探討真菌類免疫調節蛋白質改善歐洲紫杉醇引起的不良副作用

歐洲紫杉醇 (商品名：TaxotereR) 為臨床上常見的化療藥物之一。Docetaxel會造成骨髓抑制 (Myelosuppression) 包括嗜中性白血球低下症 (Neutropenia) 和貧血 (Anemia)。真菌類免疫調節蛋白 (Fungal Immunomodulatory Proteins，FIP) 自金針菇 (Flammulina velutipes) 及松杉靈芝 (Ganoderma tsugae) 純化與選殖得來，分別命名為 FIP-fve與FIP-gts。過去文獻指出，FIP-fve與FIP-gts皆具有免疫調節及抗腫瘤能力。本實驗欲探討FIP-fve與FIP-gts對Docetaxel引起的不良副作用之影響。從動物實驗之血液常規檢查結果顯示 (Control v Docetaxel)，Docetaxel組出現嗜中性白血球低下 (白血球：6.29 ± 1.19 × 103/μL v 3.40 ± 0.74 × 103/μL) 及貧血 (紅血球：9.19 ± 0.52 × 106/μL v 8.06 ± 0.43 ×106/μL)。FIP-gts可減緩Docetaxel所誘發的白血球低下 (Docetaxel v Docetaxel與FIP-gts共同處理，3.40 ± 0.74 × 103/μL v 5.81 ± 1.64 × 103/μL)。透過組織染色發現 (Control v Docetaxel v Docetaxel與FIP-fve共同處理 v Docetaxel與FIP-gts共同處理)，腸絨毛長度分別為 (570.38 ± 68.66 μm v 365.85 ± 19.21 μm v 474.65 ± 6.8 μm v 506.60 ± 17.87 μm)。結果顯示，FIP-fve與FIP-gts 可顯著減緩Docetaxel所造成的腸損傷。且FIP-fve與FIP-gts 可有效減緩Docetaxel所誘發的骨髓中脂肪細胞數目(100 ± 22.04% v 275.27 ± 45.31% v 154.84 ± 22.35% v 138.71 ± 12.35%)。使用3D微電腦斷層掃描 (Micro-CT) 進行骨小樑定量分析 (Control v Docetaxel)，Docetaxel會顯著降低骨體積百分比 (21.82 ± 1.10% v 11.35 ± 3.32%)、骨小樑數目 (3.12 ± 0.26 mm-1 v 1.66 ± 0.46 mm-1)、骨表面密度 (12.20 ± 0.99 mm-1 v 6.82 ± 1.67 mm-1) 及增加骨小樑間距(0.21 ± 0.02 mm v 0.38 ± 0.11 mm)，表示Docetaxel會造成骨損傷及增加骨質疏鬆的風險。FIP-fve與FIP-gts 可顯著減緩Docetaxel所降低的骨體積百分比、骨小樑數目及骨表面密度。經由酵素連結免疫吸附分析法 (ELISA) 發現，相較於Docetaxel組 (72.88 ± 39.19 ρg/mL)，Docetaxel結合FIP-gts共同處理組 (145.14 ± 76.74 ρg/mL) 小鼠血漿中G-CSF含量顯著較高。在人類週邊血單核球細胞實驗中也發現FIP-fve與FIP-gts均可刺激G-CSF之mRNA表現。另外，FIP-fve與FIP-gts 均可抑制A459肺癌細胞之存活。透過RT-PCR 與ELISA發現，FIP-fve與FIP-gts 均可以減少A459參與肺癌誘導骨吸收病變的單核細胞趨化蛋白-1 (MCP-1)表現量，顯示FIP-fve與FIP-gts 可能可減緩肺癌所誘導的骨病變。本研究首次發現FIP-fve及FIP-gts具有減緩Docetaxel所誘發的不良副作用之功效。因此，我們認為FIP-fve及FIP-gts具有發展成Docetaxel輔助藥物之潛力。

Docetaxel (TaxotereR) is usually applied in routine chemotherapy. However, the most common side effects of Docetaxel is myelosuppression, include neutropenia and anemia. Fungal Immunomodulatory Proteins of Flammulina velutipes and Ganoderma tsugaeare were called FIP-fve and FIP-gts, respectively. In previous study, both FIP-fve and FIP-gts have immunomodulatory and anti-cancer ability. This study is aimed to investigate the protective function of FIP-fve and FIP-gts on docetaxel-induced adverse side effects. In animal model, complete blood count (Control v Docetaxel) reveals neutropenia (WBC：6.29 ± 1.19 × 103/μL v 3.40 ± 0.74 × 103/μL) and anemia (RBC：9.19 ± 0.52 × 106/μL v 8.06 ± 0.43 × 106/μL). Moreover, FIP-gts but not FIP-fve can reverse docetaxel-induced neutropenia (Docetaxel v Docetaxel combined with FIP-gts，3.40 ± 0.74 × 103/μL v 5.81 ± 1.64 × 103/μL). As compared to Control group (570.38 ± 68.66 μm), histological sections of intestine shows that villous length was decrease in Docetaxel group (365.85 ± 19.21 μm), Docetaxel combined with FIP-fve (474.65 ± 6.8 μm) and Docetaxel combined with FIP-fve (506.60 ± 17.87 μm)), respectively. Consequences, FIP-fve and FIP-gts significantly decreased docetaxel-induced intestine damage. Moreover, numbers of adipocyte (% of control) was significantly increased in Docetaxel group (275.27 ± 45.31%) as compared to Control group (100 ± 22.04%) in bone marrow. FIP-fve (154.84 ± 22.35%) and FIP-gts (138.71 ± 12.35%) can decrease docetaxel-induced adipocyte in bone marrow. Quantification of trabecular bone (Control v Docetaxel) percent bone volume (21.82 ± 1.10% v 11.35 ± 3.32%), trabecular number (3.12 ± 0.26 mm-1 v 1.66 ± 0.45 mm-1), bone surface density (12.20 ± 0.99 mm-1 v 6.82 ± 1.67 mm-1), and trabecular separation (0.21 ± 0.02 mm v 0.379 ± 0.109 mm) analyzed by on 3D micro-CT analysis. This is reason that Docetaxel induced bone damage and increase osteoporosis risk. Moreover FIP-fve and FIP-gts significantly reversed docetaxel-decreased percent bone volume, trabecular number and bone surface density. On the enzyme-linked immunosorbent assay (ELISA), total plasma G-CSF concentration was significantly increased in Docetaxel + FIP-gts group (145.14 ± 76.74 ρg/mL) as compared to Docetaxel group (72.88 ± 39.19 ρg/mL). Both of FIP-fve and FIP-gts can stimulate G-CSF mRNA expression in human peripheral blood mononuclear cells (hPBMCs) by RT-PCR and inhibit A549 cell survival by MTT assay. Both of FIP-fve and FIP-gts reduce the mRNA expression of monocyte chemotactic protein-1 (MCP-1), which promotes lung cancer-induced bone resorptive lesions in A549 lung cancer cells by RT-PCR and ELISA. This is the first study to reveal the novel function of FIP-fve and FIP-gts in mitigating docetaxel-induced adverse side effects. Therefore, we suggest that FIP-fve and FIP-gts may be potential adjuvants of docetaxel.