[Mechanism of Guizhi Gancao Decoction against myocardial ischemia-reperfusion injury based on network pharmacology and experimental verification].

This study employed network pharmacology to investigate the effect of Guizhi Gancao Decoction(GGD) on myocardial ischemia-reperfusion injury(MI/RI) in rats and decipher the underlying mechanism. Firstly, the chemical components and targets of GGD against MI/RI were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), SwissTargetPrediction, and available articles. STRING and Cytoscape 3.7.2 were used to establish the protein-protein interaction(PPI) network for the common targets, and then Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were carried out for the core targets. The "drug-active component-target-pathway" network was built. Furthermore, molecular docking between key active components and targets was conducted in AutoDock Vina. Finally, the rat model of MI/RI was established, and the myocardial infarction area was measured. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were employed to detect cardiomyocyte pathology and ultrastructural changes. Western blot was employed to determine the expression of related proteins in the myocardial tissue. A total of 75 chemical components of GGD were screened out, corresponding to 318 targets. The PPI network revealed 46 core targets such as tumor protein p53(TP53), serine/threonine kinase 1(AKT1), signal transducer and activator of transcription 3(STAT3), non-receptor tyrosine kinase(SRC), mitogen-activated protein kinase 1(MAPK1), MAPK3, and tumor necrosis factor(TNF). According to GO and KEGG enrichment analyses, the core targets mainly affected the cell proliferation and migration, signal transduction, apoptosis, and transcription, involving advanced glycation end products-receptor(AGE-RAGE), MAPK and other signaling pathways in cancers and diabetes complications. The molecular docking results showed that the core components of GGD, such as licochalcone A,(+)-catechin, and cinnamaldehyde, had strong binding activities with the core target proteins, such as MAPK1 and MAPK3. The results of animal experiments showed that compared with the model group, GGD significantly increase superoxide dismutase, decreased malondialdehyde, lactate dehydrogenase, and creatine kinase-MB, and reduced the area of myocardial infarction. HE staining and TEM results showed that GGD pretreatment restored the structure of cardiomyocytes and alleviated the pathological changes and ultrastructural damage of mitochondria in the model group. In addition, GGD significantly down-regulated the phosphorylation of c-Jun N-terminal kinase and p38 and up-regulate that of extracellular regulated kinases 1/2 in the myocardial tissue. The results suggested that GGD may exert the anti-MI/RI effect by regulating the MAPK signaling pathway via the synergistic effects of Cinnamomi Ramulus and Glycyrrhizae Radix et Rhizoma.

[Chaihu Sanshen Capsules protect rats from myocardial ischemia reperfusion injury via PKCßâ ¡/NOX2/ROS signaling pathway].

This study aims to explore the pathogenesis of myocardial ischaemia reperfusion injury(MIRI) based on oxidative stress-mediated programmed cell death and the mechanism and targets of Chaihu Sanshen Capsules in treating MIRI via the protein kinase Cß(PKCßⅡ)/NADPH oxidase 2(NOX2)/reactive oxygen species(ROS) signaling pathway. The rat model of MIRI was established by the ligation of the left anterior descending branch. Rats were randomized into 6 groups: sham group, model group, clinically equivalent-, high-dose Chaihu Sanshen Capsules groups, N-acetylcysteine group, and CGP53353 group. After drug administration for 7 consecutive days, the area of myocardial infarction in each group was measured. The pathological morphology of the myocardial tissue was observed by hematoxylin-eosin(HE) staining. The apoptosis in the myocardial tissue was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). Enzyme-linked immunosorbent assay(ELISA) was employed to measure the le-vels of indicators of myocardial injury and oxidative stress. The level of ROS was detected by flow cytometry. The protein and mRNA levels of the related proteins in the myocardial tissue were determined by Western blot and real-time quantitative PCR(RT-qPCR), respectively. Compared with the sham group, the model group showed obvious myocardial infarction, myocardial structural disorders, interstitial edema and hemorrhage, presence of a large number of vacuoles, elevated levels of myocardial injury markers, myocardial apoptosis, ROS, and malondialdehyde(MDA), lowered superoxide dismutase(SOD) level, and up-regulated protein and mRNA le-vels of PKCßⅡ, NOX2, cysteinyl aspartate specific proteinase-3(caspase-3), and acyl-CoA synthetase long-chain family member 4(ACSL4) in the myocardial tissue. Compared with the model group, Chaihu Sanshen Capsules reduced the area of myocardial infarction, alleviated the pathological changes in the myocardial tissue, lowered the levels of myocardial injury and oxidative stress indicators and apoptosis, and down-regulated the mRNA and protein levels of PKCßⅡ, NOX2, caspase-3, and ACSL4 in the myocardial tissue. Chaihu Sanshen Capsules can inhibit oxidative stress and programmed cell death(apoptosis, ferroptosis) by regulating the PKCßⅡ/NOX2/ROS signaling pathway, thus mitigating myocardial ischemia reperfusion injury.

