黄原胶注射液的研制及其对实验性骨关节炎疗效和作用机制的研究
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摘要
骨关节炎(osteoarthritis, OA)是以关节软骨的破坏和骨质增生为特点的最常见的慢性骨关节病,是导致老年人关节疼痛及功能障碍的主要原因之一。OA的发病机制尚不清楚,一般认为是由多因素参与的复杂病变过程,与年龄、创伤、炎症、职业、肥胖、代谢和遗传等有关。OA的治疗方法主要包括手术、药物和辅助治疗3种。其中,药物治疗是早中期OA的首选方法。多年基础和临床研究证明,关节腔内注射玻璃酸钠(sodium hyaluronate, SH)对OA的疗效显著。关节腔内注射SH可起到润滑和缓冲作用,减少关节组织间摩擦,而且SH还可以减轻和缓解疼痛。但SH不稳定,易被体内酶和自由基降解,作用时间较短,反复的关节腔内注射还可能造成关节感染。因此寻找黏弹性高、稳定性更好的药物关节腔内注射治疗OA具有重要意义。黄原胶(xanthan gum, XG)是一种经发酵生产的微生物胞外杂多糖,相对分子质量(relative molecular mass, Mr)为2×106-2×107。由于XG与SH都属于多糖类药物,性质相近,黏弹性高,关节腔内注射XG也可起到润滑和缓冲作用。而且XG不易被体内酶和自由基降解,稳定性更好,在关节腔的存留时间更长,可减少注射次数,避免关节感染。因此XG有可能成为治疗OA新药。本课题确定了XG工业化发酵工艺和注射用XG原料药制备工艺,生产出高Mr、高质量的注射用XG原料药；确定了XG注射液配方,制备了高质量的XG注射液；通过体内和体外实验评价了XG注射液的安全性；制备了体内和体外OA模型,考察了XG注射液对实验性OA的疗效和作用机制。
1注射用黄原胶原料药的制备工艺及质量控制
目的：确定XG工业化发酵工艺和注射用XG原料药制备工艺,建立注射用XG原料药质量控制方法。方法：以发酵液黏度及产胶量为考察指标,在10L发酵罐中对碳源浓度和接种量进行优化,确定最终发酵工艺条件,并进行50L规模的中试放大以及10t规模的工业化试产。先确定注射用XG原料药制备的主要步骤,然后通过正交试验考察影响每个步骤纯化效果的关键因素,确定最佳制备工艺。参考国内外已有的药用辅料级XG质量标准及与XG性质、用途相似的注射用SH原料药质量标准,确定注射用XG原料药质量检查项目及标准草案。结果：优化后的发酵培养基及工业化发酵工艺条件为：淀粉5%,硫酸铵0.1%,豆饼粉0.3%,柠檬酸0.1%,硫酸镁0.01%,碳酸钙0.02%,接种量5%,温度28.5℃,pH7.0。注射用XG原料药制备的主要步骤为：XG粗品制备,粗品溶解,硅藻土吸附,除菌过滤,酶解,活性炭吸附,除碳过滤,微孔滤膜精滤,醇沉,干燥得精制品。主要的质量检查项目为：性状、鉴别、检查(溶液的澄清度与颜色、酸碱度、黏度、丙酮酸、吸光度、Mr与Mr分布、蛋白质含量、氮含量、异丙醇残留量、干燥失重、灰分、重金属、砷盐、铅含量、细菌内毒素和微生物限度)和含量测定。质量检查结果表明,注射用XG原料药各项指标均达到或优于已有的XG质量标准及与XG性质、用途相似的注射用SH原料药的质量标准,达到注射用原料药要求。结论：确定了XG工业化发酵工艺,在放大试产过程中XG的产量及质量稳定。确定了注射用XG原料药制备工艺,制备了符合注射用要求的高质量、低蛋白质含量的XG原料药。确定了注射用XG原料药质量检查项目及相应的标准。
2黄原胶注射液的制备工艺及质量控制
目的：确定XG注射液配方及制备工艺,建立XG注射液质量控制方法。方法：通过配方筛选确定XG注射液的配方及配制工艺。通过热稳定性考察,确定XG注射液的灭菌方法。采用间羟基联苯比色法测定XG注射液的含量,并对该方法进行方法学验证。参考与XG性质、用途相似的SH注射液质量标准,确定质量检查项目及标准。结果：XG注射液的配方成分为：1%黄原胶、0.5%氯化钠、0.44%磷酸二氢钠和0.7%磷酸氢二钠。XG注射液的灭菌方法为：121℃灭菌15min。间羟基联苯比色法可快速准确地测定XG注射液的标示量百分含量,该法操作简单且可靠性好。主要的质量检查项目为：性状、鉴别、检查(pH值、吸光度、渗透压摩尔浓度、Mr与Mr分布、细菌内毒素及注射剂项下的各项目检查)和含量测定。质量检查结果表明,XG注射液的各项考察指标均达到或优于与XG性质、用途相似的SH注射液质量标准。结论：确定了XG注射液的配方、制备工艺及含量测定方法,制备了高质量XG注射液。确定了XG注射液质量检查项目及标准,该质量控制方法简单准确,可全面反映产品质量。
3黄原胶注射液的安全性评价
目的：评价兔膝骨关节腔注射XG的安全性及XG对兔软骨细胞的细胞毒性。方法：在兔膝骨关节腔注射2%、1%和0.5%(w/v)的XG注射液及等量生理盐水(normal saline, NS),注射剂量为兔每千克体重0.1mL,每周注射1次,连续5周。检测兔膝关节宽度、血液学和血液生化参数并观察肝、肾、膝关节组织病理学变化。采用机械分离法和酶消化法从兔膝关节软骨中分离软骨细胞,在含10%胎牛血清的DMEM/F12培养液中培养细胞,采用甲苯胺蓝、番红-O和Ⅱ型胶原免疫荧光法鉴定软骨细胞。经不同浓度XG(10-2000μg/mL)作用后,采用四甲基偶氮唑盐(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide, MTT)法检测软骨细胞增殖,用酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)试剂盒检测细胞培养液中基质金属蛋白酶(matrix metalloproteinase, MMP)和基质金属蛋白酶组织抑制剂1(tissue inhibitor of metal protease-1,TIMP-1)的含量。结果：与NS组比,不同剂量XG组兔膝关节宽度、血液学及血液生化参数等均无明显改变(P>0.05)；肝、肾及关节组织未见明显病理改变(P>0.05)。本课题成功分离和培养了兔软骨细胞,培养的细胞形态及生长情况均符合软骨细胞特征。细胞被甲苯胺蓝染成蓝色,被番红-O染成红色且在胞浆及胞膜Ⅱ型胶原免疫荧光染色阳性表达,表明培养的细胞为软骨细胞。兔软骨细胞在含10-2000μg/mL的XG培养液中生长活跃,与在不含XG培养液中培养的细胞比,细胞增殖率并未有明显变化(P>0.05),培养液中MMP-1、-3、-13及TIMP-1的含量也未发生明显改变(P>0.05)。结论：在兔膝关节腔注射XG(0.5%-2%)0.1mL/kg,每周1次,连续5周,全身及局部未见明显毒性作用。XG(10-2000μg/mL)不会对体外培养的兔软骨细胞产生明显细胞毒作用。
4黄原胶注射液对实验性骨关节炎的药效和作用机制研究
目的：制备体内和体外OA模型,考察XG对实验性OA的疗效和作用机制。方法：将实验兔随机分为4组,分别在第1、3和5d于右膝关节腔注射2%、5%、10%(w/v)木瓜蛋白酶和0.03mol/L L-半胱氨酸混合溶液0.1mL/kg以及等量NS(空白对照组)。整个实验过程观察兔状态并记录体重和膝关节宽度。首次注射后第2、4、6周将兔分批处死,取造模侧股骨髁、胫骨平台及滑膜做大体及组织病理学观察,并进行评分,评价不同浓度木瓜蛋白酶引起的兔膝OA模型。将实验大鼠随机分为4组,于左膝关节腔注射含碘乙酸钠(monosodium iodoacetate, MIA)3mg、2mg、1mg的NS溶液60μL及等量NS(空白对照组)。造模后分别于第3、7、14、21、28、35、42、49d进行大鼠疼痛行为学测试,包括承重测试和机械缩足阈值(paw withdrawal threshold, PWT)测试。处死大鼠后取造模侧股骨髁和胫骨平台做大体及组织病理学观察,并进行评分,评价不同剂量MIA引起的大鼠膝OA模型。将实验兔右膝关节腔注射2%木瓜蛋白酶造模。造模后将兔分为3组：NS组兔右膝关节腔注射NS,0.1mL/kg,1次/周,连续5周；SH组兔右膝关节腔注射1%SH,0.1mL/kg,1次/周,连续5周；XG组兔在第2和4周右膝关节腔注射1%XG,0.1mL/kg,在第1、3和5周注射等量NS；所有兔左膝关节腔注射NS,0.1mL/kg,1次/周,连续5周,作为假手术组(sham组)。整个实验过程观察动物状态,测量造模侧膝关节宽度。治疗结束后用ELISA法检测关节滑液中白介素1p(interleukin-1, IL-1β)、肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)及前列腺素E2(prostaglandin E2, PG E2)的含量,用硝酸还原酶法检测关节滑液中一氧化氮(nitric oxide, NO)的含量,对股骨髁和胫骨平台软骨及滑膜做大体及组织病理学观察并评分,用二甲基亚甲蓝法测定关节软骨中糖胺聚糖(glycosaminoglycan, GAG)的含量,用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleoitidyl transferase mediated nick end labeling, TUNEL)评价关节软骨中的细胞凋亡率,用免疫组化及Western-blot法测定关节软骨中MMP及TIMP-1的蛋白表达,通过RT-PCR法测定关节软骨中MMP及TIMP-1的mRNA含量,综合评价XG注射液对兔膝OA的药效和作用机制。用MIA3mg诱导大鼠OA模型。造模后将大鼠分为4组,分别为注射XG2次组,注射SH2次组,注射SH5次组和注射NS对照组。造模后第14、35d,XG2次组与SH2次组分别注射XG和SH注射液(1%,w/v)0.1mL,第21、28、42d注射等量NS。SH5次组与NS组于造模后第14、21、28、35、42d分别注射SH和NS。采用大鼠模型研究中的方法对各组疼痛指标及关节软骨破坏程度进行评估,评价XG对大鼠OA关节软骨的保护作用及对疼痛的缓解作用。采用小鼠扭体试验和毛细血管通透性试验评价XG对小鼠的镇痛作用及对腹腔毛细血管通透性的影响。用IL-1β(10ng/mL)诱导体外培养的兔软骨细胞作为OA的体外模型,给予不同浓度XG(10-2000μg/mL)干扰后,采用MTT法检测软骨细胞增殖,采用ELISA法检测细胞培养液中MMP及TIMP-1含量,评价XG对兔OA软骨细胞的保护作用。