低频超声促透通络止痛中药对不同程度膝骨性关节炎软骨基质的影响
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摘要
目的：
1.通过动物实验研究通络止痛中药在低频超声促透法下对大鼠不同程度膝关节骨关节炎(Knee osteoarthritis KOA)的膝关节功能、软骨组织学以及软骨细胞外基质的影响,阐述其作用机制。
2.对不同程度KOA患者进行通络止痛中药的低频超声促透治疗,观察治疗前后患者软骨细胞外基质相关生物标志物,HSS功能评分、VAS疼痛评分和(SF-36)中文版健康调查简易量表的变化,进一步探讨这一外治法的临床疗效以及作用机制。
方法：
1.动物实验：采用大鼠前交叉韧带切断的经典方法制备膝骨关节炎大鼠模型,左膝为手术侧,右膝为对照侧。84只清洁级全雄SD大鼠随机分为3组：空白对照组(18只)、超声促透组(33只)和中药外敷组(33只)。所有大鼠予以造模,术前检测行为学并记录Lequesne MG评分,应用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清中Ⅱ型胶原羧基端肽((cterminal crosslinking telopeptide of type II collagen, CTX-Ⅱ))、软骨寡聚基质蛋白(cartilage oligomeric matrix protein, COMP)及基质金属蛋白酶-3(matrix metalloproteinases-3, MMP-3)含量。空白对照组正常饲养,术后2、4、8周时分别随机选取6只大鼠观察其行为学改变并记录LequesneMG评分,应用ELISA法检测血清中CTX-Ⅱ. COMP及MMP-3含量；处死后观察大鼠膝关节软骨大体改变,股骨内侧髁软骨及滑膜的组织形态学改变,根据软骨Mankin's组织学评分予以评价,并根据评分结果确定大鼠膝骨关节炎的病变程度。超声促透组和中药外敷组在术后2、4、8周时各随机选取11只大鼠进行干预处理,超声促透组使用超声电导仪对术侧膝关节促透通络止痛中药,中药外敷组仅使用中药外敷。1次／日,30min,连续治疗2周。2周后处死,标本采集及数据记录同空白对照组。
3.临床实验：临床纳入80例符合膝关节骨关节炎诊断标准且中医辩证为瘀血阻络证的患者,并根据HSS评分将其严重程度分为优、良、中、差四级,每一级患者20例,随机分为2组：中药促透组(10例)和盐水促透组(10例)。所有患者均服用基础中成药盘龙七片进行活血通络治疗,中药促透组患者予以超声促透通络止痛中药治疗,生理盐水促透组予以超声促透生理盐水治疗,4次／周,30min,连续治疗四周。采用ELISA法测定治疗前后患者血清内CTX-Ⅱ、COMP及MMP-3含量的变化,并在治疗前、治疗后采集VAS评分、HSS评分以及SF-36评分,评估各组临床疗效。
结果：
1.动物实验：
1.1行为学Lequesne MG评分：空白对照组造模后2、4、8周时大鼠行为学明显改变,Lequesne MG评分逐渐增高；超声促透组与空白对照组相比,Lequesne MG评分在2、4、8周组时降低,有统计学差异(p<0.05)；与中药外敷组相比,Lequesne MG评分在4周时下降明显,两两相比有统计学差异(p<0.05)。
1.2软骨大体观察：空白对照组造模后2、4、8周时软骨大体观察明显改变,软骨退变程度增加；超声促透组与空白对照组相比,软骨大体观察在2、4周组发生改变,退变程度减轻；与中药外敷组相比无明显改变。
1.3软骨组织学Mankin' s评分：空白对照组造模后2、4、8周时软骨组织形态学发生明显改变,Mankin' s评分的增加与时间成正相关。超声促透组与空白对照组相比,软骨组织形态学发生改变,Mankin' s评分2、4周组降低,2周时差异最为显著(p<0.01),4周时存在差异(p<0.05)；与中药外敷组相比,Mankin' s评分2周时下降明显(p<0.05)。
1.4滑膜组织形态学：空白对照组造模后2、4、8周时滑膜组织形态学明显改变；超声促透组与空白对照组和中药外敷组相比,滑膜组织形态学2、4、8周组改变,滑膜炎症反应降低。
1.5CTX-Ⅱ、COMP、MMP-3
