Expression Of NLRP3 Inflammasome Research Articles

CB2R activation ameliorates late adolescent chronic alcohol exposure-induced anxiety-like behaviors during withdrawal by preventing morphological changes and suppressing NLRP3 inflammasome activation in prefrontal cortex microglia in mice

BackgroundChronic alcohol exposure (CAE) during late adolescence increases the risk of anxiety development. Alcohol-induced prefrontal cortex (PFC) microglial activation, characterized by morphological changes and increased associations with neurons, plays a critical role in the pathogenesis of anxiety. Alcohol exposure increases NLRP3 inflammasome expression, increasing cytokine secretion by activated microglia. Cannabinoid type 2 receptor (CB2R), an essential receptor of the endocannabinoid system, regulates microglial activation and neuroinflammatory reactions. We aimed to investigate the role of CB2R activation in ameliorating late adolescent CAE-induced anxiety-like behaviors and microglial activation in C57BL/6J mice. MethodsSix-week-old C57BL/6J mice were acclimated for 7 days and then were administered alcohol by gavage (4 g/kg, 25 % w/v) for 28 days. The mice were intraperitoneally injected with the specific CB2R agonist AM1241 1 h before alcohol treatment. Anxiety-like behaviors during withdrawal were assessed by open field test and elevated plus maze test 24 h after the last alcohol administration. Microglial activation, microglia-neuron interactions, and CB2R and NLRP3 inflammasome-related molecule expression in the PFC were measured using immunofluorescence, immunohistochemical, qPCR, and Western blotting assays. Microglial morphology was evaluated by Sholl analysis and the cell body-to-total cell size index. Additionally, N9 microglia were activated by LPS in vitro, and the effects of AM1241 on NLRP3 and N9 microglial activation were investigated. ResultsAfter CAE, mice exhibited severe anxiety-like behaviors during withdrawal. CAE induced obvious microglia-neuron associations, and increased expression of microglial activation markers, CB2R, and NLRP3 inflammasome-related molecules in the PFC. Microglia also showed marked filament retraction and reduction and cell body enlargement after CAE. AM1241 treatment ameliorated anxiety-like behaviors in CAE model mice, and it prevented microglial morphological changes, reduced microglial activation marker expression, and suppressed the microglial NLRP3 inflammasome activation and proinflammatory cytokine secretion induced by CAE. AM1241 suppressed the LPS-induced increase in NLRP3 inflammasome-related molecules, IL-1β release, and M1 phenotype markers (iNOS and CD86) in N9 cell, which was reversed by CB2R antagonist treatment. ConclusionsCAE caused anxiety-like behaviors in late adolescent mice at least partly by inducing microglial activation and increasing microglia-neuron associations in the PFC. CB2R activation ameliorated these effects by preventing morphological changes and suppressing NLRP3 inflammasome activation in PFC microglia.

Asiatic acid and andrographolide reduce hippocampal injury through suppressing neuroinflammation caused by Salmonella typhimurium infection.

Damage caused by Salmonella is not only limited to the gastrointestinal tract, but also occurs in the central nervous system (CNS). The aim of this study was to explore the protective effects of asiatic acid (AA) and andrographolide (AD) on the CNS through simulating common infection in mice by oral administration of Salmonella typhimurium (S. typhimurium). The results showed that the neurons in the hippocampus of mice were damaged after S. typhimurium invaded CNS in mice, and the inflammation was increased, which was manifested by the increased expression of inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, interferon (IFN)-γ and IL-12b and the activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasomes. The damage and inflammatory response of mouse hippocampal neurons were effectively reduced by AA or AD pretreatment. Furthermore, we observed the significant activation of microglia after S. typhimurium infection. AA and AD attenuated S. typhimurium -induced hippocampal injury by reducing the inflammatory response on microglia. The findings suggest that the AA and AD protect CNS from injury caused by S. typhimurium infection through inhibiting over expression of multiple neuroinflammatory mediators and NLRP3 inflammasome in mice.

Investigation of He's Yang Chao recipe against oxidative stress-related mitophagy and pyroptosis to improve ovarian function.

