丹滲注射液關(guān)節(jié)內(nèi)注射預(yù)防關(guān)節(jié)粘連的實(shí)驗(yàn)研究

1、丹滲注射液關(guān)節(jié)內(nèi)注射預(yù)防關(guān)節(jié)粘連的實(shí)驗(yàn)研究        【摘要】     目的探討預(yù)防性關(guān)節(jié)內(nèi)注射丹參注射液對關(guān)節(jié)粘連的影響。方法24只日本雄性大耳白兔隨機(jī)分為3組，每組8只，分別為鹽水對照組（A組）、玻璃酸鈉對照組（B組）、丹參治療組（C組）。手術(shù)切開右后膝關(guān)節(jié)，搜刮髕上囊前壁，切除大部分髕上囊后壁滑膜和髕下脂肪墊，術(shù)畢用長腿管形石膏固定右后肢膝關(guān)節(jié)于伸直位。每只動(dòng)物采用的藥物品種及劑量分別是：A組注生理鹽水0.3 ml、B組注玻璃酸鈉0.3 ml、C組注丹滲注

2、射液0.3 ml，共4次。4周后空氣栓塞處死動(dòng)物，行關(guān)節(jié)活動(dòng)度測量、關(guān)節(jié)腔大體觀察、髕上囊滑膜組織的組織學(xué)觀察和總膠原含量檢測。結(jié)果關(guān)節(jié)活動(dòng)度B、C組大于A組（P<0.01),B、C組之間差異無顯著性（P>0.05);關(guān)節(jié)粘連計(jì)分B、C組均于小A組（P<0.01），B、C組之間差異無顯著性（P>0.05);髕上囊前壁滑膜病變組織學(xué)計(jì)分B、C、組均小于A組（P<0.01），B、C組之間差異無顯著性（P>0.05)；髕上囊前壁滑膜組織總膠原含量B、C組小于A組（P<0.01)。結(jié)論丹參注射液關(guān)節(jié)內(nèi)注射能有效預(yù)防關(guān)節(jié)粘連，且與玻璃酸鈉的效果類似，這可能與丹參

3、通過多種途徑減少組織的膠原含量和/或降低組織損傷修復(fù)的病理反應(yīng)有關(guān)。     【關(guān)鍵詞】  關(guān)節(jié)粘連預(yù)防 丹參關(guān)節(jié)內(nèi)注射 膝關(guān)節(jié)兔    Prophylactic effects of intra-articular injection of  salvia miltiorrhiza on joint adhesions in rabbit knee jointsDUAN Kan(段戡), YUAN Changshen (袁長深), YAO Hongyi(姚弘毅), LIU Yuxiang (劉

4、宇翔), LIU Zhengzhi(劉政治)Department of Musculoskeletal diseases,the first Affiliated Hospital of Guangxi TCM University,Dongge Road 89-9,Nanning,530023,China    Abstract:ObjectiveTo investigate the prophylactic effects of intra-articular injection of salvia miltiorrhiza on joint adhesion

5、. MethodsTwentyfour Japanese male rabbits were randomly divided into 3 groups.All right hind knees were made in this way: the auterior wall of suprapatellar bursae was scraped, the posterior wall of suprapatellar bursae and the infrapatellar fat pad were cut off. Each knee was injected with physiolo

6、gical saline 0.3 ml for group A, sodium hyaluronate injection 0.3 ml for group B, and salvia miltiorrhiza injection 0.3 ml for group C. All right hind knees were immobilized in the extension position with a long leg plaster cast for 4 weeks, and injected weekly.ResultsAfter 4 weeks, the joint moveme

7、nt range in group B or C was larger than that in group A (P<0.01), and there was no significant difference （P>0.05）compared to group B and group C. The scoring of joint adhesions in group B or C was smaller than that in group A (P<0.01), and there was no significant difference（P>0.05）com

8、pared to group B and group C. The scoring of histological changed of the synovial membrane in group B or C was smaller than that in group A (P<0.01), and there was no significant difference（P>0.05）compared to group B and group C.Total collagens content of the synovial membrane in group B or C

9、was smaller than that in group A (P<0.01), while that of group C was less than in group B(P<0.01). ConclusionSimilarly to sodium hyaluronate, salvia miltiorrhiza can effectively prevent joint adhesions.Key words:joint (intraarticular) adhesions/prevention;   salvia  miltiorrhiza

10、/intraarticular  injection;  knee joints/rabbitsChinese Library Classification Number:R687    Document Code：A    Article ID：1005-8478（2009）22-1739-04The joint adhesion is a common complication caused by periarticular injuries, postoperative joints or immobiliz

11、ation joints, which is the jointinternal reason to cause fibroanchylosis. Once joint adhesion is completed, it becomes difficult to treat.Therefore, preventing joint adhesion becomes very important. It was found that Salvia miltiorrhiza injection could prevent joint adhesions in ours preliminary stu

