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標題: | 探討gamma-胺基丁酸對Vhlh基因剔除小鼠腎臟病
與免疫調節的影響 Effects of gamma-aminobutyric acid on renal disease and immunomodulation in Vhlh gene-knockout mice |
作者: | Hsun-Yi Huang 黃薰儀 |
指導教授: | 林璧鳳(Bi-Fong Lin) |
關鍵字: | GABA,VHL基因,腎臟疾病,巨噬細胞,調節型T細胞, gamma-aminobutyric acid,Von Hippel-Lindau,renal disease,macrophages,regulatory T cells, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 臺灣末期腎臟疾病的盛行率為全球第一,急性腎臟病、慢性腎臟病及洗腎人口數也居高不下,因此有效延緩或降低腎臟發炎及損傷程度刻不容緩。已知gamma-aminobutyric acid (GABA) 除了為一抑制型神經傳遞物質外,也具有降低發炎與調節免疫的作用,然而GABA對急、慢性腎病的免疫調節機轉尚不明確。因本研究室在繁殖Vhlhdel/del小鼠過程得到的Vhlh+/del 小鼠,有較WT C57BL/6小鼠容易誘發腎損傷的特性,及繁殖所得的腎臟VHL基因缺失 (Vhlhdel/del) 小鼠,有自發產生腎炎腎癌及生命期只有3個月的特性。因此欲利用會導致腎炎及腎癌的
馬兜鈴酸 (AA) 誘導Vhlh+/del 小鼠急性腎臟病,並利用Vhlhdel/del小鼠作為慢性腎病的動物模式,探討GABA對急性與慢性腎炎的影響及免疫調節作用。 首先利用AA誘導Vhlh+/del 小鼠急性腎臟病。將11~13週齡的Vhlh+/del 小鼠分別以AIN-93飼料 (Con組、AA組;4 mg/kg BW)、含GABA米飼料 (Gr-AA組) 與含GABA純物質的飼料 (G-AA組) 餵飼2週後犧牲。結果發現,GABA可顯著降低AA誘導腎臟損傷、腎纖維化程度、尿蛋白肌酸酐比值、血清肌酸酐、血清尿素氮與血清IL-6濃度,同時也能降低巨噬細胞與調節型T細胞浸潤於腎臟中的比例,並減少腎臟組織中MCP-1與CCR2表現量及MCP-1與TGF-β的濃度,提升脾臟細胞分泌IFNγ與IL-17A的能力。續以腎臟細胞探討GABA護腎機轉,發現GABA可顯著降低AA誘導腎小管細胞中ROS生成、NF-κB轉錄活性及發炎因子COX-2基因表現量,也可顯著降低MCP-1、IL-6與TGF-β的分泌量,此抗發炎機轉在腎間質細胞中也同樣被證實。綜合上述,推測GABA可透過影響腎臟細胞中ROS-NF-κB路徑,減緩AA誘導Vhlh+/del 小鼠腎臟發炎情形,減緩腎臟急性發炎反應及纖維化的狀況。 在自發性腎炎腎癌的Vhlhdel/del小鼠實驗中,將5週齡離乳後的Vhlhdel/del小鼠分組,分別以AIN-93飼料 (Con組)、含GABA米飼料 (Gr組) 與含GABA純物質的飼料 (G組) 餵飼10週,於15週齡時犧牲,分別探討GABA對Vhlhdel/del小鼠慢性腎炎的免疫調節機轉,及對Vhlhdel/del小鼠腎臟細胞中代謝路徑改變 (metabolic reprogramming) 的影響。首先,在GABA對慢性腎炎的免疫調節機轉結果中發現,餵飼GABA不僅可以回復Vhlhdel/del小鼠血清與腎臟中的GABA含量,也可顯著延長小鼠的生命期。再者,GABA也可顯著降低亮細胞產生數目、腎小管損傷指標KIM-1、腎纖維化程度及全身性發炎指標IL-6,減少巨噬細胞、M1巨噬細胞、調節型T細胞浸潤,降低MCP-1、TGF-β與IL-17A等細胞激素產生。另外也發現了GABA具降低小鼠脾臟細胞分泌IFNγ、IL-17A、IL-10、TGF-β與IL-6,增加TNF-α的能力。而GABA的免疫調節機轉,分別於動物及細胞實驗中均證實,GABA可降低MCP-1與CCR2表現量,減少單核球浸潤,此與GABA減緩Vhlhdel/del小鼠腎臟發炎反應相關。 GABA對Vhlhdel/del小鼠腎臟細胞中代謝路徑的影響結果顯示,GABA可顯著調節腎臟組織中glutathione代謝、tryptophan代謝及circadian rhythm相關基因表現量。在glutathione代謝路徑中發現,GABA可顯著降低Vhlhdel/del小鼠腎臟細胞中GST與GPx酵素活性,並增加VHL缺失腎小管細胞中氧化自由基的生成;在tryptophan代謝路徑中發現,GABA可顯著降低Ido1與Ido2基因表現量,推測此與調節免疫細胞活性有關;在circadian rhythm結果中發現,GABA可顯著回復與細胞凋亡及增生相關的Per2基因表現量,並降低細胞增生指標ki67基因表現量。 綜合上述,無論是AA誘導之Vhlh+/del小鼠或是自發性腎炎之Vhlhdel/del小鼠,GABA不僅可以調節腎臟中巨噬細胞與調節型T細胞浸潤,也可減輕腎臟發炎媒介物質的產生,並透過調節全身性免疫細胞活性,進而減輕腎臟損傷、腎臟發炎及腎臟纖維化的作用。再者,GABA也可透過調節Vhlhdel/del小鼠腎臟中的代謝路徑,改變細胞中的氧化壓力、抑制細胞增生及降低免疫抑制作用。因此本研究顯示GABA可透過多重護腎機轉,減緩腎臟病的疾病程度。 Taiwan has the highest global prevalence of end-stage renal disease (ESRD), with the number of new patients suffering from ESRD increasing annually. Therefore, preventing or reducing renal inflammation and injury is important. GABA is not only a CNS neurotransmitter, but also has several physiological functions, such as immunomodulatory effects and renal protective abilities. However, the mechanism by which GABA regulates renal inflammation remains unclear. Von Hippel-Lindau gene knock-out (Vhlhdel/del) mice can spontaneously develop fibrosis, inflammation and hyperplastic clear-cell lesions in the kidney and have an average life span of approximately 3 months. In addition, renal injury is easily induced in Vhlh+/del mice. To explore the immunomodulatory effects of GABA, aristolochic acid (AA)-induced acute kidney disease (AKD) Vhlh+/del mice and Vhlhdel/del mice were used as animal models of AKD and chronic kidney disease (CKD). To generate AA-induced AKD Vhlh+/del mice, 11-13-week-old Vhlh+/del mice were fed an AIN-93 diet (control; Con), AIN-93 diet with AA (AA), GABA rice diet with AA (Gr-AA), or GABA-supplemented AIN-93 diet with AA (G-AA) for 2 weeks. Both the Gr-AA and G-AA diets significantly decreased the urine protein/urine creatinine ratio, serum creatinine, BUN, and interleukin (IL)-6 