Fig 1: Effect of 25-HC and 25-HC@DDAB in the rescued survival rate of septic mice model. (a) Time-course survival rate of the cecal ligation and puncture (CLP) mice model after the treatment of 25-HC and 25-HC@DDAB (n = 10/each group). (b) Histological analysis of lung tissue from CLP mice models. The administration of 25-HC@DDAB reduced the blood vessel rupture and lung tissue damage. Representative images from each group are shown (n = 5). Scale bar, 100 µm. (c) Changes of inflammation-related signatures in mice models after the administration of 25-HC and 25-HC@DDAB after the CLP treatment. The in vivo signatures includes serum 25-HC level (n = 10), vascular permeability (n = 5), ICAM-1 (n = 5) level in lung tissue, leukocyte (n = 5) and neutrophil migration (n = 5) in bronchoalveolar lavage (BAL), and SREBP2 activity (n = 10) in lung tissue. (d) Changes of septic markers in the blood of 25-HC and 25-HC@DDAB treated CLP mouse models. The markers include CRP, LDH, ALT, AST, BUN, and creatinine (n = 5). (e) Changes of SREBP2-related mRNA expression after the treatment of 25-HC and 25-HC@DDAB. The mRNAs related to SREBP2 are Nox2, Nrlp3, IL1b, Mcp1, Icam1, and Srebp2 (n = 5). (f) Changes of cytokine levels after the treatment of 25-HC and 25-HC@DDAB in CLP mouse models. The cytokines include IL-1β, IL-6, IL-8, and TNF-α (n = 5). Statistical analysis was performed using a two-tailed unpaired t-test. *P < 0.05, **P < 0.01.
Fig 2: Inhibition of SREBP-2 and NF-κB prevents the inflammatory cytokines and mRNA levels related to SREBP-2. a Activation level of SREBP-2 in COVID-19 ICU patients’s PBMCs by the treatment of NF-κB inhibitor (SN50) and SREBP-2 inhibitor (Fatostatin A) (**p < 0.01). b, c Level of IL-1β (b) and TNF-α (c) in COVID-19 ICU patients’s PBMCs by the treatment of SN50 and Fatostatin A (**p < 0.01). d–i Changed level of mRNA after the treatment of SN50 and Fatostatin A. d SREBF2, e IL-1β, f TNF-α, g SCAP, h INSIG1, and i SIRT1 (**p < 0.01)
Fig 3: SREBP-2 C-term reflects the severity of infectious diseases, and thus can be used as a diagnostic marker. a Level of SREBP-2 C-term in COVID-19 patients’ plasma (**p < 0.01). b Level of SREBP-2 C-term in survival and deceased patients’ plasma (**p < 0.01). c Level of SREBP-2 C-term in the plasma of pneumonia, sepsis, and septic shock patients (**p < 0.01). d, e Level of lactate dehydrogenase (LDH) (d) and C-reactive protein (CRP) (e) in nonICU and ICU COVID-19 patients (**p < 0.01). f Computed tomography (CT) images of COVID-19 patients’ lung depending on the level of SREBP-2 C-term (**p < 0.01, n.s. not significant)
Fig 4: SREBP-2 activation is critical for the vascular inflammatory responses via cholesterol release and cytokine expression. a Western blot analysis of N-term and C-term of SREBP-2 in whole cell lysate (WCL) and supernatant (Sup.) after the LPS stimulation (1 μg/ml). b Time-dependent NF-κB activation after the LPS stimulation (1 μg/ml) (**p < 0.01). c Time-dependent secretion of SREBP-2 C-term in WCL and Sup. after the LPS stimulation (1 μg/ml) (**p < 0.01). d Filipin staining after the LPS stimulation (Scale bar: 200 μm). e Western blot analysis of ATP-binding cassette transporter (ABCA1) in human umbilical vein endothelial cell (HUVEC) after the LPS stimulation (1 μg/ml). f Effect of SREBP-2 knockdown in the suppression of cytokine production. g Transendothelial permeability of HUVEC after the inhibition or overexpression (O/E) of relevant signaling (*p < 0.05, **p < 0.01)
Fig 5: Analysis of patients’ blood revealed the SREBP-2 as a severity diagnostic marker for COVID-19. a Activation level of SREBP-2 with respect to the severity of COVID-19 (**p < 0.01). b Activation level of SREBP-2 in survival and deceased patients of COVID-19 (**p < 0.01). c Activation level of NF-κB with respect to the severity of COVID-19 (**p < 0.01). d Activation level of NF-κB in survival and deceased patients of COVID-19 (**p < 0.01). e Level of IL-1β in the COVID-19 patients’ plasma (**p < 0.01). f Level of TNF-α in the COVID-19 patients’ plasma (**p < 0.01). g Relative viability of PBMCs obtained from COVID-19 patients with respect to the culture time in vitro (**p < 0.01). h Activation level of SREBP-2 in PBMCs obtained from COVID-19 patients (**p < 0.01, n.s. not significant)
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