Increased Chondrocyte Apoptosis in Kashin-Beck Disease and Rats Induced by T-2 Toxin and Selenium Deficiency

Objective To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease（KBD） and in an established T-2 toxin-and selenium（Se） deficiency-induced rat model. Methods Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase d UTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and m RNA levels were determined by real-time reverse transcription polymerase chain reaction. Results Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and m RNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet. Conclusion T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.

Inhibitory Effects of SRT1720 on the Apoptosis of Rabbit Chondrocytes by Activating SIRT1 via p53/bax and NF-κB/PGC-1αPathways

SRT1720, a new discovered drug, was reported to activate silent information regulator 1（SIRT1） and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside（SNP）（2.5 mmol/L） and divided into five groups： 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group（0 μmol/L SRT1720）. MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α（PGC-1α） were detected by Western blotting and the expression levels of SIRT1, type Ⅱ collagen, and aggrecan m RNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the m RNA expression levels of type Ⅱ collagen and aggrecan increased（P〈0.05）, and the expression levels of p53, NF-κB and bax decreased（P〈0.05）. It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.

Experimental Study on the Suppression of Sodium Nitroprussiate-lnduced Chondrocyte Apoptosis by Tougu Xiaotong Capsule(透骨消痛胶囊)-Containing Serum

Objective：To study the mechanism of action of Tougu Xiaotong Capsule（透骨消痛胶囊,TGXTC） ex vivo in suppressing chondrocyte（CD） apoptosis induced by sodium nitroprussiate（SNP）.Methods：Thirty New Zealand rabbits,2 months old,were randomized by lottery into five groups,six in each：the blank group treated with saline,the positive control group treated with Zhuanggu Guanjie Pill（壮骨关节丸,70 mg/kg）,and the three experimental groups,EGA,EGB,and EGC,treated with low dose（35 mg/kg）,moderate dose（70 mg/kg）,and high dose（140 mg/kg） of TGXTC,respectively.All treatments were administered via gastrogavage twice a day for 3 days.Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared.CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining.SNP of various final concentrations（0,0.5,1.0,and 2.0 mmol/L） was used to induce CD apoptosis,and the dosage-effect relationship of SNP in inducing CD apoptosis was determined.Serum samples from the blank,control,and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP.Cell apoptosis was determined by Hoechst 33342 staining,viability of CDs was quantified by MTT,CD apoptosis rate was determined by annexin V-FITC/PI staining,levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR,and contents of caspase-3 and caspase-9 proteins were determined by colorimetry.Results：CD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner.The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study.Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis,decrease in p53 mRNA expression,inhibition of catalytic activities of caspase-3 and caspase-9,and increase in Bcl-2 mRNA expression when compared with the serum from the blank group（P0.05）.Conclusion：TGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression,and inhibition of caspase-3 and caspase-9 catalytic activities.