UNVEILING THE PROTECTIVE MECHANISMS OF PUERARIN AGAINST ACUTE LUNG INJURY: A COMPREHENSIVE EXPLORATION OF THE ROLES AND MECHANISMS OF MST1/ERS SIGNALING.

ABSTRACT: Objectives: Puerarin, the principal active constituent extracted from Pueraria, is believed to confer protection against sepsis-induced lung injury. The study aimed to elucidate the role and mechanism of Mst1/ERS in puerarin-mediated protection against acute lung injury (ALI). Methods: Monolayer vascular endothelial cell permeability was assessed by gauging the paracellular flow of FITC-dextran 40,000 (FD40). ELISA was employed for the quantification of inflammatory cytokines. Identification of target proteins was conducted through western blotting. Histological alterations and apoptosis were scrutinized using hematoxylin-eosin staining and TUNEL staining, respectively. The ultrastructure of the endoplasmic reticulum was observed via transmission electron microscopy. Results: Puerarin significantly protected mice from LPS-induced ALI, reducing lung interstitial width, neutrophil and lymphocyte infiltration, pulmonary interstitial and alveolar edema, and lung apoptosis. Puerarin treatment also markedly attenuated levels of TNF-α and IL-1ß in both alveolar lavage fluid and serum. Furthermore, puerarin significantly attenuated LPS-induced increases in Mst1, GRP78, CHOP, and Caspase12 protein expression and blunted LPS-induced decrease in ZO-1 protein expression in lung tissues. Puerarin obviously reduced endoplasmic reticulum expansion and vesiculation. Similarly, puerarin significantly mitigated the LPS-induced reduction in HUVEC cell viability and ZO-1 expression. Puerarin also attenuated LPS-induced increase in apoptosis, TNF-α and IL-1ß, FD40 flux, and Mst1, GRP78, CHOP, and Caspase12 expression in HUVEC cells. Nevertheless, the inhibitory impact of puerarin on vascular endothelial cell injury, lung injury, and endoplasmic reticulum stress (ERS) was diminished by Mst1 overexpression. Conclusion: These findings demonstrated that the Mst1/ERS signaling pathway played a pivotal role in the development of LPS-induced vascular endothelial cell dysfunction and ALI. Puerarin exhibited the ability to attenuate LPS-induced vascular endothelial cell dysfunction and ALI by inhibiting the Mst1/ERS signaling pathway.

Metformin Protects Against Acute Kidney Injury Induced by Lipopolysaccharide via Up-Regulating the MCPIP1/SIRT1 Pathway.

In the present study, we aimed to explore the effect and underlying mechanism of metformin on lipopolysaccharide (LPS)-induced acute kidney injury (AKI). A total of 24 BALB/C mice were randomly divided into four groups: control group, LPS group and metformin group (50 or 100 mg/kg). The histological changes and cell apoptosis in kidney tissues were detected by hematoxylin-eosin staining and terminal-deoxynucleotidyl transferase-mediated nick end labeling assay, respectively. Enzyme-linked immunosorbent assay was applied to determine serum levels of blood urea nitrogen (BUN), kidney injury molecule-1 (Kim-1), creatinine (Cre), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Western blotting analysis were carried out to confirm the expressions of monocyte chemotactic protein-inducible protein 1 (MCPIP1), silent information regulator sirtuin 1 (SIRT1), and NF-κB p65 (acetyl K310). Compared with the control group, the mice in LPS group had glomerular capillary dilatation, renal interstitial edema, tubular cell damage and apoptosis. The serum levels of BUN, KIM-1, Cre, TNF-α, and IL-1ß in LPS group were significantly higher than those in control group. Moreover, LPS also elevated the expressions of MCPIP1 and NF-κB p65 (acetyl K310) but decreased the expression of SIRT1 in kidney tissues. However, metformin distinctly decreased LPS-induced renal dysfunction, the serum levels of BUN, KIM-1, Cre, TNF-α, and IL-1ß. In addition, metformin markedly increased the expressions of MCPIP1 and SIRT1 but decreased the expression of NF-κB p65 (acetyl K310) in kidney tissues. Metformin prevented LPS-induced AKI by up-regulating the MCPIP1/SIRT1 signaling pathway and subsequently inhibiting NF-κB-mediated inflammation response.

