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Background: Pathological cardiac hypertrophy can lead to heart failure and is one of the leading causes of death globally. Understanding the molecular mechanism of pathological cardiac hypertrophy will contribute to the treatment of heart failure. DUBs (deubiquitinating enzymes) are essential to cardiac pathophysiology by precisely controlling protein function, localization, and degradation. This study set out to investigate the role and molecular mechanism of a DUB, USP25 (ubiquitin-specific peptidase 25), in pathological cardiac hypertrophy. Methods: The role of USP25 in myocardial hypertrophy was evaluated in murine cardiomyocytes in response to Ang II (angiotensin II) and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of heart failure patients. Liquid chromotography with mass spectrometry/mass spectrometry analysis combined with Co-IP was used to identify SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A), an antihypertrophy protein, as an interacting protein of USP25. To clarify the molecular mechanism of USP25 in the regulation of SERCA2a, we constructed a series of mutant plasmids of USP25. In addition, we overexpressed USP25 and SERCA2a in the heart with adenoassociated virus serotype 9 vectors to validate the biological function of USP25 and SERCA2a interaction. Results: We revealed increased protein level of USP25 in murine cardiomyocytes subject to Ang II and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of patients with heart failure. USP25 deficiency aggravated cardiac hypertrophy and cardiac dysfunction under Ang II and transverse aortic constriction treatment. Mechanistically, USP25 bound to SERCA2a directly via its USP (ubiquitin-specific protease) domain and cysteine at position 178 of USP25 exerts deubiquitination to maintain the stability of the SERCA2a protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby maintaining calcium handling in cardiomyocytes. Moreover, restoration of USP25 expression via adenoassociated virus serotype 9 vectors in USP25 −/− mice attenuated Ang II-induced cardiac hypertrophy and cardiac dysfunction, whereas myocardial overexpression of SERCA2a could mimic the effect of USP25. Conclusions: We confirmed that USP25 inhibited cardiac hypertrophy by deubiquitinating and stabilizing SERCA2a.

Myocardial injury and cardiovascular dysfunction are serious consequences of sepsis and contribute to high mortality. Currently, the pathogenesis of myocardial injury in sepsis is still unclear, and therapeutic approaches are limited. In this study, we investigated the protective effect of emodin on septic myocardial injury and the underlying mechanism. Lipopolysaccharide (LPS)-induced C57BL/6 mice and cardiomyocytes were used as models of sepsis in vivo and in vitro, respectively. The results showed that emodin alleviated cardiac dysfunction, myocardial injury and improved survival rate in LPS-induced septic mice. Emodin attenuated the levels of inflammatory cytokines and cardiac inflammation induced by LPS. Emodin reduced NOD-like receptor protein 3 (NLRP3) and Gasdermin D (GSDMD) expression in the heart tissue of LPS-induced septic mice. In vitro, emodin alleviated LPS-induced cell injury and inflammation in cardiomyocytes by inhibiting NLRP3 inflammasome activation. In addition, an NLRP3 inhibitor was used to further confirm the function of the NLRP3 inflammasome in LPS-induced myocardial injury. Taken together, our findings suggest that emodin improves LPS-induced myocardial injury and cardiac dysfunction by alleviating the inflammatory response and cardiomyocyte pyroptosis by inhibiting NLRP3 inflammasome activation, which provides a feasible strategy for preventing and treating myocardial injury in sepsis.

Obesity-induced cardiomyopathy (OIC) is an increasingly serious global disease caused by obesity. Chronic inflammation greatly contributes to the pathogenesis of OIC. This study aimed to explore the role and mechanism of tabersonine (Tab), a natural alkaloid with antiinflammatory activity, in the treatment of OIC. High fat diet (HFD)-induced obese mice were administered with Tab. The results showed that Tab significantly inhibit inflammation, myocardial fibrosis, and hypertrophy to prevent heart dysfunction, without the alteration of body weight and hyperlipidemia, in HFD-induced obese mice. H9c2 cells and primary cardiomyocytes stimulated by palmitic acid (PA) were used to explore the molecular mechanism and target of Tab. We examined the effect of Tab on key proteins involved in HFD/PA-induced inflammatory signaling pathway and found that Tab significantly inhibits TAK1 phosphorylation in cardiomyocytes. We further detected the direct interaction between Tab and TAK1 at the cellular, animal, and molecular levels. We found that Tab directly binds to TAK1 to inhibit TAK1 phosphorylation, which then blocks TAK1-TAB2 interaction and then NF-κB pro-inflammatory pathway in cultured cardiomyocytes. Our results indicate that Tab is a potential agent for the treatment of OIC, and TAK1 is an effective therapeutic target for this disease.

