- 學位論文
補充滅活益生菌 (Clostridium butyricum 與 Lactobacillus reuteri) 對順鉑誘導腎損傷、腸道受損與腸道菌相失衡之影響
Effects of inactivated probiotics (Clostridium butyricum and Lactobacillus reuteri) supplement on cisplatin-induced renal damage, intestinal injury and gut microbiota dysbiosis
摘要
Lactobacillus reuteri 及 Clostridium butyricum 已被證實為可改善許多疾病之益生菌，然而近年益生菌在臨床上出現安全性的疑慮，便興起滅活型益生菌在臨床效益的可行性探討，文獻指出滅活型益生菌對於腸道黏膜炎及腎臟疾病具有改善效果，但滅活型益生菌是否對於化療藥物順鉑 (Cisplatin) 誘導之腎、腸毒性與腸道菌相失衡具有改善效果，目前尚不清楚，因此，本研究欲探討補充 UV 滅活型益生菌 (Lactobacillus reuteri + Clostridium butyricum; UV-killed LCs) 對順鉑治療後腸、腎損傷及腸道菌相失衡之影響。我們以 Wistar 大鼠於順鉑施打前10天以管餵給予 UV-killed LCs 直至實驗第24天，並於實驗第 1、8、15、19、22 天施打順鉑 5 mg/kg，大鼠於第 25 天犧牲。結果顯示：UV-killed LCs 可以 (1) 顯著減輕腎臟病理特徵及腎功能指標；顯著降低腎臟 NOX4、MDA 表達，並提升抗氧化酵素活性以及 Nrf2、HO-1 表現；顯著抑制腎臟 IL-33/ST2 發炎信號路徑及其下游 MAPK/NFB 與細胞激素及趨化因子，並提升 IL-10 含量；顯著減少腎臟 Bax、p53、Cleaved caspase-3 與 NLRP3/Caspase-1 表現；顯著降低腎臟纖維化指標 Fibronectin、-SMA、-catenin 之表現；顯著減少腎臟 T 細胞 (CD4)、巨噬細胞 (F4/80) 表現。(2) 增加食物利用率、促進胃排空；保護迴腸腺窩、絨毛、潘氏細胞、杯狀細胞完整性與消化酵素活性；顯著抑制腸道 TLR4/MyD88/MAPK/NFB 發炎信號路徑與 MPO 活性；維持迴腸細胞增生能力；顯著增加結腸長度與 IL-10 表達；增加結腸緊密蛋白 ZO-1、Occludin、Claudin-1、黏液素表現以及總短鏈脂肪酸含量。(3) 腸道菌相分析中，UV 滅活型的 LCs 可增加物種多樣性與豐富度，並提升菌相中 Lachnospiraceae NK4A136 group、Prevotellaceae UCG 001 的相對豐富度，及減少 Escherichia-Shigella 的潛力，綜合上述實驗結果，滅活型益生菌明顯改善腎功能、調降腎臟和腸道發炎，這可能透過促進恢復腸道微生物群結構組成，維持腸道屏障功能，並以腸腎軸線的調節來調降發炎信號路徑。
並列摘要
Lactobacillus reuteri and Clostridium butyricum are probiotics that have been proven to improve many diseases. However, in recent years, the safety of live probiotics is very concerning in the clinical practice. Hence, the study of feasibility of inactivated probiotics in clinical benefits have raised concerns. The literature indicated that inactivated probiotics has improving effect on the intestinal mucositis and kidney disease. However, it is not clear whether inactivated probiotics have a beneficial influence on cisplatin-induced nephrotoxicity, gastrointestinal toxicity and gut dysbiosis. The study aims to explore the impact of supplementation with UV-inactivated probiotics (Lactobacillus reuteri and Clostridium butyricum; UV-killed LCs) on intestinal, renal injury and the composition of the microbiota after cisplatin treatment. Wistar rats were gavaged with UV-killed LCs for 10 days prior to cisplatin injection until the end of the experiment on day 24. Cisplatin (5 mg/kg; i.p.) induced on day1, 8, 15, 19 and 22. Then all rats in this study were sacrificed at the 25th day. Our results showed that UV-killed LCs can significantly alleviate renal pathological features and renal function markers, and reduce the expression of renal NOX4 and MDA when compared to the cisplatin-treated group. UV-killed LCs elevated the activity of antioxidant enzyme and the expression of Nrf2 and HO-1 in the kidneys. In addition, UV-killed LCs significantly inhibited renal IL-33/ST2 inflammatory signaling pathways and its downstream MAPK/NFκB signaling pathway, cytokines /chemokines production and increased IL-10 content. The treatment of UV-killed LCs could decline renal Bax, p53, cleaved caspase-3 and NLRP3/Caspase-1 mRNA expression. UV-killed LCs significantly ameliorated renal fibrosis as demonstrated by the decreased expression level of Fibronectin, α-SMA, -catenin and decreased the protein expression of T cell (CD4) and macrophage (F4/80) in the kidney. UV-killed LCs significantly improved gastric emptyting, feeding efficiency and protected cisplatin-induced intestinal mucositis and attenuated the decline in digestive enzymes activity. Additionally, UV-killed LCs significantly inhibited ileum TLR4/MyD88/MAPK/NFB inflammatory signaling pathways and MPO activity, stimulated the proliferation of intestinal epithelia, significantly increased colon length and IL-10 expression. UV-killed LCs had the ability to increase expression of tight junction proteins (ZO-1, Occludin, Claudin-1), mucin in colon tissue, and fecal total short-chain fatty acid concentrations. Using next-generation sequencing analysis, our results revealed the administration of UV-killed LCs increased species diversity and richness while increased the relative abundance of Lachnospiraceae NK4A136 group and Prevotellaceae UCG 001 after cisplatin treatment. Of note, there were trends of abundant decrease in Escherichia-Shigella in the UV-killed LCs group compared with the cisplatin group, although these differences were not significant. Based on the above results, we can conclude that inactivated LCs can significantly improve the renal function, alleviate renal and intestinal inflammation. The mechanism of inactivated LCs alleviates cisplatin-induced intestinal and kidney damage may be mediated by intestinal-kidney axis, which promoted the restoration of a healthy gut microbiota composition and promote its diversity, maintained the intestinal barrier function and downregulated inflammatory-related signaling pathway activation.