Objective: To investigate the therapeutic effect of N- acetylglucosamine on experimental osteoarthritis in rabbits.
Methods: Anterior cruciate ligament transection (ACLT) was induced in the laboratory. In the first study, rabbits (6 rabbits in each group) were injected with glucose or normal saline three times a week from 1 week after operation. In the second study, rabbits (8 in each group) were injected with glucose (or once or twice a week) or normal saline. In the third study, rabbits (7 rabbits in each group) were injected with glucose, hyaluronic acid or normal saline twice a week. Eight weeks after death, the knee joint was evaluated macroscopically and histologically by ACLT.
Results: The experiment of intramuscular injection in the treatment of knee arthritis in glucose rabbits showed no slight cartilage protection, but showed anti-inflammatory activity. Conversely, intra-articular glucose management twice a week can reduce cartilage degeneration. In addition, intra-articular glucose also inhibits synovitis. Weekly intra-articular injection of glucose has not proved to be effective. In the treatment of hyaluronic acid, the therapeutic effect of cartilage protecting glucose is better than that of tackifier.
Conclusion: Intra-articular glucose has chondroprotective effect, and its anti-inflammatory activity has been verified.
Abbreviation: ACLT, anterior cruciate ligament transection; Polysaccharide and glycosaminoglycan; Global land cover network, glucose, glucose, N- acetylglucosamine, arthritis, osteoarthritis.
Keywords: N- acetylglucosamine; Cartilage; Knee osteoarthritis
Osteoarthritis is the most common joint disease, characterized by progressive cartilage degeneration and subchondral bone changes. At present, OA is used to treat chronic synovitis. 13 is a limited symptom management, analgesic and anti-inflammatory drug, and a thickening device. Glucosamine (Global Land Cover Network) is widely used to treat arthritis and improve disease activities. Some people think that the global land cover network has delayed the gradually narrowing joint space 8, and improved the biomechanical OA knee joint 9. 8, 9, 10, 1 1.
The mechanism of global land cover network guiding anti-arthritis activities remains to be determined. The limitation of studying the global land cover network in vitro lies in the anti-inflammatory and cartilage protection. The main influence of the global land cover network is to observe the low concentration of millimoles, 12, 13, which is a factor of oral global land cover network. Although it can be expected that the global land cover network and its derivatives have the greatest anti-arthritis effect, the sugar concentration in joints is 103, 104. 14 16.
In this study, N- acetylglucosamine was selected as the first choice for intra-articular treatment. This is based on our previous view that glucose inhibits interleukin 1 more effectively than the global land cover network. Activation of human articular chondrocytes 13 in vitro In addition, when the concentration exceeds 10 mmol/L, the global land coverage network significantly reduces the feasibility of culturing chondrocytes in vitro, and 17 and 18 constitute potentially complex intra-articular administration. Conversely, even if the concentration is higher than 50 mmol/L, glucose will not induce chondrocyte death.
In this study, anterior cruciate ligament transection (ACLT) caused by glucose-induced knee arthritis in rabbits was analyzed. Three independent experiments were carried out. The first research report examined the efficacy of intramuscular injection in controlling blood sugar. The second study determined the optimal frequency of intra-articular injection of glucose for anti-arthritis activities. The third study compared the efficacy of intra-articular glucose and intra-articular hyaluronic acid, which is a viscous support device currently used to treat knee osteoarthritis.
Materials and methods
reagent
Glucose was purchased from Sigma (St. Louis, Missouri). It was dissolved in physiological saline, disinfected and filtered through a 0.22 micron filter (acton Corning Company). Sterile glucose solution stored in sodium hyaluronate (Hyalgan) at 4℃ was purchased by Sanofi Fort Santa de Laba (new york).
animal
New Zealand white rabbits, aged 8 12 months, weighing 3.74±4.2kg, with closed epiphysis, were all used in the experiment. All the studies were conducted in accordance with the scientific guidelines for experimental animals in the United States, and were approved by the Scripps Institute and the Animal Review Committee of the University of California, San Diego. * * * for 57 rabbits.
ACLT
Unilateral or bilateral ACLT showed medial arthroscopy. 19 was used in each group of experiments. All animals remain independent and free to move. These animals were operated on eight weeks after being killed. Previously published data showed that most cartilage degeneration of ACLT rabbits developed at this time. 20,265,438+0.