Renal fibrosis is a crucial pathological feature of hypertensive renal disease (HRD). In-depth analysis of the pathogenesis of fibrosis is of great significance for the development of new drugs for the treatment of HRD. USP25 is a deubiquitinase that can regulate the progression of many diseases, but its function in the kidney remains unclear. We found that USP25 was significantly increased in human and mouse HRD kidney tissues. In the HRD model induced by Ang II, USP25−/− mice showed significant aggravation of renal dysfunction and fibrosis compared with the control mice. Consistently, AAV9-mediated overexpression of USP25 significantly improved renal dysfunction and fibrosis. Mechanistically, USP25 inhibited the TGF-β pathway by reducing SMAD4 k63-linked polyubiquitination, thereby suppressing SMAD2 nuclear translocation. In conclusion, this study demonstrates for the first time that the deubiquitinase USP25 plays an important regulatory role in HRD.

Schisandra is a well-known traditional Chinese medicine in East Asia. As a traditional Chinese medicine derivative with Schisandra chinensis as raw material, bicyclol is well known for its significant anti-inflammatory effect. Chronic inflammation plays a significant part in obesity-induced cardiomyopathy. Our purpose was to explore the effect and mechanism of bicyclol on obesity-induced cardiomyopathy.Mice fed with a high-fat diet (HFD) and cardiomyocytes stimulated by palmitic acid (PA) were used as models of obesity-related cardiomyopathy in vivo and in vitro, respectively. The therapeutic effect of bicyclol on pathological changes such as myocardial hypertrophy and fibrosis was evaluated by staining cardiac tissue sections. PCR was used to detect inflammatory factors in H9c2 cells and animal heart tissue after bicyclol treatment. Then, we used western blotting to detect the expression levels of the myocardial hypertrophy related protein, myocardial fibrosis related protein, NF-κB and MAPK pathways.Our results indicated that bicyclol treatment significantly alleviates HFD-induced myocardial inflammation, fibrosis, and hypertrophy by inhibiting the MAPK and NF-κB pathways. Similar to animal level results, bicyclol could significantly inhibit PA-induced inflammation and prevent NF-κB and MAPK pathways from being activated.Our results showed that bicyclol has potential as a drug to treat obesity-induced cardiomyopathy.

Chronic and persistent obesity can lead to various complications, including obesity cardiomyopathy. Inhibition of the inflammatory response is an effective measure for the intervention of obesity cardiomyopathy. Numerous studies indicate that costunolide (Cos) can reduce inflammation. However, the role of Cos in obesity cardiomyopathy and its molecular targets remains unknown.We aimed to clarify potential cardioprotective effects and mechanism of Cos against obesity cardiomyopathy.The model of obesity cardiomyopathy was established by feeding mice with a high-fat diet for 24 weeks. Cos at 10 and 20 mg/kg or vehicle (1% CMCNa solution) was administered once every two days via oral gavage from the 17th to 24th week. Body weight, heart weight/tibia length, cardiac function, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers, inflammatory factors were assessed. The targets of Cos were predicted through molecular docking. Pull-down assay and biolayer interferometry were used to confirm the target of Cos.Cos effectively reduces obesity-induced cardiomyocyte inflammation, cardiac hypertrophy and fibrosis, thereby improving cardiac function. We confirmed that Cos can interact with TAK1 and inhibit downstream NF-κB pathway activation by blocking the formation of the TAK1/TAB2 complex, thus inhibiting inflammatory cytokine release in cardiomyocytes.Our results demonstrated that Cos significantly improved myocardial remodeling and cardiac dysfunction against obesity cardiomyopathy by reducing myocardial inflammation. Therefore, Cos may serve as a promising therapeutic agent in obesity cardiomyopathy.

