Study On The Role And Mechanism Of Metformin In The Treatment Of Pulmonary Hypertension

【Objective】 Pulmonary Aterial Hypertension(PAH)is one of the common severe complications of congenital heart disease(CHD).The main pathological mechanism of PAH is pulmonary smooth muscle proliferation and vascular remodeling.PAH is the end-stage complications in many cardiovascular disease.It has important clinical significance to study its development mechanism and seek new drug treatment targets.The latest research shows that pulmonary vascular endothelial cells in AMPK(AMP dependent protein kinase,Adenosine 5 "-monophosphate(AMP)-activated protein kinase,AMPK)plays an important role in the pathological process of PAH.Metformin(MET)is one of the most commonly used antidiabetic drug.It inhibits glycogen output and promote skeletal muscle glucose uptake.It is also an agonist of the AMPK pathway.This project aims to explore the possibility of AMPK as a new therapeutic target for PAH,and to explain the complete process of metformin for PAH treatment further.【Methods】 Choose 24 healthy male SD rats aged 6~8 weeks,weighing 180g-250 g.The Rats were randomly divided into six groups(n=8): normal control group(1month group and 2 months group),monocrotaline injection group(1 month group and 2 months group),and treatment group(MCT+MET 1 month group and 2months group).In the MCT Group,PAH was induced by intraperitoneal injection of MCT(Monocrotaline)in rats(60mg/kg),while control group was injected intraperitoneally with equal volume of normal saline;While the MCT+MET group were given metformin suspension(100mg/kg·day-1)by gavage.The hemodynamic parameters of heart and lung were detected in different groups of rats,and the hemodynamics was detected by Doppler ultrasound in 30 days(1 month group)and60 days(2 months group)respectively;The mean pulmonary artery pressure(m PAP),right ventricular systolic pressure(RVSP),pulmonary artery systolic pressure(SBP)and pulmonary arterial diastolic pressure(DBP)were measured by right heart floating catheter after B-ultrasound examination;After the euthanasia,the right ventricular hypertrophy index(RV/LV+S)was measured by heart tissue.H&E staining,Masson staining and Sirius red staining were conducted to detect the remodeling of arteriole in the heart and lung tissue.Western blot of homogenate of lung tissue and immunohistochemistry were conducted to examine the protein level of related molecular pathways.【results 】 In the 1 month group and 2 months group,their own MCT group compared with control group respectively.MCT rats’ m PAP and RVSP were increased in varying degrees.HE staining showed that the pulmonary micro vascular smooth muscle have different degrees of proliferation;Histological analysis of Masson staining and Sirius red staining showed that pulmonary artery was obviously muscular and type I collagen was obviously proliferated.The determination of the degree of arteriosarcoma of the lung and the quantitative analysis of collagen around the blood vessels showed arterial arteriosclerosis and hyperplasia of blood vessels.The above changes were more obvious in the 1 month group.The hemodynamics and histological analysis of rats in 1 month and 2 months were shown that the rats in the doses of 60 mg/kg of MCT injection in 1 month group and2 months group have self-healing tendencies,and 2 months group is significant.MCT rats were given Metformin suspension gavage(100 mg/kg/day-1).The two groups(1 month group and 2 months group)MCT+MET rats’ m PAP and RVSP reduced to some extent.H&E staining analysis showed that the proliferation of small pulmonary arterial smooth muscle was not obvious.The analysis of Masson staining and Sirius red staining revealed the improvement of vascularization and collagen fibrosis in the MCT+MET group.In addition,the effect of MET was not obvious in group of 2 months,which may be related to its self-healing tendency.Finally,the results of WB verify that metformin’s effect is consistent with the activation of AMPK and its downstream ACC(Acetyl-Co A Carboxylase,ACC).The AMPK correlation pathways of the self-healing MCT rats were also activated in varying degrees compared to the Control group.Immunohistochemistry also confirms this result.Immunohistochemistry also showed that the rat pulmonary hypertension model induced by wild lilium was infiltrated by inflammatory factors,and metformin alleviated the reaction.【conclusion】1.The AMPK pathway of the MCT model was activated in different degrees due to the difference in feeding time.2.The MET has a definite therapeutic effect on the pulmonary hypertension rats with MCT model.In the course of disease,the efficacy of metformin decreased over time.The activity of AMPK pathway was positively correlated with the effect of metformin.【Objective】Pulmonary Aterial Hypertension(PAH)is one of the most common complications of congenital heart disease(CHD).The main pathological mechanism of PAH is the proliferation of pulmonary arterioles smooth muscle and vascular remodeling.According to a new study,the AMPK(Adenosine 5‘-monophosphate(AMP)-activated protein kinase,AMPK)pathway agonist-metformin has a therapeutic effect on PAH.Adenosine activated protein kinase(AMPK)is an important protein kinase,which mainly coordinates metabolism and energy needs.It also has important relationship with mitochondrial homeostasis.This study attempts to elucidate the mechanism of metformin to relieve PAH.【Methods】 Choose 24 healthy male SD rats aged 6~8 weeks,weighing 180g-250 g.The Rats were randomly divided into three groups(n=8): normal control group(group Control),monocrotaline injection group(group MCT),and treatment group(group MCT+MET).In the MCT Group,PAH was induced by intraperitoneal injection of MCT(Monocrotaline)in rats(60mg/kg),while control group was injected intraperitoneally with equal volume of normal saline;While the MET group were given metformin suspension(100mg/kg·day-1)by gavage.Rats were killed 30 days after MCT injection(1 month group).Using frozen section for reactive oxygen species(reactive oxygen species,ROS)and mitochondrial membrane potential(JC-1,Δψm)were tested by immunofluorescence.The ultrastructure and mitochondrial morphology of lung tissue were observed by electron microscope.WB detects mitochondrial related proteins such as Cytochrome C(Cyto C).【results】 After the MCT rats were given metformin(Metformin,MET)in the 1month group,it was proved in the first part that MCT did relieve the pulmonary hypertension in different degrees.ROS was detected by frozen section,and the fluorescence intensity was analyzed by immunofluorescence.The results showed that the ROS formation in MCT+MET group was lower than that in MCT group;Mitochondrial membrane potential is semi quantitative detection;JC-1(red /green fluorescence intensity,fluorescence intensity,Δψm)compare the relative ratio.The value of MCT group was higher,and the red fluorescence intensity was stronger,indicating that the mitochondrial membrane potential was increased,and the cell was in the stage of apoptosis inhibition and vascular remodeling;On the contrary,the ratio of MCT+MET treatment group was larger.Indicating that the intensity of red fluorescence was stronger.On the contrary,the membrane potential of MCT+MET treatment group was lower than that of MCT group,but it was not normal,either.To observe the mitochondrial morphology and ultrastructure of lung tissue using electron microscopy,Control group was not found in mitochondria arranged disorderly,no edema and vacuolization,and mitochondrial cristae arranged orderly;MCT group showed mitochondrial derangement,visible mitochondria edema,vacuoles,and cristae structure destroyed;In group MCT+MET,mitochondrial structure and internal cristae were relatively normal,and no edema or vacuoles were observed.WB assay showed that the expression of Cyto C in MCT group was increased,and the mitochondrial damage was more serious,but the increase in MET group was not obvious.【conclusion】1.The treatment of pulmonary hypertension can reduce the mitochondrial damage in the lung tissue and may be associated with promoting apoptosis.2.The MET treatment of pulmonary hypertension can maintain the normal morphology of mitochondria in the lung tissues.