腎臟保健複合乳酸桿菌應用於慢性腎臟病貓之功效性及其腸道保健機能性之探討

Abstract

慢性腎臟病 (chronic kidney disease, CKD) 的特徵為逐漸損失腎臟功能，且損傷是無法修復的。此疾病於年老貓隻群體中的盛行率高達80%，由此可知CKD是一個十分重要的議題等待被解決的。在宿主腸道中某些嗜蛋白質的微生物，特別是Escherichia coli，傾向產生會導致CKD惡化的尿毒素前驅物。因此經由改變腸道微生物組成或許會是減緩CKD進程一個新穎的輔助性治療及預防的策略。複合乳酸桿菌 (Lactobacillus mixture, Lm) 是實驗室先前所篩選出的益生菌組合，包含了Lacticaseibacillus paracasei subsp. paracasei MFM 18和Lactiplantibacillus plantarum subsp. plantarum MFM 30-3。Lm在先前的CKD小鼠模型中已被證實具有腎臟保健的功效，因此在第一部份我們將透過給予含有Lm的益生菌寵物零食證實其對於CKD家貓的功效。此外，近年來越來越多臨床或體內試驗皆指出炎症性腸炎 (inflammatory bowel disease, IBD) 是CKD的風險因子。4–23%的IBD患者會產生腎臟或泌尿系統相關的併發症，包含腎結石 (nephrolithiasis)、腎小管間質性腎炎 (tubulointerstitial nephritis)、腎絲球腎炎 (glomerulonephritis) 以及類澱粉沉積症 (amyloidosis)。除此之外，低劑量的Lm在CKD小鼠試驗中具有改善腸道滲漏的能力。故在第二部分的試驗中，我們將應用Lm在IBD小鼠模型，並進一步探討其增進腸道屏障功能的可能機制。 在CKD貓隻的臨床試驗中，我們將Lm油噴於市售貓隻飼料上，並以小包裝的方式製作成益生菌寵物零食。益生菌寵物零食提供三種不同口味，給予較挑食的CKD貓隻更多選擇。藉由益生菌寵物零食的方式，我們希望能減緩貓隻食用膠囊時受到的緊迫。此益生菌寵物零食符合所有關於寵物飼料安全與化學安定性的規定。而零食上Lm的穩定性也有被量測，其能夠於室溫中穩定存在至少一個月以上。CKD貓隻在試驗期間需每日食用含有Lm的益生菌寵物零食持續8周，並且每4周採集一次糞便、尿液以及血液樣本以進行後續分析。結果顯示，所有的CKD貓隻在Lm零食干預後都有較低或維持的血漿肌酸酐含量，且超過50%的受試CKD貓隻有觀察到有較低的三甲胺-N-氧化物 (trimethylamine N-oxide, TMAO)、硫酸吲哚酚 (indoxyl sulfate, IS) 和硫酸苯酯 (phenyl sulfate, PS) 含量。而受試貓隻的胃口跟活動力也都有獲得了改善。在糞便菌相分析中，益生菌寵物零食可以顯著地增加細菌多樣性並且調節腸道菌相組成，特別是增加了Lactobacillus跟Blautia caecimuris，且減少Enterobacteriaceae、Peptostreptococcaceae、Blautia hominis及Blautia coccoides。這些結果表明，給予CKD貓隻含有Lm的益生菌寵物，藉此調節其腸道菌相組成以及減少尿毒素含量，最終達到臨床上減緩CKD的進展，並增進CKD貓隻生活品質之功效。 在研究的第二個部分，我們給予C57BL/6的雌性小鼠兩周的Lm，並在第7至13天時將牠們的飲用水換成含有2.5% DSS的飲用水以誘導結腸炎產生。結果顯示，Lm能夠減緩DSS造成的糞便潛血、疾病活動指數 (disease activity index, DAI) 增加、腸道滲漏以及較高的組織學病理評分。尤其是高劑量的Lm組，擁有較長的結腸長度、較重的體重、較低的促炎因子含量，表示高劑量的Lm有較佳的調節免疫系統與腸道保護的能力。然而，低劑量的Lm也比高劑量Lm展現出促進緊密連接蛋白的能力。推測Lm可能的作用機制為經由調節盲腸菌相組成，降低Enterobacteriaceae且提高Lactobacillus之含量，並促進丁酸之生成以促進腸道屏障功能。整體而言，高劑量之Lm更能有效減緩結腸炎。 綜上所述，Lm為一個具有多功能之乳酸菌組合。通過本研究，我們闡明含有Lm的益生菌寵物零食能夠減緩貓隻的CKD病程發展，且Lm能夠減輕小鼠結腸炎的嚴重程度。此外，透過分析貓隻腸道菌群與DSS誘導結腸炎小鼠模型，提供更多機制的探討。

