SSBP1 Inhibits Ang ?-induced Myocardial Fibrosis By Regulating P53

Background:Heart failure is the end-stage symptom of many cardiovascular diseases and the leading cause of death.Ventricular remodeling accompanies heart failure from beginning to the end.The main pathological changes of ventricular remodeling are myocardial hypertrophy and myocardial fibrosis(MF).MF is difficult to reverse.The degree of MF determines the prognosis of patients.Myocardial fibroblasts are the main effector cells of myocardial fibrosis,and their excessive proliferation and dysfunction of collagen synthesis are the initial factors of MF.The main role of RAAS is a neurohumoral mechanism used to regulate cardiovascular disease and plays an important role in promoting the occurrence and development of MF.Angiotensin II(Ang II)can activate the TGF-?1 signal,increase collagen synthesis and play a key role in myocardial remodeling.Valsartan can specifically bind to ATI receptors.It can block the functions of promoting cell proliferation,regulating body fluids,releasing aldosterone and constricting blood vessels caused by AT1 receptor.The process of myocardial fibrosis is complex.There are multiple pathways and molecular signals involved in MF.Single-stranded DNA binding protein 1(SSBP 1)is mainly involved in DNA damage response and DNA repair.SSBP1 is located in the mitochondria of eukaryotic cells.It can inhibit the TGF-?/Smad 3 and transduction pathway and play a certain inhibitory effect on activation of myocardial fibroblasts.P53 is a transcription factor that mainly plays a role in maintaining mitochondrial function and regulating cell proliferation and differentiation.Some studies have found that SSBP1 can enhance the stability of P53 by acetylation.Therefore,we hypothesized that SSBP1/P53 might be a new target for inhibiting myocardial fibrosis.Part one:Mouse myocardial fibrosis model was preparedObjective:To prepare a mouse myocardial fibrosis model using Ang ?Methods:C57BL/6 mice,23 of them,aged 10 weeks,were implanted with a osmotic minipump subcutaneously.mice of the sham group(n=12)were infused with an equal volume of acetic acid;the experimental group(n=11)was infused with Ang ? solution,1.44 ?g/g/day.After 2week,heart function of the mice in two groups was examined using echocardiography.An echocardiographic system was used to measure heart rate,inte rventricular septal thickness diastolic(IVSTD),left ventricular end posterior wall dimension diastolic(LVPWD),fractional shortening(FS)and ejection fraction(EF),via a 12-MHz transducer.The systolic blood pressure of the mice was detected using tail cuff methods.the mice were sacrificed and the hearts were immediately harvested with the heart tissues being fixed in 4%paraformaldehyde.The heart sections were rehydrated and stainedusingpicrosirus red dye.The stained collagen was observed with an inverted microscope in a blinded manner,and the stainingarea was quantified by Image J software.Results:In the experimental group,the Ang ? could increase systolic blood pressure,increase the weight of the heart and thicken the walls of the ventricles.However,in the short term(2 weeks),there was no effect on the left ventricular ejection fraction and short axis shortening rate in mice.The collagen fiber staining area of myocardial tissue sections in the experimental group was 19.4%and was 42.1%in the control group.If the SSBP1 protein content of the control group was set as 1,the experimental group is significantly reduced by 0.46.(P<0.05).Part Two Valsartan Inhibits Ang ?-induced myocardial fibrosis through SSBP1Objective:The first purpose is to verify whether SSBP1 inhibits MF.The second purpose is to determine whether valsartan inhibits MF by SSBP1.Methods:The C57BL/6J mice were subcutaneously implanted using an osmotic mini-pump.The mice in the model group(n=11)and valsartan group(n=11)were infused with Ang? with a dosage of 1.44 ?g/g/day(diluted in 10 mM acetic acid)for 2 weeks.The mice of the sham group(n=12)were infused with an equal volume of acetic acid;moreover,the mice in the valsartan group were given valsartan diluted in water(40 mg/kg/day)after Ang? infusion,whereas the mice in the model group were fed with only water.After Ang II and valsartan treatment,the mice were sacrificed and the hearts were immediately harvested with the heart tissues being fixed in 4%paraformaldehyde.After decalcification and dehydration,the heart samples were embedded in paraffin and cut as 5-?m sections,and the sections were rehydrated and stained using picrosirus red dye The stained collagen was observed with an in-verted microscope in a blinded manner,and the staining area was quantified by Image J softwareFibroblasts culturing:?Detect the expression levels of COL1A1,COL3A1,SSBP1 protein(WB method)and mRNA(qRT-PCR method)in myocardial tissue of each group.? Prepare myocardial tissue mitochondrial suspension.The content of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in the mouse hearts was examined using a biochemical analysis kit based on the manufacturer's protocol?The mouse adult cardiac fibroblasts(MCFs)were isolated.Briefly,The cells were passed for three passages,which were used for the following experiments.For Ang? treatment,the MCFs were treated Ang? for 24 hours.To block the ac tivity of AT1R,the MCFs were pretreated with 1.0 ?mol/L valsartan for 12 hours before treatment with Ang?.blank control;Ang? 0.5umol/l group;Ang ? 1.0umol/1;Ang? 1.0umol/1+valsartan 1.0umol/l group;WB method Groups COL1A1,COL3A1,NOX1,NOX4 and SSBP1 proteins.Results:?The expression of Nox1,Nox4,COL1A1,and COL3A1 was increased by Ang II and recovered by valsartanhe.?Ang ? can abate SSBP1 expression.SSBP1 expression was restored in mice or cells exposed to valsartan?The activities of citrate synthase,complex ? and complex ? in the valsartan group were enhanced compared with the experimental group.The levels of NOX1 mRNA and NOX4 mRNA decreased.? The higher the Ang ? concentration,the higher the protein expression levels of COL1A1,COL3A1,NOX1 and NOX4,and the lower the protein expression levels of SSBP1.Part Three:The Role of P53 and SSBP1 on Ang II-stimulated myocardial fibroblast proliferation and collagen synthesisObjective:The aim was to determine whether SSBP1 could inhibit the proliferation of cardiac fibroblasts induced by Ang? and the expression of collagen.By regulating P53.Methods:Isolate and culture myocardial fibroblasts.The SSBP1 expression was manipulated via SSBP1 shRNA and pcDNA3.1/SSBP1 plasmids,while the p53 ex-pression was enhanced via Ad-p53 GFP infection.? Silencing/up-regulating SSBP1,with or without Ang? stimulation,the MTT assay was used to detect fibroblast proliferation.WB method was used to detect collagen COL1A1,COL3A1;NOX1,NOX4 protein expression.? Silencing/up-regulating SSBP1 gene,WB method was used to detect P53 protein ? Up-regulated the P53 gene,and with or without Ang? stimulation,the MTT assay was used to detect fibroblast proliferation.WB method was used to detect COL1A1,COL3A1,NOXl,and NOX4 expression.Results:?The knockdown of SSBP1 increased cardiac fibroblast proliferation,collagen expression,and decreased p53 expression,which was impeded via SSBP1 overexpression.?Ang? increased the expression of Nox 1 and Nox 4,and the regulation of SSBP1 gene level had no effect on it.?The forced expression of p53 abated the fibroblast proliferation and collagen expression that was induced by Ang?.Graphical Abstract:?The expression of SSBP1 was inhibited,in Ang ?-induced myocardial fibrosis.?Valsartan can inhibited collagen expression and ROS activation,through increasing expression of SSBP1 antagonism MF,by Ang ?induced.?SSBP1 abated the fibroblast proliferation and collagen expression that was induced by Ang? via the P53 protein.