NF-κB Pathway Activation During Endothelial-to-Mesenchymal Transition In A Rat Model Of Doxorubicin-induced Cardiotoxicity

PurposeWe want to reveal the closely connection between endothelial-tomesenchymal(EndoMT)and doxorubicin-induced myocardial fibrosis by establishing a SD rat model of doxorubicin-induced cardiotoxicity.In addition,The activation of NF-κB signaling pathway during endothelialto-mesenchymal(EndoMT)provides a theoretical basis for the search of a novel therapeutic targets for doxorubicin-induced cardiomyopathy.Method1.To establish a doxorubicin-induced cardiotoxicity rat model:sprague-daw(SD)rats rats aged 10 weeks and weighing 150-200 g were selected.The rats were randomly divided into four groups named a control group and three doxorubicin treatment groups respectively(doxorubicin treatment groups for one week,two weeks and six weeks),with 12 rats in each group,half male and half female,a total of 48 rats.In the experimental group,doxorubicin hydrochloride at a concentration of2mg/kg was intraperitoneally injected into SD rats weekly,and assessed after 1,2,and 6 weeks respectively.The control group treated with intraperitoneal injection at the same amount of saline.In the control group,heart was extracted immediately after rats was killed,while in the experimental group,heart was extracted after continuous injection of 1W,2W and 6W respectively.2.Endothelial primary cells isolation:the aorta of heart tissue from the control group and the three experimental groups was cut into fragments with scissors,and the tissue was enzymolysised with 1 mg/m L collagenase I for 45 minutes,then double volume phosphate buffer solution was added in order to remove collagenase I.The mixture of cells and tissues was then digested at 37℃ for 45 minutes with 0.25% trypsin and stirred it every 15 minutes.After 45 minutes,the digestion was terminated by adding twice the original volume of the digestion stop medium(including 15% FBS and85% DMEM).The Cell and tissue mixture were cultured in a culture dish and placed in a 37-degree cell incubator until the next day.The primary endothelial cells were purified by magnetic bead separation when the primary cells were adherent to the dish and in good condition.3.Hematoxylin-eosin(H&E)and Masson trichrome staining were used to detect the pathomorphic changes of myocardial tissue and cardiac vessels in SD rats.4.The hydroxyproline content in the heart tissues of SD rats treated with doxorubicin was used to quantify the collagen content of using a hydroxyproline analysis kit.5.The ultrastructural changes of cardiac myocytes,vascular cells and organelles treated with doxorubicin were observed by electron microscopy.6.Immunofluorescence double staining was used to verify EndoMT in myocardium tissue and cells respectively in vitro and in vivo.Expression of NF-κB P65 in myocardium was also detected by immunohistochemistry.In addition,protein levels of CD31,α-SMA and Collagen-I in myocardial tissue were detected by Western blotting.Results1.After intraperitoneal injection of doxorubicin(2mg/kg,once a week),all doxorubicin-treated rats showed hair loss,abdominal distention(Supplement data-S1),and listless.A total of seven rats died when we have not finished the experiment.2.Hematoxylin-eosin(H&E)and Masson trichrome staining were performed on heart tissue sections extracted from SD rats in three experimental groups treated with doxorubicin.The results showed that with the accumulation of doxorubicin dose in rats,the degree of vascular injury and heart fibrosis was gradually enhanced.There were significant pathomorphological changes such as the gradual widening of myocardial fiber gap,myocardial lysis,myocardial fibrosis,myocardial vascular endothelial injury and increased permeability,and more red blood cells exuded to the gaps in the myocardial fibers.3.Under electron microscope,In the doxorubicin-1W group,the cardiac muscle filaments were disorderly arranged and a few autophagic vacuoles and swollen mitochondria with widened cristae were detected.The nuclei of the vascular ECs were swollen.As accumulative dose,In the doxorubicin-6W group,the cardiac muscle filaments were swollen.We observed more swollen mitochondria,some of which were damaged and had more widened cristae.The vascular ECs were more swollen as were their nuclei.4.The EndoMT process was verified by immunofluorescence double staining with vascular endothelial cells extracted from doxorubicin-treated heart tissue.The results showed that the percentage of vessels with CD31/α-SMA co-expression in the doxorubicin-1W and-2W groups showed an increasing tendency when compared to that in the control group,but the difference was not statistically significant(p>0.05).In contrast,the doxorubicin-6W group had a significantly higher percentage of vessels with CD31/αSMA co-expression than the control group(p<0.05).In addition,immunofluorescence double staining of vascular endothelial cells extracted from heart tissue showed that CD31 expression was decreased and α-SMA expression was increased in the doxorubicin-6W group compared with the control group.At the same time,protein levels of CD31,α-SMA and collagen I were measured by Western Blot.The results showed that compared with the control group,CD31 expression levels were significantly lower in the doxorubicin-6W group,while the levels of α-SMA and Collagen-I were significantly increased.5.The activation of NF-κB signaling pathway was verified by immunofluorescence double staining of rat heart tissue and vascular endothelial cells extracted from it.In the control group,few endothelial cells expressed P65,while in the doxorubicin treated groups,the proportion of endothelial cells expressing P65 increased.The percentage of p65 and CD31 co-expressing in the nucleus of doxorubicin-6W group was higher than that in the control group.In addition,the expressions of P65 and P-P65 were detected in the extracted vascular endothelial cells(ECs),and it was observed that only a few ECs in the control group expressed p65 and PP65 at the same time,mainly located in the cytoplasm.In the doxorubicin-6W group,many cells expressed both P65 and P-P65,and mainly located in the nucleus.Conclusion1.We established a doxorubicin-induced cardiotoxicity rat model,which showed a time-dependent increase in fibrosis severity.2.Vascular injury and autophagy activation contributed to the pathogenesis of doxorubicininduced cardiotoxicity.3.We found that the NF-κB pathway was activated during EndoMT in this model.