Hydrogen peroxide induced oxidative stress in the spleen of metallothionein Ⅰ/Ⅱ knockout mice

doi:10.3969/j.issn.2095-4344.2013.07.019

Abstract

Abstract:

BACKGROUND: In the early stage of cerebral ischemia-reperfusion, there is a tremendous amount of inflammatory factor expression in the spleen. These inflammatory factor cause oxidative stress damage which leads to cell apoptosis in the spleen after cerebral ischemia-reperfusion.
OBJECTIVE: To investigate the effect of exogenous hydrogen peroxide on spleen cell viability and the effect of N-acetyl-L-cytokine on protection of induced oxidative stress in the spleen of metallothionein-Ⅰ/Ⅱ knockout mice.
METHODS: Metallothionein-Ⅰ/Ⅱ knockout mice spleen cell suspension was prepared and treated with various concentrations of hydrogen peroxide (0.1, 0.2, 0.5, 1, 2 mmol/L) for 2 hours, and the cell viability was detected by MTT colorimetric method. Based on the cells and mitochondria level, hydrogen peroxide induced spleen cells were divided into six group: control group, N-acetyl-L-cytokine group, 0.5 mmol/L hydrogen peroxide group, 1 mmol/L hydrogen peroxide group, N-acetyl-L-cytokine+0.5 mmol/L hydrogen peroxide group, N-acetyl-L-cytokine+1 mmol/L hydrogen peroxide group. Then, the cell viability was detected by MTT colorimetric method, lactate dehydrogenate activity were assayed by microplate reader; mitochondrial permeability transition pore was evaluated by ultraviolet spectrophotometer after 2 hours.
RESULTS AND CONCLUSION: With the increase of the concentration of hydrogen peroxide, the spleen cell viability was significantly decreased (P < 0.01), and 0.2, 0.5, 1, 2 mmol/L hydrogen peroxide groups had the greatest reduction. Compared with the control group, N-acetyl-L-cytokine could significantly increase spleen cell viability (P < 0.01), decrease lactate dehydrogenate activity (P < 0.01) and decrease the opening state of mitochondrial permeability transition pore (P < 0.01). Compared with 0.5 mmol/L hydrogen peroxide group and 1 mmol/L hydrogen peroxide group, N-acetyl-L-cytokine+0.5 mmol/L hydrogen peroxide group and N-acetyl-L-cytokine+1 mmol/L hydrogen peroxide group could also increase spleen cell viability (P < 0.01), decrease lactate dehydrogenate activity (P < 0.01) and decrease the opening state of mitochondrial permeability transition pore (P < 0.01), respectively. These findings suggest that with the increase of the concentration of hydrogen peroxide, the spleen cell viability is significantly decreased in a dose-dependent manner, especially for 0.2, 0.5, 1, 2 mmol/L hydrogen peroxide; N-acetyl-L-cysteine can relieve the oxidative stress damage induced by hydrogen peroxide in metallothionein-Ⅰ/Ⅱ knockout mice spleen cells, through reducing the lactate dehydrogenase release and the opening state of mitochondrial permeability transition pore, and increasing spleen cell viability.

Key words: tissue construction, cytology experiments in tissue construction, metallothionein, metallothionein-Ⅰ/Ⅱ knockout mice, spleen, apoptosis, N-acetyl-L-cysteine, hydrogen peroxide, oxidative stress, mitochondrial permeability transition pore, cerebral ischemia/reperfusion, lactate dehydrogenase, the National Natural Science Foundation of China

CLC Number:

R318

He Jing, Wei Si-yu, Liu Feng-yong, Ye Jing, Shen Bing-ling, Yao Xiao-mei. Hydrogen peroxide induced oxidative stress in the spleen of metallothionein Ⅰ/Ⅱ knockout mice[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(7): 1243-1250.

Figures/Tables 4

References

[1] Thirumoorthy N, Shyam SA, Manisenthil Kumar KT, et al. A review of metallothionein isoforms and their role in pathophysiology. World J Surg Oncol. 2011;9:54.

[2] Inoue k, Takano H, Shimada A, et al. Metallothionein as an anti-in?ammatory mediator. Mediators of In?ammation. 2009; 2009:101659.

[3] Yao X，Miao W, Li M, et al. Protective effect of albumin on VEGF and brain edema in acute isehemia in rats. Neurosei Lett. 2010;472(3):179-183．

[4] Wang M, Wang YM, Li HD, et al. Zhonghua Laonian Xinnaoxueguanbing Zazhi. 2011;13(2):164-167. 王敏,王永明,李海东,等.白蛋白治疗对小鼠脑缺血早期脑组织Toll样受体4和骨髓分化因子88 mRNA表达的影响[J].中华老年心脑血管病杂志,2011,13(2):164-167.

[5] Offner H, Vandenbark AA, Hurn PD. Effect of experimental stroke on peripheral immunity: CNS ischemia induces profound immunosuppression. Neuroscience. 2009;158(3): 1098-1111.

[6] Kim CH, Vaziri ND, Rodriguez-Iturbe B. Integrin expression and H2O2 production in circulating and splenic leukocytes of obese rats. Obesity (Silver Spring). 2007; 15(9):2209-2216.

