Myocardial Pathological Changes Research Articles

Effect of moxibustion at "Xinshu" (BL15) and "Feishu" (BL13) on expression of TRPV1 and CGRP in the myocardial tissue of chronic heart failure rats.

To observe the effect of moxibustion at "Xinshu" (BL15) and "Feishu" (BL13) on transient receptor potential vanilloid type 1(TRPV1), calcitonin gene-related peptide (CGRP), and serum interleukin-10 (IL-10) in the myocardial tissue of rats with chronic heart failure (CHF), so as to explore its underlying mechanisms in improvement of CHF. Male SD rats were randomly divided into the normal, model, moxibustion, capsaicin, moxibustion + capsaicin, and moxibustion + solvent groups, with 10 rats in each group. The CHF model was established by permanent ligation of the anterior descending branch of the left coronary artery. Mild moxibustion was applied to bilateral BL13 and BL15 for 30 min once daily for 4 weeks. Rats in the capsaicin group were smeared with capsaicin in the acupoint area once a day for 4 weeks. For rats of the moxibustion + capsaicin and moxibustion + solvent groups, capsaicin and solvent were applied to the acupoint area before moxibustion for 4 weeks, respectively. The ejection fraction (EF) and left ventricular fractional shortening rate (FS) were examined by echocardiography. HE staining was used to observe the myecardial morphological structure. The mRNA and protein expression levels of TRPV1, CGRP and galectin-3 (Gal-3) in myocardial tissue were detected by real-time quantitative PCR and Western blot, respectively. The content of IL-10 in serum was detected by ELISA. After modeling, the pathological changes of myocardium (as cardiac muscle fiber disorder, inflammatory cell infiltration, etc.) were obvious, and the EF, FS, serum IL-10, protein and mRNA exspression of TRPV1 and CGRP were significantly decreased (P<0.01) in the model group compared with the normal group, while the protein and mRNA exspression of Gal-3 were significantly up-regulated (P<0.01). Following the interventions, the above-mentioned indexes were all reversed in moxibustion, capsaicin, and moxibustion + capsaicin groups (P<0.01), and the effect of moxibustion + capsaicin was the best (P<0.05, P<0.01). Moxibustion can reduce myocardial injury and improve cardiac function in CHF rats, which may be related to its effects in up-regulating the expression of TRPV1 and CGRP, and down-regulating the expression of Gal-3 to alleviate myocardial fibrosis.

Shexiang Tongxin Dropping Pill Promotes Angiogenesis through VEGF/eNOS Signaling Pathway on Diabetic Coronary Microcirculation Dysfunction.

To study the effect of Shexiang Tongxin Dropping Pill (STDP) on angiogenesis in diabetic cardiomyopathy mice with coronary microcirculation dysfunction (CMD). According to a random number table, 6 of 36 SPF male C57BL/6 mice were randomly selected as the control group, and the remaining 30 mice were injected with streptozotocin intraperitoneally to replicate the type 1 diabetes model. Mice successfully copied the diabetes model were randomly divided into the model group, STDP low-dose group [15 mg/(kg·d)], medium-dose group [30 mg/(kg·d)], high-dose group [60 mg/(kg·d)], and nicorandil group [15 mg/(kg·d)], 6 in each group. The drug was given by continuous gavage for 12 weeks. The cardiac function of mice in each group was detected at the end of the experiment, and coronary flow reserve (CFR) was detected by chest Doppler technique. Pathological changes of myocardium were observed by hematoxylin-eosin staining, collagen fiber deposition was detected by masson staining, the number of myocardial capillaries was detected by platelet endothelial cell adhesion molecule-1 staining, and the degree of myocardial hypertrophy was detected by wheat germ agglutinin staining. The expression of the vascular endothlial growth factor (VEGF)/endothelial nitric oxide synthase (eNOS) signaling pathway-related proteins in myocardial tissue was detected by Western blot. Compared with the model group, medium- and high-dose STDP significantly increased the left ventricular ejection fraction and left ventricular fraction shortening (P<0.01), obviously repaired the disordered cardiac muscle structure, reduced myocardial fibrosis, reduced myocardial cell area, increased capillary density, and increased CFR level (all P<0.01). Western blot showed that high-dose STDP could significantly increase the expression of VEGF and promote the phosphorylation of vascular endothelial growth factor receptor 2, phosphoinositide 3-kinase, protein kinase B, and eNOS (P<0.05 or P<0.01). STDP has a definite therapeutic effect on diabetic CMD, and its mechanism may be related to promoting angiogenesis through the VEGF/eNOS signaling pathway.

