海南医科大学学报

目的：探讨黄精多糖(polygonatum sibiricum polysaccharides,PSP)对裸鼠前列腺癌PC-3细胞移植瘤生长及磷脂酰肌醇-3-羟激酶/蛋白激酶B(PI3K/Akt)和核因子κB(NF-κB)信号通路中关键蛋白的影响。方法：选择雄激素非依赖型前列腺癌PC-3细胞株接种于BALB/c裸鼠皮下组织，构建模型，并分为5组，每组6只，然后根据人与裸鼠体表面积换算给药量。对照组予生理盐水，环磷酰胺组(CTX)予30 mg·kg^-1·d^-1,PSP组分为低剂量组100 mg·kg^-1·d^-1、中剂量组200 mg·kg^-1·d^-1、高剂量组400 mg·kg^-1·d^-1,0.2 mL灌胃/次/只，隔天1次，持续30 d。每3 d测量记录一次裸鼠体重和瘤体积，分析PSP对移植瘤的生长情况、大小及裸鼠生长状态的影响。以末次给药4 h后的瘤体积计算抑瘤率，使用原位末端标记法(TUNEL)和流式细胞术观测瘤细胞凋亡情况，细胞增殖试剂盒-8(CCK8)检测脾细胞增殖活力，乳酸脱氢酶(LDH)释放法检测自然杀伤(NK)细胞活性，酶联免疫吸附(ELISA)法检测血清肿瘤坏死因子α和白细胞介素-2、-6(TNF-α、IL-2、IL-6)的水平，免疫组织化学(IHC)法分析裸鼠移植瘤组织中PI3K、Akt、NF-κB P65蛋白及磷酸化(p-PI3K、p-Akt、NF-κB p-P65)蛋白和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达。结果：模型建立初期，各组裸鼠精神状态良好，活动自如且频繁，进食稳定，对外界刺激反应灵敏，但随着瘤体生长均出现活动度、进食、反应减少等改变。与对照组相比，CTX组和PSP组裸鼠抑瘤率，流式细胞术中瘤细胞凋亡率，IHC中Caspase-3水平均升高(P<0.05或P<0.001);CTX组和中、高剂量组Tunel中瘤细胞凋亡率，中、高剂量组LDH中NK细胞活力、ELISA中IL-6水平，高剂量组CCK8中脾细胞活力，PSP组TNF-α、IL-2水平均升高(P<0.05);CTX组和PSP组瘤体积和IHC中p-P65水平，CTX组ELISA中TNF-α、IL-2水平和IHC中p-PI3K、p-Akt水平，中、高剂量组IHC中p-PI3K、p-Akt水平均降低(P<0.05)。与CTX组相比，PSP组裸鼠瘤体积，LDH法中NK细胞活力，ELISA中TNF-α、IL-2水平和IHC中p-P65水平均升高(P<0.05)；中、高剂量组CCK8中脾细胞活力、ELISA中IL-6水平，低剂量组IHC中p-PI3K、p-Akt水平均升高(P<0.05);PSP组抑瘤率和Tunel中瘤细胞凋亡率，低、中剂量组IHC中Caspase-3表达均降低(P<0.01或P<0.001)。与低剂量组相比，中、高剂量组裸鼠抑瘤率，Tunel和流式细胞术中瘤细胞凋亡率，ELISA中TNF-α、IL-2、IL-6水平，IHC中Caspase-3表达均升高(P<0.05)；中、高剂量组瘤体积和IHC中p-PI3K、p-Akt水平，高剂量组p-P65水平均降低(P<0.05或P<0.001)。与中剂量组比较，高剂量组裸鼠抑瘤率，Tunel和流式细胞术中瘤细胞凋亡率，ELISA中TNF-α、IL-2、IL-6水平，IHC中Caspase-3水平均升高(P<0.05)；高剂量组瘤体积，IHC中p-PI3K、p-Akt、p-P65水平均降低(P<0.05)。结论：PSP可抑制荷瘤裸鼠PC-3细胞增殖并诱导其凋亡，其机制可能是通过降低PI3K、Akt、P65蛋白的活化水平抑制PI3K/Akt、NF-κB信号通路的激活以及促进凋亡因子Caspase-3的表达实现的，同时可能与其调节免疫细胞的活性和血清细胞因子TNF-α、IL-2、IL-6的水平有关。

Objective: To investigate the impacts of Polygonatum sibiricum polysaccharides(PSP) on the growth of PC-3 cell transplanted tumors in nude mice and key proteins in the phosphatidylinositol-3-hydroxy kinase/protein kinase B(PI3K/Akt) and nuclear factor-κB(NF-κB) signaling pathways. Methods: PC-3 androgen-independent prostate cancer cell line was selected and inoculated into the subcutaneous tissue of BALB/c nude mice to establish a model. The mice were divided into 5 groups, with 6 mice in each group, and the drug dosage was adjusted based on the conversion of body surface area between humans and nude mice. The control group was given physiological saline, cyclophosphamide(CTX) group was given 30 mg·kg^-1·d^-1, low-dose group was given PSP at 100 mg·kg^-1·d^-1, medium-dose group was given PSP at 200 mg·kg^-1·d^-1, high-dose group was given PSP at 400 mg·kg^-1·d^-1. Each nude mouse was administered 0.2 mL by gavage every other day for 30 days. Every three days, the body weight and tumor volume of the nude mice were recorded to analyze the effect of PSP on the growth and size of the transplanted tumors. Tumor volume was measured 4 hours after the last drug administration to calculate the tumor inhibition rate(TIR). Observation of cell apoptosis in tumor