NH4Cl Treatment Prevents Doxorubicin-induced Myocardial Dysfunction In Mice Through P38MAPK Pathway

Background: Doxorubicin is a widely used spectrum antitumor drug,but the life-threatening side-effect of cardiotoxicity limits its clinical application.Up to now,only dexrazoxane can be used to prevent doxorubicin-induced myocardial toxicity.Unfortunately,dexrazoxane is only able to prevent the occurrence of cardiac dysfunction but not cure the already existing myocardial dysfunction.In view of this,finding new drugs which can effectively inhibit the toxicity of doxorubicin become an important target for oncologist as well as cardiologist.Ammonium chloride,as a regulator in PH value which is cheap and easy to obtain can regulate the p H value and has an extremely important role in metabolism.In this study,we revealed the protective effect of NH4 CL and relevant mechanism involved in NH4 CL protection against DOX-induced cardiomyocyte death and size expanded.Method:8 week old male C57/BL6 mice(N=32)were randomly divided into 4 groups: Control group(distilled water and intraperitoneal injection of saline,N=8),Ammonium chloride group(Ammonium chloride and intraperitoneal injection of saline,N=8),Doxorubicin Group(distilled water and intraperitoneal injection of doxorubicin,N=8),Doxorubicin + Ammonium chloride group(Ammonium chloride and intraperitoneal injection of doxorubicin,N=8).The mice were administered DOX(5 mg/kg)or normal saline(NS)by intraperitoneal injection once weekly for 2 weeks.Where indicated,NH4Cl(0.28 M)was added to the drinking water one week before the start of DOX treatment sustained during the injection of DOX.We sacrificed the mice and performed additional experiments 7 days after the final injection.Hearts were removed from mice anaesthetized with saturated 3-bromoethanol intraperitoneally.Materials for the following studies: 1.pathological staining: H&E and Masson staining were used to detect the level of inflammation,myocardial hypertrophy and fibrosis in myocardium.The rate of myocardium hypertrophy was detected by WGA staining.Immunohistochemical Mac-2 staining was used to further detect the inflammation of myocardial cells.2.Transmission Electron Microscope(TEM): The level of autophagy in myocardial cells was detected by TEM and the autophagic vacuoles in myocardial cells were observed.3.TUNEL method: this method was used to detect the apoptosis level of cardiac myocytes in each group.4.Caspse 3 enzyme activity assay: To further detect the level of apoptosis in cardiac myocytes.5.Western bloting(WB): P38 MAPK pathway related protein: P38 MAPK pathway related protein: rabbit anti-P38MAPK(1:1000,Cell Signaling Technologies),anti-p-p38MAPK(1:1000,Cell Signaling Technologies),anti-HSP27(1:1000,R&D system),anti-p-HSP27(1:1000,R&D system),apoptosis related protein: anti-BCL-2(1:1000,Abcam),anti-BAX(1:1000,Abcam);Autophagy related protein: anti-Becline1(1:1000 Abcam)and Gapdh antibody(1:1000,sigma).6.Real time quantitative polymerase chain reaction(Real-time quantitative polymerase chain reaction,q-PCR): inflammation related RNA:IL-1 beta,beta Interleukin-1;cardiac function related RNA:ANP,atrial natriuretic peptide;BNP brain,natriuretic peptide and GAPDH glyceraldehyde 3-phosphate dehydrogenase.,reference:Result: 1.cardiac function assessment Compared with control group,the cardiac function of doxorubicin group significantly deteriorated and the EF value decreased significantly.Compared with the adriamycin group,the EF value of Ammonium chloride + doxorubicin group was significantly improved(P<0.005).q PCR results showed that the ANP,BNP levels in doxorubicin group increased significantly compared with the control group(P<0.05),and the expression level of them decreased significantly after ammonium chloride addition.2.Ventricular hypertrophy A cross-sectional view of the heart showed a significant increase in cardiac volume in heart failure compared with the control group,After the use of NH4 CL,the heart volume was significantly reduced.(P<0.05)WGA staining showed that the cross-sectional area of myocardial cells in Doxorubicin group was significantly higher than that in control group(P<0.05).After using NH4 CL,the area was significantly reduced(P<0.05).3.The level of inflammation and fibrosis of myocardium 3.1 Inflammation H&E staining showed that the control group of mice with myocardial fibers arranged neatly,the cell size is moderate,no interstitial infiltration of inflammatory cells and no adriamycin-related apoptotic cardiomyocytes in the interstitium.Compared with the control group,doxorubicin group of mice myocardial cell structure disorder,gap widened,interstitial inflammatory cells appear a lot of infiltration and a large number of apoptotic cells were found in interstitial cells.After the treatment of NH4 CL,compared with doxorubicin group,the mouse myocardial structure arranged neat,interstitial narrowing;inflammatory cell infiltration and apoptosis of cells were significantly reduced.q PCR results showed that after injection of doxorubicin,IL-1β,a specific marker of inflammation has elevated(P<0.05).After the treatment of NH4 CL,the level of it was close to normal(P<0.05)3.2 Fibrosis Masson staining showed that compared with the control group,doxorubicin group showed severe fibrosis,after using NH4 CL,the level was close to normal(P<0.05).4.Apoptosis and autophagy 4.1TUNEL Compared with the control group,the apoptosis level of myocardial cells in doxorubicin group increased significantly(P<0.05)and the apoptosis level of myocardial cells decreased significantly after the treatment of NH4CL(P<0.05).4.2 CASPSE3 enzyme activity Compared with the control group,CASPASE3 enzyme activity in doxorubicin group was significantly increased.While after the treatment of NH4 CL,the Caspase3 enzyme activity was significantly declined(P<0.05)4.3 Western bloting Western bloting results showed that compared with the control group,the apoptosis related protein of BAX in doxorubicin group increased and BCL2 decreased(P<0.05).After addition of ammonium chloride,the level of BAX decreased while the BCL2 level increased.4.4 Electron microscope Electron microscopy showed that compared with the control group,the level of autophagy and autophagic vacuoles increased in doxorubicin group,however,after the treatment of ammonium chloride,autophagic vacuoles and the level of autophagy decreased.WB was used to further explore the protective effect of ammonium chloride in doxorubicin-induced myocardial toxicity and to explore the function of P38 MAPK in this process.(P<0.05)5.Western blotingConclusion: 1.Ammonium chloride may attenuate myocardial toxicity caused by doxorubicin,2.Ammonium chloride protects the myocardial toxicity induced by adriamycin through P38 MAPK pathway.