结果：对兔OA模型研究结果表明,各组兔造模侧膝关节宽度均于首次注射木瓜蛋白酶后第2周显著增长,并随木瓜蛋白酶剂量的增高而增大,但之后各组均有不同程度缓解；软骨和滑膜的破坏程度随木瓜蛋白酶剂量的增高和造模时间的延长而加重。对大鼠OA模型研究结果表明,关节腔注射MIA会引起大鼠后足支撑力的显著下降,也会导致PWT明显降低,且此两项指标的降低程度与MIA的用量呈正相关。其中MIA3mg与MIA2mg所引起的疼痛行为改变最为显著,疼痛可维持至第49d,而MIA1mg引起的疼痛会逐渐缓解；大鼠造模侧膝关节破坏程度则随MIA剂量的增高和造模时间的延长而加重。对兔OA的药效评价结果表明,与NS组比,兔膝骨关节腔注射XG能保护软骨,抑制兔膝OA中软骨降解,降低膝关节宽度、关节滑液中IL-1β、TNF-α、PG E2、NO的含量及关节软骨中MMP-1、-3含量,提高OA关节软骨中软骨细胞存活率、GAG含量及TIMP-1表达量。而且,注射XG5周2次与注射SH5周5次对关节软骨的保护效果无显著性差异。对大鼠OA的药效评价结果表明,与NS组比,大鼠骨关节腔注射XG对大鼠OA具有较长效的疼痛缓解作用,对关节软骨具有保护作用,可减轻软骨降解,延缓OA进程。小鼠实验结果表明,与NS组比,腹腔注射XG能显著延长醋酸致小鼠疼痛的潜伏期,减少扭体次数；而且XG能显著抑制醋酸引起的小鼠毛细血管通透性的增加。体外实验结果表明,IL-1β能诱发软骨细胞损伤,抑制软骨细胞增殖,并提高培养液中MMP-1、-3和-13的含量,降低培养液中TIMP-1的含量。XG(100-2000μg/mL)能保护体外培养的软骨细胞,减轻IL-1β对软骨细胞的损伤,降低培养液中MMP-1、-3和-13的含量,提高细胞增殖存活率及培养液中TIMP-1的含量,并且XG在100-1000μg/mL浓度范围内的保护效果呈剂量依赖关系。结论：兔膝关节腔内注射2%、5%、10%的木瓜蛋白酶和大鼠膝关节腔内注射1.7%、3.3%、5%的MIA均可制备不同严重程度的OA模型,这两种模型建模周期短且重现性好。药效学评价结果表明,XG能减轻醋酸致小鼠疼痛并抑制小鼠毛细血管通透性的增加；XG也能保护体外培养的软骨细胞,减轻IL-1β对软骨细胞的损伤；膝骨关节腔注射XG可保护木瓜蛋白酶诱导的兔OA软骨和MIA诱导的大鼠OA软骨,减轻炎症,缓解疼痛,延缓实验性OA进程。而且,5周给药2次与SH5周给药5次的治疗效果无显著性差异,因此用XG治疗OA可减少关节腔给药次数。
Osteoarthritis (OA), also known as degenerative joint disease, is one of the most common joint disorders. It is characterized by the degradation and loss of articular cartilage. OA is one of the main cause of joint pain and functional disorders of elderly people. Multiple factors are known to affect the progression of OA, including increasing age, trauma, inflammation, occupation, obesity, metabolism and genetic factor. Current treatments for OA involve surgical treatment, pharmacotherapy and adjuvant therapy. Pharmacotherapy is recommended as the first line therapy on early and mid-term OA. Intro-articular (IA) injection of sodium hyaluronate (SH) is indicated as an effective treatment for OA due to its lubricating and cushioning properties. Furthermore, IA injection of SH can reduce joint pain. However, SH will be quickly degraded in vivo by hydrolytic or enzymatic reactions because of its instability. Therefore, a compound which is similar to SH in the structure and function, but with a longer effect in the joint, is needed. Xanthan gum (XG) is a natural microbial extracellular heteropolysaccharide. Typically, XG is made from fermentation of Xanthomonas campestris in well-agitated fermenter. The relative molecular mass (Mr) of XG distribution ranges from2×106to20×106. XG is similar to SH in rheology and viscosity. XG solution is very stable in wide ranges of pH, ionic concentration and temperature. Furthermore, XG will not be easily degraded in vivo. IA injection of XG is probably an effective therapeutic method for OA due to it's high viscosity. XG injection is also likely to have a long-lasting protective effect on articular cartilage to avoid numerous injections. In the present study, we determined the industrialized fermentation process of XG, obtained the XG with good quality and high Mr, evaluated the safety of IA injection of XG into knee joint of rabbit and the preliminary cytotoxicity of XG on rabbit chondrocytes, evaluated in vivo and in vitro pharmacodynamics and action mechanism of XG on experimental OA.
1Preparation and quality control of the bulk drug of xanthan gum
Objective:To determine the industrialized fermentation process of XG, obtain good quality and high Mr of XG and establish a quality standard which can control the samples. Methods:To optimize the carbon sources concentration and inoculum concentration in10L fermentation tank through investigating fermentation broth viscosity and XG yield. After determining the optimal fermentation condition, the fermentation processe in50L and10t fermentation tank were investigated. We firstly determined the main purification steps, and then investigated the key factors of each step through the orthogonal test, determined the optimal preparation process of XG eventually. We referred to the quality standard of the bulk drug of XG in ChP, USP, EP and SH to determine the method of quality control. Results:The optimal medium components were defined as:starch5%, ammonium sulfate0.1%, bean cake power0.3%, citric acid0.1%, MgSO40.01%, CaCO30.02%, inoculum concentration5%, temperature28.5℃, pH7.0. The preparation process consisted of the preparation and redissolution of raw XG samples, diatomite adsorption, cake filtration, enzymolysis, active carbon