Mechanism of Shexiang Tongxin Dropping Pills in treating diabetic cardiomyopathy based on network pharmacology and animal experiments

This study aimed to explore the mechanism of Shexiang Tongxin Dropping Pills(STDP) in treating diabetic cardiomyopathy(DCM) based on network pharmacology, molecular docking, and animal experiments. BATMAN, TCMSP, and GeneCards were searched for the active ingredients and targets of STDP against DCM. STRING and Cytoscape were used to build the protein-protein interaction(PPI) network and &quot;drug-active ingredient-target&quot; network. Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis of the targets were carried out based on DAVID. The molecular docking of key receptor proteins with corresponding active ingredients was performed using AutoDock Vina. The rat model of DCM was established by a high-fat diet combined with intraperitoneal injection of streptozotocin. Rats were assigned into control, model, low-(20 mg·kg~(-1)) and high-dose(40 mg·kg~(-1)) STDP, and metformin(200 mg·kg~(-1)) groups. After 8 weeks of continuous administration, the cardiac function, myocardial pathological changes, and myocardial collagen fiber deposition of rats in each group were detected by echocardiography, hematoxylin-eosin(HE) staining, and Sirius red staining, respectively. The myocardial hypertrophy was detected by WGA staining. The expression levels of p38 mitogen-activated protein kinase(p38), phosphorylation-p38(p-p38), c-Jun N-terminal kinase(JNK), phosphorylation-JNK(p-JNK), caspase-3, and C-caspase-3 in the myocardial tissue of rats in each group were measured by Western blot. The network pharmacology predicted 199 active ingredients and 1 655 targets of STDP and 463 targets of DCM. One hundred and thirty-four potential targets of STDP for treating DCM were obtained, and the AGE-RAGE signaling pathway in diabetic complications was screened out. Molecular docking results showed that miltirone, dehydromiltirone, and tryptanthrin had strong binding affinity with RAGE. The results of animal experiments confirmed that STDP effectively protected the cardiac function of DCM rats. Compared with the DCM model group, the STDP groups showed significantly down-regulated protein levels of p-p38, p-JNK, and C-caspase-3. To sum up, STDP may protect the cardiac function of DCM rats by regulating the AGE-RAGE signaling pathway.

CRIF1 attenuates doxorubicin-mediated mitochondrial dysfunction and myocardial senescence via regulating PXDN.

CR6-interacting factor 1 (CRIF1), a multifunctional protein that affects mitochondrial function and cell senescence, plays a regulatory role in heart-related diseases. However, whether CRIF1 participates in myocardial senescence by regulating mitochondrial function remains unclear. Doxorubicin (DOX)-induced C57BL/6 mice to construct mouse myocardial senescence model, and the myocardial function indicators including lactate dehydrogenase (LDH) and Creatine kinase isoform MB (CK-MB) were assessed. The expression of CRIF1 was detected by western blot. Myocardial pathological changes were examined by transthoracic echocardiography and haematoxylin and eosin (H&E) staining. Cell senescence was detected by SA-β-gal staining. JC-1 staining was used to detect mitochondrial membrane potential. Biochemical kits were used to examine oxidative stress-related factors. Additionally, AC16 cardiomyocytes were treated with DOX to mimic the cellular senescence model in vitro. Cell activity was detected by cell counting kit-8 (CCK-8) assay. Co-immunoprecipitation (CO-IP) was used to verify the relationship between CRIF1 and peroxidasin (PXDN). The CRIF1 expression was significantly decreased in DOX-induced senescent mice and AC16 cells. Overexpression of CRIF1 significantly ameliorated DOX-induced myocardial dysfunction and myocardial senescence. Additionally, CRIF1 overexpression attenuated DOX-induced oxidative stress and myocardial mitochondrial dysfunction. Consistently, CRIF1 overexpression also inhibited DOX-induced oxidative stress and senescence in AC16 cells. Moreover, CRIF1 was verified to bind to PXDN and inhibited PXDN expression. The inhibitory effects of CRIF1 overexpression on DOX-induced oxidative stress and senescence in AC16 cells were partly abolished by PXDN expression. CRIF1 plays a protective role against DOX-caused mitochondrial dysfunction and myocardial senescence partly through downregulating PXDN.

Astragaloside IV-induced BMSC exosomes promote neovascularization and protect cardiac function in myocardial infarction mice via the miR-411/HIF-1α axis

