补肾益肝活血方对大鼠膝骨关节炎疗效探讨及软骨影响实验研究
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摘要
目的:探讨补肾益肝活血方对大鼠膝骨关节炎的疗效作用及对关节软骨的影响。方法:3月龄SD雌性大鼠54只,随机分成模型组45只及空白组9只,模型组采用右后膝关节腔注射4%木瓜蛋白酶溶液进行诱导膝骨关节炎模型。经确认模型建立成功后,再次随机分组,分为中药(高、中、低剂量)组、阳性对照组、模型组及空白组各8只大鼠。3个中药组分别予以补肾益肝活血方煎煮液(低剂量组5 g/kg,中剂量组10 g/kg,高剂量组20 g/kg)灌服,阳性对照组予以双醋瑞因胶囊溶液0.027 g/m L浓度灌胃,模型组及空白组均以0.9%生理盐水灌胃。给药体积为10 m L/kg,共4周。给药结束后,处死大鼠,并通过检测血清炎症因子、肉眼及光镜下观察软骨组织、软骨组织的免疫组化分析等进行探讨。结果:(1)血清炎症因子检测:中药高剂量组、中剂量组大鼠血清中IL-1β、TNF-α含量较模型组低(P<0.05);随给药浓度增加,IL-1β、TNF-α降低明显(P<0.05)。(2)肉眼及光镜下观察:中药高剂量组、中剂量组、低剂量组以及阳性对照组在肉眼和光镜下观察,软骨的退变程度相比于模型组均有不同程度改善(P<0.05),中药高剂量组与阳性对照组对比差异不明显(P>0.05)。(3)软骨组织的免疫组化分析:在各组软骨组织中MMP-3、EFEMP-1均有不同程度阳性表达;模型组及各药物干预组MMP-3、EFEMP-1阳性表达均高于空白组;MMP-3的阳性表达在中药高、中剂量组及阳性对照组要低于中药低剂量组(P<0.05);中药高、中剂量组及阳性对照组EFEMP-1阳性表达高于中药低剂量组(P<0.05)。结论:补肾益肝活血方能有效治疗大鼠膝骨关节炎,能改善关节软骨退变,且存在一定的剂量依赖关系。
Objective:To evaluate the effect of Bushen Yigan Huoxue Decoction on knee osteoarthritis in rats,and its effect on articular cartilage. Methods:Fifty-four SD female rats randomly divided into model group 45,the blank group 9,model group using 4% posterior knee joint cavity injection of papain solution to induce model of knee osteoarthritis. After the success of the confirmation model,the group was randomly divided into TCM(high,medium and low dose)group,positive control group,model control group and blank group. Three Chinese traditional medicine groups respectively for Bushen Yigan Huoxue Decoction(low dose group of 5 g/kg,medium dose group of 10 g/kg,high dose group of 20 g/kg),the positive control group was treated with the concentration of 0.027 g/m L of diacetin,model control group and blank group with 0.9% saline lavage,dosing volume for 10 m L/kg,a total of 4 weeks. At the end of the drug,rats were executed and to do some exploration by detecting inflammatory cytokines,observing cartilage tissue under the naked eye and under the microscope and immunohistochemical analysis of cartilage tissue. Results:(1)Serum inflammatory factor detection:the content of IL-1β and TNF-α in the serum of the high-dose group and the medium-dose group were lower than that in the model group(P<0.05). As the concentration of the drug increased,the decrease of IL-1β and TNF-α was significant(P<0.05).(2)Observing under the naked eye and light microscope:Under the naked eye and light microscope observation,the cartilage degeneration degree of high dose group,middle dose group,low dose group and positive control group were improved to varying degrees compared with model group(P<0.05),Chinese medicine high dose group and positive control group showed no obvious difference(P>0.05).(3)The immunohistochemical analysis of cartilage tissues:MMP-3 and EFEMP-1 in the cartilage tissues of each group had different levels of positive expression. The MMP-3,EFEMP-1 positive expression of the model group and the drug intervention group were higher than the blank group. The positive expression of MMP-3 was lower than the low-dose group(P<0.05)in the high dose group,medium dose group and the positive control group. The expression of EFEMP-1 in high dose group,medium dose group and positive control group was higher than that in low-dose group(P<0.05). Conclusion:Bushen Yigan Huoxue Decoction can effectively treat the knee osteoarthritis in rats,can improve the degeneration of articular cartilage,and there is a certain dosedependent relationship.