adsorption, cake filtration, fine filtration with microporous membrane filter, precipitation using isopropanol and stoving. The main items of quality standard involved characters, identification, tests (clear degree and color, pH, viscosity, pyruvic acid, absorbance at257nm and280nm, Mr and Mr distribution, protein content, nitrogen content, limit of isopropyl alcohol, loss on drying, ash content, heavy metals, arsenic, lead, endotoxin level, microbial enumeration tests) and assay. The results of quality tests indicated that all the test items were in accordance with the regulations. Conclusion:We determined the industrialized fermentation process of XG. The yield and quality of XG were stable in a large-scale production. XG with good quality and high Mr was obtained. The preparation process was simple, practical, and suitable for large-scale industrial production. Furthermore, we established a quality standard which can control XG.
2Preparation and quality control of xanthan gum injection
Objective:To prepare XG injection and establish the methods of quality control. Methods:The composition of formula and manufacturing technique were designed and the sterilization condition was investigated. XG injection was determined by3-phenylphenol colorimetric method. XG injection was compared with the national standard and the internal control standard of SH injection. Results:The formula composed of XG1%, sodium chloride0.5%, sodium dihydrogen phosphate0.44%and sodium hydrogen phosphate0.7%. XG injection was sterilized at121℃for15minutes. The main items of quality standard involved characters, identification, tests (pH, absorbance at257nm and280nm, osmolarity, Mr and Mr distribution, endotoxin level, and other items belong to the injection quality standard) and assay. The results of quality tests indicated that all the test items were in accordance with the regulations. Conclusion:The preparing technique was stable, simple and feasible. The quality standard could cotrol the quality of XG injection.
3Safety evaluation of xanthan gum injection
Objective:To evaluate the safety of intra-articular injection of xanthan gum (XG) into knee joint of rabbit and the preliminary cytotoxicity of XG on rabbit chondrocytes. Methods:Rabbits were intra-articularly injected with0.1mL/kg of0.5%,1%,2%XG or0.9%NaCl in knee joints once a week for5weeks. The width of knee joint and the hematological and biochemical parameters were examined before and after treatment. The histopathological changes in liver, kidney and knee joint were observed. Rabbit chondrocytes were obtained through mechanical separation and enzymatic digestion, and cultured in DMEM/F12medium containing10%fetal bovine serum (FBS). The morphology of chondrocytes was observed by inverted phase microscopy, and chondrocytes were identified by histological examination of toluidine blue, safranin-O and type Ⅱ collagen immunofluorescence staining. After treatment with XG (10-2000μg/mL), the proliferation of cells was determined using MTT assay and the levels of MMP-1,-3,-13and TIMP-1proteins in media were determined using commercially available ELISA kits according to the manufacturer's instructions. Results:Compared to NS group, the width of knee joint and the hematological and biochemical parameters showed no significant change in XG groups with different doses, and no obvious histopathological changes were detected in liver, kidney and knee joint. The cells morphology and growth rhythm were in accordance with the chondrocytes' character. When stained with toluidine blue, safranin-O and anti-type Ⅱ collagen antibody, the results were positive. The color of the chondrocytes stained by toluidine blue was blue and by safranin-O was red. Type Ⅱ collagen immunofluorescence positive signals which presented with red fluorescence were localized in cytoplasm and cell membrane. Chondrocytes could proliferate actively in the presence of XG. XG did not significantly affect chondrocytes viability and the production of MMP-1,-3,-13and TIMP-1in chondrocytes in doses ranging from10to2000μg/mL. XG displayed no cytotoxicity to rabbit chondrocytes. Conclusion:When0.1mL/kg of0.5%-2%XG were intra-articularly injected into rabbit knee joint once a week for5weeks, there is no significant systemic and topical toxicity. XG (10-2000μg/mL) displayed no cytotoxicity to rabbit chondrocytes.