tissue using TdT-mediated dUTP nick end labeling(TUNEL) and flow cytometry. The proliferation activity of spleen cells was detected by Cell Counting Kit-8(CCK8). The activity of natural killer cells(NKc) was detected by lactate dehydrogenase(LDH) release assay. Using enzyme-linked immunosorbent assay(ELISA) to detect the levels of tumor necrosis factor-α, interleukin-2, and interleukin-6(TNF-α, IL-2, IL-6). Immunohistochemistry(IHC) was performed to analyze the expression of PI3K, Akt, NF-κB P65, and phosphorylated protein(p-PI3K, p-Akt, NF-κB p-P65) and caspase-3 in the transplanted tumor tissues of nude mice. Results: In the early stage of model establishment, nude mice were in good spirits, with free and frequent movement, stable food intake, and sensitive responses to external stimuli. However, with the growth of the tumor, all nude mice showed poor mental state, reduced activity and diet. Compared to the control group, TIR, apoptosis rate of PC-3 cells(ARoPc) in flow cytometry, and caspase-3 expression(IHC) increased in the CTX and PSP groups(P<0.05, P<0.001). ARoPc in TUNEL(CTX group), NK cell vitality(LDH), IL-6 levels(ELISA) in medium-and high-dose PSP groups, spleen cell proliferation(CCK8) in high-dose PSP, and TNF-α and IL-2 levels in PSP groups were also increased(P<0.05). Tumor volume and p-P65(IHC) in CTX and PSP groups, along with TNF-α and IL-2 levels(ELISA), p-PI3K, and p-Akt(IHC) in CTX, and p-PI3K and p-Akt(IHC) in medium-and high-dose PSP groups decreased(P<0.05). Compared to the CTX group, the PSP group showed increased tumor volume, NKc vitality(LDH), TNF-α, IL-2 levels(ELISA), and p-P65(IHC)(P<0.05). Spleen cell vitality(CCK8) and IL-6 levels(ELISA) in medium-and high-dose groups, as well as p-PI3K and p-Akt levels(IHC) in the low-dose PSP group, were also increased(P<0.05). Tumor IR and ARoPc(TUNEL, flow cytometry) in the PSP group, along with caspase-3 levels(IHC) in low-and medium-dose groups, were decreased(P<0.01, P<0.001). In medium-and high-dose PSP groups, tumor IR, ARoPc(TUNEL, flow cytometry), TNF-α, IL-2, IL-6 levels(ELISA), and caspase-3 levels(IHC) increased compared to the low-dose group(P<0.05), while tumor volume, p-PI3K, p-Akt(IHC) in these groups, and p-P65(high-dose) were decreased(P<0.05, P<0.001). The high-dose group showed increased tumor IR, ARoPc(TUNEL, flow cytometry), TNF-α, IL-2, IL-6 levels(ELISA), and caspase-3 levels(IHC) compared to the medium-dose group(P<0.05), with reductions in tumor volume and p-PI3K, p-Akt, and p-P65 levels(IHC)(P<0.05). Conclusion: PSP can inhibit the proliferation of PC-3 cells and induce their apoptosis. The mechanism may be related to inhibiting the PI3K/Akt and NF-κB signaling pathways by reducing the activation of PI3K, Akt, and P65 proteins, and promoting the expression of Caspase-3. It may be also related to regulating the activity of immune cells, and levels of TNF-α, IL-2, and IL-6.