Objective:To evaluate the effect of Bushen Yigan Huoxue Decoction on knee osteoarthritis in rats,and its effect on articular cartilage. Methods:Fifty-four SD female rats randomly divided into model group 45,the blank group 9,model group using 4% posterior knee joint cavity injection of papain solution to induce model of knee osteoarthritis. After the success of the confirmation model,the group was randomly divided into TCM(high,medium and low dose)group,positive control group,model control group and blank group. Three Chinese traditional medicine groups respectively for Bushen Yigan Huoxue Decoction(low dose group of 5 g/kg,medium dose group of 10 g/kg,high dose group of 20 g/kg),the positive control group was treated with the concentration of 0.027 g/m L of diacetin,model control group and blank group with 0.9% saline lavage,dosing volume for 10 m L/kg,a total of 4 weeks. At the end of the drug,rats were executed and to do some exploration by detecting inflammatory cytokines,observing cartilage tissue under the naked eye and under the microscope and immunohistochemical analysis of cartilage tissue. Results:(1)Serum inflammatory factor detection:the content of IL-1β and TNF-α in the serum of the high-dose group and the medium-dose group were lower than that in the model group(P<0.05). As the concentration of the drug increased,the decrease of IL-1β and TNF-α was significant(P<0.05).(2)Observing under the naked eye and light microscope:Under the naked eye and light microscope observation,the cartilage degeneration degree of high dose group,middle dose group,low dose group and positive control group were improved to varying degrees compared with model group(P<0.05),Chinese medicine high dose group and positive control group showed no obvious difference(P>0.05).(3)The immunohistochemical analysis of cartilage tissues:MMP-3 and EFEMP-1 in the cartilage tissues of each group had different levels of positive expression. The MMP-3,EFEMP-1 positive expression of the model group and the drug intervention group were higher than the blank group. The positive expression of MMP-3 was lower than the low-dose group(P<0.05)in the high dose group,medium dose group and the positive control group. The expression of EFEMP-1 in high dose group,medium dose group and positive control group was higher than that in low-dose group(P<0.05). Conclusion:Bushen Yigan Huoxue Decoction can effectively treat the knee osteoarthritis in rats,can improve the degeneration of articular cartilage,and there is a certain dosedependent relationship.
引文
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[4]施玲玲,陈东煜.膝关节骨关节炎的中医药治疗现状[J].中国中医骨伤科杂志,2014,22(7):72-74.
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[7]季卫锋,施伟峰,陈林,等.补肾活血法防治大鼠膝骨性关节炎的实验研究[J].中国骨伤,2012,25(3):246-250.
[8]潘雪璟,王伟钢.膝骨关节炎的中医研究进展[J].风湿病与关节炎,2016(12):74-77.
[9]纪红,杨进宝.中医辨证治疗膝关节骨性关节炎的临床疗效观察[J].中医临床研究,2015(19):107-108.
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[11]王平乐.补肾活血治疗骨性关节炎92例[J].中国民族民间医药,2010(6):116.
[12]王清玉,李良华,王和鸣,等.补肾活血方治疗膝关节骨性关节炎100例[J].中国中医骨伤科杂志,2013(4):10-11.
[13]张利,寇久社,张鸿,等.补肾活血汤治疗膝骨性关节炎疗效观察[J].陕西中医,2012(8):1002-1003.
[14]潘慧琴.补肾壮筋汤防治骨关节炎的临床分析[J].中国伤残医学,2014(5):68-69.