Primary ovarian insufficiency (POI) is a common gynecological disease with serious ramifications including low pregnancy rate and low estrogen symptoms. Traditional Chinese medicine is regarded as an effective treatment for POI. However, the therapeutic mechanism of it is unclear. In this study, a mouse model of primary ovarian insufficiency was established by intraperitoneal injection of cyclophosphamide (CTX) and He's Yang Chao Recipe (HSYC) concentrate was used for intragastric administration. Serum hormone levels (Anti-Müllerian Hormone, Estradiol, Progesterone, Luteinizing Hormone and Follicle Stimulating Hormone) and Oxidative Stress (OS) related products, superoxide dismutase (SOD), GSH-Px, and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Pathological changes in ovarian tissue were evaluated by hematoxylin and eosin staining, and flow cytometry was used to determine reactive oxygen species content and mitochondrial membrane potential levels in granulosa cells. Mitochondrial distribution and morphology were investigated using immunofluorescence staining. The level of mitophagy was evaluated by LC3 immunofluorescence staining and autophagosome counts using electron microscopy. Western blotting and qPCR were used to detect the expression of proteins and genes related to mitophagy and the NLRP3 inflammasome. After HSYC treatment, the ovarian damage was milder than in the CTX group. Compared with the CTX group; SOD, GSH-Px, and the total antioxidant capacity were significantly increased, while MDA and ROS were decreased in the HSYC treatment groups. Furthermore, mitochondrial distribution and membrane potential levels were improved after HSYC treatment compared to the CTX group. After the HSYC treatment, the LC3 fluorescent intensity and autophagosome counts were decreased. Similarly, mitophagy related markers PINK1, Parkin, LC3, and Beclin1 were decreased, while p62 was significantly increased, compared with the CTX groups. The mRNA and protein expression of NLRP3 inflammasome, NLRP3, caspase-1, GSDMD, IL-18, and IL-1β were significantly decreased in the HSYC treatment groups. This is the first study in molecular mechanisms underlying HSYC against granulosa cell injury in POI. HSYC protects ovaries from CTX-induced ovarian damage and oxidative stress. HSYC enhanced ovarian function in mice with primary ovarian insufficiency by inhibiting PINK1-Parkin mitophagy and NLRP3 inflammasome activation.

Aurantiamide suppresses the activation of NLRP3 inflammasome to improve the cognitive function and central inflammation in mice with Alzheimer's disease.

This study was aimed at exploring the mechanism by which aurantiamide (Aur) targeted NLRP3 to suppress microglial cell polarization. The 7-month-old APP/PS1 mice and C57BL/6 mice were applied to be the study objects, and Aur was administered intragastrically to APP/PS1 mice at 10mg/kg and 20 mg/kg. The changes in the neurocognitive function of mice were measured by Morris Water Maze (MWM) test. In the in vitro experiments, the mouse BV2 cells were employed as the study objects, which were subject to treatment with 10μM and 20 μM Aur and induced with LPS and IFN-γ in order to activate BV2 cells and induce their M1 polarization. Aur was found to suppress the M1 polarization of mouse microglia, reduce central neuroinflammation, and improve the cognitive function in mice. Meanwhile, Aur suppressed the activation and the expression of NLRP3 inflammasome. The results of experiments in vitro demonstrated that Aur inhibited the activation and M1 polarization of BV2 cells. Aur targets NLRP3 and suppresses the activation of NLRP3 inflammasome.

Uric acid promotes myocardial infarction injury via activating pyrin domain-containing 3 inflammasome and reactive oxygen species/transient receptor potential melastatin 2/Ca2+pathway

Cardiomyocytes injury has been considered as a key contributor for myocardial infarction (MI). Uric acid (UA) can induce cardiomyocytes injury, which is closely related to NLRP3 activation and inflammatory factor generation. However, the mechanism how UA modulates cardiomyocytes remains elusive. Western blotting and qRT-PCR were applied for measuring protein and mRNA expression, respectively. ROS production and Ca2+ influx were measured by flow cytometry. Patch clamp technique was used for measuring transient receptor potential melastatin 2 (TRPM2) channel. Ligation of left anterior descending for 2 h was performed to induce MI animal model. The rats were treated by different concentration of uric acid. The artery tissues were stained by HE and collected for measurement of NLRP3 and inflammatory factors. Supplementation of UA significantly promoted apoptosis, and augmented the expression of intercellular adhesion molecule-1, chemoattractant protein-1, vascular cell adhesion molecule-1, and NLRP3 inflammasome. Knockdown of NLRP3 reversed the influence of UA on MI by decreasing collagen deposition, fibrotic area, apoptosis. The expression of NLRP3 inflammasome increased markedly after treatment of UA. UA activated ROS/TRPM2/Ca2+ pathway through targeting NLRP3. UA activated NLRP3 inflammasome and augments inflammatory factor production, which in turn exacerbates cardiomyocytes injury. Knockdown of NLRP3 reversed the influence of UA on apoptosis and cell cycle. UA may promote cardiomyocytes injury through activating NLRP3 inflammasome and ROS/TRPM2 channel/Ca2+ pathway.

