| Part I The protective effect of palmatine on cardiotoxicity induced by doxorubicin in miceObjective: To study the protective role of palmatine(PLT)in the acute cardiotoxicity of doxorubicin(DOX),and to explore its possible mechanism through a series of experiments.Methods: Sixty adult C57BL/6 mice(all male),were randomly divided into the following groups: saline group,DOX group,PLT 25 mg/kg group,PLT 50 mg/kg group and dexrazoxane(DEX)group.Among them,the latter 4 groups were injected with 15mg/kg DOX to establish an acute cardiotoxicity model.The PLT 25 mg/kg group,PLT 50mg/kg group and DEX group were given 25 mg/kg PLT,50 mg/kg PLT and 150 mg/kg DEX,respectively.The body weight,heart weight and cardiac function in each group were detected;serum creatine kinase isoenzymes(CK-MB),cardiac isoforms of troponin I(c Tn I)and lactate dehydrogenase(LDH)content in each group were detected;The levels of interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor α(TNF-α)and interferon-γ(IFN-γ)in myocardial tissue and serum of mice in each group were detected;the content of total superoxide dismutase(T-SOD),catalase(catalase,CAT),4-hydroxynonenal(4-HNE)and malondialdehyde(MDA)in each group were detected;the expression of DHE and apoptosis index in each group were detected;the expression of Sirt1 and nuclear factor-κB(NF-κB)proteins in the myocardium of mice was detected.Result:1.Compared with the normal saline group,the cardiac function of DOX group was significantly decreased,the myocardial injury was significantly increased,and the related indexes of myocardial inflammation,myocardial oxidative stress and promoting myocardial apoptosis were significantly increased.2.Compared with DOX group,the body weight,heart weight and cardiac function of DEX group were significantly increased,and myocardial injury was significantly alleviated.3.Compared with DOX group,the indexes related to myocardial injury in PLT group were significantly improved,mainly as follows:1)PLT can increase the body weight and heart weight in DOX-treated mice,and reduce the contents of myocardial CK-MB,LDH and c Tn I.In addition,PLT can increase LVEF and LVFS in DOX-treated mice;2)PLT can reduce the expression of IL-1β,IL-6,IFN-γ and TNF-α in DOX-treated mice;3)PLT can increase the expression of T-SOD and CAT and reduce the content of 4-HNE,MDA and DHE in DOX-treated mice.In addition,PLT treatment significantly attenuated myocardial apoptosis after DOX insults;4)PLT can increase the expression of Sirt1 and reduce the phosphorylation level of NF-κB in DOX-treated mice.Conclusion: PLT can reduce cardiac inflammation,inhibit oxidative stress and cardiomyocyte apoptosis,and improve DOX-inducedd cardiotoxicity in mice.PLT treatment increased the expression of Sirt1 protein and decreased the expression of p-NF-κB protein in the myocardium of DOX mice.Part II The protective effect of palmatine on doxorubicin-induced cardiotoxicity was verified in vitroObjective: To study the protective role and mechanism of PLT in cardiotoxicity induced by DOX in vitro.Methods: Primary rat cardiomyocytes(NRCMs)was isolated and cultured.After successful culture,CCK-8 method was used to detect the effects of different concentrations of PLT on NRCMs.After the detection,the cells were divided into the following groups:PBS group,DOX stimulation group,DOX+PLT 5μmol/L group,DOX+PLT 50μmol/L group and DOX+PLT 100μmol/L group.The NRCMs were stimulated by DOX and different concentrations of PLT respectively.The contents of CK-MB,LDH and c Tn I in each group were detected;The content of IL-1β,IL-6,IFN-γ and TNF-α in each group were detected;the contents of T-SOD,CAT,4-HNE and MDA in each group were detected;the protein expressions of Bcl-2,Bax and c-caspase3 in five groups were detected;the proteins of Sirt1 p-NF-κB and NF-κB in five groups were detected.Result:1.CCK-8 results showed that the viability of NRCMs was not affected when the concentration of PLT was less than 100μmol/L.However,when PLT > 100μmol/L,the viability of NRCMs decreased gradually with the increase of PLT concentration.2.Compared with PBS group,myocardial injury indexes of DOX group were significantly increased,oxidative stress,inflammatory response and pro-apoptotic indexes of DOX group were significantly increased,while inhibition of apoptosis indexes were significantly decreased,Sirt1 protein expression was significantly decreased,and p-NF-κB protein expression was significantly increased.3.Compared with DOX group,the indexes related to myocardial injury were significantly improved in all concentration groups of PLT,mainly as follows:1)PLT can increase the content of CK-MB,LDH and c Tn I in NRCMs;2)PLT can reduce the expression of IL-1β,IL-6,IFN-γ and TNF-α in NRCMs;3)PLT can increase the expression of T-SOD and CAT in NRCMs,and reduce the content of 4-HNE and MDA;4)PLT can increase the expression of Sirt1 in NRCMs and reduce the phosphorylation level of NF-κB.Conclusion: When the concentration of PLT was less than or equal to 100 μmol/L,the viability of primary rat cardiomyocytes was not affected.PLT can reduce DOX-induced inflammatory response,oxidative stress and cardiomyocyte apoptosis,and improve DOX-indecd cardiotoxicity.PLT treatment increased the expression of Sirt1 protein and decreased the phosphorylation level of NF-κB in primary rat cardiomyocytes induced by DOX.Part III The role of Sirt1 in palmatine-mediated cardioprotectionObjective: To further confirm the role of the Sirt1 signaling pathway in PLT-mediated cardioprotection.Methods: NRCMs was isolated and cultured,and then transfected with si-Sirt1 to knock down the expression of Sirt1.Western Blots and q PCR were used to detect the effect of Sirt1 knockdown.The contents of CK-MB,LDH and c Tn I in each group were detected;The content of IL-1β,IL-6,IFN-γ and TNF-α in each group were detected;the contents of T-SOD,CAT,4-HNE and MDA in each group were detected;The expression of Bcl-2,Bax and c-caspase3 in five groups were detected.Result:1.Compared with the Scr group,the expression of Sirt1 protein and m RNA in cardiomyocytes transfected with si-Sirt1 decreased significantly.2.Compared with PBS+Scr group,myocardial injury index,inflammation index,oxidative damage index and apoptosis related index of cardiomyocytes in DOX+Scr group were significantly increased,while the indexes of inhibition of apoptosis were significantly decreased.3.Compared with DOX+Scr group,myocardial injury index,inflammation index,oxidative damage index and apoptosis-promoting index of cardiomyocytes in DOX+PLT+Scr group were significantly decreased,while anti-apoptosis indexes were significantly increased.4.Compared with DOX+PLT+Scr group,the related indexes of myocardial injury in DOX+PLT+ Si-SIRT1 group were significantly increased,mainly as follows:1)Sirt1 knockdown increased the levels of CK-MB,LDH and c Tn I in NRCMs and restricted the cardioprotective effects of PLT;2)Sirt1 knockdown increased the expression of IL-1β,IL-6,IFN-γ and TNF-α in NRCMs and restricted the cardioprotective effects of PLT;3)Sirt1 knockdown decreased the expression of T-SOD and CAT in NRCMs,and increased the content of 4-HNE and MDA;Conclusion: Inhibition of Sirt1 expression by gene knockdown technique can reverse the cardioprotective effect of PLT.The Sirt1/NF-κB pathway plays a central role in PLT-mediated protection against DOX-induced cardiotoxicity. |
