南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (11): 2340-2349.doi: 10.12122/j.issn.1673-4254.2025.11.06
收稿日期:2025-04-23
出版日期:2025-11-20
发布日期:2025-11-28
通讯作者:
刘柳青
E-mail:liuliuqing@ahtcm.edu.cn
作者简介:刘柳青,博士,讲师,E-mail: liuliuqing@ahtcm.edu.cn
基金资助:
Liuqing LIU1(
), Kun WANG1, Xueqing WANG2, Bingxin DU3
Received:2025-04-23
Online:2025-11-20
Published:2025-11-28
Contact:
Liuqing LIU
E-mail:liuliuqing@ahtcm.edu.cn
摘要:
目的 研究枸杞多糖(LBP)对顺铂损伤的卵巢颗粒细胞的保护作用及其潜在机制。 方法 采用顺铂(2.5 µg/mL,24 h)诱导人颗粒细胞样肿瘤细胞系(KGN)建立KGN损伤模型,采用不同浓度LBP(100、500、1000 mg/L)干预受损KGN,各组细胞分别用细胞计数试剂盒-8检测细胞活力,流式细胞术检测凋亡率,ELISA法检测抗苗勒管激素(AMH)水平,透射电镜观察超微结构,Western blotting检测凋亡相关蛋白(Bax、caspase-3、Bcl-2)及PI3K/AKT信号通路的表达情况,RT-qPCR法检测微小RNA(miR)-23a表达情况。通过慢病毒转染构建miR-23a过表达/敲低模型,验证LBP的作用机制。 结果 顺铂抑制KGN活力(P<0.01),诱导凋亡(P<0.01),降低AMH水平(P<0.01),造成染色质凝聚、细胞核固缩、细胞质空泡化等超微结构异常,并增加促凋亡基因Bax、caspase-3的表达(P<0.01),减少抑凋亡基因Bcl-2的表达(P<0.05),下调PI3K/AKT信号通路的表达与活化(P<0.01),上调miR-23a的含量(P<0.01)。LBP干预可不同程度地逆转上述损伤,中剂量LBP作用24 h可改善KGN细胞活力、凋亡率、内分泌功能和超微结构(P<0.01)。中剂量LBP通过下调miR-23a(P<0.01)激活PI3K/AKT通路(P<0.01),抑制Bax、caspase-3(P<0.01),上调Bcl-2(P<0.05)。miR-23a过表达削弱LBP的保护作用,而敲低miR-23a则增强其疗效(P<0.01)。 结论 LBP通过抑制miR-23a表达,激活PI3K/AKT通路,抑制顺铂诱导的KGN凋亡,为保护卵巢功能提供了潜在治疗策略。
刘柳青, 王坤, 王雪晴, 杜冰心. 枸杞多糖通过下调miR-23a减轻顺铂诱导的颗粒细胞损伤[J]. 南方医科大学学报, 2025, 45(11): 2340-2349.
Liuqing LIU, Kun WANG, Xueqing WANG, Bingxin DU. Lycium barbarum polysaccharides alleviates cisplatin-induced granulosa cell injury by downregulating miR-23a[J]. Journal of Southern Medical University, 2025, 45(11): 2340-2349.
