Chronic Colitis Upregulates MicroRNAs That Inhibit Brain-derived Neurotrophic Factor In The Heart Of Adult Rat

Objectives Ulcerative colitis and Crohn’s disease are classified as chronic inflammatory bowel diseases(IBD)with known extraintestinal manifestations.The interplay between heart and gut in IBD has previously been noted,but the mechanisms remain elusive.Taking the rat heart as the research target,using different colitis models in rats identified miRNA colitis models that mediate molecular remodeling and lead to heart damage in rats.Our study observed whether chronic colitis changed the miRNA spectrum in adult heart from different levels of animal model.Our study observed Whether the change of miRNA was related to the change of functional protein.Methods 1.Making animal models ⑴Chronic colitis(DSS)group: Dextran sodium sulfate(DSS)was given to adult rats for 5 days followed by 9 days with normal drinking water for 4 cycles over 8 weeks.⑵ Acute colitis(TNBS)group : Rats were lightly anesthetized with isoflurane(4% for induction and 1%for maintenance)and 250 μl of trinitrobenzene sulfonic acid(TNBS)in phosphate buffered saline containing 40% ethanol was injected intrarectally via a catheter,advanced to 8 cm into the colon.Control rats received one time intracolonical injection of 250 μl of saline.2.The heart size and function of rats were measured by echocardiography,the body weight and colon length were measured,and the histological characteristics of rat heart and colon were analyzed by H & E staining.The activities of myeloperoxidase(MPO)in colonic mucosa and submucosa were measured,and IL-1β,Matrix metalloproteinase(MMP)7,MMP9,IL18.in colonic mucosa was measured by real-time quantitative reverse transcriptase polymerase chain reaction(RT-qPCR).3.Subcellular isolation of rat heart tissue was carried out,and the levels of brain-derived neurotrophic factor(BDNF)in rat serum and cardiac cytoplasmic / nuclear components were quantitatively detected by enzyme-linked immunosorbent assay(ELISA),Serum B-type natriuretic peptide(BNP)was determined by enzyme immunoassay(EIA).COL3A1 in rat heart was detected by immunohistochemistry.The apoptotic cells in rat heart were detected by terminal deoxynucleotidyl transferase(TDT)-mediated d UTP nick end labeling(TUNEL).BDNF m RNA in colonic mucosa and heart of DSS rats was measured by RT-qPCR.4.MiRNA microarray was used to detect the heart tissue of rats in DSS group and control group.The target genes of all significantly changed miRNAs in DSS induced colitis was predicted by org.micro RNA,miRDB and miRWalk The levels of mir-1b,let-7d and mir-155-5p miRNAs in heart tissue were measured by RT-qPCR.5.α-Actin was used as internal reference,and the glycogen synthase kinase-3 β(glycogen synthase kinase-3 β,GSK-3 β),B-cell lymphoma-2(Bcl-2),phosphorylated protein kinase B(phospho Akt),signal transducer and activator of transcription(STAT3),embryonic lethal abnormal vision like 1(elavl1),cleaved caspase 3,Cleaved caspase 7,type III collagen α-1(Collagen Ⅲ alpha-1,COL3A1)in heart tissue was detected by Western blot.Results The DSS rats demonstrated a significant decline in LVEF(30%)and accompanying increases in left ventricular mass(36%)and size compared to controls calculated on M-mode images from transthoracic echocardiography.An increase in serum levels of BNP(2.4-fold)was found in chronic colitis rats in serum BNP.H&E staining of the heart sections showed cell swelling,irregular nuclear pattern,and enlarged spaces between muscle fibers in DSS rats.MPO activity and IL-1 β,MMP7 and mmp9 m RNA levels in colonic mucosa increased significantly.The overall mucosal structure of the colon was significantly changed and the surface epithelial structure was lost.Micro RNA data showed that a total of 56 miRNAs were significantly increased in the heart through colitis,of which 8 miRNAs increased due to colitis were predicted to be regulators of BDNF.Then,the increased expression of mir-1b,let-7d and miR-155 in heart tissue was confirmed by RT-qPCR.Heart GSK-3 β,BCL2,p Akt(s133),β-Catenin,STAT3,COL3A1 and ELAVL1 increased,the levels of BDNF protein in heart and serum decreased significantly,and BDNF in heart and systemic circulation decreased in colitis induced by TNBS too.COL3A1 accumulates in the heart of rats with chronic colitis.Conclusions Colon length was significantly shortened,LVEF was significantly decreased,left ventricular weight and size were also increased,serum BNP level was increased,MPO activity and IL-1β in colonic mucosa were increased in DSS chronic colitis rat model.The levels of Mmp 7 and Mmp9 m RNA were increased significantly.H & E staining of colon and heart showed significant changes in the structure of overall mucosa and cardiomyocytes.The levels of miR-1b,let-7d and miR-155-5p in DSS colitis heart tissue were increased,the levels of BDNF in serum and heart were decreased