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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (6): 632-637.doi: 10.3736/jcim20110609

• Original Experimental Research • Previous Articles     Next Articles

Andrographolide inhibits extracellular signal-regulated kinase 1/2 signaling pathway in activated macrophages

Lin-hua Qin1(), Jiao Lü1, Lin Kong2, Yun-xing Shi1, Yong-ping Li1, Guo-zhong Zhou1, Zhi-wu Zheng1, Lin Li1, Xing-ying Ji1   

  1. 1. Department of Digestive Diseases, the 411 Hospital of Peopled Liberation Army, Shanghai 200081, China
    2. Shanghai Health Science Center, Chinese Academy of Sciences, Shanghai 200025, China
  • Received:2011-02-28 Accepted:2011-04-14 Online:2011-06-20 Published:2011-06-15
  • Contact: Qin Lin-hua E-mail:ando_8171@yahoo.com.cn

Objective: To investigate the effects of andrographolide on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and tumor necrosis factor-α (TNF-α) expression in lipopolysaccharide (LPS)-activated macrophages.
Methods: LPS-activated mouse peritoneal macrophages were cultured in media with different concentrations of andrographolide. Cytotoxicity of andrographolide was detected by cell counting kit-8. The macrophages were lysed, and then expressions of phosphorylated ERK1/2, JNK and p38 and nuclear factor-κB inhibitor (IκBα) protein were detected by Western blotting and TNF-α mRNA expression was detected by reverse transcription-polymerase chain reaction. Supernatants of the macrophages were used to detect content of TNF-α protein by enzyme-linked immunosorbent assay.
Results: Andrographolide at 1-100 μg/mL showed no cytotoxicity on LPS-activated mouse peritoneal macrophages. Andrographolide inhibited ERK1/2 phosphorylation in LPS-activated murine peritoneal macrophages, which was concentration-dependent (P<0.01). Andrographolide at 1-25 μg/mL had no effects on phosphorylation levels of JNK and p38 and IκBα degradation in LPS-stimulated mouse peritoneal macrophages. In activated macrophages, TNF-α expression was inhibited by 12 μg/mL andrographolide and 20 μmol/L PD98059 (inhibitor of ERK1/2 signaling pathway) at both mRNA expression and protein secretion levels.
Conclusion: In LPS-activated macrophages, andrographolide may inhibit the expression of TNF-α by inhibiting ERK1/2 signaling pathway.

Key words: andrographolide, extracellular signal-regulated MAP kinases, mitogen-activated protein kinases, tumor necrosis factor-α, macrophage activation

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Figure 1

Andrographolide inhibited ERK1/2 phophorylation in LPS-stimulated mouse peritoneal macrophages **P<0.01, vs M. Mouse peritoneal macrophages were first treated with andrographolide for 1 h. After further culture with 0.5 μg/mL lipopolysaccharide (LPS) or without LPS for 1 h, phosphorylated ERK1/2 (A), total ERK1/2 (A), phosphorylated p38 (B), total p38 (B), phosphorylated JNK (C) and total JNK (C) were detected by Western blotting; after culture with 0.5 μg/mL LPS or without LPS for 15 min, IκBα (D) and β-actin (D) were detected by Western blotting. b: blank; M: 0.1% dimethyl sulfoxide; ERK1/2: extracellular signal-regulated kinase 1/2."

Figure 2

Both andrographolide and PD98059 decreaded mRNA level of TNF-α **P<0.01, vs M. Mouse peritoneal macrophages were first treated with 12 μg/mL andrographolide or 20 μmol/L PD98059 for 1 h. After culture with 0.5 μg/mL lipopolysaccharide (LPS) or without LPS for 1 h, tumor necrosis factor-α (TNF-α) mRNA was detected by reverse transcription-polymerase chain reaction and phosphorylated ERK1/2 and total ERK1/2 was detected by Western blotting. b: blank; M: 0.1% dimethyl sulfoxide. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ERK1/2: extracellular signal-regulated kinase 1/2."

Figure 3

Both andrographolide and PD98059 inhibited secretion of TNF-α protein **P<0.01, vs M. Mouse peritoneal macrophages were first treated with 12 μg/mL andrographolide or 20 μmol/L PD98059 for 1 h. After further culture with 0.5 μg/mL lipopolysaccharide (LPS) or without LPS for 24 h, tumor necrosis factor-α (TNF-α) protein in the supernatants was detected by enzyme-linked immunosorbent assay."

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