25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

25-hydroxycholesterol (25-HC) plays a role in the regulation of cell survival and immunity. However, the effect of 25-HC on myocardial ischemia/reperfusion (MI/R) injury remains unknown. Our present study aimed to investigate whether 25-HC aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. The overlapping differentially expressed genes (DEGs) in MI/R were identified from the GSE775, GSE45818, GSE58486, and GSE46395 datasets in Gene Expression Omnibus (GEO) database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the database of Annotation, Visualization and Integration Discovery (DAVID). The protein-protein interaction (PPI) network of the overlapping DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These bioinformatics analyses indicated that cholesterol 25-hydroxylase (CH25H) was one of the crucial genes in MI/R injury. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate MI/R injury. Western blot and RT-qPCR analysis demonstrated that CH25H was significantly upregulated in OGD/R-stimulated H9C2 cardiomyocytes. Moreover, knockdown of CH25H inhibited the OGD/R-induced pyroptosis and nod-like receptor protein 3 (NLRP3) inflammasome activation, as demonstrated by cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), RT-qPCR, and western blotting assays. Conversely, 25-HC, which is synthesized by CH25H, promoted activation of NLRP3 inflammasome in OGD/R-stimulated H9C2 cardiomyocytes. In addition, the NLRP3 inhibitor BAY11-7082 attenuated 25-HC-induced H9C2 cell injury and pyroptosis under OGD/R condition. In conclusion, 25-HC could aggravate OGD/R-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cells.

circMYO9A_006 inhibits expression of cardiac hypertrophy-related pro-teins in cardiomyocytes by translating protein MYO9A-208aa / 中国病理生理杂志

AIM:To investigate the effect of circular RNA MYO9A-006(circMYO9A_006)on hypertrophic phenotype of cardiomyocytes and the underlying mechanism.METHODS:The effect of adenovirus-mediated overexpres-sion of circMYO9A_006 on the expression of hypertrophy-related proteins,including β-myosin heavy chain(β-MHC),skeletal muscle actin alpha 1(ACTA1)and atrial natriuretic peptide(ANP),was evaluated in neonatal mouse ventricular cardiomyocytes(NMVCs).Moreover,a neonatal rat ventricular cardiomyocyte(NRVC)model of phenylephrine(PE)-in-duced hypertrophy was established.The effect of circMYO9A_006 overexpression on NRVC size was ascertained using Phalloidin-iFluor 647 staining method.Dual-luciferase reporter assay was employed to measure the activity of potential in-ternal ribosome entry sites(IRES)in circMYO9A_006.The translation and intracellular location of the circMYO9A_006-translated protein,MYO9A-208aa,were verified using Western blot.To investigate the role of MYO9A-208aa in the ef-fect of circMYO9A_006 on the cardiomyocyte hypertrophic phenotype,we prepared and used the following adenoviruses:the recombinant circMYO9A_006-ORF adenovirus to express MYO9A-208aa,the recombinant circMYO9A_006-ATG-mut adenovirus that does not express MYO9A-208aa,the recombinant circMYO9A_006 adenovirus,and the adenovirus vector control.These were then employed to infect NRVCs.RESULTS:Successful adenovirus-mediated overexpression of circMYO9A_006 was observed in NMVCs.The increased expression of circMYO9A_006 notably reduced the expres-sion of hypertrophy-related proteins in NMVCs(P<0.01).Concurrently,overexpression of circMYO9A_006 substantially reduced the expression of hypertrophy-associated proteins and diminished the size of PE-induced NRVCs(P<0.05).Dual-luciferase reporter assay identified the activity of 2 IRES in circMYO9A_006.Western blot results indicated that circ-MYO9A_006 could produce the MYO9A-208aa protein with an anticipated molecular weight of 28 kD in NRVCs,primari-ly found in the cytoplasm.Elevated expression of both circMYO9A_006 and MYO9A-208aa consistently reduced the ex-pression of hypertrophy-associated proteins(P<0.01),and counteracted the enlarged size of PE-induced NRVCs(P<0.05).However,increased expression of circMYO9A_006-ATG-mut did not counteract the PE-induced hypertrophic phe-notype of NRVCs.CONCLUSION:circMYO9A_006 attenuates the hypertrophic phenotype of cardiomyocytes by synthe-sizing the MYO9A-208aa protein.

Construction of recombinant adenovirus vector of Uhrf1 gene and its role in DNA damage repair of cardiomyocytes / 中国病理生理杂志

AIM:To construct a recombinant adenovirus vector carrying the mouse ubiquitin-like with plant homeodomain and RING finger domains 1(Uhrf1)gene,validate the expression of Uhrf1 in neonatal mouse cardiomyo-cytes and explored its role in hydrogen peroxide(H2O2)-induced DNA damage.METHODS:The mouse Uhrf1 gene cod-ing sequence was amplified by polymerase chain reaction(PCR),digested,and inserted into the pADM-CMV-C-FH vec-tor to create the recombinant adenoviral plasmid ADM-Uhrf1.Following transfection into HEK293T cells,we generated re-combinant adenoviral particles,amplified,purified,and determined the titer.Neonatal mouse cardiomyocytes were infect-ed at an multiplicity of infection(MOI)of 50,UHRF1 protein expression was validated via Western blot and immunofluo-rescence staining.H2O2-induced DNA damage was explored along with adenovirus-mediated Uhrf1 overexpression to inves-tigate its role in DNA damage repair.RESULTS:ADM-Uhrf1 virus titer,determined by capsid immunofluorescence as-say,was 1.8×1013 pfu/L.Western blot confirmed a significant increase in UHRF1 protein expression(P＜0.05),with im-munofluorescence indicating predominant nuclear localization.Uhrf1 overexpression effectively inhibited the expression of the DNA damage marker,phosphorylated H2AX protein(γH2AX)(P＜0.01).CONCLUSION:We successfully con-structed a recombinant adenoviral vector carrying the mouse Uhrf1 gene,facilitating Uhrf1 overexpression in neonatal mouse cardiomyocytes.Furthermore,this overexpression effectively alleviated DNA damage in cardiomyocytes.

