潛力益生菌株降低尿毒素硫酸吲哚酚於順鉑誘發急性腎損傷大鼠之研究

Abstract

硫酸吲哚酚為累積於慢性腎臟疾病患者體內之尿毒素之一，其親蛋白質的特性使此類尿毒素難以藉血液透析治療有效被去除，可能導致嚴重的腎功能衰退及心血管疾病等併發症，甚至造成死亡。吲哚為硫酸吲哚酚之前驅物，由可產生色胺酸酶之腸道細菌如 Escherichia coli等代謝色胺酸於腸道生成，再由肝臟代謝形成硫酸吲哚酚。本研究之目的為篩選具降低硫酸吲哚酚潛力之益生菌並探討其對血液硫酸吲哚酚濃度、腎功能及腸道菌組成影響及可能之機制。 首先經體外及動物試驗篩選出混合3株乳酸菌之Pm-1（Lactobacillus plantarum、 L. paracasei 及 Streptococcus thermophilus）較單一菌株更具有良好之硫酸吲哚酚移除潛力。動物試驗利用腹腔注射cisplatin誘導之急性腎損傷之大鼠模式探討Pm-1降低尿毒素之潛力，結果發現，餵食Pm-1之大鼠處理組可顯著降低cisplatin造成之血液中硫酸吲哚酚濃度升高及顯著降低腸道中之E. coli菌數，然而於腎功能相關之血液生化值則無顯著影響。 為了降低大量發酵之成本，本研究利用反應曲面法開發Pm-1共同最適化之取代培養基，以序列二次規劃法（sequential quadratic programming, SQP）進行最適化，經過氮、碳源篩選及反應曲面之建立等步驟，顯示使用糖蜜酵母粉，麥芽糊精及磷酸氫二鉀作為取代培養基之主原料配方可使所培養之菌數分別達到 9.01、8.71及8.94 log/mL，並且僅為商業培養基MRS成本之20%。 本研究亦比較延長餵食Pm-1之天數對cisplatin誘導之急性腎損傷大鼠模式之影響，結果顯示，藉由長期餵食 Pm-1 之試驗組中，其降低血液與肝、腎之硫酸吲哚酚濃度之能力較佳，根據實驗結果推測Pm-1可能藉由吸附或代謝腸道中之尿毒素前驅物、調控腸道菌組成及降低氧化壓力之機制而具有預防腎功能衰退之潛力。

Numerous molecules that are either excreted or metabolized by the kidney accumulated in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS) is a protein bound uremic toxin, which is unable to effectively eliminate by haemodialysis (HD). The accumulation of protein-bound uremic toxins has been suggested to be related to complications and mortality in HD patients. Indole, the precursor of IS, are produced by intestinal bacteria, such as Escherichia coli, from tryptophan. Since uremic toxins might be reduced by modulating microbiota in the colon, probiotics seem to be potential to reduce the concentration of intestinal nitrogenous metabolites. Thus, the aim of this study was to find potential probiotics which may improve renal function by reducing IS production. Three strains, Lactobacillus plantarum, L. paracasei and Streptococcus thermophilus, were selected to have the ability to reduce indoxyl sulphate in vitro. Combination of strains (Pm-1) demonstrated a better IS removing ability than individual strains. We further investigated the uremic toxin-reducing probiotics by the cisplatin-induced acute kidney injury (AKI) rat model. Results indicated that oral administration of Pm-1 in cisplatin-induced acute kidney injury model significantly suppressed the accumulation of IS in the serum. Oral administration of Pm-1 also decreased in the number of E. coli and coliforms in feces. We further optimized growth medium by response surface methodology (RSM) with sequential quadratic programming (SQP). Results indicated that the media with sugar yeast, maltodextrin and dipotassium as major component could yield the highest cell numbers to 9.01、8.71 and 8.94 log/mL, respectively. The cost of medium was reduced to less than 20% when compared with commercial MRS medium. Our findings suggest that long term treatment with the strain combination Pm-1 might be a useful approach to decreasing IS accumulation in the serum and kidney. The possible mechanisms involved might include the binding/metabolizing IS in intestine and/or the modulation of bacterial growth in colon by Pm-1, both of which could decrease IS accumulation. The stimulation of anti-inflammatory cytokines and release of oxidative stress by Pm-1 might also be involved in the amelioration of AKI symptoms.