1.5.12、4周时,超声促透组与空白对照组相比,CTX-Ⅱ含量明显降低,二者相比有显著差异(P<0.01)；8周时,超声促透组与空白对照组相比,CTX-Ⅱ含量降低,二者相比有统计学差异；(P<0.05)；与中药外敷组相比,CTX-Ⅱ含量降低明显,2、4、8周时均存在差异(P<0.05)。
1.5.2空白对照组大鼠2、4、8周时COMP逐渐增长；2、4周时,超声促透组与空白对照组相比含量明显减少(P<0.01)；4周时,超声促透组与中药外敷组相比,含量降低明显,二者相比有统计学差异(P<0.05)；8周时,超声促透组空白对照组相比无明显差异。基质金属蛋白酶-3(MMP-3)含量
1.5.32周时,超声促透组和空白对照组相比MMP-3含量降低,二者有统计学差异(P<0.05)；4周时超声促透组与空白对照组相比,MMP-3含量显著降低(P<0.01),与中药外敷组相比,含量降低明显,二者相比有统计学差异(P<0.05)；8周时,超声促透组含量少于空白对照组和中药外敷组,但无统计学差异。
2.临床实验结果：
2.1HSS评分：评分为优的患者,中药促透组治疗后评分显著增高,与治疗前相比有显著差异(P<0.01),疗效优于盐水促透组(P<0.05)。评分为良的患者,中药促透组患者治疗后HSS评分增高,与治疗前相比有统计学差异(P<0.05)。
2.2VAS评分：评分为优、良、中度的患者,中药促透组治疗后VAS评分降低,与治疗前存在统计学差异(P<0.05)。中药促透组所有患者治疗后VAS评分下降明显,与治疗前存在显著差异(P<0.01)。
2.3SF-36生存调查表：在生理机能、生理职能、社会功能与一般健康这四部分,优、良两组中药促透组患者治疗后评分增高,与治疗前相比有统计学差异(P<0.05)；躯体疼痛和精神健康这两部分中,优、良、中三组的中药促透组患者治疗后评分均高于治疗前(P<0.05)。
2.4CTX-Ⅱ、COMP、MMP-3
2.4.1与治疗前相比,中药促透组治疗后Ⅱ型胶原羧基端肽(CTX-Ⅱ)含量有所降低,同时在优、良、中三组中,中药促透组治疗后Ⅱ型胶原羧基端肽(CTX-Ⅱ)含量降低,但相互比较无统计学差异(P>0.05)。
2.4.2中药促透组中良、中两组患者治疗后与治疗前相比,COMP含量明显降低有统计学差异(P<0.05),优组治疗后COMP含量下降,但二者无统计学差异(P>0.05)。总体统计,COMP含量治疗后明显降低,与治疗前相比存在统计学差异(P<0.05)。
2.4.3中药促透组治疗后MMP-3含量明显降低,与治疗前相比存在统计学差异(P<0.05)；中药促透组优组治疗后MMP-3含量降低,与治疗前含量存在差异(P<0.05),良和中度的患者治疗后MMP-3含量下降,但二者相比无统计学差异(P>0.05)。
结论：
1.切除前交叉韧带可以造成膝关节的生物力学失衡,引起软骨退行性变进而引起KOA的发生,这一方法所造大鼠KOA模型的病变程度与时间成正相关,可以很好地模拟人类KOA的发展,制备出不同程度的KOA模型。
2.低频超声促透通络止痛中药对大鼠膝骨关节炎模型的起效作用可能是通过减缓软骨的退行性变,尤其是减缓软骨细胞外基质的降解,降低滑膜炎症所致,从而减轻膝关节局部炎性反应,改善患肢功能,其效果明显优于普通外敷法,但对于严重的膝骨关节炎作用并不明显。
3.低频超声促透通络止痛中药可以降低膝骨关节炎患者血清内CTX-Ⅱ、COMP及MMP-3含量,减轻软骨细胞外基质的降解从而减缓软骨的退行性变；能够显著改善患者VAS评分、HSS评分以及SF-36评分,尤其是能显著改善患者精神健康和躯体疼痛等,但对于严重的膝骨关节炎患者无明显疗效。
Objective:
1. Through animal experiment,we observed the influence on knee function, cartilage histology and cartilage extracellular matrix of rats with varying degrees of knee osteoarthritis (Knee osteoarthritis KOA) with Tongluozhitong medicine by low-frequency ultrasound transdermal penetration,and described the mechanism of action.
