This study investigated the effects and underlying mechanisms of vanillic acid(VA) against cardiac fibrosis(CF) induced by isoproterenol(ISO) in mice. Male C57BL/6J mice were randomly divided into control group, VA group(100 mg·kg~(-1), ig), ISO group(10 mg·kg~(-1), sc), ISO + VA group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig), ISO + dynamin-related protein 1(Drp1) inhibitor(Mdivi-1) group(10 mg·kg~(-1), sc + 50 mg·kg~(-1), ip), and ISO + VA + Mdivi-1 group(10 mg·kg~(-1), sc + 100 mg·kg~(-1), ig + 50 mg·kg~(-1), ip). The treatment groups received the corresponding medications once daily for 14 consecutive days. On the day after the last administration, cardiac functions were evaluated, and serum and cardiac tissue samples were collected. These samples were analyzed for serum aspartate aminotransferase(AST), lactate dehydrogenase(LDH), creatine kinase-MB(CK-MB), cardiac troponin I(cTnI), reactive oxygen species(ROS), interleukin(IL)-1β, IL-4, IL-6, IL-10, IL-18, and tumor necrosis factor-α(TNF-α) levels, as well as cardiac tissue catalase(CAT), glutathione(GSH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC) activities, and cytochrome C levels in mitochondria and cytoplasm. Hematoxylin-eosin, Masson, uranium acetate and lead citrate staining were used to observe morphological and mitochondrial ultrastructural changes in the cardiac tissues, and myocardial injury area and collagen volume fraction were calculated. Flow cytometry was applied to detect the relative content and M1/M2 polarization of cardiac macrophages. The mRNA expression levels of macrophage polarization markers [CD86, CD206, arginase 1(Arg-1), inducible nitric oxide synthase(iNOS)], CF markers [type Ⅰ collagen(Coll Ⅰ), Coll Ⅲ, α-smooth muscle actin(α-SMA)], and cytokines(IL-1β, IL-4, IL-6, IL-10, IL-18, TNF-α) in cardiac tissues were determined by quantitative real-time PCR. Western blot was used to detect the protein expression levels of Coll Ⅰ, Coll Ⅲ, α-SMA, Drp1, p-Drp1, voltage-dependent anion channel(VDAC), hexokinase 1(HK1), NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), caspase-1, cleaved-caspase-1, gasdermin D(GSDMD), cleaved N-terminal gasdermin D(GSDMD-N), IL-1β, IL-18, B-cell lymphoma-2(Bcl-2), B-cell lymphoma-xl(Bcl-xl), Bcl-2-associated death promoter(Bad), Bcl-2-associated X protein(Bax), apoptotic protease activating factor-1(Apaf-1), pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, poly(ADP-ribose) polymerase-1(PARP-1), and cleaved-PARP-1 in cardiac tissues. The results showed that VA significantly improved cardiac function in mice with CF, reduced myocardial injury area and cardiac index, and decreased serum levels of AST, CK-MB, cTnI, LDH, ROS, IL-1β, IL-6, IL-18, and TNF-α. VA also lowered MDA and MPO levels, mRNA expressions of IL-1β, IL-6, IL-18, and TNF-α, and mRNA and protein expressions of Coll Ⅰ, Coll Ⅲ, and α-SMA in cardiac tissues, and increased serum levels of IL-4 and IL-10, cardiac tissue levels of CAT, GSH, SOD, and T-AOC, and mRNA expressions of IL-4 and IL-10. Additionally, VA ameliorated cardiac pathological damage, inhibited myocardial cell apoptosis, inflammatory infiltration, and collagen fiber deposition, reduced collagen volume fraction, and alleviated mitochondrial damage. VA decreased the ratio of F4/80~+CD86~+ M1 cells and the mRNA expressions of CD86 and iNOS in cardiac tissue, and increased the ratio of F4/80~+CD206~+ M2 cells and the mRNA expressions of CD206 and Arg-1. VA also reduced protein expressions of p-Drp1, VDAC, NLRP3, ASC, caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-1β, IL-18, Bad, Bax, Apaf-1, cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP-1, and cytoplasmic cytochrome C, and increased the expressions of HK1, Bcl-2, Bcl-xl, pro-caspase-3, pro-caspase-9 proteins, as well as the Bcl-2/Bax and Bcl-xl/Bad ratios and mitochondrial cytochrome C content. These results indicate that VA has a significant ameliorative effect on ISO-induced CF in mice, alleviates ISO-induced oxidative damage and inflammatory response, and its mechanism may be closely related to the inhibition of Drp1/HK1/NLRP3 and mitochondrial apoptosis signaling pathways, suppression of myocardial cell inflammatory infiltration and collagen fiber deposition, reduction of collagen volume fraction and CollⅠ, Coll Ⅲ, and α-SMA expressions, thus mitigating CF.
Animals ; Isoproterenol/adverse effects* ; Male ; Mice ; Signal Transduction/drug effects* ; Vanillic Acid/administration & dosage* ; Dynamins/genetics* ; Mice, Inbred C57BL ; Fibrosis/genetics* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Myocardium/metabolism* ; Humans