A NEW APPROACH TO ASSISTED REPRODUCTIVE TECHNOLOGIES PREPARATION IN WOMEN WITH ENDOMETRIOSIS ASSOCIATED INFERTILITY USING PROBIOTICS.

Aim: To examine the expression Nlrp3-inflammasome in the blood of women with endometriosis-associated infertility using assisted reproductive technologies with included probiotic and estimate a new approach with included probiotic for preparation to assisted reproductive technologies. Materials and methods: For the purpose of the research we examined 30 infertile women. Control group consisted of 10 women with tubal infertility due to an inflammatory process in the anamnesis, in whom, as a result of a complex clinical and laboratory examination, no other diseases were detected and in terms of their health they could be equated with practically healthy women (age from 21 to 42 years, the average age was 29.75 years) and who did not take probiotic. The main group consists from 20 women with external genital endometriosis were included in assisted reproductive technologies. Patients from main group took probiotic produced by Unic Biotech Ltd, India. 1 tablet twice a day with 10×109 Lactobacillus during 1 month in complex preparation (treatment) before assisted reproductive technologies. The indices of Nlrp3 - Inflammasome determined before and after preparation. The study was performer in Bukovinian State Medical University and Centre of Reproductive Medicine. To analyze the expression of the NLPP3-inflammasome gene and determine the relative normalized expression of NLPP3 mRNA, we used the polymerase chain reaction with reverse transcription in real time (RT-PCR). The object for molecular genetic studies by the RT-PCR method was the fraction of mononuclear cells isolated from the whole blood of patients with endometriosis. The value of p (authenticity difference) was determined by Student's table-Fischer. Differences between contrasting averages were considered significant at p <0.05. Results: Analized the results of our research stated that in main group expression of Nlrp-3 inflammasome was 24,43, which is significantly higher than after preparation (0,70 accordingly). In control group expression of Nlrp3-inflammasome was 0,54. Expression of Nlrp-3 inflammasome increased in patients before preparation more than 34 times compare with patients after preparation accordingly. Conclusions: consequently, after using probiotic, the increased level of Nlrp3-inflammasome sharply decreases, indicating the effectiveness and the possibility of use in the programme for assisted reproductive technologies preparation.

Dimethyl fumarate attenuates MSU-induced gouty arthritis by inhibiting NLRP3 inflammasome activation and oxidative stress.

Dimethyl fumarate (DMF) has shown anti-inflammatory and antioxidant activities. However, the effects of DMF on gouty arthritis remain elusive, and the underlying mechanism is not understood. In this study, we aim to investigate the role of DMF in gouty arthritis. Mice were gavage with DMF for consecutive 7 days at two different doses (10 mg/kg/day or 30 mg/kg/day, once daily) in advance and then monosodium sodium urate (MSU) was injected into their joint to establish an acute gout mice model. The pain and swelling of the hind paw in mice were determined. The production of pro-inflammatory cytokine in the paw tissues was assessed by Elisa and the inflammatory infiltration of the joint was determined by hematoxylin and eosin (H&E) staining. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the tissues were measured by commercial kits. In addition, the expression of nuclear factor kappa B (NF-κB) and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome and downstream genes were detected by PCR and Western blot. Furthermore, LPS-primed murine macrophages Raw 264.7 cells were treated with different concentrations of DMF (2 μM, 5 μM, 10 μM) for 2 h, and then challenged with MSU (200 μg/mL) for other 12 h to observe the effect of DMF on cell viability via cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) levels in the supernatant of culture medium. Immunofluorescent staining was used to detect the NLRP3 inflammasome activation and reactive oxygen species (ROS) production in vitro. Caspase-1 activity was measured by corresponding assay kits both in vivo and in vitro. DMF attenuated pain and swelling in MSU-induced gout mice by decreasing pro-inflammatory cytokine production and inflammatory cell infiltration, as well as improved oxidative stress. Moreover, DMF inhibited the activation of NF-κB and NLRP3 inflammasome and subsequent expression of caspase-1, interleukin-1β (IL-1β), and IL-18 at both mRNA and protein levels. Meanwhile, DMF suppressed NLRP3 inflammasome expression and ROS production in LPS and MSU-stimulated Raw 264.7 cells, thereby protecting the cells from inflammatory injury. DMF serves as a new approach for the treatment of MSU-induced gouty arthritis by suppressing NLRP3 inflammasome activation and oxidative stress.