significantly,and the markers of myocardial hypertrophy and myocardial fibrosis were increased.BDNF was also reduced in the heart and systemic circulation of TNBS induced colitis in rats.Objectives This study found that chronic colitis can damage cardiac function through the rat colitis model,but the specific mechanism of its damage to cardiomyocytes is unclear.Acute DSS colitis rats were treated with Interleukin IL-1β neutralizing antibody.H9c2 cardiomyocytes were treated with different inflammatory mediators.H9c2 cardiomyocytes were transfected with different miRNAs,BDNF proteins or rat BDNF specific siRNA.We observed how chronic colitis changes the miRNA spectrum in adult heart through functional proteins at different levels of molecule,cell and whole and explored the mechanism of chronic colitis damaging heart function so as to provide a new theoretical basis for the treatment of heart damage caused by IBD.Methods 1.Animal experiments: ⑴IL-1β antibody was used for treatment of acute DSS colitis group:Rats were treated with 5% DSS in the drinking water for 7 days.IL-1β antibody(25 μg/kg in 200 μl of saline)was administrated daily by intraperitoneal injection for 7 days.Rats in control groups were subjected to daily intraperitoneal injection of 200 μl saline.⑵With IL-1 β or tumor necrosis factor(TNF)-α Treated rats:Rats were anesthetized with isoflurane(4% for induction and 1% for maintenance)and given recombinant IL-1β or TNF-α(Gene Script,10 μg/kg)by tail vein injection.Control group received saline e(n= 8 per group).2.H9c2 cells were cultured.H9c2 were transfected with miR-1b,let-7d and miR-155.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was used as internal reference.The BDNF protein level of H9c2 cells after three transfections was detected by Western blot test.H9c2 cells were transfected with BDNF protein or rat BDNF specific siRNA,and the BDNF m RNA of H9c2 cells knocked out by siRNA was determined by RT-qPCR.The viability of H9c2 cells was detected by 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide(3-(4,5)-dimethylthiadiazo(-z-y1)-3,5-di-phenyltetrazolium romide,MTT)colorimetry.H2O2 induced apoptosis of H9c2 cells.GAPDH was used as internal reference.Cleaved caspase 3,cleaved caspase 7 and phosphorylated histone H2AX(phosphorylated histone,phosphorylated-h)2A X(γ H2AX)were detected by Western blot.With inflammatory mediators H2O2 and TNF-α 、 H9c2 cells were treated with different concentration IL-1β or with low-dose and high-dose LPS for 24 hours.With 18 S ribosomal RNA as the endogenous control,the miR-155 level of H9c2 cardiomyocytes was quantitatively measured by Taq Man based RT-qPCR.3.Adult rats were treated with recombinant rat IL-1β Or TNF-α of IL-1β neutralizing antibody was used in rats with acute DSS colitis.The levels of miR-155 and BDNF in rat myocardium were evaluated by RT-qPCR.Results 1.With inflammatory mediators H2O2,TNF-α,IL-1 β or low-dose and high-dose LPS treatment of H9c2 cells,IL-1β was found Significantly to up-regulate the level of miR-155 in H9c2 cells.Adult rats were treated recombinant IL-1 β or TNF-α.IL-1 β(not TNF-α)Increased miR-155 in adult rat hearts(compared with control and TNF-α(P < 0.01).IL-1 β neutralizing antibody was used simultaneously in DSS rats.IL-1β neutralizing antibody negated the DSS-induced upregulation of miR-155 in the adult at heart(p<0.01 vs.vehicle-treated DSS rats).More importantly,downregulation of BDNF by DSS was concurrently abrogated by administration of IL-1β neutralizing antibody.2.H9c2 cardiac myofibrobast cells were transfected with miR-1b,Let-7d,and miR-155.All three miRNAs significantly downregulated BDNF protein expression compared to miRNA negative control(22%,15%,and 43%,respectively,vs.control.p<0.05),with miR-155 demonstrating the most significant effect(p<0.05 vs.miR-1b and Let-7d).This effect was augmented by concurrent transfection of cells with all three miRNAs(p<0.01 vs.miR-1b and Let-7d).Recombinant BDNF protein had no effect on BDNF m RNA levels,the rat BDNF-specific siRNA was successfully transfected into H9c2 rat cardiomyoblast cells with a significant reduction in BDNF m RNA(70%,p<0.01).Cell proliferation quantified by MTT colorimetric assay was significantly increased in the cells treated with recombinant BDNF protein compared to control cells(31%,p<0.05).A significant reduction in cell proliferation was noted in the BDNF knockdown group(35%,p<0.01 vs.si Control group).Apoptosis in H9c2 cells was induced through the administration of H2O2,manifested through cleavage of Caspase-3 and elevation of phosphorylated histone H2AX(γH2AX);the changes were mitigated by BDNF overexpression.Conclusions Proinflammatory cytokine IL-1 β Increased miR-155 expression and decreased BDNF levels in adult rat hearts.Mir-1b,let-7d and miR-155 inhibited BDNF in H9c2 cardiomyocytes.BDNF improved apoptosis of H9c2 cardiomyocytes induced by H2O2.