Intramuscular injection of glucose
From 1 week after operation, glucose was injected into muscle three times a week for 7 weeks. The dose of glucosamine is 200mg/kg per injection. The control group was injected with the same amount of normal saline.
Intraarticular injection of glucose and hyaluronic acid
Intraarticular injection of glucose began at 65438 0 weeks after operation and lasted for 7 weeks. Rabbits were injected with 1-2 times, and the weekly glucose was 0.3 ml for each knee joint. The single dose of glucose injection is 80 mg per injection. The control animals were injected with joint saline twice a week (combined with 0.3 ml). Hyaluronic acid (3 mg/0.3 ml per joint) was injected into the joint cavity, starting from 1 week after operation and lasting for 7 weeks. Synovial fluid analysis was performed in three animals with complete synovial fluid exudation (two animal control groups and one hyaluronic acid group). The joint fluid culture of three animals was negative.
Evaluation of knee joint with hair morphology
The hair morphology of knee joint, including tibial plateau and femoral condyle, was evaluated by double-blind method.
The distal femur and proximal tibia were obtained so that the axial length of the bone was 3.54 cm. Each sample on the articular cartilage surface was covered with a solution, including phosphate buffer (1:5 ratio) of Indian ink (eberhard Flower, Lewisburg, Tennessee). The solution in ink is to remove the mild imprint with a paper towel moistened with phosphate buffer in advance. Subsequently, all the joint photos and digital images are analyzed.
The evaluation results of graded articular cartilage are as follows:
1 (complete surface) The surface has a normal appearance, and no ink remains.
The second (minimum jitter) surface keeps the ink as elongated spots or light gray patches.
The tertiary (open flutter) area has a velvety appearance and retains the ink as black patches.
Grade 4 (erosive) cartilage loss exposes bones.
digital video
At high altitude, the femoral and tibial condyles on the articular surface were gently stripped of dry and clean loose tissues. Each trunk is close to the optical substitute. Picture (resolution: 60 pixels/mm2; Screen magnification: 20 times) Using Canon EOS D30 digital camera, the femoral condyle was obtained with a macro lens of 100 mm at a distance of about 12 cm. The millimeter scale is included in the precise scale of the captured image. Then the scale image is projected on the femoral condyle of the three-dimensional model. Three-dimensional surface lesions detect the edge lesions of interactive conspiracy. A digital image of the tibial articular surface was obtained as described above. Three-dimensional projection is not used, because the tibial surface is relatively flat, and there is no big difference between two-dimensional measurement and three-dimensional measurement.
Histologically graded knee joint
The distal femur and proximal tibia from rabbit knee joint were fixed in 10% buffered formalin, and decalcified in -2 decalcifier (ThermoShandon, Pittsburgh, CA) and paraffin embedded block. Lateral sagittal plane and medial femoral condyle and tibial coronal plane were used for histological analysis.
To evaluate the content of glycosaminoglycan sulfate (mucopolysaccharide) in stained tissue sections and the O/ fast green of saffron.
Histological evaluation of synovitis is based on the proliferation of synovium and the existence of villous structure. Synovium is attached to tibial plateau, lateral condyle of femur and medial condyle alone. The single-layer fluff-free structure of synovial lining is considered normal (grade 0). The contents of hypertrophic synovium and synovial villi are considered to be abnormal (1 grade).
statistical analysis
Fisher exact test and two-tailed t-test are used for statistical analysis of experimental data: two samples are paired (digital data analysis).
result
Intramuscular glucose and experimental osteoarthritis in rabbits
Anti-arthritis active glucose is an independent evaluation group experiment. Firstly, the management of a series of experimental activities is analyzed. Large doses of glucose are injected by muscle. The therapeutic effect of intramuscular injection was evaluated in 6 bilateral glucose ACLT rabbits and 6 bilateral ACLT rabbits who received intramuscular injection of normal saline. Hair morphological analysis showed that there was no significant difference between the tibial plateau and femoral condyle and the animals in the treatment group and the control group with glucose cartilage injury. Histological examination of knee joint showed that there was no statistically significant difference in chondroitin sulfate mucopolysaccharide content, cartilage integrity and therapeutic cloning of chondrocytes between glucose injected into rabbits and normal saline. Compared with the control group, the synovial diffusion of rabbits treated with glucose showed a downward trend. Overall, intramuscular injection of glucose did not prove the efficacy of cartilage protection in experimental osteoarthritis in rabbits, but it slightly reduced synovitis.