| Group | CDDP exposure duration(h) | |||
|---|---|---|---|---|
| 0 | 12 | 24 | 48 | |
| NC | 1.00±0.00 | 1.00±0.00 | 1.00±0.00 | 1.00±0.00 |
| CDDP 1.25 | 0.97±0.15 | 0.87±0.10 | 0.65±0.07a | 0.61±0.07a |
| CDDP 2.5 | 0.98±0.14 | 0.77±0.09b | 0.50±0.03a | 0.41±0.04a |
| CDDP 5 | 0.94±0.12 | 0.65±0.08a | 0.42±0.03a | 0.28±0.04a |
| CDDP 10 | 0.97±0.10 | 0.55±0.10a | 0.32±0.03a | 0.25±0.06a |
表1 顺铂处理0 、12 、24 、48 h后各组细胞活力
Tab.1 KGN cell viability after cisplatin treatment at different concentrations for 0, 12, 24, and 48 h (n=6, Mean±SD)
| Group | CDDP exposure duration(h) | |||
|---|---|---|---|---|
| 0 | 12 | 24 | 48 | |
| NC | 1.00±0.00 | 1.00±0.00 | 1.00±0.00 | 1.00±0.00 |
| CDDP 1.25 | 0.97±0.15 | 0.87±0.10 | 0.65±0.07a | 0.61±0.07a |
| CDDP 2.5 | 0.98±0.14 | 0.77±0.09b | 0.50±0.03a | 0.41±0.04a |
| CDDP 5 | 0.94±0.12 | 0.65±0.08a | 0.42±0.03a | 0.28±0.04a |
| CDDP 10 | 0.97±0.10 | 0.55±0.10a | 0.32±0.03a | 0.25±0.06a |
| Group | LBP exposure duration (h) | ||
|---|---|---|---|
| 12 | 24 | 48 | |
| NC | 1.00±0.00 | 1.00±0.00 | 1.00±0.00 |
| MC | 0.71±0.04a | 0.59±0.06a | 0.52±0.07a |
| LBP-L | 0.72±0.04 | 0.74±0.03c | 0.58±0.07e |
| LBP-M | 0.80±0.09 | 0.85±0.06c | 0.74±0.04c |
| LBP-H | 0.76±0.11 | 0.70±0.06f | 0.65±0.04df |
表2 LBP处理12、24、48 h后各组细胞活力
Tab.2 KGN cell viability after LBP treatment for 12, 24, and 48 h (n=6, Mean±SD)
| Group | LBP exposure duration (h) | ||
|---|---|---|---|
| 12 | 24 | 48 | |
| NC | 1.00±0.00 | 1.00±0.00 | 1.00±0.00 |
| MC | 0.71±0.04a | 0.59±0.06a | 0.52±0.07a |
| LBP-L | 0.72±0.04 | 0.74±0.03c | 0.58±0.07e |
| LBP-M | 0.80±0.09 | 0.85±0.06c | 0.74±0.04c |
| LBP-H | 0.76±0.11 | 0.70±0.06f | 0.65±0.04df |
| Group | Apoptosis rate (%) |
|---|---|
| NC | 5.27±0.22 |
| MC | 45.70±1.14a |
| LBP-L | 29.29±0.52ce |
| LBP-M | 16.55±0.02c |
| LBP-H | 27.82±0.28ce |
表3 各组KGN凋亡率
Tab.3 Apoptosis rate of KGN cells in different groups (n=3, Mean±SD)
| Group | Apoptosis rate (%) |
|---|---|
| NC | 5.27±0.22 |
| MC | 45.70±1.14a |
| LBP-L | 29.29±0.52ce |
| LBP-M | 16.55±0.02c |
| LBP-H | 27.82±0.28ce |
| Group | AMH (pg/mL) |
|---|---|
| NC | 370.05±14.23 |
| MC | 45.99±2.23a |
| LBP-L | 155.49±9.19ce |
| LBP-M | 209.59±11.30c |
| LBP-H | 182.18±6.44ce |
表4 各组AMH水平
Tab.4 AMH levels in KGN cell in each group (n=6, Mean±SD)
| Group | AMH (pg/mL) |
|---|---|
| NC | 370.05±14.23 |
| MC | 45.99±2.23a |
| LBP-L | 155.49±9.19ce |
| LBP-M | 209.59±11.30c |
