Effect of Zhengqingfengtongning on the expression of aggrecanase-1 (ADAMTs-4)and aggrecanase-2(ADAMTs-5)in the cartilage tissue of knee joint in rabbit models of osteoarthritis / 中国比较医学杂志

Objective To analyze the effect of a Chinese medicine Zhengqingfengtongning on the expression of aggrecanase-1(ADAMTs-4)and aggrecanase-2(ADAMTs-5)in cartilage tissue of knee joint in rabbit models of osteoarthritis,and to study the mechanism of action of Zhengqingfengtongning in treatment for osteoarthritis. Methods 32 New Zealand rabbits were randomly divided into the observation group and the model group, and 16 healthy New Zealand rabbits were chosen as the blank group. The blank group did not receive the intervention treatment, the model group received physiological saline in gastric gavage, the observation group was given the Zhengqingfengtongning by gastric gavage. The soft tissue samples of knee joint in the three groups were taken at 4 weeks after drug intervention. The joint morphology,joint fluid and articular cartilage were observed by histopathology. The ADAMTs-4 and ADAMTs-5 mRNA were detected by RT-PCR. The data were then statistically analyzed. Results The grades of articular cartilage and Mankin's score of the model group were significantly higher than those of the observation group and blank group,the grades of articular cartilage and Mankin's score of the observation group were significantly higher than those of the blank group, and the difference between the two groups was statistically significant(P< 0.05). The ADAMTs-4 and ADAMTs-5 mRNA expressions of the model group were significantly higher than those of the observation group and blank group. The ADAMTs-4 and ADAMTs-5 mRNA expressions of the observation group were significantly higher than those of the blank group, and the difference between the two groups was statistically significant(P< 0.05). Conclusions The Zhengqingfengtongning can improve the degree of joint lesions in osteoarthritis animal models,which may be related to inhibiting the expression of ADAMTs-4 and ADAMTs-5.

Role of 1α,25(OH)2D3in the metabolism of aggrecan and aggrecanase in chondrocytes of rat articular cartilage / 中国实验动物学报

Objective To investigate the role of vitamin D in the synthesis and degradation of aggrecan in rat articular chondrocytes at cellular level. Methods Rat articular chondrocytes were stimulated by IL-1α, IL-1β and TNF-α, respectively. Normal and inflammatory chondrocytes were treated with different doses of vitamin D, respectively. CCK8, Flow cytometry, real time-PCR and western blot analysis were used to examine the proliferation activity and apoptosis level of chondrocytes, and the expression of aggrecan, ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels. Results IL-1α,IL-1β and TNF-α significantly decreased the proliferation activity and increased the apoptosis level of the chondrocytes. Furthermore, IL-1α, IL-1β and TNF-α significantly decreased the expression of aggrecan, and increased the expressions of ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels in the chondrocytes. 1α,25 (OH)2D3supplementation significantly increased the proliferation activity and decreased the apoptosis level of chondrocytes stimulated by IL-1α, IL-1β and TNF-α in a dose-dependent manner, but not affected the normal chondrocytes. Meanwhile, 1α,25(OH)2D3also significantly increased the expression of aggrecan, and decreased the expressions of ADAMTS-4 and ADAMTS-5 at both mRNA and protein levels in the chondrocytes under inflammatory conditions. Conclusions Vitamin D may promote the anabolism of aggrecan and inhibit aggrecanase activity in chondrocytes under inflammatory conditions, which may impact overall protection for articular cartilage.

EXPERIMENTAL STUDY ON LENTIVIRUS-MEDIATED MULTI-GENES CO-TRANSFECTION IN BONE MARROW MESENCHYMAL STEM CELLS FOR TREATMENT OF KNEE OSTEOARTHRITIS IN CYNOMOLGUS MONKEY / 中国修复重建外科杂志

OBJECTIVE: To observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA). METHODS: BMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×107 cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR. RESULTS: No significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P<0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P<0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P<0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P<0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P<0.05). CONCLUSIONS: Lentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.

Expression of matrix aggrecanase 2 and a tissue inhibitor of metalloproteinases in degenerated intervertebral discs / 中华物理医学与康复杂志

Objective To observe the expression of aggrecanase 2 and a tissue inhibitor of metalloproteinase 3 (TIMP-3) in degenerate human lumbar intervertebral discs and their role in degeneration of the nucleus pulposus.Methods Pfirrmann classification was used to class degenerate intervertebral discs observed through MRI.They were divided into three groups:a control group (Pfirrmann grade Ⅰ-Ⅱ),a degeneration group (Pfirrmann grade Ⅲ-Ⅳ),and a severe degeneration group (Pfirrmann grade Ⅴ).A total of 45 cases accepted lumbar spine surgery for removing nucleus pulposus specimens.Each group contained 15 cases.After formalin-fixation and paraffin embedding,immunohistochemistry was used to detect aggrecanase 2 and TIMP-3 expression in the nucleus pulposus cells.Results The percentages of cells positive for aggrecanase 2 were (13.58 ± 7.76) ％,(33.48 ± 13.95) ％ and (56.00 ± 18.39) ％ in the control,degeneration and severe degeneration groups respectively.These differences had statistical significance.The percentages of cells positive for TIMP-3 were (34.78 ± 13.80) ％,(46.77 ± 10.98) ％ and (50.65 ± 16.45) ％,and these differences were again statistically significant.The aggrecanase 2/TIMP-3 ratios were also significantly different.Conclusion As the degree of degeneration of the nucleus pulposus increased,the expression of aggrecanase 2 and TIMP-3 rose,which indicates that both changes were closely connected with the degeneration.Their ratio was correlated with the degree of degeneration of the nucleus pulposus.

SKI306X inhibition of glycosaminoglycan degradation in human cartilage involves down-regulation of cytokine-induced catabolic genes

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.

Regulation of aggrecanase-1 and aggrecanase-2 expressions by hypoxia and cytokines in rheumatoid fibroblast-like synoviocytes / 대한내과학회지

BACKGROUND: While aggrecanases (aggrecanase-1 and aggrecanase-2) are substantially responsible for cartilage aggrecan breakdown in rheumatoid arthritis (RA), not much information is available on the regulation or expression of the two key aggrecanases in rheumatoid fibroblast-like synoviocytes (FLS). The aim of this study is to determine the effect of hypoxia and several cytokines on the expression of the aggrecanases in rheumatoid FLS. METHODS: FLS obtained from RA patients were cultured under hypoxic condition for 24 hours. Quantitative polymerase chain reaction assays for mRNA expression of aggrecanase-1 and aggrecanase-2 were performed on FLS cultured under hypoxia. Additionally, to see the effect of various cytokines, same experiments were conducted after treating FLS with IL-1beta, IL-6, EGF, TGF-beta and TNF-alpha, compared with control. RESULTS: Hypoxia significantly increased both aggrecanase-1 and -2 mRNA expression in rheumatoid FLS compared with normoxia. IL-1beta, IL-6, EGF, TGF-beta and TNF-alpha upregulated the mRNA expression of aggrecanase-1. Both EGF and TGF-beta upregulated the mRNA expression of aggrecanase-2, but TNF-alpha significantly downregulated the mRNA expression of aggrecanase-2. CONCLUSIONS: These results showed upregulation of aggrecanase-1 and -2 by hypoxia and differential regulation of aggrecanase-1 and -2 by various cytokines in rheumatoid FLS. It suggests that hypoxia and cytokines enhance the aggrecanase activity of RA FLS and contribute to joint destruction.