| 摘要: | 本研究採用自體免疫性腎臟損傷的加速型腎毒血清(nephrotoxic serum, NTS)腎炎小鼠模型探討其損傷進程下的機轉與生物標記，根據模型進程分為正常組(Normal; W1)、IgG注射組(Control; W2)、IgG注射加NTS注射後7天(Day 7; W3)與14天(Day 14; W4)等四組。利用NMR分析非標靶性代謝體學，結果發現包含糖解作用、酮體代謝、眾多胺基酸的代謝路徑以及能量利用終點的檸檬酸循環均會隨著NTS腎炎的病程發生顯著的改變，根據L-乳酸的增加與上游胺基酸表現量的減少，推測代謝路徑的改變和瓦式效應(Warburg effect)有關，因此胺基酸的補充可能為有效改善疾病的方法。Receptor-interacting kinase 3 (RIP3)蛋白僅表現於Day 7組的腎臟組織中，顯示雖然NTS腎炎中會活化細胞程序性壞死路徑，但並非影響代謝物表現的關鍵。在標靶性代謝體學的部分，以LC-MS/MS方法測定醣化終產物Nε-(carboxyethyl)lysine (CEL)、Nε-(carboxymethyl)lysine (CML)和methylglyoxal-derived hydroimidazolone 1 (MG-H1)，三者在Normal組尿液中含量分別為2.33 ± 0.28、2.77 ± 0.30與1.14 ± 0.10 μg/12 hr，在其他三組皆有1.5到2倍的含量上升，而在Normal組血清中三者的濃度分別為34.59 ± 11.56、59.83 ± 7.70與27.68 ± 3.35 ng/mL，隨著加速型NTS腎炎模型進程，三者的表現量會逐漸上升，最終在Day 14組有4.6到5.8倍之間的增加。此外，從tumor necrosis factor-α (TNF-α)和glyoxalase 1 (GLO1)的結果可知在疾病階段會活化發炎反應與乙二醛?系統，但是隨NTS腎炎進展，TNF-α會逐步上升而GLO1則逐步下降，推測免疫反應造成TNF-α增加，TNF-α會抑制GLO1，使得甲基乙二醛轉向形成醣化終產物，最終醣化終產物的堆積造成腎臟進一步的損傷。最後，本研究建立顯微鏡式圓二色光譜檢測系統，利用蛋白質經過醣化後會改變圓二色性的特性，以此檢測方法能在Day 14組發現圓二色性訊號的發生變化，代表此檢測法能成為影像醫學以及人工智能診斷的另一個應用方向。
This study used an accelerated nephrotoxic serum (NTS) nephritis mouse model, an autoimmune kidney disease, to discover the mechanism and biomarkers of immune kidney disease. According to the progression of nephritis, four groups, including normal (Normal; W1), IgG injection (Control; W2), 7 days after NTS injection (Day 7; W3) and 14 days after NTS injection (Day 14; W4), were divided. The results of NMR-based metabolomic study showed that glycolysis, synthesis and degradation of ketone bodies, many amino acids pathway and citrate cycle were significantly affected by the progression of NTS nephritis. According to the increase of L-lactic acid and the decrease of upstream amino acids expression, the change of pathway could be related to the Warburg effect. Then, amino amino acid supplement would mitigate the progression of nephritis. In the kidney tissue, receptor-interacting kinase 3 (RIP3) was only found in the Day 7 group. The result indicated that necroptosis was activated in NTS nephritis, but it was not the key point of affecting the expression of metabolites. The advanced glycation end products (AGEs), including Nε-(carboxyethyl)lysine (CEL), Nε-(carboxymethyl)lysine (CML) and methylglyoxal-derived hydroimidazolone 1 (MG-H1), were analyzed by LC-MS/MS method. The amount of CEL, CML and MG-H1 in the urine of normal group were 2.33 ± 0.28、2.77 ± 0.30, and 1.14 ± 0.10 μg/12 hr, respectively. CEL, CML and MG-H1 in other groups were 1.5 to 2 times higher than those in normal group. In the serum, CEL, CML and MG-H1 in normal group were 34.59 ± 11.56、59.83 ± 7.70, and 27.68 ± 3.35 ng/mL. With the progression of NTS nephritis, the AGEs were gradually increased, and had 4.6 to 5.8 times increase in Day 14 group. Depends on the increase of tumor necrosis factor-α (TNF-α) and glyoxalase 1 (GLO1), inflammation and glyoxalase system were activated under disease stage. However, with the progression of NTS nephritis, TNF-α gradually increased, and GLO1 gradually decreased. Because the expression of GLO1 might be suppressed by TNF-α, methylglyoxal turned to form AGEs. The accumulation of AGEs led to more serious kidney damage. Futhermore, this study established a microscope-type circular dichroism (CD) system. Because the glycation protein leads to CD change, the system could determine the level of kidney damage. The kidney tissue of Day 14 group showed significant diffence of CD level by our CD system. The system provides another application for medical imaging and artificial intelligence diagnosis. |