2. Patients with varying degrees of knee osteoarthritis were therapied by Tongluozhitong medicine through low-frequency ultrasound transdermal penetration.We observed the changes of biomakers of cartilage extracellular matrix,VAS score,HSS function score and the Chinese version of Easy Scale Health Survey (SF-36), and investgated the clinical efficacy and mechanisms of the external treatment.
Method:
1. Animal experiment:rat models of osteoarthritis were prepared by cutting anterior cruciate ligament,;Left knees of rats were modeling while right knees were control.84male SD rats were randomly divided into three groups:control group(18), the group of ultrasonic transdermal penetration(33) and herbal topical groups(33).All rats were prepared and detected about the behavior by recording Lequesne MG score,and CTX-Ⅱ,COMP and MMP-3in serum were detected by ELISA.The control group were normal feeding,and were randomly selected to observe changes of behavior and CTX-Ⅱ,COMP and MMP-3at2/4/8weeks after surgery.After killed,the control group were observed on General observation of knee cartilage,and synovium and medial femoral condylar cartilage morphology and were evaluated by cartilage modified Mankin's score and determine the degree of rats on KOA. Ultrasonic transdermal penetration group and herbal topical group were randomly selected for intervention treatment at the2/4/8weeks after surgery. Ultrasonic transdermal penetration group was transparentted with Tongluozhitong herbal to The operating knee by ultrasound and conductivity meter while the herbs topical group were just intervened by topical herbal.1/d,30min,2weeks.2weeks after death, the data was recorded which was as same as the control group.
2.Clinical trials:80cases of knee osteoarthritis in line with western medicine and TCM syndrome diagnostic criteria of the blood stasis syndrome were divided into four categories,excellent, good, fair and poor according to the severity of HSS score,and each category was20patients and randomly divided into2groups:group of transdermal penetration of Chinese medicine and group of transdermal penetration of saline,10patients in each group.All patients took Chinese medicine Panlong seven basis to get Huoxuetongluo therapy. The group of transdermal penetration of Chinese medicine were therapied by Tongluozhitong medicine through ultrasound penetration while The group of transdermal penetration of saline were therapied by saline through ultrasound penetration,4times/week,30min,4weeks of continuous treatment. CTX-Ⅱ, COMP and MMP-3in serum of patients were measured by ELISA and VAS score, HSS score, and SF-36scores were collected before and after the treatment, to evaluate the clinical efficacy of the various groups.
Results:
1. Animal experiment
1.1Behavior and Lequesne MG score
Behavior control group changed obviously at2,4,8week after modeling,and Lequesne MG score increasted gradually.Ultrasonic transdermal penetration group compared with the control group, Lequesne MG score decreased at2,4and8week, there was significant difference (P<0.05); compared with the herbs topical group,Lequesne MG score decreased at2,4and8week, there was significant difference (P<0.05).
1.2General observation of cartilage
Cartilage general observation of control group changed obviously,and the degree of cartilage degeneration aggravated.Ultrasonic transdermal penetration group compared with the control group, general observation of cartilage changed at2,4week,and the degree of cartilage degeneration eased;and there was no significant difference between Ultrasonic transdermal penetration group and the herbs topical group,
1.3Cartilage tissue morphology and Mankin's score
Cartilage tissue morphology of control group changed obviously at2,4,8week after modeling;Mankin's score increasted and positively related to time.Ultrasonic transdermal penetration group compared with the control group, cartilage tissue morphology changed,and Mankin's score decreased at2,4week.There was significant difference (P<0.01) at2weeks and general difference (P<0.05) at4weeks.Compared with the herbs topical group,Mankin's score of Ultrasonic transdermal penetration group decreased sharply at2weeks,(P<0.05).
1.4Synovial tissue morphology
Synovial tissue morphology of control group changed obviously at2,4,8weekS.Ultrasonic transdermal penetration group compared with the control group and herbs topical group, Synovial tissue morphology changed at2,4,8week.
1.5CTX-Ⅱ、COMP、MMP-3
1.5.1CTX-Ⅱ
Compared with the control group,CTX-Ⅱ of ultrasonic transdermal penetration group decreased sharply at2,4weeks and there was significant difference (P<0.01),and it aslo decreased at8weeks (P<0.05).Compared with the herbs topical group,CTX-II of ultrasonic transdermal penetration group decreased at2,4weeks and there was significant difference at2,4,8weeks (P<0.05).
