| BACKGROUND and OBJECTIVE:Cardiovascular remodeling is a series of inappropriate ventricular structure and vascular structure changes resulting from body’s response to a variety of cardiovascular damage.The pathogenesis of cardiovascular remodeling is complex and involves multiple systems such as the sympathetic nervous system and the renin-angiotensin-aldosterone system(RAAS),and in this process,various mechanisms such as inflammatory responses,autophagy,and oxidative stress are involved.Metalloproteinase 9(MMP-9),a member of the matrix metalloproteinase family(MMPs)family,is involved in physiological processes such as embryo implantation and angiogenesis,Previous studies have found that MMP-9 is significantly higher in a variety of cardiovascular diseases,when MMP-9 gene knockout or reduced expression can reduce cardiovascular remodeling development,while increased MMP-9 activity or expression further aggravates the occurrence of ventricular remodeling.It suggests that MMP-9 is involved in the process of the development of ventricular remodeling.Smooth muscle actin(SMA)can be used as a response indicator of the degree of ventricular remodeling during ventricular remodeling,and increased SMA protein expression indicates aggravated remodeling.Previous studies have found that a rat model of ventricular remodeling can be constructed by infusion of MMP-9,and the model is accompanied by an increase in angiotensin converting enzyme(ACE),angiotensin Ⅱ(Ang Ⅱ)and blood pressure,suggesting that MMP-9 induced ventricular remodeling may be mediated by RAAS.Subsequently,we used knockout AT-1 receptor(Agtrla knockout)mice,and infusion of MMP-9 was also found to cause cardiovascular remodeling in mice,suggesting that MMP-9 has a pro-ventricular remodeling effect independent of RAAS,but the specific mechanism is not yet fully understood.Autophagy is the process by which the cell itself removes damaged organelles from the cell and converts them into new energy substances.P62 is a key protein in the regulation of autophagy,and p62 protein can regulate autophagy by regulating the Kelch-like epichlorohydrin-related protein-1(Keap1)-nuclear factor E2-related factor 2(Nrf2)pathway.Studies have confirmed that when p62 accumulates in large amounts,it leads to elevated Keapl expression,which negatively regulates Nrf2;decreasing Nrf2 expression inhibits autophagy.Microtubule-associated protein 1A/1B-light chain 3(LC3Ⅱ),as a marker of autophagy levels,can reflect autophagy levels.When autophagy was inhibited,LC3Ⅱ was decreased and the binding of mitochondrial outer membrane receptor protein(TOM20)to LC3Ⅱ was reduced.Both MMP-9 and autophagy can lead to the development of ventricular remodeling,but it is unknown whether ventricular remodeling induced by MMP-9 regulates autophagy through autophagy and through what pathway.Based on the existence of regulation of autophagy by p62,this study intends to further investigate whether MMP-9 induces cardiovascular remodeling through the p62/Keap1-Nrf2 pathway.Since Ang Ⅱ also regulates p62 and is also involved in autophagy regulation,and our previous study found that MMP-9 can increase Ang Ⅱ,in order to avoid the effect of Ang Ⅱ-induced cardiovascular remodeling,this study intends to carry out a study in wild-type mouse basal AT1 receptor knockout mice.That is,wild-type mice and Agtr1a knockout mice were used to introduce MMP-9 by injection of adeno-associated virus(AAV9)vector to induce high MMP-9 expression in mice to investigate the mechanism of MMP-9 induced cardiovascular remodeling.To further confirm whether MMP-9 affects the p62/Keap1-Nrf2 pathway through the p62 intermediate link,wild-type mouse embryonic fibroblasts and p62 knockout mouse embryonic fibroblasts were planned to be used to highly express MMP-9 by transfecting MMP-9 DNA plasmid and observe Keapl and Nrf2 protein expression and autophagy changes downstream of the pathway.At present,the systemic therapeutic drugs for cardiovascular remodeling caused by increased MMP-9 have not been marketed in clinical practice.Fufang Zhen Zhuo Tiao ZhiCapsule(FTZ)is a clinical experience formula developed by Professor Guo Jiao using the theory of"regulating the liver and enshu",which is mainly composed of eight herbs including Ligustrum