马达加斯加猴欢喜茎皮对红细胞膜的保护作用。
Protective effects of stem bark of Harungana madgascariensis on the red blood cell membrane.
机构信息
Department of Biochemistry, Faculty of Sciences, University of Dschang, Dschang, Cameroon.
出版信息
BMC Complement Altern Med. 2013 May 10;13:98. doi: 10.1186/1472-6882-13-98.
BACKGROUND
Anemia is a condition that has multiple origins. One such origin is the destruction of red blood cells' (RBCs) membrane induced by free radicals. Treatment of anemia could therefore be enhanced by the use of free radicals' scavengers potentially found in some medicinal plants. In this study, the protective effect of Harungana madagascariensis on the RBCs' membrane physiology was investigated in vitro and in vivo.
METHODS
In vitro hemolytic anemia was induced by incubation of fresh human RBCs with carbontetrachloride (CCl4) in Olive oil (Oo). Relaxation times of protons excited at 20 MHz (Carr-Purcell-Meiboom-Gill pulse sequence) in the absence or presence of paramagnetic Mn2+ ions (T2i for "extracellular" water and T2a for "intracellular" water, respectively) were determined at several temperatures (25-37°C) via Nuclear Magnetic Resonance (NMR) on a Bruker Minispec spectrometer. Water exchange times (Te) were consequently calculated using the Conlon-Outhred equation: 1/Te = (1/T2a) - (1/T2i). Morphological characteristics (mean cell volume, V, and cell surface area, A) were determined by photonic microscopy and the RBCs' diffusional water permeability (Pd) was calculated as Pd = (1/Te)(Va/A), where Va is the aqueous volume in the RBC and is about 0.7 of the cell volume (V). The activation energy of the diffusional process (Ea) for the respective temperature range was estimated using the Arrhenius modified equation k = A(T/T0)ne-Ea/RT. Inhibition of the water diffusion induced by incubation with para-chloro-mercuribenzoic acid (PCMB) at 25, 30 and 37°C was calculated as I(%) = [(Pd control - Pd sample)/Pd control]*100.To investigate the protective influence of the extract on the RBC membrane, inhibition of the water permeability was evaluated on membranes pre-incubated with the Harungana madagascariensis extract. Male rats were used in in vivo investigations. Malondialdehyde (MDA) and cholesterol in the RBC membrane were estimated by induction of lipid peroxidation while the antioxidant properties of catalase (CAT) and superoxide dismutase (SOD) on the membrane were evaluated in regard to their antioxidant properties on the membrane.
RESULTS
T2a significantly decreased at each temperature. Te results were higher in both RBCs and RBCs + extract groups incubated with PCMB compared to non-incubated controls, but differences were not statistically significant. A high percentage (73.81 ± 7.22) of RBCs pre-incubated with the extract presented the regular biconcave shape. Inhibition by PCMB of the RBCs' membrane water permeability was increased at 30°C and decreased in the presence of extract (25°C and 37°C), while Ea decreased from 30.52 ± 1.3 KJ/mol to 25.49 ± 1.84 KJ/mol. Presence of the Harungana madagascariensis extract normalized the SOD and CAT activities as well as the MDA and membrane cholesterol concentrations altered by the CCl4-induced oxidative stress.
CONCLUSION
Harungana madagascariensis could protect the RBCs' membrane through its antioxidative properties.
背景
贫血是一种由多种原因引起的病症。其中一种原因是自由基导致红细胞（RBC）膜的破坏。因此，通过使用可能存在于某些药用植物中的自由基清除剂来治疗贫血，可以增强治疗效果。在这项研究中，研究了马达加斯加哈伦加纳（Harungana madagascariensis）对红细胞膜生理学的体外和体内保护作用。
方法
通过在橄榄油（Oo）中孵育新鲜人 RBC 与四氯化碳（CCl4），在体外诱导溶血性贫血。在不存在或存在顺磁 Mn2+离子（分别为“细胞外”水的 T2i 和“细胞内”水的 T2a）的情况下，通过 Bruker Minispec 光谱仪上的核磁共振（NMR），在多个温度（25-37°C）下测定激发质子的弛豫时间（20 MHz 的 Carr-Purcell-Meiboom-Gill 脉冲序列）。随后使用 Conlon-Outhred 方程计算水交换时间（Te）：1/Te = (1/T2a) - (1/T2i)。通过光子显微镜测定红细胞形态特征（平均细胞体积，V 和细胞表面积，A），并根据以下公式计算红细胞扩散水通透性（Pd）：Pd = (1/Te)(Va/A)，其中 Va 是 RBC 中的水体积，约为细胞体积（V）的 0.7。使用修正后的 Arrhenius 方程 k = A(T/T0)ne-Ea/RT 估算各自温度范围内扩散过程的活化能（Ea）。在 25、30 和 37°C 下用对氯汞苯甲酸（PCMB）孵育时，通过计算水扩散的抑制率，将抑制率定义为 I(%) = [(Pd control - Pd sample)/Pd control]*100。为了研究提取物对红细胞膜的保护作用，评估了在预先用哈伦加纳（Harungana madagascariensis）提取物孵育的膜上，水通透性的抑制作用。雄性大鼠用于体内研究。通过诱导脂质过氧化来评估红细胞膜中的丙二醛（MDA）和胆固醇，同时评估膜上过氧化氢酶（CAT）和超氧化物歧化酶（SOD）的抗氧化性能，以评估其对膜的抗氧化性能。
结果
在每个温度下，T2a 均显著降低。与未孵育对照相比，用 PCMB 孵育的 RBC 和 RBC+提取物组的 Te 结果均较高，但差异无统计学意义。用提取物预先孵育的红细胞中，有 73.81 ± 7.22%呈现出规则的双凹形。PCMB 对 RBC 膜水通透性的抑制作用在 30°C 时增加，而在存在提取物时（25°C 和 37°C）降低，而 Ea 从 30.52 ± 1.3 KJ/mol 降低至 25.49 ± 1.84 KJ/mol。哈伦加纳（Harungana madagascariensis）提取物的存在使 SOD 和 CAT 活性以及由 CCl4 诱导的氧化应激改变的 MDA 和膜胆固醇浓度正常化。
结论
哈伦加纳（Harungana madagascariensis）可通过其抗氧化特性保护 RBC 膜。
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