红景天苷对人肺癌A549细胞裸鼠移植瘤生长的影响及对MAPK/ERK1/2信号通路的调控作用

Effect of Salidroside on Growth of Human Lung Cancer A549 Cell Transplanted Tumor in Nude Mice and Its Regulation on MAPK/ERK1/2 Signaling Pathway

目的:研究红景天苷对人肺癌A549细胞裸鼠移植瘤生长的作用,并研究其对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)/细胞外信号调节激酶(extracellular signal regulated kinase,ERK1/2)信号通路的调控作用。方法:培养人肺癌A549细胞制备浓度为3×10^7 mL^-1的细胞悬液,取200μL皮下注射于裸鼠右前肢腋窝皮下。待注射部位出现肿瘤结节时,为建模成功。挑出肿瘤结节大小均为5 mm左右裸鼠共40只,随机分为5组:阴性对照组、阳性对照组、红景天苷高剂量组、红景天苷中剂量组和红景天苷低剂量组,每组8只。阳性对照组裸鼠腹腔注射3 mg·kg^-1顺铂,每3天注射1次;红景天苷高剂量组、中剂量组、低剂量组裸鼠分别腹腔注射红景天苷50 mg·kg^-1、25 mg·kg^-1、12.5 mg·kg^-1,每天1次;阴性对照组裸鼠注射等体积的生理盐水,每天1次。连续给药2周。取肿瘤组织称取质量,计算瘤体生长抑制率;通过苏木精伊红(HE)染色观察瘤体组织病理变化;通过末端标记法观察肿瘤细胞凋亡情况并计算凋亡指数;通过RT-qPCR检测肿瘤细胞中ERK1/2、细胞周期蛋白D1(cyclinD1)、c-Myc、Bcl-2和Bcl-2相关蛋白X(Bax)mRNA相对表达量;通过蛋白免疫印迹检测肿瘤细胞中ERK1/2、cyclinD1、c-Myc、Bcl-2和Bax蛋白表达水平及p-ERK1/2水平。结果:各组瘤质量比较显示:阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05);其中组间两两比较显示:阴性对照组>红景天苷低剂量组>阳性对照组和红景天苷中剂量组>红景天苷高剂量组。各组抑瘤率比较显示:阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05);组间两两比较显示:红景天苷高剂量组>阳性对照组和红景天苷中剂量组>红景天苷低剂量组>阴性对照组。HE染色结果比较显示:阴性对照组肿瘤组织可见明显异型细胞,细胞核分裂象多,细胞密度大且排列无序,阳性对照组和红景天苷各剂量组表现出核分裂象减少,细胞死亡,其中红景天苷高剂量组与其他组比较,细胞核分裂象最少,细胞密度最低、坏死最严重。各组细胞凋亡数和凋亡指数比较显示:阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05),其中组间两两比较显示:红景天苷高剂量组>阳性对照组和红景天苷中剂量组>红景天苷低剂量组>阴性对照组。RT-qPCR和蛋白免疫印迹结果比较显示,各组ERK1/2 mRNA和蛋白表达量比较,差异无统计学意义(P>0.05);cyclinD1、c-Myc、Bcl-2 mRNA和蛋白表达水平及p-ERK1/2水平结果显示,阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05),其中组间两两比较显示:阴性对照组>红景天苷低剂量组>阳性对照组和红景天苷中剂量组>红景天苷高剂量组。Bax mRNA和蛋白表达水平结果显示,阳性对照组和红景天苷中剂量组比较,差异无统计学意义(P>0.05),其余各组间比较,差异均有统计学意义(P<0.05),两两比较:红景天苷高剂量组>阳性对照组和红景天苷中剂量组>红景天苷低剂量组>阴性对照组。结论:红景天苷可抑制人肺癌A549细胞裸鼠移植瘤的生长,破坏肿瘤细胞结构,促进肿瘤细胞凋亡,推测其机制可能与抑制MAPK/ERK1/2信号通路,上调Bax mRNA和蛋白表达水平,下调cyclinD1、c-Myc、Bcl-2 mRNA和蛋白表达水平,降低p-ERK1/2水平有关。

