基于蛋白质组学探讨准噶尔阿魏抗结肠癌的作用机制

Investigation on anti-colon cancer action mechanism of Junggar Ferula based on proteomics

目的通过动物实验分析准噶尔阿魏乙醇提取物(简称醇提物)的体内抗结肠癌活性,基于蛋白质组学探讨准噶尔阿魏醇提物治疗结肠癌可能涉及的药理作用机制。方法建立结肠癌CT26荷瘤小鼠模型,随机分为正常对照组、模型组、顺铂组及准噶尔阿魏醇提物低、中、高剂量组,连续给药12 d,取材并计算各组抑瘤率及脏器指数。取模型组和抑瘤率>40%的准噶尔阿魏醇提物剂量组小鼠肿瘤组织标本,利用串联质谱标记(TMT)技术筛选准噶尔阿魏醇提物组与模型组间的差异蛋白,分析涉及的生物功能、关键作用靶标及信号通路。结果准噶尔阿魏醇提物高、中、低剂量组的抑瘤率分别为(63.82±4.01)%、(38.17±3.78)%、(36.25±3.43)%,准噶尔阿魏醇提物高剂量组的抑瘤率明显高于准噶尔阿魏醇提物中、低剂量组(P<0.05)。与正常对照组相比,顺铂组及准噶尔阿魏醇提物高、中、低剂量组小鼠的脾重、肾重及胸腺重均明显减小(P<0.05);准噶尔阿魏醇提物高、中、低剂量组间小鼠的脾重、肾重及胸腺重比较,差异均无统计学意义(P>0.05)。与正常对照组相比,准噶尔阿魏醇提物高剂量组小鼠的脾指数、胸腺指数明显减小(P<0.05),肾指数差异无统计学意义(P>0.05);与正常对照组相比,准噶尔阿魏醇提物中剂量组小鼠的脾指数、肾指数、胸腺指数均明显减小(P<0.05);与正常对照组相比,准噶尔阿魏醇提物低剂量组小鼠的肾指数、胸腺指数明显减小(P<0.05),脾指数差异无统计学意义(P>0.05);准噶尔阿魏醇提物高剂量组小鼠的肾指数明显高于准噶尔阿魏醇提物中、低剂量组(P<0.05)。蛋白质组学筛选出642个差异蛋白,其中FN1、FLNC、HSPH1、HSP90AA1等蛋白排名在前10;COG分析结果显示差异蛋白共参与了582个生物功能,主要涉及翻译后修饰及蛋白转运(15.8%)、信号转导机制(15.1%)、转录(9.1%)等;KEGG富集分析显示差异蛋白参与的信号通路主要涉及PI3K-Akt信号通路、细胞外基质-受体相互作用、癌症中的蛋白聚糖、补体和凝血级联反应、TNF信号通路、AMPK信号通路、甘氨酸丝氨酸和苏氨酸代谢等。结论动物实验显示准噶尔阿魏醇提物的抗结肠癌活性好,蛋白质组学分析揭示了准噶尔阿魏治疗结肠癌可能的关键靶标及分子机制,FN1、FLNC、HSPH1、HSP90AA1等可能是其中的关键蛋白。

Objective To analyze the in vivo anti-colon cancer activity of Junggar Ferula ethanol extract by the animal experiment,and to explore its possibly involved pharmacological mechanisms in treating colon cancer based on proteomics.Methods The mouse model of colon cancer CT26 bearing tumor was established,randomly divided into the normal control group,model group,cisplatin group and Junggar Ferula ethanol extract low,medium and high doses groups.Continuous medication lasted for 12 d,The samples were taken,and the tumor inhibition rate and organ index were calculated in each group.The mice tumor tissue samples were taken from the model group and the Junggar Ferula ethanol extract dose groups with the tumor inhibition rate>40%.The tandem mass labeling(TMT)technology was used to screen the differential proteins between the Junggar Ferula ethanol extract group and the model group,the involved biological functions,key action targets and signaling pathways were analyzed.Results The tumor inhibition rates of the Junggar Ferula ethanol extract high,medium and low dose groups were(63.82±4.01)%,(38.17±3.78)%and(36.25±3.43)%respectively.The tumor inhibition rate of the Junggar Ferula ethanol extract high dose group was significantly higher than that of the Junggar Ferula ethanol extract medium and low dose groups(P<0.05).Compared with the normal control group,the spleen weight,kidney weigh and thymus weight of mice in the cisplatin group and the Junggar Ferula ethanol extract high,medium and low dose groups were significantly decreased(P<0.05).There was no statistically significant difference in the spleen weight,kidney weight and thymus weight among the Junggar Ferula ethanol extract high,medium and low dose groups(P>0.05).The spleen index and thymus index in the Junggar Ferula ethanol extract high dose group was significantly decreased(P<0.05),while the difference in kidney index was not statistically significant(P>0.05);compared with the normal control group,the spleen index,kidney index,and thymus index of mice in the Junggar Ferula ethanol extract medium dose group were significantly decrease(P<0.05);compared with the normal control group,the kidney index and thymus index the Junggar Ferula ethanol extract low dose group were significantly decreased(P<0.05),while the difference in the spleen index was not statistically significant(P>0.05);the renal index of mice in the Junggar Ferula ethanol extract high dose group was significantly higher than that in the Junggar Ferula ethanol extract medium and low dose groups(P<0.05).Proteomic screened 642 differential proteins,in which FN1,FLNC,HSPH1,HSP90AA1 and other proteins ranked in the top 10;the COG analysis results showed that differential proteins were involved in 582 biological functions,mainly involving post-translational modifications and protein transport(15.8%),signal transduction mechanisms(15.1%),transcription(9.1%),etc;the KEGG enrichment analysis showed that the signaling pathways involved in differential proteins mainly involve PI3K-Akt signaling pathway,extracellular matrix receptor interaction,proteoglycans in cancer,complement and coagulation cascade reactions,TNF signaling pathway,AMPK signaling pathway,glycine serine and threonine metabolism,etc.Conclusion The animal experiment shows that the anti-colon cancer activity of Junggar Ferula ethanol extract is good.The proteomic analysis reveals the possible key target and molecular mechanism of Junggar Ferula ethanol extract in treating colorectal cancer.FN1,FLNC,HSPH1 and HSP90AA1 may be its key proteins.