柴胡皂苷D对甲状腺乳头状癌细胞TPC-1生物学行为的影响

doi:10.3877/cma.j.issn.2095-1221.2022.04.002

目的

探讨柴胡皂苷D对体外甲状腺乳头状癌（PTC）细胞TPC-1增殖、侵袭、迁移、活性氧和凋亡的影响。

方法

不同浓度（0、5、10、20 μmol/L）柴胡皂苷D干预TPC-1细胞，CCK-8法检测细胞增殖率；Annexin-V-FITC/PI双染试剂盒检测细胞凋亡率；活性氧试剂盒检测细胞活性氧；Transwell小室法检测细胞侵袭、迁移能力；Western blot法检测凋亡蛋白剪切的半胱天冬酶3 （cleaved caspase 3）、B淋巴细胞瘤-2基因（Bcl-2）、Bcl2相关X蛋白（Bax）和侵袭迁移相关蛋白E钙黏蛋白（E-cadherin)和N钙黏蛋白（N-cadherin）及Wnt/β连环蛋白（β-catenin）信号通路相关蛋白Wnt3a和β-catenin的表达量。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与0 μmol/L柴胡皂苷D干预相比，（5、10、20 μmol/L）柴胡皂苷D干预TPC-1细胞存活率[24 h：（88.07±0.75）%、（76.06±0.56）%、（65.80± 1.66）%比（100.00±1.00）%；48 h：（72.40±0.35）%、（52.26± 0.22）%、（41.30±0.17）%比（100.00± 1.53）%；72 h：（56.50±0.22）%、（40.26±0.21）%、（31.10± 0.14）%比（100.00±0.14）%]、细胞侵袭率[（65.43±1.32）%、（49.46±0.31）%、（22.48±0.85）%比（100.00±1.25）%]、迁移率[（67.45±0.96）%、（52.42±0.39）%、（42.12±1.10）%比（100.00±2.00）%]、Bcl-2蛋白表达量（0.51±0.02、0.31±0.02、0.22±0.02比0.62±0.01）、Wnt3a表达量（0.56±0.06、0.37±0.01、0.26±0.02比0.72±0.03）、β-catenin表达量（0.59±0.02、0.40±0.03、0.14±0.03比0.82±0.03）和N-cadherin表达量（0.49±0.05、0.34 ±0.03、0.14± 0.03比0.62±0.05）均降低，细胞活性氧含量增加，凋亡率[（12.36±1.32）%、（23.15±2.27）%、（36.65±3.22）%比（7.32±1.25）%]、Bax蛋白表达量（0.45±0.02、0.51±0.01、0.79±0.02比0.32±0.01）、cleaved caspase-3蛋白表达量（0.26±0.02、0.37±0.02、0.40±0.02比0.13±0.01）和E-cadherin表达量（0.20±0.02、0.37±0.02、0.46±0.05比0.12±0.01）均增加（P均< 0.05）。

结论

柴胡皂苷D能够抑制PTC细胞的生物学行为。

关键词: 甲状腺乳头状癌, 柴胡皂苷D, 凋亡, 侵袭迁移

Objective

To explore the effect of saikosaponin D on the proliferation, invasion, migration, reactive oxygen species and apoptosis of TPC-1 papillary thyroid carcinoma (PTC) cells in vitro.

Methods

The cells were treated with 0、5、10、20 μmol/L saikosaponin D. The cell proliferation rate was detected by CCK-8 assay. Annexin V conjugate (Annexin V-FITC) and propidium iodide (PI) double staining kit was used to detect cell apoptosis rate. Reactive oxygen detection kit was used to detect cell reactive oxygen species. Transwell chamber method was used to detect cell invasion and migration ability. Western blot was used to detect the expression of apoptosis proteins including cleaved caspase-3, B-cell lymphoma-2 (Bcl-2) , Bcl2 associated X protein (Bax) , invasion and migration related proteins E-cadherin, N-cadherin and Wnt/β-catenin signaling pathway related proteins Wnt3a and β-catenin. At the same time, one-way ANOVA was used to compare multiple groups and the LSD-t test was used for pairwise comparison between groups.