Efficacy of Danlou tablet on myocardial ischemia/ reperfusion injury assessed by network pharmacology and experimental verification.

OBJECTIVE: To investigate the potential pharmacological mechanism of Danlou tablet (, DLT) with a long-term clinical application in the treatment of myocardial ischemia/reperfusion (I/R) injury through network pharmacology, molecular docking and experimental verification. METHODS: The main chemical ingredients in DLT were retrieved from the Traditional Chinese Medicine (TCM) System Pharmacology Database, the TCM information database, the bioinformatics analysis tool for molecular mechanism of TCM, and HERB database. Disease targets of I/R were accessed from the databases of Online Mendelian Inheritance in Man, GeneCards, Therapeutic Target Database, and DisGeNET database. The overlaying genes of DLT and I/R were obtained from the Venny online platform. The core targets and protein-protein interaction network were constructed and analyzed via the Search Tool for the Retrieval of Interacting Genes Proteins database and Cytoscape software. Furthermore, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by the Metascape platform. Based on the results, the component-target-pathway network was constructed and drafted via the Cytoscape software and the platform of Bioinformatics. Furthermore, we performed molecular docking to predict the binding information between chemical molecules and target proteins. Finally, oxygen-glucose deprivation/recovery (OGD/R)-induced H9c2 cardiomyocytes were used to validate the results of network pharmacology in vitro. RESULTS: A total of 189 active chemical components in DLT and 849 correlative targets of I/R were screened. Of note, 133 overlaying genes found from the Venny online platform were concentrated into 28 core genes. Furthermore, the GO and KEGG pathway enrichment analysis presented that DLT might participate in 42 types of GO molecular functions, 747 types of GO biological processes, 19 types of GO cellular components, and 140 kinds of pathways to treat I/R. In the component-target-pathway network, the indirect relationship between herbs and their possible effective pathways was clarified. Based on the molecular docking, we speculated that Baicalein-prostaglandin G/H synthase 2 (PTGS2) with -3.24 kcal/mol, Luteolin-heat shock protein 90 alpha family class A member 1 (HSP90AA1) with -3.22 kcal/mol, Baicalein-HSP90AA1 with -3.13 kcal/mol, and Quercetin-HSP90AA1 with -3.05 kcal/mol possessed the strongest binding force of less than -3 kcal/mol, sequentially. Experimental verification showed that Quercetin, Luteolin, and Baicalein could increase the relative cell viability of OGD/R-stimulated cardiomyocytes, probably by suppressing PTGS2, and activating HSP90AA1 and estrogen receptor 1 expression. CONCLUSIONS: We predicted the potential active compounds as the material basis of DLT that may provide a new approach to elucidate the novel pharmacological mechanism underlying the treatment of cardiac I/R damage.

Effect of electroacupuncture on ventricular structure and function in rats with myocardial ischemia-reperfusion injury. / ç”µé’ˆå¯¹å¿ƒè‚Œç¼ºè¡€å†çŒæ³¨æŸä¼¤å¤§é¼ å¿ƒå®¤ç»“æž„å’ŒåŠŸèƒ½çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on changes of ventricular structure and function in rats with myocardial ischemia-reperfusion injury (MIRI), so as to explore its potential mechanisms underlying improvement of ventricular remodeling after MIRI. METHODS: Forty male SD rats were randomly divided into 4 groups：sham operation group, model group, EA group and medication (sacubactril valsartan, LCZ696) group, with 10 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery and reperfusion. EA (2 Hz/100 Hz, 2 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once every other day for 21 d. Rats of the medication group received gavage of LCZ696 (60 mg·kg-1·d-1). After the intervention, echocardiography was used to detect the ejection fraction (EF) and fractional shortening (FS) of the left ventricle, and the contents of serum tumor necrosis factor-α(TNF-α), vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1) were assayed by enzyme-linked immunosorbent assay. The pathological changes of myocardial tissue were observed after HE staining. The Masson staining was used to evaluate the myocardial collagen deposition and myocardial fibrosis. The mRNA expression levels of collagen Ⅰ and Ⅲ and connective tissue growth factor (CTGF) in the myocardial tissue were detected by quantitative real-time PCR, and the expression levels of IL-1ß and IL-18 were detected by Western blot. RESULTS: In contrast to the sham operation group, the EF and FS levels of the left ventricle were ob-viously decreased (P<0.001), while the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, the expression levels of IL-1ß and IL-18 were significantly increased (P<0.001, P<0.000 1, P<0.05, P<0.01) in the model group. Compared with the model group, the EF and FS levels were remarkably increased (P<0.01), whereas the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, and the expression levels of IL-1ß and IL-18 were significantly down-regulated (P<0.001, P<0.01, P<0.05) in both the medication and EA groups. No significant differences were found between the EA and medication groups in all the indexes mentioned above. CONCLUSIONS: EA can improve the left-ventricular fibrosis and function, delay or reverse ventricular remodeling in MIRI rats, which may be related to its functions in down-regulating myocardial inflammatory response and mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF.

