Evaluation on the effects of berberine administrations on cytokine secretions by BALB/c mouse splenocytes using real time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods

Abstract

In Chinese medicine, bitter foods are described as “heat-removing” agents for reducing fever and have been widely used in therapeutic application. The major bitter compounds in bitter foods are alkaloids, such as aloperine, amygdalin, berberine, crotaline and naringenin. The bitter compounds are suggested to have multiple physiological functions, including anti-pyretic, anti-nociceptive and anti-tumor activities. However, there are few discussions on immunomodulatory effects of bitter compounds. Therefore, the objective of this study was to investigate the anti-inflammatory effects of five selected bitter compounds including aloperine, amygdalin, berberine, crotaline and naringenin administrations on cytokine secretions by BALB/c mouse splenocytes under four different in vitro experimental models. The potent bitter compound, berberine, was further selected to evaluate its immunomodulatory mechanism using real time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) methods. In this study, there were four different in vitro experimental models used to detect the immunomodulatory effects of bitter compound administrations on BALB/c primary splenocytes. In the spontaneous splenocyte culture model, the primary splenocytes were just co-cultured with samples. In the inflammatory model, the lipopolysaccharide (LPS)-stimulated splenocytes were co-cultured with samples. In the preventive model, splenocyte cultures were first incubated with samples, then washed out samples, and finally stimulated with LPS. In the repair model, splenocyte cultures were first stimulated with LPS, then washed out LPS, and finally treated with samples. In the first part, the results showed that five selected bitter compounds including aloperine, amygdalin, berberine, crotaline and naringenin, significantly (P<0.05) decreased the productions of pro-inflammatory cytokines by spontaneous splenocytes, such as interleukin (IL)-6 or tumor necrosis factor alpha (TNF-α). The levels of IL-6 and TNF-α significantly decreased in the inflammatory and preventive models at moderate concentrations of the selected bitter compounds expect crotaline. In addition, the ratios of pro-/anti- inflammatory cytokine, displayed as either IL-6/IL-10 or TNF-α/IL-10, also significantly diminished in both inflammatory and preventive models by the selected bitter compounds expect crotaline. It is suggested that the selected bitter compounds expect crotaline might have anti-inflammatory effects in vitro. Among the five selected bitter compounds, berberine has a significant effect on inhibiting the inflammation induced by LPS. Therefore, berberine was further administered to splenocyte cultures to detect its regulation on gene expression. In the second part, an investigation was further done to clarify the effect of berberine on cytokine mRNA expressions of primary splenocyte. Results showed that berberine had no significant effect on the mRNA expressions of IL-6, IL-10 and TNF-α, suggesting that berberine administration might affect cytokine expression via post-transcriptional regulation. However, berberine administration could down-regulate the gene expression ratio of Th2 (IL-5)/Th1 (IL-2) cytokines in the inflammatory model. The results suggested that berberine administration might have potential effects on modulating the Th2/Th1 immune balance toward the Th1 pole in vitro. In conclusion, berberine administration at moderate concentrations may significantly decrease the inflammation induced by LPS in vitro, via decreasing the ratio of pro-/anti-inflammatory cytokine (IL-6/IL-10 or TNF-α/IL-10). Besides, it is suggested that berberine could modulate immune responses through down-regulating the gene expression ratio of Th2/Th1 cytokines.

傳統中醫普遍認為苦味食物具有良好的”退火”功效，已經被廣泛運用在食療保健上。苦豆鹼(aloperine)、苦杏仁苷(amygdalin)、黃連素(berberine)、野百合鹼(crotaline)以及柚苦苷(naringenin)等，為苦味食物中主要的苦味生物鹼，研究證實其具有許多生理活性，包含退燒、鎮痛以及抗癌等活性，但對苦味物質的免疫調節功能，至今仍無完整研究，因此本篇主要研究目的為，選取五種苦味物質：苦豆鹼、苦杏仁苷、黃連素、野百合鹼以及柚苦苷，在動物體外四種不同實驗模式下，探討其抗發炎活性，並從其中選出黃連素，利用即時聚合酶連鎖反應及酵素免疫連結分析法評估苦味物質黃連素對BALB/c小鼠脾臟細胞分泌細胞激素之影響，以探討其免疫調節功能。本研究使用四種不同體外實驗來評估苦味物質對BALB/c小鼠初代脾臟細胞之免疫調節作用，實驗設計如下：單獨添加樣品與初代脾臟細胞共同培養(單獨添加樣品模式)、以內毒素脂多醣(lipopolysaccharide, LPS)刺激脾臟細胞同時添加樣品與細胞共同培養(發炎模式)、樣品先與脾臟細胞培養，洗去樣品，再加入LPS刺激脾臟細胞(預防模式)以及先以LPS刺激脾臟細胞，洗去LPS，再加入樣品與脾臟細胞培養(恢復模式)。 第一部分研究結果顯示，單獨添加五種苦味物質苦豆鹼、苦杏仁苷、黃連素、野百合鹼以及柚苦苷與脾臟細胞共同培養，可顯著抑制促發炎細胞激素介白質(interleukin, IL)-6或腫瘤壞死因子-α (tumor necrosis factor alpha, TNF-α)分泌量。脾臟細胞在發炎及預防的模式下，添加苦豆鹼、苦杏仁苷、黃連素以及柚苦苷可顯著抑制促發炎細胞激素分泌量，並調節促發炎IL-6 (TNF-α)/抗發炎細胞激素IL-10分泌量的比值，顯示苦味物質具有抗發炎的潛力，而五種苦味物質中又以黃連素抗發炎的能力最顯著，故以黃連素探討其對初代脾臟細胞細胞激素基因表現量之影響。 第二部分研究黃連素在不同細胞培養模式下對脾臟細胞細胞激素mRNA表現量的影響，發現黃連素對IL-6、IL-10及TNF-α mRNA的表現無顯著調控作用，推測黃連素對脾臟細胞細胞激素分泌之調控，可能屬於轉錄後調節作用(post-transcriptional regulation)。另外，在發炎模式下，黃連素可以下調Th2 (IL-5)/Th1 (IL-2)細胞激素mRNA表現量的比值，顯示黃連素具有調節免疫的功能，可使免疫平衡傾向Th1反應。 綜合本實驗結果發現，黃連素可以藉由降低促發炎/抗發炎細胞激素(IL-6/IL-10或TNF-α/IL-10)分泌量的比值，而減緩LPS所誘導的發炎反應。除此之外，黃連素可以下調Th2/Th1細胞激素mRNA表現量的比值，進而調節小鼠脾臟細胞的免疫反應變化。