lucidum,Atractylodes macrocephala,and Sanqi,and is clinically used to treat diseases with abnormal glucose and lipid metabolism.Previous studies have found that FTZ can treat hyperlipidemia,diabetes,and prevent in-stent restenosis by regulating blood lipids and blood glucose,improving insulin resistance,reducing inflammatory factors,and oxidative stress injury;and reduce white fat cross-sectional area thereby reducing body fat accumulation and improving atherosclerosis.MMP-9 can be secreted by inflammatory cells such as macrophages and neutrophils,so we envisage that FTZ may have some efficacy in reducing MMP-9 expression or participating in autophagy regulation,while improving cardiovascular remodeling.For example,FTZ can improve cardiovascular remodeling induced by high MMP-9,which will provide a new understanding of the pathogenesis of cardiovascular remodeling and provide theoretical support for the therapeutic targets of integrated traditional Chinese and western medicine in the treatment of cardiovascular remodeling.Therefore,we intend to add FTZ to intervene high MMP-9 mice and investigate the ameliorative effect of FTZ on cardiovascular remodeling induced by high MMP-9.Materials and Methods:Animal experiment1.experimental animals:homozygous mice of wild-type and knockout types of the F8 generation were obtained using homozygote mating.2.Grouping and treatment of experimental animals:48 WT and 48 KO mice were randomly divided into wild-type and knockout control groups,model group,FTZ low and high dose groups according to body weight and male and female stratification,with a total of 8 groups,6 mice in each group.AAV9 unloaded control group:wild homozygotes(WT Control)and knockout homozygotes(KO Control),100 μl of AAV9 unloaded virus was injected into the tail vein.AAV9-MMP-9 model group:wild homozygote(WT MMP-9)and knockout homozygote(KO MMP-9);AAV9-MMP-9FTZ low-dose group:wild homozygote(WT Low Dose)and knockout homozygote(KO Low Dose);AAV9-MMP-9FTZ high-dose group:wild homozygote(WT High Dose)and knockout homozygote;(KO High Dose)AAV9-MMP-9100 μl was injected into the tail vein of each group above.FTZ intervention was performed at the end of the 8th week after AAV9 injection:the WT Low Dose group and KO LowDose group were given FTZ 3 g/kg/d,1 ml,by gavage;the WT High Dose group was given FTZ 6 g/kg/d,1 ml,by gavage;the WT Control group and KO Control group were given equal dose of vehicle by gavage once a day for 6 weeks.3.Animal experimental observation items:On the first day of the experiment,the baseline blood pressure of the mice was measured by the tail-cuff method,the body weight was monitored,and then AAV9-MMP-9 was injected.Blood pressure changes were measured on day 56th(at the end of week 8th);on day 57th(week 9),the mice were administered according to groups and treatment methods,followed by daily changes in body weight and food intake of the mice,and blood pressure of the mice was measured every two weeks.On the 98th day(at the end of the 14th week),cardiac ultrasound was measured under isoflurane anesthesia to detect left ventricular anterior and posterior wall thickness,left ventricular systolic and diastolic diameters,end-systolic and end-diastolic ventricular volumes and cardiac function at each stage of systole and diastole;then fasting for 12 hours,0.3%pentobarbital sodium solution 0.01-0.02ml/g was intraperitoneally injected for anesthesia,orbital venous plexus blood was selected for blood sampling,followed by sampling to detect various indicators,heart separation and weighing,weighing index(LVWI,LVWI=weight g/body weight g)was calculated,myocardial pathological section HE and Masson were used to observe myocardial pathological changes and collagen changes,respectively,imagePro-plus6.0 software was used to calculate myocardial cell cross-sectional area and myocardial collagen volume fraction;MMP-9 immunofluorescence staining was used to observe MMP-9 distribution in myocardium;immunofluorescence colocalization(LC3II/TOM20)was used to observe myocardial autophagy;abdominal aorta and mesenteric artery sections HE and Masson staining were used to observe vascular cavity wall ratio and collagen volume fraction,imagePro-plus6.0 software was used to analyze and calculate white