Inhibition of high mobility group protein attenuates spinal astrocyte injury in rats after oxygen glucose deprivation/reoxygenation

doi:10.3969/j.issn.2095-4344.0836

Abstract

Abstract:

BACKGROUND: Many investigations focus on the inhibition of high mobility group protein (HMGB1) for improving the functional recovery of spinal cord in mice, but how to culture spinal cord astrocytes in vitro and inhibit the expression of HMGB1 for attenuating oxygen glucose deprivation/reoxygenation (OGD/R) injury is rarely reported.
OBJECTIVE: To investigate the effect of HMGB1 inhibition on spinal cord astrocytes after OGD/R in vitro.
METHODS: Astrocytes were isolated from rat spinal cord, and were then cultured and identified. (1) The rat primary spinal cord astrocytes were subjected to 6-hour OGD, followed by 6-, 12-, and 24-hour reoxygenation, respectively. The rat primary spinal cord astrocytes cultured in the normal medium were used as controls. (2) The rat spinal cord astrocytes were divided into five groups: control group (normal medium); 6-hour OGD/24-hour R group; 6-hour OGD/24-hour R plus HMGB1 shRNA group; 6-hour OGD/24-hour R plus non-targeting shRNA group; 6-hour OGD/24-hour R plus ethyl pyruvate group. The expression and release of HMGB1 in astrocytes were determined by western blot assay and ELISA. The cell injury and survival rate were assessed by lactate dehydrogenase and MTT assay. The morphological changes of the cells were observed under light microscope.
RESULTS AND CONCLUSION: The expression and release of HMGB1 protein was increased after OGD/R (P < 0.01), and reached the highest value at reoxygenation for 24 hours (P < 0.01). The expression level of HMGB1 in the HMGB1 shRNA and ethyl pyruvate groups was signficnatly decreased, suggesting that specific RNA interference and ethyl pyruvate could effectively inhibit the expression of HMGB1. The leakage rate of lactate dehydrogenase was increased and cell survival rate was decreased after OGD/R, and cell injury was the most serious at reoxygenation for 24 hours (P < 0.01). Compared with the 6-hour OGD/24-hour R group, the leakage rate of lactate dehydrogenase in the HMGB1 shRNA and ethyl pyruvate groups was significantly reduced. There were cell swelling, decomposition and other damage changes after OGD/R, while HMGB1 shRNA and ethyl pyruvate RNA could significantly reduce cell injury. Our findings imply that HMGB1 plays an important role in the occurrence and development of spinal cord astrocyte injury, and inhibition of HMGB1 can alleviate spinal cord astrocyte injury in rats after OGD/R.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Spinal Cord Injuries, High Mobility Group Proteins, Tissue Engineering

CLC Number:

R318

Song Jun-lai1, Li Man2, Sun Lin1, Ma Xun1, Lü Cong1, He Ya-jun1. Inhibition of high mobility group protein attenuates spinal astrocyte injury in rats after oxygen glucose deprivation/reoxygenation [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(28): 4537-4543.

Figures/Tables 2

2.1 星形胶质细胞的培养与鉴定在光学显微镜下观察成熟的脊髓星形胶质细胞，细胞成宽大扁平状，形状多不规则，有多个突起，互相连接，细胞质丰富、核大质浅，偶有小胶质细胞等杂细胞，采用免疫荧光的化学方法，S100β染色阳性可鉴定其为星形胶质细胞，细胞纯度达95%以上，图1。 2.2 Western blot检测星形胶质细胞HMGB1的表达与对照组(0.20±0.02)相比，细胞氧糖剥夺6 h/复氧6 h (0.38±0.03)后HMGB1的表达显著升高(P < 0.01)，复氧培养至12 h时(0.38±0.03)，继续升高，培养至24 h时(0.61±0.03)，达到最高值(P < 0.01)。与氧糖剥夺6 h/复氧24 h组相比，特异性RNA干扰组(0.15±0.02)及丙酮酸乙酯组(0.29±0.02)HMGB1的表达量明显降低，说明特异性的HMGB1 shRNA及丙酮酸乙酯能抑制细胞中HMGB1表达，见图2-4。 2.3 ELISA检测细胞上清中HMGB1的质量浓度与对照组相比，细胞氧糖剥夺/复氧后细胞上清中的HMGB1的质量浓度显著升高(P < 0.01)，并随着复氧时间延长，其质量浓度逐渐升高，复氧至24 h时，达到最高值(P < 0.01)。与氧糖剥夺6 h/复氧24 h组相比，特异性RNA干扰组及丙酮酸乙酯组HMGB1的质量浓度明显降低，说明特异性的HMGB1 shRNA和丙酮酸乙酯能明显抑制细胞中HMGB1的表达和释放，见表1。 2.4 星形胶质细胞氧糖剥夺/复氧后乳酸脱氢酶漏出率的变化与对照组细胞相比，细胞氧糖剥夺/复氧后，乳酸脱氢酶漏出率均明显增加，并且随着复氧时间延长乳酸脱氢酶漏出率逐渐升高，复氧培养至24 h时达到最高值。与氧糖剥夺6 h/复氧24 h组相比，特异性RNA干扰组及丙酮酸乙酯组细胞的乳酸脱氢酶漏出率明显下降，而非特异性RNA干扰组乳酸脱氢酶漏出率无明显差异，图5。 2.5 星形胶质细胞氧糖剥夺/复氧后细胞存活率的变化与对照组细胞相比，细胞氧糖剥夺/复氧后，细胞存活率明显降低，并且随着复氧时间延长细胞存活率逐渐下降，复氧培养至24 h时细胞存活率达到最低(P < 0.01)，与与氧糖剥夺6 h/复氧24 h组相比，特异性RNA干扰组及丙酮酸乙酯细胞的存活率明显升高(P < 0.01)，而非特异性RNA干扰组存活率无明显差异，图6。 2.6 光镜下观察氧糖剥夺/复氧后的星形胶质细胞显微镜下观察，对照组细胞生长状态良好，胞浆丰富饱满，形态多样，呈多边形，扁平状，连接较为紧密。星形胶质细胞经氧糖剥夺6 h复氧24 h后，出现边缘皱缩，折光性增高，胞体略增大，胞内形成少量的颗粒样物质，突起增粗缩短，少量细胞呈凝固性坏死。特异RNA干扰及丙酮酸乙酯组可见细胞肿胀明显减轻，胞内颗粒样物质明显减少，所以从微观角度看抑制掉HMGB1能改善星形胶质细胞氧糖剥夺/复氧后损伤的效果，见图7。"

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