[Effects of electroacupuncture pretreatment on GABAA receptor of fastigial nucleus and sympathetic nerve activity in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To observe the effects of electroacupuncture (EA) pretreatment on cardiac function, sympathetic nerve activity, indexes of myocardial injury and GABAA receptor in fastigial nucleus in rats with myocardial ischemia reperfusion injury (MIRI), and to explore the neuroregulatory mechanism of EA pretreatment in improving MIRI. METHODS: A total of 60 male SD rats were randomly divided into a sham operation group, a model group, an EA group, an agonist group and an agonist+EA group, 12 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery. EA was applied at bilateral "Shenmen" (HT 7) and "Tongli" (HT 5) in the EA group and the agonist+EA group, with continuous wave, in frequency of 2 Hz and intensity of 1 mA, 30 min each time, once a day for 7 consecutive days. After intervention, the MIRI model was established. In the agonist group, the muscone (agonist of GABAA receptor, 1 g/L) was injected in fastigial nucleus for 7 consecutive days before modeling, 150 µL each time, once a day. In the agonist+EA group, the muscone was injected in fastigial nucleus 30 min before EA intervention. The data of electrocardiogram was collected by PowerLab standard Ⅱ lead, and ST segment displacement and heart rate variability (HRV) were analyzed; the serum levels of norepinephrine (NE), creatine kinase isoenzyme MB (CK-MB) and cardiac troponin I (cTnI) were detected by ELISA; the myocardial infarction area was measured by TTC staining; the morphology of myocardial tissue was observed by HE staining; the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were detected by immunohistochemistry and real-time PCR. RESULTS: Compared with the sham operation group, in the model group, ST segment displacement and ratio of low frequency to high frequency (LF/HF) of HRV were increased (P<0.01), HRV frequency domain analysis showed enhanced sympathetic nerve excitability, the serum levels of NE, CK-MB and cTnI were increased (P<0.01), the percentage of myocardial infarction area was increased (P<0.01), myocardial fiber was broken and interstitial edema was serious, the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were increased (P<0.01). Compared with the model group, in the EA group, ST segment displacement and LF/HF ratio were decreased (P<0.01), HRV frequency domain analysis showed reduced sympathetic nerve excitability, the serum levels of NE, CK-MB and cTnI were decreased (P<0.01), the percentage of myocardial infarction area was decreased (P<0.01), myocardial fiber breakage and interstitial edema were lightened, the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were decreased (P<0.01). Compared with the EA group, in the agonist group and the agonist+EA group, ST segment displacement and LF/HF ratio were increased (P<0.01), HRV frequency domain analysis showed enhanced sympathetic nerve excitability, the serum levels of NE, CK-MB and cTnI were increased (P<0.01), the percentage of myocardial infarction area was increased (P<0.01), myocardial fiber breakage and interstitial edema were aggravated, the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were increased (P<0.01). CONCLUSION: EA pretreatment can improve the myocardial injury in MIRI rats, and its mechanism may be related to the inhibition of GABAA receptor expression in fastigial nucleus, thereby down-regulating the excitability of sympathetic nerve.

Dexmedetomidine Mitigates Myocardial Ischemia/Reperfusion-Induced Mitochondrial Apoptosis through Targeting lncRNA HCP5.

Our study aimed to explore the function and mechanism of Dexmedetomidine (Dex) in regulating myocardial ischemia/reperfusion (I/R)-induced mitochondrial apoptosis through lncRNA HCP5. We demonstrated Dex suppressed I/R-induced myocardial infarction and mitochondrial apoptosis in vivo. Dex induced the expression of lncRNA HCP5 and MCL1, inhibited miR-29a expression and activated the JAK2/STAT3 signaling. Dex attenuated hypoxia/reoxygenation (H/R)-induced mitochondrial apoptosis by upregulating lncRNA HCP5 in cardiomyocytes. Overexpression of lncRNA HCP5 sponged miR-29a to suppress H/R-induced mitochondrial apoptosis. Knockdown of miR-29a also alleviated cardiomyocyte apoptosis by upregulating MCL1. Overexpression of lncRNA HCP5 activated the JAK2/STAT3 signaling through sponging miR-29a and enhancing MCL1 expression in cardiomyocytes. Dex mitigated myocardial I/R-induced mitochondrial apoptosis through the lncRNA HCP5/miR-29a/MCL1 axis and activation of the JAK2/STAT3 signaling.

Lupeol Alleviates Myocardial Ischemia-Reperfusion Injury in Rats by Regulating NF-[Formula: see text]B and Nrf2 Pathways.