1.5.2COMP
COMP of the control group increased gradually at2,4,8weeks.Compared with the control group,COMP of ultrasonic transdermal penetration group decreased sharply at2,4weeks and there was significant difference (P<0.01).Compared with the herbs topical group,COMP of ultrasonic transdermal penetration group decreased at4weeks (P <0.05).There was no difference between the control group and ultrasonic transdermal penetration group.
1.5.3MMP-3
Compared with the control group,MMP-3of ultrasonic transdermal penetration group decreased at2weeks and there was difference (P<0.05);compared with the control group,MMP-3of ultrasonic transdermal penetration group decreased sharply at4weeks(P <0.01). Compared with the herbs topical group,MMP-3of ultrasonic transdermal penetration group decreased at4weeks (P<0.05).At8weeks,MMP-3of ultrasonic transdermal penetration group was less than the control group and the herbs topical group,but there was no difference.
2.Clinical results
2.1HSS score
In patients rated as excellent, HSS score of transdermal penetration of Chinese medicine group increasted obviously,and there was significant difference(P<0.01),and the efficacy was better than group of transdermal penetration of saline(P<0.05).In patients rated as good, HSS score of transdermal penetration of Chinese medicine group increasted obviously,and there was significant difference compared to pretreatment.
2.2VAS score
In patients rated as excellent,good and fair,VAS score of transdermal penetration of Chinese medicine group decreasted obviously after treatment,and there was significant difference compared to pretreatment.(P<0.05).VAS score of all patients in group of transdermal penetration of Chinese medicine increasted obviously after treatment,and there was significant difference compared to pretreatment.(P<0.01).
2.3the MOS36item short from health survey, SF-36:In patients rated as excellent and good,the score of SF-36of transdermal penetration of Chinese medicine group on Physical Functioning,Role-Physical,Social Functioning and General Health increasted obviously after treatment.,and there was significant difference compared to pretreatment.(P<0.05). In patients rated as excellent, good and fair,The score of SF-36of transdermal penetration of Chinese medicine group on Bodily Pain and Mental Health increasted obviously after treatment.,and there was significant difference compared to pretreatment.(P<0.05).
2.4CTX-Ⅱ、COMP、MMP-3
2.4.1CTX-Ⅱof transdermal penetration of Chinese medicine group decreasted after treatment;and in patients rated as excellent,good and fair,CTX-Ⅱ of transdermal penetration of Chinese medicine group decreasted after treatment,but there was no significant difference compared to pretreatment(P>0.05).
2.4.2COMP of transdermal penetration of Chinese medicine group decreasted obviously after treatment,and there was significant difference compared to pretreatmentand (P<0.05).In patients rated as good and fair,COMP of transdermal penetration of Chinese medicine group decreasted after treatment,and there was significant difference compared to pretreatment (P<0.05);In patients rated as excellent,COMP of transdermal penetration of Chinese medicine group decreasted after treatment,but there was no significant difference compared to pretreatment(P>0.05).
2.4.3MMP-3of transdermal penetration of Chinese medicine group decreasted obviously after treatment,and there was significant difference compared to pretreatmentand (P<0.05).In patients rated as excellent,COMP of transdermal penetration of Chinese medicine group decreasted after treatment,and there was significant difference compared to pretreatment (P<0.05).In patients rated as good and fair,MMP-3of transdermal penetration of Chinese medicine group decreasted after treatment,but there was no significant difference compared to pretreatment(P>0.05).
Conclusion:
1. Cutting anterior cruciate ligament of the knee can make biomechanical imbalance, cause cartilage degeneration and led to the occurrence of KOA.The degree of KOA in rat models through this approach has positive correlation with time.This kind of model can be well simulated to development of human KOA and this approach can cause different degrees of KOA models.
2.Onset of effect to rat KOA models by Low-frequency ultrasound transdermal penetrating Tongluozhitong medicine may be slowing cartilage degeneration,especially slowing the degradation of the extracellular matrix of cartilage, reducing synovitis so that it can relieve knee inflammatory and improve limb function, which is better than ordinary topical method, but not obvious to severe knee osteoarthritis.
3.low-frequency ultrasound transdermal penetration of Tongluozhitong medicine can reduce the CTX-Ⅱ, COMP and MMP-3levels in serum of patients with knee osteoarthritis, slow the degradation of extracellular matrix and thus slow down the degeneration of cartilage.It is able to significantly improve VAS score, HSS score and SF-36scores of patients, especially in mental health and physical pain, but with no significant effect to severe knee osteoarthritis.