| LBP-H | 182.18±6.44ce |
| Group | Bax | Caspase-3 | Bcl-2 |
|---|---|---|---|
| NC | 0.21±0.01 | 0.14±0.00 | 0.42±0.04 |
| MC | 0.45±0.02a | 0.42±0.01a | 0.09±0.02b |
| LBP-L | 0.39±0.01ce | 0.37±0.00ce | 0.25±0.02c |
| LBP-M | 0.28±0.03c | 0.27±0.00c | 0.38±0.04d |
| LBP-H | 0.36±0.02ce | 0.32±0.00ce | 0.28±0.00d |
表5 各组KGN凋亡相关蛋白相对表达量
Tab.5 Relative expression levels of apoptosis-related proteins in KGN cells in each group (n=3, Mean±SD)
| Group | Bax | Caspase-3 | Bcl-2 |
|---|---|---|---|
| NC | 0.21±0.01 | 0.14±0.00 | 0.42±0.04 |
| MC | 0.45±0.02a | 0.42±0.01a | 0.09±0.02b |
| LBP-L | 0.39±0.01ce | 0.37±0.00ce | 0.25±0.02c |
| LBP-M | 0.28±0.03c | 0.27±0.00c | 0.38±0.04d |
| LBP-H | 0.36±0.02ce | 0.32±0.00ce | 0.28±0.00d |
| Group | p-PI3K | PI3K | p-AKT | AKT |
|---|---|---|---|---|
| NC | 0.25±0.01 | 0.47±0.01 | 0.47±0.01 | 0.48±0.01 |
| MC | 0.06±0.01a | 0.16±0.01a | 0.21±0.01a | 0.33±0.01a |
| LBP-L | 0.09±0.01ce | 0.24±0.02f | 0.29±0.01ce | 0.41±0.01ce |
| LBP-M | 0.16±0.01c | 0.38±0.00c | 0.42±0.02c | 0.46±0.01c |
| LBP-H | 0.12±0.01ce | 0.31±0.00ce | 0.35±0.01ce | 0.43±0.01ce |
表6 各组KGN PI3K/AKT信号通路蛋白相对表达量
Tab.6 Relative protein expression levels of the PI3K/AKT signaling pathway in KGN cells in each group (n=3, Mean±SD)
| Group | p-PI3K | PI3K | p-AKT | AKT |
|---|---|---|---|---|
| NC | 0.25±0.01 | 0.47±0.01 | 0.47±0.01 | 0.48±0.01 |
| MC | 0.06±0.01a | 0.16±0.01a | 0.21±0.01a | 0.33±0.01a |
| LBP-L | 0.09±0.01ce | 0.24±0.02f | 0.29±0.01ce | 0.41±0.01ce |
| LBP-M | 0.16±0.01c | 0.38±0.00c | 0.42±0.02c | 0.46±0.01c |
| LBP-H | 0.12±0.01ce | 0.31±0.00ce | 0.35±0.01ce | 0.43±0.01ce |
图5 Western blotting法检测各组p-PI3K、PI3K、p-AKT、AKT的表达
Fig.5 Expressions of p-PI3K, PI3K, p-AKT and AKT in KGN cells in each group detected by Western blotting.
| Group | MiR-23a |
|---|---|
| NC | 1.00±0.02 |
| MC | 2.50±0.13a |
| LBP-L | 1.89±0.05ce |
| LBP-M | 1.56±0.06c |
| LBP-H | 1.66±0.08c |
表7 各组KGN miR-23a表达量
Tab.7 miR-23a expression levels in KGN cells in each group (n=6, Mean±SD)
| Group | MiR-23a |
|---|---|
| NC | 1.00±0.02 |
| MC | 2.50±0.13a |
| LBP-L | 1.89±0.05ce |
| LBP-M | 1.56±0.06c |
| LBP-H | 1.66±0.08c |
| Group | MiR-23a |
|---|---|
| NC | 1.00±0.09 |
| inhibitor-NC | 1.02±0.07 |
| inhibitor | 0.43±0.02ag |
| mimic-NC | 0.98±0.06 |
| mimic | 1.65±0.04ah |
表8 慢病毒转染对miR-23a表达的影响
Tab.8 Effect of lentiviral transfection on miR-23a expression in KGN cells (n=6, Mean±SD)
| Group | MiR-23a |