Anti-arthritis active intra-articular glucose
As the largest biological activity, glucose is considered as a low millimolar concentration in vitro. On June 5438+3, 2003, we examined the experiment of highly effective intra-articular glucose in the treatment of osteoarthritis. The experiment includes three groups of rabbits. In the first group, rabbits were injected with glucose twice a week; In the second group, rabbits were injected with glucose once a week. The third group of rabbits received intra-articular saline. The period of treatment was 7 weeks.
The morphological analysis of hair showed that compared with the severity of osteochondrosis in the control group, the treatment of femoral condyle twice a week had a tendency to reduce intra-articular glucose (P 0.11); Weekly intra-articular glucose management did not show significant benefits (Figure 1A). Similar results show that in the analysis of tibial plateau, twice-weekly injection of glucose has a beneficial therapeutic trend, and phosphorus is "0. 18" (Figure 1B). Did not achieve statistical significance, probably because the group is small.
Results Macroscopically, the cartilage lesions analyzed by digital image were confirmed. The average (standard deviation) control area of femoral condylar cartilage lesion was14.34 (12.86) mm2v4.40 (7.57) mm2. Rabbits were treated twice a week (P 0.02). Weekly glucose administration did not significantly reduce the cartilage lesion area (13.74 (12.75438+0) square millimeter and phosphorus "0.88".
Generally speaking, articular cartilage lesions of tibial plateau are smaller than femoral condyle, which probably reflects the protective effect. Although rabbits with glucose lesion less than 50% (3.76 (6.69) mm2v 7.90 (8.44) mm2) in the control group were treated twice a week, there was no significant difference between the two groups due to the limited number of experimental animals (P "There was no significant difference between the weekly treatment of rabbits and glucose, but compared with the control group (the average cartilage lesion area was 9.59 (6.31)
Finally, twice-weekly intra-articular glucose management can reduce the severity of cartilage degeneration more than once-weekly intra-articular glucose and saline injection.
Compared with intra-articular glucose, intra-articular hyaluronic acid
The third study report of each goal confirmed the independent effect of intra-articular injection of glucose twice a week on cartilage protection, and compared the curative effects. Intra-articular injection of glucose hyaluronic acid is used to treat knee OA.2325 To this end, unilateral intra-articular injection of glucose (7 rabbits), hyaluronic acid (7 rabbits) or normal saline (7 rabbits) in ACLT was performed twice a week.
Hair morphology analysis showed that the trend of femoral condyle improved rickets. Compared with the control group and hyaluronic acid treatment group, phosphorus was "0. 10" (fig. 2a).
Examination of the tibial plateau showed significant chondroprotective glucose activity, because only 1/7 rabbits developed chondromalacia, while rabbits in saline injection group on June 7th and hyaluronic acid treatment group on May 7th showed chondromalacia (Figure 2b). The effect of chondrocytes on glucose statistics was better than that of normal saline group (P "0.0 13) and hyaluronic acid treatment group (P" 0.034).
Digital image analysis shows that the femoral condyle (Figure 3a) and tibial plateau (Figure 3b) are treated with glucose and animal hyaluronic acid, showing greater efficacy.
In short, the results of the third series of experiments confirmed that the therapeutic effect of glucose injection was controlled twice a week, and its superior cartilage protection activity was compared with that of intra-articular hyaluronic acid treatment.
Histological analysis
The knee joints of 7 control rabbits and 6 glucose-treated rabbits (from the third research report) were used for histological analysis. A rabbit was excluded from the glucose treatment group because of the determination of inclusion bodies in articular cartilage.
