Norcantharidin inhibits the expression of extracellular matrix and TGF-β1 in HK-2 cells induced by high glucose independent of calcineurin signal pathway.

Abstract

Norcantharidin (NCTD) was shown in our previous studies to attenuate renal tubulointerstitial fibrosis in rat models with diabetic nephropathy (DN). The aim of this study was to determine the effects of NCTD on the expression of extracellular matrix (ECM) and TGF-β1 in HK-2 cells stimulated by high glucose and on calcineurin (CaN)/NFAT pathway. Whether or not the antifibrotic effect of NCTD on renal interstitium was dependent on its inhibition of CaN pathway was also investigated. Experimental concentrations of NCTD were verified by cytotoxic test and MTT assay. HK-2 cells were transfected with CaN small interference RNA (siRNA). The mRNA and protein expressions of FN, ColIV, TGF-β1, and CaN in HK-2 cells were detected by real-time PCR and western blot. The CaN/NFAT pathway was examined by indirect immunofluorescence and western blot. Our study revealed that NCTD concentrations over 5 mg/l had overt cytotoxicity on HK-2 cells. Meanwhile, both 2.5 and 5 mg/l NCTD inhibited HK-2 cell proliferation (P < 0.05). NCTD inhibited the upregulation of FN, ColIV, and TGF-β1 of HK-2 cells stimulated by high glucose (P < 0.05), and also significantly downregulated the expression of CaN mRNA and protein in HK-2 cells (P < 0.05). In addition, not only was the nuclear translocation of NFATc inhibited, but its protein level in the nucleus was also reduced. Following CaN siRNA transfection, CaN mRNA and protein expression were significantly decreased. In contrast, the protein levels of FN, ColIV, and TGF-β1 increased in HK-2 cells stimulated by high glucose (P < 0.05). However, NCTD treatment downregulated their expression. These results indicated that NCTD could decrease the expression of ECM and TGF-β1 in HK-2 cells stimulated by high glucose, downregulate CaN expression, and block the CaN/NFAT signaling pathway. However, the effect of NCTD on inhibition of the expression of ECM and TGF-β1 was not associated with its inhibition of the CaN/NFAT pathway.

• Authors: Ying Li, Qiong Chen, Fu-You Liu, You-Ming Peng, Shuai Wang, Ji Li, Jun Li, Shao-Bin Duan, Lin Sun, Guang-Hui Ling, Jun-Hui Luo
• Journal: Laboratory investigation; a journal of technical methods and pathology (Lab Invest) Vol. 91 Issue 12 Pg. 1706-16 (Dec 2011) ISSN: 1530-0307 [Electronic] United States
• PMID: 21931301 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bridged Bicyclo Compounds, Heterocyclic
• Collagen Type IV
• Fibronectins
• RNA, Messenger
• RNA, Small Interfering
• TGFB1 protein, human
• Transforming Growth Factor beta1
• norcantharidin
• Calcineurin
• Glucose

Topics

• Bridged Bicyclo Compounds, Heterocyclic (pharmacology, therapeutic use)
• Calcineurin (metabolism)
• Cell Line
• Cell Proliferation (drug effects)
• Collagen Type IV (metabolism)
• Diabetic Nephropathies (drug therapy, etiology, metabolism)
• Drug Evaluation, Preclinical
• Epithelial Cells (drug effects)
• Extracellular Matrix (drug effects, metabolism)
• Fibronectins (metabolism)
• Glucose (adverse effects)
• Humans
• RNA, Messenger (metabolism)
• RNA, Small Interfering (metabolism)
• Signal Transduction (drug effects)
• Transforming Growth Factor beta1 (metabolism)