Propofol protects H9C2 cells against hypoxia/reoxygenation injury through miR-449a and NR4A2.
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ABSTRACT:
SUBMITTER: Qian Q
PROVIDER: S-EPMC8406901 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial's structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.Methods and resultsThe H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.ConclusionsDuring myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect.
| S-EPMC4854406 | biostudies-literature
Project description:Myocardial infarction (MI) is identified as the myocardial necrosis due to myocardial ischemia/reperfusion (I/R) injury and remains a leading cause of mortality. C1q/TNF-related protein 13 (CTRP13) is a member of CTRP family that has been found to be involved in coronary artery disease (CAD). However, the role of CTRP13 in MI remains unclear. We aimed to explore the functional role of CTRP13 in H9c2 cells exposed to hypoxia/reoxygenation (H/R). Our results demonstrated that H/R stimulation significantly decreased the expression of CTRP13 in H9c2 cells. H/R-induced an increase in ROS production and reductions in activities of SOD and CAT were prevented by CTRP13 overexpression but were aggravated by CTRP13 silencing. Moreover, CTRP13 overexpression could reverse the inductive effect of H/R on caspase-3 activity and bax expression, as well as the inhibitory effect of H/R on bcl-2 expression in H9c2 cells. However, CTRP13 silencing presented opposite effects with CTRP13 overexpression. Furthermore, CTRP13 overexpression enhanced the H/R-stimulated the expression levels of p-AMPK and nuclear Nrf2, and Nrf2 transcriptional activity. However, inhibition of AMPK reversed the CTRP13-mediated activation of Nrf2/ARE signaling and the cardiac-protective effect in H/R-exposed H9c2 cells. Additionally, silencing of Nrf2 reversed the protective effects of CTRP13 against H/R-stimulated oxidative stress and apoptosis in H9c2 cells. Finally, recombinant CTRP13 protein attenuated myocardial I/R-induced injury in rats. Taken together, these findings indicated that CTRP13 protected H9c2 cells from H/R-stimulated oxidative stress and apoptosis via regulating the AMPK/Nrf2/ARE signaling pathway. Our results provided evidence for the therapeutic potential of CTRP13 in myocardial I/R injury.
| S-EPMC8335840 | biostudies-literature
Project description:Trichosanthis Pericarpium (TP) is a traditional Chinese medicine for treating cardiovascular diseases. In this study, we investigated the effects of TP aqueous extract (TPAE) on hypoxia/reoxygenation (H/R) induced injury in H9c2 cardiomyocytes and explored the underlying mechanisms. H9c2 cells were cultured under the hypoxia condition induced by sodium hydrosulfite for 30 min and reoxygenated for 4 h. Cell viability was measured by MTT assay. The amounts of LDH, NO, eNOS, and iNOS were tested by ELISA kits. Apoptotic rate was detected by Annexin V-FITC/PI staining. QRT-PCR was performed to analyze the relative mRNA expression of Akt, Bcl-2, Bax, eNOS, and iNOS. Western blotting was used to detect the expression of key members in the PI3K/Akt pathway. Results showed that the pretreatment of TPAE remarkably enhanced cell viability and decreased apoptosis induced by H/R. Moreover, TPAE decreased the release of LDH and expression of iNOS. In addition, TPAE increased NO production and Bcl-2/Bax ratio. Furthermore, the mRNA and protein expression of p-Akt and eNOS were activated by TPAE pretreatment. On the contrary, a specific inhibitor of PI3K, LY294002 not only inhibited TPAE-induced p-Akt/eNOS upregulation but alleviated its anti-apoptotic effects. In conclusion, results indicated that TPAE protected against H/R injury in cardiomyocytes, which consequently activated the PI3K/Akt/NO signaling pathway.