Results

Compared with 0 μmol/L saikosaponin D, 5、10、20 μmol/L saikosaponin D significantly decreased survival rate (24 h: 88.07±0.75, 76.06±0.56, 65.80±1.66 vs 100.00±1.00. 48 h: 72.40±0.35, 52.26±0.22, 41.30±0.17 vs 100.00±1.53. 72 h: 56.50±0.22, 40.26±0.21, 31.10±0.14 vs 100.00±0.14) , cell invasion rate [ (65.43±1.32) %, (49.46±0.31) %, (22.48±0.85) % vs (100.00±1.25) %], mobility[ (67.45±0.96) %, (52.42±0.39) %, (42.12± 1.10) % vs (100.00±2.00) %], Bcl-2 protein expression (0.51±0.02, 0.31±0.02, 0.22±0.02 vs 0.62±0.01) , Wnt3a expression (0.56±0.06, 0.37±0.01, 0.26±0.02 vs 0.72±0.03) , β-catenin expression (0.59±0.02, 0.40±0.03, 0.14±0.03 vs 0.82±0.03) , N-cadherin expression (0.49±0.05, 0.34±0.03, 0.14±0.03 vs 0.62±0.05) in TPC-1 cells. Compared with 0 μmol/L saikosaponin D, 5、10、20 μmol/L saikosaponin D significantly increased the content of reactive oxygen species in cells and the apoptosis rate[ (12.36±1.32) %, (23.15±2.27) %, (36.65±1.32) %vs (7.32±1.25) %], Bax protein expression (0.45±0.02, 0.51±0.01, 0.79±0.02 vs 0.32±0.01) , cleaved caspase-3 protein expression (0.26±0.02, 0.37±0.02, 0.40±0.02 vs 0.13±0.01) and E-cadherin expression (0.20±0.02, 0.37±0.02, 0.46±0.05 vs 0.12±0.01) (all P < 0.05) .

Conclusion

Saikosaponin D can inhibit the biological behavior of PTC cells.

Key words: Papillary thyroid carcinoma, Saikosaponin D, Apoptosis, Invasion and migration

表1 柴胡皂苷D作用于TPC-1细胞后细胞凋亡率及cleaved caspase-3、Bax、Bcl-2蛋白相对表达量（±s）

分组	实验次数	细胞凋亡率	Bax	Bcl-2	cleaved caspase-3
0 μmol/L	3	7.32 ± 1.25	0.32 ± 0.01	0.62 ± 0.01	0.13 ± 0.01
5 μmol/L	3	12.36 ± 1.32^a	0.45 ± 0.02^a	0.51 ± 0.02^a	0.26 ± 0.02^a
10 μmol/L	3	23.15 ± 2.27^ab	0.51 ± 0.01^ab	0.31 ± 0.02^ab	0.37 ± 0.02^ab
20 μmol/L	3	36.65 ± 3.22^abc	0.79 ± 0.02^abc	0.22 ± 0.02^abc	0.40 ± 0.02^abc
F值		192.332	288.673	163.462	212.899
P值		< 0.001	< 0.001	< 0.001	< 0.001

表1 柴胡皂苷D作用于TPC-1细胞后细胞凋亡率及cleaved caspase-3、Bax、Bcl-2蛋白相对表达量（±s）

分组	实验次数	细胞凋亡率	Bax	Bcl-2	cleaved caspase-3
0 μmol/L	3	7.32 ± 1.25	0.32 ± 0.01	0.62 ± 0.01	0.13 ± 0.01
5 μmol/L	3	12.36 ± 1.32^a	0.45 ± 0.02^a	0.51 ± 0.02^a	0.26 ± 0.02^a
10 μmol/L	3	23.15 ± 2.27^ab	0.51 ± 0.01^ab	0.31 ± 0.02^ab	0.37 ± 0.02^ab
20 μmol/L	3	36.65 ± 3.22^abc	0.79 ± 0.02^abc	0.22 ± 0.02^abc	0.40 ± 0.02^abc
F值		192.332	288.673	163.462	212.899
P值		< 0.001	< 0.001	< 0.001	< 0.001