1.通过动物实验研究通络止痛中药在低频超声促透法下对大鼠不同程度膝关节骨关节炎(Knee osteoarthritis KOA)的膝关节功能、软骨组织学以及软骨细胞外基质的影响,阐述其作用机制。
2.对不同程度KOA患者进行通络止痛中药的低频超声促透治疗,观察治疗前后患者软骨细胞外基质相关生物标志物,HSS功能评分、VAS疼痛评分和(SF-36)中文版健康调查简易量表的变化,进一步探讨这一外治法的临床疗效以及作用机制。
方法：
1.动物实验：采用大鼠前交叉韧带切断的经典方法制备膝骨关节炎大鼠模型,左膝为手术侧,右膝为对照侧。84只清洁级全雄SD大鼠随机分为3组：空白对照组(18只)、超声促透组(33只)和中药外敷组(33只)。所有大鼠予以造模,术前检测行为学并记录Lequesne MG评分,应用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清中Ⅱ型胶原羧基端肽((cterminal crosslinking telopeptide of type II collagen, CTX-Ⅱ))、软骨寡聚基质蛋白(cartilage oligomeric matrix protein, COMP)及基质金属蛋白酶-3(matrix metalloproteinases-3, MMP-3)含量。空白对照组正常饲养,术后2、4、8周时分别随机选取6只大鼠观察其行为学改变并记录LequesneMG评分,应用ELISA法检测血清中CTX-Ⅱ. COMP及MMP-3含量；处死后观察大鼠膝关节软骨大体改变,股骨内侧髁软骨及滑膜的组织形态学改变,根据软骨Mankin's组织学评分予以评价,并根据评分结果确定大鼠膝骨关节炎的病变程度。超声促透组和中药外敷组在术后2、4、8周时各随机选取11只大鼠进行干预处理,超声促透组使用超声电导仪对术侧膝关节促透通络止痛中药,中药外敷组仅使用中药外敷。1次／日,30min,连续治疗2周。2周后处死,标本采集及数据记录同空白对照组。
3.临床实验：临床纳入80例符合膝关节骨关节炎诊断标准且中医辩证为瘀血阻络证的患者,并根据HSS评分将其严重程度分为优、良、中、差四级,每一级患者20例,随机分为2组：中药促透组(10例)和盐水促透组(10例)。所有患者均服用基础中成药盘龙七片进行活血通络治疗,中药促透组患者予以超声促透通络止痛中药治疗,生理盐水促透组予以超声促透生理盐水治疗,4次／周,30min,连续治疗四周。采用ELISA法测定治疗前后患者血清内CTX-Ⅱ、COMP及MMP-3含量的变化,并在治疗前、治疗后采集VAS评分、HSS评分以及SF-36评分,评估各组临床疗效。
结果：
1.动物实验：
1.1行为学Lequesne MG评分：空白对照组造模后2、4、8周时大鼠行为学明显改变,Lequesne MG评分逐渐增高；超声促透组与空白对照组相比,Lequesne MG评分在2、4、8周组时降低,有统计学差异(p<0.05)；与中药外敷组相比,Lequesne MG评分在4周时下降明显,两两相比有统计学差异(p<0.05)。
1.2软骨大体观察：空白对照组造模后2、4、8周时软骨大体观察明显改变,软骨退变程度增加；超声促透组与空白对照组相比,软骨大体观察在2、4周组发生改变,退变程度减轻；与中药外敷组相比无明显改变。
1.3软骨组织学Mankin' s评分：空白对照组造模后2、4、8周时软骨组织形态学发生明显改变,Mankin' s评分的增加与时间成正相关。超声促透组与空白对照组相比,软骨组织形态学发生改变,Mankin' s评分2、4周组降低,2周时差异最为显著(p<0.01),4周时存在差异(p<0.05)；与中药外敷组相比,Mankin' s评分2周时下降明显(p<0.05)。
1.4滑膜组织形态学：空白对照组造模后2、4、8周时滑膜组织形态学明显改变；超声促透组与空白对照组和中药外敷组相比,滑膜组织形态学2、4、8周组改变,滑膜炎症反应降低。
1.5CTX-Ⅱ、COMP、MMP-3
1.5.12、4周时,超声促透组与空白对照组相比,CTX-Ⅱ含量明显降低,二者相比有显著差异(P<0.01)；8周时,超声促透组与空白对照组相比,CTX-Ⅱ含量降低,二者相比有统计学差异；(P<0.05)；与中药外敷组相比,CTX-Ⅱ含量降低明显,2、4、8周时均存在差异(P<0.05)。