200 BERBERINE ASSOCIATED TO DAPAGLIFLOZIN SYNERGISTICALLY REDUCES CARDIAC CELL APOPTOSIS DURING EXPOSURE TO TRASTUZUMAB THROUGH INDUCTION OF PAMPK AND RECUTION OF NLRP3, IL6 AND LEUKOTRIENES LEVELS

Abstract Background Trastuzumab has improved the prognosis in patients with HER2-positive breast cancer, but it can induce left ventricular dysfunction with reduced ejection fraction or HF during treatment. Dapagliflozin is a SGLT2i with cardio-renal benefits. In the DAPA-HF (Dapagliflozin And Prevention of Adverse-outcomes in Heart Failure) trial, the sodium-glucose cotransporter 2 inhibitor dapagliflozin decreased the risk of worsening HF events and cardiovascular death in patients with HF and reduced ejection fraction. Berberine is a nutraceutical compound characterized by multiple metabolic effects in patients with/without cardiovascular diseases. Recent preclinical systematic review indicated that Berberine significantly reduces myocardial infarct size and the incidence of ventricular arrhythmia, improves cardiac function, ameliorates myocardial apoptosis. Purpose To investigate on the potential cardioprotective properties of Berberine associated to SGLT2i Dapagliflozin against HER-2 blocking agent-induced cardiotoxicity Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of trastuzumab (200 nM) alone or co-incubated with Berberine (200 mM) or Dapagliflozin (50 nM) or both in combination for 48 h. After the incubation period, we performed the following tests: cell viability, apoptosis, expression of NLRP3 inflammasome, methylglyoxal and leukotrienes-B4. Expression of pAMPK was analyzed through western blot. Moreover, quantification of IL-6 was performed through ELISA method. Results Berberine and Dapagliflozin increased significantly the cell viability of cardiomyocytes exposed to Trastuzumab. When combined, Berberine and Dapagliflozin increased synergistically cell viability of cardiac cells (p&amp;lt;0.001 vs Trastuzumab). Cell apoptosis was reduced of 32.5, 41.8 and 72.7% for berberine, dapagliflozin and both combined (vs trastuzumab group). Methylglyoxal,a marker of AGEs, was strongly reduced compared to untreated cells. Western blot analysis clearly demonstrate that pAMPK was induced by berberine and Dapagliflozin, improving mitochondrial metabolism. No significant changes in Leukotriene B4 expression were seen. Intracellular levels of IL-6 were reduced of 46.3, 62.7 and 86.3% for berberine, dapagliflozin and both combined (vs trastuzumab group). Conclusion Berberine and Dapagliflozin exerts significant cardioprotective effects in cardiac cells exposed to the HER2-bloking agent Trastuzumab. Berberine and Dapagliflozin in combination induces an anti-inflammatory phenotype to myocardial cells through the reduction of biomarkers involved in heart failure and apoptosis.

201 CTLA-4 AND PD-1 BLOCKING AGENTS INCREASES SYSTEMIC SDF-1, CARDIAC DAMPS FIBRONECTIN EDA, S-100 CALGRANULIN, GALECTINE-3 AND NLRP-3/MYD-88 CHEMOKINE PATHWAYS