fat cell cross-sectional changes in mice.ImagePro-Plus 6.0 software was used for analysis and calculation;MMP-9,TIMP-1,cholesterol,triacylglycerol,fasting insulin,and fasting blood glucose contents in plasma were measured by ELISA and glucose and lipid metabolism changes were observed by calculating the insulin resistance index;MMP-9 mRNA in myocardium was measured by qPCR;and MMP-9,p62,Keap1,Nrf2,LC3II,and SMA protein contents in myocardium were measured by Western blot.Cell experiments1.CellWild-type mouse embryonic fibroblasts(MFEs p62+/+)and p62 knockout 2.Cell experimental grouping and treatment:MFEs p62+/+and MFEs p62-/-were equally divided into two groups,the control group(control group)and the MMP-9 high expression group(MMP-9 group).In the control group,15 μl of unloaded transfectant was added to the culture medium,and in the MMP-9 group,an equal volume of transfectant mixture(5 μg MMP-9 DNA plasmid+15 μl transfectant)was added to the culture medium,followed by 24 hours of culture of cells of the two genotypes in the incubator,and cellular proteins were extracted for subsequent detection.3.Cell experimental observation items:MMP-9 protein expression was measured by Western blot to verify the effect of MMP-9 DNA plasmid transfection;Keapl and Nrf2 protein expression was measured to observe the effect of MMP-9 on p62 knockout cells;LC3Ⅱ protein expression was measured to observe autophagy.Results:Animal experiment1.Baseline:There was no difference in initial body weight and blood pressure between the groups,and the initial blood pressure of knockout mice was lower than that of wild-type miceThere was no significant difference in the initial body weight level between the groups(p>0.05);the initial blood pressure(systolic blood pressure,diastolic blood pressure,mean arterial pressure)of knockout mice was lower than that of wild-type mice(p<0.01),while there was no significant difference in the initial blood pressure between the two genotypes of mice(p>0.05).2.Modeling:high expression of MMP-9 increased food intake and blood pressure in mice,with no significant change in body weight(1)Changes in body weight and food intake of mice in each group with high expression of MMP-9On 1st-56th day,there was no significant change in body weight in mice with the two genotypes of high MMP-9 expression,and there was an upward trend in food intake compared with the control group(p>0.05).(2)High expression of MMP-9 increases blood pressureOn 56th day,systolic blood pressure,mean arterial pressure,and diastolic blood pressure were significantly increased in the WT MMP-9 group compared with the WT Low Dose group and the WT High Dose WT Control group(p<0.001;p<0.001;p<0.05);systolic blood pressure and mean arterial pressure were significantly increased in the KO MMP-9 group,KO Low Dose group,and KO High Dose KO Control group(p<0.001);there was no significant difference in their own blood pressure between wild-type and knockout mice.3.Mice in the model group had increased food intake,decreased food intake after FTZ intervention,and no difference in body weightThe food intake of the wild-type model group was significantly increased compared with the control group.Four weeks before FTZ intervention,there was no difference in food intake in the model group(p>0.05);6 weeks after FTZ intervention,food intake was reduced in the intervention group compared with the model group(p<0.05).There was no significant difference in WT body weight after FTZ intervention.After 2 weeks of FTZ intervention,there was no significant difference in food intake among the groups of knockout mice;after 4 weeks of FTZ intervention,the food intake was significantly increased in the model group compared with the control group(p<0.05),and there was no difference in food intake in the model group(p>0.05);after 6 weeks of FTZ intervention,the food intake was decreased in the KO High Dose model group compared with the control group(p<0.05).There was no significant change in KO body weight after FTZ intervention.4.Blood pressure increased in the model group and decreased after FTZ interventionSystolic blood pressure,diastolic blood pressure,and mean arterial pressure were significantly higher in the WT MMP-9 group compared with the WT Control