| Objective: To investigate the protective effect and molecular mechanism of total propolis flavonoids(TFP)on isoproterenol(ISO)-induced myocardial hypertrophy in rats and H9C2 cells.Methods:(1)In vivo experiment: SD rats were randomly divided into 5 groups,blank group,model group,compound Danshen tablet positive group,and propolis total flavonoids low-dose group(FL group)and propolis total flavonoids high-dose group(FH group),10 in each group.Except for the Blank group,all other groups were injected with ISO at 20,10,and 5 mg/kg per day,respectively,to establish the model,and from the 11 th to the 14 th day,ISO was injected at 3 mg/kg.The state of the rat was observed and recorded and the growth curve was drawn;the echocardiogram of the rat was detected;the cardiac mass index and cardiac coefficient of the rat were detected;the myocardial cell apoptosis was detected by flow cytometry;HE and Masson staining were used to observe the rat The cardiac pathological conditions of the rats were detected by the kit;the contents of TG,T-CHO,LDL-C,HDL-C and ATP in the serum of the rats were detected by the kit;the contents of FFA and c AMP in the serum and heart tissue of the rats were detected by the Elisa kit;the contents of FFA and c AMP were detected by the kit Serum T-SOD,GSH activity,MDA and Glu contents;16S r DNA amplicon sequencing to detect rat fecal flora;non-targeted metabolomics to detect related metabolites in rat serum;RT-PCR to detect rat heart The expression levels of related genes in tissues;Western blot was used to detect the effects of related protein expression levels in rat heart tissue.(2)In vitro experiments: H9C2 cells were cryopreserved,recovered and cultured;RTCA cell real-time monitoring system was used to detect the proliferation level of H9C2cells;the dose and time of ISO damage to H9C2 cells were detected;H9C2 cells were grouped and divided into blank group(Blank),model group(Model),total propolis flavonoids low-dose group(TFPL)and propolis total flavonoids high-dose group(TFPH);ISO modeling was used for all groups except the Blank group;MTT and CCK8 methods were used to detect the survival rate of injured H9C2 cells;the morphology of H9C2 cells was observed by inverted microscope;H9C2 cells were detected by confocal laser microscopy Intracellular calcium ion H9C2 cells;kit to detect T-SOD,GSH activity and MDA content in H9C2 cells;kit to detect TG,The contents of T-CHO,Glu and ATP;the protein concentration in H9C2 cells was detected by BCA method;the content of FFA in the supernatant and pellet of H9C2 cells was detected by Elisa method;AMPK,PPARɑ,PPARγ,GS,PKA,Beclin1,LC3 B and Cas-9 protein fluorescence intensity;RT-PCR method was used to detect the expression levels of related genes in H9C2 cells;Western blot method was used to detect the expression levels of related proteins in H9C2 cells.Results:(1)In vivo experiment: The rats in the Blank group ate and drank normally,moved freely,breathed steadily,and their body weight increased rapidly.With the progress of the experiment,the rats in the Model group gradually developed symptoms of myocardial hypertrophy,which gradually deteriorated.For decreased food intake,discomfort,decreased activity,shortness of breath,partial hair loss,slow weight gain,loose stools,unformed,low urine output,the FL group and FH group also had the above symptoms,but the onset time was later,and the degree of lighter.The EF% and FS% of the rats in the FL group and the FH group were significantly increased,and the LVIDd and LVIDs of the rats in the FH group were significantly decreased.Rats in the FH group gained faster weight,decreased VW,and decreased VW/BW index.The total apoptotic rate of cardiomyocytes in FL group and FH group was significantly decreased.The degree of myocardial cell injury,fiber structure and morphology in the FH group were significantly improved.The contents of TG,T-CHO and LDL-C in the serum of the rats in the FH group decreased,while the contents of HDL-C and ATP in the serum of the rats in the FH group decreased and increased.In the FH group,the content of c AMP in serum was decreased,and the content of c AMP in heart tissue was significantly increased.The content of FFA in serum and heart tissue of rats in FH group decreased.The activities of GSH,T-SOD and Glu content in serum of rats in FL group and FH group were increased,and the content of MDA was decreased.At the phylum level,the richness of Firmicutes was down-regulated,the richness of Bacteroidetes was up-regulated.After FH treatment,the ratio of Firmicutes to Bacteroidetes increased significantly.This study found that in the heat map of KEGG function distribution,the main functions included membrane transport,carbohydrate metabolism,amino acid metabolism,energy metabolism and lipid metabolism.In the COG function distribution heatmap,the main functions are carbohydrate transport and metabolism,amino acid transport and metabolism,cell wall/membrane/envelope biogenesis,energy production and conversion.Based on the Lef Se analysis results of the KEGG function and COG predictions,this study finally determined lipid metabolism and energy production and conversion.Through metabolome Venn analysis,the distribution of metabolites among the three groups was observed,and 38 valuable differential metabolites were finally found by the analysis,and the separation of differential metabolites was clear in each comparison.After TFP intervention,the above 38 differential metabolites tended to be close to normal levels,indicating that TFP has a certain regulatory effect on these differential metabolites.In this study,9 biological pathways were identified by KEGG pathway enrichment