Nicorandil alleviates cardiac remodeling and dysfunction post -infarction by up-regulating the nucleolin/autophagy axis.

OBJECTIVE: The present study aimed to investigate whether the drug nicorandil can improve cardiac remodeling after myocardial infarction (MI) and the underlying mechanisms. METHODS: Mouse MI was established by the ligation of the left anterior descending coronary artery and H9C2 cells were cultured to investigate the underlying molecular mechanisms. The degree of myocardial collagen (Col) deposition was evaluated by Masson's staining. The expressions of nucleolin, autophagy and myocardial remodeling-associated genes were measured by Western blotting, qPCR, and immunofluorescence. The apoptosis of myocardial tissue cells and H9C2 cells were detected by TUNEL staining and flow cytometry, respectively. Autophagosomes were observed by transmission electron microscopy. RESULTS: Treatment with nicorandil mitigated left ventricular enlargement, improved the capacity of myocardial diastolic-contractility, decreased cardiomyocyte apoptosis, and inhibited myocardial fibrosis development post-MI. Nicorandil up-regulated the expression of nucleolin, promoted autophagic flux, and decreased the expressions of TGF-ß1 and phosphorylated Smad2/3, while enhanced the expression of BMP-7 and phosphorylated Smad1 in myocardium. Nicorandil decreased apoptosis and promoted autophagic flux in H2O2-treated H9C2 cells. Autophagy inhibitors 3-methyladenine (3MA) and chloroquine diphosphate salt (CDS) alleviated the effects of nicorandil on apoptosis. Knockdown of nucleolin decreased the effects of nicorandil on apoptosis and nicorandil-promoted autophagic flux of cardiomyocytes treated with H2O2. CONCLUSIONS: Treatment with nicorandil alleviated myocardial remodeling post-MI through up-regulating the expression of nucleolin, and subsequently promoting autophagy, followed by regulating TGF-ß/Smad signaling pathway.

The role of PSGL-1 in pathogenesis of systemic inflammatory response and coagulopathy in endotoxemic mice.

INTRODUCTION: Endotoxemia often results in systemic inflammatory response syndrome (SIRS), coagulation disturbance and acute lung injury (ALI), and such a condition is associated with the activation of platelets, leukocytes and vascular endothelial cells (VECs). P-selectin glycoprotein ligand 1 (PSGL-1) is a key regulatory molecule in the activation of platelets, leukocytes and VECs. However, it still remains largely unexplored whether PSGL-1 plays an important role in SIRS, coagulation dysfunction and ALI of endotoxemia. In the present study, we aimed to study the role of PSGL-1 in above-mentioned situations using endotoxemic mice. MATERIALS AND METHODS: An endotoxemia model was established in BALB/c mice via lipopolysaccharide (LPS) administration. Moreover, the mice were simultaneously injected with PSGL-1 antibody for intervention. The survival rate, morphologic changes of lung tissues, platelet-leukocyte adhesion, tissue factor expression on leukocytes, fibrinogen deposition in lung tissues, serum levels of inflammatory factors and the activation of VECs were determined. RESULTS: The results showed that the aggregation and recruitment of platelets and leukocytes in lung tissues, the expression of tissue factor on leukocytes, the serum levels of inflammatory factors, the activation of VECs, and the fibrinogen deposition in lung tissues were increased in endotoxemic mice, which were significantly alleviated by administration of PSGL-1 antibody. Moreover, blockade of PSGL-1 markedly increased survival rate, and alleviated coagulation disturbance and lung injury in endotoxemic mice. CONCLUSIONS: Taken together, PSGL-1 played an important role in pathogenesis of SIRS and coagulation dysfunction and ALI in endotoxemic mice.

Puerarin Protects Against LPS-Induced Vascular Endothelial Cell Hyperpermeability via Preventing Downregulation of Endothelial Cadherin.

