槟榔碱通过断裂转导蛋白样增强子4对裸鼠骨肉瘤细胞自噬和凋亡的影响实验研究

Effect of arecoline on autophagy and apoptosisin nude mice osteosarcoma cells via TLE4

目的:探究槟榔碱通过断裂转导蛋白样增强子4(TLE4)对大鼠骨肉瘤细胞自噬和凋亡的影响。方法:选取SPF级裸鼠建立骨肉瘤移植瘤模型。将建模成功的60只裸鼠随机分为模型组,槟榔碱低、中、高剂量组(分别腹腔注射10、20、50 mg/kg槟榔碱),TLE4抑制剂组(腹腔注射3 mg/kg TAK-242)和顺铂组(腹腔注射2 mg/kg顺铂),每组10只,隔日注射1次,连续注射14 d。将未建模的10只裸鼠作为对照组。对照组和模型组每天注射同体积0.9%氯化钠溶液。观察移植瘤体积,取瘤体进行称重。实时荧光定量PCR(RT-PCR)检测骨肉瘤组织TLE4 mRNA表达,免疫组织化学染色检测病理学变化和TLE4蛋白表达。Western blot检测重组人自噬效应蛋白(Beclin-1)、微管相关蛋白1轻链3Ⅰ(LC3Ⅰ)、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、家蚕隔离体蛋白1(p62)、B淋巴细胞瘤-2(Bcl-2)、Bcl相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)蛋白表达。结果:与模型组比较,槟榔碱低、中、高剂量组以及TLE4抑制剂组和顺铂组TLE4、移植瘤体积和重量、Beclin-1、LC3Ⅱ/LC3Ⅰ、LC3Ⅱ、Bcl-2降低,Bax、Caspase-3、LC3Ⅰ升高(均P<0.05)。与槟榔碱低剂量组比较,槟榔碱中、高剂量组以及TLE4抑制剂组和顺铂组TLE4、移植瘤体积和重量、Beclin-1、LC3Ⅱ/LC3Ⅰ、LC3Ⅱ、Bcl-2降低,Bax、Caspase-3、P62、LC3Ⅰ升高(均P<0.05)。与槟榔碱中剂量组比较,槟榔碱高剂量组和TLE4抑制剂组TLE4、移植瘤体积和重量、LC3Ⅱ/LC3Ⅰ、Bcl-2降低,Bax、P62、LC3Ⅰ升高(均P<0.05)。与槟榔碱高剂量组比较,TLE4抑制剂组和顺铂组Beclin-1、LC3Ⅱ/LC3Ⅰ、LC3Ⅱ升高,P62、LC3Ⅰ、Bax、Caspase-3降低(均P<0.05)。TLE4抑制剂组和顺铂组以上指标比较差异无统计学意义(均P>0.05)。结论:槟榔碱可能通过抑制TLE4表达影响骨肉瘤细胞自噬和凋亡,从而抑制骨肉瘤的生长。

Objective:To investigate the effect of arecoline on autophagy and apoptosis in nude mice osteosarcoma cells via transducin-like enhancer of split 4(TLE4).Methods:Nude mouse xenograft osteosarcoma models were established using SPF-grade nude mice.Sixty successful model mice were randomly divided into model group,low-,medium-,and high-dose arecoline groups(intraperitoneal injection of 10,20,and 50 mg/kg arecoline,respectively),TLE4 inhibitor group(intraperitoneal injection of 3 mg/kg TAK-242),and cisplatin group(intraperitoneal injection of 2 mg/kg cisplatin).Each group contained 10 mice.Injections were given every other day for a total of 14 days.Ten unmodeled mice served as the control group.The control group and the model group received daily injections of an equal volume of 0.9%sodium chloride solution.The tumor volumes were observed,and the tumors were weighed at the end of the experiment.RT-qPCR was used to detect the expression of TLE4 mRNA in osteosarcoma tissue.Immunohistochemistry was used to examine the histopathological changes and TLE4 expression in the tissues.Western blot was employed to detect the protein expression levels of LC3Ⅰ,LC3Ⅱ,p62,Bax,Bcl-2,and Caspase-3.Results:Compared with the model group,the TLE4,tumor volume and weight,Beclin-1,LC3Ⅱ/LC3Ⅰratio,LC3Ⅱ,and Bcl-2 levels were significantly reduced,while the levels of Bax,Caspase-3,and LC3Ⅰwere significantly increased in the low-,medium-,and high-dose arecoline groups,the TLE4 inhibitor group,and the cisplatin group(all P<0.05).Compared with the low-dose arecoline group,the TLE4,tumor volume and weight,Beclin-1,LC3Ⅱ/LC3Ⅰratio,LC3Ⅱ,and Bcl-2 levels were significantly reduced,while the levels of Bax,Caspase-3,p62,and LC3Ⅰwere significantly increased in the medium-and high-dose arecoline groups,the TLE4 inhibitor group,and the cisplatin group(all P<0.05).Compared with the medium-dose arecoline group,the TLE4,tumor volume and weight,LC3Ⅱ/LC3Ⅰratio and Bcl-2 levels were significantly reduced,while the levels of Bax、P62、LC3Ⅰwere significantly increased in the high-dose arecoline group and the TLE4 inhibitor group(all P<0.05).Compared with the high-dose arecoline group,the Beclin-1、LC3Ⅱ/LC3Ⅰ、LC3Ⅱlevels were significantly increased,whereas the levels of P62、LC3Ⅰ、Bax、Caspase-3 were significantly decreased in the TLE4 inhibitor group and the Cisplatin group(all P<0.05).There was no significant difference between the TLE4 inhibitor group and the cisplatin group regarding these indicators(all P>0.05).Conclusion:Arecoline can inhibit the growth of osteosarcoma,and its mechanism may involve suppressing the expression of TLE4 in osteosarcoma cells,thereby regulating autophagy and apoptosis.