Background Multipotent mesenchymal stromal cells (MSCs) have recently been reported to promote vasculogenesis by differentiating into endothelial cells and releasing numerous cytokines and paracrine factors. However, due to low cell activity, their potential for clinical application is not very satisfactory. This study aimed to explore the effects and mechanisms of 1,25-dihydroxyvitamin D (1,25(OH)2D3) on the vasculogenesis of MSCs. Methods MSCs were isolated from the femurs and tibias of rats and characterized by flow cytometry. After treatment with different concentrations of 1,25(OH)2D3 (0 µM, 0.1 µM and 1 µM), the proliferation of MSCs was analyzed by Cell Counting Kit-8 (CCK-8), and the migratory capability was measured by Transwell assays and cell scratch tests. Capillary-like structure formation was observed by using Matrigel. Western blotting was used to detect the expression of FLK-1 and vWF to investigate the differentiation of MSCs into endothelial cells. Western blotting and gelatin zymography were used to detect the expression and activities of VEGF, MMP-2 and MMP-9 secreted by MSCs under the influence of 1,25(OH)2D3. Finally, the VDR antagonist pyridoxal-5-phosphate (P5P) and the PI3K/AKT pathway inhibitor LY294002 were utilized to test the phosphorylation levels of key kinases in the PI3K/AKT pathway by Western blotting and the formation of capillary-like structures in Matrigel. Results The proliferation and migratory capability of MSCs and the ability of MSCs to form a tube-like structure in Matrigel were enhanced after treatment with 1,25(OH)2D3. Moreover, MSCs treated with 1,25(OH)2D3 showed high expression of vWF and Flk-1. There was a significant increase in the expression of VEGF, MMP-2 and MMP-9 secreted by MSCs treated with 1,25(OH)2D3, as well as in the activity of MMP-2 and MMP-9. The phosphorylation level of AKT increased with time after 1,25(OH)2D3 treatment, while LY294002 weakened AKT phosphorylation. In addition, the ability to form capillary-like structures was reduced when the VDR and PI3K/AKT pathways were blocked. Conclusion This study confirmed that 1,25(OH)2D3 treatment can strengthen the ability of MSCs to promote vasculogenesis in vitro, and the mechanism may be related to the activation of the PI3K/AKT pathway.

Coronary heart disease can be effectively prevented by alleviating atherosclerotic plaque progression. Ox-LDL-induced inflammatory response in macrophages is a critical factor in the pathophysiology of atherosclerosis. It is well known that circular RNAs (circRNAs) are associated with the progression of several human diseases, such as coronary artery diseases, by sponging microRNAs (miRNAs), but the function and hidden mechanisms of circRNAs in macrophage inflammation and lipid metabolism remain unclear. In our study, we established an ox-LDL-stimulated macrophage model and used microarray to detect circRNA expression in macrophages. The results revealed distinct profiles of circRNA expression across the ox-LDL-stimulated macrophage group and the control group. Among them, hsa_circ_0007478 was upregulated in ox-LDL-stimulated macrophages, accompanied by reduced miR-765 and increased EFNA3 expression. Activation of NLRP3 inflammasome and IL-1β in macrophages was decreased following silencing of hsa_circ_0007478 or transfection of miR-765 mimics. In addition, we demonstrated that as a direct target gene of miR-765, the expression of EFNA3 regulated NLRP3 inflammasome and IL-1β levels in macrophages. Besides, hsa_circ_0007478 promoted EFNA3 expression by acting as a miR-765 sponge. We further showed that hsa_circ_0007478/miR-765/EFNA3 axis could also be involved in the inhibition of the lipid metabolism and foam cells formation in ox-LDL-macrophages. Taken together, these findings suggest that Hsa_circ_0007478 may be a potential molecular target against the inflammatory response and foam cells during atherosclerosis.

Doxorubicin (Dox), as a widely used anthracycline antitumor drug, can cause severe cardiotoxicity. Cardiomyocyte death and inflammation are involved in the pathophysiology of Dox-induced cardiotoxicity (DIC). Gasdermin D (GSDMD) is known as a key executioner of pyroptosis, which is a pro-inflammatory programmed cell death. We aimed to investigate the impact of GSDMD on DIC and systematically reveal its underlying mechanisms. Our findings indicated that Dox induced cardiomyocyte pyroptosis in a GSDMD-dependent manner by utilizing siRNA or overexpression-plasmid technique. We then generated GSDMD global knockout mice via CRISPR/Cas9 system and found that GSDMD deficiency reduced Dox-induced cardiomyopathy. Dox induced the activation of inflammatory caspases, which subsequently mediated GSDMD-N generation indirectly. Using molecular dynamics simulation and cell-free systems, we confirmed that Dox directly bound to GSDMD and facilitated GSDMD-N-mediated pyroptosis. Furthermore, GSDMD also mediated Dox-induced mitochondrial damage via Bnip3 and mitochondrial perforation in cardiomyocytes. These findings provide fresh insights into the mechanism of how Dox-engaged GSDMD orchestrates adverse cardiotoxicity and highlight the prospects of GSDMD as a potential target for DIC.