1.5.2空白对照组大鼠2、4、8周时COMP逐渐增长；2、4周时,超声促透组与空白对照组相比含量明显减少(P<0.01)；4周时,超声促透组与中药外敷组相比,含量降低明显,二者相比有统计学差异(P<0.05)；8周时,超声促透组空白对照组相比无明显差异。基质金属蛋白酶-3(MMP-3)含量
1.5.32周时,超声促透组和空白对照组相比MMP-3含量降低,二者有统计学差异(P<0.05)；4周时超声促透组与空白对照组相比,MMP-3含量显著降低(P<0.01),与中药外敷组相比,含量降低明显,二者相比有统计学差异(P<0.05)；8周时,超声促透组含量少于空白对照组和中药外敷组,但无统计学差异。
2.临床实验结果：
2.1HSS评分：评分为优的患者,中药促透组治疗后评分显著增高,与治疗前相比有显著差异(P<0.01),疗效优于盐水促透组(P<0.05)。评分为良的患者,中药促透组患者治疗后HSS评分增高,与治疗前相比有统计学差异(P<0.05)。
2.2VAS评分：评分为优、良、中度的患者,中药促透组治疗后VAS评分降低,与治疗前存在统计学差异(P<0.05)。中药促透组所有患者治疗后VAS评分下降明显,与治疗前存在显著差异(P<0.01)。
2.3SF-36生存调查表：在生理机能、生理职能、社会功能与一般健康这四部分,优、良两组中药促透组患者治疗后评分增高,与治疗前相比有统计学差异(P<0.05)；躯体疼痛和精神健康这两部分中,优、良、中三组的中药促透组患者治疗后评分均高于治疗前(P<0.05)。
2.4CTX-Ⅱ、COMP、MMP-3
2.4.1与治疗前相比,中药促透组治疗后Ⅱ型胶原羧基端肽(CTX-Ⅱ)含量有所降低,同时在优、良、中三组中,中药促透组治疗后Ⅱ型胶原羧基端肽(CTX-Ⅱ)含量降低,但相互比较无统计学差异(P>0.05)。
2.4.2中药促透组中良、中两组患者治疗后与治疗前相比,COMP含量明显降低有统计学差异(P<0.05),优组治疗后COMP含量下降,但二者无统计学差异(P>0.05)。总体统计,COMP含量治疗后明显降低,与治疗前相比存在统计学差异(P<0.05)。
2.4.3中药促透组治疗后MMP-3含量明显降低,与治疗前相比存在统计学差异(P<0.05)；中药促透组优组治疗后MMP-3含量降低,与治疗前含量存在差异(P<0.05),良和中度的患者治疗后MMP-3含量下降,但二者相比无统计学差异(P>0.05)。
结论：
1.切除前交叉韧带可以造成膝关节的生物力学失衡,引起软骨退行性变进而引起KOA的发生,这一方法所造大鼠KOA模型的病变程度与时间成正相关,可以很好地模拟人类KOA的发展,制备出不同程度的KOA模型。
2.低频超声促透通络止痛中药对大鼠膝骨关节炎模型的起效作用可能是通过减缓软骨的退行性变,尤其是减缓软骨细胞外基质的降解,降低滑膜炎症所致,从而减轻膝关节局部炎性反应,改善患肢功能,其效果明显优于普通外敷法,但对于严重的膝骨关节炎作用并不明显。
3.低频超声促透通络止痛中药可以降低膝骨关节炎患者血清内CTX-Ⅱ、COMP及MMP-3含量,减轻软骨细胞外基质的降解从而减缓软骨的退行性变；能够显著改善患者VAS评分、HSS评分以及SF-36评分,尤其是能显著改善患者精神健康和躯体疼痛等,但对于严重的膝骨关节炎患者无明显疗效。
Objective:
1. Through animal experiment,we observed the influence on knee function, cartilage histology and cartilage extracellular matrix of rats with varying degrees of knee osteoarthritis (Knee osteoarthritis KOA) with Tongluozhitong medicine by low-frequency ultrasound transdermal penetration,and described the mechanism of action.
