Effect Of Piperine On The Expression Of Inflammatory Cytokines And Antimicrobial Peptides In Human Colonic Carcinoma Cell Lines

Aim:Piperine is a major alkaloid isolated from fruits of black pepper(Piper nigrum Linn) and long pepper(Piper longum Linn). It has been exhibited pharmacological activities on several diseases, including gastrointestinal disorders. However, few studies have focused on its effect on anti-inflammatory bowel diseases and the molecular mechanisms underlying its anti-inflammatory activities. The present study therefore aimed to examine the anti-inflammatory effect and the underlying mechanisms of piperine, by detecting the expression of inflammatory cytokines and antimicrobial peptides under piperine treatment, as well as its influences on NF-κB(nuclear factor-κB) and MAPK(mitogen-activated protein kinase) pathways in lipopolysaccharide(LPS) or Staphylococcus aureus cells(SAC)-stimulated SW480, HT-29 and Caco-2 cells, an in vitro experimental cell model for human inflammatory bowel disease(IBD). Methods:1. The effect of piperine on cell proliferation was evaluated by WST-1 assay. The expression of inflammatory cytokines was measured by cytometric beeds array(CBA) combined with flow cytometry analysis; Reverse Transcription-Quantitative PCR(RT-q PCR) was used to analyze the messenger RNA(m RNA) levels of inflammatory cytokines. The regulation of piperine on NF-κB pathway and MAPKs(ERK、p38 and JNK) pathways was determined by Western blotting. The CBA kit and RT-q PCR were used again to detect the effect of SB203580(the inhibitor of p38 MAPK pathway) and SP600125(the inhibitor of JNK pathway) on the secretion of IL-8 and the expression of IL-8 m RNA.2. RT-q PCR was also used to analyze the regulatory effect of piperine on the m RNA expression of human defensins, including HD-5(human alpha-defensin-5), HD6(human alpha-defensin-6) and HBD-1(human beta-defensin-1), and regenerating islet-derived protein(Reg) Ⅲγ.Results:1. Piperine inhibited the proliferation of SW480, HT-29 and Caco-2 cells in a dose-dependent manner, and the 50% inhibiting concentration(IC50) values were 147.6 mM, 328.5 mM and 155.1 mM, respectively, suggesting that piperine had low cytotoxicity on these cell lines. Piperine dose-dependently suppressed LPS-induced secretion of IL-8 protein and the expression of IL-8 m RNA, but not that of IL-10. Western blotting results showed that piperine attenuated the JNK(c-Jun N-terminal kinase) and p38 MAPK signaling, instead of NF-κB pathway and ERK(extracellular signal-regulated kinase) pathway. CBA and RT-q PCR assays showed that both the secretion of IL-8 protein and the expression of IL-8 m RNA upon LPS stimulation were attenuated by SB203580 and SP600125.2. RT-q PCR showed that LPS and SAC greatly suppressed the expression of human defensin DEFA5 and DEFA6 m RNA while piperine significantly counteracted the inhibitory effect of LPS and SAC on the expression of DEFA5 and DEFA6 m RNAs, instead of DEFB1 and Reg IIIγ m RNA. Conclusion:1. Piperine could attenuate the inflammatory response in colonic epithelial-like cells via down-regulating the MAPK signaling and thus the expression of IL-8, suggesting its potential application in anti-inflammation therapy.2. Piperine might exert its anti-colitis effect through antagonizing the inhibitory effect of LPS and SAC on the expression of defensins in human intestinal epithelial cells.