Human amniotic MSCs-mediated anti-inflammation of CD206hiIL-10hi macrophages alleviates isoproterenol-induced ventricular remodeling in mice.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammation which makes them suitable for the treatment of various diseases. OBJECTIVE: This study aimed to explore the therapeutic effect and molecular mechanism of hAMSCs in ventricular remodeling (VR). METHODS: hAMSCs were characterized by a series of experiments such as flow cytometric analysis, immunofluorescence, differentiative induction and tumorigenicity. Mouse VR model was induced by isoproterenol (ISO) peritoneally, and the therapeutic effects and the potential mechanisms of hAMSCs transplantation were evaluated by echocardiography, carboxy fluorescein diacetate succinimidyl ester (CFSE) labeled cell tracing, histochemistry, qRT-PCR and western blot analysis. The co-culturing experiments were carried out for further exploring the mechanisms of hAMSCs-derived conditioned medium (CM) on macrophage polarization and fibroblast fibrosis in vitro. RESULTS: hAMSCs transplantation significantly alleviated ISO-induced VR including cardiac hypertrophy and fibrosis with the improvements of cardiac functions. CFSE labeled hAMSCs kept an undifferentiated state in heart, indicating that hAMSCs-mediated the improvement of ISO-induced VR might be related to their paracrine effects. hAMSCs markedly inhibited ISO-induced inflammation and fibrosis, seen as the increase of M2 macrophage infiltration and the expressions of CD206 and IL-10, and the decreases of CD86, iNOS, COL3 and αSMA expressions in heart, suggesting that hAMSCs transplantation promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages. Mechanically, hAMSCs-derived CM significantly increased the expressions of CD206, IL-10, Arg-1 and reduced the expressions of iNOS and IL-6 in RAW264.7 macrophages in vitro. Interestingly, RAW264.7-CM remarkably promoted the expressions of anti-inflammatory factors such as IL-10, IDO, and COX2 in hAMSCs. Furthermore, the CM derived from hAMSCs pretreated with RAW264.7-CM markedly inhibited the expressions of fibrogenesis genes such as αSMA and COL3 in 3T3 cells. CONCLUSION: Our results demonstrated that hAMSCs effectively alleviated ISO-induced cardiac hypertrophy and fibrosis, and improved the cardiac functions in mice, and the underlying mechanisms might be related to inhibiting the inflammation and fibrosis during the ventricular remodeling through promoting the polarization of CD206hiIL-10hi macrophages in heart tissues. Our study strongly suggested that by taking the advantages of the potent immunosuppressive and anti-inflammatory effects, hAMSCs may provide an alternative therapeutic approach for prevention and treatment of VR clinically.

Sugarcane leaf polysaccharide exerts a therapeutic effect on cardiovascular diseases through necroptosis.

Background: Necroptosis, a novel form of programmed cell death wherein the necrotic morphology is characterized by swelling of the cells, rupture of the plasma membrane, and dysfunction of the organelle, has been always observed in cardiovascular diseases. Sugarcane leaf polysaccharide (SLP) are primary components present in sugarcane leaves that exert cardiovascular protective effects. However, the positive effect of SLP and underlying mechanisms in myocardial ischemia-reperfusion (MI/R) remain unexplored. Aim: In this study, the protective effects of SLP on MI/R injury were investigated under in vitro and in vivo conditions. Methods: The protective effects of SLP on MI/R injury were assessed using tertiary butyl hydrogen peroxide (TBHP)-stimulated-H9c2 cells in the in vitro assay and using Sprague Dawley rats in the in vivo assay. Results: In vitro, SLP significantly reversed TBHP-induced H9c2 cell death by inhibiting necroptosis and oxidative stress. SLP exerted antioxidant activity through the Nrf2/HO-1 pathway. SLP suppressed necroptosis by decreasing phosphorylation of RIP1, RIP3, and MLKL in TBHP-stimulated H9c2 cells. In vivo, SLP attenuated MI/R injury by decreasing the myocardial infarct area; increasing myeloperoxidase and superoxide dismutase levels; and reducing malondialdehyde, interleukin-6, and tumor necrosis factor-α levels.

Discovery of New Heterocyclic/Benzofuran Hybrids as Potential Anti-Inflammatory Agents: Design, Synthesis, and Evaluation of the Inhibitory Activity of Their Related Inflammatory Factors Based on NF-κB and MAPK Signaling Pathways.