|---|---|
| NC | 1.00±0.09 |
| inhibitor-NC | 1.02±0.07 |
| inhibitor | 0.43±0.02ag |
| mimic-NC | 0.98±0.06 |
| mimic | 1.65±0.04ah |
| Group | Cell viability (n=6) | Apoptosis rate (%, n=3) |
|---|---|---|
| NC | 1.00±0.00 | 5.50±0.53 |
| MC | 0.41±0.03a | 44.49±0.38a |
| LBP | 0.74±0.06c | 18.20±0.84c |
| inhibitor-NC+LBP | 0.76±0.08c | 17.32±0.23c |
| inhibitor+LBP | 0.88±0.09c | 13.12±0.39cei |
| mimic-NC+LBP | 0.76±0.04c | 17.64±0.35c |
| mimic+LBP | 0.64±0.09d | 27.62±0.73cej |
表9 miR-23a对KGN细胞活力和凋亡率的影响
Tab.9 Effect of miR-23a overexpression and knockdown on KGN cell viability and apoptosis rate (Mean±SD)
| Group | Cell viability (n=6) | Apoptosis rate (%, n=3) |
|---|---|---|
| NC | 1.00±0.00 | 5.50±0.53 |
| MC | 0.41±0.03a | 44.49±0.38a |
| LBP | 0.74±0.06c | 18.20±0.84c |
| inhibitor-NC+LBP | 0.76±0.08c | 17.32±0.23c |
| inhibitor+LBP | 0.88±0.09c | 13.12±0.39cei |
| mimic-NC+LBP | 0.76±0.04c | 17.64±0.35c |
| mimic+LBP | 0.64±0.09d | 27.62±0.73cej |
| Group | AMH (pg/mL) |
|---|---|
| NC | 358.92±16.15 |
| MC | 54.23±5.93a |
| LBP | 217.87±8.79c |
| inhibitor-NC+LBP | 212.64±7.93c |
| inhibitor+LBP | 259.52±6.73cei |
| mimic-NC+LBP | 210.80±8.02c |
| mimic+LBP | 163.54±5.61cej |
表10 miR-23a对KGN分泌AMH的影响
Tab.10 Effect of miR-23a overexpression and knockdown on AMH secretion in KGN cells (n=6, Mean±SD)
| Group | AMH (pg/mL) |
|---|---|
| NC | 358.92±16.15 |
| MC | 54.23±5.93a |
| LBP | 217.87±8.79c |
| inhibitor-NC+LBP | 212.64±7.93c |
| inhibitor+LBP | 259.52±6.73cei |
| mimic-NC+LBP | 210.80±8.02c |
| mimic+LBP | 163.54±5.61cej |
| Group | MiR-23a |
|---|---|
| NC | 1.00±0.05 |
| MC | 2.49±0.13a |
| LBP | 1.58±0.10c |
| inhibitor-NC+LBP | 1.58±0.09c |
| inhibitor+LBP | 1.20±0.05cei |
| mimic-NC+LBP | 1.59±0.07c |
| mimic+LBP | 1.99±0.12cej |
表11 慢病毒转染对LBP调节miR-23a表达的影响
Tab.11 Effect of lentiviral transfection on LBP-mediated regulation of miR-23a expression in KGN cells (n=6, Mean±SD)
| Group | MiR-23a |
|---|---|
| NC | 1.00±0.05 |
| MC | 2.49±0.13a |
| LBP | 1.58±0.10c |
| inhibitor-NC+LBP | 1.58±0.09c |
| inhibitor+LBP | 1.20±0.05cei |
| mimic-NC+LBP | 1.59±0.07c |
| mimic+LBP | 1.99±0.12cej |
| Group | p-PI3K | PI3K | p-AKT | AKT |
|---|---|---|---|---|
| NC | 0.34±0.02 | 0.49±0.02 | 0.43±0.02 | 0.47±0.00 |
| MC | 0.10±0.01a | 0.19±0.01a | 0.12±0.00a | 0.37±0.01a |
| LBP | 0.20±0.01c | 0.27±0.04c | 0.27±0.02c | 0.42±0.01c |