Rat cardiac fibroblasts inhibit gap junction between cardiomyocytes through increasing matrix metalloproteinase 2 activity / 中国病理生理杂志

AIM:To investigate the effect of conditioned medium from hypoxia/reoxygenation(H/R)-treated rat cardiac fibroblasts(CFs)on gap junction between cardiomyocytes and determine whether its mechanism is related to matrix metalloproteinase 2(MMP2)activity.METHODS:(1)H9c2 cells were randomly divided into five groups:con-trol group,normal group,ARP-100 group,H/R group,and H/R+ARP-100 group.Scrape loading/dye transfer assay was used to assess the gap junction function.Western blot was used to detect the expression and phosphorylation levels of Cx43.Gelatin zymography assay was performed to measure MMP2 activity.(2)SD rats were randomly divided into control group,ARP-100 group,ischemia-reperfusion(I/R)group,and I/R+ARP-100 group,with 8 rats in each group.Micro-electrode array technology was used to record the type and duration of arrhythmia.Immunohistochemistry experiment was performed to assess expression levels and distribution of Cx43 in myocardial tissues.RESULTS:Compared with the con-trol group,the H/R group showed decreased protein expression of Cx43(P＜0.01),narrowed distance of lucifer yellow dif-fusion(P＜0.01),and increased MMP2 activity(P＜0.01).ARP-100 attenuated H/R-induced gap junction dysfunction(P＜0.05).The arrhythmia score was also reduced after perfusion with ARP-100(P＜0.01).CONCLUSION:H/R-treated rat CFs can inhibit gap junction between cardiomyocytes,and its mechanism may involve increased MMP2 activity.

Silencing YTHDF2 improves Ang Ⅱ-induced hypertrophy and apoptosis of primary neonatal rat cardiomyocytes / 中华老年心脑血管病杂志

Objective To investigate the effect of YTH domain family protein 2(YTHDF2)on an-giotensin Ⅱ(Ang Ⅱ)-induced hypertrophy and apoptosis of primary neonatal rat cardiomyocytes.Methods The expression level of YTHDF2 was detected in the primary neonatal rat cardiomyo-cytes with or without Ang Ⅱ stimulation(AngⅡ group and normal group).The cells were divid-ed into blank group(transfected with siRNA+PBS),siYTHDF2 group(transfected with siYTHDF2+PBS),model group(siRNA+Ang Ⅱ)and experimental group(siYTHDF2+Ang Ⅱ)to investi-gate the effects of silencing YTHDF2 on the hypertrophy and apoptosis of cardiomyocytes.West-ern blotting and RT-qPCR were used to detect the expression of YTHDF2 at protein and mRNA levels,and RT-qPCR was employed to measure the mRNA levels of myocardial hypertrophic related genes atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and beta-myosin heavy chain(β-MHC),and cardiomyocyte apoptosis related genes Bax and B lymphocytoma 2 gene(Bcl-2).The surface area of cardiomyocytes was observed by α-actin immunofluorescence staining.Cardiomyocyte apoptosis was observed by TUNEL staining,and the binding relationship between YTHDF2 and Bcl-2 was verified by immunoprecipitation.Results The expression of YTHDF2 at protein and mRNA levels were significantly higher in the AngⅡ group than the nor-mal group(1.49±0.03 vs 0.97±0.09,1.50±0.08 vs 1.00±0.07,P＜0.05).Compared with the blank group,the surface area of cardiomyocytes was notably enlarged,apoptotic rate was obvi-ously increased,the mRNA levels of ANP,BNP,β-MHC and Bax were significantly increased,and that of Bcl-2 was remarkably decreased in the model group(P＜0.05).The experimental group obtained decreased surface area and apoptotic rate of cardiomyocytes,lower mRNA levels of ANP,BNP,β-MHC and Bax,and increased mRNA expression of Bcl-2(P＜0.05).Conclusion Silencing YTHDF2 can alleviate Ang Ⅱ-induced hypertrophy and apoptosis in primary neonatal rat cardiomyocyte,and YTHDF2 inhibits the expression of Bcl-2 by binding to it.

