香草扶正合剂对结肠癌荷瘤小鼠免疫抑制状态及NF-<i>κ</i>B/p65、NSD2、PD-L2表达的影响

程权; 石占利; 陆莉莉; 傅华洲

doi:10.3969/j.issn.1671-038X.2024.12.13

香草扶正合剂对结肠癌荷瘤小鼠免疫抑制状态及NF-κB/p65、NSD2、PD-L2表达的影响

- 1.
  西湖大学医学院附属杭州市第一人民医院中医科(杭州，310006)
- 2.
  浙江省中西医结合医院重症医学科
- 3.
  杭州市拱墅区朝晖街道社区卫生服务中心全科
基金项目:
国家中医药管理局第七批全国老中医药专家学术经验继承工作建设项目(No：国中医药人教函〔2022〕76号)；浙江省医药卫生科技计划项目(No：2024KY1323)；浙江省名老中医专家传承工作室建设项目(No：GZS2020029)

详细信息

通讯作者: 程权，E-mail：hzallanqcheng@163.com

中图分类号: R735.35

收稿日期: 2024-02-18

刊出日期: 2024-12-15

Effects of Xiangcao Fuzheng Mixture on tumor invasion and metastasis and NF-κB/p65, NSD2, PD-L2 protein expression in colon cancer mice

- 1.
  Department of Traditional Chinese Medicine, Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, Hangzhou, 310006, China
- 2.
  Department of Intensive Care Unit, Zhejiang Integrated Traditional and Western Medicine Hospital
- 3.
  Department of General Practice, Chaohui Community Health Service Center, Gongshu District, Hangzhou