Abstract Background Immune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases.In this study, pro-inflammatory and pro-fibrotic effects of short-term ICIs therapy in preclinical models were analyzed. Methods Firstly, in a human in vitro model, human cardiomyocytes co-cultured with hPBMC were exposed to ICIs (with CTLA-4 or PD-1 blocking agents, at 200 nM) for 72h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA-4 or PD-1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100,Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF-kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100- Calgranulin, Fibronectin and Galectine-3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF-1, IL-1β and IL-6 were analyzed before, during and after ICIs therapy. Results Radial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF-kB expression shows that short-term anti-CTLA-4 or anti-PD-1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin-3, pro-collagen 1-α and MMP-9 were increased after treatment with all ICIs. Both anti-CTLA-4 or anti-PD-1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL-1β, TNF-α and IL-6. Systemic levels of SDF-1, IL-1β and IL-6 were increased during and after treatment with ICIs. Conclusions Short therapy with PD-1 and CTLA-4 blocking agents increases vascular expression of NF-kB, systemic SDF-1, IL-1β, IL-6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro-inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy. The overall picture of the study suggests new putative biomarkers of ICIs-mediated systemic and myocardial damages potentially useful in clinical cardioncology.

205 SGLT-2 INHIBITOR DAPAGLIFLOZIN REDUCES ANTHRACYCLINE-MEDIATED CARDIOMYOPATHY IN PRECLINICAL MODELS THROUGH REDUCTION OF MYOCARDIAL NF-KB, MYD-88 AND NLRP3 EXPRESSION: A BIOCHEMICAL AND HISTOLOGICAL STUDY

Abstract Background Doxorubicin-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently been shown to be of therapeutic value in patients with type 2 diabetes, chronic kidney disease, and heart failure with reduced ejection fraction (HFrEF), conditions that commonly coexist and are interrelated pathophysiologically. Purpose We hypothesized that Dapagliflozin (an SGLT2i), administered during doxorubicin, could improve cardiac function in preclinical models Methods Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO-DAPA, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, DAMPs (galectine 3 and calgranulinS100), pAMPK, NF-kB, and 13 chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through ELISA and western blot methods. Results DAPA improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88, DAMPs and NF-kB in cardiac tissues was seen in DOXO-DAPA group compared to DOXO mice (p&amp;lt;0.001). Cardiac expression of IL-1β, IL-6, TNF-α, G-CSF and GM-CSF were significantly reduced after treatment with DAPA indicating anti-inflammatory properties. Expression of pAMPK was strongly enhanced in DAPA-DOXO compared to DOXO group. Levels of Calgranulin S100 and galectine-3 were strongly enhanced in DOXO group; on the other hand their expression were reduced by 47.7 and 52.3% in DAPA-DOXO group vs DOXO (p&amp;lt;0.005). Hystology evaluation indicate reduction NF-kB expression in myocardial and renal tissue in DOXO-DAPA vs DOXO. Conclusion In this preclinical study, DAPA is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. The overall picture of the study pushes the use of DAPA in prevention of cardiomyopathies induced by anthracyclines in cancer patients.

Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis.

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain‑containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010pg/mL and 2738pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

Altered expression of NLRP3 inflammasome in pediatric patients with primary immune thrombocytopenia and its relation to disease activity

Altered expression of NLRP3 inflammasome in pediatric patients with primary immune thrombocytopenia and its relation to disease activity

Abstract 12406: Sglt2i Dapagliflozin Associated to Berberine Reduces Cardiac Cell Apoptosis and Necrosis During Exposure to Her-2 Blocking Agents Through Induction of Pampk and Reduction of Inflammasome, Il-6, Methylglyoxal and Leukotrienes-B4 Levels