Myocardial remodeling is one of the main lesions in the late stage of chronic heart failure and seriously affects the prognosis of patients. Continuous activation of the renin-angiotensin-aldosterone system (RAAS) contributes to the development of myocardial remodeling greatly, and angiotensin II (Ang II), its main constituent, can directly lead to cardiac remodeling through an inflammatory response and oxidative stress. Since Ang II-induced myocardial remodeling is closely related to inflammation, we tried to explore whether the anti-inflammatory drug oridonin (Ori) can reverse this process and its possible mechanism. Our study investigated that hypertrophy and fibrosis can be induced after being treated with Ang II in cardiomyocytes (H9c2 cells and primary rat cardiomyocytes) and C57BL/6J mice. The anti-inflammatory drug oridonin could effectively attenuate the degree of cardiac remodeling both in vivo and vitro by inhibiting GSDMD, a key protein of intracellular inflammation which can further activate kinds of inflammation factors such as IL-1β and IL-18. We illustrated that oridonin reversed cardiac remodeling by inhibiting the process of inflammatory signaling through GSDMD. After inhibiting the expression of GSDMD in cardiomyocytes by siRNA, it was found that Ang II-induced hypertrophy was attenuated. These results suggest that oridonin is proved to be a potential protective drug against GSDMD-mediated inflammation and myocardial remodeling.

Pyroptosis serves a crucial function in various types of ischemia and reperfusion injuries. Oridonin, a tetracycline diterpene derived from Rabdosia rubescens, can significantly inhibit the aggregation of NLRP3-mediated inflammasome. This experiment is aimed at investigating the effect of oridonin on pyroptosis in mice cardiomyocytes. Based on the models of myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R), Evans Blue/TTC double staining, TUNEL staining, and Western blotting were applied to determine the effects of oridonin on myocardial damage, cellular activity and signaling pathways involved in pyroptosis. During I/R and H/R treatments, the extent of gasdermin D-N domains was upregulated in cardiomyocytes. Apart from that, oridonin improved cell survival in vitro and decreased the myocardial infarct size in vivo by also downregulating the activation of pyroptosis. Finally, the expression levels of ASC, NLRP3 and p-p65 were markedly upregulated in cardiomyocytes after H/R treatment, whereas oridonin suppressed the expression of these proteins. The present experiment revealed that myocardial I/R injury and pyroptosis can be alleviated and inhibited by oridonin pretreatment via NF-κB/NLRP3 signaling pathway, both in vivo and in vitro. Therefore, oridonin may serve as a potentially novel agent for the clinical treatment of myocardial ischemia-reperfusion injuries.

Background Emodin has recently been reported to have a powerful antiinflammatory effect, protecting the myocardium against ischemia/reperfusion (I/R) injury. Pyroptosis is a proinflammatory programmed cell death that is related to many diseases. The present study investigated the effect of emodin on pyroptosis in cardiomyocytes. Materials and methods Sprague Dawley rats were randomly divided into sham, I/R, and I/R+Emodin groups. I/R model was subjected to 30 minutes' ligation of left anterior descending coronary artery, followed by 2 hours of reperfusion. Cardiomyocytes were exposed to hypoxic conditions for 1 hour and normoxic conditions for 2 hours. The level of the pyroptosis was detected by Western blot, real-time PCR analysis, and ELISA. Results The level of gasdermin D-N domains was upregulated in cardiomyocytes during I/R or hypoxia/reoxygenation (H/R) treatment. Moreover, emodin increased the rate of cell survival in vitro and decreased the myocardial infarct size in vivo via suppressing the levels of I/R-induced pyroptosis. Additionally, the expression of TLR4, MyD88, phospho-IκBα, phospho-NF-κB, and the NLRP3 inflammasome was significantly upregulated in cardiomyocytes subjected to H/R treatment, while emodin suppressed the expression of these proteins. Conclusion This study confirms that emodin treatment was able to alleviate myocardial I/R injury and inhibit pyroptosis in vivo and in vitro. The inhibitory effect of emodin on pyroptosis was mediated by suppressing the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, emodin may provide an alternative treatment for myocardial I/R injury.

