基于网络药理学与实验研究探讨β-葡聚糖治疗S180腹水瘤的作用机制

Mechanism of β-glucan for S180 ascites tumor based on network pharmacology and experimental study

目的基于网络药理学对β-葡聚糖治疗S180腹水瘤的作用靶点进行筛选,并用S180腹水瘤小鼠模型对相关作用靶点进行实验验证。方法①通过Swiss Target Prediction、Geen Cards及OMIM数据库对β-葡聚糖活性成分进行筛选并对S180腹水瘤相关靶点进行预测;运用String数据库构建蛋白互作网络图,并结合生物信息分析软件(Cytoscape 3.8.3)构建成分靶点互作网,预测β-葡聚糖的作用靶点;利用R Studio进行GO及KEGG信号通路分析。②采用CCK-8法检测β-葡聚糖对S180细胞活性的影响。③建立S180腹水瘤皮下实体瘤模型,并给予顺铂(腹腔注射顺铂0.2 mg·kg^(-1))、不同剂量β-葡聚糖(50 mg·kg^(-1)、100 mg·kg^(-1)、200 mg·kg^(-1)灌胃),21 d后处死小鼠,对各组抑瘤率及脾脏指数进行比较;采用ELISA法检测荷瘤小鼠血清中TNF-α、IFN-γ、IL-2、IL-4、IL-6水平;Western blot法检测Bcl-2、Bax、VEGFA、JAK2及STAT3蛋白表达,对富集得到的通路进行验证,研究β-葡聚糖治疗肿瘤的作用机制。结果①收集得到β-葡聚糖作用靶点33个与腹水瘤靶点3116个,并得到交集靶点12个,对交集靶点进行蛋白互作发现VEGFA、STAT3等8个关键靶点,KEGG分析发现β-葡聚糖通过PI3K-Akt、MAPK等炎性通路治疗S180腹水瘤。②CCK-8实验结果显示β-葡聚糖对S180细胞无直接抑制作用。③体内实验显示,与模型组比较,β-葡聚糖中、高剂量组小鼠瘤质量均明显减少(P<0.05,P<0.01)。与模型组比较,β-葡聚糖能明显提高小鼠的IL-2、TNF-α、IFN-γ水平,降低IL-4、IL-6的水平。Western blot结果显示β-葡聚糖能明显上调Bax表达并下调Bcl-2表达(P<0.05,P<0.01),引起VEGF/JAK2/STAT3的表达明显下调(P<0.05,P<0.01)。结论β-葡聚糖可通过作用于JAK与Bcl-2靶点干预PI3K-Akt通路且能够影响VEGF/JAK/STAT3调控EGFR通路,抑制肿瘤的增长,促进肿瘤的凋亡,从而对腹水瘤发挥治疗作用。

Objective To screen the targets ofβ-glucan for S180 ascites tumor by network pharmacology and to verify the related action targets on the S180 ascites tumor mouse model by experimental.Methods①Theβ-glucan active ingredients were screened and S180 ascites tumor-related targets were predicted by Swiss Target Predictio,Geen Cards and OMIM databases,respectively.The protein-protein interaction network(PPI)was constructed with STRING database,and the component-target interaction network was constructed by the biological information analysis software(Cytoscape 3.8.3)to predict the action targets of β-glucan.GO and KEGG signaling pathways were analyzed with R Studio.②CCK-8 method was used to detect the effect of β-glucan on S180 cell activity.③The S180 ascites tumor model was established as a subcutaneous solid tumor.The mice were administrated cisplatin(intraperitoneal injection of cisplatin 0.2 mg·kg^(-1))and different doses of β-glucan(50,100,200 mg·kg^(-1) intragastrically),and sacrificed 21 days later.The tumor inhibition rate and spleen index were compared among the groups.The serum levels of TNF-α,IFN-γ,IL-2,IL-4,and IL-6 in tumor-bearing mice were detected by ELISA.Western blot was used to detect the expression of Bcl-2,Bax,VEGFA,JAK2 and STAT3 proteins to validate the enrichmentobtained pathway and determine the mechanism of β-glucan in the treatment of tumors.Results①Among the 33 β-glucan action targets and 3116 ascites tumor targets collected,12 were intersecting targets,and 8 key targets such as VEGFA and STAT3 were found through protein interaction with the intersecting targets.KEGG analysis showed thatβ-glucan treated S180 ascites tumor through PI3K-Akt,MAPK and other inflammatory pathways.②CCK-8 experiment showed that β-glucan had no direct inhibition on S180 cells.③In vivo experiments showed that compared with the model group,the tumor mass of the β-glucan medium and high dose groups were significantly reduced(P<0.05,P<0.01).β-glucan obviously increased the levels of IL-2,TNF-α,and IFN-γ,while reduced the levels of IL-4 and IL-6.Western blot showed thatβ-glucan significantly up-regulated the expression of Bax,down-regulated the expression of Bcl-2,and VEGF/JAK2/STAT3(P<0.05,P<0.01).Conclusion β-glucan can interfere with PI3K-Akt pathway by acting on JAK and Bcl-2 targets and affect VEGF/JAK/STAT3 to regulate EGFR pathway,inhibit tumor growth and promote tumor apoptosis,thereby exerting therapeutic effects on ascite tumors.