| Osteoarthritis (OA) is one of the most common joint diseases, mainly occurred in the shoulder, knee, hip, elbow, ankle, spine, etc. And the knee osteoarthritis (KOA) occurred most frequently. The WHO predicts that Chinese OA patients will reach150million by2015, and China will become one of the countries that have most OA patients in the world. Incidences of disability and even death are rising year by year due to aging, muscle atrophy and KOA. As China has gradually entered an aging society, KOA has become an important public health problem that affects the quality of life of elderly Chinese.KOA develops as articular cartilage degeneration, joint swelling, stiffness, pain even loss of function. Currently, methods that treat KOA are mainly surgical, drug and adjuvant therapy. At the early and middle stage of KOA, drugs and adjuvant therapy are commonly used clinically to alleviate patients’ pain and improve joint function-Medications treating KOA can be divided into anti-inflammatory drugs, painkillers, viscoelastic supplements. In1997, the U.S. Food and Drug Administration approved the use of viscoelastic supplements to treat the symptoms of KOA. The American Rheumatism Association and the European League Against Rheumatism also recognized viscoelastic supplements as a non-surgical treatment to reduce pain, increase joint function and treat OA symptoms. Currently hyaluronic acid (HA) or cross-linked hyaluronic acid are the most common viscoelastic supplements, but the action mechanism are not clear and HA is easily degraded due to enzymes and free radicals. What’s more, HA has a short half-life (the reported longest time is8.8days) so that patients need continuous administration. Therefore, it has more important significance that finding a new viscoelastic supplement which is similar to HA in function, but with higher stablity and longer period of administration. Xanthan gum (XG) is obtained through the aerobic fermentation by Xanthomonas campestris. And XG has many excellent features such as water solubility, thickening ability, suspension power, stability and rheological property. Currently XG is primarily used as a slow-release material which mixes with non-steroidal anti-inflammatory drugs to make oral medications that treat OA. And there is no injection used clinically, and the mechanism of XG treating OA is unclear. In vivo and in vitro, the pharmacodynamic effect and mechanism of XG injection that treats KO A will be studied from multi levels, including molecular, cellular and tissue levels. So it can provide the theoretical foundation for future clinical studies and commercial production of XG injection.1. Preparation of injectable XG active pharmaceutical ingredientObjective To prepare injectable grade XG. Methods Strains:Xanthomonas campestris pv. Campestris58. Seed medium:sucrose2%, peptone0.5%, yeast extract0.1%, pH=7. Fermentation medium:corn starch6%, soybean meal0.25%, NaCl0.5%, MgSO40.25%, K2HPO40.25%,(NH4)2SO40.1%, pH=7.5. Strains were inoculated in the fermentation medium after two times of activation, then were cultured for72h at30℃,230r/min. After fermentation, XG is obtained through filtration, sterilization, alcohol precipitation, reconstitution diatomite adsorption, activated carbon adsorption, microporous membrane filtration, alcohol precipitation, vacuum-drying and crush. Finally the injectable grade XG was evaluated according to the quality standards. Results The injectable grade XG was prepared according to the above methods. The relative molecular weight (Mw) of XG was3.384×106. The XG yield reached9.13g/L. And the products meet the evaluation criteria. Conculsion The fermentation, separation and purification conditions of injectable grade XG were screened. And the products meet the quality standards.2. Experiments of injectable XG that treat KOA in vivoObjective To establish OA model in vivo, and evalutate the effect and action mechanism of XG on this model. Methods After anterior cruciate ligament transaction and meniscectomy, New Zealand White rabbits were divided randomly into four groups. HA injection was used as positive control, and the animals in control and model group were intra-articularly injected with NaCl0.9%(w/v). The efficacy and mechanism of XG were evaluated through general observation, histological evaluation, mRNA and protein expression. Results4weeks after surgery, the animal model of OA was established successfully.(1)13weeks after administration, the general observation showed that compared with the model group, cartilage and synovial lesions of the right knees were improved in HA group and XG groups. And the gross scores were significantly lower (P<0.05). But there was no significant difference between XG group and HA group (P>0.05).(2)The histological evaluation showed that, compared with the model group, Mankin’s scores of HA and XG group were obviously lower (P<0.05), but no significant difference between XG and HA group was observed. Compared with the model group, the colour fading of toluidine blue stain was reduced in XG and HA treated groups. And it suggested that the aggrecan loss of the cartilage of model animals was improved.(3) The mRNA expression of several factors showed that, compared with the model group, XG and HA increased the mRNA expression of TIMP-1, type II collagen and aggrecan significantly. Furthermore, there was no significant difference between the effect of XG and HA. Compared with the model group,1time of treatment with2%XG once and HA could significantly reduce the mRNA expression of aggrecanase, but the effect of2times of treatment with1%XG was not obvious. Compared with the model group, XG and HA could significantly reduce the mRNA expression of IL-1β, and the effect of HA and1%XG was higher than that of2%XG. Compared with the model group, XG and HA could significantly reduce the mRNA expression of MMP-1, and the effect of1%XG was higher than that of HA and2%XG). Compared with the model group, XG and HA could significantly reduce the mRNA expression of MMP-3and iNOS, and there was no significant difference between the effect of XG and HA.