Cathepsin B aggravates atherosclerosis in ApoE-deficient mice by modulating vascular smooth muscle cell pyroptosis through NF-κB / NLRP3 signaling pathway.

Atherosclerosis (AS) is a chronic inflammatory disease involving cell death and inflammatory responses. Pyroptosis, a newly discovered pro-inflammatory programmed cell death process, exacerbates inflammatory responses. However, the roles of cathepsin B (CTSB) in pyroptosis and AS remain unclear. To gain further insight, we fed ApoE-/- mice a high-fat diet to investigate the effects and mechanisms of CTSB overexpression and silencing on AS. We also explored the specific role of CTSB in vascular smooth muscle cells (VSMCs) in vitro. The study revealed that high-fat diet led to the formation of AS plaques, and CTSB was found to increase the AS plaque lesion area. Immunohistochemical and TUNEL/caspase-1 staining revealed the existence of pyroptosis in atherosclerotic plaques, particularly in VSMCs. In vitro studies, including Hoechst 33342/propidium iodide staining, a lactate dehydrogenase (LDH) release assay, detection of protein indicators of pyroptosis, and detection of interleukin-1ß (IL-1ß) in cell culture medium, demonstrated that oxidized low-density lipoprotein (ox-LDL) induced VSMC pyroptosis. Additionally, CTSB promoted VSMC pyroptosis. Ox-LDL increased the expression of CTSB, which in turn activated the NOD-like receptor protein 3 (NLRP3) inflammasome and promoted NLRP3 expression by facilitating nuclear factor kappa B (NF-κB) p65 nuclear translocation. This effect could be attenuated by the NF-κB inhibitor SN50. Our research found that CTSB not only promotes VSMC pyroptosis by activating the NLRP3 inflammasome, but also increases the expression of NLRP3.

Dapagliflozin Attenuates NLRP3/Caspase-1 Signaling Pathway-Mediated Pyroptosis of Vascular Smooth Muscle Cells by Downregulating CTSB.

Background: Atherosclerosis is a chronic inflammatory disease. Pyroptosis triggers and amplifies the inflammatory response and plays an important role in atherosclerosis. Cathepsin B (CTSB) can promote atherosclerosis and activate NOD-like receptor protein 3 (NLRP3) to mediate pyroptosis. Dapagliflozin (DAPA) can inhibit cell pyroptosis to improve atherosclerosis. This study aimed to explore the effect of DAPA on oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis of vascular smooth muscle cells (VSMCs) and its underlying mechanism. Objective: We aimed to investigate the effect of DAPA on ox-LDL-induced pyroptosis of VSMCs in mice and its underlying mechanism. Methods: VSMCs were transfected with CTSB-overexpressing and -silencing lentiviral vectors. VSMCs were treated with different concentrations of ox-LDL (0, 50, 100 and 150 µg/ml ). Then, Hoechst 33342/PI double staining, interleukin (IL)-1ß and lactate dehydrogenase (LDH) release assay were used to detect cell pyroptosis. Western blotting was used to detect pyroptosis indicators protein, based on which the appropriate concentration of ox-LDL was selected. After VSMCs were treated with different concentrations of DAPA (0.1 µM, 1.0 µM, 5.0 µM, 10 µM, 25 µM and 50 µM), the proliferative activity of VSMCs was detected using Cell Counting Kit-8 (CCK8) assay. After VSMCs were pretreated with different DAPA concentrations (0.1 µM, 1.0 µM, 5.0 µM and 10 µM) for 24 hours and then treated with 150 µg/mL ox-LDL for 24 hours, the effects of different concentrations of DAPA on pyroptosis of VSMCs were detected, based on which the appropriate DAPA concentration was selected. After lentivirus transfected VSMCs were treated with 150 µg/mL ox-LDL for 24 hours, the effects of overexpression and silencing of CTSB in pyroptosis were observed. On the basis of DAPA (0.1 µM)- and ox-LDL(150 µg/mL)-treated VSMCs, overexpression and silencing of CTSB were used to observe the effects of DAPA and CTSB on ox-LDL-mediated VSMCs pyroptosis. Results: (1) VSMCs stably transfected with CTSB-overexpressing and -silencing lentiviruses were obtained; 150 µg/mL was the optimal concentration of ox-LDL for inducing pyroptosis of VSMCs, and 0.1 µM was the optimal concentration of DAPA for ameliorating pyroptosis of VSMCs. (2) Ox-LDL-induced pyroptosis of VSMCs was worsened by CTSB overexpression but suppressed by CTSB silencing. (3) DAPA attenuated ox-LDL-induced pyroptosis of VSMCs through downregulating CTSB and NLRP3. (4) Overexpression of CTSB based on DAPA intervention aggravated ox-LDL-induced pyroptosis of VSMCs. Conclusion: DAPA attenuates NLRP3/caspase-1 pathway-mediated pyroptosis of VSMCs through downregulating CTSB.

