中性胞苷/阳离子脂质递送猪脾提取物(PAT)发挥强效抗肿瘤作用

doi:10.5246/jcps.2025.01.002

中国药学(英文版) ›› 2025, Vol. 34 ›› Issue (1): 14-27.DOI: 10.5246/jcps.2025.01.002

中性胞苷/阳离子脂质递送猪脾提取物(PAT)发挥强效抗肿瘤作用

于小桐¹^,²^,^#, 吴静¹^,^#, 张琦¹, 洪佳美¹, 于晶¹, 关注¹, 尹艳慧², 杨振军¹^,^*()

^1. 北京大学药学院天然药物及仿生药物全国重点实验室, 北京 100191
^2. 北京大学基础医学院免疫学系卫生部医学免疫学重点实验室, 北京 100191

收稿日期:2024-09-21 修回日期:2024-10-11 接受日期:2024-12-01 出版日期:2025-02-20 发布日期:2025-02-20
通讯作者: 杨振军

Enhanced antitumor efficacy of fresh pig spleen extracts (PAT) encapsulated with cytidinyl and cationic lipid nanocomplexes

Xiaotong Yu¹^,²^,^#, Jing Wu¹^,^#, Qi Zhang¹, Jiamei Hong¹, Jing Yu¹, Zhu Guan¹, Yanhui Yin², Zhenjun Yang¹^,^*()

¹ State Key Laboratory of Natural & Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
² Department of Immunology, School of Basic Medical Sciences, Key Laboratory of Medical Immunology of Ministry of Health, Peking University, Beijing 100191, China

Received:2024-09-21 Revised:2024-10-11 Accepted:2024-12-01 Online:2025-02-20 Published:2025-02-20
Contact: Zhenjun Yang
About author:
^# These authors contributed equally to this paper.
Supported by:
The Contract Grant from Zhejiang Fengan Biopharmaceutical Co., Ltd, Zhejiang Province, China.

摘要/Abstract

摘要：

我国自古以来一直使用中药来治疗和预防疾病, 包括中草药和动物药物, 有研究表明中草药汤剂中的miRNA也是其发挥作用的关键成分之一。根据古书《本草突经》记载, 猪脾是一种中药, 煮熟后可食用, 也可以用于中药组方。本研究针对主要由寡核苷酸和肽组成的新鲜猪脾提取液口服冻干粉PAT, 以中性胞苷脂材DNCA和阳离子脂质CLD混合包载(PAT/Mix), 发现其可以促进A549细胞的早期和晚期细胞凋亡, 显著抑制A549 和 BCPAP 细胞的增殖, 瘤内注射可显著降低A549肺腺癌小鼠的肿瘤体积和重量。转录组学和蛋白质组学分析显示, PAT/Mix可能通过沉默A549细胞中的DEPDC1 mRNA进而影响细胞过程和代谢来发挥抗肿瘤生物活性, 也能促进骨髓树突状细胞(BMDC)的成熟, 有利于其对肿瘤抗原的加工和呈递。PAT/Mix有望成为一种同时具有免疫调节和抗肿瘤作用的药物。

关键词: 猪脾提取物, miRNA, DNCA, CLD, 抗肿瘤治疗

Abstract:

Traditional Chinese Medicine (TCM) has been utilized in China for thousands of years to treat and prevent various diseases, with recent research highlighting microRNAs (miRNAs) in herbal decoctions as key bioactive components. The oral lyophilized powder of the pig spleen extracts (PAT) composed of oligonucleotides and peptides extracted from fresh pig spleen and delivered via a lipid mixture containing neutral cytidinyl lipid DNCA and cationic lipid CLD (Mix), has demonstrated significant antitumor effects against A549 and BCPAP cells, promoting early and late apoptosis by approximately 20% in A549 cells. Intratumoral administration of PAT/Mix in A549-bearing mice significantly reduced both tumor volume and weight. Transcriptomic and proteomic analyses suggested that PAT/Mix exerted its antitumor activity by silencing DEPDC1 mRNA in A549 cells, thereby influencing cellular processes and metabolic pathways. Additionally, PAT/Mix was found to promote the maturation of bone marrow dendritic cells (BMDCs), enhancing antigen processing and presentation. These findings indicated that PAT/Mix held promise as a therapeutic agent with both immunomodulatory and antitumor properties, owing not only to its peptide components but also to miRNAs, which played a critical therapeutic role.

