基于IGF/MMP信号通路研究健脾利湿化瘀方有效拆方对前列腺癌细胞侵袭、迁移的影响

Influence of the effective decomposed in Nourishing Spleen Removing Dampness and Eliminating Blood-stasis Decoction on the invasion,migration and apoptosis of the human prostate cancer cells based on the IGF/MMP signaling pathway

关键词:  健脾利湿化瘀方  前列腺癌  细胞增殖  细胞迁移

Key words:  Nourishing Spleen Removing Dampness and Eliminating Blood-stasis Decoction  prostate cancer  cell proliferation  cell migration

摘要:

[目的] 在健脾利湿化瘀方临床疗效的基础上，结合前期动物实验研究，初步运用前列腺癌细胞增殖实验初筛健脾利湿化瘀方的最佳作用拆方；再基于胰岛素样生长因子（IGF）/基质金属蛋白酶（MMP）信号通路探究作用机制，为进一步研究全方作用机制奠定基础。[方法] 用四甲基噻唑蓝（MTT）法检测健脾利湿化瘀方各拆方组含药血清，包括健脾扶正组（黄芪、刺五加、补骨脂）、化瘀散结组（姜黄、大黄、王不留行）、清热解毒组（白英、蛇六谷、车前草），对前列腺癌PC3和DU-145细胞的增殖抑制率，并取化瘀散结组药物作为进一步研究用药；通过Transwell、划痕实验评估化瘀散结组药物影响细胞的侵袭、迁移情况；用实时荧光定量聚合酶链式反应（Real-Time PCR）方法检测前列腺活化间质细胞中基质细胞衍生因子-1（SDF-1）、胰岛素样生长因子-1（IGF-1）、MMP、纤维粘连蛋白（FN）基因的mRNA转录水平的变化情况；用蛋白免疫印迹（Western Blot）法检测化瘀散结组药物对前列腺活化间质细胞系胞SDF-1、IGF-1、MMP、FN蛋白表达的影响。[结果] 不同拆方组中，化瘀散结药物组对PC-3、DU-145抑制率最高，且随着药物浓度增加，分别从18.77%、58.89%增加至87.30%，呈一定剂量依赖性（P<0.05）；细胞迁移实验中化瘀散结组药物与阴性对照组相比，DU-145侵袭细胞数分别为（7.33±2.51）、（22.00±2.65）个（P=0.002），PC-3侵袭细胞数为（3.67±0.58）、（22.67±7.77）个（P<0.05），差异均有统计学意义；划痕实验结果显示化瘀散结组药物PC-3、DU-145细胞24 h迁移距离分别为（330.12±13.15）、（453.34±42.74） μm，阴性对照组迁移距离分别为（525.40±6.58）、（759.75±40.88） μm，P值分别为0.01、0.018，差异均有统计学意义。化瘀散结组药物明显降低Vimentin、SM22、SMM的蛋白表达，而Calcyclin的表达与阴性对照组比较差异无统计学意义。化瘀散结组药物对IGF-1、MMP基因相对表达量均在高剂量组中较低，降低SDF-1、IGF、MMP蛋白表达。[结论] 化瘀散结药物组能够有效地抑制前列腺癌细胞的增殖、侵袭、迁移，其机制可能与化瘀散结药物下调IGF/MMP信号通路有关。

Abstract:

[Objective] On the basis of the clinical efficacy of the Nourishing Spleen Removing Dampness and Eliminating Blood-stasis Decoction,combined with the previous animal experiment research,this paper preliminarily screened the best action of Nourishing Spleen Removing Dampness and Eliminating Blood-stasis Decoction through the proliferation experiment of prostate cancer cells. Based on the IGF/MMP signaling pathway,the mechanism of action was explored to lay a foundation for further research on the mechanism of action of all directions.[Methods] We separated compound prescription and then use MTT method to detect cells proliferation inhibition rate from the Jianpi Fuzheng group (Astragalus Nembranaceus,Acanthopanax,Malaytea Scurfpea fruit),the Huayu Sanjie group (Turmeric,Rhubarb,Cowherb seed) and the Qingre Jiedu group (Lyratum Thunb,Snake Six Valley,Plantain). Then through the PC-3 and Du-145 cells proliferation inhibition rate,we choose the best drug part as the further study. Cell invasion and migration were evaluated by transwell and scratch test. The mRNA transcription levels of SDF-1,IGF-1,MMPS and FN genes mRNA were detected by Real-Time PCR. Western blot was used to detect the effects of the Huayu Sanjie group on the protein expressions of SDF-1,IGF-1,MMPS and FN in prostatic activated stromal cell lines.[Results] Among the different un-prescription groups,the inhibition rate of PC-3 and DU-145 was the highest in the removing stasis group,and it increased from 18.77%,58.89% to 87.30%. With the increase of drug concentration,it shows a dose dependence (P<0.05). In the cell migration experiment,compared with the control group,the number of DU-145 invasion cells was 7.33±2.51,22.00±2.65 (P=0.002),and the number of PC-3 invasion cells was 3.67±0.58,22.67±7.77(P<0.05),with statistically significant differences. The scratch test results showed that the migration distance of PC-3 and DU-145 cells in the removing stasis group was (330.12+13.15),(453.34±42.74) μm,and that of the control group was (525.40±6.58),(759.75±40.88) μm,the P values were 0.01 and 0.018 relatively,and the differences were statistically significant. The expression of Vimentin,SM22 and SMM was significantly decreased in the Huayu Sanjie group,while the expression of Calcyclin was not statistically different from that of the control group. The relative expression levels of IGF-1 and MMP genes in the Huayu Sanjie group were lower than those in the high-dose group. Western blot also showed that the drugs in the Huayu Sanjie group reduced the protein expressions of SDF-1,IGF and MMP.[Conclusion] The Huayu Sanjie drugs group can effectively inhibit the proliferation,invasion and migration of prostate cancer cells,and the mechanism may be related to the down-regulation of IGF/MMP signaling pathway by Huayu Sanjie drugs.