This study focused on investigating the mechanism of the astragaloside IV-induced bone marrow mesenchymal stem cell exosome (AS-IV-MSC-exo)/microRNA(miR)-411/HIF-1α axis in affecting vascular neovascularization and protecting cardiac function in myocardial infarction (MI) mice. Exosomes (MSC-exo and AS-IV-MSC-exo) were separated by differential centrifugation and then characterized. MI mouse models were established by left anterior descending coronary artery ligation. Echocardiography was used to evaluate cardiac function. HE staining and Masson staining were performed to observe myocardial histopathology. Capillary density in the myocardium via immunohistochemistry and quantified the expression of vascular endothelial growth factor (VEGF) via RT-qPCR. The expression of miR-411 and HIF-1α was tested by RT-qPCR and western blot and the targeting relationship of miR-411 and HIF-1α was verified by bioinformatics website and dual luciferase reporter gene assay. Exosomes with lipid bi-layer membrane structure, expressing exosomal surface marker proteins, and being taken up by cardiomyocytes could be successfully isolated utilizing ultracentrifugation. Intramyocardial injection of MSC-exo could restore cardiac function, decrease myocardial pathological changes and collagen deposition, and promote neovascularization in MI mice; the effect of AS-IV-MSC-exo was more significant. The ability of AS-IV-MSC-exo to restore cardiac function, lower myocardial pathological changes and collagen deposition, and promote neovascularization in MI mice was diminished when miR-411 expression in AS-IV-MSC-exo was reduced. Mechanistically, miR-411 was found to target and inhibit HIF-1α expression. Overexpression of HIF-1α impaired the impact of AS-IV-MSC-exo on improving cardiac function and promoting neovascularization in MI mice. AS-IV-MSC-exo improves cardiac function and promoted neovascularization via the miR-411/HIF-1α axis, thereby ameliorating MI.

Inhibition of the STIM1/Orai1 Signaling Pathway by Glycine Betaine Mitigates Myocardial Hypertrophy in Spontaneous Hypertension Rats.

Spontaneous hypertension is a leading risk factor for cardiovascular diseases morbidity and mortality. Glycine betaine (GB) is a natural vitamin that has the potential to lower blood pressure. This work attempted to investigate the role and mechanisms of GB in spontaneous hypertension. Spontaneously hypertensive rats (SHRs) were administrated with 100, 200, or 400 mg/kg of GB by gavage or combined with by injection of lentivirus-mediated STIM1 overexpression vector. The heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart weight/body weight (HW/BW) of rats were monitored. The pathological changes in myocardium were examined by hematoxylin and eosin staining and Masson staining. The expression of genes and proteins was detected by quantitative real-time PCR, western blotting, and immunohistochemistry. GB at 200 and 400 mg/kg reduced the HR, SBP, DBP and HW/BW in SHRs. GB decreased the cross-sectional area and fibrotic area in the myocardium and downregulated the expression of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) in the myocardium of SHRs. It indicated that GB treatment effectively alleviated myocardial hypertrophy in SHRs. Additionally, GB treatment repressed the expression of stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1) in the myocardium of SHRs. STIM1 overexpression reversed GB treatment-mediated inhibition of myocardial hypertrophy in SHRs. In conclusion, GB repressed STIM1/Orai1 signaling pathway, which contributed to alleviating myocardial hypertrophy in SHRs. Thus, our study provides a theoretical basis for GB as an antihypertensive drug.

Exosomes from Adipose-Derived Mesenchymal Stem Cells Protect Against Cyclophosphamide-Induced Cardiotoxicity in Rats.

A certain dosage of cyclophosphamide (CYP) in clinical applications contributes to severe cardiotoxicity. Herein, this study explored the impact of adipose-derived mesenchymal stem cell (AdMSC)-exosomes (Exos) on CYP-induced cardiotoxicity.AdMSCs and AdMSCs-Exos were isolated and identified. CYP was utilized for developing a cardiotoxicity rat model, after which blood was collected and then the serum contents of cardiac injury-related indexes (creatine kinase-MB, lactate dehydrogenase, aspartate aminotransferase, and alkaline phosphatase) were detected with enzyme-linked immunosorbent assay kits. Oxidative stress (OS)-related indicators were measured with the corresponding kits. Myocardial pathological changes and collagen fibrosis were tested with hematoxylin-eosin and Masson staining, and apoptosis-related and autophagy-related proteins in rat cardiac tissues with immunohistochemistry and Western blot assays, respectively.AdMSCs and AdMSCs-Exos were successfully isolated. AdMSCs-Exos could target rat hearts. AdMSCs-Exos improved cardiac function and diminished the content of the cardiac injury-related indexes in CYP rats. In addition, AdMSCs-Exos reduced CYP-induced cardiac fibrosis, OS, apoptosis, and autophagy in rats.AdMSCs-Exos alleviated CYP-induced cardiotoxicity in rats via the repression of OS, apoptosis, and autophagy.

HDAC3 Impairs Cardiac Function in Chronic Heart Failure Rats via Mediating MicroRNA-26b-3p to Target High Mobility Group AT-Hook 2.