More Information

Corresponding author: CHENG Quan, E-mail: hzallanqcheng@163.com

Received Date: 18 February 2024

Publish Date: 15 December 2024

摘要

摘要: 目的探讨香草扶正合剂(XCFZ)对结肠癌荷瘤小鼠免疫抑制状态及肿瘤组织核转录因子-κB(NF-κB)/p65、核受体结合SET结构域蛋白2(NSD2)、程序性死亡配体2(PD-L2)表达的影响。方法采用HT29结肠癌细胞株培养移植法建立结肠癌荷瘤小鼠模型，随机分为模型组、5-氟尿嘧啶(5-FU)组、XCFZ低剂量组、XCFZ中剂量组、XCFZ高剂量组和联合组，每组各10只；造模第2天开始，中药组每天以XCFZ低、中、高剂量灌胃，化疗组以5-FU腹腔注射，联合组以XCFZ中剂量和5-FU腹腔注射联合给药，模型组按照等量生理盐水注射给药，称取小鼠体质量、瘤质量和去瘤后体质量，并计算抑瘤率和脾胸腺指数，肿瘤组织苏木精-伊红染色，流式细胞术检测CD3⁺、CD4⁺及CD8⁺阳性细胞数的变化情况，计算CD4⁺/CD8⁺比例，酶联免疫吸附试验(ELISA)法检测小鼠血清趋化因子受体4(CXCR4)、人整合素αVβ5(ITGαVβ5)、转化生长因子-β(TGF-β)、白细胞介素-10(IL-10)细胞因子表达水平；Western blot检测肿瘤组织NF-κB/p65、NSD2和PD-L2蛋白水平。结果与模型组比较，联合组抑瘤率最为显著，其次为5-FU组、XCFZ中剂量组(P＜0.01)。与5-FU组比较，XCFZ低、中、高剂量组及联合组去瘤后体质量升高(P＜0.05)。与5-FU组比较，XCFZ低、中、高剂量组和联合组脾脏指数升高(P＜0.05，P＜0.01)。与XCFZ中剂量组比较，XCFZ低、高剂量组，5-FU组和联合组脾和胸腺指数降低(P＜0.05，P＜0.01)。XCFZ各组肿瘤细胞核仁固缩，细胞坏死和核碎裂现象亦较多。5-FU组肿瘤细胞较小，核分裂现象和异型细胞少见，坏死碎裂细胞较多。与XCFZ中剂量组比较，XCFZ高、低剂量组、5-FU组及联合组CD8⁺含量显著降低(P＜0.01)。与5-FU组比较，XCFZ高、中、低剂量组及联合组CD4⁺/CD8⁺显著降低(P＜0.05，P＜0.01)，其中XCFZ中剂量组CD4⁺/CD8⁺比值最低(P＜0.05，P＜0.01)。与5-FU组比较，联合组肿瘤转移因子CXCR4、ITGαVβ5、TGF-β及IL-10表达水平明显降低(P＜0.05，P＜0.01)。与XCFZ中剂量组比较，XCFZ高、低剂量组CXCR4、ITGαVβ5、TGF-β、IL-10表达水平显著升高(P＜0.05，P＜0.01)。与XCFZ中剂量组比较，XCFZ高、低剂量组NF-κB/p65、NSD2和PD-L2表达升高(P＜0.05，P＜0.01)。与5-FU组比较，联合组抑制NF-κB/p65、NSD2和PD-L2表达效果更优(P＜0.01)。结论 XCFZ可抑制结肠癌荷瘤小鼠肿瘤生长，且中剂量效果最优，降低血清肿瘤转移因子水平，改善免疫抑制状态，同时下调NF-κB/p65、NSD2、PD-L2相关蛋白的表达，从而抑制结肠癌的侵袭转移。
- 香草扶正合剂 /
- 结肠癌 /
- 免疫抑制 /
- NF-κB/p65 /
- NSD2 /
- PD-L2
Abstract: Objective To investigate the effect of Xiangcao Fuzheng Mixture(XCFZ) on immunosuppression and the expression of nuclear factor-kappa B(NF-κB)/p65, nuclear receptor binding SET domain-protein 2(NSD2) and programmed cell death-ligand 2(PD-L2) in colon cancer tumor-bearing mice. Methods HT29 colon cancer cell line was cultured and transplanted to establish colon cancer tumor-bearing mice models. The mice were divided into several groups, including a model group, a 5-fluorouracil(5-FU) group, and XCFZ groups with low, medium, and high doses, as well as a combination group, with 10 mice per group. From the second day of modeling, the Chinese medicine group was treated with low, medium and high doses of XCFZ by gavage, the chemotherapy group was treated with intraperitoneal injection of 5-FU, the combination group was treated with medium dose of XCFZ combined with intraperitoneal injection of 5-FU, and the model group was injected with the same amount of normal saline. The body weight, tumor weight and body weight after tumor removal were measured, and the tumor inhibition rate and spleen and thymus index were calculated. The changes of CD3⁺, CD4⁺and CD8⁺ cells were detected by flow cytometry, and the CD4⁺/CD8⁺ratio was calculated. The expression levels of CXCR4, ITGαVβ5, TGF-β and IL-10 in serum of mice were detected by ELISA. The protein levels of NF-κB/p65, NSD2 and PD-L2 in tumor tissues were detected by Western blot. Results Compared with the model group, the combined group showed the most significant tumor inhibition rate, followed by the 5-FU group and the medium dose XCFZ group(P < 0.01). Compared with 5-FU group, the body weight of XCFZ low, medium, and high dose groups and XCFZ combined group increased after tumor removal(P < 0.05). Compared with 5-FU group, the spleen index of XCFZ low, medium and high dose groups and XCFZ combined group were increased(P < 0.05, P < 0.01). Compared with the XCFZ medium dose group, the spleen and thymus indexes of the XCFZ low and high dose groups, 5-FU group and combination group were decreased(P < 0.05, P < 0.01). Karyopyknosis, cell necrosis and nuclear fragmentation were more common in XCFZ group. In the 5-FU group, the tumor cells were smaller, the mitotic phenomenon and atypical cells were less, and the necrotic and fragmented cells were more. Compared with the XCFZ medium dose group, CD8⁺ cells in the XCFZ high and low dose groups, 5-FU group and combination group were significantly decreased(P < 0.01). Compared with 5-FU group, CD4⁺/CD8⁺ in XCFZ high, medium, low dose groups and combination group were significantly decreased(P < 0.05, P < 0.01), and the ratio in XCFZ medium dose group was the lowest(P < 0.05, P < 0.01). Compared with the 5-FU group, the expression levels of CXCR4, ITGαVβ5, TGF-β and IL-10 in the combined group were significantly decreased(P < 0.05, P < 0.01). The expression levels of CXCR4, ITGαVβ5, TGF-β and IL-10 in the high and low dose XCFZ groups were significantly higher than those in the medium dose XCFZ group(P < 0.05, P < 0.01). Compared with the medium dose XCFZ group, the high and low dose XCFZ groups had significant increases in the expression of NF-κB/p65, NSD2 and PD-L2(P < 0.05, P < 0.01). Compared with the 5-FU group, the combined group had a better inhibitory effect on the expression of NF-κB/p65, NSD2 and PD-L2(P < 0.01). Conclusion XCFZ can inhibit the tumor growth of colon cancer bearing mice, and the medium dose has the best effect. XCFZ can reduce the serum level of tumor metastasis factors, improve the immunosuppressive state, and down-regulate the expression of NF-κB/p65, NSD2, and PD-L2 related proteins, thereby inhibiting the invasion and metastasis of colon cancer.
- Xiangcao Fuzheng Mixture /
- colon cancer /
- immunosuppression /
- NF-κB/p65 /
- NSD2 /
- PD-L2