MiR-31a-5p aggravates apoptosis in myocardial ischemia by targeting HIF-1α / 中国胸心血管外科临床杂志

@#Objective To investigate the expression of miR-31a-5p in myocardial infarction (MI) mice and its potential mechanism. Methods A dataset was downloaded from the gene expression database, and miR-31a-5p and its predicted target gene hypoxia-inducible factor-1α (HIF-1α) were screened using bioinformatics methods. The MI model was established by ligating the left anterior descending branch of the coronary artery in C57BL/6J male mice which were randomly divided into sham and MI groups (n=6 in each group). The in vitro hypoxic cell model was induced by treatment of H9c2 cells with cobalt chloride (CoCl2) and divided into a control group, a model group, a NC group, a miR-31a-5p mimic group and a miR-31a-5p inhibitor group. The degree of myocardial tissue fibrosis was stained by Masson and analyzed. The expression levels of miR-31a-5p and HIF-1α mRNA in mouse myocardial tissues and H9c2 cells were detected by qRT-PCR. Western blotting was used to detect the expression levels of B-cell lymphoma 2 (Bcl-2), cleaved-caspase 3 apoptotic protein in mouse myocardial tissues and HIF-1α and apoptotic protein in H9c2 cells, respectively. The dual luciferase reporter gene assay was used to verify the targeting relationship between miR-31a-5p and HIF-1α. Results Masson staining showed significantly increased fibrosis in MI mice (P<0.000 1); miR-31a-5p, cleaved-caspase 3 were significantly elevated and Bcl-2 was decreased in MI mice and CoCl2 treated H9c2 (P<0.05). The results of dual luciferase reporter assay showed that the relative luciferase activity of miR-31a-5p mimic cotransfected with HIF-1α-3'-UTR WT plasmid was reduced (P<0.000 1); miR-31a-5p mimic decreased HIF-1α expression and increased apoptotic protein levels in CoCl2 induced H9c2 cells (both P<0.05), while miR-31a-5p exerted the opposite effect. Conclusion miR-31a-5p can aggravate apoptosis in myocardial ischemia by targeting HIF-1α.

Function of Cortactin/N-cadherin signal axis in pathological myocardial hypertrophy / 中国药理学通报

Aim To investigate the regulatory effect of Cortaetin on pathological myocardial hypertrophy induced by isoprenaline (ISO) and the underlying mechanism. Methods ISO was used to stimulate neonatal rat cardiomyocytes for 24 h, and myocardial hypertrophy model was established at the cellular level. C57BL/6 mice were injected subcutaneously with ISO for one week to establish myocardial hypertrophy model at animal level. RT-qPCR was used to detect the changes of mRNA and Western blot was used to detect the changes of relative protein content. Immunofluorescence was used to measure the subcellular location of Cortaetin and the change of its expression. The overex-pression of Cortaetin by adenovirus infection and the knockdown of Cortaetin by transfection of small interfering RNA were studied. Results On the cellular and animal levels, ISO-induced myocardial hypertrophy models were successfully established, and it was observed that ISO caused the decrease of Cortaetin and N-cadherin protein levels. Overexpression of Cortaetin could reverse the decrease of N-cadherin protein level and myocardial hypertrophy caused by ISO. Knockdown of Cortaetin showed the opposite effect. Conclusion Cortaetin, in combination with N-cadherin, may play a role in combating myocardial hypertrophy by enhancing the connections between cardiomyocytes.

Effect and mechanism of protein kinase N1 on the proliferation of mouse cardiomyocytes / 新乡医学院学报

Objective To explore the effect and mechanism of protein kinase N1(PKN1)on the proliferation of mouse cardiomyocytes.Methods Two 1-day-old mice were anesthetized with isoflurane,and their cardiomyocytes were isolated and divided into the control group and the interference group.The cardiomyocytes in the interference group were transfected with PKN1 fragments,while the cardiomyocytes in the control group were transfected with control fragments.According to the random number table method,10 mice were divided into the normal group and the observation group,with 5 mice in each group.The mice in the observation group were injected with PKN1 adenovirus in situ in the heart,while the mice in the normal group were injected with empty adenovirus in situ in the heart.The Ki-67 positive expression in myocardial cells and tissues of mice in the four groups was detected by immunofluorescence assay,indicating the proliferation ability of cardiomyocytes.The PKN1 mRNA expression in cardiomyocytes of mice in the control group and interference group was measured by real-time fluorescence quan-titative polymerase chain reaction.The expression of PKN1 and cyclin D1 proteins in cardiomyocytes of mice in the control group and interference group was determined by Western blot.Results The positive expression rates of Ki-67 in myocardial cells of mice in the interference group was significantly lower than that in the control group(t=11.201,P＜0.01);the positive expression rate of Ki-67 in myocardial tissue of mice in the observation group was significantly lower than that in the normal group(t=11.851,P＜0.01).The relative expression level of PKN1 mRNA in cardiomyocytes of mice in the interference group was significantly lower than that in the control group(t=7.022,P＜0.01).The relative expression levels of PKN1 and cyclin D1 proteins in cardiomyocytes of mice in the interference group were significantly lower than those in the control group(t=5.762,6.884;P＜0.01).Conclusion The decreased expression of PKN1 in mouse cardiomyocytes can inhibit the expression of cyclin D1 protein,thereby restraining cardiomyocyte proliferation.