FNDC5/PPARa Pathway Alleviates THP-1-derived Macrophage Pyroptosis and Its Mechanism.

Context: Atherosclerosis (AS) is a chronic inflammatory disease. Pyroptosis is a newly discovered, pro-inflammatory cell death that can trigger and amplify the occurrence and progression of AS. Researchers are still uncertain about the anti-atherosclerotic mechanism of "fibronectin type III domain-containing protein 5" (FNDC5). Objective: The study aimed to investigate the ability of FNDC5-mediated, "peroxisome proliferator activated receptor alpha" (PPARa) to inhibit oxidized low-density lipoprotein (ox-LDL)-induced, THP-1-derived macrophage pyroptosis and to determine a potential molecular mechanism at the cellular level. Design: The research team performed a laboratory study. Setting: The study took place in the Department of Cardiovascular Medicine at the Affiliated Hospital of Guzhou Medical University at the Medical Research Institute at Guizhou Medical University in Guiyang, Guizhou, China. Outcome Measures: The research team: (1) constructed and stably transfected FNDC5 gene-overexpressing and FNDC5 gene-silencing lentiviral vectors into THP-1 cells; (2) observed the cell morphology under an inverted fluorescence microscope and screened the stably transfected THP-1 cells with puromycin; (3) verified the transfection efficiency using quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot; (4) used phorbol to induce THP-1 cells into macrophages; (5) cultured the THP-1-derived macrophages with different concentrations of ox-LDL-25, 50, 75, and 100 µg/ml-for 24 h; (6) performed Hoechst 33342/ propidium iodide (PI) double staining and examined lactate dehydrogenase (LDH) and interleukin-1 beta (IL-1ß) activity to determine the effects of ox-LDL on THP-1-derived macrophage pyroptosis; (7) selected the optimal ox-LDL concentration; (8) divided the THP-1-derived macrophages into seven groups: NC group (no ox-LDL intervention), ox-LDL group, PBS group, Mock1 group, Ad-FNDC5 group, Mock2 group, and Sh-FNDC5 group; (9) examined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and "nuclear factor kappa-light chain enhancer of activated B cells P65" (NF-κB P65), and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining, respectively; (10) treated the THP-1-derived macrophages with FNDC5 expression with GW6471, a specific PPARα antagonist; (11) determined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and NF-κB P65, and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining and detection of the LDH and IL-1ß activity, respectively. Results: With the stably transfected THP-1 cells with FNDC5 overexpression or silencing the ox-LDL-induced, THP-1-derived, macrophage pyroptosis occurred in a concentration-dependent manner. Compared with the ox-LDL, phosphate buffered saline (PBS), Mock1, and Mock2 groups, the Ad-FNDC5 group had a significant increase in expression of FNDC5 and of peroxisome proliferator activated receptor alpha (PPARa) proteins (P < .05). The "nuclear factor kappa-light chain enhancer of activated B cells P65: (NF-κB P65), NOD-like receptor thermal protein domain associated protein 3, (NLRP3), Caspase-1, gasdermin D (GSDMD, IL-1ß and IL-18 protein expressions, percentage of PI-positive cells, LDH activity, and IL-1ß activity decreased significantly (P < .05); the results in the Sh-FNDC5 group were opposite to those in the Ad-FNDC5 group. 3. Intervention with GW6471 (PPARa antagonist) in the stably transfected THP-1-derived macrophages with FNDC5 overexpression abolished the protective effect of FNDC5 against ox-LDL-induced THP-1-derived macrophage pyroptosis. Conclusions: Irisin/PPARa inhibited THP-1-derived macrophage pyroptosis and inflammation and delayed AS by inhibiting the NF-κB/NLRP3 pathway.

