| Background: Doxorubicin(DOX)is a potent anthracycline chemotherapeutic drug.DOX-induced cardiotoxicity(DIC)limits its application in cancer treatment,as this complication is detrimental and fatal.Reactive oxygen species(ROS)production,autophagic dysfunction and apoptosis are crucial factors related to DIC.Previous studies have shown that SIRT4 is associated with cardiac energy metabolism,mitochondrial dysfunction and cardiac cell death.In the hypoxia induced cardiomyocyte injury model,the expression of SIRT4 decreased,and overexpression of SIRT4 increased the activity of mitochondrial membrane potential and inhibited cardiomyocyte apoptosis.However,little is known about the correlations of SIRT4 and DIC.Previous studies found that the expression of SIRT4 decreased in DIC cell model.Combined with the literature and previous results,we speculate that SIRT4 is involved in the occurrence and development of DIC and plays a protective role.Therefore,this subject intends to carry out research from three aspects levels.Firstly,the DIC model was established,using gene transfection and interfering with the expression of SIRT4,and exploring the role of SIRT4 in DIC through in vivo and in vitro experiments;Secondly,the specific mechanism of SIRT4 inhibiting DIC was clarified by q PCR,Western blotting,immunohistochemistry and immunostaining,so as to provide new ideas for reducing DIC.Part Ⅰ: The relationship between SIRT4 expression and DICObjective: To explore the correlation between SIRT4 and DIC.Methods:(1)Establishment of DOX chronic myocardial injury mouse model We established the mouse model of myocardial injury by intraperitoneal injection of DOX(5mg / kg/week,intraperitoneal injection for 4 weeks,total dose 20 mg / kg)into c57BL/6J mice.The NS-treated groups injected with an equivalent amount of NS for comparison.The cardiac function of mice was measured by small animal Doppler ultrasound(left ventricular ejection fraction LVEF% and left ventricular short axis shortening LVFS%);The levels of serum cardiaclactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)were detected by biochemical analyzer;HE staining,immunofluorescence and TUNEL staining were used to evaluate the success of DIC animal model.(2)The expression of SIRT4 and apoptosis related proteins Bax and Bcl2 detected by quantitative PCR and Western blotting.(3)Establishment of DIC cell model H9C2 cardiomyocytes incubated with DOX(1um)for 24 hours to establish the myocardial injury cell model.CCK8 and TUNEL staining used to evaluate the success of DIC cell model.(4)Quantitative PCR and Western blotting used to detect the m RNA and protein of SIRT4 gene and the expression of apoptosis related proteins Bax and Bcl2.Result:(1)Compared with the control group,after intraperitoneal injection of DOX in C57BL/6J mice,Echocardiography showed that LVEF% and LVFS% decreased,the levels of LDH and CK-MB in serum increased significantly,the arrangement of muscle fibers in myocardial tissue was disordered,and cardiomyocyte apoptosis increased.(2)The results of quantitative PCR and Western blotting showed that the expression of SIRT4 m RNA and protein in the heart tissue of DOX treated group was lower than that of the control group.(3)In the DIC cell model,compared with the control group,the activity of H9C2 cardiomyocytes in DOX treatment group gradually decreased and the apoptosis increased.(4)The results of quantitative PCR and Western blotting showed that the expression of SIRT4 m RNA and protein in H9C2 cardiomyocytes in DOX treatment group decreased compared with the control group.Conciusion: In DIC model,the transcription and protein expression of SIRT4 decreased,and they negatively correlated.Part Ⅱ: Study on the role of SIRT4 in DICObjective: To clarify the scientific hypothesis that SIRT4 may participate in the occurrence and development of DIC by regulating autophagy and apoptosis.Methods:(1)To overexpress SIRT4 in the heart,the mice in each group injected with AAV9-SIRT4,AAV9-NC and AAV9-sh-SIRT4 at a dose of 1 × 1012 particles via the tail vein.Three weeks after AAV9 injection,we divided the mice into five groups: NS,NS +AAV9-SIRT4,DOX + AAV9-NC,DOX + AAV9-SIRT4,and DOX +AAV9-sh-SIRT4.By measuring the body weight of mice,biochemical detection of myocardial injury indexes(LDH,CK-MB),cardiac ultrasound,HE staining used to evaluate the effects of overexpression of SIRT4 on cardiac function and cardiac psychology in DIC animal model.