Esketamine alleviates hypoxia/reoxygenation injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration

Abstract Objective This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. Methods The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 μM) or TRPV1 inhibitor capsazepine (1 μM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. Results After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. Conclusion ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

Tanshinone Ⅱ A ameliorates ischemia/reperfusion injury in H9c2 cardio-myocytes by activating SIRT1-AMPK pathway via miR-155-5p / 中国免疫学杂志

Objective:To explore the mechanism of Tanshinone ⅡA(TⅡA)in improving ischemia/reperfusion(I/R)injury of H9c2 cardiomyocytes by activating Sirtuin 1(SIRT1)-adenosine 5'-monophosphateactivated protein kinase(AMPK)pathway through miR-155-5p.Methods:H9c2 cells were cultured in vitro and I/R damage model was established.After modeling,H9c2 cells were randomly divided into model group,TⅡA group,TⅡA+miR-NC group,TⅡA+miR-155-5p mimics group,10 μmol/L TⅡA was added for intervention after transfection,and the H9c2 cells supplemented with DMSO were used as control group.qRT-PCR was used to detect expression level of miR-155-5p;MTT method was used to analyze cell proliferation ability;flow cytometry was used to evaluate cell apoptosis;ELISA was used to determine the levels of TNF-α,IL-4,IL-10,IL-17,lactate dehydrogenase(LDH),malo-ndialdehyde(MDA)and superoxide dismutase(SOD);Western blot was used to detect relative expressions of SIRT1,AMPK and p-AMPK proteins.Results:Compared with control group,expression of miR-155-5p in model group was increased,cell viability was decreased,apoptosis rate and expressions of TNF-α,IL-17,LDH and MDA were increased,while expressions of IL-4,IL-10,SOD,SIRT1 and p-AMPK were decreased(P<0.05);compared with model group,expression of miR-155-5p in TⅡA group was reduced,cell viability was increased,apoptosis rate and expressions of TNF-α,IL-17,LDH and MDA were decreased,while expressions of IL-4,IL-10,SOD,SIRT1 and p-AMPK were increased(P<0.05);compared with TⅡA group and TⅡA+miR-NC group,expression of miR-155-5p in TⅡA+miR-155-5p mimics group was increased,cell viability was decreased,apoptosis rate and expressions of TNF-α,IL-17,LDH and MDA were increased,while expressions of IL-4,IL-10,SOD,SIRT1 and p-AMPK were decreased(P<0.05).Conclusion:TⅡA can improve I/R injury of H9c2 cardiomyocytes by down-regulating miR-155-5p,and its mechanism may be related to the activation of SIRT1-AMPK pathway.

Effects of tropomyosin 3 on pyroptosis of cardiomyocytes and fibroblast activation induced by hypoxia/reoxygenation in rats / 中华危重病急救医学

Objective:To explore the role of tropomyosin 3 (TPM3) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte pyroptosis and fibroblast activation.Methods:Rat cardiomyocytes (H9c2 cells) were treated with H/R method to simulate myocardial ischemia/reperfusion (I/R) injury, and cell proliferation activity was evaluated with cell counting kit-8 (CCK8). The expression of TPM3 mRNA and protein was detected by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. H9c2 cells with stable TPM3-short hairpin RNA (shRNA) expression were constructed and treated with H/R (hypoxia for 3 hours, and reoxygenation for 4 hours). The expression of TPM3 was measured by RT-qPCR. The expressions of TPM3, pyroptosis-related proteins including caspase-1, NOD-like receptor protein 3 (NLRP3) and Gasdermin family proteins-N (GSDMD-N) were measured by Western blotting. The expression of caspase-1 was also observed by immunofluorescence assay. The levels of human interleukins (IL-1β, IL-18) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) to elucidate the effect of sh-TPM3 on pyroptosis of cardiomyocytes. Rat myocardial fibroblasts were incubated with the above cell supernatant, and the expressions of human collagen Ⅰ, collagen Ⅲ, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase inhibitor 2 (TIMP2) were detected by Western blotting to determine the effect of TPM3-interfered cardiomyocytes on the activation of fibroblasts under H/R conditions.Results:Compared with the control group, H/R treatment for 4 hours significantly decreased the survival rate of H9c2 cells [(25.81±1.90)% vs. (99.40±5.54)%, P < 0.01], promoted the expression of TPM3 mRNA and protein [TPM3/GAPDH (2 -ΔΔCt): 3.87±0.50 vs. 1, TPM3/β-Tubulin: 0.45±0.05 vs. 0.14±0.01, both P < 0.01], and promoted the expressions of caspase-1, NLRP3, GSDMD-N, and the enhanced release of cytokines IL-1β and IL-18 [cleaved caspase-1/caspase-1: 0.89±0.04 vs. 0.42±0.03, NLRP3/β-Tubulin: 0.39±0.03 vs. 0.13±0.02, GSDMD-N/β-Tubulin: 0.69±0.05 vs. 0.21±0.02, IL-1β (μg/L): 13.84±1.89 vs. 4.31±0.33, IL-18 (μg/L): 17.56±1.94 vs. 5.36±0.63, all P < 0.01]. However, compared with the H/R group, sh-TPM3 significantly weakened the promoting effects of H/R on these proteins and cytokines [cleaved caspase-1/caspase-1: 0.57±0.05 vs. 0.89±0.04, NLRP3/β-Tubulin: 0.25±0.04 vs. 0.39±0.03, GSDMD-N/β-Tubulin: 0.27±0.03 vs. 0.69±0.05, IL-1β (μg/L): 8.56±1.22 vs. 13.84±1.89, IL-18 (μg/L): 9.34±1.04 vs. 17.56±1.94, all P < 0.01]. In addition, the expressions of collagen Ⅰ, collagen Ⅲ, TIMP2, and MMP-2 in myocardial fibroblasts were significantly increased by the cultured supernatants from the H/R group (collagen Ⅰ/β-Tubulin: 0.62±0.05 vs. 0.09±0.01, collagen Ⅲ/β-tubulin: 0.44±0.03 vs. 0.08±0.00, TIMP2/β-tubulin: 0.73±0.04 vs. 0.20±0.03, TIMP2/β-Tubulin: 0.74±0.04 vs. 0.17±0.01, all P < 0.01). However, these boosting effects were weakened by sh-TPM3 (collagen Ⅰ/β-Tubulin: 0.18±0.01 vs. 0.62±0.05, collagen Ⅲ/β-Tubulin: 0.21±0.03 vs. 0.44±0.03, TIMP2/β-Tubulin: 0.37±0.03 vs. 0.73±0.04, TIMP2/β-Tubulin: 0.45±0.03 vs. 0.74±0.04, all P < 0.01). Conclusion:Interference with TPM3 can alleviate H/R-induced cardiomyocyte pyroptosis and fibroblast activation, suggesting that TPM3 may be a potential target of myocardial I/R injury.

