Luteolin improves myocardial cell death induced by serum from rats with spinal cord injury

doi:10.12307/2024.729

Abstract

Abstract:

BACKGROUND: Cardiac dysfunction due to spinal cord injury is an important factor of death in patients with spinal cord injury; however, the specific mechanism is still not clear. Therefore, revealing the mechanism of cardiac dysfunction in spinal cord injury patients is of great significance to improve their quality of life and survival rate.

OBJECTIVE: To investigate the mechanism of luteolin in improving serum-induced myocardial cell death in spinal cord injury rats.

METHODS: Allen’s impact instrument was used to damage the spine T9-T11 of male SD rats to establish a spinal cord injury model meanwhile a sham operation group was set as the control group. The serum of rats of each group was collected. H9c2 cells were divided into a blank control group, a sham operated rat serum group, a spinal cord injury rat serum group and a luteolin pretreatment group. The cells in blank control group were only cultured with ordinary culture medium. The cells in the sham operated rat serum group were treated with medium containing 10% serum from sham operated rat. The cells in the spinal cord injury rat serum group were treated with medium containing 10% serum from spinal cord injury rat. The cells in the luteolin pretreatment group were precultured with a final concentration of 20 μmol/L luteolin for 4 hours and then changed to a medium containing 10% rat serum from spinal cord injury rat. After 24 hours of culture, the survival rate of each group of H9c2 cells was measured by CCK-8 assay. Western blot assay was used to detect the expression of autophagy related protein LC3 and p62 in H9c2 cells in each group.

RESULTS AND CONCLUSION: Compared with the blank control group, there was no significant change in cell survival rate in the sham operated rat serum group (P > 0.05). Compared with the sham operated rat serum group, the cell survival rate (P < 0.01) and the expression of LC3 protein (P < 0.05) in spinal cord injury rat serum group was significantly reduced, and the expression of p62 protein was significantly increased (P < 0.05). Compared with the spinal cord injury rat serum group, the survival rate of cells in the luteolin pretreatment group significantly increased (P < 0.000 1); the expression of LC3 protein significantly increased (P < 0.05), and the expression of p62 protein significantly decreased (P < 0.05). The results indicate that luteolin may improve myocardial cell death induced by serum from rats with spinal cord injury by promoting autophagy.

Key words: spinal cord injury, serum, H9c2 cell, autophagy, luteolin, cardiac dysfunction

CLC Number:

Zhang Wenwen , Xu Mengru , Tian Yuan , Zhang Lifei , Shi Shu , Wang Ning , Yuan Yuan , Wang Li , Hao Haihu. Luteolin improves myocardial cell death induced by serum from rats with spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(1): 38-43.

Figures/Tables 1

2.1 大鼠后肢功能 BBB 评分 5 名实验人员分别在脊髓损伤建模成功后 12，24，72 h 对大鼠后肢运动功能进行盲法 BBB 评分，见图 1。 2.2 脊髓损伤 72 h 后大鼠心肌细胞的自噬受到抑制为了明确大鼠脊髓损伤后心肌细胞自噬的变化情况，在大鼠脊髓损伤 12，24，72 h 取心肌组织，采用 PCR 检测 LC3 mRNA 的表达水平。与假手术组相比，脊髓损伤大鼠心肌组织中的 LC3 mRNA 表达降低，且在脊髓损伤 24，72 h 降低显著 (P < 0.05)。进一步采用 Western blot 检测自噬相关蛋白的表达，与假手术组相比，脊髓损伤大鼠心肌组织中自噬标志蛋白 LC3 的表达降低，自噬底物 p62 的表达升高。以上结果表明，脊髓损伤后大鼠心肌细胞的自噬受到抑制，且在脊髓损伤 72 h 后自噬水平降低最为显著 (P < 0.05)，见图 2。 2.3 脊髓损伤大鼠血清可降低 H9c2 心肌细胞的存活率为了检测脊髓损伤大鼠血清对心肌细胞的存活率是否有影响，使用含体积分数 10% 脊髓损伤大鼠血清的培养基处理 H9c2 心肌细胞 12，24，36，48 h，利用 CCK-8 法检测细胞存活率，以假手术大鼠血清处理细胞作为对照。如图 3 所示，在脊髓损伤大鼠血清处理 H9c2 细胞 24 h 时，细胞存活率较对照组显著降低 (P < 0.000 1)。因此采用 24 h 的处理时间进行后续实验。 2.4 确定木犀草素处理H9c2心肌细胞的安全浓度为了确定木犀草素处理 H9c2 心肌细胞的最适浓度，分别使用 10- 100 μmol/L 木犀草素处理细胞 24 h，采用 CCK-8 检测细胞存活率。如图 4 所示，与空白对照组相比，10，20 μmol/L 木犀草素对细胞存活率无明显影响 (P > 0.05)，而用 30， 50，100 μmol/L 木犀草素处理 H9c2 细胞后，细胞存活率显著降低 (P < 0.05)。因此采用 20 μmol/L 木犀草素进行后续实验。 2.5 木犀草素可改善脊髓损伤大鼠血清诱导的心肌细胞存活率与空白对照组相比，使用假手术大鼠血清处理细胞后，细胞存活率无明显改变；使用脊髓损伤大鼠血清处理细胞后，细胞存活率显著降低 (P < 0.01)；进一步使用木犀草素预处理 H9c2 细胞 4 h，再用脊髓损伤大鼠血清处理细胞 24 h，细胞存活率明显回升 (P < 0.000 1)，见图 5。以上结果提示木犀草素可改善脊髓损伤大鼠血清对心肌细胞造成的损伤。 2.6 木犀草素可改善脊髓损伤大鼠血清诱导的心肌细胞自噬抑制如图 6 所示，与假手术大鼠血清处理细胞相比，脊髓损伤大鼠血清处理的 H9c2 细胞 LC3 蛋白表达显著降低 (P < 0.05)，p62 蛋白表达显著升高 (P < 0.05)。进一步使用 20 μmol/L 木犀草素对 H9c2 细胞预处理 4 h，再使用脊髓损伤大鼠血清处理细胞 24 h，H9c2 细胞 LC3 蛋白表达升高 (P < 0.05)，p62 蛋白表达降低 (P < 0.05)。以上结果表明木犀草素可以改善脊髓损伤大鼠血清诱导的心肌细胞自噬抑制。 "

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