12、dy1. The author have carried out the further research to prevent joint adhesion by improving the model method, increasing injection frequency and adjusting observation target.1  Materials and MethodsThe experiment groups:Twentyfour adult Japanese white rabbits (male: weighting 2.02.5kg, provide

13、d by experimental animal center of Guangxi TCM university and the licence number is SYXK Gui 20030001) were randomly divided into 3 groups: group A (physiological saline control group), group B (sodium hyaluronate control group), group C (salvia militiorrhiza treatment group), each group of 8, numbe

14、ring respectively.Modeling and processing: The chemistry pure urethane was matched into 25% solution with 0.9% physiological saline. All rabbits were anesthetic by auricular vein injection in according to 1g·kg-1 weighing (4ml·kg-1 weighing). Referencing Ye Genmao et al2, the knee joint of

15、 the right hind limb was treated as follows: after having cut off the body wool around the knee joint, iodophor was used for disinfection of skin. Then patellar medial of right knee joint was incised approximately 3 cm in sterile condition. Cut joint capsule, turnovered patellar outward and scraped

16、the anterior wall of suprapatellar bursae after that. Meanwhile, the anterior wall of suprapatellar bursae was stripped from the femur and the infrapatellar fat pad behind patellar ligaments was cut off. Then the joint was punctured after having sutured the articular capsule and the skin. Finally, t

17、he medicine was injected into the joint cavity after pulling out intraarticular hemorrhage. Each rabbit was injected with: physiological saline 0.3 ml for group A, sodium hyaluronate injection 0.3 ml for group B(Shandong bausch and Lomb Freda Pharmaceutical Limited Company, number: 060530022), and s

18、alvia miltiorrhiza injection 0.3 ml for group C (Shanghai First Biochemistry Medicine Limited Company, number: 060804). After operation, the right knee was immobilized in continuous extension position with a long leg plaster cast for 4 weeks, and injected weekly. Anterior gypsum of the knee joint wa

19、s opened into a window in order to inject medicine. The opposite limb didnt given the intervention. All rabbits were raised in separate cages. In this period, all rabbits were given intramuscular injection Gentamicin 20,000 U, 2 times per day, a total of 3days. After four weeks, the rabbits were exe

20、cuted with air embolism. Every rabbits joint movement range was measured after demolishing gypsum. Then the joint cavity was roughly observed, and the synovial membrane of the anterior wall of suprapatellar bursae (in duplicate) was gained for histologic evaluation and total collagens content.2 

21、; Observations and methodsJoint movement range measuring: When the experiment was finished, all joint movement range of rabbits was measured. Fixed right thigh near on desk, pulled the knee joint with the 500g pulling force under 10cm place for 5 minute, and measured the knee joint flexure angle wit

22、h a long handle joint activity measuring instrument. That is namely for joint activity.Joints adhesion roughly observing: Joint movement range measured, the joint cavity was observed. Classified by the grading system introduced by Ye Genmao et al3, the degree of adhesion was divided into 5 grades an

23、d respectively counted 04 scoring. Grade 0: no adhesion scores 0. Smooth synovial, suprapatellar bursa and the joint cavity size is normal; Grade 1: weak adhesion scores 1. Synovial membrane is mild dropsy, suprapatellar bursa reduces approximately 1/4, and slight filiform adhesion disperses in the

24、joint cavity; Grade 2: mild adhesion scores 2. Synovial membrane is moderate dropsy, suprapatellar bursa reduces approximately 1/2, and much filiform adhesion disperses in the joint cavity; Grade 3: moderately dense adhesion scores 3. Synovial membrane is the obvious dropsy, suprapatellar bursa redu

25、ces approximately 3/4, and much comparative filiform adhesion disperses in the joint cavity; Grade 4: dense and fibrous adhesion scores 4. suprapatellar bursa is nearly vanishing, compact adhesion in joint cavity.Histology evaluation: After roughly observed, one anterior wall of suprapatellar bursa

26、tissue was immediately cut. The selected specimens were fixed in 4% paraformaldehyde solution for 2448h. Then, the specimen was dehydrated in alcohol series, embedded in paraffin and made into a 3umthick sliced section. The sections were stained using hematoxyline and eosin(HE), and were histologica

27、lly analyzed. Suprapatellar bursa tissue was classified by pathological changed to divided into 4 grades, and respectively counted 03 scoring. Grade 0: the synovial membrane integrity, the cell arrangement is neat, nonhyperemia dropsy, infiltrates without the inflammatory cells; Grade 1: the synovia

28、l hyperemia dropsy, the surface layer cell proliferation; Grade 2: the synovial cell assumes papillary proliferation, the blood vessel proliferation in synovial and the granulation tissue forming; Grade 3: the granulation tissue fibrosis in synovial, the partner has been infiltrated by the lymphocyt

29、e and the cytoplasmic cell.Total collagens content determination:The other specimens 35 mg stored in -20 refrigerator would be strictly completed by the hydroxyproline test according to laboratory procedure by providing Nanjing Jiancheng biological research institute (number:20061116). The total col