Histone deacetylase 3 (HDAC3) has been studied in chronic heart failure (CHF), while the regulatory mechanism of HDAC3 on the development of CHF in regulating microRNA (miR)-26b-3p/high mobility group AT-hook 2 (HMGA2) axis has not been extensively investigated. This study aimed to probe the effects of HDAC3, miR-26b-3p and HMGA2 on CHF. CHF rat models were established using aortic coarctation. HDAC3, miR-26b-3p and HMGA2 levels in CHF rats were examined. Thereafter, the CHF rats were injected with relative oligonucleotides and plasmids of HDAC3, miR-26b-3p and HMGA2 to detect the cardiac function, inflammatory reaction, myocardial tissue pathological changes, and cardiomyocyte apoptosis. The binding relationship between miR-26b-3p and HMGA2 and the interaction between HDAC3 and miR-26b-3p were validated. HDAC3 and HMGA2 were elevated, while miR-26b-3p was decreased in CHF rats. The reduced HDAC3 or HMGA2 or enriched miR-26b-3p attenuated cardiac dysfunction, inflammatory reaction, myocardial tissue pathological changes and cardiomyocyte apoptosis in CHF rats, while the reduction of miR-26b-3p exerted the opposite effects. Furthermore, the inhibition of the miR-26b-3p or elevation of HMGA2 reversed the effect of reduced HDAC3 on mitigating CHF progression. Mechanically, miR-26b-3p targeted HMGA2 and HDAC3 bound to miR-26-3p. Downregulation of HDAC3 relieves cardiac function in CHF rats via mediating miR-26b-3p/HMGA2 axis. This study provides novel theory references and a distinct direction for the therapy strategies of CHF.

Syndecan-1: From a Promising Novel Cardiac Biomarker to a Surrogate Early Predictor of Kidney and Liver Injury in Patients with Acute Heart Failure.

(1) Background: Acute heart failure (HF) represents a complex clinical syndrome burdened by increased mortality and a high rate of systemic complications. Although natriuretic peptides (e.g., NT-proBNP) currently represent the diagnostic and prognostic gold standard in acute HF, those molecules do not accurately reflect all the pathophysiological mechanisms involved in the progression of this pathology when determined independently. Therefore, the current paradigm tends to focus on a multi-marker approach for the risk stratification of patients with acute HF. Syndecan-1 is a less studied biomarker in cardiovascular diseases; its assessment in patients with acute HF being potentially able to reflect the myocardial pathological changes, such as fibrosis, inflammation, endothelial dysfunction or global wall stress. (2) Methods: We conducted a single center prospective study that enrolled 173 patients (120 patients admitted for acute HF, compared to 53 controls with stable chronic HF). A complete standardized clinical, echocardiography and laboratory evaluation was performed at admission, including serum samples for the determination of syndecan-1 by the enzyme-linked immunosorbent assay (ELISA) method. (3) Results: The serum concentration of syndecan-1 was significantly higher in patients with acute HF, compared to controls [121.4 (69.3-257.9) vs. 72.1 (41.4-135.8) ng/mL, p = 0.015]. Syndecan-1 was a significant predictor for the diagnosis of acute HF, expressed by an area under the curve (AUC) of 0.898, similar to NT-proBNP (AUC: 0.976) or cardiac troponin (AUC: 0.839). Moreover, syndecan-1 was independently associated with impaired kidney and liver function at admission, being also a predictor for early, subclinical organ dysfunction in patients with normal biological parameters at admission. When included in the multi-marker model, syndecan-1 levels influenced mortality more significantly than NT-proBNP or troponin. A multivariable regression including syndecan-1, NT-proBNP and troponin provided additional prognostic value compared to each independent biomarker. (4) Conclusions: Syndecan-1 can be considered a promising novel biomarker in acute HF, exhibiting adequate diagnostic and prognostic value. Additionally, syndecan-1 can be used as a surrogate biomarker for non-cardiac organ dysfunction, as its highs levels can accurately reflect early acute kidney and liver injury.

Ginkgo biloba extract protects against diabetic cardiomyopathy by restoring autophagy via adenosine monophosphate-activated protein kinase/mammalian target of the rapamycin pathway modulation.

Studies demonstrated that Ginkgo biloba extract (GBE) played a cardioprotective role in diabetic conditions. Impaired autophagy is one of the mechanisms underlying diabetic cardiomyopathy (DCM). The effect of GBE on autophagy has been observed in several diseases; however, whether GBE can ameliorate DCM by regulating autophagy remains unclear. Here, we investigated the effect of GBE on DCM and the potential mechanisms regarding autophagy using a streptozotocin (STZ)-induced diabetic rat model and a high-glucose (HG)-stimulated H9C2 cell model. We demonstrated that GBE attenuated metabolic disturbances, improved cardiac function, and reduced myocardial pathological changes in diabetic rats. Impaired autophagy as well as dysregulation of the adenosine monophosphate-activated protein kinase/ mammalian target of the rapamycin (AMPK/mTOR) signaling pathway were observed in diabetic hearts, as evidenced by the reduced conversion of LC3B-I to LC3B-II along with excessive p62 accumulation, decreased AMPK phosphorylation, and increased mTOR phosphorylation, which could be reversed by GBE treatment. In vitro, GBE reduced the apoptosis induced by HG in H9C2 cells by activating AMPK and inhibiting mTOR to restore autophagy. However, this effect was inhibited by the AMPK inhibitor Compound C. In conclusion, the ameliorative effect of GBE on DCM might be dependent on the restoration of autophagy through modulation of the AMPK/mTOR pathway.

