Soluble dispersive mixture of chicken collagen type Ⅱ delays the morphological changes of articular cartilage in rats with osteoarthritis:Comparison among different oral doses and excipients

BACKGROUND: Soluble dispersive mixture of domestic chicken collagen type Ⅱ (SmCC Ⅱ ) has been proven to be safe and effective for rheumatoid arthritis (RA) treatment while there are some similar articular cartilage degradation and autoimmune pathogenesis between osteoarthritis (OA) and RA, so it is worth studying whether SmCC Ⅱ is effective for the precaution or treatment of OA.OBJECTIVE: To observe the prophylactic and therapeutic effects of SmCC Ⅱ on rat OA and analyze the concomitant matrix metalloproteinase (MMPs) and tissue protease changes in osteoarthritic rats.DESIGN: Randomized and controlled observation.SETTING: Department of Clinical Pharmacy, Sixth People's Hospital, Shanghai Jiao Tong University.MATERIALS: Totally 258 rats of clean grade and either gender, aged 6 weeks were involved in this experiment. SmCC Ⅱ (white powder, Batch No.00031004) was provided by Professor Ren Geng-Fu from Shanghai Engineering Institute of Herbal Biomedicine. The 20 mg/L and 80 mg/L solution of SmCC Ⅱ effective component were prepared before use.While 0.25% excipient (mannitol, product of Jiangsu Tianyuan Medical Co., Ltd) solution was prepared by 200 g/L mannitol dissolved in sterile saline solution.METHODS: The study was carried out in the Whole Animal Laboratory and Experimental Center, Sixth People's Hospital,Shanghai Jiao Tong University between March 2003 and February 2006. ①Prophylactic study: Totally 132 rats were randomized into 5 groups: OA group (n =36): treated with sterile saline solution orally by a 16G syringe, 1mL/d; Low- and high-dose SmCC Ⅱ groups (n =24, respectively): treated with 20 mg/L and 80 mg/L SmCC Ⅱ solution orally, 1 mL/d;Excipient group (n =24): treated with 2.5g/L mannitol, 1 mL/d. OA models were surgically induced in these 4 groups by Hilth's method; Normal group (n =24): Rats without operation were treated with sterile saline solution orally, 1 mL/d. All the rats were administrated on the day of modeling. ②Therapeutic study: Totally 126 rats were randomized into 5 groups, grouping, administration and rat number in different groups were the same as those in prophylactic study,respectively, except for the n =30 in OA group. In addition, all the rats were adminstrated for 8 weeks since 6 weeks after operation. The knee joints of right hind limb of all the rats were taken off after 1-week treatment. And sample was cut open along coronal incision for use. ③Morphological study of articular cartilage was conducted by HE staining and Mankin score was calculated to evaluate the severity of arthritis, immunohistochemical studies of matrix metalloproteinase (MMP)-13, MMP-9 and cathepsin K (Cath K) were carried out by ABC method in situ and the percentage of positive-stained chondrocytes were calculated while the mRNA level for MMP-13, MMP-9, Cath K and the tissue inhibitor of metalloproteinase 1 (TIMP1) were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method.MAIN OUTCOME MEASURES: ①The morphological changes of articular cartilage in different groups in prophylactic or therapeutic study.②The level of MMP-13, MMP-9, Cath K and their corresponding mRNA levels.RESULTS: All the 258 rats were involved in the final analysis. ①Prophylactic study: Apparent degeneration appeared in the rats of OA group and the mankin score in OA group was higher than that in high- or low- SmCC Ⅱ groups [(6.44±0.81), (2.75±0.85), (2.70±0.81) points respectively, P ＜ 0.05], the mRNA and positive-stained chondrocyte percentage of MMP-13, MMP-9 and Cath K in OA group was higher than that in high- or low-dose SmCC Ⅱ group, respectively (P ＜0.05-0.01) while the TIMP1mRNA level in OA group was not significantly higher than that in high or low SmCC Ⅱ group (P＞ 0.05). There were no significant changes on the level of MMP-13, MMP-9 and Cath K between excipient and OA group (P ＞ 0.05). ②Therapeutic study: The Mankin's score of OA group was higher than high or low SmCC Ⅱ group [(6.96±1.02), (3.08±0.45), (4.00±0.94) respectively, P ＜ 0.05-0.01] while the mRNA and positive-stained chondrocyte percentage of MMP-13, MMP-9 and Cath K in OA group was higher than that in high- or low-dose SmCC Ⅱ group,respectively (P ＜ 0.05-0.01) while the TIMP1mRNA level in OA group was not significantly higher than that in high- or low-dose SmCC Ⅱ group (P 0.05).CONCLUSION: SmCC Ⅱ can delay the degradation of articular cartilage of OA rats and maybe effective for OA prevention or treatment. The mechanism maybe related to SmCC Ⅱ reducing the synthesis of MMP-13, MMP-9 and Cath K at transcriptional level.