Anti-inflammatory effects of enzyme-treated asparagus extract and its constituents in hepatocytes

Abstract

Background: Asparagus (Asparagus officinalis L.) is one of the most ancient vegetables, and it is rich in asparagine. Enzyme-treated asparagus extract (ETAS™; Amino Up Chemical Co., Ltd., Sapporo, Japan) is the final product of enzyme-treatment of asparagus stems and subsequent extraction. Two constituents were purified from ETAS and identified: 5-hydroxymethyl-2-furfural (HMF), an abundant constituent, and (S)-asfural, a novel constituent, which is a derivative of HMF. ETAS has been reported to increase the expression of heat shock proteins (HSPs), which are essential for the repair or removal of defective proteins. The expression of Hsp family genes is regulated by the transcription factor heat shock factor 1 (HSF1). It is unknown whether ETAS and its constituents elicit anti-inflammatory effects, such as the suppression of nitric oxide (NO), an inflammatory mediator synthesized by inducible nitric oxide synthase (iNOS) in interleukin (IL)-1β-treated hepatocytes.

Objective: To examine the anti-inflammatory effects of ETAS, we treated rat hepatocytes with ETAS, or its constituents (S)-asfural or HMF, and IL-1β and then analyzed the expression of the iNOS gene and other genes involved in inflammation.

Methods: Primary cultured rat hepatocytes were prepared by collagenase perfusion. ETAS, (S)-asfural, or HMF was added to the medium with IL-1β and incubated at 37 °C. When necessary, an inhibitor of HSF1 was added. NO in the medium was measured by the Griess method, and the half-maximal inhibitory concentration (IC₅₀) values were determined. To analyze the mRNA expression, a reverse transcription-quantitative polymerase chain reaction was performed. Antibody arrays were used to determine the levels of cytokines and chemokines in the medium.

Results: ETAS suppressed NO production in IL-1β-treated hepatocytes without causing cytotoxicity. ETAS decreased the levels of both iNOS mRNA and the antisense transcript, whereas it increased the levels of Hsf1 mRNA and Hsp70 mRNA. ETAS also suppressed the production of pro-inflammatory cytokines and chemokines in hepatocytes. When (S)-asfural and HMF were added to the medium, they suppressed NO production and iNOS gene expression. The IC₅₀ value of (S)-asfural was approximately 3-fold lower than that of HMF. In contrast, (S)-asfural increased the levels of Hsf1 mRNA. Interestingly, the KRIBB11, an inhibitor of HSF1, reduced the expression of the iNOS gene. When both (S)-asfural and KRIBB11 were added, the level of iNOS mRNA was lower than when (S)-asfural alone was added.

Conclusion: ETAS and its constituents (S)-asfural and HMF suppressed NO production and the expression of pro-inflammatory cytokines and chemokines, thus showing anti-inflammatory effects. Our data suggest the possibility that the increased HSF1 level is involved in suppression of NO by ETAS and its constituents, although HSF1 is essential for the expression of the iNOS gene.

Keywords: nitric oxide, inducible nitric oxide synthase, inflammation, heat shock factor, asparagus