In the present study, we aimed to investigate the effects of puerarin on the hyperpermeability of vascular endothelial cells induced by lipopolysaccharide (LPS) and its underlying mechanisms. Human umbilical vein endothelial cells (HUVECs) were pre-incubated with puerarin (25, 50, and 100 µM) for 1 h, and then exposed to LPS (1 µg/mL). The monolayer permeability of endothelial cells was assessed by measuring the paracellular flux of FITC-dextran 40,000 (FD40). The expression of vascular endothelial cadherin (VE-cadherin) in HUVECs was examined by Western blotting analysis. A total of 18 mice were randomly assigned into three groups as follows: control group, LPS group, and puerarin group. The pulmonary W/D ratio (wet-to-dry weight ratios) was calculated, and the lung morphology was examined. The levels of TNF-α and IL-1ß in cell supernatant and mouse serum were determined by ELISA. Compared with the control group, LPS obviously increased the flux of FD40 and the monolayer permeability, raised the levels of TNF-α and IL-1ß in cell supernatant, and reduced the VE-cadherin expression in HUVECs. However, puerarin (25, 50, and 100 µM) was able to relieve such LPS-induced increase in flux of FD40 and then reduce the hyperpermeability. Puerarin decreased the levels of TNF-α and IL-1ß in cell supernatant and increased the VE-cadherin expression in HUVECs (P < 0.05). Moreover, LPS obviously increased the levels of TNF-α and IL-1ß in mouse serum and elevated the pulmonary W/D ratios, resulting in lung injury. However, all of above-mentioned LPS-induced changes were improved by puerarin pre-treatment. Puerarin could alleviate LPS-induced hyperpermeability in endothelial cells via preventing downregulation of endothelial cadherin.

Clopidogrel reduces lipopolysaccharide-induced inflammation and neutrophil-platelet aggregates in an experimental endotoxemic model.

Platelet activation contributes to organs failure in inflammation and plays an important role in endotoxemia. Clopidogrel inhibits platelet aggregation and activation. However, the role of clopidogrel in modulating inflammatory progression of endotoxemia remains largely unexplored. Therefore, we investigated the role of clopidogrel on the activation of platelet and leukocytes in lipopolysaccharide (LPS)-induced inflammation in mice. Animals were treated with clopidogrel or vehicle before LPS induction. The expression of neutrophil-platelet aggregates and platelet activation and tissue factor was determined. Immunofluorescence was used to analyze platelet-leukocyte interactions and tissue factor (TF) expression on leukocytes. Clopidogrel pretreatment markedly decreased lung damage, inhibited platelet-neutrophil aggregates and TF expression. In addition, clopidogrel reduced thrombocytopenia and affected the number of circulating white blood cell in endotoxemia mice. Moreover, clopidogrel also reduced platelet shedding of CD40L and CD62P in endotoxemic mice. Taken together, clopidogrel played an important role through reducing platelet activation and inflammatory process in endotoxemia.

Puerarin prevents vascular endothelial injury through suppression of NF-κB activation in LPS-challenged human umbilical vein endothelial cells.

OBJECTIVE: In the present study, we aimed to explore the effects of puerarin on vascular endothelial cell injury induced by lipopolysaccharide (LPS) and its underlying mechanisms. METHODS: The cell viability and morphological changes were assessed using the cell counting kit-8 (CCK-8) assay and 4´,6-diamidino-2-phenylindole (DAPI) staining, respectively. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), monocyte/macrophage chemotactic protein-1 (MCP-1), IL-8, intercellular cell adhesion molecule-1 (ICAM-1), thrombomodulin (TM) and plasminogen activator inhibitor-1 (PAI-1) in cell culture supernatant were determined by the enzyme-linked immunosorbent assay (ELISA). The neutrophils adhesion to endothelial cells were examined by myeloperoxidase activity assay. The nuclear translocation of nuclear factor-κB p65 (NF-κB p65) was assessed by immunofluorescence analysis. RESULTS: Compared with the control group, LPS challenge significantly injured human umbilical vein endothelial cells (HUVECs) and increased the levels of TNF-α, IL-1ß, MCP-1, IL-8, ICAM-1, TM and PAI-1 in the cell culture supernatants. The neutrophils adhesion to endothelial cells were significantly increased in LPS-challenged HUVECs. Moreover, LPS challenge increased the nuclear translocation of NF-κB p65. However, puerarin pre-treatment attenuated the vascular endothelial injury and reduced the levels of TNF-α, IL-1ß, MCP-1, IL-8, ICAM-1, TM and PAI-1 in cell supernatants of LPS-challenged HUVECs. In addition, the neutrophils adhesion to HUVECs induced by LPS were also decreased by puerarin pre-treatment. Furthermore, puerarin pre-treatment reduced the nuclear translocation of NF-κB p65 elicited by LPS. CONCLUSIONS: Puerarin prevented LPS-induced vascular endothelial injury, the mechanism of which might be related to the suppression of NF-κB activation and subsequently altered levels of inflammatory factors and coagulation-related factors.