Cardiovascular disease is a global health problem. Previous studies revealed that it involves acute myocardial infarction and ischemia-reperfusion (I/R) injury. The mechanism of myocardial I/R injury is complex. But recognizing its mechanisms will bring important clinical significance. Lupeol is widely found in Chinese medicinal herbs and has been shown to have a variety of bio-activities. However, the pharmacological action of lupeol in the progress of myocardial ischemia-reperfusion injury (MIRI) is unclear. This study used a rat myocardial I/R model and the morphological changes in myocardium were determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The expression levels of IL-10, IL-1[Formula: see text], TNF-[Formula: see text], and IL-6 were assessed by quantitative real-time PCR (qRT-PCR) and ELISA. The expression levels of MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH) level and inflammatory cytokines were quantified using ELISA. The cellular apoptotic rate was determined by TUNEL staining. The findings showed that lupeol significantly decreased myocardial infarction after I/R and ameliorated I/R-induced myocardial inflammation, apoptosis, and oxidative stress. Furthermore, our results suggested that lupeol protected against MIRI-induced myocardial infarction through modulation of NF-[Formula: see text]B and Nrf2 signaling pathways. In summary, this study first clarified the cardioprotective effects of lupeol against I/R-induced myocardial infarction in rats, which could be due to its anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Our study also highlighted a mechanism of NF-[Formula: see text]B and Nrf2 signaling, through which lupeol could be a promising agent in protecting against I/R-induced myocardial infarction.

[Effect of electroacupuncture preconditioning on expression of Gasdermin D, Caspase-1 and IL-1ß in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture(EA) preconditioning on expression of Caspase-1, Gasdermin D(GSDMD) and interleukin-1ß(IL-1ß) in myocardial tissue of myocardial ischemia reperfusion injury (MIRI) rats in order to explore its underlying mechanisms in resisting MIRI. METHODS: Forty male rats were randomly divided into 4 groups: normal control (normal), sham operation (sham), MIRI model and EA groups. The MIRI model was established by ligation of the left anterior descending branch of the left coronary artery for 30 min and perfusion. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once a day for 3 consecutive days. The echocardiography was used to analyze the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and left ventricular ejection fraction (LVEF, by using Teichholz formula) 4 h after modeling. The myocardial TTC staining was used to observe the proportion of the infarct area, and Western blot was used to detect the expression levels of GSDMD, Caspase-1, IL-1ß proteins in the myocardium. RESULTS: Compared with the normal group, the immunoactivity of GSDMD was increased in the sham group (P<0.05). Compared with the sham group, the LVEF was significantly decreased (P<0.000 1), while the myocardial infarction area, immunoactivity of GSDMD, and the expression levels of Caspase-1, GSDMD and IL-1ß proteins were considerably increased in the model group (P<0.000 1, P<0.001). In comparison with the model group, the decreased ejection fraction and the increased myocardial infarction area, and Caspase-1, GSDMD and IL-1ß expression were reversed in the EA group (P<0.001, P<0.000 1, P<0.01). CONCLUSION: EA preconditioning may ameliorate myocardial injury in MIRI rats which may be associated with its function in down-regulating the expression of myocardial Caspase-1 protein to reduce cardiomyocyte pyroptosis.

Electroacupuncture preconditioning alleviates myocardial ischemia-reperfusion injury through the hypothalamic paraventricular nucleus- interposed nucleus nerve pathway.

OBJECTIVE: To explore whether the paraventricular nucleus (PVN) participates in regulation of the anti-myocardial ischemia-reperfusion injury (MIRI) effect of electroacupuncture (EA) and whether this is achieved through the PVN-interposed nucleus (IN) neural pathway. METHODS: The modeling method of myocardial ischemia reperfusion injury was achieved by ligating the left anterior descending coronary artery in Sprague-Dawley rats. We used the Powerlab multi-channel physiological recorder system to record electro-cardiograms and analyze the changes in ST segment displacement; 2,3,5-Triphenyltetrazolium chloride staining was used to observe the percentage of myocardial infarction areas. Detecting cardiac troponin I (cTnI), lactate dehydrogenase (LDH) in serum was done with an enzyme-linked immunosorbent assay kit. Morphological changes in the myocardium were detected in each group with hematoxylin-eosin staining of paraffin sections. Detection of c-fos protein expression in the PVN of the hypothalamus was done with the immune-ofluorescence method. The Plexon multi-channel acquisition system recorded PVN neuron discharges and local field potentials in each group of rats. Offline Sorter software was used for cluster analysis. Neuro Explorer software was used to perform autocorrelation, raster and frequency characteristics and spectral energy analysis of neuron signals in each group. RESULTS: Compared with the MIRI model group, the areas of myocardial infarction in the EA group were significantly reduced; the expression of cTnI, LDH in serum was decreased significantly. The firing frequency of pyramidal cells in the PVN was significantly increased and the spectrum energy map showed energy was reduced, c-fos expression in PVN was reduced, this indicated that neuronal activity in the PVN participates in the effect of EA improving myocardial injury. In addition, we used the kainic acid method to lesion the IN and observed that the effect of EA was weakened. For example, the area of myocardial infarction of lesion IN + EA group in rats was significantly increased compared with that resulting from EA group, the expression of cTnI, LDH in serum was significantly increased, the firing frequency of pyramidal cells in the PVN was significantly reduced. A spectral energy diagram shows that the energy after damage was higher than that of EA group. At the same time, the expression of c-fos in the PVN increased again. CONCLUSION: Our results indicated that the PVN-IN nerve pathway may participate as an effective pathway of EA to improve the effect of myocardial injury.