Chronic kidney disease (CKD) is characterized by a substantial loss of kidney function and cannot be recovered. Since the prevalence of this disease in the geriatric feline population is up to 80%, this is an important issue waiting to be solved. Some proteolysis bacteria in the host’s intestine, especially Escherichia coli, tend to produce uremic toxins precursors, which progress CKD. Thus, microbiota-based strategies might be a novel auxiliary therapeutic and preventative way. Lactobacillus mixture (Lm, Lacticaseibacillus paracasei subsp. paracasei MFM 18 and Lactiplantibacillus plantarum subsp. plantarum MFM 30-3) was previously selected in our lab and proven to have kidney-protective effects in CKD mice model. Hence, we first clinically verified Lm’s function in CKD cats by treating probiotic pet treats that contain Lm. Additionally, there is increasing both clinical and in vivo pieces of evidence proving that inflammatory bowel disease (IBD) is a risk factor for CKD. Four to twenty-three% of IBD patients will develop kidney- or urinary system-related complications, including nephrolithiasis, tubulointerstitial nephritis, glomerulonephritis, and amyloidosis. Furthermore, the low dose of Lm had the ability to improve gut leakage in CKD mice. Thus, in the second part of my study, we applied Lm in IBD mice model to investigate the possible mechanism of Lm in enhancing gut barrier function. For CKD feline clinical trial, Lm was oil-sprayed on commercial cat feed, and packed in small packages as probiotic pet treats. Three different flavors of pet treats were developed to offer more choices for picky CKD cats. By providing probiotic pet treats, we hope to alleviate stress for cats when administrating capsules. The probiotic pet treats met all the requirements of pet feed safety and chemical stability. The stability of Lm in treats at room temperature was also determined and required for at least a month. CKD cats were then fed with Lm-containing probiotic pet treats daily for 8 weeks. The fecal, urine, and blood samples were collected every 4 weeks. The results demonstrated that all the CKD cats had lower or maintain levels of plasma creatinine after probiotic pet treats intervention. Furthermore, more than 50% of the tested CKD cats were observed a reduction in serum trimethylamine N-oxide (TMAO), indoxyl sulfate (IS), and phenyl sulfate (PS). The appetite and physical activity of the tested cats were also improved. The fecal microbiota results showed that probiotic pet treats significantly increased bacteria diversity and modified the composition of gut microbiota by increasing Lactobacillus and Blautia caecimuris, and decreasing Enterobacteriaceae, Peptostreptococcaceae, Blautia hominis, and Blautia coccoides. The finding suggested that probiotic pet treats which contain Lm not only have the ability to reduce the progression of CKD clinically, but also can enhance the life quality of CKD cats by modifying gut microbiota and decreasing uremic toxins. In the second part of the study, C57BL/6 female mice were treated with Lm for 2 weeks, and changed their drinking water to contain 2.5% of dextran sulfate sodium (DSS) from day 7 to 13 to induce colitis. The results showed that Lm can mitigate fecal bleeding, increasing disease activity index (DAI), gut leakage, and higher histological score caused by DSS. Especially, high dose of Lm group (HD group) has longer colon length, higher body weight, and lower pro-inflammatory factors, showing that high dose of Lm has better ability to mediate the immune system and gut-protective ability. However, low dose of Lm (LD) also shows a potential ability to improve tight junction protein. The possible mechanism of Lm is to modify the microbiota in the cecum, especially decreasing Enterobacteriaceae and increasing Lactobacillus, and to up-regulate the level of butyric acid to enhance the gut barrier functionality. To sum up, high dose of Lm have better ability to mitigate colitis. In conclusion, Lm is a multi-functional probiotic mixture. Through the present study, we demonstrated that Lm-containing probiotic pet treats possessed an alleviating CKD effect in felines. Besides, Lm has the ability to mitigate the severity of colitis in mice. We also provide mechanism insights via analysis of the feline gut microbiome and DSS-induced colitis mouse model.