The loss of chondroitin sulfate mucopolysaccharide in femoral medial condyle and tibial plateau is also similar to the effect of glucose treatment and control (see table 1). Compared with the control animals, glucose treatment on the lateral femoral condyle of rabbits showed a tendency to reduce the loss of sulfated mucopolysaccharide (P 0.056). The review shows that the trend of cartilage integrity is cartilage protective activity on tibial plateau, and phosphorus is "0. 10" (see table 1).
discuss
At present, the drug treatment of osteoarthritis is limited to relieving pain and improving the viscoelasticity of synovial fluid, but these measures have no structural adjustment activity. The results of recent multi-center clinical trials show that oral global land cover network can reduce losses. Although OA Cartilage Joint.8 is widely used in patients with global land cover network and office automation, there is only limited data, and its activity is in experimental arthritis. Treatment of experimental arthritis in rabbits with oral OA lesions. 26, morphologically improved global land cover network, injecting chymopapain into joints caused by experimental arthritis. The content of sulfated glycosaminoglycan in oral cartilage preserved by global land cover network deteriorated significantly, but the pathological mechanism of osteoarthritis joints in global land cover network is still unclear. At the concentration of millimoles, it is impossible to achieve oral administration with the best experimental research activities. The concentration of 12 16 can be injected into the joint cavity. Based on our previous point of view, this shows that glucose with anti-inflammatory activity and cytotoxicity is superior to the global land cover network in vitro. 13, 18 The purpose of this study was to investigate the anti-arthritis activity of high-dose glucose injection in rabbits and joints.
ACLT induced by experimental arthritis in rabbits. This is the most widely used model to study the treatment of diseases. OA.28 improved ACLT leads to biomechanical abnormalities of knee joint, including adding front drawer and increasing pronation during extension and 90 flexion. Similar to OA knee 28, the most serious cartilage degeneration in rabbits occurred in the medial femoral condyle, followed by condyle 28, which was not involved in the field of mild and moderate arthritis of tibial plateau ACLT.
Experimental osteoarthritis in rabbits treated by intramuscular injection of glucose did not prove the protective effect of cartilage, but showed a trend of decreasing synovitis. In two independent studies, intra-articular glucose management twice a week has been shown to reduce cartilage degeneration. Weekly intra-articular injection of glucose failed to prove the therapeutic effect.
Inflammatory changes and ACLT models, including joint effusion and synovial hyperplasia, are important mechanisms of joint pain in patients OA.301. In this study, we observed the anti-inflammatory activity of intra-articular glucose, which decreased in severe nodular synovitis. Observing the anti-arthritis effect of glucose can partly lead to the ability to stop the inflammatory reaction. It directly affects the reaction of glucose to chondrocytes, including inhibiting the expression of inducible nitric oxide synthase and oxygenase. 13 may prevent cell death and cartilage matrix degradation. Glucosamine also inhibits the influence of inflammatory reaction on chronic peritoneal dialysis. In addition, oral glucose has been successfully used to control inflammation in children with Crohn's disease and ulcerative colitis. 33
Among other glucose properties, it is a stimulating effect, which contributes to its anti-arthritis activity. Synthesis of hyaluronic acid in human articular chondrocytes and synovial fibroblasts. 34 increased the production of hyaluronic acid. After contact with glucose, this is also considered to be the reason why glucose solution is supplemented to human peritoneal mesothelial cells and fibroblasts. Chronic peritoneal dialysis accumulates polysaccharides between peritoneum and reduces peritoneal permeability. 36
Our research includes comparing the anti-arthritis effects of glucose and hyaluronic acid. According to the experimental results and limited observation under office automation microscope, hyaluronic acid has mild cartilage protection and anti-inflammatory activity in human body. 3739 and hyaluronic acid, glucose physiological saline managed by internal joints has a shorter half-life (glucose and hyaluronic acid per hour are measured in minutes (Sambo Yellow, unpublished data)). However, experimental arthritis shows better disease glucose modification activity than hyaluronic acid. This discovery opens up a way for designing and optimizing the pharmacokinetic parameters of glucose preparation to obtain better therapeutic effect of OA.
No adverse reactions of systemic or local blood glucose control were found in this study. The published data also show that glucose has excellent safety for human body. Adult healthy volunteers have good tolerance to intravenous glucose up to100 g/h. There was no significant change in serum glucose and ammonia concentrations. Oral or rectal administration of glucose in children with inflammatory bowel disease did not produce adverse reactions. 33
In a word, this study confirmed the modification and anti-inflammatory activity of pre-n- acetylglucosamine on glucose and diseases.
Express gratitude/gratitude
We thank Clayton-Achermann for his excellent technical help and histological analysis, and Dr. James Koziol for his help and statistical analysis of data.
This work was supported by the National Institutes of Health AG07996, AT00052 and AR46990.
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