Electroacupuncture at PC6 (Neiguan) Attenuates Angina Pectoris in Rats with Myocardial Ischemia-Reperfusion Injury Through Regulating the Alternative Splicing of the Major Inhibitory Neurotransmitter Receptor GABRG2.

Angina pectoris is the most common manifestation of coronary heart disease, causing suffering in patients. Electroacupuncture at PC6 can effectively alleviate angina by regulating the expression of genes, whether the alternative splicing (AS) of genes is affected by acupuncture is still unknown. We established a rat model of myocardial ischemia-reperfusion by coronary artery ligation and confirmed electroacupuncture alleviated the abnormal discharge caused by angina pectoris measured in EMG electromyograms. Analysis of the GSE61840 dataset established that AS events were altered after I/R and regulated by electroacupuncture. I/R decreased the expression of splicing factor Nova1 while electroacupuncture rescued it. Further experiments in dorsal root ganglion cells showed Nova1 regulated the AS of the GABRG2, specifically on its exon 9 where an important phosphorylation site is present. In vivo, results also showed that electroacupuncture can restore AS of GABRG2. Our results proved that electroacupuncture alleviates angina results by regulating alternative splicing.

LncRNA SOX2-OTinhibitionprotects against myocardialischemia/reperfusion-inducedinjury via themicroRNA-186-5p (miR-186-5p)/Yin Yang 1 (YY1)pathway.

Long noncoding RNAs (lncRNAs) exert essential effects in regulating myocardial ischemia/reperfusion (MI/R)-induced injury. This work intended to explore the functions of lncRNA SOX2-OT and its regulatory mechanism within MI/R-induced injury. In this study, gene expression was determined by RT-qPCR. Western blotting was applied for the detection of protein levels. Pro-inflammatory cytokine concentrations, cardiomyocyte viability, and apoptosis were detected via ELISA, CCK-8 and flow cytometry. In the in vitro model, SOX2-OT and YY1 were both upregulated, while miR-186-5p was downregulated. SOX2-OT knockdown attenuated oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cardiomyocyte dysregulation through relieving inflammation, promoting proliferation, and reducing apoptosis in OGD/R-treated H2C9 cells. SOX2-OT positively regulated YY1 expression via miR-186-5p. Moreover, miR-186-5p inhibition or YY1 upregulation abolished the effects of SOX2-OT blocking on the inflammatory responses, proliferation, and apoptosis of OGD/R-challenged H2C9 cells. In conclusion, our results, for the first time, demonstrated that SOX2-OT inhibition attenuated MI/R injury in vitro via regulating the miR-186-5p/YY1 axis, offering potential therapeutic targets for MI/R injury treatment.

Majonoside-R2 Postconditioning Protects Cardiomyocytes Against Hypoxia/Reoxygenation Injury by Attenuating the Expression of HIF1α and Activating RISK Pathway.

Reoxygenation of hypoxic cardiac myocytes can paradoxically induce myocardial injury and affect the recovery processes. Pharmacological postconditioning is an efficient strategy used in clinical practice that protects cardiomyocytes from hypoxia/reoxygenation (HR) injury. Natural products or foods have been known to possess effective cardioprotective properties. Majonoside-R2 (MR2) is a dominant saponin component of Vietnamese ginseng that has several biological effects. In this study, we evaluated the protective effect of MR2 on HR-stimulated cardiomyocytes and investigated the related molecular mechanisms. H9C2 cardiomyocytes were exposed to HR conditions with or without MR2 supplementation. Samples from experimental groups were used to analyze the expression of apoptosis- and activating reperfusion injury salvage kinase (RISK)-related factors in response to HR injury by using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Post-treatment, MR2 enhanced cell viability under HR conditions. We found that MR2 suppressed the expression of hypoxia-inducible factor 1-alpha (HIF1α) and transforming growth factor beta 1 (TGFß1), modulated Akt/GSK3ß/cAMP response element-binding signaling, and regulated gene expression related to apoptosis (B cell lymphoma-extra-large [Bcl-xl], Bcl-2 homologous killer [Bak], Bcl-2 associated X [Bax], and connexin 43 [Cnx43]). Thus, the present findings demonstrate that MR2 protects cardiomyocytes against HR injury by suppressing the expression of HIF1α and activating the RISK pathway.