NF-κB and MAPK are classic inflammation signaling pathways which regulate inflammation signal transmission and induce the expression of many inflammatory factors. Based on the potent anti-inflammatory activity of benzofuran and its derivatives, several new heterocyclic/benzofuran hybrids were first designed and synthesized by molecular hybridization. Their structure was confirmed by 1H NMR, 13C NMR, HRMS or X-single crystal diffraction. The anti-inflammatory activity of these new compounds was screened by compounds; compound 5d exhibited an excellent inhibitory effect on the generation of NO (IC50 = 52.23 ± 0.97 µM), and low cytotoxicity (IC50 > 80 µM) against the RAW-264.7 cell lines. To further elucidate the possible anti-inflammatory mechanisms of compound 5d, the hallmark protein expressions of the NF-κB and MAPK pathways were studied in LPS-stimulated RAW264.7 cells. The results indicate that compound 5d not only significantly inhibits the phosphorylation levels of IKKα/IKKß, IKßα, P65, ERK, JNK and P38 in the classic MAPK/NF-κB signaling pathway in a dose-dependent manner, but also down-regulates the secretion of pro-inflammatory factors such as NO, COX-2, TNF-α and IL-6. Further, the in vivo anti-inflammatory activity of compound 5d indicated that it could regulate the involvement of neutrophils, leukocytes and lymphocytes in inflammation processes, and reduce the expression of IL-1ß, TNF-α and IL-6 in serum and tissues. These results strongly suggest that the piperazine/benzofuran hybrid 5d has a good potential for developing an anti-inflammatory lead compound, and the anti-inflammatory mechanism might be related to the NF-κB and MAPK signaling pathways.

Hederasaponin C Alleviates Lipopolysaccharide-Induced Acute Lung Injury In Vivo and In Vitro Through the PIP2/NF-κB/NLRP3 Signaling Pathway.

Gene transcription is governed by epigenetic regulation that is essential for the pro-inflammatory mediators surge following pathological triggers. Acute lung injury (ALI) is driven by pro-inflammatory cytokines produced by the innate immune system, which involves the nod-like receptor 3 (NLRP3) inflammasome and nuclear factor-κB (NF-κB) pathways. These two pathways are interconnected and share a common inducer the phosphatidylinositol 4,5-bisphosphate (PIP2), an epigenetic regulator of (Ribosomal ribonucleic acid (rRNA) gene transcription, to regulate inflammation by the direct inhibition of NF-κB phosphorylation and NLRP3 inflammasome activation. Herein, we report that hederasaponin C (HSC) exerted a therapeutic effect against ALI through the regulation of the PIP2/NF-κB/NLRP3 signaling pathway. In lipopolysaccharide (LPS)/lipopolysaccharide + adenosine triphosphate (LPS+ATP)-stimulated macrophages, our results showed that HSC remarkably inhibited the secretion of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α). Moreover, HSC inhibited NF-κB/p65 nuclear translocation and the binding of PIP2 to transforming growth factor-ß activated kinase 1 (TAK1). The intracellular calcium (Ca2+) level was decreased by HSC via the PIP2 signaling pathway, which subsequently inhibited the activation of NLRP3 inflammasome. HSC markedly alleviated LPS-induced ALI, restored lung function of mice, and rescued ALI-induced mice death. In addition, HSC significantly reduced the level of white blood cells (WBC), neutrophils, and lymphocytes, as well as pro-inflammatory mediators like IL-6, IL-1ß, and TNF-α. Hematoxylin and eosin (H&E) staining results suggested HSC has a significant therapeutic effect on lung injury of mice. Interestingly, the PIP2/NF-κB/NLRP3 signaling pathway was further confirmed by the treatment of HSC with ALI, which is consistent with the treatment of HSC with LPS/LPS+ATP-stimulated macrophages. Overall, our findings revealed that HSC demonstrated significant anti-inflammatory activity through modulating the PIP2/NF-κB/NLRP3 axis in vitro and in vivo, suggesting that HSC is a potential therapeutic agent for the clinical treatment of ALI.

Tanshinone I exerts cardiovascular protective effects in vivo and in vitro through inhibiting necroptosis via Akt/Nrf2 signaling pathway.

BACKGROUND: Tanshinone I (TI) is a primary component of Salvia miltiorrhiza Bunge (Danshen), which confers a favorable role in a variety of pharmacological activities including cardiovascular protection. However, the exact mechanism of the cardiovascular protection activity of TI remains to be illustrated. In this study, the cardiovascular protective effect and its mechanism of TI were investigated. METHODS: In this study, tert-butyl hydroperoxide (t-BHP)-stimulated H9c2 cells model was employed to investigate the protective effect in vitro. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) kit. The reactive-oxygen-species (ROS) level and mitochondrial membrane potential (MMP) were investigated by the flow cytometry and JC-1 assay, respectively. While in vivo experiment, the cardiovascular protective effect of TI was determined by using myocardial ischemia-reperfusion (MI/R) model including hematoxylin-eosin (H&E) staining assay and determination of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release were detected by Enzyme-linked immunosorbent assay (ELISA). Receptor interacting protein kinase 1 (RIP1), receptor interacting protein kinase 3 (RIP3), receptor interacting protein kinase 3 (MLKL), protein kinase B (Akt), Nuclear factor erythroid 2 related factor 2 (Nrf2), Heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO-1) were determined by western blotting. RESULTS: Our data demonstrated that TI pretreatment attenuated t-BHP and MI/R injury-induced necroptosis by inhibiting the expression of p-RIP1, p-RIP3, and p-MLKL. TI activated the Akt/Nrf2 pathway to promote the expression of antioxidant-related proteins such as phosphorylation of Akt, nuclear factor erythroid 2 related factor 2 (Nrf2), quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expression in t-BHP-stimulated H9c2 cells. TI relieved oxidative stress by mitigating ROS generation and reversing MMP loss. In vivo experiment, TI made electrocardiograph (ECG) recovery better and lessened the degree of myocardial tissue damage. The counts of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), and the release of TNF-α and IL-6 were reversed by TI treatment. SOD level was increased, while MDA level was decreased by TI treatment. CONCLUSION: Collectively, our findings indicated that TI exerted cardiovascular protective activities in vitro and in vivo through suppressing RIP1/RIP3/MLKL and activating Akt/Nrf2 signaling pathways, which could be developed into a cardiovascular protective agent.

