| BackgroundIbrutinib is a novel,first-in-class oral Bruton's tyrosine kinase(BTK)inhibitor,approved by the United States Food and Drug Administration(FDA)for relapsed and refractory mantle cell lymphoma(MCL),chronic lymphocytic leukemia(CLL),and Waldenstrom's macroglobulinemia(WM).Increased incidence of AF(atrial fibrillation)and bleeding have been observed in patients undergoing treatment.In the first three large randomized trials of CLL,the incidence of AF in Ibrutinib was 5-7.7%.A recently published data summary analysis found that the risk of AF with ibrutinib was about four times higher than that of the published study.At present,it is believed that the occurrence and maintenance of AF is related to multiple wavelet reentry,focal triggering,atrial electrical remodeling,structural reconstruction,and neurohumoral factors,and it is the result of a combination of mechanisms.The most fundamental is the electrical remodeling and structural remodeling of the atria,ie the electrophysiological properties of the atrial myocytes and related proteins changed,but the specific mechanism of its occurrence has not yet been fully elucidated,and further research is needed.Wenxin Granule(WXKL)is consisted of Chinese herbal medicines of Dangshen,Huangjing,Panax,Amber and Nansong,the first nationally approved anti-arrhythmic Chinese patent medicine.A large number of studies have shown that WXKL has both anti-arrhythmic drugs of type ?,? and ?,and has regulatory effects on various ion channels.It is widely used in the treatment of various types of patients with AF.Based on this,this study will establish a model of atrial fibrillation through esophageal Burst stimulation,and to study the effect of Wenxin Granules on atrial electrophysiological and molecular regulation mechanisms of ibrutinib-induced AF in mice.Methods1 Electrocardiogram was used to detect the duration of AF in mice.The indicators of cardiac function in mice were detected by echocardiography.The degree of tissue fibrosis was observed by HE staining,Masson's staining,and Sirius red staining.Transmission electron microscope was also used to observe changes in the ultrastrcture of atrial myocytes.2 Confocal laser scanning microscopy was used to detect intracellular Ca2+ release,and Fura-4 fluorescent dye staining and MitoSOX Red staining to observe the effects of WXKL on calcium spark,calcium transient,and mitochondrial reactive oxygen species in atrial cells of ibrutinib-induced AF in mice.3 Using proteomics to detect AF protein expression,mainly by two-dimensional gel electrophoresis of protein high-throughput separation and purification of cells,and then professional computer software for image analysis,and then by mass spectrometry and protein data processing technology,analysised and identification of protein spots on the gel,and then observed the WXKL treated ibrutinib induced AF in mice in the protein expression of atrial muscle.4 Using Western blotting molecular biology methods,we selected NOX2 and NOX4 signaling pathways as study objects.To study the effect of WXKL on oxidative stress-related signaling pathways in the mice of ibrutinib induced AF,and needed further analysis its molecular mechanism for the treatment of AF.Results1 In vivo,using the esophagus electrode Burst stimulation,the results showed that WXKL can reduce the time of ibrutinib induced AF conversed to sinus rhythm in mice.From the overall cardiac morphology,cross-sectional histological examination of the heart,transmission electron microscopy observation of cell ultrastructure,and echocardiographic measurement of cardiac function indicators.The mice AF model was successful.The myocardial tissue is slightly fibrotic,and the mitochondrial ridges in the myocardial ultrastructure were slightly altered.However,the WXKL after treatment improved the degree of organization restructuring.2 Ibrutinib induced AF in mice increased in calcium concentration in atrial myocytes,triggering cell contraction,which is calcium transient.WXKL can improve the occurrence of cellular calcium sparks in ibrutinib-induced AF.So it can prove that WXKL improve myocardial contractility by regulating intracellular calcium ion concentration and father restoring normal heart rhythm.3 iTRAQ's Quantitative Proteomics Analysis performed only preliminary screening of differentially expressed proteins for ibrutinib induced AF.The results showed that some proteins may be associated with cardiovascular disease,such as Mitochondrial brown fat uncoupling protein 1,Glycerol-3-Phosphate dehydrogenase[NAD(+)],and Deoxyguanosine kinase,mitochondrial,etc.4 Ibrutinib induced AF can increase the Glycerol-3-phosphate dehydrogenase[NAD(+)]protein NADPH two subunits NOX2 and NOX4 expression in oxidative stress related signal pathway.Wenxin Keli after treatment improved the trend of protein expression.Detailed verification and evaluation of key proteins and pathways are needed,and we need to further refine them in future studies.ConclusionIn summary,under the pathological conditions of AF in vivo,Wenxin Keli can inhibit and delay the occurrence and development of myocardial tissue remodeling and electrical remodeling,regulated intracellular calcium ion disorders,and have a certain improvement effect on AF.After preliminary screening of differential proteins,the two subunits NOX2 and NOX4 in Glycerol-3-phosphate dehydrogenase[NAD(+)]were selected for validation.The study also preliminarily explored the molecular mechanism of Wenxin Keli to play a role.By reducing the expression of NOX2 and NOX4 oxidative stress related proteins,it may play a role in regulating heart rhythm.The need to further use GO functional annotation and KEGG pathway screening for differential proteins helps to understand the metabolic or molecular signaling pathways these proteins,to explore the effect of Wenxin Keli on signaling pathways and related protein expression in AF treatment.This process may provide the basis for the rationality of the next experiment design. |
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