OBJECTIVES: To explore the mechanism that mediate the therapeutic effect of quercetin on heart failure. METHODS: We searched the TCMSP and Swiss ADME databases for the therapeutic targets of quercetin and retrieved heart failure targets from the Genecards and OMIM databases. The intersecting targets were analyzed with GO and KEGG pathway analysis using DAVID database, and the key genes were identified via PPI analysis. Molecular docking between the core targets and quercetin was performed using PyMOL and AutoDock Tools. In a heart failure model established in H9C2 cardiomyocytes by treatment with isoproterenol, the effect of quercetin on the expressions of the MAPK signaling pathway was tested. RESULTS: A total of 60 intersecting targets were identified. Enrichment analysis revealed that quercetin may inhibit heart failure through the MAPK signaling pathway. The core genes, including AMPK3 and BCL-2, were identified as potential key regulators in quercetin-mediated improvement of heart failure. Cellular experiments demonstrated that quercetin significantly reduced isoproterenol-induced apoptosis of cardiomyocytes in a dose-dependent manner and obviously decreased the Bax/Bcl-2 ratio and the expression levels of caspase-3, ERK and p38 in the cells. CONCLUSIONS Quercetin improves heart failure possibly by inhibiting cardiomyocyte apoptosis through the MAPK signaling pathway.
Quercetin/pharmacology* ; Myocytes, Cardiac/drug effects* ; Heart Failure/metabolism* ; Apoptosis/drug effects* ; MAP Kinase Signaling System/drug effects* ; Rats ; Animals ; Isoproterenol

This study investigated the mechanisms and targets of Shenfu Injection in the intervention in chronic heart failure(CHF) through the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/caspase-1 signaling pathway. A CHF model was induced in rats by subcutaneous injection of isoproterenol. Model rats were randomly divided into a model group, a Shenfu Injection group, and a MCC950(NLRP3 inhibitor) group, and a blank group was also set up as a control. After 15 days of treatment, echocardiography was performed to measure cardiac function parameters [left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS)]. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), interleukin(IL)-1β, and IL-18. Hematoxylin-eosin(HE) and Masson staining were used to observe morphological changes in myocardial tissues, and Western blot was used to measure the expression levels of NLRP3/caspase-1 pathway-related proteins [NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), IL-1β, and IL-18]. The study found that isoproterenol-induced CHF in rats resulted in decreased cardiac function, worsened myocardial fibrosis, increased expression levels of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 in myocardial tissues, elevated serum inflammatory factors, and induced myocardial cell pyroptosis. Following Shenfu Injection intervention, the Shenfu Injection group showed significantly improved LVEF and LVFS, a significant decrease in NT-proBNP, a marked downregulation of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 protein expression levels, reduced serum inflammatory factors IL-1β and IL-18 expression in CHF rats, and a decrease in the rate of TUNEL-positive cells. Shenfu Injection can significantly improve cardiac function in CHF, inhibit myocardial fibrosis, and alleviate the progression of myocardial cell pyroptosis through the inhibition of the NLRP3/caspase-1 pathway.
Rats ; Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Interleukin-18/metabolism* ; Caspase 1/metabolism* ; Stroke Volume ; Isoproterenol ; Ventricular Function, Left ; Heart Failure/drug therapy* ; Fibrosis ; Drugs, Chinese Herbal

