雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.217,P=0.671); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.408)。④骨代謝標(biāo)記物mRNA表達(dá)檢測(cè)結(jié)果。藥物干預(yù)12周后,4組間BALPmRNA表達(dá)比較,差異有統(tǒng)計(jì)學(xué)意義[(1.01±0.04),(0.62±0.10),(0.78±0.13),(0.83±0.13); F=14.482,P=0.000]; 模型組、二至丸組與雌二醇組低于假手術(shù)組(P=0.000,P=0.001,P=0.007); 二至丸組與雌二醇組高于模型組(P=0.015,P=0.002),二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.385)。4組間TRACP-5bmRNA表達(dá)比較,差異有統(tǒng)計(jì)學(xué)意義[(0.98±0.04),(2.05±0.41),(1.67±0.26),(1.50±0.21); F=16.574,P=0.000]; 模型組、二至丸組與雌二醇組高于假手術(shù)組(P=0.000,P=0.000,P=0.003); 二至丸組與雌二醇組低于模型組(P=0.024,P=0.002); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.282)。4組間MMP-9mRNA表達(dá)比較,差異有統(tǒng)計(jì)學(xué)意義[(1.00±0.06),(1.86±0.17),(1.51±0.32),(1.45±0.35); F=11.684,P=0.000]; 模型組、二至丸組與雌二醇組高于假手術(shù)組(P=0.000,P=0.002,P=0.007); 二至丸組與雌二醇組低于模型組(P=0.028,P=0.010); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.643)。4組間Cath-KmRNA表達(dá)比較,差異有統(tǒng)計(jì)學(xué)意義[(0.99±0.03),(2.52±0.57),(1.85±0.27),(1.74±0.54); F=13.543,P=0.000]; 模型組、二至丸組與雌二醇組高于假手術(shù)組(P=0.000,P=0.002,P=0.005); 二至丸組與雌二醇組低于模型組(P=0.011,P=0.004); 二至丸組與雌二醇組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.663)。結(jié)論:對(duì)于絕經(jīng)后骨質(zhì)疏松模型大鼠,二至丸和戊酸雌二醇片均可增加其骨密度和骨小梁數(shù)量; 作用機(jī)制可能是通過(guò)促進(jìn)BALP表達(dá)、抑制TRACP-5b、MMP-9與Cath-K表達(dá),從而抑制骨代謝紊亂; 兩種藥物的作用相當(dāng)。
Abstract:
ABSTRACT Objective:To explore the mechanism of action of Erzhi Wan(二至丸,EZW)in inhibiting bone metabolism disorder in rats with postmenopausal osteoporosis(PMOP).Methods:Sixty 2-month-old clean-grade female SD rats were randomly divided into sham-operated group(15)and operation group(45)by using random digits table.The resection of fat around bilateral ovaries were performed on rats in sham-operated group,and the bilateral ovariectomy were performed on rats in operation group to build the PMOP rat models.At 2 weeks after the modeling,the rats in operation group were randomly subdivided into model group,EZW group and estradiol group,15 cases in each group.The rats in EZW group and estradiol group were intragastric administrated with EZW suspension and estradiol valerate suspension respectively,while the others in sham-operated group and model group were intragastric administrated with normal saline(NS),once a day for consecutive 12 weeks.After the end of drug intervention,the serum contents of bone alkaline phosphotase(BALP),tartrate-resistant acid phosphatase-5b(TRACP-5b),matrix metalloproteinase-9(MMP-9)and cathepsin K(Cath-K)were measured by using ELISA method.Three rats were randomly selected from each group and their L5 vertebraes were fetched out and fixed with 4% paraformaldehyde,and then the bone mineral density(BMD)detection and histological detection were performed.The L5 vertebraes of the other 12 rats in each group were preserved in liquid nitrogen,and the mRNA expression of bone metabolism marker were detected.The detection results were compared between the 4 groups.Results:After 12-week drug intervention,there was statistical difference in the detection result of lumbar vertebra BMD between the 4 groups(0.16+/-0.02,0.10+/-0.03,0.13+/-0.02,0.14+/-0.02 g/cm(2); F=5.800,P=0.005).The BMD were higher in sham-operated group,EZW group and estradiol group compared to model group(P=0.001,P=0.024,P=0.010).There was no statistical difference in the BMD between sham-operated group and EZW group and between sham-operated group and estradiol group(P=0.123,P=0.236),and there was no statistical difference in the BMD between EZW group and estradiol group(P=0.701).After 12-week drug intervention,the lumbar bone trabeculas of rats of sham-operated group were in a compact state and were uniformly distributed and regularly arranged,and they interlaced to form a netty structure with smooth surface and uniform interspace.The bone trabeculas of rats of model group became thin and were characterized by sparse,scattered and discontinuous distribution,and they presented with debris,rupture,rough surface and poor integrity.The bone trabeculas became thin and declined in number and characterized by sparse distribution and relatively regular arrangement in rats of EZW group and estradiol group.There was statistical difference in the area percentage of bone trabecula between the 4 groups(23.25+/-5.32,11.82+/-3.87,17.10+/-3.83,18.08+/-2.59%; F=8.144,P=0.001).The area percentage of bone trabecula was lower in model group,EZW group and estradiol group compared to sham-operated group(P=0.000,P=0.015,P=0.038),and was higher in EZW group and estradiol group compared to model group(P=0.034,P=0.014),and there was no statistical difference in the area percentage of bone trabecula between EZW group and estradiol group(P=0.677).After 12-week drug intervention,there was statistical difference in the serum