2. Patients with varying degrees of knee osteoarthritis were therapied by Tongluozhitong medicine through low-frequency ultrasound transdermal penetration.We observed the changes of biomakers of cartilage extracellular matrix,VAS score,HSS function score and the Chinese version of Easy Scale Health Survey (SF-36), and investgated the clinical efficacy and mechanisms of the external treatment.
Method:
1. Animal experiment:rat models of osteoarthritis were prepared by cutting anterior cruciate ligament,;Left knees of rats were modeling while right knees were control.84male SD rats were randomly divided into three groups:control group(18), the group of ultrasonic transdermal penetration(33) and herbal topical groups(33).All rats were prepared and detected about the behavior by recording Lequesne MG score,and CTX-Ⅱ,COMP and MMP-3in serum were detected by ELISA.The control group were normal feeding,and were randomly selected to observe changes of behavior and CTX-Ⅱ,COMP and MMP-3at2/4/8weeks after surgery.After killed,the control group were observed on General observation of knee cartilage,and synovium and medial femoral condylar cartilage morphology and were evaluated by cartilage modified Mankin's score and determine the degree of rats on KOA. Ultrasonic transdermal penetration group and herbal topical group were randomly selected for intervention treatment at the2/4/8weeks after surgery. Ultrasonic transdermal penetration group was transparentted with Tongluozhitong herbal to The operating knee by ultrasound and conductivity meter while the herbs topical group were just intervened by topical herbal.1/d,30min,2weeks.2weeks after death, the data was recorded which was as same as the control group.
2.Clinical trials:80cases of knee osteoarthritis in line with western medicine and TCM syndrome diagnostic criteria of the blood stasis syndrome were divided into four categories,excellent, good, fair and poor according to the severity of HSS score,and each category was20patients and randomly divided into2groups:group of transdermal penetration of Chinese medicine and group of transdermal penetration of saline,10patients in each group.All patients took Chinese medicine Panlong seven basis to get Huoxuetongluo therapy. The group of transdermal penetration of Chinese medicine were therapied by Tongluozhitong medicine through ultrasound penetration while The group of transdermal penetration of saline were therapied by saline through ultrasound penetration,4times/week,30min,4weeks of continuous treatment. CTX-Ⅱ, COMP and MMP-3in serum of patients were measured by ELISA and VAS score, HSS score, and SF-36scores were collected before and after the treatment, to evaluate the clinical efficacy of the various groups.
Results:
1. Animal experiment
1.1Behavior and Lequesne MG score
Behavior control group changed obviously at2,4,8week after modeling,and Lequesne MG score increasted gradually.Ultrasonic transdermal penetration group compared with the control group, Lequesne MG score decreased at2,4and8week, there was significant difference (P<0.05); compared with the herbs topical group,Lequesne MG score decreased at2,4and8week, there was significant difference (P<0.05).
1.2General observation of cartilage
Cartilage general observation of control group changed obviously,and the degree of cartilage degeneration aggravated.Ultrasonic transdermal penetration group compared with the control group, general observation of cartilage changed at2,4week,and the degree of cartilage degeneration eased;and there was no significant difference between Ultrasonic transdermal penetration group and the herbs topical group,
1.3Cartilage tissue morphology and Mankin's score
Cartilage tissue morphology of control group changed obviously at2,4,8week after modeling;Mankin's score increasted and positively related to time.Ultrasonic transdermal penetration group compared with the control group, cartilage tissue morphology changed,and Mankin's score decreased at2,4week.There was significant difference (P<0.01) at2weeks and general difference (P<0.05) at4weeks.Compared with the herbs topical group,Mankin's score of Ultrasonic transdermal penetration group decreased sharply at2weeks,(P<0.05).
1.4Synovial tissue morphology
Synovial tissue morphology of control group changed obviously at2,4,8weekS.Ultrasonic transdermal penetration group compared with the control group and herbs topical group, Synovial tissue morphology changed at2,4,8week.
1.5CTX-Ⅱ、COMP、MMP-3
1.5.1CTX-Ⅱ
Compared with the control group,CTX-Ⅱ of ultrasonic transdermal penetration group decreased sharply at2,4weeks and there was significant difference (P<0.01),and it aslo decreased at8weeks (P<0.05).Compared with the herbs topical group,CTX-II of ultrasonic transdermal penetration group decreased at2,4weeks and there was significant difference at2,4,8weeks (P<0.05).