A large number of β-adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of cardiovascular diseases and other diseases. Nonetheless, it remains unclear whether these commonly used β-AR drugs can activate downstream β- arrestin-biased signaling pathways. The objective of this study was to investigate β-arrestin2 recruitment effects of β-AR agonists and antagonists that were commonly used in clinical practice. We used TANGO (transcriptional activation following arrestin translocation) assay to detect the β-arrestin2 recruitment by β-AR ligands in HEK293 cell line (HTLA cells) stably transfected with tetracycline transactivator protein (tTA) dependent luciferase reporter and β-arrestin2-TEV fusion gene. Upon activation of β-AR by a β-AR ligand, β-arrestin2 was recruited to the C terminus of the receptor, followed by cleavage of the G protein-coupled receptors (GPCRs) fusion protein at the TEV protease-cleavage site. The cleavage resulted in the release of tTA, which, after being transported to the nucleus, activated transcription of the luciferase reporter gene. The results showed that β-AR non-selective agonists epinephrine, noradrenaline and isoprenaline all promoted β-arrestin2 recruitment at β1-AR and β2-AR. β1-AR selective agonists dobutamine and denopamine both promoted β-arrestin2 recruitment at β1-AR. β2-AR selective agonists procaterol and salbutamol promoted β-arrestin2 recruitment at β2-AR. β-AR non-selective antagonists alprenolol and pindolol promoted β-arrestin2 recruitment at β1-AR. β1-AR selective antagonists celiprolol and bevantolol showed β-arrestin2 recruitment at β1-AR. β2-AR selective antagonists butoxamine showed β-arrestin2 recruitment at β1-AR. These results provide some clues for the potential action of β-AR drugs, and lay a foundation for the screening of β-arrestin-biased β-AR ligands.
Humans ; beta-Arrestin 2/metabolism* ; HEK293 Cells ; Adrenergic beta-Agonists/pharmacology* ; Isoproterenol/pharmacology* ; Receptors, Adrenergic, beta-2/metabolism* ; Norepinephrine/pharmacology*

Cardiac hypertrophy is a common pathological process of various cardiovascular diseases and eventually develops into heart failure. This paper was aimed to study the different pathological characteristics exhibited by different mouse strains after hypertrophy stimulation. Two mouse strains, A/J and FVB/nJ, were treated with isoproterenol (ISO) by osmotic pump to induce cardiac hypertrophy. Echocardiography was performed to monitor heart morphology and function. Mitochondria were isolated from hearts in each group, and oxidative phosphorylation function was assayed in vitro. The results showed that both strains showed a compensatory enhancement of heart contractile function after 1-week ISO treatment. The A/J mice, but not the FVB/nJ mice, developed significant cardiac hypertrophy after 3-week ISO treatment as evidenced by increases in left ventricular posterior wall thickness, heart weight/body weight ratio, cross sectional area of cardiomyocytes and cardiac hypertrophic markers. Interestingly, the heart from A/J mice contained higher mitochondrial DNA copy number compared with that from FVB/nJ mice. Functionally, the mitochondria from A/J mice displayed faster O
Animals ; Cardiomegaly/chemically induced* ; Heart Failure ; Isoproterenol/toxicity* ; Mice ; Mitochondria ; Myocytes, Cardiac/metabolism*

OBJECTIVETo explore the feasibility of intraperitoneal injection of isoproterenol (ISO) to induce cardiac remodeling in FVB/N mice.

METHODSForty-eight FVB/N mice were divided into back subcutaneous saline group (subcutaneous saline group), intraperitoneal saline group, back subcutaneous ISO group (subcutaneous ISO group), and intraperitoneal ISO group according to the route of administration of saline or ISO. ISO (30 μg/g body weight/day) was given to the subcutaneous ISO group and the intraperitoneal ISO group, twice daily with an interval of 12 hours, for 14 consecutive days. The subcutaneous saline group and the intraperitoneal saline group were injected with an equal volume of saline. The left ventricular end-diastolic posterior wall thickness was measured by echocardiography, and the ratio of heart weight to tibia length was determined. Hematoxylin-eosin staining was used to determine the myocardial fiber diameter. Picric-sirius red staining was used to determine the myocardial collagen deposition area. Quantitative real-time PCR was used to measure the mRNA expression of collagen I.

RESULTSCompared with the subcutaneous ISO, subcutaneous saline, and intraperitoneal saline groups, the intraperitoneal ISO group had increased sizes of the cardiac cavity and the heart. Compared with the subcutaneous saline and intraperitoneal saline groups, the subcutaneous ISO group showed no significant changes in the gross morphology of the cardiac cavity and the heart. The intraperitoneal ISO group showed significant increases in the ratio of heart weight to tibia length, myocardial fiber diameter, left ventricular end-diastolic posterior wall thickness, myocardial collagen area percentage, and the mRNA expression of collagen I compared with the subcutaneous ISO, subcutaneous saline, and intraperitoneal saline groups (P<0.01). There were no significant differences in the above five indices between the subcutaneous ISO group and the subcutaneous saline and intraperitoneal saline groups (P>0.05). No significant difference in the mortality rate was found between the subcutaneous ISO and intraperitoneal ISO groups (P>0.05).