[15]张玉可,郭艳幸.活血利节汤治疗瘀血闭阻型膝骨关节炎临床观察[J].风湿病与关节炎,2014(12):18-19,22.
[16]张金山,程圆圆,刘健.膝骨关节炎患者血清IL-1β、TGF-β变化及相关性研究[J].江西中医学院学报,2011,23(6):29-32.
[17]林梓凌,樊粤光,赵京涛,等.补肾活血方药对膝关节骨折术后功能恢复的影响[J].中国实验方剂学杂志,2013,6(15):209.
[18]Naito K,Takahahi M,Kushida K,et al.Measurement of matrix metalloproteinases and tissue inhibitor of metallpproteinases1 in patients with knee osteoarthritis[J].Rhumatology(Oxford),1999,38(6):510.
[19]郎媛媛,孟洁,宋晓萌,等.EFEMP1通过下调MMP-7表达抑制肺癌细胞生长和侵袭[J].中国肺癌杂志,2015,18(2):92-97.
[2]王亚克,陶娟.骨关节炎的中医研究进展[J].成都中医药大学学报,2012(1):95-96.
[3]孙自力.人膝骨关节炎治疗现状及方案研究[J].中国现代医生,2015,53(15):147-151.
[4]施玲玲,陈东煜.膝关节骨关节炎的中医药治疗现状[J].中国中医骨伤科杂志,2014,22(7):72-74.
[5]LIN YS,HUANG MH,CHAI CY.Effects of helium-neon laser on the mucopoly saccharide induction in experimental osteoarthritic cartilage[J].Osteoarthritis Cartilage,2005,12:339-346.
[6]Pelletier JP,Jovanovic D,Fernandes JC,et al.Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase[J].Arthritis&Rheumatism,1998,41(7):1275-1286.
[7]季卫锋,施伟峰,陈林,等.补肾活血法防治大鼠膝骨性关节炎的实验研究[J].中国骨伤,2012,25(3):246-250.
[8]潘雪璟,王伟钢.膝骨关节炎的中医研究进展[J].风湿病与关节炎,2016(12):74-77.
[9]纪红,杨进宝.中医辨证治疗膝关节骨性关节炎的临床疗效观察[J].中医临床研究,2015(19):107-108.
[10]黄丹奇.运用中医肾主骨的理论治疗膝骨性关节炎临床研究[J].中国临床医生,2011,39(5):63-64.
[11]王平乐.补肾活血治疗骨性关节炎92例[J].中国民族民间医药,2010(6):116.
[12]王清玉,李良华,王和鸣,等.补肾活血方治疗膝关节骨性关节炎100例[J].中国中医骨伤科杂志,2013(4):10-11.
[13]张利,寇久社,张鸿,等.补肾活血汤治疗膝骨性关节炎疗效观察[J].陕西中医,2012(8):1002-1003.
[14]潘慧琴.补肾壮筋汤防治骨关节炎的临床分析[J].中国伤残医学,2014(5):68-69.
[15]张玉可,郭艳幸.活血利节汤治疗瘀血闭阻型膝骨关节炎临床观察[J].风湿病与关节炎,2014(12):18-19,22.
[16]张金山,程圆圆,刘健.膝骨关节炎患者血清IL-1β、TGF-β变化及相关性研究[J].江西中医学院学报,2011,23(6):29-32.
[17]林梓凌,樊粤光,赵京涛,等.补肾活血方药对膝关节骨折术后功能恢复的影响[J].中国实验方剂学杂志,2013,6(15):209.
[18]Naito K,Takahahi M,Kushida K,et al.Measurement of matrix metalloproteinases and tissue inhibitor of metallpproteinases1 in patients with knee osteoarthritis[J].Rhumatology(Oxford),1999,38(6):510.
[19]郎媛媛,孟洁,宋晓萌,等.EFEMP1通过下调MMP-7表达抑制肺癌细胞生长和侵袭[J].中国肺癌杂志,2015,18(2):92-97.