contents of BALP between the 4 groups(58.14+/-9.04,40.91+/-6.47,51.13+/-7.88,52.23+/-7.41 unit/l; F=5.239,P=0.008).The serum contents of BALP were higher in sham-operated group,EZW group and estradiol group compared to model group(P=0.001,P=0.034,P=0.012).There was no statistical difference in the serum contents of BALP between sham-operated group and EZW group(P=0.133)and between sham-operated group and estradiol group(P=0.286)and between EZW group and estradiol group(P=0.645).There was statistical difference in the serum contents of TRACP-5b between the 4 groups(0.71+/-0.13,0.98+/-0.22,0.79+/-0.09,0.77+/-0.07 pg/ml; F=3.874,P=0.025).The serum contents of TRACP-5b were lower in sham-operated group,EZW group and estradiol group compared to model group(P=0.004,P=0.038,P=0.022),and there was no statistical difference in the serum contents of TRACP-5b between sham-operated group and EZW group(P=0.320)and between sham-operated group and estradiol group(P=0.459)and between EZW group and estradiol group(P=0.795).There was statistical difference in the serum contents of MMP-9 between the 4 groups(1.63+/-0.23,2.01+/-0.35,1.64+/-0.25,1.58+/-0.18 pg/ml; F=3.507,P=0.034).The serum contents of MMP-9 were lower in sham-operated group,EZW group and estradiol group compared to model group(P=0.021,P=0.023,P=0.009).There was no statistical difference in the serum contents of MMP-9 between sham-operated group and EZW group(P=0.972)and between sham-operated group and estradiol group(P=0.699)and between EZW group and estradiol group(P=0.673).There was statistical difference in the serum contents of Cath-K between the 4 groups(2.55+/-0.40,4.06+/-0.87,3.07+/-0.77,2.73+/-0.69 pg/ml; F=5.539,P=0.006).The serum contents of Cath-K were lower in sham-operated group,EZW group and estradiol group compared to model group(P=0.001,P=0.024,P=0.004).There was no statistical difference in the serum contents of Cath-K between sham-operated group and EZW group(P=0.217)and between sham-operated group and estradiol group(P=0.671)and between EZW group and estradiol group(P=0.408).After 12-week drug intervention,there was statistical difference in BALP mRNA expression between the 4 groups(1.01+/-0.04,0.62+/-0.10,0.78+/-0.13,0.83+/-0.13; F=14.482,P=0.000).The BALP mRNA expressions were lower in model group,EZW group and estradiol group compared to sham-operated group(P=0.000,P=0.001,P=0.007),and were higher in EZW group and estradiol group compared to model group(P=0.015,P=0.002),and there was no statistical difference in the BALP mRNA expression between EZW group and estradiol group(P=0.385).There was statistical difference in the TRACP-5b mRNA expression between the 4 groups(0.98+/-0.04,2.05+/-0.41,1.67+/-0.26,1.50+/-0.21; F=16.574,P=0.000).The TRACP-5b mRNA expressions were higher in model group,EZW group and estradiol group compared to sham-operated group(P=0.000,P=0.000,P=0.003),and were lower in EZW group and estradiol group compared to model group(P=0.024,P=0.002),and there was no statistical difference in the TRACP-5b mRNA expression between EZW group and estradiol group(P=0.282).There was statistical difference in the MMP-9 mRNA expression between the 4 groups(1.00+/-0.06,1.86+/-0.17,1.51+/-0.32,1.45+/-0.35; F=11.684,P=0.000).The MMP-9 mRNA expressions were higher in model group,EZW group and estradiol group compared to sham-operated group(P=0.000,P=0.002,P=0.007),and were lower in EZW group and estradiol group compared to model group(P=0.028,P=0.010),and there was no statistical difference in the MMP-9 mRNA expression between EZW group and estradiol group(P=0.643).There was statistical difference in Cath-K mRNA expression between the 4 groups(0.99+/-0.03,2.52+/-0.57,1.85+/-0.27,1.74+/-0.54; F=13.543,P=0.000).The Cath-K mRNA expressions were higher in model group,EZW group and estradiol group compared to sham-operated group(P=0.000,P=0.002,P=0.005),and were lower in EZW group and estradiol group compared to model group(P=0.011,P=0.004),and there was no statistical difference in the Cath-K mRNA expression between EZW group and estradiol group(P=0.663).Conclusion:For PMOP rat models,both EZW and estradiol valerate tablets can inhibit bone metabolism disorder through promoting the expression of BALP and inhibiting the expression of TRACP-5b,MMP-9 and Cath-K,which may be the mechanisms of action in increasing the BMD and the number of bone trabecula.The two drugs are similar to each other in curative effect.

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備注/Memo

備注/Memo:
基金項(xiàng)目:國(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目(81230087),福建省自然科學(xué)基金項(xiàng)目(2015J01339),福建省衛(wèi)生系統(tǒng)中青年骨干人才培養(yǎng)項(xiàng)目(2015-ZQN-JC-32),福建省高校新世紀(jì)優(yōu)秀人才支持計(jì)劃項(xiàng)目(閩科教[2015]54號(hào)) 通訊作者:李燦東 E-mail:[email protected]
更新日期/Last Update: 2018-04-02