| Objective Acute myocardial infarction is a severe disease caused by artery obstruction,it owns high mortality,which can provide new thoughts for AMI.Acute myocardial infarction induces endoplasmic reticulum stress-mediated autophagy and apoptosis,we infers that endoplasmic reticulum stress may play an important role in the occurrence and development of acute myocardial infarction.Mzb1 is an endoplasmic reticulum protein which could binds to the endoplasmic reticulum stress chaperone GRP94,therefore,we speculates that Mzb1 may regulate endoplasmic reticulum stress progression.Mzb1 overpression could improve heart injury.Puerarin is the traditional Chinese medicine which can descline inflammatory,oxidative and reticulum stress.The literature shows that puerarin has a protective effect on experimental myocardial ischemia,and this research aims to explore the protective mechanism of puerarin on acute myocardial ischemia induced by coronary artery ligation,and provide a new basis for the clinical treatment of puerarin.Methods The research includes two part experiments.In vivo: 10-week-old SPF-grade males C57BL/6 mice randomly selected with Sham,acute myocardial infarction group(AMI)and puerarin group(50 mg/kg/day,100 mg/kg/day),10 mice in each group.After 1 week of adaptive feeding,puerarin groups were subjected to daily gavage(50mg/kg/day,100 mg/kg/day),continuous gavage for 14 days,then the mice were subjected with left coronary artery ligation for 24 h.H&E staining was used to detect myocardial structures,and DHE staining measured the ROS in the heart.Phosphorylated IRE1(p-IRE1),phosphorylated inositol-requiring enzyme 1(IRE1),chaperone heat shock protein 78(GRP78),transcription factor C/ Transcription factor C/EBP homologous protein(CHOP),Cleaved-Caspase3 and Mzb1 protein expression were examed by Western blot.In vitro,AC16 cells were randomly divided into solvent group,H2O2 group,puerarin group and Mzb1 knockdown group,MTT was used to evaluate cell viability,ROS staining detects oxygen,and TUNEL staining tests the apoptosis.Western blot monitor the expression with p-IRE1,IRE1,GRP78,CHOP,Cleaved-Caspase3,and Mzb1.Results1.In vivo:(1)Cardiac function evaluation: compared with the Sham group,the left ventricular ejection fraction(EF)and left ventricular shortening fraction(FS)of mice in the AMI group were significantly reduced(P < 0.05),the EF and FS in the puerarin group were rised than AMI(P < 0.05).(2)TTC results: in AMI group,the area of infarction was increased compared with Sham(P < 0.01),it was reduced after puerarin(P < 0.05).(3)H&E staining results: the myocardial fibers in AMI were broken and arranged disordered,and the intercellular space became wider than Sham,different concentrations of puerarin treatment improve muscle filament arrangement and restore heart structure.(4)DHE staining results: compared with the Sham group,the oxygen radical level of mice in the AMI group increased,puerarin group oxygen radical levels were reduced compared to the AMI group.(5)Apoptosis results: the protein of Cleaved-Caspase3 in the AMI group was elevated compared to the Sham(P< 0.05),but the protein descend in the puerarin(P < 0.05).(6)Results of endoplasmic reticulum stress proteins: the levels of GRP78,CHOP and p-IRE1 proteins in mice in the AMI group was upregulated than Sham(P < 0.05),in the puerarin group,the expression of GRP78,CHOP and p-IRE1 proteins was downregulated compared with the AMI group(P < 0.05).Mzb1 protein expression results: compared with the Sham group,the expression of Mzb1 protein in mice in the AMI group decreased(P < 0.05),compared with the model group,Mzb1 protein expression was elevated in mice in the AMI group.(P < 0.05).2.In vitro:(1)Evaluation of AC16 cardiomyocytes viability: there was no apparent discrepancy between Vehicle(Vec),control and the puerarin(Pue)(P > 0.05),compared with Vec group,cell viability in H2O2 group decreased significantly(P <0.05),and cell viability was significantly restored after puerarin treatment(P < 0.05).(2)Oxygen results: the percentage of positive cells in the H2O2 group increased than Vec(P < 0.01),and it decreased after puerarin treatment(P < 0.05).(3)Apoptosis results:TUNEL results: the percentage of apoptosis-positive in H2O2 rised significantly compared with Vec(P < 0.05),and the percentage decreased after puerarin(P < 0.05),Cleaved-Caspase3 protein results: Cleaved-Caspase3 in H2O2 upregulater than Vec(P <0.01),and the protein expression decreased after puerarin treatment(P < 0.05).(4)Mzb1 protein detection results: Compared with Vec,the expression of Mzb1 protein in H2O2 group decreased(P < 0.05),and the expression after puerarin treatment increased(P < 0.05).(5)Evaluation of AC16 cardiomyocyte viability after silencing Mzb1:compared with the H2O2+pue group,cell viability decreased significantly in the H2O2+pue+si-Mzb1 group(P < 0.05).(6)Results of AC16 cardiomyocytes reactive oxygen species after silencing Mzb1: compared with the H2O2+pue group,the percentage of positive cells in the H2O2+pue+si-Mzb1 group increased(P < 0.05).(7)AC16 cardiomyocyte apoptosis detection results after silencing Mzb1: TUNEL test results: Compared with H2O2+pue group,the percentage of apoptosis-positive cells in H2O2+puerarin+si-Mzb1 group increased(P < 0.05).Results of Cleaved-Caspase3protein: Compared with the H2O2+pue group,the expression of Cleaved-Caspase3 protein in the H2O2+pue+si-Mzb1 group was upregulated(P < 0.05).(8)Results of endoplasmic reticulum stress-related pathway protein detection in AC16 cardiomyocytes after silencing Mzb1: compared with Vec,the expression of GRP78,CHOP and p-IRE1 proteins in the H2O2 group was upregulated(P < 0.05),and the expression of GRP78,GRP94 and p-IRE1 proteins after puerarin treatment was downregulated(P < 0.05),compared with the H2O2+pue group,the expression of GRP78,CHOP and p-IRE1 proteins in the H2O2+pue+si-Mzb1 group was upregulated(P < 0.05).(9)The protein level of Mzb1 in AC16 cells after Tunicamycin(TM)treatment: TM inhibited the expression of Mzb1,which was recovered by purearin treatment(P < 0.05),compared to the TM+pue group,protein expression was upregulated in the Mzb1 knockdown group(P < 0.05).The consequence of endoplasmic reticulum stress proteins induced by TM: the proteins of GRP78,CHOP and p-IRE1 in the TM was fallen compared with control(P < 0.05),and the expression of GRP78,CHOP and p-IRE1 proteins after puerarin treatment was down-regulated(P< 0.05),while Mzb1 silencing blocked the function of puerarin(P < 0.05).Conclusions1.Acute myocardial infarction reduces the expression of cardiomyocyte protective molecule Mzb1,increases oxidative and endoplasmic reticulum stress in cardiomyocytes,puerarin therapy can upregulate cardiomyocyte Mzb1 expression,reduce cardiomyocyte oxidative stress and endoplasmic reticulum stress and cardiomyocyte damage and death.2.In the H2O2-induced vivo myocardial injury model,we further confirm that puerarin may inhibit reticulum stress and damage in cardiomyocytes by upregulating Mzb1 endoplasm.This study proposes a new theoretical mechanism for the myocardial protective effect of the traditional Chinese medicine puerarin in the treatment of myocardial infarction. |
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