| inhibitor-NC+LBP | 0.20±0.01c | 0.28±0.03c | 0.28±0.03c | 0.43±0.01c |
| inhibitor+LBP | 0.29±0.00cei | 0.41±0.03cei | 0.40±0.02cei | 0.46±0.01cei |
| mimic-NC+LBP | 0.21±0.01c | 0.29±0.03c | 0.27±0.03c | 0.42±0.01c |
| mimic+LBP | 0.14±0.01cej | 0.21±0.01fj | 0.14±0.01ej | 0.38±0.01ej |
表12 miR-23a对LBP调节PI3K/AKT信号通路相对表达量的影响
Tab.12 Effect of miR-23a overexpression and knockdown on LBP-mediated regulation of PI3K/AKT signaling pathway in KGN cells (n=3, Mean±SD)
| Group | p-PI3K | PI3K | p-AKT | AKT |
|---|---|---|---|---|
| NC | 0.34±0.02 | 0.49±0.02 | 0.43±0.02 | 0.47±0.00 |
| MC | 0.10±0.01a | 0.19±0.01a | 0.12±0.00a | 0.37±0.01a |
| LBP | 0.20±0.01c | 0.27±0.04c | 0.27±0.02c | 0.42±0.01c |
| inhibitor-NC+LBP | 0.20±0.01c | 0.28±0.03c | 0.28±0.03c | 0.43±0.01c |
| inhibitor+LBP | 0.29±0.00cei | 0.41±0.03cei | 0.40±0.02cei | 0.46±0.01cei |
| mimic-NC+LBP | 0.21±0.01c | 0.29±0.03c | 0.27±0.03c | 0.42±0.01c |
| mimic+LBP | 0.14±0.01cej | 0.21±0.01fj | 0.14±0.01ej | 0.38±0.01ej |
图8 Western blotting法检测miR-23a对p-PI3K、PI3K、p-AKT、AKT表达的影响
Fig.8 Effect of miR-23a overexpression and knockdown on the expressions of p-PI3K, PI3K, p-AKT and AKT detected by Western blotting.
| Group | Bax | Caspase-3 | Bcl-2 |
|---|---|---|---|
| NC | 0.15±0.01 | 0.10±0.03 | 0.50±0.01 |
| MC | 0.43±0.02a | 0.45±0.01a | 0.17±0.01a |
| LBP | 0.30±0.02c | 0.25±0.03c | 0.30±0.01c |
| inhibitor-NC+LBP | 0.30±0.02c | 0.22±0.03c | 0.30±0.01c |
| inhibitor+LBP | 0.19±0.02cei | 0.14±0.04cei | 0.45±0.01cei |
| mimic-NC+LBP | 0.32±0.02c | 0.25±0.01c | 0.32±0.02c |
| mimic+LBP | 0.40±0.03ej | 0.41±0.03ej | 0.24±0.02cej |
表13 miR-23a对LBP调节凋亡相关蛋白相对表达量的影响
Tab.13 Effect of miR-23a overexpression and knockdown on LBP-mediated regulation of apoptosis-related protein expressions in KGN cells (n=3, Mean±SD)
| Group | Bax | Caspase-3 | Bcl-2 |
|---|---|---|---|
| NC | 0.15±0.01 | 0.10±0.03 | 0.50±0.01 |
| MC | 0.43±0.02a | 0.45±0.01a | 0.17±0.01a |
| LBP | 0.30±0.02c | 0.25±0.03c | 0.30±0.01c |
| inhibitor-NC+LBP | 0.30±0.02c | 0.22±0.03c | 0.30±0.01c |
| inhibitor+LBP | 0.19±0.02cei | 0.14±0.04cei | 0.45±0.01cei |
| mimic-NC+LBP | 0.32±0.02c | 0.25±0.01c | 0.32±0.02c |
| mimic+LBP | 0.40±0.03ej | 0.41±0.03ej | 0.24±0.02cej |
图9 Western blotting法检测miR-23a对Bax、caspase-3、Bcl-2表达的影响
Fig.9 Effect of miR-23a overexpression and knockdown on expressions of Bax, caspase-3 and Bcl-2 detected by Western blotting.
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