CONCLUSIONSIntraperitoneal injection of ISO can induce cardiac hypertrophy and fibrosis in FVB/N mice.

Animals ; Atrial Remodeling ; drug effects ; Cardiovascular Diseases ; drug therapy ; metabolism ; pathology ; physiopathology ; Collagen ; metabolism ; Disease Models, Animal ; Humans ; Injections, Intraperitoneal ; Isoproterenol ; administration & dosage ; Male ; Mice ; Myocardium ; metabolism ; pathology

Increased intracellular levels of Ca²⁺ are generally thought to negatively regulate lipolysis in mature adipocytes, whereas store-operated Ca²⁺ entry was recently reported to facilitate lipolysis and attenuate lipotoxicity by inducing lipophagy. Transient receptor potential mucolipin1 (TRPML1), a Ca²⁺-permeable non-selective cation channel, is mainly expressed on the lysosomal membrane and plays key roles in lysosomal homeostasis and membrane trafficking. However, the roles of TRPML1 in lipolysis remains unclear. In this study, we examined whether the channel function of TRPML1 induces lipolysis in mature adipocytes. We found that treatment of mature adipocytes with ML-SA1, a specific agonist of TRPML1, solely upregulated extracellular glycerol release, but not to the same extent as isoproterenol. In addition, knockdown of TRPML1 in mature adipocytes significantly reduced autophagic flux, regardless of ML-SA1 treatment. Our findings demonstrate that the channel function of TRPML1 partially contributes to lipid metabolism and autophagic membrane trafficking, suggesting that TRPML1, particularly the channel function of TRPML1, is as therapeutic target molecule for treating obesity.
Adipocytes ; Glycerol ; Homeostasis ; Isoproterenol ; Lipid Metabolism ; Lipolysis ; Membranes ; Obesity

PURPOSE: Periostin mediates critical steps in gastric cancer and is involved in various signaling pathways. However, the roles of periostin in promoting gastric cancer metastasis are not clear. The aim of this study was to investigate the relevance between periostin expression and gastric cancer progression and the role of stress-related hormones in the regulation of cancer development and progression. MATERIALS AND METHODS: Normal, cancerous and metastatic gastric tissues were collected from patients diagnosed with advanced gastric cancer. The in vivo expression of periostin was evaluated by in situ hybridization and immunofluorescent staining. Meanwhile, human gastric adenocarcinoma cell lines MKN-45 and BGC-803 were used to detect the in vitro expression of periostin by using quantitative real-time polymerase chain reaction (PCR) and western blotting. RESULTS: Periostin is expressed in the stroma of the primary gastric tumors and metastases, but not in normal gastric tissue. In addition, we observed that periostin is located mainly in pericryptal fibroblasts, but not in the tumor cells, and strongly correlated to the expression of α-smooth muscle actin (SMA). Furthermore, the distribution patterns of periostin were broader as the clinical staging of tumors progressed. We also identified a role of stress-related signaling in promoting cancer development and progression, and found that isoprenaline upregulated expression levels of periostin in gastric cancer cells. CONCLUSION: These findings suggest that the distribution pattern of periostin was broader as the clinical staging of the tumor progressed and found that isoprenaline upregulated expression levels of periostin in gastric cancer cells.
Adenocarcinoma/*metabolism/pathology ; Adrenergic beta-Agonists/pharmacology ; Aged ; Blotting, Western ; Cell Adhesion Molecules/drug effects/*metabolism ; Cell Line, Tumor ; Fibroblasts/*metabolism ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; Isoproterenol/*pharmacology ; Male ; Neoplasm Staging ; RNA, Messenger/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Stomach/metabolism/pathology ; Stomach Neoplasms/*metabolism/pathology ; Up-Regulation

BACKGROUNDβ-adrenoceptors play a crucial regulatory role in blood vessel endothelial cells. Isoprenaline (ISO, a β-adrenergic agonist) has been reported to promote angiogenesis through upregulation of vascular endothelial growth factor (VEGF) expression; however, the underlying mechanism remains to be investigated. It is widely accepted that certain noncoding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), can regulate endothelial cell behavior, including their involvement in angiogenesis. Therefore, we aimed to investigate whether noncoding RNAs participate in ISO-mediated angiogenesis using human umbilical vein endothelial cells (HUVECs).