group(p<0.001);diastolic blood pressure was significantly higher in the WT MMP-9 low dose group compared with the WT control group(p<0.05).Systolic blood pressure and mean arterial pressure were significantly lower in the WT Low Dose group and the WT High Dose group compared with the MMP-9 group(p<0.001),and systolic blood pressure was significantly lower in the WT High Dose group compared with the WT Low Dose group(p<0.05).Compared with the KO Control group,the KO MMP-9 group had significantly higher systolic blood pressure and mean arterial pressure(p<0.001),and the KO Low Dose KO MMP-9 group had significantly lower resting arterial pressure(p<0.05);the KO High Dose KO MMP-9 group had significantly lower systolic blood pressure and resting arterial pressure(p<0.05;p<0.001).FTZ dose-dependently decreased blood pressure.5.Model group ventricular remodeling,FTZ can improve ventricular remodeling(1)Myocardial hypertrophy and weight index increased in the model group;FTZ could improve myocardial hypertrophy and reduce weight index①Cardiac ultrasoundCompared with the WT control group,the ventricular wall was thickened,the LV mass index was increased and the E/A ratio was decreased in the WT MMP-9 group;the KO model group showed similar findings(p<0.05).After FTZ intervention,ventricular wall hypertrophy and LV mass decreased in a dose-dependent manner in both genotype intervention groups(p<0.05).②WeighingMMP-9 weight index(LVWI)was increased in both genotypes compared with the control group(p<0.05).There was a decrease after FTZ intervention,with a significant decrease in the high-dose MMP-9 group compared with the decrease(p<0.05).(2)Myocardial tissue in the model group showed remodeling pathological changes;FTZ could improve its changes①HE staining was used to observe the changes of myocardial remodelingIn the two genotype model groups,the cross-sectional area of cardiomyocytes was increased,the myocardial fibers were disorganized,the cytoplasm was dissolved or necrotic,the boundary between cardiomyocytes was blurred,and myocardial fiber breakage was occasionally observed.After FTZ intervention,compared with the model group,the crosssectional area of cardiomyocytes was reduced(p<0.05),the myocardial arrangement was more neat,the myocardial fiber rupture was reduced,and the myocardial cells showed clear boundaries,with a more significant improvement in the high-dose group.②Masson staining was used to observe the changes of myocardial collagen volume fractionMyocardial collagen volume fraction(CVF)was significantly lower in the two genotype model groups than in the control group(p<0.05).CVF was increased in the high-dose model group with both genotypes(p<0.05);there was no difference in the low-dose model group with both genotypes(p>0.05).(3)The content of SMA,a myocardial ventricular remodeling protein,was increased in the model group,and FTZ intervention could reduce its protein expressionSMA was significantly higher in the myocardium of the two genotype model groups(p<0.05).After FTZ intervention,the expression of SMA was decreased in the WT High Dose WT MMP-9 group compared with the SMA group(p<0.05);it was decreased in the KO Low Dose KO MMP-9 group compared with the SMA group(p<0.05).6.Vascular remodeling in the model group;FTZ could improve vascular remodeling(1)The mesenteric artery cavity to wall ratio was decreased in the model group;FTZ could increase the mesenteric artery cavity to wall ratioThe ratio of mesenteric artery lumen wall was significantly reduced in the two genotype models compared with the control group(p<0.05).After FTZ intervention,the mesenteric artery lumen wall ratio was increased in the high-dose model group of both genotypes compared with the mesenteric artery lumen wall ratio(p<0.05).(2)The CVF of the thoracic aorta was decreased in the model group;FTZ increased the number of CVF in the thoracic aortaCompared with the control group,the CVF of thoracic aorta was significantly decreased(p<0.05),and the cavity wall ratio of thoracic aorta was not significantly changed.After FTZ intervention,CVF in thoracic aorta increased significantly in the high-dose model group of both genotypes compared with the control group(p<0.05).7.MMP-9 expression was increased in the myocardium and plasma of the model group,which was significantly inhibited by FTZ(1)Immunofluorescence observation of myocardial MMP-9 distributionMyocardial immunofluorescence showed that myocardial MMP-9 protein expression was increased in the model group of the two genotypes,mainly distributed in the myocardial interstitium,especially in the muscle fiber disorder;FTZ could reduce myocardial MMP-9 expression,especially in the high-dose group,without affecting the distribution.