D-柠檬烯对胶质母细胞瘤细胞增殖的抑制作用及其机制

doi:10.13481/j.1671-587X.20240308

摘要/Abstract

摘要：

目的探讨D-柠檬烯对胶质母细胞瘤（GBM）细胞增殖和凋亡的影响，并阐明其可能的作用机制。方法将GBM细胞分为对照组（0 mmol·L^－1 D-柠檬烯）和0.2、 0.4、 0.6、 0.8及1.0 mmol·L^－1 D-柠檬烯组。采用CCK-8法检测各组细胞增殖抑制率，克隆形成法检测各组细胞克隆形成率，Annexin Ⅴ-FITC/PI法检测各组细胞凋亡率，Western blotting法检测各组细胞中蛋白激酶B（AKT）、B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）和多聚二磷酸腺苷（ADP）核糖聚合酶（PARP）蛋白表达水平，免疫荧光法检测各组细胞中裂解的半胱氨酸天冬氨酸蛋白酶（cleaved Caspase）-3 蛋白表达水平。15 只建立皮下移植瘤模型小鼠随机分为空白组（0 mg·kg^－1·d^－1 D-柠檬烯）、低剂量D-柠檬烯组（200 mg·kg^－1·d^－1D-柠檬烯）和高剂量D-柠檬烯组（400 mg·kg^－1·d^－1D-柠檬烯），每组5只。计算各组小鼠体内肿瘤抑制率，HE染色和免疫组织化学染色观察各组小鼠皮下肿瘤组织形态表现，并绘制肿瘤生长曲线，免疫组织化学法检测各组小鼠皮下瘤组织中Ki67蛋白阳性表达率，TUNEL染色法检测各组小鼠肿瘤细胞凋亡情况。结果对照组细胞呈长梭形，状态良好，紧密且贴壁生长，细胞器和细胞质正常；给药48 h后，0.6 mmol·L^－1 D-柠檬烯组细胞体积减小，细胞膜虽完整但通透性增强，细胞质皱缩，细胞内部产生空泡结构，部分呈碎片状漂浮在溶液表面；0.8 和1.0 mmol·L^－1 D-柠檬烯组细胞中有明显凋亡小体生成，呈现凋亡状态。CCK-8法，与对照组比较，0.6、0.8和1.0 mmol·L^－1D-柠檬烯组U87、LN229及GL261细胞增殖抑制率均明显升高（P<0.01），0.4 mmol·L^－1D-柠檬烯组U87和GL261细胞增殖抑制率均明显升高（P<0.01）。克隆形成法，与对照组比较，0.4、0.6和0.8 mmol·L^－1D-柠檬烯组U87、LN229及GL261细胞克隆形成率均明显降低（P<0.05或P<0.01）。Annexin Ⅴ-FITC/PI法，与对照组比较，D-柠檬烯处理48 h后，0.6、0.8和1.0 mmol·L^－1 D-柠檬烯组LN229细胞凋亡率明显升高（P<0.01）。Western blotting法，与对照组比较，0.6、0.8和1.0 mmol·L^－1D-柠檬烯组LN229细胞中Bax蛋白表达水平均明显升高（P<0.01），AKT和Bcl-2蛋白表达水平均明显降低（P<0.01）；0.8和1.0 mmol·L^－1D-柠檬烯组LN229细胞中PARP蛋白表达水平均明显升高（P<0.01）。免疫荧光法，与对照组比较，0.6、0.8和1.0 mmol·L^－1D-柠檬烯组LN229细胞中cleaved Caspase-3蛋白表达水平均明显升高（P<0.01）。与空白组比较，低和高剂量D-柠檬烯组小鼠肿瘤体积均明显减小（P<0.01）。与空白组比较，低和高剂量D-柠檬烯组小鼠肿瘤质量均明显降低（P<0.05），肿瘤抑制率均明显升高（P<0.05）。空白组小鼠肿瘤细胞弥漫分布，细胞核染色加深，核浆比增大；低和高剂量D-柠檬烯组小鼠肿瘤组织出现大量肿瘤细胞变性坏死。与空白组比较，低和高剂量D-柠檬烯组小鼠肿瘤组织中Ki67蛋白阳性表达率均明显降低（P<0.01）。与空白组比较，低和高剂量D-柠檬烯组小鼠肿瘤细胞凋亡率均明显升高（P<0.01）。结论 D-柠檬烯具有抑制GBM细胞增殖的作用，其作用机制可能与调控AKT蛋白的表达并激活Caspase-3通路诱导凋亡有关。

关键词: D-柠檬烯, 胶质母细胞瘤, 细胞凋亡, 移植瘤, 蛋白激酶B

Abstract:

Objective To discuss the effect of D-limonene on the proliferation and apoptosis of the glioblastoma （GBM） cells，and to clarify its possible mechanism. Methods The GBM cells were divided into control group （0 mmol·L^-1 D-limonene） and 0.2， 0.4， 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups. CCK-8 method was used to detect the inhibitory rates of proliferation of the cells in various groups； clone formation assay was used to detect the clone formation rates of the cells in various groups； Annexin Ⅴ-FITC/PI method was used to detect the apoptotic rates of the cells in various groups； Western blotting method was used to detect the expression levels of protein kinase B （AKT）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and poly adenosine diphosphate（ADP）-ribose polymerase （PARP） proteins in the cells in various groups； imunofluorescence method was used to detect the expression levels of cleaved Caspase-3 protein in the cells in various groups. Fifteen model mice with subcutaneous tumor xenografts were randomly divided into blank group （0 mg·kg^-1·d^-1 D-limonene）， low dose of D-limonene group （200 mg·kg^-1·d^-1 D-limonene）， and high dose of D-limonene group （400 mg·kg^-1·d^-1 D-limonene）， and there were 5 mice in each group.The inhibitory rates of the tumor in vitro in various groups were calculated； HE staining and immunohistochemical staining were used to observe the morphology of subcutaneous tumor tissue of the mice in various groups and the growth curves of the tumor were drawn； immunohistochemical assay was used to detect the positive expression rates of Ki67 protein in subcutaneous tumor tissue of the mice in various groups； TUNEL staining was used to detect the apoptosis of the tumor cells in various groups. Results In control group， the cells were spindle-shaped， in good condition， growing closely and adherently， with normal organelles and cytoplasm. After treated for 48 h， the cells in 0.6 mmol·L^-1 D-limonene group showed reduced volume， intact but more permeable cell membranes， shrunken cytoplasm， internal vacuole structures， and some fragments floating in the solution. The cells in 0.8 and 1.0 mmol·L^-1 D-limonene groups exhibited significant apoptotic bodies and were in an apoptotic state. The CCK-8 results showed that compared with control group， the inhibitory rates of proliferation of the U87， LN229， and GL261 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）， the inhibitory rates of proliferation of the U87 and GL261 cells were significantly increased （P<0.01）. The clone formation assay results showed that compared with control group， the clone formation rates of the U87， LN229， and GL261 cells in 0.4， 0.6， and 0.8 mmol·L^-1 D-limonene groups were significantly decreased （P<0.05 or P<0.01）. The Annexin Ⅴ-FITC/PI results showed that compared with control group， after treated with D-limonene for 48 h， the apoptotic rates of the LN229 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of Bax proteins in the LN229 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）， while the expression levels of AKT and Bcl-2 proteins were significantly decreased （P<0.01）， the expression level of PARP protein in the LN229 cells in 0.8 and 1.0 mmol·L^-1 D-limonene group was significanthy increased（P<0.01）.The immunofluorescence results showed that compared with control group， the expression levels of cleaved Caspase-3 protein in the LN229 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）. Compared with blank group， the tumor volumes of the mice in low and high doses of D-limonene groups were significantly decreased （P<0.01）. Compared with blank group， the tumor weights of the mice in low and high doses of D-limonene groups were significantly decreased （P<0.05）， and the inhitory rates of tumor were significantly increased （P<0.05）. The tumor cells in blank group were diffusely distributed， with deepened nuclear staining and increased nucleocytoplasmic ratio； a large number of degenerated and necrotic tumor cells were observed in tumor tissue of the mice in low and high doses of D-limonene groups. Compared with blank group， the positive expression rates of Ki67 protein in tumor tissue of the mice in low and high doses of D-limonene groups were significantly decreased （P<0.01）. Compared with blank group， the apoptotic rates of tumor cells of the mice in low and high doses of D-limonene groups were significantly increased （P<0.01）. Conclusion D-limonene has the inhibitory effect on the proliferation of the GBM cells； its mechanism may be related to the regulation of AKT protein expression and the activation of the Caspase-3 pathway to induce the apoptosis.

Key words: D-limonene, Glioblastoma, Apoptosis, Transplantation tumor, Protein kinase B

中图分类号:

R739.4

王腾飞, 陈凤, 齐玲, 雷婷, 宋美慧. D-柠檬烯对胶质母细胞瘤细胞增殖的抑制作用及其机制[J]. 吉林大学学报(医学版), 2024, 50(3): 647-657.

Tengfei WANG, Feng CHEN, Ling QI, Ting LEI, Meihui SONG. Inhibitory effect of D-limonene on proliferation of glioblastoma cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2024, 50(3): 647-657.

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参考文献 21

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Group	Inhibitory rate of proliferation
Group	LN229 cells	U87 cells	GL261 cells
Control	0	0	0
0.2 mmol·L^－1 D-linonene	8.77±2.36	13.30±1.89	5.18±0.89
0.4 mmol·L^－1 D-linonene	7.10±5.79	36.15±4.11^*	12.02±4.55^*
0.6 mmol·L^－1 D-linonene	23.80±2.20^*	52.43±10.06^*	43.91±7.31^*
0.8 mmol·L^－1 D-linonene	29.24±2.63^*	49.91±4.45^*	52.68±1.95^*
1.0 mmol·L^－1 D-linonene	64.80±0.49^*	60.69±3.15^*	61.12±7.51^*

Group	Clone formation rate
Group	LN229 cells	U87 cells	GL261 cells
Control	100.00±5.99	100.00±3.96	100.00±12.63
0.4 mmol·L^－1 D-linonene	64.23±8.64^**	71.62±9.52^*	67.05±5.62^**
0.6 mmol·L^－1 D-linonene	57.93±6.79^**	58.85±7.78^**	49.43±3.68^**
0.8 mmol·L^－1 D-linonene	21.41±4.33^**	47.21±8.22^**	42.05±3.50^**

Group	Tumor weight (m/g)	Inhibitory rate of tumor (η/%)
Blank	0.45±0.23	0
Low dose of D-limonene	0.16±0.07^*	65.52±15.51^*
High dose of D-limonene	0.16±0.08^*	67.46±17.57^*

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