Study On Protective Effect And Mechanism Of Pilose Antler Peptide On Adriamycin-induced Myocardial Injury By TGF-β/smads/ERK Signaling Pathway

Objective:Pilose antler is the key medicine for warming and nourishing the heart and kidney which has the effects of generating and refining qi,strengthening the heart and veins,and promoting the repair of various tissues.Its pharmacodynamic substances are polypeptides.In this study,the protective effect of pilose antler peptide（PAP）on adriamycin（ADR）-induced myocardial injury was observed through in vivo and in vitro experiments,and to explore the correlation and mechanism with TGF-β/Smads/ERK signaling pathway.Methods:1.Extraction and purification of PAP:PAP was extracted by acetate buffer solution extraction,the protein was identified by the biuret method,the protein content was detected by the Coomassie brilliant blue assay,and PAP was separated and purified by ion-exchange chromatography and gel filtration chromatography.2.In vitro cell tests:The effects of ADR and PAP on the survival rate of H9c2 cells were detected by CCK-8 method.The experiments were divided into control group,ADR group,PAP group and combined induction group（ADR+PAP group）.Levels of lactate dehydrogenase（LDH）,Creatine kinase MB（CK-MB）and cardiac troponin（cTn）in cell supernatants were detected by ELISA.The levels of B-cell lymphoma-2（Bcl-2）,Bcl-2apoptotic factor X（Bax）and Caspase9 in H9c2 cells were detected by immunofluorescence assay.Cell apoptosis and cell cycle were detected by flow cytometry.3.In vivo tests:The model of myocardial injury on rats was replicated by intraperitoneal injection of ADR in every other day.The experiment was divided into control group,ADR group,CoQ₁₀0 group,low dose group（ADR+PAP 100 mg/kg group）,and high dose group（ADR+PAP 200 mg/kg group）.The general state of the rats in each group was observed,and the body weight（BW）of the rats was measured every 3 days.Heart weight（HW）and HW/BW index of rats in each group were measured.Powerlab data acquisition and analysis system was used to detect changes of ST segment and heart rate（HR）of electrocardiogram on rats.The myocardial specific transcription factors Nkx2.5,GATA4,ATF-2,MEF-2C,cTnT and cTnI levels in Serum and Bax,Bcl-2 and Caspase3levels in myocardial tissue were detected by ELISA.HE staining was used to detect the pathological changes of myocardium in rats.Apoptosis of cardiomyocytes was detected by TUNEL staining.4.The metabolomics study:Urine metabolites from ADR group and PAP group rats were detected by UPLC-MS/MS analysis.Quantitative analysis of metabolites were detected by multiple reaction monitoring（MRM）with triple quadrupole mass spectrometry.Principal component analysis（PCA）and other methods were used to classify the two groups of metabolites to find potential biomarkers and enrich them by KEGG method.The mRNA levels of Transforming growth factor-prime（TGF-β）,Smad7,Extracellular regulated protein kinases（ERK）,Major histocompatibility complex（MHC）and Connexin43（CX43）in H9c2cells and rats were detected by RT-PCR.The protein expression levels of TGF-β/Smads/ERK signaling were detected by western blot.Results:1.Based on the preliminary work in the laboratory,the optimal extraction process of PAP was pH=4.0 HAc-NaAc buffer,90%ethanol,centrifugation time 20min,and ion exchange chromatography and gel filtration chromatography were used to obtain two active ingredients with higher purity.The technology of extracting PAP with salt buffer solution extraction technology could guarantee the physical and chemical properties of PAP intact.2.In vitro（1）The CCK-8 analysis results showed that the IC₅₀0 of ADR-induced H9c2 cells apoptosis was at 5μM for 24 h,and PAP could promote the proliferation of H9c2 cells which the optimal conditions was at 16μg/mL for 72 h.（2）LDH test results showed that compared with the control group,the LDH in the ADR group was significantly increased（P<0.01）,and the PAP group had no significant change（P>0.05）.Compared with the ADR group,LDH in ADR+PAP group was significantly reduced（P<0.01）.（3）ELISA results showed that compared with the control group,the levels of CK-MB,cTnT,and cTnI in the ADR group were significantly increased（P<0.05 or P<0.01）,while the PAP group had no significant changes（P>0.05）.By comparison,the levels of CK-MB,cTnT and cTnI in the ADR+PAP group were significantly reduced（P<0.05 or P<0.01）.（4）Flow cytometry results showed that compared with the control group,the apoptosis rate of H9c2 cells in the ADR group was significantly increased（P<0.01）,and the number of cells in the G2/M phase was significantly increased（P<0.01）,there was no significant change in the PAP group（P>0.05）.Compared with the ADR group,the apoptosis rate in the ADR+PAP group was significantly reduced（P<0.01）,and the number of cells in the G2/M phase was significantly reduced（P<0.01）.3.In vivo（1）Compared with the control group,the rats in the ADR group were mentally debilitated,their activity was significantly reduced,movement was slow,activity was reduced,body hair was fluffy,drinking water were significantly reduced,and their BW was significantly reduced（P<0.01）.The BW of rats in CoQ₁₀0 group,PAP low and high dose groups was significantly increased（P<0.05 or P<0.01）,but still significantly lower than that in control group.（2）Compared with the control group,the HW of the rats in the ADR group was significantly reduced（P<0.05）,however the HW/BW index was significantly increased（P<0.01）.Compared with the ADR group,there was no significant change on the HW of rats in the CoQ₁₀0 group,PAP low and high dose groups（P>0.05）.However,the HW/BW index decreased significantly（P<0.05 or P<0.01）.