The features of autoimmunity in complicated atherosclerosis: A pilot study

Background: There is increasing evidence that autoimmunity plays an essential role in atherogenesis. At the same time, changes in the profile of natural autoantibodies (AAb) are characteristic not only of autoimmune, but also of all somatic diseases in general, since AAb can, without being the cause of the disease, respond to illness-related changes being a kind of bioregulators of homeostasis and/or immunological clearance factors. Methods: The enzyme immunoassays ELI-Cardio-Test was used to determine changes in serum content of a 12 natural AAb towards the autoantigens, which are the markers of pathological changes in myocardium and blood vessel walls, in 5 patients with diagnosis of severe complicated atherosclerosis and 5 healthy individuals. Results: patients with atherosclerosis were characterized by significantly more frequent and pronounced deviations in the autoimmunoreactivity against platelet membrane antigen (TrM) than healthy controls. The level of other natural AAb did not differ between study groups. Conclusions: It is not possible to distinguish between individuals without atherosclerosis and those with severe atherosclerosis, based on the overall picture of the autoimmune profile for manufacturer-selected antigens. However, the obvious discordance of groups in terms of autoimmunoreactivity to TrM merits further study. Perhaps, it is worth for a manufacturer to modify the set of autoantigens in this panel and focus more on the autoimmunity against platelet antigens as well as add the appropriate information about anti-TrM autoimmunity to the kit leaflet.

PS-B02-4: GLYCINE BETAINE ALLEVIATES MYOCARDIAL HYPERTROPHY IN SPONTANEOUS HYPERTENSION RATS BY REGULATING AMPK/NF-KAPPAB/STIM1/ORAI1 SIGNALING PATHWAY

Background: Spontaneous hypertension is one of the common cardiovascular diseases. Glycine betaine (GB) is a natural vitamin that has the potential to lower blood pressure. This work attempted to investigate the precise mechanism of action of GB in spontaneous hypertension. Methods: Spontaneous hypertension rats (SHRs) were administrated with 100, 200 or 400 mg/kg of GB by gavage, or followed by injection of lentivirus-mediated STIM1 overexpression vector. The heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart weight/body weight (HW/BW) of rats were monitored. The pathological changes of myocardium were examined by hematoxylin and eosin staining and Masson staining. The expression of gene and protein was detected by quantitative real-time PCR and western blotting. Results: GB at 200 and 400 mg/kg reduced the levels of HR, SBP, DBP and HW/BW in SHRs. GB decreased the cross-sectional area and fibrotic area in the myocardium, and down-regulated the expression of ANP and beta-MHC in the myocardium of SHRs. It indicated that GB treatment effectively alleviated myocardial hypertrophy in SHRs. Additionally, GB treatment enhanced p-AMPK expression and repressed the expression of p-NF-kappaB, STIM1 and Orai1 in the myocardium of SHRs. STIM1 overexpression reversed GB treatment-mediated inhibition of myocardial hypertrophy in SHRs. Conclusion: This study demonstrated that GB activated AMPK and repressed NF-kappaB/STIM1/Orai1 signaling pathway, which contributed to alleviate myocardial hypertrophy in SHRs. Thus, this work provides a theoretical basis for GB as an antihypertensive drug.

Effect of Dendrobine on Cardiac Dysfunction in Rats with Myocardial Infarction by Regulating Autophagy

Aim: To observe Dendrobine (Den) on rats with post-myocardial infarction cardiac dysfunction and mechanism. Materials: Dividing 27 rats as Sham, Model and Den groups, rats treated with two weeks of drug had their cardiac function and structure measured by ultrasound; their myocardial pathological changes observed by HE and Masson staining and observe apoptosis cell number by TUNEL staining; their serum activities of LDH and CK-MB detected by ELISA; myocardial autophagy protein expressions detected by WB and immunohistochemistry. Results: Model group displayed decreased cardiac function levels, enlarged area of myocardial fibrosis, more serum activities of LDH and CK-MB, increased myocardial tissue structural damage and apoptosis cell number, downregulated LAMP2 expression, and up-regulated expressions of Beclin1, LC3-II/LC3-I rate, and P62. To rat victims of myocardial infarction, Den improved cardiac function, reduced area of myocardial fibrosis, compromised activities of serum LDH and CK-MB, and relieved damage in myocardial structure, decreased apoptosis cell number in myocardial tissue, up-regulated the expressions of Beclin1, LAMP2 and LC3-II, and down-regulated P62 to promote the autophagy in myocardium damaged by myocardial infarction. Conclusion: Den alleviates post-myocardial infarction cardiac dysfunction through improvement of autophagosomes formation and autophagic flux via Beclin1/LAMP2 pathway.