Key words: Extract of pig spleen, miRNA, DNCA, CLD, Anti-tumor efficacy

Supporting: /attached/file/20250222/20250222155959_910.pdf

于小桐, 吴静, 张琦, 洪佳美, 于晶, 关注, 尹艳慧, 杨振军. 中性胞苷/阳离子脂质递送猪脾提取物(PAT)发挥强效抗肿瘤作用[J]. 中国药学(英文版), 2025, 34(1): 14-27.

Xiaotong Yu, Jing Wu, Qi Zhang, Jiamei Hong, Jing Yu, Zhu Guan, Yanhui Yin, Zhenjun Yang. Enhanced antitumor efficacy of fresh pig spleen extracts (PAT) encapsulated with cytidinyl and cationic lipid nanocomplexes[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(1): 14-27.

图/表 10

Table 1. Primers of several mRNAs used in this study.

Table 2. Sequences of siRNA used in this study.

Figure 1. The antiproliferative ability of PAT/Mix nanocomplexes in various tumor cells. (A) The cell viability of indicated cells treated with PAT (10 mg/mL) after 48 h. (B) The cell viability of indicated cells treated with PAT delivered with different ratios of DNCA to CLD (1 mg/mL). (C) The cell viability of MC-38, Hep-G2, and MCF-7 cells treated with PAT (1 mg/mL) delivered with different ratios of DNCA to CLD. (D) The cell viability of A549 and BCPAP cells treated with PAT (1 mg/mL) delivered with different ratios of DNCA and CLD. (E) The dose-response relationship of PAT/Mix nanocomplexes in A549 and BCPAP cells (DNCA/CLD, 10/10 μg/mL). (F) The cell viability of indicated cells treated with empty Mix lipids at different ratios.

Figure 2. Characteristics of various formulations. The size (A) and zeta potential (B) of indicated formulations measured by DLS. (C–D) Representative transmission electron micrographs of indicated preparations.

Figure 3. (A) The heatmap of differential genes in A549 cells treated by PBS, Mix, and PAT/Mix. (B) Histogram of functional enrichment of regulated genes and proteins of A549 cells treated with PAT/Mix. (C) The numbers of genes and proteins regulated by PAT/Mix compared to the Mix group. (D) Proteins and genes significantly down-regulated and up-regulated both in transcriptomics and proteomic analyses.

Figure 4. (A) RT-qPCR analysis of DEPDC1 mRNA in A549 cells treated with indicated formulations for 24 h (PAT, 1 mg/mL; DNCA/CLD, 10/10 μg/mL). (B–C) Apoptosis analysis and representative scatter plots of A549 cells treated with indicated formulations for 24 h (PAT, 1 mg/mL; DNCA/CLD, 10/10 μg/mL, n = 3).

Figure 5. Antitumor efficacy of PAT/Mix nanocomplexes in vivo. 3×106 A549 cells were inoculated into BALB/c-nude mice. The mice were treated with GenOpti (blank), Mix, PAT (6 mg), or PAT (6 mg)/Mix by intertumoral injection (n = 5). (A) Mean tumor volume of mice treated with indicated formulations (*P < 0.05). (B) The weight of subcutaneous tumors separated from mice at the experimental endpoint. (C) The picture of subcutaneous tumors at the experimental endpoint in nude mice. (D) Body weights of mice treated with indicated formulations.

Figure 6. (A) The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), blood urea nitrogen (BUN), creatinine, and uric acid in blood. Blood was harvested when mice reached tumor volume endpoints. Data are presented as mean ± SEM; (B) Representative H&E-stained images of the liver. No significant pathological changes were observed in the liver between the control and treatment groups (n = 3).

Figure 7. Immune factors level in A549 tumor-bearing mice treated with indicated formulations.

Figure 8. Flow cytometric quantification of CD40 expression (A) and proportion (B) in BMDCs treated with indicated formulations treated for 24 h (PAT: 1 mg/mL, n = 4). The significant differences were represented by P-values: *P < 0.05; ***P < 0.001; ****P < 0.0001.

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中性胞苷/阳离子脂质递送猪脾提取物(PAT)发挥强效抗肿瘤作用

Enhanced antitumor efficacy of fresh pig spleen extracts (PAT) encapsulated with cytidinyl and cationic lipid nanocomplexes

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