Introduction: Trastuzumab has improved the prognosis in patients with HER2-positive breast cancer, but it can induce left ventricular dysfunction with reduced ejection fraction or HF during treatment. Dapagliflozin is a SGLT2i with cardio-renal benefits. In DAPA-HF trial, the sodium-glucose cotransporter 2 inhibitor dapagliflozin decreased the risk of worsening HF events and cardiovascular death in patients with HF and reduced ejection fraction. Berberine is a nutraceutical compound characterized by multiple metabolic effects in patients with/without cardiovascular diseases. Hypothesis: To investigate on the potential cardioprotective properties of Berberine associated to SGLT2i Dapagliflozin against HER-2 blocking agent-induced cardiotoxicity Methods: Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of trastuzumab (200 nM) alone or co-incubated with Berberine (200 mM) or Dapagliflozin (50 nM) or both in combination for 48 h. After the incubation period, we performed the following tests: cell viability, apoptosis, expression of NLRP3 inflammasome, methylglyoxal and leukotrienes-B4. Expression of pAMPK was analyzed through western blot. Moreover, quantification of IL-6 was performed Results: Berberine and Dapagliflozin increased significantly the cell viability of cardiomyocytes exposed to Trastuzumab. When combined, Berberine and Dapagliflozin increased synergistically cell viability of cardiac cells (p&lt;0.001 vs Trastuzumab). Cell apoptosis was reduced of 32.5, 41.8 and 72.7% for berberine, dapagliflozin and both combined (vs trastuzumab group). Methylglyoxal,a marker of AGEs, was strongly reduced compared to untreated cells. Western blot analysis clearly demonstrate that pAMPK was induced by berberine and Dapagliflozin, improving mitochondrial metabolism. No significant changes in Leukotriene B4 expression were seen. Intracellular levels of IL-6 were reduced of 46.3, 62.7 and 86.3% for berberine, dapagliflozin and both combined (vs trastuzumab group). Conclusions: Berberine and Dapagliflozin in combination induces an anti-inflammatory phenotype to myocardial cells through the reduction of biomarkers involved in heart failure and apoptosis.

Abstract 10603: Dapagliflozin Increases Ampk, Reduces Fibrosis/Hypertrophy and Nlrp-3, Il-6 and Galectin-3 Levels in Short-Term Doxorubicin-Treated Mice Improving Cardiac Function

Introduction: Doxorubicin-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently been shown to be of therapeutic value in patients with type 2 diabetes, chronic kidney disease, and heart failure with reduced ejection fraction (HFrEF), conditions that commonly coexist and are interrelated pathophysiologically. Hypothesis: Dapagliflozin (an SGLT2i), administered during doxorubicin, could improve cardiac function in preclinical models Methods: Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), DAPA at 12 mg/kg (DAPA, n=6) or doxorubicin combined to DAPA (DOXO-DAPA, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, DAMPs (galectine 3 and calgranulinS100) , pAMPK, NF-kB, and 13 chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through ELISA and western blot methods. Results: DAPA improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88, DAMPs and NF-kB in cardiac tissues was seen in DOXO-DAPA group compared to DOXO mice (p&lt;0.001). Cardiac expression of IL-1β, IL-6, TNF-α, G-CSF and GM-CSF were significantly reduced after treatment with DAPA indicating anti-inflammatory properties. Expression of pAMPK was strongly enhanced in DAPA-DOXO compared to DOXO group. Levels of Calgranulin S100 and galectine-3 were strongly enhanced in DOXO group; on the other hand their expression were reduced by 47.7 and 52.3% in DAPA-DOXO group vs DOXO (p&lt;0.005). Hystology evaluation indicate reduction in hypertrophy, fibrosis and cardiac necrosis in DOXO-DAPA vs DOXO. Conclusions: DAPA is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. The overall picture of the study pushes the use of DAPA in prevention of cardiomyopathies induced by anthracyclines in cancer patients.

Abstract 12402: Sacubitril-Valsartan Increases Pampk and Reduces Inflammasome, Myddosome, Il-6 and Galectin-3 Levels in Short-Term Doxorubicin-Treated Mice Improving Longitudinal Strain and Ejection Fraction

Introduction: Doxorubicin-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sacubitril-valsartan (LCZ 696) is a combination drug, made up of neprilysin inhibitor sacubitril and angiotensin II receptor blocker valsartan, used for the treatment of heart failure in patients with a reduced ejection fraction. Hypothesis: We hypothesized that LCZ 696, administered during doxorubicin, could improve cardiac function Methods: Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), LCZ-696 at 60 mg/kg (LCZ, n=6) or doxorubicin combined to LCZ-696 (DOXO-LCZ, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, DAMPs (galectine 3 and calgranulinS100) , pAMPK, NF-kB, and 13 chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through ELISA and western blot methods. Results: LCZ 696 improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88, DAMPs and NF-kB in cardiac tissues was seen in DOXO-LCZ group compared to DOXO mice (p&lt;0.001). Cardiac expression of IL-1β, IL-6, TNF-α, G-CSF and GM-CSF were significantly reduced after treatment with LCZ-696 indicating anti-inflammatory properties. Expression of pAMPK was strongly enhanced in LCZ-696-DOXO compared to DOXO group. Levels of Calgranulin S100 and galectine-3 were strongly enhanced in DOXO group; on the other hand their expression were reduced by 47.7 and 52.3% in LCZ-696-DOXO group vs DOXO (p&lt;0.005). Conclusions: LCZ-696 is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. The overall picture of the study pushes the use of Sacubitril-valsartan in prevention of cardiomyopathies induced by anthracyclines in cancer patients.

