| Objective:To investigate mechanism of myocardial fibrosis induced by X-ray in Wistar neonatal rats,elucidate effect and potential molecular mechanism of extract of Radix Angelica Sinensis and Radix Hedysari(RAS-RH)on myocardial fibrosis and changes of ultrastructure induced by X-ray,and define effect of RAS-RH with multi-target and multi-pathway for providing experimental basis on the treatment of radiation-induced myocardial fibrosis,and offer new methods and strategies for clinical treatment of radiation-induced myocardial fibrosis.Methods:Primary myocardial fibroblasts(CFs)were cultured to establish a cell model of radiation-induced myocardial fibrosis induced by X-ray.Proliferation,apoptosis and relative proteins of myocardial fibroblast after X-ray radiation were verified by CCK-8,immunofluorescence staining,Western blot.High-throughput transcriptome sequencing was used to predict and analysis target genes expression.Network pharmacology and molecular docking techniques were used to analyze active components,core targets,and molecular docking ability of Radix Angelica Sinensis and Radix Hedysari.Effects of different doses of RAS-RH on proliferation rate and mobility of myocardial fibroblasts after X-ray irradiation and expression of proteins were verified by CCK-8,Wound healing,Western blot.Ultrastructure and cytoskeletal structure of CFs after X-ray radiation were observed by Transmission electron microscopy,the expression of immunofluorescence in myocardial fibroblasts and Western blot were used to detect the related cytoskeletal proteins to observe the effect of RAS-RH on the ultrastructure of myocardial fibroblasts after X-ray irradiation.The effect of RAS-RH on X-ray-induced cardiac fibroblasts was used to analyze differentially expressed genes and enriched pathways by High-throughput transcriptome sequencing,which were compared with previous experiments.HE and Masson staining,immunofluorescence identification,and Western blot were used to detect effect of RAS-RH on cardiac fibrosis in rats after X-ray irradiation in vivo expriment.Results:1.The purity of primary CFs was 95% after identification by Vimentin protein.Proliferation of CFs was promoted at 48 hours after X-ray irradiated with a single dose of 1Gy,(p <0.05);compared with the control group,the apoptosis rate decreased at 48 hours after irradiation in the model.Immunofluorescence staining was used to localize the expression of TGF-β1 andα-SMA in CFs after irradiation;The protein expression of TGF-β1,Samd3,and Col1 in the model were significantly increased compared with the control group(p <0.05).Analysis of high-throughput transcriptome sequencing results showed that 112 differentially expressed m RNAs,45 genes were up-regulated and 67 genes were down-regulated,mainly including mitochondrial inner membrane transferase 17b(Timm17b),basal cell adhesion molecule(Bcam),mitochondrial ribosomal protein(Mrpl30),matrix metallopeptidase 11(Mmp11),etc.GO enrichment was mainly divided into four major categories,including fibrosis,inflammation,autophagy,organelles,etc.KEGG enrichment analysis identified 24 pathways,including immune response process,inflammatory response,and cancer pathway,etc.2.125 main active components of Angelica sinensis,43 main active compounds of Radix Hedysari,and 12 common active components were selected,and 28 core protein targets associated with radiation-induced myocardial fibrosis via Network Pharmacology.Active components of Angelica sinensis and Radix Hedysari,including AKT,PIK3 CA,MMP9,etc,involving 82 important signaling pathways.Pathways mainly affected including cancer pathways,and PI3K-Akt signaling pathways,etc.Molecular docking technique was used to predict the binding ability of Angelica sinensis and Radix Hedysari core active components to key targets AKT,MMP9,MMP2,and m TOR,and the results revealed that mistletoe bombesin,medi pterostilbene,grapefruit bombesin,and glycyrrhizin components had strong binding ability with AKT,MMP9,MMP2,and m TOR.3.Compared with the model group,9 μg/m L RAS-RH decreased cell proliferation after 48hours(p <0.01)in a dose-dependent manner.Compared with the control group,the wound healing rate of myocardial fibroblasts after X-ray irradiation in the model group was significantly increased(p <0.05);compared with the model group,the cell wound healing rate was significantly decreased in RAS-RH group;compared with the control group,the protein levels of TGF-β1,Samd3,and Col1 in the model group were increased;compared with the model group,the protein levels of TGF-β1,Smad3,and Col1 in RAS-RH group were significantly decreased.Transmission electron microscopy showed that the results of the control group that the edge of the cell membrane was intact;number of mitochondria was abundant and evenly distributed;autophagosomes were individual.The model cells were edematous as a whole,the pseudopodia and processes disappeared;the number of mitochondria was more,autolysosomes were abundant.RAS-RH group showed moderate edema as a whole,with a large area of damage at the edge of the cell membrane;the number of autolysosomes was small.In the control group,the microfilaments of cardiac fibroblasts were arranged in parallel and neatly clear.Compared with the control group,the myocardial fibroblast volume became smaller,the adhesion force was enhanced,F-actin was thickened and knotted;compared with the model group,the arrangement of myocardial fibroblast microfilaments in RAS-RH showed disorganized broken filaments,the microfilaments were detached,and the adhesion force was significantly reduced.4.The effect of RAS-RH on myocardial fibroblasts after X-ray radiation was test by High-throughput transcriptome sequencing.Results beween the control group and the model group were consistent with those of the first expriment.After analysis of the model group and the RAS-RH group,it was found that transcriptome gene expression was closely related to integrins,cytoskeleton(microtubules,intermediate filaments),mitochondria,and lysosomes,and had an intervention effect on the regulation of cytoskeleton,the positive and negative regulation of integrins,and mitochondrial and lysosomal functions.Cancer pathway and PI3K/AKT pathway were the highest value in the model group and RAS-RH group,which was consistent with the results of experiment 2.5.8Gy was used as the X-ray radiation dose to establish radiation-induced myocardial fibrosis in Wistar rats,and 50 mg/kg/day dose of RAS-RH was given to intervene myocardial fibrosis in rats after X-ray radiation.In HE staining,compared with the control group,myocarditis exudation occurred more significantly after X-ray irradiation in the model group;compared with the model group,the exudation of inflammatory substances in the myocardium was reduced in the RAS-RH group.In Masson staining,compared with the control group,the myocardial tissue texture of the model group was disorganized and disordered,and collagen increased into fibrosis;compared with the model group,the myocardial tissue texture of RAS-RH group was relatively neat.RAS-RH inhibited the immunofluorescent expression of TGF-β1,Smad3,and Col1 in rat hearts after irradiation.Compared with the control group,protein expressions of TGF-β1,PI3K、AKT were increased after X-ray radiation in the rat heart,while protein expressions of TGF-β1,PI3 K,and AKT were significantly decreased in the RAS-RH group compared with the model group(p <0.05).Conclusion:RAS-RH can attenuate radiation-induced myocardial fibrosis by TGF-β1/Smad3 pathway and regulate autophagy through PI3K/Akt pathway,ameliorate the remolding of cytoskeleton and ultrastructure after X-ray radiation,and alleviate fibrosis after radiation-induced cardiac injury in rats. |
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