TGF?1 Is Regulated By LPS/TLR4 And Contributes To Myocardial Fibrosis Of Constrictive Pericarditis

Objective:Constrictive Pericarditis(CP)is a ventricular diastolic filling abnormality caused by loss of pericardial elasticity.The inelastic pericardium limits the volume of ventricle,the pressure of atrial and venous increase,stroke volume and cardiac output are significantly reduced,and patients may experience heart failure,exertional dyspnea,edema,arrhythmia,or even sudden death.Exudation,fibrosis mainly involves visceral pericardium in early stage,severe fibrosis,calcification and adhesion appear in chronic phase.Abnormal systolic function is accompanied by a decrease in CP contractile function,which is associated with myocardial fibrosis.Myocardial fibrosis may be an important factor affecting CP treatment and prognosis.The mechanism of early CP myocardial fibrosis is the basis and key to treatment and prognosis.TGF?1 promotes transdifferentiation of mesenchymal mesenchymal cells and cardiac fibroblasts to myofibroblasts,and then phosphorylation of Smads leads to abnormal expression and imbalance of?SMA,COL?/?and extracellular matrix,which may be the key factor inducing myocardial fibrosis of CP.LPS/TLR4 can up-regulate TGF?1 expression in a variety of ways,TGF?1/Smads pathway may be the mechanism leading to myocardial fibrosis in CP model rats.In this study,a rat model was used to explore the method of preparing a CP model without injecting inflammatory agent into the thoracic cavity.The model was validated in two ways:pericardial elasticity loss and ventricular diastolic abnormalities.Based on the successfully prepared rat CP model,two-dimensional speckle tracking imaging(STI)technique was used to further identify and evaluate myocardial damage induced by CP through various motion indicators including longitudinal,circumferential,radial stain and torsion of left ventricular.Molecular biology techniques were used to further detect the expression of LPS/TLR4-TGF?1related markers in myocardial tissue of CP rat model.Cardiac fibroblasts(CFs)were used to further validate the possible mechanism by which LPS/TLR4 upregulates TGF?1/Smads in CP-induced myocardial fibrosis in primary CFCs.Methods:1.Preparation and validation of rat model of constrictive pericarditis:(1)Male Wistar rats were divided into 4 groups according to different treatment methods:blank control group(N),LPS treatment group(LPS),pure talc powder treatment group(TP),LPS+talcum powder treatment group(LPS+TP).Each group was randomly divided into 6 rats.The corresponding agent causing inflammation is injected into the pericardial cavity through the anterior chest wall.(2)After 8 weeks,echocardiography and pathology were performed.(3)Using gross specimens,HE,Masson,and Sirius red staining were used to observe the thickening and fibrosis of pericardium in the four groups.(4)Real-time PCR was used to detect the m RNA expression of?SMA,COL?and COL?;and western blotting was used to detect the protein expression of?SMA in the pericardial tissues in all samples.2.Evaluation of myocardial injury in rats with constrictive pericarditis by two-dimensional speckle tracking imaging:(1)Male Wistar rats were divided into 2 groups:13 in the control group(N)and 17 in the model group(CP).In the CP group,the inflammatory agent was injected into the pericardial cavity through the anterior chest wall,while the N group was anesthetized,but no pericardial injection was performed.(2)After 8 weeks of feeding,echocardiographic scans were performed using a GE Vivid E9 ultrasound system with an M12S probe(9MHz to 12 MHz)and a rodent program,and dynamic images of each slice were stored for analysis at the GE Echo PAC workstation.Routine parameters including chamber diameter,area,left ventricular ejection fraction,mitral E'and E/E',torsion,circumferential,longitudinal,radial strain and strain rate were measured.(3)Gross specimens,HE,Masson,and Sirius red staining were used to observe thickening and fibrosis of pericardium in these two groups.3.LPS/TLR4 up-regulates TGF 1/Smads to promote myocardial fibrosis in rats with constrictive pericarditis:(1)The expression of TGF?1 in visceral pericardium and myocardial tissue of rats in N group and CP group was detected by immunohistochemistry.(2)Real-time quantitative PCR was used to detect the m RNA expression of TLR4,TGF?1,?SMA,COL?,COL?,MMP1/2/8/9/13 and TIMP1/2 in the free wall tissues of the two groups.(3)Western Blot was used to detect protein expression of TLR4,TGF?1,Smad2/3,p-Smad2/3 and?SMA.(4)Neonatal rat CFs were primary cultured and identified by immunofluorescence detection of VIM.Three treatment groups of(1)DMSO-LPS-LPS+CLII095,(2)DMSO-LPS-LPS+LY2157299-TGF?1,(3)DMSO-TGF?1-TGF?1+SIS3 were used.(5)Real-time PCR,Western Blot and immunofluorescence were used to detect related indicators.Results:1.Preparation and validation of rat model of constrictive pericarditis:(1)The parietal pericardium of the N group was thin and transparent.The HE stained smear pericardium showed a monolayer cell structure.Masson stained with fine blue staining and SR staining with fine red staining.Pericardium in LPS group had slight opacity and thickening.The stained local visceral pericardium was slightly thickened,and there was no statistically significant difference with N group.Masson staining was blue staining,SR staining was red staining,and the positive staining area ratio was not statistically different.In TP group,talc powder particles were scattered in the parietal pericardium,which had low transparency,turbidity and thickening.There was a little dot-like adhesion with the surface of the