探討氧化鈰奈米粒子對於間質性膀胱炎/膀胱痛症的治療效果

Abstract

背景: 間質性膀胱炎(Interstitial Cystitis, IC)，又稱膀胱痛症(Bladder Pain Syndrome, BPS)，是一種膀胱慢性發炎症狀，患者飽受骨盆疼痛和膀胱壓力導致的尿頻、尿急、漏尿的痛苦。研究發現，氧化壓力和活性自由基(Reactive Oxygen Species, ROS) 是間質性膀胱炎的誘因之一。而氧化鈰奈米粒子(Cerium Oxide Nanoparticles, CONP)具有可自我再生循環的抗氧化能力，具有抗細胞凋亡、抗發炎的效果，近年作為抗氧化物被廣泛研究。與目前臨床研究治療間質性膀胱炎的抗氧化物質，如，維他命D、兒茶素、褪黑激素等不同，CONP的抗氧化能力更持久。在本研究中通過施加ICR公鼠環磷酰胺(Cyclophosphamide，CYP)作為慢性膀胱疼痛的動物實驗的誘導劑。CYP可經由小鼠肝臟代謝成丙烯醛 (Acrolein)導致IC的發生。而細胞實驗則選取可在細胞培養液中自行水解生成丙烯醛的4-氫過氧環磷酰胺(4-hydroper-oxycyclophosphamide, 4-HC)誘導人類尿路上皮細胞T24產生氧化壓力。目的: 探討氧化鈰奈米粒子作為抗氧化劑，對由氧化損傷造成的間質性膀胱炎症狀是否具有治療效果。材料與方法: 利用金屬鹽水解法合成CONP，通過SEM、TEM分析粒徑大小；EDX進行成分分析；DLS方法測定CONP於溶液中的分散粒徑；XRD分析結晶構造；XPS進行表面元素價態分析，氮氣吸附法測定粒子的比表面積。細胞實驗組別分為: (1) 控制組; (2) 4-HC誘導組:以37.5 μM 4-HC加入細胞培養液4小時以誘導細胞產生氧化壓力; (3) CONP pre-treat組別:在4-HC誘導前施加5 μg/mL CONP 24小時，驗證CONP對於4-HC產生之氧化傷害的還原效果; (4) only CONP組:CONP的施加對於細胞毒性的探討。根據ISO-10993-5標準，利用WST-1 assay 和Live/Dead assay驗證CONP材料的生物相容性及各組別細胞存活率；DCFDA assay驗證CONP對細胞氧化損傷的還原效果；qRT-PCR驗證發炎反應及細胞凋亡相關基因表現。動物實驗分為: (1) 控制組：腹腔注射滅菌PBS 6次 (2) CYP誘導組: IP 注射80 mg/kg 之CYP藥物4次 (3) pre-treat CONP組：先IP 注射30 mg/ kg之CONP 2次後，與CYP誘導組同時間給予4次CYP藥物。犧牲當天通過Voiding spot assay，收集各組別小鼠3小內的排尿濾紙，拍照、量化以評估CONP的施加對尿頻的改善狀況。動物犧牲後取膀胱組織以H E染色組織切片觀察，並利用TEM拍攝膀胱表皮細胞的排列狀況；SEM觀察膀胱粘膜層的恢復。通過血液生化分析確認CONP是否造成肝腎毒性。結果與討論：合成之CONP符合ISO-10993-5標準對生醫材料生物相容性之要求規範。在細胞實驗中，預先施加5 μg/mL CONP 可有效緩解由37.5 μM的4-HC引發的T24細胞內ROS的累積，恢復細胞活性，且對由4-HC上調之IL-6、TNF-α等相關發炎、氧化損傷基因有顯著抑製作用。動物實驗中，組織染色切片及相應的SEM、TEM的結果證明CONP的施加恢復了膀胱表皮細胞間的緊密連接且幫助內皮粘膜結構恢復。血液生化分析結果證實CONP不會造成肝腎毒性。結論：氧化鈰奈米粒子可有效緩解膀胱表皮細胞之氧化損傷，緩解間質性膀胱炎引起的膀胱組織發炎及功能性缺失，是一種潛在的、可用於治療間質性膀胱炎的生物醫學材料。

Background: Interstitial Cystitis (IC), also known as Bladder Pain Syndrome (BPS), is a chronic inflammatory condition in which patients suffer from pelvic pain and bladder pressure, frequent urination, urgency, and nocturia. Oxidative stress and reactive oxygen species (ROS) have been confirmed to be one of the reasons for IC. Antioxidants in clinical studies such as vitamin D, catechins, and melatonin have poor therapeutic effects due to their short-term antioxidant capacity. Cerium Oxide Nanoparticles (CONP) have self-regenerating antioxidative, anti-apoptotic and anti-inflammatory properties, and have been widely studied as antioxidants in recent years. Contrary to the current clinical research on the treatment of interstitial cystitis with antioxidant substances, such as vitamin D, catechins, melatonin, etc., CONP's antioxidant capacity may more durable. Cyclophosphamide (CYP), which can be metabolized by the liver to product acrolein, inducing interstitial cystitis both in humans and mice, was used as an inducer for in vivo study. While 4-hydroperoxycyclophosphamide (4-HC), forming a precursor of acrolein in the medium, was used to induce the cell to generate ROS and apoptosis in vitro. Purpose: To verify the therapeutic effects of cerium oxide nanoparticles for interstitial cystitis caused by oxidative damage. Materials and Methods: CONP was synthesized by the mantel-salt hydrolysis method, and the particle size was analyzed by SEM and TEM and EDX were used for the component analysis. The hydrodynamic size of CONP was determined by the DLS method. XRD was used to analyze the crystal structure, while XPS was used to analyze the surface element valence state. The specific surface area was determined by a nitrogen adsorption method. In vitro, human urothelial cells T24, were cultured and divided into (1) control group, (2) 4-HC group: added 37.5 μM 4-HC, 4 hours to induce cell oxidative stress, (3) pre-treat CONP group: applied 5 μg/mL CONP for 24 hours before 37.5 μM 4-HC 4-hours induction, (4) only CONP group: only applied 5μg/mL CONP for 24 hours. The biocompatibility of CONP was verified by WST-1 assay and Live/Dead assay, following ISO-10993-5 standard. DCFDA assay analyzed the antioxidative effects of CONP. q-RTPCR verified the inflammatory response and antioxidative related gene expression. In vivo, evaluate the improvement of CONP to the frequency of urination by a 3-hour void spot assay. The bladder tissue morphology was taken and observed with HE stained and TEM. The recovery of the bladder mucosa was observed by SEM. Results and discussion: The synthesized CONP meets the ISO-10993-5 standard for biocompatibility of biomedical materials. In cell experiments, pre-treated cells with 5 μg/mL CONP can effectively alleviate the accumulation of ROS caused by 37.5 μM 4-HC, meanwhile, inhibit IL-6, TNF-α, and other inflammatory or oxidative damage related genes expression. In animal experiments, the HE tissue staining sections and the corresponding TEM proved that the application of CONP restored the tight junctions between bladder epidermal cells. SEM images showed that CONP helped restore the structure of bladder endothelial mucosa. The results of blood biochemical analysis confirmed that CONP does not cause liver and kidney toxicity. Conclusion: Cerium oxide nanoparticles can effectively alleviate oxidative damage of T24 cells and alleviate bladder tissue inflammation and dysfunctional caused by interstitial cystitis, which is a potential biomaterial for the treatment of interstitial cystitis.