Objective:To study the effect of salidroside on the growth of transplanted tumor of human lung cancer A549 cells in nude mice and its regulation on mitogen activated protein kinase(MAPK)/extracellular signal regulated kinase(ERK1/2)signal pathway.Method:Human lung cancer A549 cells were cultured to prepare a cell suspension with a concentration of 3×10^7 mL^-1.200μL of cell suspension was subcutaneously injected into the right forelimb armpit of nude mice to the dorsal side.When tumor nodules appear at the injection site,the modeling is successful.Forty nude mice with tumor nodules of about 5 mm in size were selected and randomly divided into 5 groups:negative control group,positive control group,salidroside high dose group,salidroside medium dose group and salidroside low dose group,with 8 mice in each group.Positive control group nude mice received intraperitoneal injection of 3 mg·kg^-1 cisplatin once every 3 days.The nude mice in high dose group,medium dose group and low dose group were injected intraperitoneally with salidroside 50 mg·kg^-1,25 mg·kg^-1 and 12.5 mg·kg^-1 once a day respectively.Nude mice in negative control group were injected with equal volume of normal saline once a day.Continuous administration for 2 weeks.The tumor tissue was weighed and the tumor growth inhibition rate was calculated.Histopathological changes of tumor were observed by hematoxylin eosin(HE)staining.The apoptosis of tumor cells was observed by end labeling method and the apoptosis index was calculated.The relative expression of ERK1/2,cyclinD1,c-Myc,Bcl-2 and Bcl-2 related protein X(Bax)mRNA in tumor cells was detected by RT-qPCR.The expression levels of ERK1/2,cyclinD1,c-Myc,Bcl-2,Bax and p-ERK1/2 in tumor cells were detected by western blot.Result:The comparison of tumor quality in each group showed that there was no significant difference between positive control group and salidroside medium dose group(P>0.05),while there was significant difference between other groups(P<0.05).The comparison between the two groups shows:negative control group>salidroside low dose group>positive control group and salidroside medium dose group>salidroside high dose group.The comparison of tumor inhibition rates among the groups showed that there was no significant difference between the positive control group and the salidroside medium dose group(P>0.05),while there was significant difference between the other groups(P<0.05).The comparison between the two groups showed that:salidroside high-dose group>positive control group and salidroside medium-dose group>salidroside low-dose group>negative control group.Comparison of HE staining results showed that the negative control group had obvious atypical cells in tumor tissue,with many mitotic phases,large cell density and disordered arrangement.The positive control group and salidroside dosage groups showed decreased mitotic phases and cell death.Compared with other groups,salidroside high dosage group had the least mitotic phases,the lowest cell density and the most serious necrosis.The comparison of apoptosis number and apoptosis index in each group showed that there was no significant difference between the positive control group and the salidroside medium dose group(P>0.05),while there was significant difference between the other groups(P<0.05).the comparison between the two groups showed that the salidroside high dose group>the positive control group and the salidroside medium dose group>the salidroside low dose group>the negative control group.he comparison of RT-qPCR and western blot results showed that there was no significant difference in ERK1/2 mRNA and protein expression between the groups(P>0.05).The results of cyclinD1,c-Myc,Bcl-2 mRNA and protein expression level and p-ERK1/2 level showed that there was no significant difference between positive control group and salidroside medium dose group(P>0.05),while there was significant difference between other groups(P<0.05).the comparison between the two groups showed:negative control group>salidroside low dose group>positive control group and salidroside medium dose group>salidroside high dose group.The results of Bax mRNA and protein expression showed that there was no significant difference between the positive control group and the salidroside medium dose group(P>0.05),while there was significant difference between the other groups(P<0.05).the comparison between the two groups was:salidroside high dose group>positive control group and salidroside medium dose group>salidroside low dose group>negative control group.Conclusion:Salidroside can inhibit the growth of transplanted tumor of human lung cancer A549 cells in nude mice,destroy the structure of tumor cells,and promote apoptosis of tumor cells.It is speculated that its mechanism may be related to inhibiting MAPK/ERK1/2 signal pathway,up-regulating Bax mRNA and protein expression level,down-regulating cyclinD1,c-Myc,Bcl-2 mRNA and protein expression level,and reducing p-ERK1/2 level.