Study On The Mechanism And Significance Of Capsaicin Affecting Autophagy In Glioma

Objective:To explore the role and relationship of capsaicin and autophagy in glioma and its mechanism and clinical significance.Methods:1.Immunohistochemical Elivision~TMM plus method was used to detect the expression of autophagy-related proteins SQSTM1/p62,LC3B and mTOR in 151 human glioma tissues(the experimental group)and 20 cases of edema brain tissues(the control group).Combined with retrospectively clinical-pathologic parameters to analyze the relationship between the above factors and prognosis of patients(overall survival time,recurrence-free survival time).2.Experimental group 1:The expressions of autophagy-related proteins SQSTM1/p62and Beclin1 were observed in human glioblastoma T98G and human astrocytoma U373cell lines in the use of autophagy inducing agent rapamycin(control group),capsaicin and rapamycin and capsaicin combined,respectively.3.Experimental group 2:To construct mTOR expression plasmid that inhibit autophagy,the experiment was divided into(1)blank control group(2)empty carrier group(3)mTOR plasmid group(4)capsaicin group(5)mTOR plasmid and capsaicin group.Then T98G and U373 cell lines were transiently transfected with Lpofectamin3000.CCK-8 kit method was used to detect the proliferation of cells in the five groups;western blotting was used to detect autophagy-related proteins mTOR,SQSTM1/p62,ATG4B,ATG5,and Beclin1 in the five groups;real-time quantitative PCR was used to detect the expression of SQSTM1/p62,Beclin-1,ATG5 and LC3-II genes in the five groups.Results:1.Immunohistochemistry results showed:the expression of autophagy-related proteins SQSTM1/p62 and LC3B is higher in high-grade gliomas than in low-grade gliomas,meanwhile the expression of mTOR is lower in high-grade gliomas than in low-grade gliomas.These data mean autophagy in high-grade glioma is stronger than that of low-grade glioma.2.Speraman statistical analysis results showed:LC3B,SQSTM1/p62 and mTOR were significantly correlated with overall survival(OS)time and recurrence-free survival(RFS)time(P<0.05).The OS time and RFS time of patients with high expression of LC3B and SQSTM1/p62 were both shorter than patients with low expression of LC3B and SQSTM1/p62,patients with low expression of mTOR had shorter OS time and RFS time than patients with high mTOR expression.3.The results of the protein imprinting experiment showed:the expression of SQSTM1/p62 and Beclin1 was increased by capsaicin and autophagy inducer rapamycin in T98G and U373 cells,capsaicin and rapamycin played synergistic role in autophagy.4.The results of CCK8 cell dynamic experiment and Western Blot showed:Capsaicin can weaken the proliferation of glioma cells,accompanied by increasing expression of autophagy proteins SQSTM1/p62,ATG4B,ATG5and Beclin1 and decreasing expression of mTOR;overexpression of mTOR can increase the proliferation ability of glioma cells,accompanied with decreased expression of autophagy-related proteins SQSTM1/p62,ATG4B,ATG5 and Beclin1;the expression of mTOR in plasmid and capsaicin group was higher compared to the blank control group autophagy-related protein SQSTM1/p62,ATG4B and ATG5,and the expression of Beclin1 and mTOR was lower compared to the blank control group.5.qRT-PCR results showed:The SQSTM1/p62,Beclin-1,ATG5 and LC3-II were significantly increased in mRNA level when the capsaicin was affected.In the mTOR plasmid group SQSTM1/p62,Beclin-1,ATG5 and LC3-II genes were all decreased;compared with the blank control group,the expression mRNA level of SQSTM1/p62in the mTOR plasmid+capsaicin group was slightly lower,and the expression mRNA levels of Beclin-1,ATG5 and LC3-Ⅱwere significantly increased.All these results indicating that capsaicin may play a role through target mTOR.Conclusions:1.Autophagy promotes the malignant progression of human gliomas;2.Capsaicin is an autophagy inducer;3.Capsaicin may play a induce autophagic role through the mTOR target.