基于AKT/mTOR/p70S6K信号通路探讨双海散对卵巢癌裸鼠的影响OA

Objective To investigate the effects of Shuanghai Powder on ovarian cancer in nude mice via the protein kinase B(AKT)/mammalian target of rapamycin(mTOR)/p70 ribosomal protein S6 kinase(p70S6K)signaling pathway.Methods Fifty-four female BALB/c-nu/nu homozygous nude mice were randomly divided into a blank group(group K,n=9)and a modeling group(n=45).An ovarian cancer animal model was established by axillary injection of the human ovarian cancer cell line A2780.The 45 successfully modeled mice were randomly assigned into five groups(n=9 each):model group(group M),Shuanghai Powder low-dose group(SH-L,3.69 g/kg),Shuanghai Powder medium-dose group(SH-M,7.38 g/kg),Shuanghai Powder high-dose group(SH-H,14.76 g/kg),and cisplatin group(3 mg/kg).All groups were treated for 14 days.Body weight and tumor volume were measured once every two days.After the last administration,the mice were sacrificed,and tumors were excised and weighed.HE staining was used to observe morphological changes of ovarian cancer tissues.Immunohistochemistry(IHC)was performed to detect the protein expression intensity of phosphorylated AKT(p-AKT),phosphorylated mTOR(p-mTOR),and phosphorylated p70S6K(p-p70S6K)in mouse ovarian cancer tissues.Western blot was used to measure protein expression of AKT,p-AKT,mTOR,p-mTOR,p70S6K,p-p70S6K,B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cysteine-aspartate specific protease 3(Caspase-3),and phosphorylated Bcl-2-associated death promoter(p-Bad),while RT-qPCR was employed to determine mRNA expression of AKT,mTOR,p70S6K,Bcl-2,myeloid cell leukemia-1(Mcl-1),Bax,and Bcl-2 interacting mediator of cell death(Bim).Results During the treatment period,the survival rate of nude mice in each Shuanghai Powder group was higher than that in the cisplatin group.Compared with group K,the body weight in the cisplatin group was significantly decreased from day 3 onward(P<0.001),and that in group M was significantly decreased from day 9 onward(P<0.05).Compared with group M,the body weight in the cisplatin group was significantly decreased from day 5 onward(P<0.001),while no significant difference was observed in the body weight of each Shuanghai Powder group(P>0.05).Tumor weight was reduced in both the SH-H group and the cisplatin group(P<0.05 and P<0.01,respectively).Tumor volume was significantly reduced in the cisplatin group from day 7 onward and in the SH-H group from day 9 onward(P<0.05 and P<0.01,respectively).Obvious histopathological changes were observed in tumor tissues of group M,and improvements were observed to varying degrees after intervention in each treatment group.IHC results showed that,compared with group M,the protein expression of p-AKT,p-mTOR,and p-p70S6K in the SH-H group was significantly reduced(P<0.05,P<0.01).Western blot showed that compared with group M,there were no significant differences in the protein expression of AKT,mTOR,and p70S6K among the treatment groups(P>0.05).In the SH-M and SH-H groups,the protein expression of p-AKT,p-mTOR,and p-p70S6K,their ratios to the corresponding total proteins,as well as Bcl-2 and p-Bad protein expression decreased(P<0.05,P<0.01),while that of Bax and Caspase-3 increased(P<0.01).In the SH-L group,only the protein expression of p-p70S6K and p-Bad decreased(P<0.05,P<0.01),and that of Caspase-3 increased(P<0.01).RT-qPCR results showed that compared with group M,there were no significant differences in the mRNA expression levels of AKT,mTOR,and p70S6K among the treatment groups(P>0.05).In the SH-M group,only Bcl-2 mRNA expression decreased(P<0.01),and Bim mRNA expression increased(P<0.01).In the SH-H group,Bcl-2 and Mcl-1 mRNA expression decreased(P<0.01),while Bax and Bim mRNA expression increased(P<0.05,P<0.01).In the SH-L group,no significant differences were observed in any of the indicators(P>0.05).Conclusion Shuanghai Powder can inhibit tumor growth in ovarian cancer-bearing nude mice,and its mechanism may be related to inhibiting the phosphorylation of the AKT/mTOR/p70S6K pathway and regulating the expression of apoptosis-related proteins and genes.