To investigate the effect of Astragaloside Ⅳ on high-glucose-induced cardiomyocytes pyroptosis / 中华老年医学杂志

Objective:To investigate the effect of Astragaloside Ⅳ on high glucose-induced cardiomyocyte pyroptosis.Methods:H9c2 cells were cultured in vitro and divided into control group(5.5 mmol/L glucose), high glucose group(33.3 mmol/L glucose), Astragaloside Ⅳ group(33.3 mmol/L glucose+ 100μmol/L Astragaloside Ⅳ), and NLRP3 inhibitor group(33.3 mmol/L glucose+ 1μmol/L MCC950). Cell counting kit 8(CCK-8)was used to detect the activity of H9c2 cells.Lactate dehydrogenase(LDH)kit was used to detect the content of LDH in cell supernatant.Superoxide anion fluorescent probe(DHE)was used to detect the level of intracellular reactive oxygen species(ROS). Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the mRNA and protein expression levels of pyroptosis-related genes.Immunofluorescence was used to detect the fluorescence intensity of NLRP3.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of inflammatory factors in cell supernatant.Results:When the concentration of Astragaloside Ⅳ was 100 μmol/L, it could significantly inhibit the decrease of cardiomyocyte viability induced by high glucose( P<0.01)and reduce LDH release( P<0.01). Compared with the control group, the level of ROS was increased( P<0.01), the mRNA and protein expressions of pyroptosis-related molecules were up-regulated( P<0.01 for all), the fluorescence intensity of NLRP3 was increased( P<0.01), and the levels of inflammatory factors in the cell supernatant were increased in the high glucose group( P<0.01). Compared with the high glucose group, the ROS level was decreased( P<0.01), the mRNA and protein expressions of pyroptosis-related molecules were down-regulated( P<0.05 or P<0.01), the fluorescence intensity of NLRP3 was decreased( P<0.01), and the levels of inflammatory factors in cell supernatant were decreased( P<0.05 or P<0.01)in Astragaloside Ⅳ group and inhibitor group. Conclusions:Astragaloside Ⅳ plays a protective role in high glucose-induced cardiomyocyte injury by inhibiting NLRP3/Caspase-1 signaling pathway and inhibiting pyroptosis.Moreover, it can improve the anti-inflammatory and antioxidant properties in cell models.

Liraglutide ameliorates high glucose-induced oxidative stress injury in rat H9c2 cells through modulation of SIRT1 / 中华内分泌代谢杂志

Objective:To investigate the effect of liraglutide(LRG) on high glucose-induced oxidative stress injury in(H9c2) cardiomyocytes and its underlying mechanisms.Methods:A high glucose treatment was applied to H9c2 cells for 24 hours to establish an in vitro model of myocardial cell injury. Different concentrations of liraglutide(10, 100, 1000 nmol/L) were administered for intervention. Cell viability was evaluated using the CCK-8 assay, and changes in cell morphology were observed under an inverted microscope. After 24 hours of liraglutide(100 nmol/L) intervention following high glucose treatment, the levels of lactate dehydrogenase(LDH), superoxide dismutase(SOD), and malondialdehyde(MDA) in the cell supernatant were measured. RT-PCR and Western blotting were used to detect the mRNA and protein levels of silent information regulator factor 1(SIRT1) and forkhead box protein O1(FOXO1). Western blotting was also used to assess the acetylation level of FOXO1 protein. Small interfering RNA(siRNA) technology was employed to silence SIRT1 in H9c2 cells to confirm its role in the study. Results:Compared to the control group, the high glucose group showed decreased cell viability, cell structure damage, increased levels of LDH and MDA in the cell supernatant, decreased SOD levels, aggravated oxidative stress, decreased SIRT1 expression, and increased acetylation level of FOXO1(all P<0.05). Compared to the high glucose group, liraglutide intervention resulted in increased cell viability, improved cardiac cell morphology, reduced oxidative stress levels, increased SIRT1 expression, and decreased acetylation level of FOXO1(all P<0.05). When SIRT1 was downregulated, the protective effects of liraglutide were weakened(all P<0.05). Conclusions:Liraglutide has a protective effect against high glucose-induced oxidative stress injury in H9c2 cells, which may be associated with the upregulation of SIRT1 expression.