30、lagens content was converted according to 13.4% hydroxyprolines in the collagen.Statistical methods:All data was analyzed with SPSS11.0 software and represented as ±s.3  ResultsGeneral conditions:All animals health condition was good with no diarrhea, no death in formal experimental period

31、. Different extent swelling occurred in partial animals in the affected side foot fixed after 3 days (loosen farend partial gypsum for severe situation) was vanishing after 1 week. Patched promptly or the replaced the gypsum which the rabbits gnawed and damaged seriously to ensure fixation effective

32、 every day.Joint movement range measurement: Table 1 shows each group of the joint movement range. The statistical result showed that the joint movement range of the used medicine groups (group B, C) is bigger than that of the physiological saline control group (group A)（P<0.01）. There was no sig

33、nificant difference compared to group B and group C（P>0.05）(Table 1).Table 1  groupsnumberJoint（略）Joints adhesions roughly observe: Table 1 shows each group of joint adhesions scoring. The statistical result showed that the joint adhesions scoring of the used medicine groups (group B, C) is

34、smaller than that of the physiological saline control group (group A)（P<0.01）,which indicated that joint adhesions degree of the used medicine each group is lighter than the physiological saline control group, and there was no significant difference compared to group B and group C（P>0.05）Histo

35、logy evaluation:  Histology evaluation such as graph（graph:1-3）；Table 1 shows each group of synovial membrane pathological changed score. The statistical result showed that the pathological changed scoring of the used medicine groups (group B, C) is smaller than that of the physiological saline

36、 control group (group A)（P<0.01）, which indicated that the synovial membrane pathological changed degree of the used medicine each group of is lighter than that of the physiological saline control group; and there was no significant difference compared to group B and group C（P>0.05）Total colla

37、gens content: Table 1 shows each group of total collagens content of the synovial membrane. The statistical result showed that after 4 weeks, the total collagens content of the used medicine groups (group B, C) is lower than that of the physiological saline control group (group A) （P<0.01）, and T

38、he group C is lower than group B（P<0.01）,which indicated that the intraarticular injection of salvia miltiorrhiza on joint adhesions in experimental rabbits can be able to reduce significantly the total collagens content of adhesion tissues compared to sodium hyaluronate injection.(Fig.1-3)4 

39、; DiscussionThe joint adhesions formation as tissue fibrosis is the repaired matter of intraarticular injured tissue, which starts from inflammatory response with the infiltration of neutral granular cell，the migration of macrophage, then the appearance of fibroblast and deposition of collagen. Afte

40、r that, it results to a series of adhesion pathological changes of intraarticular tissues and causes to different extent flexionextension dysfunction of knee joint3.With clear chemical constitution, longer clinical application, The salvia miltiorrhiza has the multifunction of improving the microcirc

41、ulation, antiinflammation, antioxidation, antifibrosis, and promoting to repair the injured tissues and so on4. It can prevent all kinds of tissues and organs adhesion, such like epidural scar adhesion. Although there was no significant difference compared treatment group (salvia militiorrhiza) and

42、control group (sodium hyaluronate) in the joint movement range and the joint adhesions scoring (it meant they had similar effect to prevent and control joint adhesion), total collagens content of the synovial membrane in treatment group (salvia militiorrhiza) was less than sodium hyaluronate control

43、 group. The salvia militiorrhiza could reduce the synovium lesions score, the same as sodium hyaluronate, which indicated that the main mechanism on salvia militiorrhiza preventing joint adhesions.The salvia miltiorrhiza can prevent joint adhesions probably by three ways: First, the salvia miltiorrh

44、iza can improve microcirculation and enhance tissue collagenase activity, reduce collagen deposition and promote collagen degradation and reabsorption. In general observation, collagen fibers group A were thick, disorder and irregular, and collagen fibers in group B. C were smaller, disorder and reg

45、ular in this experiment, which indicated the preventable mechanism might be relative to oversynthesis of  inhibiting collagen. Second, the salvia miltiorrhiza can prevent adhesion by remarkably suppressing fibroblast multiplication and reducing extracellular matrix of collagen synthesis5.The re

46、search also demonstrated that the salvia miltiorrhiza can remarkably reduce synovial membrane pathological change score, and reduce damaged synovial membrane to repair later period granulation tissues fibrosis and scar formation. Third, Some people considered that joint fibrosis should closely corre

47、late to T cell induced immune response6, but salvia's antiinflammation and immunity control action4 are possibly the reason to reduce synovial membrane pathological change score.Lastly, hydroxyproline is a collagenogenic of collagen, which can reflect metabolism. The collagen contents permanent

48、hydroxyproline, and the collagen content converted by hydroxyproline content is a crucial factor to mechanical strength of the repair of injured tissues, as well as one effective target to measure adhesion degree. The salvia miltiorrhiza can reduce collagen synthesis by dropping 3Hhydroxyproline incorporating to prevent effectively joint from adhesion.In summary, the salvia