山奈酚阻滞JAK2/STAT3通路抑制宫颈癌细胞的增殖及糖酵解的作用研究

Inhibition of Proliferation and Glycolysis of Cervical Cancer Cells by Kaempferol Blocking JAK2/STAT3 Pathway

探究山奈酚对人宫颈癌细胞增殖、黏附、糖酵解及Janus激酶2(JAK2)/信号转导和转录启动因子3(STAT3)信号通路的调控作用。体外培养人宫颈癌HeLa细胞,分为对照组(不做干预)、10、20、40、80μmol/L山奈酚组,采用细胞计数试剂盒-8(CCK-8)法筛选出细胞活力接近50%的山奈酚(40μmol/L)用于后续实验。而后取对数生长期的HeLa细胞,分为对照组、实验组(40μmol/L山奈酚)、阳性药物组(20μg/mL 5-氟尿嘧啶)、抑制剂组(40μmol/L山奈酚+50μmol/L JAK2/STAT3信号通路抑制剂AG490)和激活剂组(40μmol/L山奈酚+0.5μmol/L JAK2/STAT3信号通路激活剂Colivelin)。干预24 h,采用5-乙炔基-2′脱氧尿嘧啶核苷(EdU)法、细胞黏附实验、乳酸检测试剂盒、葡萄糖检测试剂盒、蛋白免疫印迹(WB)法对细胞增殖、黏附能力、乳酸含量、葡萄糖消耗水平及JAK2/STAT3信号通路关键蛋白表达水平进行分析。40μmol/L山奈酚干预后细胞活力接近50%,故选取此浓度进行后续实验。与对照组相比,实验组和阳性药物组细胞增殖率、黏附数、乳酸含量、葡萄糖消耗水平、p-JAK2及p-STAT3蛋白表达量均显著降低(P<0.05);与实验组相比,抑制剂组细胞增殖率、黏附数、乳酸含量、葡萄糖消耗水平、p-JAK2及p-STAT3蛋白表达量进一步显著降低(P<0.05),而激活剂组上述指标变化趋势与抑制剂组相反(P<0.05)。山奈酚可通过阻断JAK2/STAT3通路的信号转导从而抑制人宫颈癌HeLa细胞的增殖、黏附及糖酵解等生物学行为。

To explore the regulatory effects of kaempferol on the proliferation,adhesion,glycolysis and Janus kinase 2(JAK2)signal transducer and activator of transcription 3(STAT3)signaling pathway of human cervical cancer cells,human cervical cancer HeLa cells were cultured in vitro and divided into control group(without intervention),10,20,40,80μmol/L kaempferol group,Cell counting kit⁃8(CCK⁃8)was used to detect cell viability,and kaempferol(40μmol/L)for subsequent experiments.Then,HeLa cells in logarithmic growth phase were divided into control group and experimental group(40μmol/L kaempferol),positive drug group(20μg/mL 5⁃fluorouracil),inhibitor group(40μmol/L kaempferol+50μmol/L JAK2/STAT3 signal pathway inhibitor AG490)and activator group(40μmol/L kaempferol+0.5μmol/L JAK2/STAT3 signaling pathway activator Colivelin).After 24 hours of intervention,5⁃acetyl⁃2′deoxyuridine(EdU)method,cell adhesion test,lactic acid test kit,glucose test kit,and protein immunoblotting(WB)method were used to analyze cell proliferation,adhesion ability,lactate content,glucose consumption level,and JAK2/STAT3 signal pathway related protein expression level.40μmol/L kaempferol caused nearly 50%cell viability after intervention,so this concentration was chosen for subsequent experiments.Cell proliferation rate,adhesion number,lactate content,glucose consumption level,protein expression levels of p⁃JAK2 and p⁃STAT3 were significantly decreased in the experimental and positive drug groups compared with the control group(P<0.05);Cell proliferation rate,adhesion number,lactate content,glucose consumption levels p⁃JAK2 and p⁃STAT3 protein expression levels were further significantly reduced in the inhibitor group compared to the experimental group(P<0.05);However,the change trend of the above indicators in the activator group was opposite to that in the inhibitor group(P<0.05).Kaempferol can inhibit the proliferation,adhesion,glycolysis and other biological behaviors of human cervical cancer HeLa cells by blocking the signal transduction of JAK2/STAT3 pathway.