Puerarin inhibits expression of tissue factor induced by oxidative low-density lipoprotein through activating the PI3K/Akt/eNOS pathway and inhibiting activation of ERK1/2 and NF-κB.

AIMS: The present study aimed to investigate whether puerarin regulated tissue factor (TF) expression induced by oxidative low-density lipoprotein (ox-LDL), an independent risk factor for atherosclerosis, and its mechanisms. MAIN METHODS: TF expression at the mRNA level was determined by reverse transcription-quantitative polymerase chain reaction, and its expression at the protein level, as well as other target proteins, was assessed by western blotting. Nitric oxide (NO) production was measured by a nitrate reduction method. KEY FINDINGS: Results demonstrated that treatment with ox-LDL (50mg/l) for 24h significantly increased (P<0.01) TF expression at the mRNA and protein levels in human umbilical vein endothelial cells (HUVECs). Such an ox-LDL exposure also triggered the dephosphorylation of Akt, resulting in a reduction of NO production and activated the extracellular signal-regulated kinase (ERK)1/2 and nuclear factor (NF)-κB signaling pathways. Pre-treatment with puerarin (50-200µM) for 1h significantly attenuated the ox-LDL-induced TF expression, augmented the phosphorylation of Akt, with a resultant increase of the NO production, and inhibited the activation of ERK1/2 and NF-κB (P<0.01). However, this beneficial effect of puerarin (100µM) was abolished by LY294002 (10µM), an inhibitor of phosphoinositide 3-kinase (PI3K), or NG-nitro-L-arginine methyl ester (100µM), an inhibitor of NO synthase. SIGNIFICANCE: These results suggested that puerarin suppressed TF expression in HUVECs through activating the PI3K/Akt/endothelial nitric oxide synthase signaling pathway and inhibiting the activation of ERK1/2 and NF-κB. These findings suggested that puerarin possessed certain anticoagulation and may be a potential novel therapeutic drug for thrombosis in coronary artery disease.

Effect of pravastatin on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine in rat aorta.

AIM: To investigate the effects of pravastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine (LPC), the major component of oxidized low-density lipoprotein, in rat thoracic aorta. METHODS: Both the endothelium-dependent relaxation response to acetylcholine and the endothelium-independent relaxation response to sodium nitroprusside of aortic rings were measured by recording isometric tension after the rings were exposed to LPC in the absence or presence of pravastatin to estimate the injury effect of LPC and the protective effect of pravastatin on the aortic endothelium, respectively. RESULTS: Exposure of aortic rings to LPC (1-10 micromol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation to acetylcholine, but did not affect endothelium-independent relaxation in response to sodium nitroprusside. Pre-incubation of aortic rings with pravastatin (0.3-3 mmol/L) for 15 min and then co-incubation of the rings with LPC (3 micromol/L) for another 30 min significantly attenuated the inhibition of endothelium-dependent relaxation induced by LPC. This protective effect of pravastatin (1 mmol/L) was abolished by NG-nitro-L-arginine methyl ester (30 micromol/L), an inhibitor of nitric oxide synthase, but not by indomethacin (10 micromol/L), an inhibitor of cyclooxygenase. Moreover, protein kinase C inhibitor chelerythrine (1 micromol/L) the superoxide anion scavenger superoxide dismutase (200 kU/L), and the nitric oxide precursor L-arginine (3 mmol/L) also improved the impaired endothelium-dependent relaxation induced by LPC, similar to the effects of pravastatin. CONCLUSION: Pravastatin can protect the endothelium against functional injury induced by LPC in rat aorta, a fact which is related to increasing nitric oxide bioavailability.