(2)The effect of overexpression of SIRT4 on cardiomyocyte apoptosis in DIC mice evaluated by TUNEL staining and Western blotting.(3)H9C2 cardiomyocytes overexpressing SIRT4,empty vector and knocking down SIRT4 expression constructed by lentiviral vector lentiviral,and then incubated with 1umol / L DOX for 24 hours to establish cardiomyocyte injury model.The experiment divided into five groups: control,DOX + Lv-NC,DOX + Lv-SIRT4,DOX + Lv-sh-NC,DOX + Lv-sh-SIRT4,The effect of overexpression of SIRT4 on apoptosis in DIC cell model evaluated by TUNEL staining and Western blotting.(4)The effect of overexpression of SIRT4 on autophagy in DIC mice model evaluated by immunofluorescence staining and Western blotting.(5)The effect of overexpression of SIRT4 on autophagy in DIC cell model evaluated by immunofluorescence staining and Western blotting.Result:(1)At the end of the experimental cycle,no experimental animals died.The body weight of DOX + AAV9-sh-SIRT4 group and DOX + AAV9-NC group was significantly lower than that of DOX + AAV9-SIRT4 group and control group.The levels of CK-MB and LDH in DOX intervention group were significantly higher than NS group.The levels of CK-MB and LDH in DOX + AAV9-sh-SIRT4 group and DOX + AAV9-NC group were significantly higher than those in DOX + AAV9-SIRT4 group.Echocardiographic results showed that overexpression of SIRT4 improved the decline of cardiac function caused by DOX.For example,compared with other groups,LVEF% and LVFS% in DOX + AAV9-SIRT4 group increased.Compared with DOX + AAV9-sh-SIRT4 group and DOX + AAV9-NC group,DOX induced myocardial atrophy was also reversed and cardiomyocytes increased significantly in DOX + AAV9-SIRT4 group.In addition,histological examination also showed that DOX + AAV9-SIRT4 group also reduced DOX induced vacuolar degeneration of cardiomyocytes.After the experiment,we also verified the expression of SIRT4 in the hearts of mice in each group.SIRT4 was highly expressed in NS + AAV9-SIRT4 group and DOX + AAV9-SIRT4 group,while the expression level of SIRT4 decreased in DOX + AAV9-NC group and DOX + AAV9-sh-SIRT4 group,but there was no change in the expression level of SIRT4 in NS group.(2)The Bcl2 / Bax ratio in DOX intervention group was significantly lower than that in NS group,and the Bcl2 / Bax ratio in DOX + AAV9-sh-SIRT4 and DOX + AAV9-NC groups was significantly lower than that in DOX + AAV9-SIRT4 group.TUNEL staining used to detect cardiomyocyte apoptosis.The results showed that the number of TUNEL positive cells in DOX intervention group was significantly higher than that in NS group,and the number of TUNEL positive cells in DOX + AAV9-SIRT4 group was significantly lower than that in DOX + AAV9-sh-SIRT4 and DOX + AAV9-NC groups.(3)We further verified by cell experiments in vitro that overexpression of SIRT4 can reduce DOX induced cardiomyocyte apoptosis.After H9C2 cells in different groups were exposed to 1u M DOX for 24 h,Western blottingting analysis showed that the Bcl2 / Bax ratio decreased after DOX exposed,which was more obvious in H9C2 cells in SIRT4 knockout group,while the Bcl2 / Bax ratio of H9C2 cells in DOX + Lv-SIRT4 group increased significantly,and the difference between groups was statistically significant.TUNEL staining showed that the number of positive cells in DOX group was significantly higher than that in control group.The number of TUNEL positive cells in DOX + Lv-sh-SIRT4 group and DOX + Lv-NC group was significantly higher than that in DOX + Lv-SIRT4 group.It is suggested that overexpression of SIRT4 can reduce DOX induced cardiomyocyte apoptosis.