[7] Liu B, Jian Z, Li Q, et al. Baicalein protects Human melanocytes from H2O2-induced apoptosis via inhibiting mitochondria-dependent caspase activation and the p38 MAPK pathway. Free Radic Biol Med. 2012;53(2):183-193.

[8] Li XB, Zhang HC, Guo ZK. Zhongguo Zuzhi Gongcheng Yanjiu. 2012;16(1):22-26. 刘新宾,张红超,郭子宽.骨髓间充质干细胞条件培养液对H2O2损伤大鼠心肌细胞的保护[J].中国组织工程研究, 2012,16(1): 22-26.

[9] Gu DS, Li W, Gu WW. Zhongguo Zuzhi Gongcheng Yanjiu. 2011;15(44):8281-8284. 顾东曙,刘闻,顾为望.静脉输注过氧化氢溶液上调小鼠脾脏和外周血CD4+Foxo3+调节性T细胞[J].中国组织工程研究与临床康复,2011,15(44):8281-8284.

[10] Yang P, Peairs JJ, Tano R, et al. Oxidant-mediated Akt activation in human RPE cells. Invest Ophthalmol Vis Sci. 2006;47(10):4598-606.

[11] Yoshino F, Yoshida A, Okada E, et al. Dental resin curing blue light induced oxidative stress with reactive oxygen. J Photochem Photobiol B. 2012;114:73-78.

[12] Nakamura R, Lnoue K, Fujitani Y, et al. In vitro study of the effect of nanoparticle-rch diesel exhaust particles on IL-18 production in splenocytes. J Toxicol Sci. 2011;36(6):823-827.

[13] Wu Y, Wang D, Wang X, et al. Caspase 3 is activated through caspase 8 instead of caspase 9 during H2O2-induced apoptosis in HeLa cells. Cell Physiol Biochem. 2011;27(5): 539-546.

[14] Ru Y, Yin L, Sun H, et al. A micropreparation of mitochondria from cells using magnetic beads with immounoaf?nity. Anal Biochem. 2012;421(1):219-226.

[15] Wang DM, Dong J, Qi ZT, et al. Xian Tiyu Xueyuan Xuebao. 2011;28(4):471-475. 王冬梅,董军,漆正堂,等.动对骨骼肌线粒体通透性转换孔及细胞凋亡的影响[J].西安体育学院学报,2011,28(4):471-475.

[16] Petronilli V, Penzo D, Scorrano L, et al. The mitochondrial permeability transition, release of cytochrome c and cell death. J Biol Chem. 2001;267(15):12030-12034.

[17] Ajmo CT Jr, Vernon DO, Collier L, et al. The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res. 2008;86(10):2227-2234.

[18] Izci Y. Splenectomy may be a prophylactic treatment for cerebral ischemia? Med Hypotheses. 2010;75(4):347-349.

[19] Yu X, Guo J, Fang H, et al. Basal metallothionein-Ⅰ/Ⅱ protects against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures. Toxicology. 2011;282(1-2):16-22.

[20] Ghanizadeh A. Gold implants and increased expression of metallothionein-Ⅰ/Ⅱ as a novel hypothesized therapeutic approach for autism. Toxicology. 2011; 283(1):63-64.

[21] Hamdi Y, Kaddour H, Vaudry D, et al. The octadecaneuropeptide ODN protects astrocytes against hydrogen peroxide-induced apoptosis via a PKA/MAPK- dependent mechanism. PLoS One. 2012;7(8):e42498.

[22] Baumeister P, Huebner T, Reiter M, et al. Reduction of oxidative DNA fragmentation by ascorbic acid, zinc and N-acetylcysteine in nasal mucosa tissue cultures. Anticancer Res. 2009;29(11):4571-4574.

[23] Khouri R, Novais F, Santana G, et al. DETC induces Leishmania parasite killing in human in vitro and murine in vivo models: a promising therapeutic alternative in Leishmaniasis. PLoS One. 2010;5(12):e14394.

[24] Xie M, Roy R. Increased levels of hydrogen peroxide induce a HIF-1-dependent modi?cation of lipid metabolism in AMPK compromised C. elegans Dauer Larvae. Cell Metab. 2012; 16(3):322-335.

[25] Wu JP, Chen HC, Li MH. Bioaccumulation and toxicodynamics of cadmiumt of reshwater planarian and the protective effect of N-Acetylcysteine. Arch Environ Contam Toxicol. 2012;63(2):220-229.

[26] Yao X, Li M, He J, et al. Effect of early acute high concentrations of iodide exposure on mitochondrial superoxide production in FRTL cells. Free Radic Biol Med. 2012; 52(8):1343-1352.

[27] Li M, Li LY, Chen ZP, et al. Tianjin Yiyao. 2010;38(3): 212-215. 李敏,李兰英,陈组培,等.过氧化氢对FRTL细胞线粒体膜电位和超氧化物生成的影响[J].天津医药,2010,38(3):212-215.

[28] Kinnally KW, Peixoto PM, Ryu SY, et al. Is mPTP the gate keeper for necrosis, apoptosis, or both? Biochim Biophys Acta. 2011;1813(4):616-622.

[29] Drahota Z, Milerova M, Endlicher R, et al. Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress. Physiol Res. 2012;61(Suppl 1):S165-172.

[30] Loor G, Kondapalli J, Lwase H, et al. Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion. Biochim Biophys Acta. 2011;1813(7):1382-1394.

[31] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.