Oleuropein Attenuates Lipopolysaccharide-Induced Acute Kidney Injury In Vitro and In Vivo by Regulating Toll-Like Receptor 4 Dimerization.

Acute kidney injury (AKI) is a common critical illness that involves multiple systems and multiple organs with a rapid decline in kidney function over short period. It has a high mortality rate and presents a great treatment challenge for physicians. Oleuropein, the main active constituent of Ilex pubescens Hook. et Arn. var. kwangsiensis Hand.-Mazz. displays significant anti-inflammatory activity, although oleuropein's therapeutic effect and mechanism of action in AKI remain to be elucidated. The present study aimed to further clarify the mechanism by which oleuropein exerts effects on inflammation in vitro and in vivo. In vitro, the inflammatory effect and mechanism were investigated through ELISA, Western blotting, the thermal shift assay, co-immunoprecipitation, and immunofluorescence staining. Lipopolysaccharide (LPS) induced acute kidney injury was employed in an animal model to investigate oleuropein's therapeutic effect on AKI and mechanism in vivo. The underlying mechanisms were investigated by Western blot analysis of kidney tissue. In LPS-stimulated macrophages, our data demonstrated that oleuropein significantly reduced the expression of inflammatory mediators like NO, IL-6, TNF-α, iNOS, and COX-2. Moreover, oleuropein inhibited NF-κB/p65 translocation, and had a negative regulatory effect on key proteins in the NF-κB and MAPK pathways. In addition, the thermal shift and co-immunoprecipitation assays revealed that oleuropein played an essential role in binding to the active sites of TLR4, as well as inhibiting TLR4 dimerization and suppressing the binding of TLR4 to MyD88. Oleuropein markedly alleviated LPS induced acute kidney injury, decreased serum creatinine and blood urea nitrogen (BUN) levels and proinflammatory cytokines. More importantly, the TLR4-MyD88-NF-κB/MAPK pathways were confirmed to play an important role in the oleuropein treatment of AKI. In this study, oleuropein exhibited excellent anti-inflammatory effects by regulating TLR4-MyD88-NF-κB/MAPK axis in vitro and in vivo, suggesting oleuropein as a candidate molecule for treating AKI.

Nuezhenide Exerts Anti-Inflammatory Activity through the NF-κB Pathway.

BACKGROUND: Nuezhenide (NZD), an iridoid glycoside isolated from Ilex pubescens Hook. & Arn. var. kwangsiensis Hand.-Mazz., used as a traditional Chinese medicine for clearing away heat and toxic materials, displays a variety of biological activities such as anti-tumor, antioxidant, and other life-protecting activities. However, a few studies involving anti-inflammatory activity and the mechanism of NZD have also been reported. In the present study, the anti-inflammatory and antioxidative effects of NZD are illustrated. OBJECTIVE: This study aims to test the hypothesis that NZD suppresses LPS-induced inflammation by targeting the NF-κB pathway in RAW264.7 cells. METHODS: LPS-stimulated RAW264.7 cells were employed to detect the effect of NZD on the release of cytokines by ELISA. Protein expression levels of related molecular markers were quantitated by western blot analysis. The levels of ROS, NO, and Ca2+ were detected by flow cytometry. The changes in mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were observed and verified by fluorescence microscopy. Using immunofluorescence assay, the translocation of NF-κB/p65 from the cytoplasm into the nucleus was determined by confocal microscopy. RESULTS: NZD exhibited anti-inflammatory activity and reduced the release of inflammatory cytokines such as nitrite, TNF-α, and IL-6. NZD suppressed the expression of the phosphorylated proteins like IKKα/ß, IκBα, and p65. Besides, the flow cytometry results indicated that NZD inhibited the levels of ROS, NO, and Ca2+ in LPS-stimulated RAW264.7 cells. JC-1 assay data showed that NZD reversed LPS-induced MMP loss. Furthermore, NZD suppressed LPS-induced NF-B/p65 translocation from the cytoplasm into the nucleus. CONCLUSION: NZD exhibits anti-inflammatory effects through the NF-κB pathway on RAW264.7 cells.