[Effect of electroacupuncture preconditioning on myocyte apoptosis in myocardial ischemia-reperfusion injury rats based on FXR/SHP pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on left-cardiac function, contents of serum TNF-α and IL-6 and expression of myocardial farnesoid X receptor(FXR), small heterodimer partner (SHP), apoptosis inducing factor (AIF) and heat shock proteins 70 (HSP70) genes in myocardial ischemia-reperfusion injury (MIRI) rats, so as to explore its mechanisms underlying improvement of ischemic myocardial injury. METHODS: Forty male Wistar rats were randomly divided into normal control, sham operation, MIRI model and EA pretreatment groups, with 10 rats in each group. Rats of the sham operation group received exposure of the thorax and heart. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery (LAD). EA (2 Hz/100 Hz and 1 mA) was applied to bilateral "Neiguan" (PC6), "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day for 7 days. The left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP)and maximal rates of rise and fall of left ventricular pressure (±dp/dtmax) were detected, the contents of serum TNF-α and IL-6 were detected by using enzyme-linked immunosorbent assay (ELISA), and the expression of FXR, SHP, AIF and HSP70 apoptotic genes in the myocardial tissue were measured by fluorescent quantitative RT-PCR. RESULTS: Compared with the normal control group, the LVEDP, contents of serum TNF-α and IL-6, and the expression levels of myocardial FXR, SHP, AIF and HSP70 mRNAs were significantly increased (P<0.05), while LVSP and ±dp/dtmax levels were obviously decreased in the model group (P<0.05). In comparison with the model group, MIRI-induced increases of LVEDP, TNF-α and IL-6 contents, and FXR, SHP and AIF mRNA expression and decreases of ±dp/dtmax and LVSP levels were reversed(P<0.05), except HSP70 mRNA expression with significantly increased (P<0.05) in the EA pretreatment group. CONCLUSION: EA pretreatment can protect the left ventricular function of the ischemic heart in MIRI rats, which may be related to its effects in reliving peripheral inflammation and regulating the expression levels of apoptosis-related factors FXR, SHP, AIF and HSP70 in the myocardium.

[Relationship between programmed cell death and myocardial ischemia-reperfusion injury and new perspectives of traditional Chinese medicine intervention].

Myocardial ischemia-reperfusion injury(MIRI) is an urgent problem in clinical treatment. As cardiomyocytes are terminal cells, MIRI-induced cardiomyocyte death will irreversibly damage the structure and function of the heart. In previous studies, apoptosis was considered to be the only way to regulate cell death, while necrosis could not be regulated. However, current studies have shown that cell necrosis could also be regulated, which was collectively called programmed cell death(PCD). Regulated cell death is actively mediated through molecular pathways, so there is the possibility of inhibiting this signaling to reduce MIRI. At present, PCD mainly includes apoptosis, autophagy, necrosis, pyroptosis and ferroptosis. As a unique treature in China, traditional Chinese medicine has the advantages of multiple pathways, multiple targets, low toxicity, less side effects and low economic costs. With the in-depth study of the efficacy of traditional Chinese medicine against MIRI, it has been confirmed that traditional Chinese medicine could regulate PCD to reduce MIRI. Therefore, this paper focuses on the relationship between PCD and MIRI, and new studies on intervention with relevant traditional Chinese medicine, with the aim to provide new MIRI prevention and treatment methods from the perspective of "intervention of PCD".

Cryptotanshinone Attenuates Ischemia/Reperfusion-induced Apoptosis in Myocardium by Upregulating MAPK3.

ABSTRACT: Chinese people have used the root of Salvia miltiorrhiza Bunge (called "Danshen" in Chinese) for centuries as an anticancer agent, anti-inflammatory agent, antioxidant, and cardiovascular disease drug. In addition, Danshen is considered to be a drug that can improve ischemia/reperfusion (I/R)-induced myocardium injury in traditional Chinese medicine. However, Danshen is a mixture that includes various bioactive substances. In this study, we aimed to identify the protective component and mechanism of Danshen on myocardium through network pharmacology and molecular simulation methods. First, cryptotanshinone (CTS) was identified as a potential active compound from Danshen that was associated with apoptosis by a network pharmacology approach. Subsequently, biological experiments validated that CTS inhibited ischemia/reperfusion-induced cardiomyocyte apoptosis in vivo and in vitro. Molecular docking techniques were used to screen key target information. Based on the simulative results, MAPKs were verified as well-connected molecules of CTS. Western blotting assays also demonstrated that CTS could enhance MAPK expression. Furthermore, we demonstrated that inhibition of the MAPK pathway reversed the CTS-mediated effect on cardiomyocyte apoptosis. Altogether, our work screened out CTS from Danshen and demonstrated that it protected cardiomyocytes from apoptosis.

