靈芝多醣PSG與蛋白LZ8調節免疫與其機制之研究

Abstract

靈芝 (Ganoderma lucidum (Fr.) Karst) 為中國傳統藥用真菌，具有抗腫瘤、免疫調節、抗病毒、抗菌、抑制血小板凝集等功效，其中靈芝多醣PSG及蛋白LZ8皆有文獻指出與靈芝調節免疫之活性相關。過往之研究多著重於探討靈芝多醣PSG之功效，然而靈芝多醣PSG中含有5 - 10％ 之蛋白質，同時多醣在化學上難以定性與定量，因此靈芝多醣與蛋白質成分調節免疫之對象與機制值得進一步探討。本研究分別以純化自靈芝菌絲體之天然LZ8 (nLZ8)、大腸桿菌 E. coli 重組之LZ8 (rLZ8)、雙鶴極品靈芝多醣PSG及以TCA法去除蛋白質所得之deproteinated-PSG (dePSG) 為樣本，探討靈芝多醣與蛋白對免疫調節功效是否具不同貢獻。在各樣本之生化特性方面，以螢光法及酚硫酸法分析各樣本中蛋白質與多醣含量，結果發現PSG粉末中含有4 - 8％ 之蛋白質及18％ 之多醣，而nLZ8中蛋白與多醣比為6:1；同時利用 LZ8 單株抗體進行西方轉漬分析，結果發現PSG中含有LZ8蛋白。在免疫調節活性方面，以小鼠脾細胞及腹腔巨噬細胞為對象，測定各樣本活化細胞之效果，結果發現rLZ8蛋白可促進小鼠脾細胞增生，誘導其釋出IFN-γ，並可顯著提高IFN-γ與IL-2 mRNA表現量，且與dePSG共同處理具有加成之效果，但多醣PSG對小鼠脾細胞並無顯著之活化效果。此外，以MACS磁珠純化法分離T細胞、T-depleted細胞、B細胞及B-depleted細胞，發現LZ8可活化T細胞，而PSG可活化B細胞。另一方面，多醣PSG可誘導小鼠腹腔巨噬細胞產生NO及 TNF-α，並可提高細胞中iNOS, TNF-α, IL-1β, IL-6, IL-12及IL-18 mRNA 表現量，但去蛋白後此活化效應顯下降，而LZ8也可提高巨噬細胞中IL-1β, IL-6, IL-12及IL-18 mRNA表現量，但無法誘發iNOS及TNF-α 基因表現及NO及TNF-α 產生。在免疫調節路徑方面，以 TLR2-/- 及 TLR4-/- 小鼠為試驗平台，分析各樣本對其腹腔巨噬細胞之作用機制，結果發現多醣PSG可活化TLR2-/- 小鼠腹腔巨噬細胞，但無法活化TLR4-/- 小鼠腹腔巨噬細胞，顯示PSG需經由TLR4以活化巨噬細胞；而 LZ8 活化脾細胞之機制與TLR2及TLR4無關。上述結果說明靈芝中的免疫調節蛋白LZ8與多醣PSG在調節免疫上各扮演不同角色，LZ8主要作用於T淋巴球細胞，對巨噬細胞亦有作用，而多醣則主要經由TLR4活化巨噬細胞與B淋巴球細胞。本研究結果顯示靈芝調節免疫的活性由多醣與蛋白共同貢獻，但兩者作用的對象與機制各不相同，此發現同時提供未來靈芝研究上一個新的方向。

Ganoderma lucidum (Fr.) Karst, a traditional medicinal fungus in China, had been shown to possess potent anti-tumor, immunomodulatory, anti-viral, anti-pathogen, hypoglycemic, and inhibiting platelet aggregation effects. Both the polysaccharides (PSG) and LZ8 protein from G. lucidum have been reported to have immunomodulatory effects. Although most studies focused on the biological effect of PSG, PSG contains 5-10％ proteins which is hard to be chemically defined. The objectives of this study were to clarify the immunomodulatory roles and related mechanisms of G. lucidum polysaccharides (PSG) and proteins (LZ8). Four different G. lucidum samples, including native LZ8 (nLZ8) extracted from the mycelia, E. coli expressed recombinant LZ8 (rLZ8), PSG, and TCA deproteinated-PSG (dePSG) were prepared for the studies. PSG was a protein-bound polysaccharide consisting of 16 - 18% polysaccharides and 4 - 8% peptides, and the ratio of proteins to polysaccharides in nLZ8 is 6:1. Western blotting reveals that PSG contained LZ8 by LZ8 monoclonal antibody analysis. Additionally, rLZ8 markedly enhanced murine splenocytes proliferation, increased IFN-γ release, and up-regulated the mRNA expression of IFN-γ and IL-2 in vitro. This activation could be further enhanced by treating with both LZ8 and dePSG. rLZ8 was further demonstrated to activate T lymphocytes, and PSG was found only to activate B lymphocytes but not T lymphocytes. On the other hand, PSG induced murine peritoneal macrophages to produce nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α), and to increase the mRNA expression of iNOS, TNF-α, IL-1β, IL-6, IL-12p40, and IL-18. Moreover, rLZ8 was found to increase the mRNA expression of IL-1β, IL-6, IL-12p40, and IL-18 by murine peritoneal macrophages but was incapable to induce NO and TNF-α production. Furthermore, peritoneal macrophages from TLR4-/- mice, but not TLR2-/- mice, were hyporesponsive in TNF-α secretion to PSG, suggesting that PSG activated macrophages through TLR4. Taken together, this study demonstrated that LZ8 and PSG played different roles on modulating immune cells through distinctive mechanisms. In conclusion, LZ8 activated T lymphocytes mainly but PSG activated macrophages and B lymphocytes. Polysaccharide and protein components were conjugated together in natural G. lucidum preparations and hereby activated the immune of the host synergistically.