(2)MMP-9 mRNA and MMP-9 protein expression were increased in the myocardium of the model group;FTZ could reduce MMP-9 mRNA and MMP-9 protein expression in the myocardiumMMP-9 mRNA and MMP-9 protein expression were significantly increased in the myocardium of the two genotype models compared with the control group(p<0.05;p<0.001,respectively).After FTZ intervention,MMP-9 mRNA and MMP-9 protein were significantly decreased in the two genotypes compared with the model group;among them,wild-type decreased in a dose-dependent manner,and no dose-dependent difference was observed in knockout type.(3)MMP-9,TIMP-1,and MMP-9/TIMP-1 were increased in the plasma of the model group,and FTZ could reduce their levelsThe levels of MMP-9,TIMP-1 and MMP-9/TIMP-1 were significantly increased in the two genotype models compared with the control group(p<0.05).After FTZ intervention,the plasma levels of MMP-9,TIMP-1,and MMP-9/TIMP-1 decreased in a dose-dependent manner in the two genotype intervention groups compared with the model group(p<0.05).8.p62 and Keap1 protein expression increased and Nrf2 protein expression decreased in the model group;FTZ significantly reversed it(1)Changes in myocardial p62,Keap1,and Nrf2 protein amountsThe expression of p62 and Keap1 was increased and the expression of Nrf2 was decreased in the two genotype models compared with the control group(p<0.05).After FTZ intervention,the expression of p62 and Keapl was decreased and the expression of Nrf2 protein was increased in the intervention group compared with the model group(p<0.05);among them,the wild-type decreased in a dose-dependent manner,and no dosedependent difference was observed in the knockout type.(2)Correlation analysis between MMP-9 and p62In both genotypes,MMP-9 was positively correlated with p62 protein expression:r=0.406,p<0.05 in the WT group;r=0.435,p<0.05 in the KO group.9.Level of autophagyThe level of LC3Ⅱ protein,which reflects autophagy,was decreased and LC3Ⅱ binding to the mitochondrial outer membrane receptor(TOM20)protein was reduced in the model group;the level of LC3Ⅱ protein increased and LC3Ⅱ binding to the mitochondrial outer membrane receptor(TOM20)protein increased after FTZ intervention.Among them,wildtype LC3Ⅱ protein levels increased in a dose-dependent manner,and no dose-dependent difference was observed in the knockout type.10.Abnormal glucose and lipid metabolism was observed in the model group,and FTZ could ameliorate abnormal glucose and lipid metabolism(1)Plasma cholesterol(CH),triacylglycerol(TG),fasting blood glucose(GLU),fasting insulin(INS)and insulin resistance index were increased in the two genotype models compared with the control group(p<0.05).FTZ intervention dose-dependently reduced its levels(p<0.05).(2)The cross-sectional area of white fat was increased in the two genotype models compared with the control group(p<0.05);FTZ intervention could reduce the cross-sectional area of white fat,and the improvement was more significant in the high-dose group.Cell experiments1.MMP-9 DNA plasmid transfection into MEFs p62+/+and MEFs p62-/-groups had good transfection results2.MMP-9 DNA plasmid transfection resulted in increased Keapl protein expression and decreased Nrf2 protein expression levels in the p62+/+group of MEFs(p<0.05);MMP-9 DNA plasmid transfection had no significant effect on Keap1 or Nrf2 protein expression in the p62-/group of MEFs(p>0.05).3.MMP-9 DNA plasmid transfection decreased LC3Ⅱ protein levels in the p62+/+group of MEFs(p<0.05);MMP-9 DNA plasmid transfection had no significant effect on LC3Ⅱ protein levels in the p62-/-group of MEFs(p>0.05).CONCLUSION:MMP-9 may lead to cardiovascular remodeling by up-regulating the p62/Keap1-Nrf2 pathway,which can be ameliorated by FTZ. |
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