Protocatechualdehyde protects oxygen-glucose deprivation/reoxygenation-induced myocardial injury via inhibiting PERK/ATF6α/IRE1α pathway.

Endoplasmic reticulum (ER) stress has been considered as a promising strategy in developing novel therapeutic agents for cardiovascular diseases through inhibiting cardiomyocyte apoptosis. Protocatechualdehyde (PCA) is a natural phenolic compound from medicinal plant Salvia miltiorrhiza with cardiomyocyte protection. However, the potential mechanism of PCA on cardiovascular ischemic injury is largely unexplored. Here, we found that PCA exerted markedly anti-apoptotic effect in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9c2 cells (Rat embryonic ventricular H9c2 cardiomyocytes), which was detected by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH), Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) assays. PCA also obviously protected cardiomyocytes in myocardial fibrosis model of mice, which was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. Transcriptomics coupled with bioinformatics analysis revealed a complex pharmacological signaling network especially for PCA-mediated ER stress on cardiomyocytes. Further mechanism study suggested that PCA suppressed ER stress via inhibiting protein kinase R-like ER kinase (PERK), inositol-requiring enzyme1α (IRE1α), and transcription factor 6α (ATF6α) signaling pathway through Western blot, DIOC6 and ER-Tracker Red staining, leading to a protective effect against ER stress-mediated cardiomyocyte apoptosis. Taken together, our observations suggest that PCA is a major component from Salvia miltiorrhiza against cardiovascular ischemic injury by suppressing ER stress-associated PERK, IRE1α and ATF6α signaling pathways.

Relationship between programmed cell death and myocardial ischemia-reperfusion injury and new perspectives of traditional Chinese medicine intervention / 中国中药杂志

Myocardial ischemia-reperfusion injury(MIRI) is an urgent problem in clinical treatment. As cardiomyocytes are terminal cells, MIRI-induced cardiomyocyte death will irreversibly damage the structure and function of the heart. In previous studies, apoptosis was considered to be the only way to regulate cell death, while necrosis could not be regulated. However, current studies have shown that cell necrosis could also be regulated, which was collectively called programmed cell death(PCD). Regulated cell death is actively mediated through molecular pathways, so there is the possibility of inhibiting this signaling to reduce MIRI. At present, PCD mainly includes apoptosis, autophagy, necrosis, pyroptosis and ferroptosis. As a unique treature in China, traditional Chinese medicine has the advantages of multiple pathways, multiple targets, low toxicity, less side effects and low economic costs. With the in-depth study of the efficacy of traditional Chinese medicine against MIRI, it has been confirmed that traditional Chinese medicine could regulate PCD to reduce MIRI. Therefore, this paper focuses on the relationship between PCD and MIRI, and new studies on intervention with relevant traditional Chinese medicine, with the aim to provide new MIRI prevention and treatment methods from the perspective of "intervention of PCD".

ATP-Sensitive Potassium Channels Mediate the Cardioprotective Effect of Panax notoginseng Saponins against Myocardial Ischaemia-Reperfusion Injury and Inflammatory Reaction.

Inflammatory response during myocardial ischemia reperfusion injury (MIRI) is essential for cardiac healing, while excessive inflammation extends the infarction and promotes adverse cardiac remodeling. Understanding the mechanism of these uncontrolled inflammatory processes has a significant impact during the MIRI therapy. Here, we found a critical role of ATP-sensitive potassium channels (KATP) in the inflammatory response of MIRI and its potential mechanism and explored the effects of Panax Notoginseng Saponins (PNS) during this possess. Rats underwent 40 min ischemia by occlusion of the left anterior descending (LAD) coronary artery and 60 min of reperfusion. PNS was treated at the corresponding time point before operation; 5-hydroxydecanoate (5-HD) and glybenclamide (Gly) (or Nicorandil (Nic)) were used as pharmacological blocker (or nonselective opener) of KATP. Cardiac function and pathomorphology were evaluated and a set of molecular signaling experiments was tested. KATP current density was measured by patch-clamp. Results revealed that in MIRI, PNS pretreatment restored cardiac function, reduced infarct size, and ameliorated inflammation through KATP. However, inhibiting KATP by 5-HD and Gly significantly reversed the effects, including NLRP3 inflammasome and inflammatory mediators IL-6, MPO, TNF-α, and MCP-1. Moreover, PNS inhibited the phosphorylation and nuclear translocation of NF-κB in I/R myocardium when the KATP was activated. Importantly, PNS promoted the expression of subunits and activation of KATP. The study uncovered KATP served as a new potential mechanism during PNS modulating MIRI-induced inflammation and promoting injured heart recovery. The manipulation of KATP could be a potential therapeutic approach for MIRI and other inflammatory diseases.