METHODSWe evaluated VEGF-A messenger RNA (mRNA) and protein levels in ISO-treated HUVECs by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To establish whether noncoding RNAs are associated with ISO-mediated angiogenesis, we measured expression of the miRNAs miR-210, miR-21, and miR-1, as well as that of the lncRNAs growth arrest-specific transcript 5 (GAS5), maternally expressed 3 (MEG3), and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in HUVECs exposed to ISO. Furthermore, to ascertain its importance in ISO-mediated angiogenesis, we constructed the HUVECs with overexpressing miR-210 and detected the subsequent expression of VEGF-A and noncoding RNAs. All statistical analyses were performed using SPSS 16.0 software. Intergroup comparisons were carried out by one-way analysis of variance.

RESULTSVEGF-A mRNA levels were elevated in the ISO group (1.57 ± 0.09) compared to those in the control group (P < 0.01). Moreover, concentrations of VEGF-A in culture supernatants significantly differed between the control (113.00 ± 19.21 pg/ml) and ISO groups (287.00 ± 20.27 pg/ml; P< 0.01). Expression of miR-1, miR-21, and miR-210 was higher (3.89 ± 0.44, 2.87 ± 087, and 3.33 ± 1.31, respectively) in ISO-treated cells than that in controls (P < 0.01), whereas that of GAS5 and MEG3 (0.22 ± 0.10 and 0.58 ± 0.16, respectively) was lower as a result of ISO administration (P < 0.05). There was no significant difference in the expression of MALAT1 between the groups. Interestingly, miR-210 overexpression heightened the levels of VEGF-A and miR-21 (5.87 ± 1.24 and 2.74 ± 1.15, respectively; P< 0.01) and reduced those of GAS5 and MEG3 (0.19 ± 0.01 and 0.09 ± 0.05, respectively; P< 0.01).

CONCLUSIONSISO-mediated angiogenesis was associated with altered expression of miR-210, miR-21, and the lncRNAs GAS5 and MEG3. The effects of miR-210 on the expression of VEGF-A and noncoding RNAs were similar to those of ISO, indicating that it might play an important role in ISO-mediated angiogenesis.

Cell Line ; Cell Survival ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Isoproterenol ; pharmacology ; MicroRNAs ; genetics ; physiology ; Neovascularization, Pathologic ; genetics ; RNA, Long Noncoding ; genetics ; Real-Time Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effect of edaravone on oxidative stress and myocardial fibrosis induced by isoproterenol in rats.

METHODSFifty male SD rats were randomly divided into 5 groups, including a control group, a myocardial fibrosis model (established by injections of isopropyl adrenaline for 10 days) group, and 3 edaravone groups with edaravone treatment at low, medium, or high doses for 14 days. After the treatments, the rats were examined for the degree of myocardial fibrosis, left ventricular mass index (LVMI), collagen volume fraction (CVF), and myocardial contents of collagen I (Col I), collage III (Col III), hydroxyproline (Hyp), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO); The expression of transforming growth factor-β1 (TGF-β1) in the myocardial tissues was examined by immunofluorescence assay and Western blotting.

RESULTSCompared with the control rats, the rat models of myocardial fibrosis showed significantly increased CVF and LVMI (P=0.000), which were lowered by edaravone treatments in a dose-dependent manner (P<0.05). The myocardial contents of Col I, Col III and Hyp also increased in the model group (P=0.000) and were lowered dose-dependently by edaravone; the contents of MDA was higher (P=0.000) and SOD and NO were lower in the model group (P=0.000), and edaravone treatments obviously increased SOD and NO contents (P<0.05). The model rats showed significantly increased myocardial expression of TGF-β1 (P=0.000), which was markedly lowered by edaravone treatments (P=0.000). The myocardial content of MDA was positively correlated while SOD and NO were negatively with LVMI, CVF, Col I, Col III and Hyp; TGF-β1 was positively correlated with LVMI, CVF, Col I, Col III, Hyp and MDA but negatively with SOD and NO.

CONCLUSIONEdaravone can relieve oxidative stress and inhibit TGF-β1 activation to ameliorate myocardial fibrosis in rats.

Animals ; Antipyrine ; analogs & derivatives ; pharmacology ; Cardiomyopathies ; chemically induced ; drug therapy ; Collagen ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; Heart ; drug effects ; Hydroxyproline ; metabolism ; Isoproterenol ; Male ; Malondialdehyde ; metabolism ; Myocardium ; pathology ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Transforming Growth Factor beta1 ; metabolism