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图 1 小鼠肿瘤组织病理学观察(苏木精-伊红染色，×400)

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图 2 各组小鼠肿瘤组织NF-κB/p65、NSD2、PD-L2蛋白条带

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表 1 小鼠瘤质量、抑瘤率和去瘤后体质量的比较 X±S

组别	瘤质量/g	抑瘤率/%	去瘤后体质量/g
模型组(n=10)	1.61±0.16	0	19.41±0.72
XCFZ低剂量组(n=10)	1.52±0.19¹⁾⁴⁾⁵⁾	36.38±4.07²⁾⁴⁾⁶⁾	19.95±1.07³⁾⁴⁾⁵⁾
XCFZ中剂量组(n=10)	1.28±0.21²⁾	52.12±3.15²⁾	22.06±0.89²⁾
XCFZ高剂量组(n=10)	1.35±0.16²⁾³⁾⁵⁾	38.29±5.46²⁾⁴⁾⁶⁾	20.32±1.08¹⁾³⁾⁵⁾
5-FU组(n=10)	0.94±0.18²⁾⁴⁾	60.72±3.10²⁾⁴⁾	17.27±1.02¹⁾⁴⁾
联合组(n=10)	0.75±0.17²⁾⁴⁾⁵⁾	69.14±2.58²⁾⁴⁾⁶⁾	19.50±0.84⁴⁾⁵⁾
与模型组比较，¹⁾P＜0.05，²⁾P＜0.01；与XCFZ中剂量组比较，³⁾P＜0.05，⁴⁾P＜0.01；与5-FU组比较，⁵⁾P＜0.05，⁶⁾P＜0.01。

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表 2 XCFZ对各组小鼠脾脏及胸腺指数的影响 mg/g，X±S

组别	脾脏指数	胸腺指数
模型组(n=10)	8.11±0.31	2.26±0.24
XCFZ低剂量组(n=10)	8.78±1.43⁴⁾⁵⁾	2.35±0.31⁴⁾⁵⁾
XCFZ中剂量组(n=10)	13.68±2.10²⁾	3.84±0.32²⁾
XCFZ高剂量组(n=10)	12.04±1.65²⁾³⁾⁶⁾	3.09±0.70²⁾³⁾⁶⁾
5-FU组(n=10)	7.32±1.02¹⁾⁴⁾	1.66±0.76¹⁾⁴⁾
联合组(n=10)	10.26±1.54¹⁾⁴⁾⁶⁾	2.81±0.62¹⁾⁴⁾⁶⁾
与模型组比较，¹⁾P＜0.05，²⁾P＜0.01；与XCFZ中剂量组比较，³⁾P＜0.05，⁴⁾P＜0.01；与5-FU组比较，⁵⁾P＜0.05，⁶⁾P＜0.01。

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表 3 XCFZ对各组小鼠脾脏T淋巴细胞亚型的影响 X±S