(4) The protein expression of several factors showed that, compared with the control group, iNOS and MMP-13protein expression of model group significantly increased, and the protein expression of aggrecan decreased significantly. Compared with the model group, XG could significantly reduce the expression of iNOS and MMP-13, and enhance the expression of aggrecan. But the difference between the effect of HA and XG was not significant. Conclusion When XG were intra-articularly injected into the right knee of rabbit, the viscoelasticity of synovial fluid increased, and XG became a joint lubricant and cushion, so that the articular cartilage and synovial membrane could self-repair. At this time, the levels of some enzymes catalyzing the degradation of cartilage matrix were reduced in vivo, such as iNOS, MMP-1,3,13and aggrecanase. Expression of some enzymes that had anabolic effect increased in vivo, such as aggrecan and TIMP. This process allowed the decrease of degradation of extracellular matrix such as type II collagen and proteoglycan, and the increase of their synthesis. As a result, the chondrocytes were protected and the metabolism of joint was promoted. What’ more, inflammatory cytokines and oxygen free radicals decreased significantly in synovial fluid so that the apoptosis of chondrocytes was reduced. Eventually, the anabolism of cartilage exceeded catabolism. The cartilage got self-repair and the development of KOA was delayed.3. Experiments of injectable XG that treat KOA in vitroObjective To establish OA model in vitro, and evalutate the effect and action mechanism of XG on this model. Methods New Zealand rabbits were sacrificed to isolate rabbit chondrocytes. Cell proliferation and cytotoxicity were measured by MTT cell morphology. The cell viability was determined by live/dead cell staining kits. The cell cycle was analyzed using propidum iodide (PI) staining by flow cytometer. LPS (10μg/mL) was added into rabbit chondrocytes to establish KOA model in vitro, and different concentrations of XG (50,100,200μg/mL) were added. The effect of XG on reactive oxygen species (ROS) was measured by fluorence microplate reader and flow cytomety. The effect of XG on the expression of IL-1β, TNF-a, PGE2, NO, MDA, SOD was determined by test kits. What’s more, the mRNA and protein expression of partial factors were determined through RT-PCR and Western blotting. Results (1) Rabbit primary chondrocyte were cultured after digestion by trypsin and collagenase type Ⅱ. Toluidine blue stained cells as purple.(2) Compared with control group, chondrocytes proliferated actively treated with50,100,200μg/mL XG. XG was not toxic on cell at the concentration of0-2000μg/mL.(3) Compared with control group, XG did not affect cell cycle at the concentration of 50-200ug/mL.(4) Compared with model group, XG and HA can reduce ROS expression induced by LPS, but no significant differences were observed between the effect of HA and XG. Compared with XG and HA, Vc had a stronger ability to reduce ROS expression.(5) Compared with control group, the expression of NO, MDA increased and the activity of SOD decreased in model group. Compared with model group, XG reduced significantly the expression of NO, but the effect of XG was lower than that of HA. XG can decrease the level of MDA, and the effect of200μg/mL XG was better than that of HA. The expression of SOD of LPS-induced rabbit chondrocyte increased after XG treatment.(6) Compared with control group, the expression of IL-1β, TNF-a, PGE2increased in model group. Compared with model group, XG and HA reduced significantly the expression of IL-1β, TNF-a and PGE2. The activity of200μg/mL XG to reduce PGE2was higher than that of100μg/mL XG, and the activity of HA was higher than that of50μg/mL XG. The activity of100and200μg/mL XG to decrease TNF-a was higher than that of50μg/mL XG. and HA. The activity of HA to decrease IL-1β was higher than that of XG.(7) The mRNA expression of several factors showed that, compared with LPS group, HA significantly increased the mRNA expression of TIMP-1. Compared with LPS group,200μg/mL XG and100μg/mL HA significantly increased the mRNA expression of aggrecan, and no significant difference was observed between the effect of XG and HA. Compared with LPS group, XG and HA significantly increased the mRNA expression of type II collagen, and there was no significant difference among the effect of different doses of XG, and between the effect of XG and HA. Compared with LPS group, XG and HA could significantly reduce the mRNA expression of aggrecanase and MMP-1, and there was no significant difference among the effect of different doses of XG, and between the effect of XG and HA. Compared with LPS group, XG and HA could significantly reduce the mRNA expression of MMP-3, and50μg/mL XG showed the strongest effect. What’s more, with the increasing of XG concentration, the effect of XG decreased, and there was difference between the effect of HA and200μg/mL XG. Compared with LPS group, XG and HA could significantly reduce the mRNA expression of IL-1β, and100μg/mL XG and HA showed the greatest effect. There was difference among the effect of50,200μg/mL XG and HA. Compared with LPS group, XG and HA significantly reduced the mRNA expression of iNOS, and HA showed the greatest effect. There was difference among50,100,200μg/mL XG and HA, but no difference among the effect of different concentation of XG was observed.(8) The protein expression of several factors showed that, compared with the control group, LPS group significantly increased the protein expression of iNOS and MMP-13, and reduced the protein expression of aggrecan. Compared with LPS group, XG and HA could significantly increase the expression of aggrecan and reduce MMP-13expression, but no difference among the effect of different concentration of XG and between the effect of XG and HA was observed.100,200μg/mL XG could decrease iNOS expression, and there was difference between the effect of XG and HA. Conclusion The stimulation of chondrocytes by LPS induced significant production of ROS and NO, up-regulated iNOS and MMPs expression, and enhanced the degradation of collagen and aggrecan. The results of the experiments showed that XG could inhibit the expression of iNOS and MMPs, so that reduce the expression of NO, and prevent the degradation of the extracellular matrix (ECM) and enhance the expression of SOD. This indicates that XG is a safe and effective chondrocyte protective agent... |
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