（3）ECG test results showed that compared with the control group,the ST segment and HR of rats in the ADR group were significantly increased（P<0.01）.Compared with the ADR group,the ST segment of the ECG and HR in the CoQ₁₀0 group,PAP low and high dose groups were significantly reduced（P<0.05 or P<0.01）.（4）ELISA test results showed that:（1）Compared with the control group,the levels of cTnT and cTnI in the serum of ADR group were significantly increased（P<0.01）.Compared with the ADR group,the serum of PAP low and high dose groups cTnT and cTnI levels were significantly reduced（P<0.05 or P<0.01）.（2）Compared with the control group,the levels of myocardial specific transcription factors Nkx 2.5,GATA4 and MEF-2C of rats in the serum of ADR group were significantly reduced（P<0.05 or P<0.01）.Compared with the ADR group,the levels of Nkx 2.5,GATA4 and MEF-2C in the serum of rats in the CoQ₁₀0 group,PAP low and high dose groups were significantly increased（P<0.05or P<0.01）.There was no significant change in the level of ATF-2 in each group（P>0.05）.（3）Compared with the control group,the levels of Bax and Caspase3 in the heart of ADR group were significantly increased,and the levels of Bcl-2 were significantly reduced（P<0.01）.Compared with the ADR group,the levels of Bax and Caspase3 on heart of rats in the CoQ₁₀0 group and PAP low and high dose groups were significantly decreased,and the levels of Bcl-2 were significantly increased（P<0.05 or P<0.01）.（5）The HE staining results showed that compared with the control group,the myocardial tissue of the ADR group showed typical myocardial damage,disordered muscle bundles,inflammatory cell infiltration,diffuse edema,and the pathological score was significantly increased（P<0.01）.Compared with the ADR group,the degree of myocardial injury and the pathological score of the rats in the CoQ₁₀,PAP low and high dose group were reduced significantly（P<0.01）.（6）The results of TUNEL staining showed that compared with the control group,the myocardial tissue of the ADR group had obvious apoptosis,dark brown areas of various sizes appeared,and the myocardial cell muscle bundles were disorderly arranged and the apoptosis area was increased significantly（P<0.01）.Compared with the ADR group,the muscle bundles of the myocardial tissue of the CoQ₁₀0 group,PAP low and high dose groups were relatively neatly arranged,and the apoptosis area was reduced significantly（P<0.01）.4.（1）A total of 121 differential metabolites were screened from metabolomics studies,including amino acids and their metabolites,benzene and its derivatives,nucleotides and their metabolites,and organic acids and their derivatives.It is related to Purine metabolism,Bile secretion and cAMP signaling pathway.（2）RT-PCR results showed that compared with the control group,the expression levels of TGF-β₁,β-MHC mRNA of H9c2 cells in the ADR group were significantly increased,and the expression levels of Smad7,α-MHC,and Cx43 mRNA were significantly reduced（P<0.05 or P<0.01）.There was no significant changes in the PAP group（P>0.05）.Compared with the ADR group,the expression levels of TGF-β₁,β-MHC mRNA in H9c2 cells in the ADR+PAP group were significantly reduced,and the expression levels of Smad7,α-MHC and Cx43 mRNA were significantly increased（P<0.05 or P<0.01）.Compared with the control group,the expression levels of TGF-β₁ andβ-MHC mRNA in heart of the ADR group were significantly increased,and the expression levels of Smad7,α-MHC and Cx43 mRNA were significantly reduced（P<0.01）.Compared with the ADR group,the expression levels of TGF-β₁ mRNA in the heart of the CoQ₁₀group,PAP low and high dose groups were significantly reduced,and the expression levels of Smad7 and Cx43 mRNA were significantly increased（P<0.05 or P<0.01）.The expression levels ofα-MHC and Cx43 mRNA in the heart of rats in the CoQ₁₀0 group and the high-dose PAP group were significantly increased（P<0.05）.The expression levels ofβ-MHC mRNA in the heart of the high-dose PAP group were significantly reduced（P<0.05）.（3）Western blot results showed that compared with the control group,the expression levels of TGF-β₁,Smad4,p-Smad2,p-Smad3,and p-ERK proteins were significantly increased in H9c2 cells in the ADR group,and the levels of Smad7 and PKC proteins were significantly reduced（P<0.05 or P<0.01）.Except for the Smad7 protein level of H9c2 cells in the PAP group,the other proteins were not significantly changed（P>0.05）.Compared with the ADR group,TGF-β₁,Smad4 of H9c2 cells in the ADR+PAP group,p-Smad2,p-Smad3 and p-ERK protein expression levels were significantly reduced（P<0.05 or P<0.01）,Smad7protein expression levels were significantly increased（P<0.01）,and Smad5 and PKC protein expression levels were not significantly changed（P>0.05）.Compared with the control group,the expression levels of TGF-β₁,Smad4,p-Smad2,p-Smad3 and p-ERK proteins in heart of ADR group were significantly increased（P<0.01）.Smad7 and PKC protein expression levels were significantly reduced（P<0.01）.Compared with the ADR group,TGF-β₁,Smad4,p-Smad2,p-Smad3 and p-ERK in heart of the CoQ₁₀0 group,low PAP and high-dose groups on protein expression level were significantly reduced（P<0.05 or P<0.01）,and the expression levels of Smad7 and PKC protein were significantly increased（P<0.01）Conclusion:PAP can improve the pathological state of ADR-induced myocardial injury by inhibiting the TGF-β/Smads/ERK signaling pathway,inhibit myocardial cell apoptosis,and slow down myocardial cell cycle tissues.The mechanism may be related to the regulation of Purine metabolism,Bile secretion,and cAMP signaling pathway related to metabolic pathways.