探討羅伊氏乳酸桿菌的培養上清液在體內和體外對5-氟尿嘧啶誘導血液毒性之影響

5-氟尿嘧啶 (5-FU) 是一種常見的化療藥物，在臨床上常引起患者出現血液毒性，如嗜中性球減少症，以及腸道黏膜炎、腹瀉等副作用。許多益生菌已被證實具有緩解化療副作用的能力，然而，益生菌之活菌在臨床上可能會引起化療患者腸道細菌轉位，從而導致敗血症的風險。因此，我們擬探討益生菌及其無細胞培養上清液是否具有緩解5-FU誘導的白血球低下症的功效。本研究對Wistar大鼠進行L. reuteri活菌及其培養上清液與濃縮之培養上清液管餵14天，隨後連續五天進行50 mg/kg 5-FU的腹腔注射。另外，我們利用RAW264.7巨噬細胞探討L. reuteri之培養上清液對顆粒性白血球群落刺激因子 (G-CSF) 產量的刺激效果及其介導的信號通路。動物實驗研究結果：(1) 無細胞培養上清液及其濃縮之培養上清液能夠顯著緩解5-FU引起的白血球數量減少，其中L. reuteri活菌表現出回復趨勢。(2) 相較於5-FU組，濃縮上清液與活菌具增加大鼠血中G-CSF的濃度，培養上清液則不具此效果。細胞實驗結果顯示：L. reuteri培養上清液具有 (1) 能刺激巨噬細胞的活性，如增加誘發型一氧化氮合成酶 (iNOS) 和環氧合酶-2 (COX-2) 蛋白的表達，顯著增加一氧化氮 (NO) 的產量，並增加促發炎因子如：腫瘤壞死因子-α (TNF-α) 、白介素-1β (IL-1β) 和白介素-6 (IL-6) 的mRNA表達、(2) 顯著增加G-CSF mRNA的表達及G-CSF的分泌。(3) L. reuteri培養上清液能以時間和劑量依賴的方式刺激RAW264.7細胞分泌G-CSF，(4) G-CSF的分泌與MAPK信號通路 (包括ERK和p38) 相關，(5) 在5-FU共同處理下，L. reuteri培養上清液仍能刺激G-CSF的產生。綜合以上結果，L. reuteri培養上清液具有緩解5-FU誘導血液毒性的潛力，可能通過MAPK信號通路刺激G-CSF的產生。

5-fluorouracil (5-FU) is a commonly used chemotherapy drug that frequently causes hematologic toxicities such as neutropenia, as well as side effects like intestinal mucositis and diarrhea in clinical settings. Probiotics have been proven to alleviate chemotherapy side effects. However, there are clinical concerns that live probiotics may lead to bacterial translocation in the intestines of chemotherapy patients, potentially causing sepsis. Therefore, we aim to investigate whether Lactobacillus reuteri and its cell-free culture supernatant (SN) and concentrated culture supernatant (cSN) can mitigate 5-FU-induced leukopenia. In this study, Wistar rats were orally administered L. reuteri, SN or cSN for 14 days, followed by intraperitoneal injections of 50 mg/kg 5-FU for five consecutive days. Additionally, we used RAW264.7 macrophages to examine the effect of SN from L. reuteri on granulocyte colony-stimulating factor (G-CSF) production and the signaling pathways involved.Based on the results of animal experiments: (1) SN and cSN were able to significantly restore the reduced white blood cell counts caused by 5-FU, with L. reuteri showing a trend of recovery. (2) Compared to the 5-FU group, the cSN and live L. reuteri showed a trend of increase the concentration of G-CSF in the blood of rats, while the SN does not have this effect.The results of cell experiments showed: (1) The SN of L. reuteri was able to stimulate the activity of macrophages, such as increasing the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, significantly increasing the production of nitric oxide (NO), and increasing the mRNA expression of pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). (2) The SN of L. reuteri was able to significantly increase the mRNA expression and secretion of G-CSF. (3) The SN of L. reuteri was able to stimulate the secretion of G-CSF by RAW264.7 cells in a time- and dose-dependent manner. (4) The secretion of G-CSF was related to the MAPK signaling pathway (including ERK and p38). (5) Even in the presence of 5-FU, the SN of L. reuteri still stimulated the production of G-CSF.In summary, the SN of L. reuteri has the potential to alleviate the hematological toxicity induced by 5-FU, possibly by stimulating the production of G-CSF through the MAPK signaling pathway.