| S-EPMC6222483 | biostudies-literature
Project description:Astragaloside IV (AS/IV) is one of the extracted components from the traditional Chinese medicine Astragalus which has been demonstrated to have potential capacity for anti-inflammation activity and for treating cardiovascular disease. Our purpose was to determine the function and underlying molecular mechanism of AS/IV in hypoxia/reoxygenation (H/R) injured in cardiomyocytes. Differentially expressed genes (DEGs) were screened using bioinformatic analysis, and the molecular targeting relationship was verified by the dual-luciferase report system. H/R injured cardiomyocytes were employed to explore the effect of AS/IV. QRT-PCR and Western blot analysis were applied to detect the expression of mRNA and proteins, respectively. Additionally, superoxide dismutase (SOD), lactic dehydrogenase (LDH) and MDA (malondialdehyde) levels were detected to determine the oxidative damage. Cell viability was assessed by CCK-8, and flow cytometry was used to evaluate cell apoptosis ratio. TGFBR1 and TLR2 were selected as DEGs. Additionally, AS/IV could enhance cell proliferation and upregulated miR-101a expression, which suppressed TGFBR1 and TLR2 expression in H/R injured cardiomyocytes. Moreover, the results of Western blot exhibited that the downstream genes (p-ERK and p-p38) in the MAPK signaling pathway were suppressed, which meant AS/IV could inhibit this pathway in H/R injured cardiomyocytes. Overall, this study demonstrated AS/IV could attenuate H/R injury in human cardiomyocytes via the miR-101a/TGFBR1/TLR2/MAPK signaling pathway axis, which means that it could serve as a possible alternate for H/R treatment.
| S-EPMC7272390 | biostudies-literature
Project description:Long noncoding RNAs (lncRNAs) participate in various biological processes and cardiovascular diseases. Recently, a novel lncRNA XR_596701 was found to be differentially expressed in obstructive sleep apnea (OSA)-induced myocardial tissue compared to normal myocardial tissues. However, the pathological effect and regulatory mechanism of XR_596701 in intermittent hypoxia (IH)-mediated cardiomyocytes damage have not been studied. The subcellular localization of XR_596701 was determined by fluorescence in situ hybridization (FISH). Gene expressions of XR_596701 and miR-344b-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in IH-induced H9c2 cells. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) staining assay. Cell apoptosis was detected by Hoechst 33342/PI staining and immunofluorescence (IF). Apoptotic protein of H9c2 cells was measured by western blot. The direct interaction between XR_596701 and miR-344b-5p as well as miR-344b-5p and Fas apoptotic inhibitory molecule 3 (FAIM3) were examined using dual-luciferase reporter assay. The significance of XR_596701 and miR-344b-5p on cell proliferation and apoptosis was evaluated by using gain-of-function and loss-of-function approaches. XR_596701 was upregulated, while miR-344b-5p downregulated in IH-induced H9c2 cells. Functionally, suppression of XR_596701 and overexpression of miR-344b-5p inhibited cell proliferation and promoted cell apoptosis in H9c2 cells. The roles of XR_596701 were achieved by sponging miR-344b-5p. And the function of miR-344b-5p was reversed by targeting FAIM3. Additionally, FAIM3 mediated IH-induced H9c2 cells damage by XR_596701. XR_596701 was serve as a novel lncRNA that indicated protective roles on proliferation and apoptosis of IH-induced H9c2 cells through the miR-344b-5p/FAIM3 axis.
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Project description:Tripartite motif (TRIM) 38 is a ubiquitin E3 protein ligase that is involved in various intracellular physiological processes. However, the role of TRIM38 in myocardial ischaemia/reperfusion (I/R) injury remains to be elucidated. We aimed to establish an in vitro cellular hypoxia/reperfusion (H/R) model to explore the role and potential mechanisms of TRIM38 in H9c2, a rat cardiomyoblast cell line. Recombinant adenoviruses for silencing or overexpressing TRIM38 were constructed and transfected into H9c2 cells. Western blotanalysisshowed that TRIM38 expression was significantly decreased after H/R injury. Functionally, TRIM38 expression relieved inflammatory responses and oxidative stress, and inhibited H/R-induced apoptosis in H9c2 cells. Mechanistically, TRIM38 overexpression inhibited H/R-induced transforming growth factor beta-activated kinase 1 (TAK1)/nuclear factor-kappa B (NF-κB) pathway activity in H9c2 cells. The opposite results were observed after TRIM38 knockdown. Furthermore, H/R-induced injury aggravated by TRIM38 deficiency in H9c2 cells was reversed upon treatment with 5Z-7-oxozeaenol, a TAK1 inhibitor. Therefore, TRIM38 reduction attenuated the anti-apoptotic capacity and anti-inflammatory potential of H/R-stimulated H9c2 cells by activating the TAK1/NF-κB signalling pathway. Specifically, TRIM38 alleviated H/R-induced H9c2 cell injury by promoting TNF receptor-associated factor 6 degradation, which led to the inactivation of the TAK1/NF-κB signalling pathway. Thus, our study provides new insights into the molecular mechanisms underlying H/R-induced myocardial injuries.