Tanshinone IIA inhibits exosome-induced cardiomyocyte pyroptosis through NLRP3/caspase 1 pathway

Purpose: To investigate the effect of Salvia miltiorrhiza, a traditional Chinese medicinal plant, on exosome-induced cardiomyocyte pyroptosis. &#x0D; Methods: Pyroptosis was induced in human AC cells using exosomes. Then, the effect of Danshen (dried roots of S. miltiorrhiza) on exosome-induced pyroptosis was determined using flow cytometry. The expressions of pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA), while protein levels of cytokines were assayed by Western blotting. &#x0D; Results: Tanshinone IIA (Tan IIA), the bioactive molecule in Danshen, inhibited cardiomyocyte pyroptosis by significantly reducing the expressions of proinflammatory cytokines (p &lt; 0.001). Thus, Tan IIA reduced pyroptosis induced by cardiomyocyte-derived exosome via inhibition of the expression of NLRP3 inflammasome in human AC cells. &#x0D; Conclusion: This study has identified a potential mechanism through which Danshen functions to prevent cardiac diseases. It involves, at least in part, the inhibition of pyroptosis in cardiomyocytes. Thus, tanshinone IIA may be a pharmacologically beneficial cardioprotective compound, especially when used against heart failure.

Effects of Curcumin Supplementation on mRNA Expression of NLRP3 Inflammasome, IL-1β, and NF-κB in Hemodialysis Patients

Effects of Curcumin Supplementation on mRNA Expression of NLRP3 Inflammasome, IL-1β, and NF-κB in Hemodialysis Patients

Berberine associated to SGLT2i Dapagliflozin synergistically reduces cardiac cell apoptosis during exposure to Trastuzumab through reduction of AGEs and IL-6 and induction of pAMPK

Abstract Background Trastuzumab has improved the prognosis in patients with HER2-positive breast cancer, but it can induce left ventricular dysfunction with reduced ejection fraction or HF during treatment. Dapagliflozin is a SGLT2i with cardio-renal benefits. In the DAPA-HF (Dapagliflozin And Prevention of Adverse-outcomes in Heart Failure) trial, the sodium-glucose cotransporter 2 inhibitor dapagliflozin decreased the risk of worsening HF events and cardiovascular death in patients with HF and reduced ejection fraction. Berberine is a nutraceutical compound characterized by multiple metabolic effects in patients with/without cardiovascular diseases. Recent preclinical systematic review indicated that Berberine significantly reduces myocardial infarct size and the incidence of ventricular arrhythmia, improves cardiac function, ameliorates myocardial apoptosis. Purpose To investigate on the potential cardioprotective properties of Berberine associated to SGLT2i Dapagliflozin against HER-2 blocking agent-induced cardiotoxicity Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of trastuzumab (200 nM) alone or co-incubated with Berberine (200 mM) or Dapagliflozin (50 nM) or both in combination for 48 h. After the incubation period, we performed the following tests: cell viability, apoptosis, expression of NLRP3 inflammasome, methylglyoxal and leukotrienes-B4. Expression of pAMPK was analyzed through western blot. Moreover, quantification of IL-6 was performed through ELISA method. Results Berberine and Dapagliflozin increased significantly the cell viability of cardiomyocytes exposed to Trastuzumab. When combined, Berberine and Dapagliflozin increased synergistically cell viability of cardiac cells (p&amp;lt;0.001 vs Trastuzumab). Cell apoptosis was reduced of 32.5, 41.8 and 72.7% for berberine, dapagliflozin and both combined (vs trastuzumab group). Methylglyoxal,a marker of AGEs, was strongly reduced compared to untreated cells. Western blot analysis clearly demonstrate that pAMPK was induced by berberine and Dapagliflozin, improving mitochondrial metabolism. No significant changes in Leukotriene B4 expression were seen. Intracellular levels of IL-6 were reduced of 46.3, 62.7 and 86.3% for berberine, dapagliflozin and both combined (vs trastuzumab group). Conclusion Berberine and Dapagliflozin exerts significant cardioprotective effects in cardiac cells exposed to the HER2-bloking agent Trastuzumab. Berberine and Dapagliflozin in combination induces an anti-inflammatory phenotype to myocardial cells through the reduction of biomarkers involved in heart failure and apoptosis Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Ricerca Corrente, Ministero della Salute

Interaction between autophagy and the NLRP3 inflammasome in Alzheimer's and Parkinson's disease.

