title: Experimental study on the suppression of sodium nitroprussiate-induced chondrocyte apoptosis by Tougu Xiaotong Capsule (透骨消痛胶囊)-containing serum.
abstract: 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 (P<0.05).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Academy of Integrative Medicine of Fujian University of Traditional Chinese Medicine, Fuzhou, China.
authors.forename: Xi-hai
authors.initials: XH
authors.lastname: Li
authors.forename: Ming-xia
authors.initials: MX
authors.lastname: Wu
authors.forename: Hong-zhi
authors.initials: HZ
authors.lastname: Ye
authors.forename: Wen-lie
authors.initials: WL
authors.lastname: Chen
authors.forename: Jiu-mao
authors.initials: JM
authors.lastname: Lin
authors.forename: Liang-pu
authors.initials: LP
authors.lastname: Zheng
authors.forename: Xian-xiang
authors.initials: XX
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D055162:Biocatalysis / Q000187:drug effects; D002214:Capsules; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology*; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D008297:Male; D008954:Models, Biological; D009599:Nitroprusside; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D002214:Capsules; D004365:Drugs, Chinese Herbal; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; C528129:tougu xiaotong; D009599:Nitroprusside; D053148:Caspase 3; D053453:Caspase 9