Trimetazidine (TMZ) exerts a strong inhibitory effect on ischemia/reperfusion (I/R) injury. Inflammation plays a key role in I/R injury. We hypothesized that TMZ may protect cardiomyocytes from I/R injury by inhibiting inflammation.The left anterior descending coronary artery was ligated for 30 min followed by 6 h of reperfusion to establish a model of I/R injury. H9c2 cardiomyocytes were subjected to 2 h of hypoxia and 3 h of normoxic conditions to establish a model of hypoxia/reoxygenation (H/R) injury. We monitored the change in pyroptosis by performing Western blot analysis, microscopy and ELISA.I/R and H/R treatment stimulated gasdermin D-N domain (GSDMD-N) expression in cardiomyocytes (sham onefold vs. I/R 2.5-fold; control onefold vs. H/R 2.0-fold). Moreover, TMZ increased the viability of H9c2 cardiomyocytes subjected to H/R treatment (H/R 65.0% vs. H/R + TMZ 85.3%) and reduced the infarct size in vivo (I/R 47.0% vs. I/R + TMZ 28.3%). H/R and I/R treatment increased the levels of TLR4, MyD88, phospho-NF-κB p65 and the NLRP3 inflammasome; however, TMZ reduced the expression of these proteins. Additionally, TMZ inhibited noncanonical inflammasome signaling induced by I/R injury.In summary, TMZ alleviated pyroptosis induced by myocardial I/R injury through the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, TMZ represents an alternative treatment for myocardial I/R injury.

Background Multipotent mesenchymal stromal cells (MSCs) have recently been reported to promote vasculogenesis by differentiating into endothelial cells and releasing numerous cytokines and paracrine factors. However, due to low cell activity, their potential for clinical application is not very satisfactory. This study aimed to explore the effects and mechanisms of 1,25-dihydroxyvitamin D (1,25(OH)2D3) on the vasculogenesis of MSCs. Methods MSCs were isolated from the femurs and tibias of rats and characterized by flow cytometry. After treatment with different concentrations of 1,25(OH)2D3 (0 µM, 0.1 µM and 1 µM), the proliferation of MSCs was analyzed by Cell Counting Kit-8 (CCK-8), and the migratory capability was measured by Transwell assays and cell scratch tests. Capillary-like structure formation was observed by using Matrigel. Western blotting was used to detect the expression of FLK-1 and vWF to investigate the differentiation of MSCs into endothelial cells. Western blotting and gelatin zymography were used to detect the expression and activities of VEGF, MMP-2 and MMP-9 secreted by MSCs under the influence of 1,25(OH)2D3. Finally, the VDR antagonist pyridoxal-5-phosphate (P5P) and the PI3K/AKT pathway inhibitor LY294002 were utilized to test the phosphorylation levels of key kinases in the PI3K/AKT pathway by Western blotting and the formation of capillary-like structures in Matrigel. Results The proliferation and migratory capability of MSCs and the ability of MSCs to form a tube-like structure in Matrigel were enhanced after treatment with 1,25(OH)2D3. Moreover, MSCs treated with 1,25(OH)2D3 showed high expression of vWF and Flk-1. There was a significant increase in the expression of VEGF, MMP-2 and MMP-9 secreted by MSCs treated with 1,25(OH)2D3, as well as in the activity of MMP-2 and MMP-9. The phosphorylation level of AKT increased with time after 1,25(OH)2D3 treatment, while LY294002 weakened AKT phosphorylation. In addition, the ability to form capillary-like structures was reduced when the VDR and PI3K/AKT pathways were blocked. Conclusion This study confirmed that 1,25(OH)2D3 treatment can strengthen the ability of MSCs to promote vasculogenesis in vitro, and the mechanism may be related to the activation of the PI3K/AKT pathway.