Cytopathic effect of rotavirus NSP4 86-175 protein on rat cardiomyocytes and the possible mechanism / 中华微生物学和免疫学杂志

Objective:To investigate the cytopathic effect of amino acids 86-175 of rotavirus non-structural protein 4 (NSP4 86-175) on rat cardiomyocytes and the possible mechanism. Methods:Rat H9C2 cardiomyocytes were treated with NSP4 86-175 protein. Changes in the growth and morphology of the cells were observed. The activity of LDH in the cell culture medium was detected. Fluo-3AM was used to label intracellular free calcium ions and the concentrations of calcium ions in rat cardiomyocytes with and without NSP4 86-175 treatment were detected by confocal laser microscopy. The expression of Bax, Bcl-2, caspase-3, 78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and caspase-12 at mRNA level was detected by real-time PCR. The expression of caspase-3, caspase-8, caspase-9, GRP78, CHOP and caspase-12 at protein level was detected by Western blot. Results:Normal cardiomyocytes showed a typical myoblast-like morphology, presenting as spindle-shaped cells with clear boundaries. Obvious cytopathic effect, vacuolar degeneration, shriveled and rounded cells, and cell fragmentation were observed after the treatment with purified NSP4 86-175 protein. The activity of LDH in cell culture medium was enhanced by NSP4 86-175 protein. NSP4 86-175 protein also enhanced the fluorescence of the calcium ions in rat cardiomyocytes, promoted cell apoptosis, up-regulated the expression of apoptotic factors including caspase-3, caspase-8, caspase-9 and Bax-2, and increased the expression of classical markers of endoplasmic reticulum stress such as GRP78, CHOP and caspase-12. Conclusions:NSP4 86-175 had a cytopathic effect on rat cardiomyocytes, which might be related to the induced intracellular calcium overload, endoplasmic reticulum stress, apoptosis and necrosis. These results might be used as theoretical reference for further study on rotavirus infection and myocardial injury.

To investigate the effects of RVD1 on autophagy and apoptosis of aging rat cardiomyocytes based on MAMs / 安徽医科大学学报

Objective @#To explore the regulatory effects of ResolvinD1 (RVD1) on autophagy and apoptosis of cardiomyocytes in aging rats , and the effects of RVD1 on mitochondria⁃associated endoplasmic reticulum membranes (MAMs) of myocardial tissue . @*Methods @#Thirty 18⁃month⁃old SD rats were randomly divided into model group , low⁃dose RVD1 group and high⁃dose RVD1 group , with 10 rats in each group , and another 10 6 ⁃month⁃old SD rats were selected as control group . The low⁃dose RVD1 group and high⁃dose RVD1 group were injected with 50 ng/ml and 100 ng/ml RVD1 through the tail vein , respectively , control group and model group were injected with the same amount of phosphate buffered saline (PBS) for 21 days . The β ⁃galactosidase activity of rat myocardial tissue was determined by p ⁃nitrophenol method , the histopathological changes of rat myocardial tissue were detected by hematoxylin⁃eosin (HE) staining , and the collagen deposition in rat myocardial tissue was observed by Masson staining , apoptosis of rat myocardial cells was detected by dUTP in situ end labeling mediated by terminal deoxynucleotide transferase(TUNEL) , the expression of autophagy marker microtubule⁃associated protein 3 (LC3) was detecotide transferase(TUNEL) , the expression of autophagy marker microtubule⁃associated protein 3 (LC3) was detected by immunohistochemical S ⁃P method , the ratio of LC3 ⁃ Ⅱ/LC3 ⁃ Ⅰ and the expression of Beclin⁃1 , p62 in rat myocardial tissue were determined by Western blot , the structure of MAMs in rat myocardial tissue was observed by transmission electron microscopy , the expression levels of glucose⁃regulatory protein 75 (GRP75) , volt⁃dependent anion channel 1 (VDAC1) and mitochondrial fusion protein 2 (Mfn2) in rat myocardial tissue were determined by Western blot .@*Results @#Compared with model group , β ⁃galactosidase activity of myocardial tissue decreased in low⁃dose RVD1 group and high⁃dose RVD1 group (P < 0. 05) , myocardial fiber breakage and myocardial cell damag were significantly alleviated , collagen fiber deposition was significantly reduced , and the proportion of TUNEL positive cells in myocardial tissue decreased (P < 0. 05) , the positive rate of LC3 protein increased (P < 0. 05) , the ratio of LC3 ⁃ Ⅱ/LC3 ⁃ Ⅰ increased , the relative expression level of Beclin⁃1 protein was up⁃regulated and the relative expression level of p62 protein was down⁃regulated ( P < 0. 05) , the structure of MAMs was tighter , and the percentage of MAMs in mitochondrial circumference also increased (P < 0. 05) , the relative protein expressions of GRP75 , VDAC1 and Mfn2 were up⁃regulated (P < 0. 05) . Compared with low⁃dose RVD1 group , β ⁃galactosidase activity in high⁃dose RVD1 group further decreased ( P < 0. 05) , no obvious damage was observed in myocardial tissue , collagen fiber deposition was further decreased , the proportion of TUNEL positive cells in myocardial tissue decreased (P < 0. 05 ) , and the positive rate of LC3 protein increased ( P < 0. 05 ) , LC3 ⁃ Ⅱ/LC3 ⁃ Ⅰ ratio increased , Beclin⁃1 relative expression level was up⁃regulated and p62 relative expression level was down⁃regulated (P < 0. 05) , the structure of MAMs was more compact , and their percentage in mitochondrial circumference further increased (P < 0. 05 ) , the relative expressions of GRP75 , VDAC1 and Mfn2 were further up⁃regulated ( P <0. 05) . @*Conclusion @#RVD1 can activate autophagy , alleviate myocardial apoptosis and collagen fiber deposition, and promote mitochondria⁃associated endoplasmic reticulum membranes in aging rats .

