The Protective Effect And Mechanism Of Tangeretin On Cisplatininduced Cardiotoxicity In Vivo And In Vitro

Tangeretin is the first polymethoxy flavonoid monomer to be found and one of the most abundant polymethoxyflavones present in the peel of various citrus fruits,has anti-inflammatory,anti-cancer,and prevention of cardiovascular diseases and other pharmacological effects.In recent years,its research basis is relatively rich,and bioavailability ranks the first among the monomers of polymethoxy flavonoids.At present,cisplatin,a commonly used clinical tumor chemotherapy drug,has problems such as high toxicity and high drug resistance.Among them,cardiotoxicity is one of the most serious side effects affecting patients with malignant tumors,which has a serious impact on the life and survival of chemotherapy patients.Therefore,it is more necessary to find natural compounds that reduce the toxic and side effects of chemotherapy drugs and cooperate with anti-tumor.This experiment explored the effects of polymethoxyflavonoid tangeretin on cisplatin-induced heart injury and related molecular mechanisms from in vivo and in vitro.First,Establishment of cisplatin-induced acute cardiotoxicity model in vivo,detect myocardial enzyme indicators,use histopathological staining and western blotting to explore the morphological changes and injury mechanisms of cardiac tissue;at the same time,establish the injury model of H9c2 cardiomyocytes induced by cisplatin in vitro,and analyze the molecular mechanism of tangeretin improving cisplatin induced cardiotoxicity from AMPK signaling pathway and mitochondria.It includes the following two aspects:1.The protective effect of tangeretin on cisplatin-induced cardiotoxicity in miceExperimental method: The mice were randomly divided into four groups: the control group(n=8),the model group(n=8),tangeretin 50 mg/kg + cisplatin(n=8),tangeretin 100 mg/kg + cisplatin(n=8).The treatment group was given different doses of tangeretin solution every day for 4 consecutive weeks.Except for the control group,the mice in each group were injected intraperitoneally with cisplatin,3 mg/kg,once every 2 days,7 times in a row,respectively on the 14 th,16th,18 th,20th,22 th,24th and26 th days,for 4 weeks.Experimental results: The levels of myocardial index enzymes troponin T(c Tn T),creatine kinase(CK),creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH)in the serum of the model group were significantly higher than those in the control group(P < 0.01),and the serum levels of cardiac enzymes in the tangeretin pretreatment group were decreased(P < 0.05).Histopathological staining showed that the mouse myocardial cells had severe inflammatory infiltration,and the arrangement of muscle fibers was disordered or even broken.Compared with the model group,the cellulite in the tangeretin group was relieved.Western blot analysis confirmed that tangeretin could improve myocardial apoptosis by activating the phosphorylation of AMPK at Thr 172 to inhibit downstream apoptotic protein expression.2.The protective effect of Tangeret on the destruction of H9c2 cells induced by cisplatinBased on the study of the mechanism of tangeretin on the cardiotoxicity of cisplatin in mice,in order to further clarify the molecular mechanism of tangeretin on cisplatin-induced cardiomyocyte injury.The cell viability of the H9c2 cardiomyocytes was determined using a MTT assay to establish a cisplatin-induced cardiomyocyte injury model and screen effective protective dose of tangeretin.mitochondrial fluorescence probe,JC-1 and Hoechst 33258 staining were used to detect the relationship between mitochondrial apoptosis and cells apoptosis in cisplatin-induced cardiomyocyte injury by tangeretin pre-administration;The addition of Compound C(AMPK protein inhibitor)and AICAR(AMPK protein activator)verified that tangeretin regulates AMPK signaling pathway and cell apoptosis by immunofluorescence,cell flow cytometry and western blotting.Experimental results:The pre-administered doses of tangeretin at 1.25,2.5,and 5 μM had a significant protective effect on cardiomyocytes stimulated with 40 μM cisplatin for 24 h.The number of mitochondria gradually decreased with the prolongation of cisplatin administration,the tangeretin group showed an improvement in the number of mitochondria;the mitochondrial membrane potential of H9c2 cells was decreased(green fluorescence)in the modol group compared with the control group while the tangeretin group exhibited orange fluorescence indicating a high membrane potential.dense staining trend in Hoechst 33258 was consistent with mitochondrial apoptosis.Immunofluorescence analysis increased expression of the glucose transporter 4 on the cell membrane in the tangeretin administration group.Finally,the results of cell flow cytometry and western blotting showed that tangeretin activated AMPK protein,inhibited ACC activity and phosphorylation of p38 MAPK,and reduced the number of mitochondrial permeability transition pores and apoptosis.In summary,this study verifies that tangeretin has a protective effect on cisplatin cardiotoxicity.It reduces myocardial enzyme levels,improves cardiac tissue pathology,activates AMPK signaling pathway,increases the number of glucose transporters on cell membranes and mitochondria in myocardial cells.The number and mitochondrial membrane potential level can be achieved by inhibiting the mitochondrial permeability open pore and the expression of Caspase apoptotic protein.This research can provide a theoretical basis for the development of clinical treatment of chemotherapeutic cardiovascular diseases and related health-care drugs.