Inflammation and proliferation of vascular smooth muscle cells (VSMCs) are the key events in intimal hyperplasia. This study aimed to explore the mechanism by which long non-coding RNA (lncRNA) KCNQ1OT1 affects VSMC inflammation and proliferation in this context. A vein graft (VG) model was established in mice to introduce intimal hyperplasia. Isolated normal VSMCs were induced with platelet-derived growth factor type BB (PDGF-BB), and the cell proliferation, migration, and secretion of inflammatory factors were determined. The results showed that KCNQ1OT1 was downregulated in the VSMCs from mice with intimal hyperplasia and in the PDGF-BB-treated VSMCs, and such downregulation of KCNQ1OT1 resulted from the increased methylation level in the KCNQ1OT1 promoter. Overexpressing KCNQ1OT1 suppressed PDFG-BB-induced VSMC proliferation, migration, and secretion of inflammatory factors. In VSMCs, KCNQ1OT1 bound to the nuclear transcription factor kappa Ba (IκBa) protein and increased the cellular IκBa level by reducing phosphorylation and promoting ubiquitination of the IκBa protein. Meanwhile, KCNQ1OT1 promoted the expression of IκBa by sponging miR-221. The effects of KCNQ1OT1 knockdown on promoting VSMC proliferation, migration, and secretion of inflammatory factors were abolished by IκBa overexpression. The roles of KCNQ1OT1 in reducing the intimal area and inhibiting IκBa expression were proved in the VG mouse model after KCNQ1OT1 overexpression. In conclusion, KCNQ1OT1 attenuated intimal hyperplasia by suppressing the inflammation and proliferation of VSMCs, in which the mechanism upregulated IκBa expression by binding to the IκBa protein and sponging miR-221.

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)‐induced injury. Here, we investigated the function of miR‐21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR‐21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR‐21+ H/R) group, (miR‐21‐negative control + H/R) group, (BEZ235+ H/R) group and (miR‐21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit‐8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR‐21 expression was down‐regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR‐21 with a miR‐21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR‐21‐mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR‐21 and BEZ235. We showed that miR‐21 could inhibit H/R‐induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.

Aims Increasing evidence shows that NLRP3 inflammasome is closely associated with the progression of atherosclerosis. The purpose of the present study was to evaluate the effects of atorvastatin on NLRP3 inflammasome in PMA-stimulated THP-1 cells and explore its underlying mechanism. Methods Human monocytic THP-1 cells were pretreated with atorvastatin for 1 h and then induced by PMA for 48 h. Total protein was collected for real-time PCR and Western blot analysis. Cytokine IL-1β release was detected by ELISA assay. And the NF-κB p65 translocation was detected by cellular NF-κB translocation kit. Results It was shown that atorvastatin significantly reduced the expression of NLRP3, the cleavage of caspase-1 and IL-1β in PMA-induced THP-1 cells. Moreover, Bay (a NF-κB inhibitor) treatment greatly suppressed the expression of NLRP3, the cleavage of caspase-1 and IL-1β in PMA-induced THP-1 cells, suggesting that the activation of NF-κB pathway takes part in regulating the expression of NLRP3 inflammasome. In addition, atorvastatin markedly inhibited the up-regulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor kappa b (NF-κB) in PMA-stimulated THP-1 cells. Conclusions Atorvastatin exerts an anti-inflammatory effect by inhibiting NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB pathway in PMA-induced THP-1 monocytes.

Ischemia/reperfusion (I/R)-induced autophagy increases the severity of cardiomyocyte injury. The aim of this study was to investigate the effects of berberine, a natural extract from Rhizoma coptidis, on the I/R-induced excessive autophagy in in vitro and in vivo models. Autophagy was increased both in H9c2 myocytes during hypoxia/reoxygenation (H/R) injury and in mouse hearts exposed to I/R. And the expression level of p-AMPK and p-mTORC2 (Ser2481) were increased during H/R period. In addition, the increased autophagy level was correlated with reduced cell survival in H9c2 myocytes and increased infarct size in mouse hearts. However, berberine treatment significantly enhanced the H/R-induced cell viability and reduced I/R-induced myocardial infarct size, which was accompanied by improved cardiac function. The beneficial effect of berberine is associated with inhibiting the cellular autophagy level, due to decreasing the expression level of autophagy-related proteins such as SIRT1, BNIP3, and Beclin-1. Furthermore, both the level of p-AMPK and p-mTORC2 (Ser2481) in H9c2 myocytes exposed to H/R were decreased by berberine. In summary, berberine protects myocytes during I/R injury through suppressing autophagy activation. Therefore, berberine may be a promising agent for treating I/R-induced cardiac myocyte injury.