heart.HE staining of local visceral pericardial thickening was statistically different compared with N group,but the lesion was more limited.Masson staining showed blue staining,SR staining showed red staining,and the positive staining area had no statistical difference.The pericardium of the LPS+TP group showed obvious thickening,adhesion,turbidity and white.The heart could not be seen through the parietal pericardium.It was difficult to peel off the heart and the surface of the heart was dirty.The parietal pericardium was thickened,turbid and whitened.HE staining showed visceral pericardium was generally thickened with multi-layered cell structure.Local talc powder granules and inflammatory cell infiltration were observed.The thickness of pericardium was statistically different compared with other groups.Masson staining showed visceral pericardium is markedly thickened and blue stained,positive staining area ratio was significantly higher than N group;SR staining showed visceral pericardial thickening and red stained.The positive staining area ratio was significantly higher than that of the N group.(2)Compared with the N group,there was no significant difference in E-peak velocity of mitral valve orifice among the three methods.The E-peak velocity of lateral wall in LPS+TP group was lower than that in other three groups,but the difference was not significant.The E/E'of lateral wall in LPS+TP group was higher than that in control group,and the difference was statistically significant.(3)PCR and WB detection of parietal pericardium were only included in N group and LPS+TP group.The expression of?SMA,COL?and COL?in pericardial tissue of LPS+TP group was significantly higher than that of N group.The expression of?SMA in pericardial tissue of LPS+TP group was higher than that of N group.2.Evaluation of myocardial injury in rats with constrictive pericarditis by two-dimensional speckle tracking imaging:(1)The inner diameter and area of atrium in CP group increased as compared with that in N group;the E'_lat decreased and E/E'_lat increased in CP group.(2)Compared with N group,IVRT,UHT and TPTV increased in CP group.The global torsion angle and the rotation angle of apical free wall decreased in CP group.The rotation rate S of apical free wall and IVS decreased,the S of basal free wall decreased,and the S of IVS did not.There were no significant differences in E both basal and apical levels,but A increased both basal and apical levels,E/A decreased in apical levels.Tr E of total torsion rate were lower than those in N group.(3)Longitudinal strain and strain rate S of free wall were lower in CP group,and strain rate E of free wall was lower than that in N group.There was no significant difference in most indexes of radial strain and strain rate between the two groups,except that the radial strain rate A of IVS increased and E/A decreased.Circumferential strain was more sensitive.In CP group,the circumferential strain and strain rate S of free wall and IVS decreased,while the E,A and E/A of free wall decreased.There was no difference in most indexes of radial strain and strain rate between the two groups.There was no difference in IVS E,but A increased and E/A decreased.(4)The visceral pericardium of CP rats showed obvious thickening and fibrosis,and multiple collagen fibers were stained in the subepicardial myocardium,extending from the epicardial direction to the myocardium.3.LPS/TLR4 up-regulates TGF 1/Smads to promote myocardial fibrosis in rats with constrictive pericarditis:(1)The expression of TGF?1 was observed in the cytoplasm of the visceral pericardium and subepicardial myocytes in the CP group,and no significant expression was observed in the N group.(2)The m RNA expression of TLR4,TGFbeta 1,?SMA,COL I and COL III in free wall myocardium of rats in CP group was significantly higher than that in N group,TIMP1 and TIMP2 were increased,MMP2 was increased,MMP13/TIMP1 was decreased,and MMP2/TIMP2 was increased.(3)The protein expressions of TLR4,TGF?1,p-Smad2/3,?SMA,COL I and COL III in cardiac myocardium of rats in CP group were significantly higher than those in N group significantly.(4)Rat myocardial fibroblasts were large,polygonal,like paving stones,with large nuclei.There were many tiny vesicle-like refractions in the cytoplasm around the nuclei,and the fluorescence expression of VIM can be seen in the whole field by immunofluorescence detection.(5)After LPS treatment,the expression of TGF?1 and fibrosis markers including?SMA,COL I and COL III increased,Smad3 protein phosphorylation increased,CLI95 and LY2157299 blocked the effect of LPS by targeting TLR4 and TGF?R,respectively.After TGF?1 treatment,the increased expressions of?SMA,COL?,COL?and the phosphorylation of Smad3 protein also could be detected,and SIS3 could inhibit the effect of TGF?1 by acting on Smad3.Conclusion:1.LPS combined with talcum powder as an inflammatory agent,through the chest wall puncture injection into the pericardial cavity,without opening the chest,can successfully construct the rat CP model;model rat showed thickened,fibrotic pericardial and decreased left ventricular diastolic function,which is consistent with the diagnosis of CP.2.CP rat model showed thickened,fibrotic visceral pericardia and subepicardial myocardial fibrosis;The contraction and diastolic function of left ventricular free wall was impaired,with diastolic dysfunction in the interventricular septum indirectly.3.LPS/TLR4 up-regulated the expression of TGF?1 and Smads phosphorylated,causing the secretion of?SMA,COL I/III,and the imbalance of MMPs/TIMPs,which may lead to myocardial fibrosis in CP model rats.