基于PI3K/AKT/eNOS信号通路探讨健脾利湿化瘀方抑制前列腺癌血管生成的实验研究

An experimental study on the inhibition of angiogenesis in prostate cancer by Jianpi Lishi Huayu Prescription based on PI3K/AKT/eNOS signaling pathway

[目的]在健脾利湿化瘀方临床疗效和前期实验的基础上,继续探讨健脾利湿化瘀方及其拆方在抑制人前列腺癌PC-3细胞荷瘤小鼠血管生成方面的作用,及其对PI3K/AKT/eNOS信号通路的影响。[方法]选取6~8周雄性裸鼠36只建立人前列腺癌荷瘤小鼠模型,随机分为空白对照组、西药组、全方组、君药组、臣药组和佐药组。实验各组干预14 d后处死取血称瘤质量,计算抑瘤率;使用酶联免疫吸附实验(ELISA)检测血清中血管内皮生长因子(VEGF)、内皮型一氧化氮合成酶(eNOS);免疫组化法观察各组瘤组织中VEGF、ANG1的表达;蛋白免疫印迹法(Western Blot)检测肿瘤组织中磷脂酰肌3-羟激酶(PI3K)、丝氨酸-苏氨酸蛋白激酶(AKT)、磷酸化的AKT(p-AKT)、eNOS蛋白表达水平。[结果]各给药组瘤体积、瘤质量均低于空白对照组(P<0.01),西药组、全方组、君药组、臣药组、佐药组的抑瘤率分别为53.95%、41.05%、19.21%、27.37%、31.32%,拆方组间比较,佐药组抑瘤效果较好,但无统计学差异(P>0.05);ELISA检测显示:各组VEGF、eNOS含量均低于空白对照组,其中西药组、全方组显著降低(P<0.05或P<0.01);Western Blot结果显示:各组PI3K、eNOS的蛋白表达均显著降低(P<0.05或P<0.01),AKT蛋白表达无显著变化(P>0.05),p-AKT呈降低趋势(P<0.05),其中西药组、全方组、佐药组呈显著下降(P<0.01)。[结论]尽管与西药对比抑瘤作用较弱,但中药健脾利湿化瘀方对前列腺癌在血管生成方面具有确切的抑制作用,尤其是具有化瘀消癥散结作用的佐药组,可能与姜黄、王不留行中某些有效成分能够下调PI3K、eNOS蛋白,影响AKT磷酸化,阻断PI3K/AKT/eNOS信号通路相关。

[Objective]On the basis of the clinical efficacy and preliminary experiments of Jianpi Lishi Huayu Prescription,the function of the prescription and the parts of this prescription in inhibiting angiogenesis of PC-3 tumor-bearing mice with human prostate cancer was explored and its effect on the PI3K/AKT/eNOS signaling pathway were further discussed.[Methods]The 36 male nude rats were selected to establish a tumor-bearing mouse model of human prostate cancer PC-3 cells.They were divided into the control group,Western medicine group,Jianpi Lishi Huayu Prescription group(full-prescription),monarch group(Huangqi,Gancao),minister group(Ciwujia,Buguzhi)and assistant group(Jianghuang,Wangbuliuxing).After 14 days,sacrificed and blood was taken to weigh the tumor and the tumor inhibition rate was calculated.Serum levels of VEGF and eNOS were detected by ELISA.Immunohistochemical method was used to observe the expression of VEGF and ANG1.Western Blot was used to detect the expression levels of PI3K,AKT,p-Akt and eNOS in tumor tissues.[Results]Tumor volume and weight of each administration group was lower than that of the control group(P<0.01).Tumor inhibition rates of each group was 53.95%,41.05%,19.21%,27.37% and 31.32%.Among the different part group of the prescription,the assistant group has better anti-tumor effect,but there is no statistical difference(P>0.05).ELISA showed that the levels of VEGF and ANG1 in each group were lower than control group,Western medicine group and the full-prescription group were significantly decreased(P<0.05,P<0.01).Western Blot showed that the protein expression of PI3K and eNOS in each group was significantly decreased(P<0.05,P<0.01),while the protein expression of AKT showed no significant change(P>0.05),p-AKT showed a decreasing trend(P<0.05),in which the Western medicine group,the full-prescription group and the admixture group showed significant decreases(P<0.01).[Conclusion]Compared with Western medicine,Jianpi Lishi Huayu Prescription has weaker effect on anti-tumor,while Jianpi Lishi Huayu Prescription has a definite inhibitory effect on the angiogenesis of prostate cancer,especially in assistant groupwith the function of removing blood stasis and dispelling syndrome.This may be related to the fact that certain active ingredients in Jianghuang and Wangbuliu can down-regulate PI3K and eNOS proteins,influence AKT phosphorylation,and block PI3K/AKT/eNOS signaling pathways.