Primary percutaneous coronary intervention, or thombolytic therapy, provides effective myocardial blood reconstruction in patients with acute myocardial infarction to reduce acute myocardial ischemic injury. However, reperfusion can itself induce cardiomyocyte death, termed myocardial reperfusion injury (I/R). Hypoxia/reoxygenation (H/R) induces apoptosis and excessive autophagy among cardiomyocytes, leading to cell death. The present study investigated the effect of curcumin, a natural extract from Curcuma longa, on these two cellular processes in H9c2 myocytes. The levels of cellular apoptosis and autophagy were found to be upregulated in the H9c2 myocytes during H/R and were correlated with a reduced rate of cell survival. However, curcumin significantly suppressed the levels of H/R‑induced apoptosis (expression of annexin V) and autophagy (LC3B‑II/LC3B‑I ratio) in the H9c2 myocytes and promoted cell survival. Additionally, the expression of B‑cell lymphoma 2 (Bcl‑2) was significantly downregulated and the expression levels of Bcl‑2‑associated X protein, beclin‑1, Bcl‑2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and silent information regulation 1 (SIRT1) were significantly upregulated in myocytes following H/R injury. These effects on the expression of these proteins were reversed by curcumin treatment. These findings suggested that the protective effect of curcumin against H/R injury in the H9c2 myocytes was through the inhibition of apoptosis and autophagy by inducing the expression of Bcl‑2 and inhibiting the expression levels of Bax, beclin‑1, BNIP3 and SIRT1. Therefore, curcumin may offer a promising therapeutic approach for the treatment of cardiomyocyte injury resulting from I/R.

MiRNAs are considered as a novel class of biomarkers or treatment targets for cardiovascular diseases. Hsa-miR-6086, a novel mi-RNA, was reported to be downregulated during the differentiation of human embryonic stem cells into endothelial cells (ECs). Interestingly, CDH5 (cadherin 5), encoding a classical cadherin of the cadherin superfamily, is a cellular marker of ECs and has been reported to be a target of hsa-miR-6086. However, the role of hsa-miR-6086 in ECs is virtually unknown. Herein, we report that hsa-miR-6086 was markedly induced by TNFα stimulation in human umbilical vein endothelial cells (HUVECs), whereas CDH5 expression was greatly reduced. Importantly, TNFα-induced suppression of CDH5 expression was largely prevented by inhibiting hsa-miR-6086, and hsa-miR-6086 mimic greatly decrease CDH5 expression in HUVECs, suggesting that the induction of hsa-miR-6086 is responsible for CDH5 downregulation by TNFα. In addition, restoration of CDH5 expression level by either inhibiting hsa-miR-6086 or exogenously expressing CDH5 cDNA that is not affected by hsa-miR-6086 protected HUVECs against TNFα-induced apoptosis and cell growth inhibition. Taken together, our study reveals that hsa-miR-6086 is induced by TNFα and mediates TNFα-induced HUVEC growth inhibition through downregulating CDH5 expression. Hence, hsa-miR-6086 might be a new target for treating TNFα-induced endothelial dysfunction.

Rupture of vulnerable atherosclerotic plaques is the leading cause of acute myocardial infarction (AMI) and unstable angina pectoris (UA). However, it still lacks an effective therapy to stabilize the vulnerable atherosclerotic plaques. Numerous reports have shown that upregulation of MMP-9 (matrix metalloproteinase-9) and EMMPRIN (extracellular matrix metalloproteinase inducer) in macrophages is involved in the progression and development of vulnerable plaques. Here we evaluated the impact of curcumin on the expression of MMP-9 and EMMPRIN in macrophages. Macrophages were pretreated with curcumin or specific inhibitors (p38 MAPK inhibitor, NF-κB p65 inhibitor) for 1 hr, then cells were cultured with oxLDL for indicated time. Real-time PCR and Western blot analysis were used to evaluate the expression of mRNA and proteins. Translocation of NF-κB p65 was detected by using laser confocal microscopy. Here we showed that curcumin attenuated the MMP-9 and EMMPRIN expression in oxLDL stimulated macrophages. Further studies revealed that curcumin inhibited oxLDL induced NF-κB activation and p38 MAPK phosphorylation. These findings illustrated that curcumin can inhibit the expression of EMMPRIN and MMP-9 in oxLDL stimulated macrophages through down regulation of NF-κB and p38 MAPK signaling pathways, which might be the molecular mechanism for the anti-atherosclerotic effect of curcumin.