Shexiang Tongxin Dropping Pills improve cardiac function in type 2 diabetic rats by promoting angiogenesis

This study aims to investigate the effect of Shexiang Tongxin Dropping Pills(STDP) on angiogenesis in type 2 diabetic rats. Thirty-two healthy male SD rats were randomized into a control group(n=6) and a type 2 diabetes mellitus(T2 D) group(n=26). The T2 DM rat model was established with a high-fat diet combined with intraperitoneal injection of streptozotocin(STZ). The model rats were randomized into a model group, a metformin group(200 mg·kg~(-1)·d~(-1)), a high-dose STDP group(40 mg·kg~(-1)·d~(-1)), and a low-dose STDP group(20 mg·kg~(-1)·d~(-1)). The corresponding agents were administered continuously for 8 weeks. The cardiac function indexes were detected by ultrasound at the experiment endpoint, and the myocardial pathological changes were observed via hematoxylin-eosin(HE) staining. The myocardial collagen fiber deposition was detected by sirius red staining, the myocardial microvascular density by immunohistochemistry, and the myocardial hypertrophy by immunofluorescence assay. Western blot was employed to measure the protein levels of vascular endothelial growth factor(VEGF), phosphorylated vascular endothelial growth factor receptor 2(p-VEGFR2), angiogenin-1(Ang1), and TEK tyrosine kinase endothelial(Tie2) in the myocardial tissue. Compared with the model group, STDP improved the left ventricular ejection fraction(EF) and fractional shortening(FS)(P&lt;0.05), alleivated the arrangement disorder of myocardial cells and local myocardial fiber rupture. Sirius red staining showed that STDP, especially the high-dose group(P&lt;0.01), repaired the deposition of myocardial collagen fibers in the model group. The results of immunohistochemistry showed that the cluster of differentiation 31(CD31) expression in myocardial tissue of the STDP groups was higher than that of the model group(P&lt;0.01). The immunofluorescence results showed that STDP mitigated the hypertrophy of myocardial cells(P&lt;0.01). Furthermore, STDP up-regulated the protein levels of VEGF, p-VEGFR2, Angl, and Tie2 compared with the model group(P&lt;0.01). In conclusion, STDP can effectively promote angiogenesis and improve cardiac function in type 2 diabetic rats by up-regulating the expression of VEGF, p-VEGFR2, Ang1, and Tie2.

AMPK/Sirt1-mediated inflammation is positively correlated with myocardial fibrosis during ageing

Objectives Cardiovascular disease is the leading cause of death in the world, and it increases dramatically with ageing. The objective of this study was to elucidate age-dependent molecular changes of inflammation and its correlation with the progression of myocardial fibrosis. Methods Methods: Male SD rats aged 3, 6, 9 and 24 months were used in this study. H&E staining was used to assessed histo-morphological changes in different ages. Masson’s trichrome staining was used to evaluate myocardial fibrosis. Immunofluorescence as well as western blot was carried out to detect the expression of vimentin. Real-time PCR was used to detect the level of pro-inflammatory chemokines MCP-1, IL1β, TNFα and IL-6. Western blotting was also carried out to detect p-AMPK, Sirt1, AC-NF-κB expression. Results Myocardial pathological changes and fibrosis are positively correlated with age. Ageing rats showed an enhanced expression of inflammatory factors and the activation of cardiac fibroblasts increases. Meanwhile, the expression of p-AMPK, Sirt1 and downstream AC-NF-κB increased significantly during ageing. Furthermore, the 15–24 months of age in rats is the fastest changing stage of increased inflammation and decreased Sirt1 activity. Conclusions Ageing is an independent risk factor for the occurrence and development of myocardial fibrosis. During ageing, myocardial fibroblasts are activated, accompanied by an increase in extracellular matrix deposition. The inflammation mediated by AMPK/Sirt1/NF-κB signalling pathway is closely positively correlated with the activation of myocardial fibroblasts and the progression of myocardial fibrosis.