bFGF and IGF-1 promotes differentiation of bone mesenchymal stem cells into cardiomyocyte-like cells and its related mechanisms / 中国药理学通报

Aim To elucidate the biological effects of insulin-like growth factor-1 ( IGF-1 ) and basic fibro-blast growth factor (bFGF) alone or in combination on the differentiation of bone mesenchymal stem cells (BMSCs) into cardiomyocytes (CMs), and to explore the mechanism in the differentiation process of BMSCs into CMs induced by IGF-1 or bFGF. Methods After four weeks of BMSCs induced by induction differentiation medium ( with or without LY294002) containing IGF-1 and/or bFGF, the expression levels of proteins associated with the cardiomyogenic phenotype in BMSCs were detected via immunocytochemistry, immuno-fluorescence staining, and Western blot. Meanwhile, the expression levels of pathway related proteins were detected by Western blot. The cell ultrastructure was observed by transmission electron microscopy (TEM) . The expression levels of myocardial specific genes were measured via RT-qPCR. Results Compared with the control group, the expression levels of myocardial specific proteins and genes significantly increased in the IGF-1, bFGF and combination groups and were the highest in the coinduction group. The TEM of the conduction group showed parallel myofilaments, mitochondria, endoplasmic reticulum and so on. The additon of LY294002 decreased the expression levels of myocardial specific proteins, genes and phosphorylation Akt. Conclusions The effect of IGF-1 combined with bFGF on the differentiation of BMSCs into CMs is markedly better than that induced by IGF-1 or bFGF a-lone, and the differentiation process may depend on the PI3K/Akt signaling pathway.

Impact of LncRNA TUG1 on high glucose-induced cardiomyocyte apoptosis by regulating the miR-181b-5p/PDCD4 axis / 天津医药

Objective To investigate the impact of long non-coding RNA(LncRNA)taurine up-regulated gene 1(TUG1)on high glucose-induced cardiomyocyte apoptosis by regulating miR-181b-5p/programmed cell death protein 4(PDCD4)axis.Methods Diabetic cardiomyopathy(DCM)cell model was established in vitro with high glucose(HG,25 mmol/L glucose).AC16 cells were divided into the NG(5.5 mmol/L glucose)group,the HG group,the HG+sh-NC group,the HG+sh-TUG1 group,the HG+miR-NC group,the HG+miR-181b-5p group,the HG+sh-TUG1+anti-miR-NC group,the HG+sh-TUG1+anti-miR-181b-5p group,the HG+miR-181b-5p+pcDNA group and HG+miR-181b-5p+pc-PDCD4 group.The Cell Counting Kit-8(CCK-8)method was applied to detect cell viability.Lactate dehydrogenase(LDH)assay was applied to detect LDH release.Quantitative real-time polymerase chain reaction(qRT-PCR)was applied to detect expression levels of TUG1,miR-181b-5p and PDCD4 mRNA.Flow cytometry was applied to detect apoptosis.Western blot assay was applied to detect levels of B-cell lymphoma 2-associated X(Bax),activated caspase 3(cleaved caspase 3)and PDCD4 proteins.Caspase-Glo3 assay was applied to assess caspase 3 activity.Dual-luciferase reporter assay was applied to verify the targeting relationship between TUG1 or PDCD4 and miR-181b-5p.Results Compared with the NG group,the cell activity decreased in the HG group,and LDH release,apoptosis rate,Bax,cleaved caspase 3 expression and caspase 3 activity increased(P＜0.05),which could be antagonized by TUG1 knockdown or miR-181b-5p overexpression(P＜0.05).Inhibition of miR-181b-5p was able to alleviate the impact of TUG1 silencing on cardiomyocyte viability and apoptosis under high glucose treatment(P＜0.05).The overexpression of PDCD4 attenuated the promotion effect of miR-181b-5p up-regulation on the viability of cardiomyocytes treated with high glucose and the inhibitory effect on apoptosis.TUG1 was able to increase the expression of PDCD4 through adsorption of miR-181b-5p(P＜0.05).Conclusion TUG1 promotes high glucose-induced cardiomyocyte apoptosis by down-regulating miR-181b-5p and up-regulating PDCD4.