analysis,namely pyrimidine metabolism,glucagon signaling pathway,autophagy-other,glycosylphosphatidylinositol-anchored biosynthesis,phenylalanine tyrosine Metabolism in amino acid and tryptophan biosynthesis,central carbon cancer,autophagy-animals,glutathione metabolism,and mannose-type O-glycan biosynthesis.However,through further literature search and screening,this study obtained more information about the glucagon signaling pathway and the autophagy-animal pathway,both of which attracted our high attention.In addition,combined with the results of 16 S r DNA amplicon sequencing of rat feces,this study found a high correlation with lipid metabolism.Therefore,by analyzing two omics approaches,this study finally identified lipid metabolism,energy metabolism and autophagy pathways.Western blot and RT-PCR results showed that the protein and gene expression levels of CPT1 A,AMPK and PPARα in the myocardial tissue of the Model group were decreased,and the protein and gene expression levels of ACC and PPARγ were increased.The protein and gene expression levels of CPT1 A,AMPK and PPARα in the FH group The protein and gene expression levels of ACC and PPARγ were significantly increased and decreased.The protein and gene expression levels of ADCY1,ADCY2,β1AR,β2AR,GS,PKA and CREB in the myocardial tissue of the Model group decreased,and the FH group showed the opposite trend.The protein and gene expression levels of Beclin1,LC3 B and Bcl-2 in the myocardial tissue of the Model group were significantly decreased,and the protein and gene expression levels of Cas-9 and BAX were increased.The FH group showed the opposite trend.The relative expression levels of ANP,BNP,α-MHC and β-MHC genes in tissues were decreased.(2)In vitro experiments: In this study,the number of H9C2 cells was determined to be10,000 cells/well for the following experiments.Finally,the concentration of ISO and the modeling time were determined to be 40 μmol/L for 24 h.The results of the MTT assay to detect the survival rate of H9C2 cells were as follows: TFP was pre-protected for 24 hours,and the doses were 1 mg/m L(TFPL)and 4 mg/m L(TFPH).The results of the CCK8 method were consistent with the results of the MTT method.The number of H9C2 cells in the Blank group was higher,and the cell body was mainly spindle-shaped.The number of H9C2 cells in the Model group was relatively reduced,and the cell body was shrinking,and some cells became round.The number of H9C2 cells in the TFPH group increased significantly,and the cell body was shrinking.Significant improvement.The intracellular calcium fluorescence intensity level of H9C2 cells in TFPH group decreased significantly.The area of H9C2 cells in the Model group increased significantly,the cell outlines tended to expand,and the phenomenon of obvious myocardial hypertrophy occurred.The H9C2 cells in the TFPH group improved significantly,and the cell surface area decreased.The ROS fluorescence intensity of H9C2 cells in TFPL group and TFPH group decreased significantly.The activities of T-SOD and GSH in H9C2 cells in TFPH group were significantly increased,and the content of MDA was decreased.The contents of TG and T-CHO in H9C2 cells in the TFPH group were significantly decreased,while the contents of Glu and ATP were increased.The intracellular protein concentration of H9C2 in TFPL group and TFPH group was significantly decreased.The content of FFA in the supernatant and pellet of H9C2 cells in the TFPH group decreased.The fluorescence intensity of AMPK and PPARɑ proteins in H9C2 cells in TFPH group increased,the fluorescence intensity of PPARγ protein in H9C2 cells in TFPH group decreased,the fluorescence intensities of GS and PKA proteins in H9C2 cells in TFPL and TFPH groups increased,and the fluorescence intensities of Beclin1 and LC3 B proteins in H9C2 cells in TFPH group increased The fluorescence intensity of Cas-9 protein in H9C2 cells in the TFPH group was significantly decreased.The relative expression levels of ANP,BNP,α-MHC and β-MHC genes in H9C2 cells in TFPH group were significantly decreased,the relative expression levels of ACC protein and genes in H9C2 cells in TFPH group were significantly decreased,and the relative expression levels of CPT1 A,AMPK and PPARα in H9C2 cells in TFPL and TFPH groups The relative expression levels of proteins and genes were significantly increased.The relative expression levels of PPARγ protein and gene in H9C2 cells in TFPH group decreased.The relative expression levels of ADCY1,ADCY2,β1AR and β2AR in H9C2 cells in TFPL and TFPH groups increased.The relative expression levels of H9C2 cells in TFPH group increased.The relative expression levels of PKA and GS proteins and genes in cells were significantly increased,the relative expression levels of Beclin1 and LC3 B proteins and genes in H9C2 cells in TFPH group increased,and the relative expression levels of Cas-9 and BAX proteins and genes in H9C2 cells in TFPH group decreased.The relative expression levels of Bcl-2 protein and gene in H9C2 cells increased.Conclusion: In this study,through in vivo and in vitro experiments,it was investigated that TFP can regulate lipid metabolism,energy metabolism,autophagy and apoptosis,increase the diversity and abundance of intestinal flora,regulate the structure of intestinal flora,and inhibit cardiomyocytes.Apoptosis,delays myocardial remodeling,improves symptoms of myocardial hypertrophy,and protects the myocardium.Key functional bacteria and potential biomarkers of cardiac hypertrophy in rats were identified and analyzed for their correlation with TFP treatment.However,its more specific mechanism of action needs further study. |
PDF Full Text Request