MicroRNA-181c-5p stimulates the development of coronary artery disease by targeting SIRT1

ObjectiveMicroRNA (miR) therapeutics is a promising approach to manage coronary artery disease (CAD). Herein, this research was aimed to explore miR-181c-5p-related mechanisms in CAD through regulating SIRT1. MethodsA CAD mouse model was established by feeding a high-fat diet in 8-week-old ApoE−/− mice. miR-181c-5p, SIRT1, and acetylated p65 levels in mouse myocardial tissues were evaluated by RT-qPCR and Western blot. Hemodynamic parameters included the maximum rising rate of the left ventricular pressure (lv + dp/dtmax) and the time values from the onset of contraction to dp/dtmax (t-dp/dtmax), while hemorheological indices included whole blood viscosity (low shear, middle shear, or high shear), plasma viscosity, hematocrit, and platelet adhesion were measured. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were detected. Mouse pathological changes, degree of fibrosis, and cardiomyocyte apoptosis in myocardial tissues were assessed by HE, Masson, and TUNEL staining, respectively. The targeting relationship between miR-181c-5p and SIRT1 was verified by bioinformatics tools, dual luciferase reporter gene assay, and RNA pull-down assays. ResultsIn myocardial tissue of CAD mice, miR-181c-5p and acetylated p65 were upregulated while SIRT1 was downregulated. Downregulating miR-181c-5p or upregulating SIRT1 effectively ameliorated CAD by improving hemodynamics and hemorheology and reducing inflammation, pathological changes, degree of fibrosis, and cardiomyocyte apoptosis in myocardial tissues of mice. miR-181c-5p targeted SIRT1, and overexpression of SIRT1 relieved upregulated miR-181c-5p-induced injuries in CAD mice. Regulating miR-181c-5p and SIRT1 affected the acetylation of p65. ConclusionDownregulation of miR-181c-5p may ameliorate myocardial pathological changes and cardiomyocyte apoptosis in CAD by upregulating SIRT1 expression and decreasing acetylated p65 levels.

Injection of Matrix Metalloproteinase-9 Leads to Ventricular Remodeling.

Objective Previous studies have found that some ventricular remodeling is accompanied by increased matrix metalloproteinase-9 (MMP-9) in vivo, and MMP-9 inhibitors can reduce ventricular remodeling. However, there is still no direct evidence that MMP-9 causes ventricular remodeling. In this study, MMP-9 was injected into rats to observe whether MMP-9 caused ventricular remodeling, thereby providing direct evidence of MMP-9-induced ventricular remodeling. Methods Forty-eight eight-week-old male Wistar rats were randomly divided, by weight, into control, low-, medium-, and high-dose MMP-9 groups and were administered normal saline or recombinant rat MMP-9 0.7, 1.4, or 2.1 ng/g, respectively, via intraperitoneal injection, twice per week. On the 28th day, six rats were randomly selected from each group (Stage I). The remaining rats continued receiving injections until the 56th day (Stage II). Echocardiography was performed to observe cardiac structure and function, and the left ventricular mass index (LVWI) was calculated. Myocardial pathological changes and the collagen volume fraction (CVF) were observed by HE and VG staining in myocardial tissue. MMP-9 levels in serum were tested using ELISA. Myocardial MMP-9 levels were measured using Western blots, and the myocardial expression levels of MMP-9 mRNA were assessed using RT-PCR. Results During Stage I, serum MMP-9 and myocardial MMP-9 mRNA levels are increased; hypertrophic cardiomyocytes, disorderly arrangement of fibers, and endochylema dissolution are observed in the medium- and high-dose groups. The left ventricular weight index (LVWI) and myocardial MMP-9 increased, and the collagen volume fraction (CVF) reduced in the high-dose group. In Stage II, the left ventricular end-diastolic volume (LVEDV) and diameter (LVIDd) are higher, and CVF decreased in the medium- and high-dose groups. Myocardial pathological lesions intensified. Serum MMP-9 in the model groups and myocardial MMP-9 in the medium- and high-dose groups are increased. Conclusions Injection of MMP-9 can lead to ventricular remodeling.

Fufang Xueshuantong Improves Diabetic Cardiomyopathy by Regulating the Wnt/β-Catenin Pathway.

Diabetic cardiomyopathy (DCM) is one of the main complications of diabetic patients and the major reason for the high prevalence of heart failure in diabetic patients. Fufang Xueshuantong (FXST) is a traditional Chinese medicine formula commonly used in the treatment of diabetic retinopathy and stable angina pectoris. However, the role of FXST in DCM has not yet been clarified. This study was conducted to investigate the effects of FXST on diabetic myocardial lesions and reveal its molecular mechanism. The rats were intraperitoneally injected with 65 mg/kg streptozotocin (STZ) to induce diabetes mellitus (DM). DM rats were given saline or FXST. The rats in the control group were intraperitoneally injected with an equal amount of sodium citrate buffer and gavaged with saline. After 12 weeks, echocardiography, heart weight index (HWI), and myocardial pathological changes were determined. The expression of transforming growth factor-beta1 (TGF-β1), collagen I, and collagen III was examined using immunofluorescence staining and western blot. The expressions of Wnt/β-catenin signaling pathway-related proteins and mRNA were detected by western blot and real-time PCR. The results showed that FXST significantly improved cardiac function, ameliorated histopathological changes, and decreased HWI in the DM rats. FXST significantly inhibited the expression of myocardial TGF-β1, collagen I, and collagen III in DM rats. Furthermore, FXST significantly inhibited the Wnt/β-catenin pathway. Taken together, FXST has a protective effect on DCM, which might be mediated by suppressing the Wnt/β-catenin pathway.

