Department of Physiology and Biophysics, School of Medicine, Health Sciences Center, A-1115, University of Louisville, Louisville, KY 40202, USA.
J Mol Neurosci. 2012 May;47(1):128-38. doi: 10.1007/s12031-011-9695-z. Epub 2012 Jan 3.
High levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy), contribute to autophagy and ischemia/reperfusion injury (I/R). Previous studies have shown that I/R injury and HHcy cause increased cerebrovascular permeability; however, the associated mechanism remains obscure. Interestingly, during HHcy, cytochome-c becomes homocysteinylated (Hcy-cyto-c). Cytochrome-c (cyto-c) transports electrons and facilitates bioenergetics in the system. However, its role in autophagy during ischemia/reperfusion injury is unclear. Tetrahydrocurcumin (THC) is a major herbal antioxidant and anti-inflammatory agent. Therefore, the objective of this study was to determine whether THC ameliorates autophagy during ischemia/reperfusion injury by reducing homocysteinylation of cyto-c in hyperhomocysteinemia pathological condition. To test this hypothesis, we employed 8-10-week-old male cystathionine-beta-synthase heterozygote knockout (CBS⁺/⁻) mice (genetically hyperhomocystemic mice). Experimental group was: CBS⁺/⁻, CBS⁺/⁻ + THC (25 mg/kg in 0.1% DMSO dose); CBS ⁺/⁻/I/R, and CBS⁺/⁻/I/R + THC (25 mg/kg in 0.1% DMSO dose). Ischemia was performed for 30 min and reperfusion for 72 h. THC was injected intra-peritoneally (I.P.) once daily for a period of 3 days after 30 min of ischemia. The infarct area was measured using 2,3,5-triphenyltetrazolium chloride staining. Permeability was determined by brain edema and Evans Blue extravasation. The brain tissues were analyzed for oxidative stress, matrix metalloproteinase-9 (MMP-9), damage-regulated autophagy modulator (DRAM), and microtubule-associated protein 1 light chain 3 (LC3) by Western blot. The mRNA levels of S-adenosyl-L-homocysteine hydrolases (SAHH) and methylenetetrahydrofolate reductase (MTHFR) genes were measured by quantitative real-time polymerase chain reaction. Co-immunoprecipitation was used to determine the homocysteinylation of cyto-c. We found that brain edema and Evans Blue leakage were reduced in I/R + THC-treated groups as compared to sham-operated groups along with reduced brain infarct size. THC also decreased oxidative damage and ameliorated the homocysteinylation of cyto-c in-part by MMP-9 activation which leads to autophagy in I/R groups as compared to sham-operated groups. This study suggests a potential therapeutic role of dietary THC in cerebral ischemia.
高同型半胱氨酸（Hcy）水平，即高同型半胱氨酸血症（HHcy），可导致自噬和缺血/再灌注损伤（I/R）。先前的研究表明，I/R 损伤和 HHcy 导致脑血管通透性增加；然而，相关机制尚不清楚。有趣的是，在 HHcy 期间，细胞色素-c 发生同型半胱氨酸化（Hcy-cyto-c）。细胞色素-c（cyto-c）在系统中转运电子并促进生物能量。然而，其在缺血/再灌注损伤期间自噬的作用尚不清楚。四氢姜黄素（THC）是一种主要的草药抗氧化剂和抗炎剂。因此，本研究旨在确定 THC 是否通过减少 HHcy 病理条件下细胞色素-c 的同型半胱氨酸化来改善 I/R 损伤期间的自噬。为了验证这一假设，我们使用了 8-10 周龄的胱硫醚-β-合酶杂合子敲除（CBS ⁺/⁻）雄性小鼠（遗传高同型半胱氨酸血症小鼠）。实验组为：CBS ⁺/⁻、CBS ⁺/⁻+THC（0.1% DMSO 剂量下 25 mg/kg）；CBS ⁺/⁻/I/R 和 CBS ⁺/⁻/I/R+THC（0.1% DMSO 剂量下 25 mg/kg）。缺血 30 分钟，再灌注 72 小时。THC 每天腹腔注射（I.P.）一次，在缺血 30 分钟后连续 3 天。用 2,3,5-三苯基氯化四唑染色测量梗死面积。通过脑水肿和 Evans Blue 外渗测定通透性。通过 Western blot 分析脑组织的氧化应激、基质金属蛋白酶-9（MMP-9）、损伤调节自噬调节剂（DRAM）和微管相关蛋白 1 轻链 3（LC3）。通过定量实时聚合酶链反应测量 S-腺苷-L-同型半胱氨酸水解酶（SAHH）和亚甲基四氢叶酸还原酶（MTHFR）基因的 mRNA 水平。通过共免疫沉淀法测定细胞色素-c 的同型半胱氨酸化。我们发现，与假手术组相比，I/R+THC 治疗组的脑水肿和 Evans Blue 渗漏减少，脑梗死面积减小。与假手术组相比，THC 还通过 MMP-9 激活降低氧化损伤并改善 I/R 组细胞色素-c 的同型半胱氨酸化，从而导致自噬。这项研究表明，膳食 THC 在脑缺血中具有潜在的治疗作用。