The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP-3) inflammasome has recently emerged as a pivotal regulator of chronic inflammation. The present study investigated the expression of NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages, and aimed to identify the effects of berberine on the inflammasome. Human monocytic THP-1 cells were pretreated with berberine for 1 h and then induced with PMA for 48 h. Total RNA and protein were collected for reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Supernatants were collected to determine IL-1β levels by using ELISA. The present study demonstrated that NLRP3 inflammasome and IL-1β were activated in PMA-induced macrophages in a time-dependent manner, whereas berberine significantly inhibited their expression in a dose-dependent manner in PMA-induced macrophages. Furthermore, berberine also suppressed the toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (Myd88)/nuclear factor (NF)-κB signaling pathway which was activated during the conversion of THP-1 cells to macrophages by PMA. In conclusion, berberine reduced NLRP3 inflammasone expression by suppressing the activation of the TLR4/Myd88/NF-κB signaling pathway in PMA-induced macrophages. This inhibitory effect may imply an important role of berberine on chronic inflammation and atherogenic progression in coronary artery disease.

Aims: In the NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) inflammasome is closely related to the progression of atherosclerosis. This study aimed to assess the effects of curcumin on NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages and explore its underlying mechanism. Methods: Human monocytic THP-1 cells were pretreated with curcumin for 1 h and subsequently induced with PMA for 48 h. Total protein was collected for Western blot analysis. Cytokine interleukin (IL)-1β release and nuclear factor kappa B (NF-κB) p65 translocation were detected by ELISA assay and cellular NF-κB translocation kit, respectively. Results: Curcumin significantly reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion in PMA-induced macrophages. Moreover, Bay (a NF-κB inhibitor) treatment considerably suppressed the expression of NLRP3 inflammasome in PMA-induced THP-1 cells. Curcumin also markedly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκB-α, and activation of NF-κB in PMA-induced macrophages. In addition, purinergic 2X7 receptor (P2X7R) siRNA was administered, and it significantly decreased NLRP3 inflammasome expression in PMA-induced macrophages. Furthermore, curcumin reversed PMA-stimulated P2X7R activation, which further reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion. Silencing of P2X7R using siRNA also suppressed the activation of NF-κB pathway in PMA-induced macrophages, but P2X7R-silenced cells did not significantly decrease the expression of TLR4 and MyD88. Conclusions: Curcumin inhibited NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB and P2X7R pathways in PMA-induced macrophages.

In coronary arteries, plaque disruption, the major acute clinical manifestations of atherosclerosis, leads to a subsequent cardiac event, such as acute myocardial infarction (AMI) and unstable angina pectoris (UA). Numerous reports have shown that high expression of MMP-9 (matrix metalloproteinase-9), MMP-13 (matrix metalloproteinase-13) and EMMPRIN (extracellular matrix metalloproteinase induce) in monocyte/macrophage results in the plaque progression and destabilization. Curcumin exerts well-known anti-inflammatory and antioxidant effects and probably has a protective role in the atherosclerosis. The purpose of our study was to investigate the molecular mechanisms by which curcumin affects MMP-9, MMP13 and EMMPRIN in PMA (phorbol 12-myristate 13-acetate) induced macrophages. Human monocytic cells (THP-1 cells) were pretreated with curcumin or compound C for 1 h, and then induced by PMA for 48 h. Total RNA and proteins were collected for real-time PCR and Western blot analysis, respectively. In the present study, the exposure to curcumin resulted in attenuated JNK, p38, and ERK activation and decreased expression of MMP-9, MMP-13 and EMMPRIN in PMA induced macrophages. Moreover, we demonstrated that AMPK (AMP-activated protein kinase) and PKC (Protein Kinase C) was activated by PMA during monocyte/macrophage differentiation. Furthermore, curcumin reversed PMA stimulated PKC activation and suppressed the chronic activation of AMPK, which in turn reduced the expression of MMP-9, MMP-13 and EMMPRIN. Therefore, it is suggested that curcumin by inhibiting AMPK-MAPK (mitogen activated protein kinase) and PKC pathway may led to down-regulated EMMPRIN, MMP-9 and MMP-13 expression in PMA-induced THP-1 cells.