4Pharmacodynamics and action mechanism evaluation of xanthan gum injection on experimental osteoarthritis
Objective:To establish experimental OA models and evaluate in vivo and in vitro pharmacodynamics and action mechanism of XG on experimental OA. Methods: New Zealand white rabbits were randomly divided into four groups and0.1mL/kg of the mixed solutions of2%,5%or10%(w/v) papain with0.03mol/L L-cysteine were intra-articularly injected into the right knee on days1,3and5respectively. The rabbits of control group were injected intra-articularly with0.1mL/kg of NaCl0.9%(w/v). The rabbits were sacrificed at2,4,6weeks respectively after the initiation of papain injections and these OA models were evaluated through recording the width of knee joint, performing the morphological observation and histological evaluation of articular cartilage and synovium. Rats were randomly divided into four groups and60μL of5%,3.3%or1.7%(w/v) monosodium iodoacetate (MIA) were intra-articularly injected into the left knee respectively. The rats of control group were intra-articularly injected with60μL of NaCl0.9%(w/v). The pain related behaviors including weight bearing and hyperalgesia to punctate mechanical stimuli were tested on days3,7,14,21,28,35,42,49. Then the rats were sacrificed and these OA models were evaluated through performing the morphological observation and histological evaluation of articular cartilage.0.1mL/kg2%papain and0.03mol/L L-cysteine was intra-articularly injectioned into the right knees of all the rabbits three times on days1,3and5, respectively. Then these animals were randomly divided into three groups.0.1mL/kg of1%XG injection was given intra-articularly into the right knees in weeks2and4, while0.1mL/kg of0.9%NaCl was injected in weeks1,3and5.0.1mL/kg of0.9%NaCl and0.1mL/kg of1%SH were injected intra-articularly into the right knees once a week for5weeks respectively. All animals were injected intra-articularly with0.1mL/kg of0.9%NaCl into the left knees on days1,3and5during the induction (papain) phase and once a week in the treatment phase. The body weight and knee joint width were detected throughout the experimental period. After treatment, the contents of IL-1β, TNF-α and PG E2in synovial fluid were measured by enzyme-linked immunosorbent assay, the content of NO was measured by nitrate reductase assay. The gross morphological observation and histological evaluation of femoral condyle, tibial plateau and synovium were performed. The glycosaminoglycan (GAG) were measured using1,9-dimethylmethylene blue (DMB) colorimetric assay. The chondrocytes apoptosis was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. The MMPs and TIMP-1protein production were measured using immunohistochemistry and Western-blot. The MMPs and TIMP-1mRNA levels were measured using RT-PCR assay. IA injections of5%MIA60μL into the left knees of all rats were performed. Then these animals were randomly divided into four groups.60μL of1%XG injection was given intra-articularly into the left knees on days14and35, while60μL of0.9%NaCl was injected on days21,28and42(IA injection of XG once every two weeks for5weeks).60μL of1%SH injection was given intra-articularly into the left knees on days14and35, while60μL of0.9% NaCl was injected on days21,28and42(IA injection of SH once every two weeks for5weeks).60μL of1%SH injection (IA injection of SH once a week for5weeks) and0.9%NaCl (NS group) were given intra-articularly into the left knees on days14,21,28,35and42. The pain related behaviors including weight bearing and hyperalgesia to punctate mechanical stimuli were tested on days3,7,14,21,28,35,42,49. Then the rats were sacrificed and these OA models were evaluated through performing the morphological observation and histological evaluation of articular cartilage. The analgesic effect of XG was also evaluated through body torsion experiment in mice and the capillary permeability experiment was carried out to observe the inhibitory effect of XG on capillary permeability. The in vitro OA model was established by adding IL-1β(10ng/mL) into the cell culture medium. After treatment with XG (10-2000μg/mL), the proliferation of cells was determined using the MTT assay and the levels of MMP-1,-3,-13, and TIMP-1proteins in media were determined using commercially available ELISA kits according to the manufacturer's instructions. Results:The results of in vivo OA models evaluation indicated that the degenerative changes were demonstrated in rabbits and rats knee joints in all experimental groups, such as thinner articular cartilage, fibrillation and destroyed cartilage matrix, and inflammation, proliferation. All these changes were much worse with increased concentration and prolonged observation time. In all MIA-treated groups, there was a significant decrement in the paw withdrawal threshold (PWT) and weight bearing of the ipsilateral limb. The hyperalgesia in rats induced by MIA3mg was stable and lasted for the whole period of the experiment, however, the hyperalgesia induced by MIA1mg could gradually alleviate. The experimental results of XG on rabbit OA showed that, compared to NS group, IA injection of XG once every two weeks for5weeks significantly decreased the severity of swelling of the knee joint, reduced the damage of cartilage surfaces and loss of safranin-O staining intensity, inhibited cells hyperplasia and infiltration of mononuclear cells in the synovium, decreased the contents of IL-1β\TNF-α, PG E2and NO in synovial fluid, inhibited chondrocytes apoptosis and MMP-1,-3protein or mRNA levels in cartilage, enhanced GAG and TIMP-1production in cartilage. Furthermore, no significant differences between the XG-treated group (IA injection of XG once every two weeks for5weeks) and the SH-treated group (IA injection of SH once a week for5weeks) were observed. The experimental results of XG on rat OA showed that, compared to NS group, IA injection of XG once every two weeks for5weeks significantly reduced the damage of cartilage surfaces and loss of safranin-O staining intensity, significantly increased the weight distribution of the ipsilateral limb and the PWT in response to the von Frey monofilaments stimulus. Furthermore, these effects were much better than those in SH-treated group (IA injection of SH once every two weeks for5weeks), but no significant differences between the XG-treated group (IA injection of XG once every two weeks for5weeks) and the SH-treated group (IA injection of SH once a week for5weeks) were observed. The results of XG on body torsion experiment in mice and the capillary permeability experiment showed that intraperitoneal injection of XG could reduce the times of torsion and inhibit capillary permeability. XG could restore chondrocytes proliferation, suppressed protein expression of MMP-1,-3, and-13, increased TIMP-1production in a dose-dependent manner in doses ranging from100to1000μg/mL in IL-1β-induced rabbit chondrocytes. Conclution:Different severities of OA were established through giving injections into the knee including IA injections of2%,5%,10%papain or5%,3.3%,1.7%MIA. These models were characterized by short period and good reproducibility. XG showed significant analgesic effect and inhibited capillary permeability. XG also exhibited protective effect on rabbit chondrocytes in the presence of IL-1β.IA injection of XG could inhibit the continuous lesions on the cartilage in experimental OA, reduce inflammation, relieve pain and delay the progression of OA. Furthermore, compared with SH, fewer delivery times of XG could get the same treatment effect under current treatment regimen.