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Project description:The creatine kinase (CK) phosphagen system is fundamental to cellular energy homeostasis. Cardiomyocytes express three CK isoforms, namely the mitochondrial sarcomeric CKMT2 and the cytoplasmic CKM and CKB. We hypothesized that augmenting CK in vitro would preserve cell viability and function and sought to determine efficacy of the various isoforms. The open reading frame of each isoform was cloned into pcDNA3.1, followed by transfection and stable selection in human embryonic kidney cells (HEK293). CKMT2- CKM- and CKB-HEK293 cells had increased protein and total CK activity compared to non-transfected cells. Overexpressing any of the three CK isoforms reduced cell death in response to 18h hypoxia at 1% O2 followed by 2h re-oxygenation as assayed using propidium iodide: by 33% in CKMT2, 47% in CKM and 58% in CKB compared to non-transfected cells (P<0.05). Loading cells with creatine did not modify cell survival. Transient expression of CK isoforms in HL-1 cardiac cells elevated isoenzyme activity, but only CKMT2 over-expression protected against hypoxia (0.1% for 24h) and reoxygenation demonstrating 25% less cell death compared to non-transfected control (P<0.01). The same cells were not protected from doxorubicin toxicity (250nM for 48h), in contrast to the positive control. These findings support increased CK activity as protection against ischaemia-reperfusion injury, in particular, protection via CKMT2 in a cardiac-relevant cell line, which merits further investigation in vivo.
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Project description:N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.
| S-EPMC4694790 | biostudies-other
Project description:Dysregulation of long non‑coding RNA (IncRNA) antisense non‑coding RNA in the INK4 locus (ANRIL) is associated with the risk of myocardial infarction (MI). Therefore, the present study aimed to determine the mechanisms underlying this association, which is currently poorly understood, to the best of our knowledge. The current study used an in vitro myocardial ischemia and reperfusion (MI/R) model, in which H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R), which demonstrated that ANRIL expression was downregulated and that ANRIL positively regulated sirtuin 1 (SIRT1) expression following H/R injury. Subsequently, it was demonstrated that ANRIL upregulated SIRT1 expression by sponging microRNA‑181a (miR‑181a). In addition, ANRIL overexpression reduced lactate dehydrogenase release and apoptosis of H9c2 cardiomyocytes exposed to H/R, indicating that ANRIL prevented H/R‑induced cardiomyocyte injury. Moreover, both miR‑181a overexpression and SIRT1 knockdown significantly decreased the protective effects of ANRIL on H/R‑induced cardiomyocyte injury, thus demonstrating that SIRT1 upregulation via sponging miR‑181a is a critical mechanism that mediates the function of ANRIL. These results provided a novel mechanistic insight into the role of ANRIL in H/R‑injured cardiomyocytes and suggested that the ANRIL/miR‑181a/SIRT1 axis may be a therapeutic target for reducing MI/R injury.
| S-EPMC8185511 | biostudies-literature
Project description:Long noncoding RNAs have been widely accepted to play important roles in acute myocardial infarction (AMI). The dysregulation of cyclin-dependent kinase inhibitor 2B antisense RNA 1 (ANRIL) was discovered in AMI patients. Nevertheless, the detailed role and molecular mechanisms of ANRIL in AMI remain indistinct. The levels of ANRIL, miR-195-5p and Bcl-2 mRNA were determined by qRT-PCR. western blot was performed to assess the expression of Bcl-2, Bax, Cyclin D1 and p21. Cell proliferation was detected by CCK-8 assay, and cell apoptosis was measured by flow cytometry. The targeted correlation between ANRIL and miR-195-5p was confirmed by the dual-luciferase reporter and RNA pull-down assays. Our data revealed that ANRIL was downregulated and miR-195-5p was upregulated in the serum of AMI patients and hypoxia/reoxygenation (H/R)-induced myocardial cells. ANRIL upregulation or miR-195-5p knockdown alleviated H/R-induced myocardial cell injury. Moreover, ANRIL sequestered miR-195-5p by acting as a sponge of miR-195-5p. ANRIL upregulated Bcl-2 expression by sponging miR-195-5p. Additionally, ANRIL overexpression alleviated H/R-induced myocardial cell injury by upregulating Bcl-2. In conclusion, our study indicated that ANRIL upregulation alleviated H/R-induced myocardial cell injury partially through sponging miR-195-5p and upregulating Bcl-2, highlighting its role as a promising mediator for new therapies for AMI treatment.
| S-EPMC9097494 | biostudies-literature