Inhibition of high mobility group box 1/nuclear factor-kappa B pathway reduces apoptosis in spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation

doi:10.3969/j.issn.2095-4344.1811

Abstract

Abstract:

BACKGROUND: Spinal cord injuries triggers the release of high mobility group box 1 (HMGB1) from nerve cells to activate the nuclear factor-kappa B (NF-κB) by binding to cell membrane surface receptor, thereby inducing a series of pathological reactions, such as spinal cord edema or inflammation. However, little is reported on whether inhibition of HMGB1/NF-κB pathway could attenuate spinal cord astrocytes apoptosis after oxygen-glucose deprivation/reoxygenation (OGD/R).
OBJECTIVE: To investigate the effect of HMGB1/NF-κB pathway on spinal cord astrocytes apoptosis after OGD/R.
METHODS: Spinal cord astrocytes of newborn Sprague-Dawley rats (provided by the Laboratory Animal Center of Shanxi Medical University, China) were cultured in vitro and an OGD/R model was established. Spinal cord astrocytes were subjected to reoxygenation 6, 12, and 24 hours. Release of HMGB1 in the culture medium was detected by ELISA. The expression of HMGB1, Bcl-2 and Bax were detected by western blot and the cell survival rate was determined by MTT. The optimal reoxygenation time was then selected for the following experiments. Spinal cord astrocytes at passage 4 were divided into normal group, OGD6h/R24h group, OGD6h/R24h+ethyl pyruvate group, OGD6h/R24h+Bay 11-7082 group. After 24 hours of reoxygenation, the release of HMGB1 was detected by ELISA, the expression of HMGB1, NF-κB, Bcl-2 and BAX was analyzed by western blot, the survival rate of astrocytes was determined by MTT, and the apoptosis of astrocytes was measured by flow cytometry.
RESULTS AND CONCLUSION: (1) The results showed that the best reoxygenation time was 24 hours, which was used for subsequent experiments. (2) Compared with the normal group, the release and expression of HMGB1 and NF-κB as well as the apoptotic rate of astrocytes were obviously increased in the OGD6h/R24h group (P < 0.05), while the survival rate of astrocytes and the ratio of Bcl-2/Bax were obviously declined in the OGD6h/R24h group (P < 0.05). Compared with the OGD6h/R24h group, the astrocyte survival rate and the ratio of Bcl-2/Bax were significantly raised (P < 0.05), while the expression of NF-κB and the apoptosis rate of astrocytes were remarkably decreased (P < 0.05) in the OGD6h/R24h+ethyl pyruvate and OGD6h/R24h+Bay 11-7082 groups. To conclude, the HMGB1/NF-κB signal pathway is involved in the regulation of apoptosis in spinal cord astrocytes after OGD/R.

Key words: spinal cord injury, astrocytes, high mobility group box B1, nuclear factor kappa B, apoptosis, oxygen-glucose deprivation/reoxygenation, Bcl-2, Bax

CLC Number:

Lü Cong, Sun Lin, Feng Haoyu, Ma Xun, He Yajun, Li Jisheng. Inhibition of high mobility group box 1/nuclear factor-kappa B pathway reduces apoptosis in spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5353-5359.

Figures/Tables 1

2.1 星形胶质细胞鉴定结果取第3代细胞，用星形胶质细胞特定表达蛋白S100β对细胞进行免疫荧光染色，染色结果为星形胶质细胞纯度为95%，见图1。 2.2 氧糖剥夺/复氧后星形胶质细胞HMGB1的表达与释放及细胞活性与凋亡程度 HMGB1表达与释放：Western blot与ELISA检测显示与正常组相比，氧糖剥夺6 h/复氧6，12，24 h组HMGB1蛋白表达与释放明显增多，且氧糖剥夺6 h/复氧24 h组中HMGB1表达与释放最多的(P < 0.05)，见图2A，B。凋亡蛋白表达：与正常组相比，氧糖剥夺6 h/复氧6，12，24 h组Bcl-2与BAX表达均增多，但Bcl-2随复氧时间增长表达逐渐减少，BAX表达随复氧时间延长表达增加，因此Bcl-2/BAX比值随时间增长降低，在复氧24 h时Bcl-2/BAX比值达到最低(P < 0.05)，见图2C。细胞存活率：与正常组相比，氧糖剥夺6 h/复氧，12，24 h组星形胶质细胞活性随时间的增加减弱，复氧24 h时脊髓星形胶质细胞存活率最低(P < 0.05)，见图2D。因此选择复氧24 h作为后续实验的时间点。 2.3 抑制HMGB1与核转录因子κB表达后氧糖剥夺6 h/复氧24 h星形胶质细胞的凋亡程度与成活率 HMGB1表达与释放：Western blot及ELISA结果显示与氧糖剥夺6 h/复氧24 h组比，氧糖剥夺6 h/复氧24 h+丙酮酸乙酯组星形胶质细胞内HMGB1的表达与释放减少(P < 0.05)，见图3A，B。核转录因子κB蛋白表达：Western blot结果显示与氧糖剥夺6 h/复氧24 h组比较，氧糖剥夺6 h/复氧24 h+Bay 11-7082组核转录因子κB表达明显减少(P < 0.05)，见图3C。凋亡蛋白表达：Western blot结果显示与氧糖剥夺6 h/复氧24 h组比较，氧糖剥夺6 h/复氧24 h+丙酮酸乙酯组与氧糖剥夺6 h/复氧24 h+Bay 11-7082组Bcl-2/BAX比值明显升高(P < 0.05)，见图3D。细胞存活率：MTT结果表明与氧糖剥夺6 h/复氧24 h组比较，氧糖剥夺6 h/复氧24 h+丙酮酸乙酯组与氧糖剥夺6 h/复氧24 h+Bay 11-7082组星形胶质细胞活性明显增强(P < 0.05)，见图3E。细胞凋亡：与氧糖剥夺6 h/复氧24 h组相比，氧糖剥夺6 h/复氧24 h+丙酮酸乙酯组与氧糖剥夺6 h/复氧24 h+ Bay 11-7082组星形胶质细胞凋亡率明显减少(P < 0.05)，见图3F。丙酮酸乙酯有效抑制HMGB1的表达，Bay 11-7082有效抑制了核转录因子κB的表达，两者分别降低了大鼠脊髓星形胶质细胞的凋亡程度，提高了星形胶质细胞的成活率。"

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