title: [Effect of norcantharidin on the expression of FN, Col IV and TGF-β1 mRNA and protein in HK-2 cells induced by high glucose].
abstract: OBJECTIVE
  To observe the effect of norcantharidin (NCTD) on the expression of mRNA and protein of fibronectin (FN), collagen IV(Col IV) and transforming growth factor-β1(TGF-β1) in human kidney proximal tubular epithelial (HK)-2 cells induced by high glucose.
  
  
  METHODS
  HK-2 cells were incubated with serum-free DMEM for 24 h to synchronize cell growth, and then the cells were divided into 4 groups: Group C (5.5 mmol/L D-glucose), Group M (5.5 mmol/L D-glucose + 24.5 mmol/L-mannitol), Group HG (30 mmol/L D-glucose), and Group HG + NCTD (30 mmol/L D-glucose + 0.5-40 mg/L NCTD). Cytotoxicity of HK-2 cells induced by high glucose of NCTD was detected by Trypan blue dye exclusive assay. The effect of NCTD on the proliferation of HK-2 cells in high glucose was determined by MTT. The cells were collected to extract total RNA and protein at 6, 24 and 48 h after the incubation. The expression of FN, Col IV and TGF-β1 mRNA was examined by RT-PCR, and FN, Col IV and TGF-β1 protein was analyzed by Western blot.
  
  
  RESULTS
  Trypan blue dye exclusive assay showed NCTD concentrations over 5 mg/L were rather toxic in HK-2 cells. The proliferation of HK-2 cells in high glucose was interrupted by interfered with 5 mg/L NCTD as measured by MTT (P<0.05). NCTD at 5 mg/L had a stronger inhibitory effect than NCTD at 2.5 mg/L. Real-time PCR and Western blot showed that the mRNA and protein expression of FN, collagen IV and TGF-β1 increased in HK-2 cells treated with high glucose (P<0.05), while that in cells treated by NCTD was dramatically inhibited (P<0.05). No change in these parameters was detected in the 30 mmol/L D-mannitol control group (P>0.05).
  
  
  CONCLUSIONS
  NCTD can downregulate FN, collagen IV and TGF-β1 mRNA and protein expression in HK-2 cells stimulated by 30 mmol/L D-glucose.
authors.affiliation: Department of Nephrology, Second Xiangya Hospital,Institute of Nephrology, Central South University, Changsha 410011, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Junhui
authors.initials: J
authors.lastname: Luo
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Lin
authors.initials: L
authors.lastname: Sun
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.forename: Xiangqing
authors.initials: X
authors.lastname: Xu
authors.forename: Youming
authors.initials: Y
authors.lastname: Peng
authors.forename: Fuyou
authors.initials: F
authors.lastname: Liu
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002460:Cell Line; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D024141:Collagen Type IV; D005353:Fibronectins; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; C069741:norcantharidin; D005947:Glucose