| BackgroundExosomes are small vesicles that are secreted by almost all cells.Exosomes secreted by Bone Marrow Mesenchymal Stem cells（BMSCs）can protect the heart by their antiinflammatory,anti-apoptotic and angiogenic effects.However,current transplantation methods do not enable exosomes to continue to function in the heart.How to make exosome act on the heart more efficiently and accurately is one of the hotspots of current research.The pericardium provides a new way of treatment with its unique anatomical characteristics.There is a lack of experimental studies on whether intracardiac injection of exosome can provide sustained relief to improve cardiac function.ObjectiveTo investigate whether exosomes derived from bone marrow mesenchymal stem cells can effectively enter cardiac tissue and treat Chronic Heart Failure（CHF）in rats after pericardial injection,and provide a new treatment for Chronic Heart Failure in clinic.Methods1.2-week-old SD rats were sacrificed by cervical dislocation method,soaked in 75%alcohol,and the femurs and tibia of both lower limbs were taken under aseptic conditions.The bone marrow cavity was rinsed,and BMSCs were isolated and purified by density gradient centrifugation.After cultured to the third generation,BMSCs were identified by immunofluorescence staining..The selected markers of mesenchymal stem cells were CD29,CD44 and CD90.2.After the isolated and purified BMSCs were cultured to the third generation,when the cell density reached about 80%,they were changed to complete culture medium containing exosome-free serum for 48 hours.Cell culture supernatant was collected and exosomes were obtained by differential ultra-high speed centrifugation.The morphology of exosomes,protein markers（CD9,CD63,Alix,GM130）and particle size and concentration of exosomes were observed by projection electron microscopy,nano-particle tracing analysis（NTA）and protein imprinting.3.The concentration of exosome was detected by BCA method,and the exosome suspension was divided into five groups: 0,50,100,150 and 200μg/ml.Rat H9C2 cells were inoculated into 96-well plates,and exosomes with different concentrations were co-cultured with rat H9C2 cells for 24 h and 48 h,respectively.The proliferation of different groups of cells was detected by cell counting kit.4.Rat H9C2 cells were inoculated into 96-well plates,and cultured for a period of time before adding doxorubicin hydrochloride solution.They were divided into control group,PBS group and exosome group.The control group was added without any reagent,and the PBS group and exosome group were added with equal volume of sterile PBS and exosome suspension,respectively.After co-culture for 24 h and 48 h,cell counting kit was used to detect whether exosomes had protective effects on cells.5.Fifty-five SD rats aged 6-8 weeks were selected and conventional fed for two weeks.The heart failure model was established by intraperitoneal injection of Doxorubicin hydrochloride.They were divided into heart failure group（HF）,exosome group（exo）and sham operation group（sham）with 15 rats in each group.The heart failure group did not receive pericardial injection,the exosome group received intracardial injection of exosome suspension,and the sham operation group received intracardial injection of normal saline.Control group（ctrl）10 patients were injected intraperitoneally with equal volume of normal saline.The left ventricular ejection fraction（LVEF）of rats was < 50% as the criterion for successful modeling.6.After successful modeling,the identified exosome suspension was injected into the pericardium of rats,and the small animal imager was used to determine whether the injected exosome existed in the pericardium.The heart tissue section was used to observe whether exosome penetrated into the heart tissue through the epicardium.7.On the 10 th day after surgery,cardiac ultrasound was performed on each group of rats.Cardiac function was measured by left ventricular ejection fraction（LVEF）,left ventricular shortening rate（LVFS）and cardiac output（CO）using m-mode ultrasound on the long axis section of the left ventricle.8.The heart tissues of the four groups were frozen and sectioned.Immunohistochemical staining was used to detect the expression of brain natriuretic peptide（BNP）and interleukin6（IL-6）in heart tissues of rats in each group.The expression of platelet-endothelial cell adhesion molecule（CD31）and vascular endothelial growth factor A（VEGFA）in heart tissue were detected by immunofluorescence staining.Masson staining technique was used to detect the content of collagen fiber in heart tissues of rats.Results1.Under light microscope,BMSCs extracted from bone marrow lumens of rats’ lower extremity long bones showed vortex-like or fish-like growth.Immunofluoresce nce results showed that mesenchymal stem cell markers CD90,CD44 and CD29 w ere all expressed in the cells,while CD45 was not.The results of immunofluoresce nce double staining showed that CD29 and CD44 were co-expressed and CD44 and CD90 were co-expressed in the extracted cells.2.Transmission electron microscopy（TEM）showed that the vesicles separated by differential speed and ultra-high speed centrifugation showed a double-layer membrane structure with a diameter of 30-150 nm.Tracking analysis of nanoparticles showed that the particle diameter in suspension reached the peak at 126.3nm.The expression of CD9,CD63 and Alix was positive and GM130 was negative.3.Cell proliferation experiment showed that different concentrations of exosomes could promote the proliferation of H9C2 cells at 24 h and 48 h,in which 100μg/ m L exosomes promoted the proliferation of H9C2 cells the most（P < 0.05）.4.Cell counting experiments showed that 100μg/m L exosomes could protect H9C2 cells from doxorubicin-induced damage at 24 h and 48 h co-culture（P < 0.01）.5.Cardiac ultrasound observation: After intraperitoneal injection of doxorubicin hydrochloride solution for 6 weeks,the left ventricular ejection fraction（LVEF）of the heart failure model group was significantly decreased or lower than 50%（P < 0.01）,while the cardiac function of the control group was not significantly changed.LVEF,LVFS and CO in the exosome group increased after pericardial injection compared with before operation（P< 0.01）.There was no significant change in cardiac function before and after operation in sham operation group and HF group.6.In vivo imaging results of small animals showed that there was a high intensity of fluorescence signal in the projection position of the heart surface in the thoracic cavity of rats 7 days after pericardial injection;The observation of cardiac tissue sections showed that exosomes penetrated into the myocardium through the epicardium.7.Immunohistochemical staining results showed that compared with the control group,BNP content in myocardial tissues of exosome group,sham group and HF group was increased,with statistical significance（P < 0.01）.The content of BNP in exosome group was significantly lower than that in sham group and HF group（P < 0.001）.The content of IL-6in the heart tissue of rats in the control group and exosome group was lower than that in the HF group and sham operation group,with statistical significance（P < 0.001）.There was no statistical difference between control group and exosome group（P > 0.05）.8.Immunofluorescence staining results showed that compared with the control group,the content of CD31 in myocardial tissue of exosome group was increased,with statistical significance（P < 0.01）.At the same time,the content of CD31 in exosome group was also higher than that in sham group and HF group（P < 0.05）,and there was no statistical difference between sham group and HF group and control group（P > 0.05）.The expression level of VEGFA in control group and exosome group was significantly higher than that in HF group and sham group（P < 0.001）.There was no statistical significance between control group and exosome group（P > 0.05）.9.Masson staining results showed that the content of collagen fiber in myocardium of rats in sham operation group and HF group was higher.The collagen fiber content in exosome group was significantly lower than that in sham operation group and HF group,and the difference was statistically significant（P < 0.001）.There was no statistical difference between the exosome group and the control group（P > 0.05）.ConclusionPericardium injection of exosomes derived from bone marrow mesenchymal stem cells can penetrate into cardiac tissue through the epicardium,which can play an antiinflammatory role in heart failure,promote angiogenesis,improve cardiac function,and reduce cardiac remodeling. |
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