Autophagy degrades phagocytosed damaged organelles, misfolded proteins, and various pathogens through lysosomes as an essential way to maintain cellular homeostasis. Autophagy is a tightly regulated cellular self-degradation process that plays a crucial role in maintaining normal cellular function and homeostasis in the body. The NLRP3 inflammasome in neuroinflammation is a vital recognition receptor in innate cellular immunity, sensing external invading pathogens and endogenous stimuli and further triggering inflammatory responses. The NLRP3 inflammasome forms an inflammatory complex by recognizing DAMPS or PAMPS, and its activation triggers caspase-1-mediated cleavage of pro-IL-1β and pro-IL-18 to promote the inflammatory response. In recent years, it has been reported that there is a complex interaction between autophagy and neuroinflammation. Strengthening autophagy can regulate the expression of NLRP3 inflammasome to reduce neuroinflammation in neurodegenerative disease and protect neurons. However, the related mechanism is not entirely clear. The formation of protein aggregates is one of the standard features of Neurodegenerative diseases. A large number of toxic protein aggregates can induce inflammation. In theory, activation of the autophagy pathway can remove the potential toxicity of protein aggregates and delay the progression of the disease. This article aims to review recent research on the interaction of autophagy, NLRP3 inflammasome, and protein aggregates in Alzheimer’s disease (AD) and Parkinson’s disease (PD), analyze the mechanism and provide theoretical references for further research in the future.

Sacubitril-valsartan activates pAMPK and reduces NLRP3, MyD88, cytokines/growth factors and DAMPs in doxorubicin-treated mice improving longitudinal strain and ejection fraction

Abstract Background Doxorubicin-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in breast cancer patients. Sacubitril-valsartan (LCZ 696) is a combination drug, made up of neprilysin inhibitor sacubitril and angiotensin II receptor blocker valsartan, used for the treatment of heart failure in patients with a reduced ejection fraction. Purpose We hypothesized that LCZ 696, administered during doxorubicin, could improve cardiac function Methods Female C57Bl/6 mice were untreated (Sham, n=6) or treated for 10 days with doxorubicin i.p at 2.17 mg/kg (DOXO, n=6), LCZ-696 at 60 mg/kg (LCZ, n=6) or doxorubicin combined to LCZ-696 (DOXO-LCZ, n=6). Ejection fraction, radial and longitudinal strain were analyzed through transthoracic echocardiography (Vevo 2100). Cardiac tissue expression of NLRP3 inflammasome, Myd88, DAMPs (galectine 3 and calgranulinS100), pAMPK, NF-kB, and 13 chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified through ELISA and western blot methods. Results LCZ 696 improved significantly the EF and prevented the reduction of radial and longitudinal strain after 10 days of treatment with doxorubicin. A reduced expression of NLRP3, MyD88, DAMPs and NF-kB in cardiac tissues was seen in DOXO-LCZ group compared to DOXO mice (p&amp;lt;0.001). Cardiac expression of IL-1β, IL-6, TNF-α, G-CSF and GM-CSF were significantly reduced after treatment with LCZ-696 indicating anti-inflammatory properties. Expression of pAMPK was strongly enhanced in LCZ-696-DOXO compared to DOXO group. Levels of Calgranulin S100 and galectine-3 were strongly enhanced in DOXO group; on the other hand their expression were reduced by 47.7 and 52.3% in LCZ-696-DOXO group vs DOXO (p&amp;lt;0.005). Conclusion In this preclinical study, LCZ-696 is able to improve cardiac function and reduce biomarkers involved in heart failure and fibrosis. The overall picture of the study pushes the use of Sacubitril-valsartan in prevention of cardiomyopathies induced by anthracyclines in cancer patients. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Ricerca Corrente, Ministero della Salute