(4)Western blotting results showed that in DOX + AAV9-sh-SIRT4 and DOX + AAV9-NC groups,the expression of LC3-II/I and Beclin increased,the level of autophagy increased,and the expression of p-mTOR and P62 decreased.However,in the heart tissue of DOX + AAV9-SIRT4 mice,the expression of p-mTOR and P62 increased,the expression of LC3-II/I and Beclin decreased,and the level of autophagy decreased.Immunofluorescence analysis showed that LC3 signal increased after DOX,especially in SIRT4 gene knockout group,while LC3 signal decreased in SIRT4 overexpression group.These results showed that compared with SIRT4 knockout mice and normal mice,the autophagy level of over expressing SIRT4 mice after DOX administration inhibited,suggesting that autophagy may be an important regulatory process of SIRT4 in the process of DIC.(5)We further verified in vitro that overexpression of SIRT4 can reduce DOX induced autophagy of cardiomyocytes.H9C2 cells in different groups exposed to 1umol / L DOX for 24 hours.Western blottingting analysis showed that the expression of p-mTOR and P62 increased and the expression of LC3-II / I and Beclin decreased in the overexpression SIRT4 group.On the contrary,in SIRT4 knockout group,the levels of p-mTOR and P62 protein decreased,and the ratios of LC3-II / I and Beclin increased.Immunofluorescence analysis showed that after DOX exposure,the fluorescence signal of LC3 increased,the signal of SIRT4 gene knockout group was more obvious,and the fluorescence signal of LC3 in overexpression SIRT4 group decreased.These results suggest that DOX activates autophagy in DIC model,and overexpression of SIRT4 can inhibit DOX induced autophagy.Conclusion:(1)SIRT4 inhibits DOX induced myocardial injury in mice;(2)SIRT4 inhibited DOX induced cardiomyocyte apoptosis in vivo and in vitro;(3)SIRT4 inhibits DOX induced cardiomyocyte autophagy in vivo and in vitro.Part Ⅲ: The mechanism of SIRT4 inhibiting DICObject: To clarify the scientific hypothesis that SIRT4 inhibits DIC through AKT /mTOR / autophagy signaling pathway.Methods:(1)To overexpress SIRT4 in the heart,the mice in each group injected with AAV9-SIRT4,AAV9-NC and AAV9-sh-SIRT4 at a dose of 1 × 1012 particles via the tail vein.Three weeks after AAV9 injection,we divided the mice into five groups: NS,NS + AAV9-SIRT4,DOX + AAV9-NC,DOX + AAV9-SIRT4,DOX + AAV9-sh-SIRT4.Western blotting used to detect the changes of AKT / mTOR signal pathway protein in heart tissue of each group to evaluate the effect of SIRT4 on the changes of AKT / mTOR signal pathway in DIC animal model.(2)H9C2 cardiomyocytes overexpressing SIRT4,empty vector and knocking down SIRT4 expression constructed by lentiviral vector lentiviral,and then incubated with 1umol / L DOX for 24 hours to establish cardiomyocyte injury model.The experiment divided into five groups: control,DOX + Lv-NC,DOX + Lv-SIRT4,DOX + Lv-sh-NC and DOX + Lv-sh-SIRT4.Western blotting used to detect the changes of AKT/ mTOR signal pathway protein in cardiomyocytes of each group to evaluate the effect of SIRT4 on the changes of AKT / mTOR signal pathway in DIC cell model.(3)In order to verify whether SIRT4 inhibits autophagy and participates in the process of DIC,this part of the experiment divided into animal experiment and cell experiment.The cell experiment divided into six groups: control,DOX + Lv-NC,DOX+ Lv-SIRT4,DOX+Lv-sh-NC,DOX + Lv-sh-SIRT4,DOX + Lv-SIRT4 + Rapamycin(autophagy activator)group.Autophagy and apoptosis detected by Western blotting and immunofluorescence.The in vivo experiment divided into five groups: NS,DOX + AAV9-NC,DOX + AAV9-SIRT4,DOX + AAV9-sh-SIRT4,DOX + AAV9-SIRT4 + Rapamycin group.Immunofluorescence,Western blotting and other methods used to detect autophagy and apoptosis related phenotypic indexes.