1.5.2COMP
COMP of the control group increased gradually at2,4,8weeks.Compared with the control group,COMP of ultrasonic transdermal penetration group decreased sharply at2,4weeks and there was significant difference (P<0.01).Compared with the herbs topical group,COMP of ultrasonic transdermal penetration group decreased at4weeks (P <0.05).There was no difference between the control group and ultrasonic transdermal penetration group.
1.5.3MMP-3
Compared with the control group,MMP-3of ultrasonic transdermal penetration group decreased at2weeks and there was difference (P<0.05);compared with the control group,MMP-3of ultrasonic transdermal penetration group decreased sharply at4weeks(P <0.01). Compared with the herbs topical group,MMP-3of ultrasonic transdermal penetration group decreased at4weeks (P<0.05).At8weeks,MMP-3of ultrasonic transdermal penetration group was less than the control group and the herbs topical group,but there was no difference.
2.Clinical results
2.1HSS score
In patients rated as excellent, HSS score of transdermal penetration of Chinese medicine group increasted obviously,and there was significant difference(P<0.01),and the efficacy was better than group of transdermal penetration of saline(P<0.05).In patients rated as good, HSS score of transdermal penetration of Chinese medicine group increasted obviously,and there was significant difference compared to pretreatment.
2.2VAS score
In patients rated as excellent,good and fair,VAS score of transdermal penetration of Chinese medicine group decreasted obviously after treatment,and there was significant difference compared to pretreatment.(P<0.05).VAS score of all patients in group of transdermal penetration of Chinese medicine increasted obviously after treatment,and there was significant difference compared to pretreatment.(P<0.01).
2.3the MOS36item short from health survey, SF-36:In patients rated as excellent and good,the score of SF-36of transdermal penetration of Chinese medicine group on Physical Functioning,Role-Physical,Social Functioning and General Health increasted obviously after treatment.,and there was significant difference compared to pretreatment.(P<0.05). In patients rated as excellent, good and fair,The score of SF-36of transdermal penetration of Chinese medicine group on Bodily Pain and Mental Health increasted obviously after treatment.,and there was significant difference compared to pretreatment.(P<0.05).
2.4CTX-Ⅱ、COMP、MMP-3
2.4.1CTX-Ⅱof transdermal penetration of Chinese medicine group decreasted after treatment;and in patients rated as excellent,good and fair,CTX-Ⅱ of transdermal penetration of Chinese medicine group decreasted after treatment,but there was no significant difference compared to pretreatment(P>0.05).
2.4.2COMP of transdermal penetration of Chinese medicine group decreasted obviously after treatment,and there was significant difference compared to pretreatmentand (P<0.05).In patients rated as good and fair,COMP of transdermal penetration of Chinese medicine group decreasted after treatment,and there was significant difference compared to pretreatment (P<0.05);In patients rated as excellent,COMP of transdermal penetration of Chinese medicine group decreasted after treatment,but there was no significant difference compared to pretreatment(P>0.05).
2.4.3MMP-3of transdermal penetration of Chinese medicine group decreasted obviously after treatment,and there was significant difference compared to pretreatmentand (P<0.05).In patients rated as excellent,COMP of transdermal penetration of Chinese medicine group decreasted after treatment,and there was significant difference compared to pretreatment (P<0.05).In patients rated as good and fair,MMP-3of transdermal penetration of Chinese medicine group decreasted after treatment,but there was no significant difference compared to pretreatment(P>0.05).
Conclusion:
1. Cutting anterior cruciate ligament of the knee can make biomechanical imbalance, cause cartilage degeneration and led to the occurrence of KOA.The degree of KOA in rat models through this approach has positive correlation with time.This kind of model can be well simulated to development of human KOA and this approach can cause different degrees of KOA models.
2.Onset of effect to rat KOA models by Low-frequency ultrasound transdermal penetrating Tongluozhitong medicine may be slowing cartilage degeneration,especially slowing the degradation of the extracellular matrix of cartilage, reducing synovitis so that it can relieve knee inflammatory and improve limb function, which is better than ordinary topical method, but not obvious to severe knee osteoarthritis.
3.low-frequency ultrasound transdermal penetration of Tongluozhitong medicine can reduce the CTX-Ⅱ, COMP and MMP-3levels in serum of patients with knee osteoarthritis, slow the degradation of extracellular matrix and thus slow down the degeneration of cartilage.It is able to significantly improve VAS score, HSS score and SF-36scores of patients, especially in mental health and physical pain, but with no significant effect to severe knee osteoarthritis.
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