1注射用黄原胶原料药的制备工艺及质量控制
目的：确定XG工业化发酵工艺和注射用XG原料药制备工艺,建立注射用XG原料药质量控制方法。方法：以发酵液黏度及产胶量为考察指标,在10L发酵罐中对碳源浓度和接种量进行优化,确定最终发酵工艺条件,并进行50L规模的中试放大以及10t规模的工业化试产。先确定注射用XG原料药制备的主要步骤,然后通过正交试验考察影响每个步骤纯化效果的关键因素,确定最佳制备工艺。参考国内外已有的药用辅料级XG质量标准及与XG性质、用途相似的注射用SH原料药质量标准,确定注射用XG原料药质量检查项目及标准草案。结果：优化后的发酵培养基及工业化发酵工艺条件为：淀粉5%,硫酸铵0.1%,豆饼粉0.3%,柠檬酸0.1%,硫酸镁0.01%,碳酸钙0.02%,接种量5%,温度28.5℃,pH7.0。注射用XG原料药制备的主要步骤为：XG粗品制备,粗品溶解,硅藻土吸附,除菌过滤,酶解,活性炭吸附,除碳过滤,微孔滤膜精滤,醇沉,干燥得精制品。主要的质量检查项目为：性状、鉴别、检查(溶液的澄清度与颜色、酸碱度、黏度、丙酮酸、吸光度、Mr与Mr分布、蛋白质含量、氮含量、异丙醇残留量、干燥失重、灰分、重金属、砷盐、铅含量、细菌内毒素和微生物限度)和含量测定。质量检查结果表明,注射用XG原料药各项指标均达到或优于已有的XG质量标准及与XG性质、用途相似的注射用SH原料药的质量标准,达到注射用原料药要求。结论：确定了XG工业化发酵工艺,在放大试产过程中XG的产量及质量稳定。确定了注射用XG原料药制备工艺,制备了符合注射用要求的高质量、低蛋白质含量的XG原料药。确定了注射用XG原料药质量检查项目及相应的标准。
2黄原胶注射液的制备工艺及质量控制
目的：确定XG注射液配方及制备工艺,建立XG注射液质量控制方法。方法：通过配方筛选确定XG注射液的配方及配制工艺。通过热稳定性考察,确定XG注射液的灭菌方法。采用间羟基联苯比色法测定XG注射液的含量,并对该方法进行方法学验证。参考与XG性质、用途相似的SH注射液质量标准,确定质量检查项目及标准。结果：XG注射液的配方成分为：1%黄原胶、0.5%氯化钠、0.44%磷酸二氢钠和0.7%磷酸氢二钠。XG注射液的灭菌方法为：121℃灭菌15min。间羟基联苯比色法可快速准确地测定XG注射液的标示量百分含量,该法操作简单且可靠性好。主要的质量检查项目为：性状、鉴别、检查(pH值、吸光度、渗透压摩尔浓度、Mr与Mr分布、细菌内毒素及注射剂项下的各项目检查)和含量测定。质量检查结果表明,XG注射液的各项考察指标均达到或优于与XG性质、用途相似的SH注射液质量标准。结论：确定了XG注射液的配方、制备工艺及含量测定方法,制备了高质量XG注射液。确定了XG注射液质量检查项目及标准,该质量控制方法简单准确,可全面反映产品质量。
3黄原胶注射液的安全性评价
目的：评价兔膝骨关节腔注射XG的安全性及XG对兔软骨细胞的细胞毒性。方法：在兔膝骨关节腔注射2%、1%和0.5%(w/v)的XG注射液及等量生理盐水(normal saline, NS),注射剂量为兔每千克体重0.1mL,每周注射1次,连续5周。检测兔膝关节宽度、血液学和血液生化参数并观察肝、肾、膝关节组织病理学变化。采用机械分离法和酶消化法从兔膝关节软骨中分离软骨细胞,在含10%胎牛血清的DMEM/F12培养液中培养细胞,采用甲苯胺蓝、番红-O和Ⅱ型胶原免疫荧光法鉴定软骨细胞。经不同浓度XG(10-2000μg/mL)作用后,采用四甲基偶氮唑盐(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide, MTT)法检测软骨细胞增殖,用酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)试剂盒检测细胞培养液中基质金属蛋白酶(matrix metalloproteinase, MMP)和基质金属蛋白酶组织抑制剂1(tissue inhibitor of metal protease-1,TIMP-1)的含量。结果：与NS组比,不同剂量XG组兔膝关节宽度、血液学及血液生化参数等均无明显改变(P>0.05)；肝、肾及关节组织未见明显病理改变(P>0.05)。本课题成功分离和培养了兔软骨细胞,培养的细胞形态及生长情况均符合软骨细胞特征。细胞被甲苯胺蓝染成蓝色,被番红-O染成红色且在胞浆及胞膜Ⅱ型胶原免疫荧光染色阳性表达,表明培养的细胞为软骨细胞。兔软骨细胞在含10-2000μg/mL的XG培养液中生长活跃,与在不含XG培养液中培养的细胞比,细胞增殖率并未有明显变化(P>0.05),培养液中MMP-1、-3、-13及TIMP-1的含量也未发生明显改变(P>0.05)。结论：在兔膝关节腔注射XG(0.5%-2%)0.1mL/kg,每周1次,连续5周,全身及局部未见明显毒性作用。XG(10-2000μg/mL)不会对体外培养的兔软骨细胞产生明显细胞毒作用。
4黄原胶注射液对实验性骨关节炎的药效和作用机制研究
目的：制备体内和体外OA模型,考察XG对实验性OA的疗效和作用机制。方法：将实验兔随机分为4组,分别在第1、3和5d于右膝关节腔注射2%、5%、10%(w/v)木瓜蛋白酶和0.03mol/L L-半胱氨酸混合溶液0.1mL/kg以及等量NS(空白对照组)。整个实验过程观察兔状态并记录体重和膝关节宽度。首次注射后第2、4、6周将兔分批处死,取造模侧股骨髁、胫骨平台及滑膜做大体及组织病理学观察,并进行评分,评价不同浓度木瓜蛋白酶引起的兔膝OA模型。将实验大鼠随机分为4组,于左膝关节腔注射含碘乙酸钠(monosodium iodoacetate, MIA)3mg、2mg、1mg的NS溶液60μL及等量NS(空白对照组)。造模后分别于第3、7、14、21、28、35、42、49d进行大鼠疼痛行为学测试,包括承重测试和机械缩足阈值(paw withdrawal threshold, PWT)测试。处死大鼠后取造模侧股骨髁和胫骨平台做大体及组织病理学观察,并进行评分,评价不同剂量MIA引起的大鼠膝OA模型。将实验兔右膝关节腔注射2%木瓜蛋白酶造模。造模后将兔分为3组：NS组兔右膝关节腔注射NS,0.1mL/kg,1次/周,连续5周；SH组兔右膝关节腔注射1%SH,0.1mL/kg,1次/周,连续5周；XG组兔在第2和4周右膝关节腔注射1%XG,0.1mL/kg,在第1、3和5周注射等量NS；所有兔左膝关节腔注射NS,0.1mL/kg,1次/周,连续5周,作为假手术组(sham组)。整个实验过程观察动物状态,测量造模侧膝关节宽度。治疗结束后用ELISA法检测关节滑液中白介素1p(interleukin-1, IL-1β)、肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)及前列腺素E2(prostaglandin E2, PG E2)的含量,用硝酸还原酶法检测关节滑液中一氧化氮(nitric oxide, NO)的含量,对股骨髁和胫骨平台软骨及滑膜做大体及组织病理学观察并评分,用二甲基亚甲蓝法测定关节软骨中糖胺聚糖(glycosaminoglycan, GAG)的含量,用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleoitidyl transferase mediated nick end labeling, TUNEL)评价关节软骨中的细胞凋亡率,用免疫组化及Western-blot法测定关节软骨中MMP及TIMP-1的蛋白表达,通过RT-PCR法测定关节软骨中MMP及TIMP-1的mRNA含量,综合评价XG注射液对兔膝OA的药效和作用机制。用MIA3mg诱导大鼠OA模型。造模后将大鼠分为4组,分别为注射XG2次组,注射SH2次组,注射SH5次组和注射NS对照组。造模后第14、35d,XG2次组与SH2次组分别注射XG和SH注射液(1%,w/v)0.1mL,第21、28、42d注射等量NS。