Mechanism of NETosis Regulating Cardiomyocyte Autophagy to Promote Acute Myocardial Infarction in Mice / 医学研究杂志

Objective To explore the correlation between acute myocardial infarction(AMI)and neutrophil extracellular traps-os-is(NETosis)in the regulation of cardiomyocyte autophagy in mice.Methods The mouse model of AMI was established in C57BL/6mice by ligation of the left anterior descending coronary artery.Mouse primary cardiomyocytes were treated with oxygen glucose deprivation(OGD)to build an in vitro model of cardiomyocyte injury.Neutrophils were treated with PMA(NETosis inducer)/DNas Ⅰ(NETosis in-hibitor),the supernatant was taken to treat OGD-induced cardiomyocytes,and rapamycin(Rap)was used to treat OGD-induced car-diomyocytes.The myocardial infarction area was detected by TTC staining;serum cTnI level was detected by enzyme-linked immunoad-sordent assay(ELISA);cardiomyocytes apoptosis was detected by TUNEL staining;NETosis marker levels in neutrophil supernatant were detected by ELISA,and related protein expression levels were detected by Western blot.Results TTC staining showed that compared with the sham-operated group,the myocardial infarction area of the mice in the model group was significantly increased,the level of cTnI in serum was significantly increased(P<0.05),the apoptosis of cardiomyocytes was increased,and the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ were significantly increased,the p62 protein expression level was significantly decreased.Compared with the control group,the expression levels of apoptosis and cleaved caspase-3 were significantly increased under OGD conditions(P<0.05).Compared with the control group,the levels of NETosis markers MPO-DNA,MPO,and NE in the neutrophil supernatant in the PMA group were signifi-cantly increased;compared with the PMA-treated group the apoptosis level of cardiomyocytes in the PMA + DNas Ⅰ group was signifi-cantly decreased;the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰwere significantly decreased,and the protein expression level of p62 was significantly increased;compared with the PMA + DNas Ⅰ group,Rap treatment could enhance the levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,and inhibit the level of p62(P<0.05).It also significantly reversed the decrease in apoptosis induced by PMA + DNas Ⅰ.Conclusion Neutrophil NETosis can promote AMI in mice by regulating cardiomyocyte autophagy,which can provide a new direction and theoretical basis for the research and development of therapeutic drugs for AMI.

Distinct mononuclear diploid cardiac subpopulation with minimal cell-cell communications persists in embryonic and adult mammalian heart / 医学前沿

A small proportion of mononuclear diploid cardiomyocytes (MNDCMs), with regeneration potential, could persist in adult mammalian heart. However, the heterogeneity of MNDCMs and changes during development remains to be illuminated. To this end, 12 645 cardiac cells were generated from embryonic day 17.5 and postnatal days 2 and 8 mice by single-cell RNA sequencing. Three cardiac developmental paths were identified: two switching to cardiomyocytes (CM) maturation with close CM-fibroblast (FB) communications and one maintaining MNDCM status with least CM-FB communications. Proliferative MNDCMs having interactions with macrophages and non-proliferative MNDCMs (non-pMNDCMs) with minimal cell-cell communications were identified in the third path. The non-pMNDCMs possessed distinct properties: the lowest mitochondrial metabolisms, the highest glycolysis, and high expression of Myl4 and Tnni1. Single-nucleus RNA sequencing and immunohistochemical staining further proved that the Myl4+Tnni1+ MNDCMs persisted in embryonic and adult hearts. These MNDCMs were mapped to the heart by integrating the spatial and single-cell transcriptomic data. In conclusion, a novel non-pMNDCM subpopulation with minimal cell-cell communications was unveiled, highlighting the importance of microenvironment contribution to CM fate during maturation. These findings could improve the understanding of MNDCM heterogeneity and cardiac development, thus providing new clues for approaches to effective cardiac regeneration.

β-catenin ameliorates myocardial infarction by preventing YAP-associated apoptosis

Abstract Objective: To explore whether the effect of β-catenin on MI and MI-induced cardiomyocyte apoptosis is YAP-dependent. Methods: The authors established an MI rat model by ligating the anterior descending branch of the left coronary artery, and an MI cell model by treating cardiomyocytes with H2O2. Results: β-catenin downregulation was observed in MI cardiac tissues and in H2O2-treated cardiomyocytes. Lentiviral-CTNNB1 was administered to MI rats to upregulate β-catenin expression in MI cardiac tissue. β-catenin recovery reduced the myocardial infarct area, fibrosis, and apoptotic cell death in MI rats. H2O2 treatment attenuated cell viability and induced cell death in cardiomyocytes, whereas β-catenin overexpression partially reversed these changes. Moreover, H2O2 treatment caused the deactivation of Yes-Associated Protein (YAP), as detected by increased YAP phosphorylation and reduced the nuclear localization of YAP. Upregulation of β-catenin expression reactivated YAP in H2O2-treated cardiomyocytes. Reactivation of YAP was achieved by administration of Mitochonic Acid-5 (MA-5) to H2O2-treated cardiomyocytes, and deactivation of YAP by CIL56 treatment in β-catenin-overexpressing H2O2-treated cardiomyocytes. MA-5 administration increased cell viability and repressed apoptosis in H2O2-treated cardiomyocytes, whereas CIL56 treatment counteracted the effects of β-catenin overexpression on cell survival and apoptosis. Conclusions: The present data indicate that β-catenin and YAP are effective treatment targets for MI, blocking the apoptotic death of cardiomyocytes.