靶向结直肠癌的小白菊内酯脂质体纳米颗粒诱导程序性坏死并改善T细胞耗竭

目的研究小白菊内酯(PTL)诱导结直肠癌细胞程序性坏死,并通过调节T细胞耗竭抑制结直肠癌的发展的机制,并通过构建其靶向脂质体改进其临床应用。方法通过CCK8实验检测不同结直肠肿瘤细胞经PTL处理后的增殖能力。利用ROS与LDH检测分析PTL诱导MC38细胞死亡的方式并进行蛋白免疫印迹分析。将24只小鼠随机分成对照组(等量生理盐水)、低剂量PTL组(5 mg/kg)与高剂量PTL组(15 mg/kg),通过在小鼠腹股沟皮下注射MC38细胞构建皮下瘤模型,给药后检测各组小鼠皮下瘤的重量与CD8^(+)T细胞的浸润情况,分析CD8^(+)T细胞耗竭表型的变化。构建PTL脂质体并对其进行表征。通过在小鼠回盲部移植肿瘤构建结直肠癌原位移植瘤模型,将32只小鼠随机分成对照组(等量生理盐水)、PTL处理组(100μg/mL)、低剂量靶向脂质体TCP-1-PTL-LNPs组(100μg/mL)和高剂量TCP-1-PTL-LNPs组(200μg/mL),尾静脉注射给药,通过免疫组化分析CD8在肿瘤中的表达情况。结果多种结直肠癌细胞(SW480、DLD1、HCT116和MC38)在PTL作用下随着药物浓度梯度增加细胞活力逐渐下降。在MC38细胞中,这一效应可被抑制剂Nec-1拮抗,同时免疫蛋白印迹分析显示经PTL处理细胞出现RIP3和MLKL的磷酸化。在小鼠皮下瘤模型中,经流式细胞术分析,与对照组或低剂量组相比,高剂量PTL组出现了肿瘤CD3^(+)CD8^(+)T细胞的浸润增加(P<0.01),同时PD1hiTIM3^(+)T细胞比例显著增加(P<0.01),而PD1loTIM3-T细胞比例显著降低(P<0.01)。在小鼠结直肠癌原位移植瘤模型中,经过PTL治疗,免疫组化结果显示CD8表达较对照组明显增加,而使用了高浓度靶向脂质体治疗的肿瘤中CD8表达更进一步增加(P<0.01)。结论小白菊内酯通过诱导细胞程序性坏死、增加肿瘤CD8^(+)T细胞浸润,改善CD8^(+)T细胞耗竭,其靶向脂质体能提高其抗肿瘤效应。

Modified Shenlingbaizhu decoction reduces intestinal adenoma formation in adenomatous polyposis coli multiple intestinal neoplasia mice by suppression of hypoxia-inducible factor 1α-induced CD4+CD25+forkhead box P3 regulatory T cells

OBJECTIVE： To test the hypothesis that modified Shenlingbaizhu decoction （MSD） attenuates the for- mation of intestinal adenomas by regulating activa- tion of CD4＋CD25＋ forkhead box P3 （FoxP3） regu- latory T cells （Tregs） by downregulation of hypox- ia-inducible factor la （HIF-la）. METHODS： Chemical fingerprints of ginsenoside Rbl, ginsenoside Rc, paeoniflorin, and dioscin in standard extractions were used as material bases of MSD. Adenomatous polyposis coli multiple intesti- nal neoplasia （ApcM＇n/＋） mice, which harbor a muta- tion in adenomatous polyposis coil, were used to host intestinal adenomas. Peripheral blood and spleen Tregs were analyzed by flow cytometry. Pro- tein expression was analyzed by immunohisto- chemistry and Western blotting. RESULTS： The number and size of intestinal adenomas were significantly reduced by MSD treatment. Mucosal thickening and the spleen size were also substantially decreased by MSD. The carcinogenesis process in Apc^min/＋ mice resembled that of human colorectal cancer. Molecular markers of neoplasms, such as 13-catenin, cyclooxygenase-2, prolif- erating cell nuclear antigen, and p53, were substantially ameliorated by MSD treatment. Moreover, MSD downregulated peripheral and spleen CD4＋ CD25＋FoxP3＋ Tregs and reduced in situ expression of CD4, CD25, and FoxP3 in intestinal adenomas. MSD also suppressed HIF-la expression in the intestinal adenomas, and HIF-la inhibition decreased expression of FoxP3 in Jurkat T cells under hypoxic conditions. CONCLUSION： MSD is a valid prescription to control the formation of intestinal adenomas in Apc^min/＋mice. It exerts anti-cancer effects partially through suppression of HIF-la that induced activation of CD4＋CD25＋FoxP3＋ Tregs in vivo and in vitro.