SH5次组与NS组于造模后第14、21、28、35、42d分别注射SH和NS。采用大鼠模型研究中的方法对各组疼痛指标及关节软骨破坏程度进行评估,评价XG对大鼠OA关节软骨的保护作用及对疼痛的缓解作用。采用小鼠扭体试验和毛细血管通透性试验评价XG对小鼠的镇痛作用及对腹腔毛细血管通透性的影响。用IL-1β(10ng/mL)诱导体外培养的兔软骨细胞作为OA的体外模型,给予不同浓度XG(10-2000μg/mL)干扰后,采用MTT法检测软骨细胞增殖,采用ELISA法检测细胞培养液中MMP及TIMP-1含量,评价XG对兔OA软骨细胞的保护作用。结果：对兔OA模型研究结果表明,各组兔造模侧膝关节宽度均于首次注射木瓜蛋白酶后第2周显著增长,并随木瓜蛋白酶剂量的增高而增大,但之后各组均有不同程度缓解；软骨和滑膜的破坏程度随木瓜蛋白酶剂量的增高和造模时间的延长而加重。对大鼠OA模型研究结果表明,关节腔注射MIA会引起大鼠后足支撑力的显著下降,也会导致PWT明显降低,且此两项指标的降低程度与MIA的用量呈正相关。其中MIA3mg与MIA2mg所引起的疼痛行为改变最为显著,疼痛可维持至第49d,而MIA1mg引起的疼痛会逐渐缓解；大鼠造模侧膝关节破坏程度则随MIA剂量的增高和造模时间的延长而加重。对兔OA的药效评价结果表明,与NS组比,兔膝骨关节腔注射XG能保护软骨,抑制兔膝OA中软骨降解,降低膝关节宽度、关节滑液中IL-1β、TNF-α、PG E2、NO的含量及关节软骨中MMP-1、-3含量,提高OA关节软骨中软骨细胞存活率、GAG含量及TIMP-1表达量。而且,注射XG5周2次与注射SH5周5次对关节软骨的保护效果无显著性差异。对大鼠OA的药效评价结果表明,与NS组比,大鼠骨关节腔注射XG对大鼠OA具有较长效的疼痛缓解作用,对关节软骨具有保护作用,可减轻软骨降解,延缓OA进程。小鼠实验结果表明,与NS组比,腹腔注射XG能显著延长醋酸致小鼠疼痛的潜伏期,减少扭体次数；而且XG能显著抑制醋酸引起的小鼠毛细血管通透性的增加。体外实验结果表明,IL-1β能诱发软骨细胞损伤,抑制软骨细胞增殖,并提高培养液中MMP-1、-3和-13的含量,降低培养液中TIMP-1的含量。XG(100-2000μg/mL)能保护体外培养的软骨细胞,减轻IL-1β对软骨细胞的损伤,降低培养液中MMP-1、-3和-13的含量,提高细胞增殖存活率及培养液中TIMP-1的含量,并且XG在100-1000μg/mL浓度范围内的保护效果呈剂量依赖关系。结论：兔膝关节腔内注射2%、5%、10%的木瓜蛋白酶和大鼠膝关节腔内注射1.7%、3.3%、5%的MIA均可制备不同严重程度的OA模型,这两种模型建模周期短且重现性好。药效学评价结果表明,XG能减轻醋酸致小鼠疼痛并抑制小鼠毛细血管通透性的增加；XG也能保护体外培养的软骨细胞,减轻IL-1β对软骨细胞的损伤；膝骨关节腔注射XG可保护木瓜蛋白酶诱导的兔OA软骨和MIA诱导的大鼠OA软骨,减轻炎症,缓解疼痛,延缓实验性OA进程。而且,5周给药2次与SH5周给药5次的治疗效果无显著性差异,因此用XG治疗OA可减少关节腔给药次数。
Osteoarthritis (OA), also known as degenerative joint disease, is one of the most common joint disorders. It is characterized by the degradation and loss of articular cartilage. OA is one of the main cause of joint pain and functional disorders of elderly people. Multiple factors are known to affect the progression of OA, including increasing age, trauma, inflammation, occupation, obesity, metabolism and genetic factor. Current treatments for OA involve surgical treatment, pharmacotherapy and adjuvant therapy. Pharmacotherapy is recommended as the first line therapy on early and mid-term OA. Intro-articular (IA) injection of sodium hyaluronate (SH) is indicated as an effective treatment for OA due to its lubricating and cushioning properties. Furthermore, IA injection of SH can reduce joint pain. However, SH will be quickly degraded in vivo by hydrolytic or enzymatic reactions because of its instability. Therefore, a compound which is similar to SH in the structure and function, but with a longer effect in the joint, is needed. Xanthan gum (XG) is a natural microbial extracellular heteropolysaccharide. Typically, XG is made from fermentation of Xanthomonas campestris in well-agitated fermenter. The relative molecular mass (Mr) of XG distribution ranges from2×106to20×106. XG is similar to SH in rheology and viscosity. XG solution is very stable in wide ranges of pH, ionic concentration and temperature. Furthermore, XG will not be easily degraded in vivo. IA injection of XG is probably an effective therapeutic method for OA due to it's high viscosity. XG injection is also likely to have a long-lasting protective effect on articular cartilage to avoid numerous injections. In the present study, we determined the industrialized fermentation process of XG, obtained the XG with good quality and high Mr, evaluated the safety of IA injection of XG into knee joint of rabbit and the preliminary cytotoxicity of XG on rabbit chondrocytes, evaluated in vivo and in vitro pharmacodynamics and action mechanism of XG on experimental OA.
