| Objective:Heart failure is a common clinical syndrome with a high mortality rate.In recent years,due to the increase of cardiovascular risk factors in the aging population,the prevalence of heart failure has been increasing year by year.In the in vitro model,compared with the traditional two-dimensional monolayer culture mode,3D organoid culture can better simulate the physiological and pathological state of organ tissues,and provide a suitable environment for the interaction between cells.Salviae Miltiorrhizae Radix et Rhizoma(Danshen)and Chuanxiong Rhizoma(Chuanxiong)reveal Chinese herbal medicines with a long history and are widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases,but there are few studies on their combination and the mechanism is not clear yet.Therefore,this research by building the 3D cardiac organoid in vitro model,discusses the Danshen and Chuanxiong medicine-guanxinning injection(GXNI)in the therapeutic effects on the heart kind of organs in vitro model,finally through the animal model in vivo experiment using the transverse aortic constriction(TAC)operation simulate heart failure disease development process to investigate the efficacy and pharmacological mechanism,to make the results more testable.This study is expected to provide a more similar organoid model for cardiovascular diseases in vitro,and provide theoretical basis for clinical application and prescription optimization of GXNI.Methods:1.The cardiac cells were co-cultured with the original ratio of primary neonatal rat heart cells into ultra-low adsorption pore plates,and self-assembled and polymerized to form 3D heart microspheres.The existence of main cells in the heart--cardiomyocytes and fibroblasts was verified by immunofluorescence of whole microspheres and slices.The concentrations of 5×103,1×104,5×104 and 1×105 heart cells were observed on day 1,2,3,7 and 14,respectively,and then the distribution of each cell was observed by immunofluorescence to determine the concentration of cardiac microspheres.Next,by observing the shape of a cardioid cultivated for 34 days and verifying its functional indicators of immunofluorescence after 34 days,the success of the established cardioid organ model was evaluated.2.In the cardiac organoid model established for the first time in vitro by phenylephrine(PE)induction,the cardiac injury disease model was formed.In order to ensure drug effectiveness,the injury should not be excessive,so it is necessary to explore different PE concentrations.The degree of hypertrophy was determined by the diameter and area of cardioid,and the beating rate of cardioid induced by PE with different concentrations was observed.Meanwhile,the effect of PE induction on mitochondrial function damage was observed by fluorescence staining,so as to determine the PE induction concentration.Next,in order to further explore the specific type of cells that play a major role in PE induced hypertrophy,cardiomyocytes,fibroblasts and endothelial cells were extracted respectively for PE induction,and their proliferation efficiency was determined by CCK8 method and immunofluorescence method to prove the specific reasons for PE induced cardioid hypertrophy.3.Through exploring the influence of Danshen-Chuanxiong medicine on the heart injury of Guanxinning injection(GXNI)composed of Traditional Chinese medicine,pharmacodynamics verification was carried out through the cardiac injury model of cardiac organ in vitro established by us.The primary heart cells were purified and analyzed to determine the concentration of GXNI by CCK8 method.Live/dead cells were verified by fluorescence staining to explore the effect of GXNI on apoptosis caused by heart injury.Next,the therapeutic effect of cardiac hypertrophy after GXNI administration was verified by morphological observation and analysis,and changes in heart diameter and area were observed.Immunofluorescence was used to verify the effect of GXNI on PE induced mitochondrial function,and real-time reverse transcription-polymerase chain reaction(RT-PCR)was used to further verify the gene expression of cardiac hypertrophy caused by cardiac injury.4.TAC was used to construct heart failure model in vivo.Four weeks after operation,the patients were randomly divided into Model group(Model),metoprolol positive group(MeTI)and GXNI treatment group,and Sham operation group(Sham).The Sham and Model groups were injected with saline daily for 28 days.The pharmacodynamic effects of GXNI were evaluated by weight,blood pressure,echocardiography,hemodynamics and pathological observation.5.Heart tissues of GXNI group and Model group were selected for transcriptome sequencing(RNA-SEQ)to obtain differentially expressed genes(DEGs).DEGs with log2 FoldChange≥1 and P ≤0.05 were carried out for core Analysis of Ingenuity Pathway Analysis(IPA)to identify the key mechanisms and targets of GXNI on heart failure.The results of IPA were verified by RT-PCR,western blotting(WB)and immunohistochemistry(IHC).Next,immunofluorescence(IF)was used to verify the IPA results in vitro through the in vitro cardiac organoid disease model we constructed previously.Results:1.The cardiac cells extracted from primary suckling rat were co-cultured to form cardiac microspheres.IF was used to verify the typical Marker of heart cells.Through the culture of different cell concentrations and different days,we found that when the initial density was 1×104,the cells in the heart-like microspheres were clearly distributed,and the oxygen concentration