Association between AT1 receptor gene polymorphism and left ventricular hypertrophy and arterial stiffness in essential hypertension patients: a prospective cohort study.

BACKGROUND: AT1 receptor gene (AGTR1) is related to essential hypertension (EH), and left ventricular hypertrophy (LVH) and arterial stiffness are common complications of EH. This study aimed to explore the association between AGTR1 genotype and LVH and arterial stiffness in EH patients. METHODS: A total of 179 EH patients were recruited in this study. Oral exfoliated cells were collected from each patient, and the genetic polymorphism of AGTR1(rs4524238) was assessed using a gene sequencing platform. The outcomes were LVH and arterial stiffness. RESULTS: Among 179 patients, 114 were with AGTR1 genotype of GG (57 males, aged 59.54 ± 13.49 years) and 65 were with AGTR1 genotype of GA or AA (36 males, aged 61.28 ± 12.79 years). Patients with AGTR1 genotype of GG were more likely to have LVH (47 [41.23%] vs. 14 [21.54%], P = 0.006) and arterial stiffness (30 [26.32%] vs. 8 [12.31%], P = 0.036). The AGTR1 polymorphism frequency was in accordance with Hardy-Weinberg equilibrium (P = 0.291). The multivariate logistic regression showed that AGTR1 genotype of GA or AA was independently associated with lower risk of LVH (OR = 0.344, 95%CI 160~0.696, P = 0.003) and arterial stiffness (OR = 0.371, 95%CI 0.155~0.885, P = 0.025) after adjusting for gender, age, and diabetes. CONCLUSION: EH patients with the AGTR1 genotype of GA or AA were at lower risk for LVH and arterial stiffness than those with the GG genotype.

Analysis of Atmospheric CO2 and CO at Akedala Atmospheric Background Observation Station, a Regional Station in Northwestern China.

Air samples were collected by flasks and analyzed via a Picarro G2401 gas analyzer for carbon dioxide (CO2) and carbon monoxide (CO) at the Akedala Atmospheric Background Station in Xinjiang, China, from September 2009 to December 2019, to analyze the changes in the characteristics of atmospheric CO2 and CO and determine the sources. The results show that the annual average CO2 concentration showed an increasing trend (growth rate: 1.90 ppm year-1), ranging from 389.80 to 410.43 ppm, and the annual average CO concentration also showed an increasing trend (growth rate: 1.78 ppb year-1), ranging from 136.30 to 189.82 ppb. The CO2 concentration and growth rate were the highest in winter, followed by autumn, spring, and summer. The CO concentration and growth rate were also the highest in winter due to anthropogenic emissions, ecosystem effects, and diffusion conditions. The main trajectories of CO2 and CO determined by the Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model were parallel to the Irtysh River valley and then passed through the Old Wind Pass. Furthermore, the main source regions of CO2 and CO at the Akedala Station were eastern Kazakhstan, southern Russia, western Mongolia, and the Xinjiang Tianshan North Slope Economic Zone of China. This study reflects the characteristics of long-term changes in CO2 and CO concentrations at the Akedala station and provides fundamental data for the studies on environmental changes and climate change in Central Asia.