1Preparation and quality control of the bulk drug of xanthan gum
Objective:To determine the industrialized fermentation process of XG, obtain good quality and high Mr of XG and establish a quality standard which can control the samples. Methods:To optimize the carbon sources concentration and inoculum concentration in10L fermentation tank through investigating fermentation broth viscosity and XG yield. After determining the optimal fermentation condition, the fermentation processe in50L and10t fermentation tank were investigated. We firstly determined the main purification steps, and then investigated the key factors of each step through the orthogonal test, determined the optimal preparation process of XG eventually. We referred to the quality standard of the bulk drug of XG in ChP, USP, EP and SH to determine the method of quality control. Results:The optimal medium components were defined as:starch5%, ammonium sulfate0.1%, bean cake power0.3%, citric acid0.1%, MgSO40.01%, CaCO30.02%, inoculum concentration5%, temperature28.5℃, pH7.0. The preparation process consisted of the preparation and redissolution of raw XG samples, diatomite adsorption, cake filtration, enzymolysis, active carbon adsorption, cake filtration, fine filtration with microporous membrane filter, precipitation using isopropanol and stoving. The main items of quality standard involved characters, identification, tests (clear degree and color, pH, viscosity, pyruvic acid, absorbance at257nm and280nm, Mr and Mr distribution, protein content, nitrogen content, limit of isopropyl alcohol, loss on drying, ash content, heavy metals, arsenic, lead, endotoxin level, microbial enumeration tests) and assay. The results of quality tests indicated that all the test items were in accordance with the regulations. Conclusion:We determined the industrialized fermentation process of XG. The yield and quality of XG were stable in a large-scale production. XG with good quality and high Mr was obtained. The preparation process was simple, practical, and suitable for large-scale industrial production. Furthermore, we established a quality standard which can control XG.
2Preparation and quality control of xanthan gum injection
Objective:To prepare XG injection and establish the methods of quality control. Methods:The composition of formula and manufacturing technique were designed and the sterilization condition was investigated. XG injection was determined by3-phenylphenol colorimetric method. XG injection was compared with the national standard and the internal control standard of SH injection. Results:The formula composed of XG1%, sodium chloride0.5%, sodium dihydrogen phosphate0.44%and sodium hydrogen phosphate0.7%. XG injection was sterilized at121℃for15minutes. The main items of quality standard involved characters, identification, tests (pH, absorbance at257nm and280nm, osmolarity, Mr and Mr distribution, endotoxin level, and other items belong to the injection quality standard) and assay. The results of quality tests indicated that all the test items were in accordance with the regulations. Conclusion:The preparing technique was stable, simple and feasible. The quality standard could cotrol the quality of XG injection.
3Safety evaluation of xanthan gum injection
Objective:To evaluate the safety of intra-articular injection of xanthan gum (XG) into knee joint of rabbit and the preliminary cytotoxicity of XG on rabbit chondrocytes. Methods:Rabbits were intra-articularly injected with0.1mL/kg of0.5%,1%,2%XG or0.9%NaCl in knee joints once a week for5weeks. The width of knee joint and the hematological and biochemical parameters were examined before and after treatment. The histopathological changes in liver, kidney and knee joint were observed. Rabbit chondrocytes were obtained through mechanical separation and enzymatic digestion, and cultured in DMEM/F12medium containing10%fetal bovine serum (FBS). The morphology of chondrocytes was observed by inverted phase microscopy, and chondrocytes were identified by histological examination of toluidine blue, safranin-O and type Ⅱ collagen immunofluorescence staining. After treatment with XG (10-2000μg/mL), the proliferation of cells was determined using MTT assay and the levels of MMP-1,-3,-13and TIMP-1proteins in media were determined using commercially available ELISA kits according to the manufacturer's instructions. Results:Compared to NS group, the width of knee joint and the hematological and biochemical parameters showed no significant change in XG groups with different doses, and no obvious histopathological changes were detected in liver, kidney and knee joint. The cells morphology and growth rhythm were in accordance with the chondrocytes' character. When stained with toluidine blue, safranin-O and anti-type Ⅱ collagen antibody, the results were positive. The color of the chondrocytes stained by toluidine blue was blue and by safranin-O was red. Type Ⅱ collagen immunofluorescence positive signals which presented with red fluorescence were localized in cytoplasm and cell membrane. Chondrocytes could proliferate actively in the presence of XG. XG did not significantly affect chondrocytes viability and the production of MMP-1,-3,-13and TIMP-1in chondrocytes in doses ranging from10to2000μg/mL. XG displayed no cytotoxicity to rabbit chondrocytes. Conclusion:When0.1mL/kg of0.5%-2%XG were intra-articularly injected into rabbit knee joint once a week for5weeks, there is no significant systemic and topical toxicity. XG (10-2000μg/mL) displayed no cytotoxicity to rabbit chondrocytes.