in the center of the spheres was sufficient,the cells had good roundness,and the heart-like rhythm was regular.After the concentration is determined,the cells can be cultured for 34 days or more.IF shows no significant difference in the arrangement,distribution and completeness of cells compared with those cultured for 7 days.Therefore,subsequent experiments will be conducted after 7 days.It proved the existence of microvessel and extracellular matrix in cardiac microspheres,which further proved that the in vitro cardiac spheroid established by us were more in line with the human body environment,and thus further proved the successful establishment of organoid.2.After PE induction with different concentrations,it was found that the diameter of cardiac microspheres was significantly different when PE concentration was 500 μmol·L-1,cardiac hypertrophy occurred,and irregular pulsation and fast or slow pulsation frequency appeared from 500 μmol·L-1.According to the analysis of high-intentioned multi-cell imaging system,the fluorescence intensity of MitoTracker and Rhod-2 heart microspheres after PE treatment was significantly increased from 500 μmol·L-1 compared with the Sham group,indicating the increase of mitochondrial mass and the significant increase of intracellular Ca2+.The fluorescence intensity of Rhodamine 123 weakened,indicating a significant decrease in mitochondrial membrane potential,with significant statistical differences compared with the Sham group.The high expression of IF CD31 and the increased absorbance of CCK8 after PE induction indicated that cardioid microsphere hypertrophy was not only caused by cardiomyocyte hypertrophy,but also caused by proliferation of endothelial cells and fibroblasts.3.The purity of each cell was found to be above 90%by purification and separation.CCK8 assay showed that the IC50 of GXNI on primary myocardial cells was 10.57 μL/mL,and the IC50 of GXNI on fibroblasts was 11.44 μL/mL.PE induced apoptosis was significantly increased by fluorescence staining of live/dead cells,and GXNI significantly reduced the proportion of dead cells.Morphological observation and analysis under light microscope showed that PE induced cardioid diameter increased significantly,and different concentrations of GXNI showed different therapeutic effects.After μL/mL GXNI treatment,cardioid diameter only decreased,and there was no statistical difference.Therefore,GXNI can reverse the pro-hypertrophy effect of PE and reduce the diameter of microspheres.Fluorescence staining detected the effect of mitochondrial function,and it was found that GXNI preadministration could reduce the abnormal increase of mitochondrial respiration to varying degrees,reduce mitochondrial quality,reduce the concentration of intracellular Ca2+,and alleviate Ca2+ overload.PCR results showed that GXNI inhibited the upregulation of ANP,BNP and β-MHC.4.In vivo,TAC was divided into groups 4 weeks after surgery and administered continuously for 28 days.Blood pressure tests showed that GXNI reduced TAC induced diastolic and systolic hypertension.Echocardiography showed that GXNI treatment significantly increased EF and FS in model group,decreased LV Vols,LV Vold,LVDs and LVDd,and enhanced cardiac function in heart failure mice.Hemodynamics showed that both+dp/dtmax and-dp/dtmax had a significant upward trend in GXNI treatment.H&E and Masson staining showed that GXNI could improve myocardial hypertrophy,myocardial fiber disorder,less collagen deposition and lower myocardial fibrosis area.5.Transcriptome sequencing of mouse heart tissues in Model and GXNI groups revealed 854 DEGs with log2 FoldChange≥1 and P≤0.05,containing 414 up-regulated and 440 downregulated genes.The IPA core analysis of the 854 DEGs was conducted to obtain the top 10 Signaling pathways,among which the third IL-17A Signaling in Fibroblasts pathway was most relevant to heart disease.At the same time,by referring to the key words of differentially expressed genes in transcriptome data and ventricular remodeling,cardiac hypertrophy and cardiac muscle fibrosis,we found the genes related to regulating ventricular remodeling leading to cardiac hypertrophy and myocardial fibrosis,and the genes with large changes in transcriptome.They Adamts4,c-Fos,Fosb,Gal,Ccl7,Ccr1,Mmp27,Mmp1 and P38.The results confirmed that 3 μL/mL GXNI can significantly reduce Adamts4,C-FOS,Gal,Ccl7,Mmp1 and P38.IHC and WB results showed that GXNI could significantly reduce the expression levels of P38,c-Fos and Mmp1.Meanwhile,IF results of cardiac organ in vitro showed that GXNI could significantly inhibit the expression of P38,C-FOS and Mmp1.Conclusion:1.Through the co-culture and self-assembly of primary suckling rat cardiac cells under low adsorption conditions to form microspheres,and through the characterization of cardiac characteristic proteins,physiological function verification,and disease model simulation,it was proved that the organoid model has similar physiological functions in human body and can achieve long-term culture for up to 34 days.2.Through 3D cardiac organoid model in vitro and TAC model in vivo,it can be proved that GXNI can effectively improve cell apoptosis,cardiac hypertrophy and myocardial fibrosis caused by ventricular remodeling.3.Il-17a Signaling in fibroblasts pathway p38/c-Fos/Mmp1 pathway was revealed and verified,which was the key pathway of GXNI to treat cardiac hypertrophy and myocardial fibrosis caused by cardiac injury. |
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