levels, and the degree of renal injury and renal fibrosis. Macrophage and regulatory T cell infiltration and MCP-1 and transforming growth factor (TGF)-β secretion were also reduced in these mice, while IFN-γ and IL-17A secretion increased in stimulated-splenocytes. The anti-inflammatory mechanism of GABA was further explored in AA/LPS-stimulated HK-2 or MES-13 cells. GABA alleviated reactive oxygen species (ROS), NF-κB, COX-2, MCP-1, and TGF-β secretion in AA/LPS-stimulated HK-2 cells, and decreased NF-κB, COX-2, MCP-1, and TGF-β secretion in AA/LPS-stimulated MES-13 cells. Taken together, these results indicated that GABA might ameliorate renal inflammation via the ROS-NF-κB pathway by reducing inflammatory cytokine secretion and lymphocyte infiltration, thus alleviating renal inflammation and injury in AA-induced AKD Vhlh+/del mice. The effects of GABA on immunomodulation in CKD Vhlhdel/del mice and metabolic reprogramming in Vhlhdel/del mice were also investigated. Five-week-old mice were fed an AIN-93 diet (Con), GABA rice diet (Gr), or GABA-supplemented AIN-93 diet (G) and their life span was followed. Another group of mice were sacrificed after 10 weeks of diet administration. Both the Gr and G diets significantly extended life spans, reverted GABA levels, and decreased the levels of urinary KIM-1, clear cell type formation, and Fn1 and Lgals3 fibrotic genes expression (typical markers of renal injury and renal fibrosis, respectively) in Vhlhdel/del mice. The serum inflammatory markers IL-6, renal MCP-1, TGF-β, Ccr2, and Mcp-1 and macrophage infiltration were significantly lower in GABA-fed mice. HK-2 cells with VHL-knockdown were used to confirm CCR2 and MCP-1 expression, monocytes migration, and cell proliferation. Lower CCR2, MCP-1, THP-1 lymphocyte migration and cell proliferation were also observed in VHL-silenced HK-2 cells treated with GABA, suggesting that GABA exerts a protective function of in CKD Vhlhdel/del mice. In addition, the effects of GABA on metabolic reprogramming in Vhlhdel/del mice were assessed. GABA regulated glutathione metabolism, tryptophan metabolism, and the circadian rhythm in Vhlhdel/del mice. For instance, GABA decreased glutathione S-transferase (GST) and glutathione peroxidase (GPx) activity for glutathione metabolism in the kidneys which might explain why ROS levels were higher in GABA-treated VHL-silenced HK-2 cells than in untreated VHL-silenced HK-2 cells. In addition, GABA reduced indoleamine 2,3-dioxygenase (Ido)-1 and Ido2 genes expression in tryptophan metabolism, thus decreasing the regulatory T cell population in the kidneys of Vhlhdel/del mice. Furthermore, GABA reverted Per2 gene expression related to the circadian rhythm and decreased the expression of Ki67, a proliferation marker, in the kidneys of Vhlhdel/del mice. In summary, GABA not only decreased macrophage and regulatory T cell infiltration, but also alleviated inflammatory and pro-fibrotic cytokines secretion in the kidneys of the AKD and CKD animal models, thus ameliorating renal inflammation, fibrosis, and renal injury. In addition, GABA regulated oxidative stress, cell proliferation, and immunosuppressive abilities by modulating metabolic reprogramming and the circadian rhythm in the kidneys of Vhlhdel/del mice. Hence, GABA is a multifunctional molecule with nephroprotective effects that has the potential to prevent and decrease the disease degree of AKD and CKD. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21159 |
DOI: | 10.6342/NTU201904229 |
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顯示於系所單位: | 生化科技學系 |
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