探討醋酸鈉改善順鉑誘導腎臟細胞損傷之機制

順鉑為一種常見的抗癌藥物，在臨床上，最常見且最嚴重的副作用為腎毒性，其中又以急性腎損傷最常發生。由於從先前在我們的實驗室發現，順鉑會使腸道菌相改變，在大鼠實驗給予順鉑的治療時補充益生菌Lactobacillus reuteri和 Clostridium butyricum之後，除了可以改善腸道菌相以外，對於腎損傷有明顯的改善，此外，分析發現糞便中的短鏈脂肪酸有明顯的上升。在本研究中，我們主要使用了三種腎臟細胞: SV40 MES-13、NRK-52E和NRK-49F細胞株，利用MTT assay發現醋酸鈉和丁酸鈉對Cisplatin誘導SV40 MES-13細胞株死亡有明顯的恢復，利用SA-β-gal staining發現醋酸鈉和丁酸鈉透過抑制細胞衰老相關蛋白β-galactocidase的活性可以觀察衰老現象的減少，使用H2DCFDA染色分析細胞內ROS在醋酸鈉和丁酸鈉的處理下也同樣有下降，利用流式細胞儀分析細胞週期分布在SV40 MES-13細胞株有明顯在G2/M期停滯，給予醋酸鈉之後則有減少，醋酸鈉也可以使細胞週期相關蛋白p21和p-Rb在SV40 MES-13細胞株有明顯的下降，使用Annexin-Ⅴ/PI staining和西方墨點法分析細胞凋亡相關蛋白在SV40 MES-13細胞株中有明顯的改善。由於有研究指出短鏈脂肪酸可以透過活化GPR41和GPR43 ligands保護腎功能，因此在研究中使用GPR41和GPR43的拮抗劑，β-hydroxybutyrate和 GLPG0974，然而在觀察細胞過早衰老、ROS的變化和細胞凋亡相關蛋白的表現量時，醋酸鈉的保護效果並沒有被抑制。在前面的實驗我們得知醋酸鈉確實對腎臟細胞有保護的功效，但是並不確定是否會影響順鉑對腫瘤抑制的效果，因此我們在動物實驗中進一步的進行驗證，使用C57BL/6母鼠注射Lewis Lung Carcinoma，並每天給與醋酸鈉每公斤1克，於施打腫瘤後5天腹腔注射順鉑每公斤10毫克，共施打4劑，並於第4劑後3天犧牲。由結果得知醋酸鈉並不會影響腫瘤抑制的效果，此外，由於在細胞實驗發現醋酸鈉可減少由Cisplatin誘導的細胞凋亡現象，因此使用IHC染色觀察腎臟組織中cleaved caspase-3的表現，也同樣有減少的趨勢。由我們的實驗結果得知醋酸鈉可以透過減少ROS產生的方式改善順鉑誘導的細胞過早衰老、細胞週期停滯與細胞凋亡，並且對於順鉑毒殺癌細胞的能力沒有影響。因此醋酸鈉可能可以作為未來治療順鉑誘導腎損傷的策略之一。

關鍵字

順鉑；醋酸鈉；腎損傷；細胞衰老；細胞凋亡

並列摘要

Cisplatin is one of effective chemotherapeutic drug in tumor against. The most widely and severely side effect is nephrotoxicity in clinical, which limit anti-tumor efficiency. Acute kidney injury(AKI) is the most common syndrome during treated with cisplatin. In our present study, we found that cisplatin would induce gut microbiota transform. Thus, we pretreated two kinds of probiotics, Lactobacillus reuteri and Clostridium butyricum, before treated with cisplatin in rat. In addition to ameliorating microbiota composition, probiotics supplement also alleviate cisplatin-induced kidney injury obviously. The quantity of short chain fatty acids increased notably which observed by cecal contents analysis. In this study, we use three different kinds of kidney cells: SV40 MES-13, NRK-52E, and NRK-49F cells, to evaluate the effects of short chain fatty acids on ameliorating cisplatin-induced kidney cells injury. We determined cisplatin concentration and found that sodium acetate and sodium butyrate increased cell viability through MTT assay. On SA-β-gal and C12FDG staining, sodium acetate and sodium butyrate alleviated cisplatin-induced premature senescence via inhibiting β-galactosidase activity. H2DCFDA staining was used to prove sodium acetate and sodium butyrate decreased cisplatin-induced ROS production. Cisplatin accumulated cell cycle in G2/M arrest and improved by sodium acetate followed by PI staining. On western blot, cisplatin upregulated p21 and p-Rb expression and downregulated by sodium acetate. Cisplatin induced apoptosis through activate caspase associated pathway followed by western blotting. Cleaved caspase-3 and cleaved caspase-7 overexpressed during cisplatin irritated which sodium acetate inhibited. We also detected apoptotic cell and quantified by Annexin-V / PI staining, our results validated that sodium acetate ameliorated cisplatin-induced apoptosis. In present research indicated that GPR41 and GPR43 were activated by SCFAs which mediated kidney injury. We use two kinds of antagonist, β-hydroxybutyrate(βOHB) and GLPG0974, estimated weather affect sodium acetate. However, our data show no difference after pre-treated βOHB or GLPG0974 in SV40 MES-13 cells follow by SA-β-gal staining, H2DCFDA staining and western blot. Long terms cisplatin develop AKI to CKD. Thus, we observed fibrosis maker, α-SMA, with western blot, but displayed no difference after cisplatin combine with sodium acetate. Our result implied that sodium acetate possessed potential advantage, but we did not understand whether the benefit affect cisplatin anti-tumor ability. Lewis Lung Carcinoma cells were injected into subcutaneous of C57BL/6 mice about 1 × 106. After 3 days tumor growth, CDDP+NaAc group received sodium acetate ( 1 g / kg, N=5, respectively ) by gavage once every day until euthanize. 5 days after tumor cells injection, CDDP and CDDP+NaAc group received cisplatin ( 10 mg / kg, N = 5, respectively ) by intraperitoneal injection once a week for four cycles. Our data expressed that sodium acetate did not affect cisplatin anti-tumor ability in vivo. Cleaved caspase-3 and α-SMA expression were analyzed by IHC staining which consistent with present results. In conclusion, our results demonstrated that sodium acetate inhibited cisplatin-induced senescence, cell cycle arrest and apoptosis depended on attenuate ROS production, may be a useful strategy on cisplatin-induced kidney injury.

並列關鍵字

cisplatin ； sodium acetate ； kidney injury ； senescence ； apoptosis

參考文獻

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探討醋酸鈉改善順鉑誘導腎臟細胞損傷之機制

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