组别	CD3⁺/%	CD4⁺/%	CD8⁺/%	CD4⁺/CD8⁺
模型组(n=10)	33.12±6.90	65.52±0.81	26.48±0.64	2.48±0.64
XCFZ低剂量组(n=10)	34.48±5.69	64.66±0.98¹⁾³⁾⁵⁾	24.73±0.78⁴⁾⁵⁾	2.73±0.58⁴⁾⁵⁾
XCFZ中剂量组(n=10)	33.11±4.71	62.70±0.89²⁾⁶⁾	32.34±0.43²⁾⁶⁾	1.89±0.43¹⁾⁶⁾
XCFZ高剂量组(n=10)	34.04±6.89	63.90±1.03¹⁾³⁾⁵⁾	28.20±0.98²⁾⁴⁾⁵⁾	2.20±0.48¹⁾³⁾⁵⁾
5-FU组(n=10)	31.32±5.65¹⁾	65.76±1.05⁴⁾	27.91±0.28¹⁾⁴⁾	2.91±0.28¹⁾⁴⁾
联合组(n=10)	34.45±4.36	63.26±0.91¹⁾⁵⁾	25.29±0.67⁴⁾⁵⁾	2.59±0.37⁴⁾⁵⁾
与模型组比较，¹⁾P＜0.05，²⁾P＜0.01；与XCFZ中剂量组比较，³⁾P＜0.05，⁴⁾P＜0.01；与5-FU组比较，⁵⁾P＜0.05，⁶⁾P＜0.01。

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表 4 XCFZ对各组小鼠血清CXCR4、ITGαVβ5、TGF-β、IL-10表达的影响 ng/L，X±S

组别	CXCR4	ITGαVβ5	TGF-β	IL-10
模型组(n=10)	36.12±3.91	78.52±4.81	236.48±34.64	95.48±8.64
XCFZ低剂量组(n=10)	35.48±2.69¹⁾³⁾⁶⁾	73.66±5.98⁴⁾⁶⁾	214.73±29.78¹⁾⁴⁾⁶⁾	86.73±10.58⁴⁾⁶⁾
XCFZ中剂量组(n=10)	30.11±1.92¹⁾⁶⁾	58.70±6.89²⁾	185.34±21.43¹⁾	61.89±10.43¹⁾
XCFZ高剂量组(n=10)	32.04±2.89¹⁾³⁾⁶⁾	62.90±7.03²⁾³⁾⁶⁾	201.20±18.98¹⁾⁴⁾⁵⁾	82.20±12.48¹⁾⁴⁾⁶⁾
5-FU组(n=10)	25.32±3.65²⁾	51.76±5.05²⁾³⁾	193.91±31.28¹⁾³⁾	57.91±11.28¹⁾⁴⁾
联合组(n=10)	21.45±2.36²⁾⁵⁾	38.26±4.91²⁾⁶⁾	152.29±30.67¹⁾⁶⁾	43.29±9.37¹⁾⁵⁾
与模型组比较，¹⁾P＜0.05，²⁾P＜0.01；与XCFZ中剂量组比较，³⁾P＜0.05，⁴⁾P＜0.01；与5-FU组比较，⁵⁾P＜0.05，⁶⁾P＜0.01。

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表 5 XCFZ对肿瘤组织NF-κB/p65、NSD2和PD-L2蛋白表达的影响 X±S

组别	NF-κB/p65	NSD2	PD-L2
模型组(n=10)	12.28±2.09	85.76±8.10	182.78±8.12
XCFZ低剂量组(n=10)	11.71±1.32¹⁾³⁾⁶⁾	82.91±7.08⁴⁾⁶⁾	175.86±9.08¹⁾⁴⁾⁶⁾
XCFZ中剂量组(n=10)	8.61±1.29²⁾	48.79±6.11²⁾	136.75±11.28²⁾
XCFZ高剂量组(n=10)	11.65±1.08¹⁾³⁾⁶⁾	56.71±8.08²⁾⁴⁾⁵⁾	173.91±10.19¹⁾⁴⁾⁶⁾
5-FU组(n=10)	7.92±2.09²⁾³⁾	45.90±5.19²⁾	119.97±9.26²⁾⁴⁾
联合组(n=10)	5.36±1.10²⁾⁶⁾	31.75±6.37²⁾⁶⁾	90.86±8.18²⁾⁶⁾
与模型组比较，¹⁾P＜0.05，²⁾P＜0.01；与XCFZ中剂量组比较，³⁾P＜0.05，⁴⁾P＜0.01；与5-FU组比较，⁵⁾P＜0.05，⁶⁾P＜0.01。

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