Dysregulation and imbalance of innate and adaptive immunity are involved in the cardiomyopathy progression.

BackgroundCardiomyopathy is known to be a heterogeneous disease with numerous etiologies. They all have varying degrees and types of myocardial pathological changes, resulting in impaired contractility, ventricle relaxation, and heart failure. The purpose of this study was to determine the pathogenesis, immune-related pathways and important biomarkers engaged in the progression of cardiomyopathy from various etiologies.MethodsWe downloaded the gene microarray data from the Gene Expression Omnibus (GEO). The hub genes between cardiomyopathy and non-cardiomyopathy control groups were identified using differential expression analysis, least absolute shrinkage and selection operator (LASSO) regression and weighted gene co-expression network analysis (WGCNA). To assess the diagnostic precision of hub genes, receiver-operating characteristic (ROC) curves as well as the area under the ROC curve (AUC) were utilized. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis and Gene Ontology (GO) analysis were conducted on the obtained differential genes. Finally, single-sample GSEA (ssGSEA) and Gene Set Enrichment Analysis (GSEA) were utilized to analyze the infiltration level of 28 immune cells and their relationship with hub genes based on gene expression profile data and all differential gene files.ResultsA total of 82 differentially expressed genes (DEGs) were screened after the training datasets were merged and intersected. The WGCNA analysis clustered the expression profile data into four co-expression modules, The turquoise module exhibited the strongest relationship with clinical traits, and nine candidate key genes were obtained from the module. Then we intersected DEGs with nine candidate genes. LASSO regression analysis identified the last three hub genes as promising biomarkers to distinguish the cardiomyopathy group from the non-cardiomyopathy control group. ROC curve analysis in the validation dataset revealed the sensitivity and accuracy of three hub genes as marker genes. The majority of the functional enrichment analysis results were concentrated on immunological and inflammatory pathways. Immune infiltration analysis revealed a significant correlation between regulatory T cells, type I helper T cells, macrophages, myeloid-derived suppressor cells, natural killer cells, activated dendritic cells and the abundance of immune infiltration in hub genes.ConclusionThe hub genes (CD14, CCL2, and SERPINA3) can be used as markers to distinguish cardiomyopathy from non-cardiomyopathy individuals. Among them, SERPINA3 has the best diagnostic performance. T cell immunity (adaptive immune response) is closely linked to cardiomyopathy progression. Hub genes may protect the myocardium from injury through myeloid-derived suppressor cells, regulatory T cells, helper T cells, monocytes/macrophages, natural killer cells and activated dendritic cells. The innate immune response is crucial to this process. Dysregulation and imbalance of innate immune cells or activation of adaptive immune responses are involved in cardiomyopathy disease progression in patients.

Effects of electroacupuncture with dexmedetomidine on myocardial ischemia/reperfusion injury in rats.

To investigate the protective effect of electroacupuncture combined with dexmedetomidine (EA + Dex) on oxidative stress injury in myocardial ischemia/reperfusion (I/R) rats. A total of 50 male Sprague-Dawley (SD) rats were randomly divided into 5 groups: sham operation (sham group); I/R group; dexmedetomidine group (Dex group); electroacupuncture group (EA group); and EA + Dex group. The myocardial I/R model was established. The EA group received EA at the Neiguan acupoint [pericardium 6 (PC6)] every day for 1 week before modeling. Rats in the EA + Dex group received EA at PC6 every day for 1 week before modeling, and intraperitoneal injection of Dex was performed 15 minutes before modeling. Dex was injected intraperitoneally in the Dex group 15 minutes before modeling. The rats were sacrificed 1 hour after reperfusion, and myocardial tissue was obtained to measure the myocardial infarction area. The myocardial tissue pathologic changes were shown by hematoxylin and eosin (HE) staining, and the superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphate (ATP), and reactive oxygen species (ROS) content in serum was determined. Compared with the sham group, the myocardial infarction area was significantly increased (P<0.01), SOD and ATP content was significantly decreased (P<0.01), and MDA and ROS content was significantly increased (P<0.01) in the I/R group; this change was significantly reduced in the Dex, EA, and EA + Dex groups (P<0.01). The indicators in the EA + Dex group were better than those in the EA and Dex groups (P<0.05). There was no significant change in the above indices in the Dex group compared with the EA group (P>0.05). EA + Dex pretreatment improved the damage of myocardial I/R by increasing SOD and ATP content and reducing the generation of MDA and ROS in an oxygen-free radical system.