[Effect of electroacupuncture preconditioning on the expressions of NF-κB p65, IκBα and IKKß in myocardial tissue of the rats with acute myocardial ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on the expressions of nuclear transcription factors-kappa B (NF-κB) p65, NF-κB inhibitor (IκB) α and IκB kinase (IKK) ß in rats with acute myocardial ischemia-reperfusion injury (MIRI) and to explore the mechanism of EA on heart meridian in relieving MIRI. METHODS: A total of 40 SD rats were randomized into a sham-operation group, a model group, an EA heart meridian group and an EA lung meridian group, 10 rats in each one. In the EA heart meridian group, acupuncture was applied to "Shenmen" (HT 7) and "Tongli" (HT 5). In the EA lung meridian group, acupuncture was applied to "Taiyuan" (LU 9) and "Lieque" (LU 7). In these two groups, EA was exerted for 20 min each time, 1 V in voltage and 2 Hz in frequency once a day. A total of 7-day EA stimulation was required before model duplication. In the model group, the EA heart meridian group and the EA lung meridian group, using ligating left anterior descending coronary artery to establish the acute MIRI models. In the sham-operation group, the chest was open, but no ligation was exerted, just the needle was penetrated through the corresponding sites for one time. The electrocardiogram (ECG) was detected and ST segment displacement was analyzed. Using Western blot method, the relative expressions of NF-κB p65, IκBα and IKKß in myocardial tissue were determined in each group. Using ELISA method, the levels of serum IL-1ß and IL-10 were determined in each group. RESULTS: Compared with the sham-operation group, ST segment displacement value was elevated 30 min after ligating and reperfusion for 120 min in the model group (P<0.05), and the value in the EA heart meridian group was lower than the model group and the EA lung meridian group (P<0.05). Compared with the sham-operation group, the expressions of NF-κB p65 and IKKß in myocardial tissue were increased (P<0.05) and the expression of IκBα reduced in the rats of the model group (P<0.05). Compared with the model group, the expressions of NF-κB p65 and IKKß in myocardial tissue were reduced (P<0.05) and the expressions of IκBα increased in the rats of the EA heart meridian group and the EA lung meridian group (P<0.05). Compared with the EA lung meridian group, the expressions of NF-κB p65 and IKKß in myocardial tissue were reduced (P<0.05) and the expression of IκBα increased in the rats of the EA heart meridian group (P<0.05). Compared with the sham-operation group, the serum level of IL-1ß was increased (P<0.05) and IL-10 reduced in the model group (P<0.05). Compared with the model group, the serum level of IL-1ß was reduced (P<0.05) and IL-10 increased in the EA heart meridian group and the level of IL-1ß was was reduced in the EA lung meridian group (P<0.05). Compared with the EA lung meridian group, the serum level of IL-1ß was reduced (P<0.05) and IL-10 increased in the EA heart meridian group (P<0.05). CONCLUSION: Electroacupuncture preconditioning at heart meridian acupoints obviously alleviates acute MIRI. IKK/IκB/NF-κB signaling pathway possibly participates in the protective mechanism of electroacupuncture preconditioning on acute MIRI.

Light Emitting Diode Therapy Protects against Myocardial Ischemia/Reperfusion Injury through Mitigating Neuroinflammation.

BACKGROUND: Neuroinflammation plays a key role in myocardial ischemia-reperfusion (I/R) injury. Previous studies showed that light-emitting diode (LED) therapy might improve M2 microglia activation and brain-derived neurotrophic factor (BDNF) expression, thereby exerting anti-inflammatory effects. Therefore, we hypothesized that LED therapy might reduce myocardial I/R injury by neuroinflammation modulation. OBJECTIVE: To explore the effect of LED therapy on myocardial I/R-induced injury and seek the underlying mechanism. METHODS: Thirty rats were randomly divided into three groups: Control group (without LED treatment or myocardial I/R, n = 6), I/R group (with myocardial I/R only, n = 12), and LED+I/R group (with myocardial I/R and LED therapy, n = 12). Electrocardiogram was recorded continuously during the procedure. In addition, brain tissue was extracted for BDNF, Iba1, and CD206 analyses, and heart tissue for myocardial injury (ischemic size and infarct size), IL-4 and IL-10 mRNA analysis. RESULTS: In comparison with the I/R group, the ischemia size and the infarct size were significantly attenuated by LED therapy in the LED+I/R group. Meanwhile, the microglia activation induced by I/R injury was prominently attenuated by LED treatment either. And it is apparent that there was also an increase in the beneficial neuroinflammation markers (BDNF and CD206) in the paraventricular nucleus (PVN) in the LED+I/R group. Furthermore, the anti-inflammatory cytokines, IL-4 and IL-10, were greatly decreased by I/R while improved by LED treatment in myocardium. CONCLUSION: LED therapy might reduce neuroinflammation in PVN and decrease myocardium injury by elevating BDNF and M2 microglia.