ABSTRACT
Objective To investigate the protective effects of carboxymethylated chitosan (CMCS) on nitric oxide (NO) induced apoptosis in rat chondrocytes, and the probable roles and mechanisms of PI3K/Akt signaling pathway in these process.Methods Rat knee articular cartilage was used as the source of chondrocytes, the cells were identified by immunohistochemical staining against collagen type Ⅱ, odium nitroprusside (SNP, 3 mmol/L) was used to establish the apoptotic models of chondrocytes.Cells were divided into four groups: the control group, the SNP-induced group, the SNP+CMCS treated group, the SNP+CMCS+PI3K inhibitor Wortmannin treated group.Cell proliferation were assessed by cell proliferation assay kit (CCK-8), the apoptotic rate of chondrocytes was determined by FCM with Annexin V-FITC/PI double staining, the expression levels of MMP-13 and TIMP-1 mRNA were detected by real-time polymerase chain reaction (PCR) analysis, the expression of Akt and p-Akt protein levels was detected by Western blotting analysis.One-way analysis of variance (ANOVA) statistical analysis was used to calculate the data.Results Three mmol/L SNP can inhibit proliferation (0.221±0.023), and the proliferation was reduced by 70％ compared with the control group (0.736±0.032, F=8.203, P=0.021);and the induced apoptosis in cultured chondrocytes could be observed.The apoptotic rate was (68.8±5.2)％.Increased MMP-3 and decreased TIMP-1 mRNA expression were observed in SNP-induced cells.After adding CMCS to SNP-induced chondrocytes, the proliferation was increased while apoptotic rate was decreased, the apoptotic rate decreased to (14.7±2.3)％.CMCS could promote the activation of p-Akt in SNP-induced chondrocytes and restore SNP-induced MMP-13 and TIMP-1 mRNA expression.Conclusion CMCS could protect apoptosis in SNP induced chon-drocvtes via activation of PI3K/Akt pathwav.
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Objective To observe the effect of intra-articular injection of CM-chitosan on nuclear factor κB (NF-κB) activation and nitric synthase expression in rat osteoarthritis cartilage,and to explore the mechanism of inhibition of joint destruction.Methods Thirty-six SD rats were randomly divided into three groups:A,B,C,12 in each group.Group A was the sham group,group B,C rats had the medial collateral ligaments cut off and part of medial meniscus were removed to establish osteoarthristis model.Group C rats were injected with 3％ carboxymethyl chitosan intra-articularly 0.15 mg/kg 5 weeks later,and then repeated injection every 1 week.Animals were sacrificed 11 weeks after surgery.The gross changes of cartilage and the expression of NF-κB (P65) were compared by immunohistochemistry,the protein expression of I-κB and P65 in nucleus were detected by Western-bloting.Inducible nitric oxide synthase (iNOS) mRNA and protein expres-sion were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western blot.The general score of cartilage was analyzed by H test,the rest data was analyzed by one-way ANOVA.Results The cartilage degeneration scores pf group C (intra-articular injection of CM-chitosan group) were significantly less than those of group B.The protein expression of NF-κB of the articular cartilage in group C (106±7)was significantly lower than group B (147±8),the I-κB degradation was inhibited significantly in group C,and the expression of iNOS mRNA and iNOS protein were reduced in OA art~icular cartilage of arthritis rat chondrocytes,therefore,it had protective effect on articular cartilage.Conclusion CMC may inhibit NF-κB signaling pathway by inhibiting the degradation of I-κB in cartilage,which reduces iNOS mRNA and protein expression in rat osteoarthritis cartilage,thereby protects rat osteoarthritis cartilage cells.
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Objective To investigate the efffect of carboxy-methyl-chitosan (CM-chitosan) on NF-kB activation and iNOS expression in IL-1β-induced rat chondrocytes, and the mechanism is explored. The statistical treatment was ANOVA. Methods The chondrocytes derived from knee joint of rats were cultured in vitro. 10 ng/ml IL-1β with or without CM-chitosan of varied concentrations (100, 200, 300 μg/ml) was added into the culture medium. Inducible NO synthase (iNOS) was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The expression of NF-κBn and NFκBc as well as I-κB were examined by Western blotting. Results CM-chitosan could significantly decrease iNOS expression of IL-1β-induced chondrocytes. Degradation of I-κB and NF-κB nuclear translocation were reversed by carbox-ymethyl-chitosan. Conclusion The study has demonstrated that CM-chitosan can inhibit the synthesis of inflammatory mediators in rat chondrocytes when stimulated with IL-1β through a NF-κB-dependent mechanism.
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Objective To investigate the way of resection of high-sacrum tumors and the reconstruction way of the sacrum. Methods From October 2001 to October 2005,7 patients with high-sacrum tumors were enrolled. After resection, the pelviclring were recormtmcted with Chinese Great Wall pedicle screw system and fibulae graft,corresponding chemotherapy and radiotherapy were given after operation. Results The short-term results were satisfactory with the lumbosacral pain reduced and the neurological function improved in different degrees, however dysuria occurred in 1 ease and 1 case cerebrospinal fluid leakageand 1 case postoperative infection and delayedunion among the 7 eases in this group. In the follow-up period of 6 months to 3 years,4 eases died for tumor recurred or metastasis. Conclusions Surgical procedure,reconstruction of the sacrum and postoperative comprehensive treatment have important effects on the prognosis. Meanwhile,it is operative key to lessen operative hemorrhage,reserve the function of caudal equine and rebuild weight high post function of the pelvis after superior sacrum tumor is removed.