4Pharmacodynamics and action mechanism evaluation of xanthan gum injection on experimental osteoarthritis
Objective:To establish experimental OA models and evaluate in vivo and in vitro pharmacodynamics and action mechanism of XG on experimental OA. Methods: New Zealand white rabbits were randomly divided into four groups and0.1mL/kg of the mixed solutions of2%,5%or10%(w/v) papain with0.03mol/L L-cysteine were intra-articularly injected into the right knee on days1,3and5respectively. The rabbits of control group were injected intra-articularly with0.1mL/kg of NaCl0.9%(w/v). The rabbits were sacrificed at2,4,6weeks respectively after the initiation of papain injections and these OA models were evaluated through recording the width of knee joint, performing the morphological observation and histological evaluation of articular cartilage and synovium. Rats were randomly divided into four groups and60μL of5%,3.3%or1.7%(w/v) monosodium iodoacetate (MIA) were intra-articularly injected into the left knee respectively. The rats of control group were intra-articularly injected with60μL of NaCl0.9%(w/v). The pain related behaviors including weight bearing and hyperalgesia to punctate mechanical stimuli were tested on days3,7,14,21,28,35,42,49. Then the rats were sacrificed and these OA models were evaluated through performing the morphological observation and histological evaluation of articular cartilage.0.1mL/kg2%papain and0.03mol/L L-cysteine was intra-articularly injectioned into the right knees of all the rabbits three times on days1,3and5, respectively. Then these animals were randomly divided into three groups.0.1mL/kg of1%XG injection was given intra-articularly into the right knees in weeks2and4, while0.1mL/kg of0.9%NaCl was injected in weeks1,3and5.0.1mL/kg of0.9%NaCl and0.1mL/kg of1%SH were injected intra-articularly into the right knees once a week for5weeks respectively. All animals were injected intra-articularly with0.1mL/kg of0.9%NaCl into the left knees on days1,3and5during the induction (papain) phase and once a week in the treatment phase. The body weight and knee joint width were detected throughout the experimental period. After treatment, the contents of IL-1β, TNF-α and PG E2in synovial fluid were measured by enzyme-linked immunosorbent assay, the content of NO was measured by nitrate reductase assay. The gross morphological observation and histological evaluation of femoral condyle, tibial plateau and synovium were performed. The glycosaminoglycan (GAG) were measured using1,9-dimethylmethylene blue (DMB) colorimetric assay. The chondrocytes apoptosis was measured using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. The MMPs and TIMP-1protein production were measured using immunohistochemistry and Western-blot. The MMPs and TIMP-1mRNA levels were measured using RT-PCR assay. IA injections of5%MIA60μL into the left knees of all rats were performed. Then these animals were randomly divided into four groups.60μL of1%XG injection was given intra-articularly into the left knees on days14and35, while60μL of0.9%NaCl was injected on days21,28and42(IA injection of XG once every two weeks for5weeks).60μL of1%SH injection was given intra-articularly into the left knees on days14and35, while60μL of0.9% NaCl was injected on days21,28and42(IA injection of SH once every two weeks for5weeks).60μL of1%SH injection (IA injection of SH once a week for5weeks) and0.9%NaCl (NS group) were given intra-articularly into the left knees on days14,21,28,35and42. The pain related behaviors including weight bearing and hyperalgesia to punctate mechanical stimuli were tested on days3,7,14,21,28,35,42,49. Then the rats were sacrificed and these OA models were evaluated through performing the morphological observation and histological evaluation of articular cartilage. The analgesic effect of XG was also evaluated through body torsion experiment in mice and the capillary permeability experiment was carried out to observe the inhibitory effect of XG on capillary permeability. The in vitro OA model was established by adding IL-1β(10ng/mL) into the cell culture medium. After treatment with XG (10-2000μg/mL), the proliferation of cells was determined using the MTT assay and the levels of MMP-1,-3,-13, and TIMP-1proteins in media were determined using commercially available ELISA kits according to the manufacturer's instructions. Results:The results of in vivo OA models evaluation indicated that the degenerative changes were demonstrated in rabbits and rats knee joints in all experimental groups, such as thinner articular cartilage, fibrillation and destroyed cartilage matrix, and inflammation, proliferation. All these changes were much worse with increased concentration and prolonged observation time. In all MIA-treated groups, there was a significant decrement in the paw withdrawal threshold (PWT) and weight bearing of the ipsilateral limb. The hyperalgesia in rats induced by MIA3mg was stable and lasted for the whole period of the experiment, however, the hyperalgesia induced by MIA1mg could gradually alleviate. The experimental results of XG on rabbit OA showed that, compared to NS group, IA injection of XG once every two weeks for5weeks significantly decreased the severity of swelling of the knee joint, reduced the damage of cartilage surfaces and loss of safranin-O staining intensity, inhibited cells hyperplasia and infiltration of mononuclear cells in the synovium, decreased the contents of IL-1β\TNF-α, PG E2and NO in synovial fluid, inhibited chondrocytes apoptosis and MMP-1,-3protein or mRNA levels in cartilage, enhanced GAG and TIMP-1production in cartilage. Furthermore, no significant differences between the XG-treated group (IA injection of XG once every two weeks for5weeks) and the SH-treated group (IA injection of SH once a week for5weeks) were observed. The experimental results of XG on rat OA showed that, compared to NS group, IA injection of XG once every two weeks for5weeks significantly reduced the damage of cartilage surfaces and loss of safranin-O staining intensity, significantly increased the weight distribution of the ipsilateral limb and the PWT in response to the von Frey monofilaments stimulus. Furthermore, these effects were much better than those in SH-treated group (IA injection of SH once every two weeks for5weeks), but no significant differences between the XG-treated group (IA injection of XG once every two weeks for5weeks) and the SH-treated group (IA injection of SH once a week for5weeks) were observed. The results of XG on body torsion experiment in mice and the capillary permeability experiment showed that intraperitoneal injection of XG could reduce the times of torsion and inhibit capillary permeability. XG could restore chondrocytes proliferation, suppressed protein expression of MMP-1,-3, and-13, increased TIMP-1production in a dose-dependent manner in doses ranging from100to1000μg/mL in IL-1β-induced rabbit chondrocytes. Conclution:Different severities of OA were established through giving injections into the knee including IA injections of2%,5%,10%papain or5%,3.3%,1.7%MIA. These models were characterized by short period and good reproducibility. XG showed significant analgesic effect and inhibited capillary permeability. XG also exhibited protective effect on rabbit chondrocytes in the presence of IL-1β.IA injection of XG could inhibit the continuous lesions on the cartilage in experimental OA, reduce inflammation, relieve pain and delay the progression of OA. Furthermore, compared with SH, fewer delivery times of XG could get the same treatment effect under current treatment regimen.
引文
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