图1 柴胡皂苷D对TPC-1细胞活性氧与凋亡的影响注：a ~ d图为荧光显微镜下观察0、5、10、20 μmol/L柴胡皂苷D干预TPC-1细胞的活性氧，绿色荧光越强表明活性氧含量越高（×100）；e ~ h图分别为0、5、10、20 μmol/L柴胡皂苷D干预TPC-1细胞的凋亡率

图2 Western blot检测cleaved caspase-3、Bax和Bcl-2蛋白相对表达量

表2 柴胡皂苷D作用于TPC-1细胞后侵袭、迁移能力与Wnt3a、β-catenin、E-cadherin、N-cadherin相对表达量（±s）

分组	实验次数	侵袭率（%）	迁移率（%）	Wnt3a	β-catenin	E-cadherin	N-cadherin
0 μmol/L	3	100.00 ± 1.25	100.00 ± 2.00	0.72 ± 0.03	0.82 ± 0.03	0.12 ± 0.01	0.62 ± 0.05
5 μmol/L	3	65.43 ± 1.32^a	67.45 ± 0.96^a	0.56 ± 0.06^a	0.59 ± 0.02^a	0.20 ± 0.02^a	0.49 ± 0.05^a
10 μmol/L	3	49.46 ± 0.31^ab	52.42 ± 0.39^ab	0.37 ± 0.01^ab	0.40 ± 0.03^ab	0.37 ± 0.02^ab	0.34 ± 0.03^ab
20 μmol/L	3	22.48 ± 0.85^abc	42.12 ± 1.10^abc	0.26 ± 0.02^abc	0.14 ± 0.03^abc	0.46 ± 0.05^abc	0.14 ± 0.03^abc
F值		203.602	165.142	187.146	225.14	175.762	182.186
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

表2 柴胡皂苷D作用于TPC-1细胞后侵袭、迁移能力与Wnt3a、β-catenin、E-cadherin、N-cadherin相对表达量（±s）

分组	实验次数	侵袭率（%）	迁移率（%）	Wnt3a	β-catenin	E-cadherin	N-cadherin
0 μmol/L	3	100.00 ± 1.25	100.00 ± 2.00	0.72 ± 0.03	0.82 ± 0.03	0.12 ± 0.01	0.62 ± 0.05
5 μmol/L	3	65.43 ± 1.32^a	67.45 ± 0.96^a	0.56 ± 0.06^a	0.59 ± 0.02^a	0.20 ± 0.02^a	0.49 ± 0.05^a
10 μmol/L	3	49.46 ± 0.31^ab	52.42 ± 0.39^ab	0.37 ± 0.01^ab	0.40 ± 0.03^ab	0.37 ± 0.02^ab	0.34 ± 0.03^ab
20 μmol/L	3	22.48 ± 0.85^abc	42.12 ± 1.10^abc	0.26 ± 0.02^abc	0.14 ± 0.03^abc	0.46 ± 0.05^abc	0.14 ± 0.03^abc
F值		203.602	165.142	187.146	225.14	175.762	182.186
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

图3 Transwell小室法检测柴胡皂苷D对TPC-1细胞侵袭和迁移的影响（结晶紫染色，×100）注：a ~ d图为倒置荧光显微镜下观察0、5、10、20 μmol/L柴胡皂苷D干预TPC-1细胞的侵袭结果；e ~ h图分别为倒置荧光显微镜下观察0、5、10、20 μmol/L柴胡皂苷D干预TPC-1细胞的迁移结果

图4 Western blot检测Wnt3a、β-catenin、E-cadherin和N-cadherin的相对表达量

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Effects of saikosaponin D on the biological behavior of papillary thyroid carcinoma TPC-1

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Effects of saikosaponin D on the biological behavior of papillary thyroid carcinoma TPC-1