(4)Based on the above results,in order to verify whether SIRT4 participates in the process of DOX myocardial injury by regulating AKT / mTOR pathway.This part of the experiment divided into animal experiment and cell experiment.The cell experiment divided into six groups: control,DOX + Lv-NC,DOX + Lv-SIRT4,DOX + Lv-sh-NC,DOX + Lv-sh-SIRT4,DOX + Lv-SIRT4 + AKTi(AKT signal pathway inhibitor)group.Autophagy and apoptosis detected by Western blotting and immunofluorescence.The in vivo experiment divided into five groups: NS,DOX + AAV9-NC,DOX + AAV9-SIRT4,DOX + AAV9-sh-SIRT4,DOX + AAV9-SIRT4 + AKTi group.Mouse cardiac ultrasound,HE staining,biochemical detection,immunofluorescence,Western blotting and other methods used to detect the phenotypic indexes related to myocardial injury,autophagy and apoptosis.Result:(1)It is worth noting that compared with DOX + Lv-NC group and DOX + Lv-sh-SIRT4 group,the number of TUNEL positive cells in DOX + Lv-SIRT4 group decreased,and this protective effect blocked by the addition of Rapamycin.The expression of Bax,Bcl2,p-mTOR,mTOR,P62,LC3-II / I and Beclin were further detected.The results showed that compared with DOX + Lv-NC group and DOX + Lv-sh-SIRT4 group,the protein expression levels of LC3-II / I and Beclin decreased,and the protein expression levels of Bcl2/BAX,P62 and p-mTOR increased in DOX + Lv-SIRT4 group.In DOX + Lv-SIRT4 + Rapamycin treatment group,the levels of LC3-II / I and Beclin protein increased,while the expression levels of Bcl2/BAX,P62 and p-mTOR protein decreased.It is suggested that overexpression of SIRT4 plays an important role in protecting cardiomyocytes from DOX induced apoptosis by inhibiting autophagy level.(2)The results showed that the number of TUNEL positive cells in DOX + AAV9-SIRT4 + Rapamycin group was equivalent to that in DOX + AAV9-NC group,which was significantly higher than that in DOX + AAV-SIRT4 group.Western blotting analysis showed that compared with DOX + AAV9-SIRT4 group,the expression levels of LC3-II / I and Beclin protein increased,and the expression levels of Bcl2/BAX,P62,p-mTOR and p-AKT protein decreased in DOX + AAV9-SIRT4 + Rapamycin group.Taken together,these findings suggest that autophagy activation can eliminate the protective effect of overexpression of SIRT4 on DOX induced myocardial injury.(3)In this study,the phosphorylation level of AKT in DOX treatment group was significantly lower than that in control group,both in vivo and in vitro,while the expression level of p-AKT in SIRT4 overexpression group increased.It is suggestes that SIRT4 can activate AKT / mTOR signal pathway.We used AKTi to block the AKT / mTOR pathway in vitro.Western blotting analysis showed that compared with DOX + Lv-SIRT4 group,the expression levels of Bax,LC3-II / I and Beclin protein in increased,and the expression levels of Bcl2,P62,p-mTOR and p-AKT protein decreased in DOX+AAV9-SIRT4+AKTi group.It is suggests that the protective effect of overexpression of SIRT4 on DOX-induced apoptosis attenuated after inhibiting AKT / mTOR pathway.(4)Similar to DOX + AAV9-SIRT4 + AKTi group,mice in DOX + AAV9-NC group showed impaired myocardial systolic function and decreased LVFS% and LVEF%.Compared with DOX + AAV9-SIRT4 group,the levels of CK-MB and LDH in DOX + AAV9-SIRT4 + AKTi group were significantly higher(P < 0.01).The number of TUNEL positive cells in DOX + AAV9-SIRT4 + AKTi group was equivalent to that in DOX + AAV9-NC group,which was significantly higher than that in DOX + AAV9-SIRT4 group.Western blotting analysis showed that compared with DOX + AAV9-SIRT4 group,the expression levels of Bax,LC3-II / I and Beclin protein increased,and the expression levels of Bcl2,P62,p-mTOR and p-AKT protein decreased in DOX+AAV9-SIRT4+AKTi group.Obviously,inhibition of AKT / mTOR pathway can eliminate the anti-apoptosis and autophagy effects of overexpression of SIRT4.In conclusion,SIRT4 inhibits DOX induced apoptosis through AKT / mTOR / autophagy pathway.Conclusion: SIRT4 alleviates DOX induced myocardial injury in vivo and in vitro through AKT / mTOR / autophagy pathway,and then inhibits the process of DIC. |
PDF Full Text Request