Fig 1: The expression of CD54 (green fluorescence) and CD206 (red fluorescence) in microglia were detected by immunofluorescence (scale bar: 100μm) and the fluorescence intensity was analyzed by Image J 1.8.0 version. The data show mean ± S.D. statistically differences between groups were shown as follows: *P < 0.05, vs. Control group; #P < 0.05, vs. lipopolysaccharide (LPS) group, n=3. Control, the control group in which cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% normal serum for 24 h; LPS, the LPS model control group in which cells were cultured in DMEM containing both 10% normal serum and stimulant (100 ng/ml LPS) for 24 h; DHZCP HD, DHZCP high dose group in which cells were preincubated with serum containing LPS for 24 h, followed incubation with DMEM containing 10% serum from rats that were treated with a high dose of DHZCP for an additional 24 h; DHZCP LD, DHZCP low dose group in which cells were incubated with serum containing LPS for 24 h, followed DMEM containing 10% serum from rats that were treated with a low dosage DHZCP for an additional 24 h.
Fig 2: Hyperglycemia induces HSP22 upregulation and endothelial injuryin vivo. Type 2 diabetes mellitus (T2DM) mice were induced with a high-fat diet (HFD; 60% of calories from fat) and injected with streptozotocin (STZ; 100 mg/kg) or buffer for 12 weeks (n = 8 per group). A) Blood glucose concentrations in control, HFD-control and diabetic mice were measured at baseline and 4, 8, and 12 weeks after diabetes induction. B) The body weights of the control, HFD-control and diabetic mice were measured at baseline and 4, 8, and 12 weeks after diabetes induction. C) Representative images of hematoxylin and eosin staining (H&E, original magnification ×40). D) Representative immunohistochemical staining images of ICAM-1, VCAM-1 and HSP22 in aortic sections (original magnification ×40). E) Quantification of positive staining for ICAM-1 and VCAM-1 in aortic rings. n = 5. F) Representative images of a gene heat map from a cytokine array. Serum was obtained from each group of mice for a murine cytokine array analysis. Unsupervised hierarchical clustering of genes was performed to generate heat maps showing the genes that were regulated by hyperglycemia (n = 3 per group). G) Quantification of the fluorescence intensities of cytokine levels in serum. H) mRNA expression of IL5, IL6, IL13, Mip3α and TGFβ1 in the aorta. I) Quantification of positive staining for HSP22 in aortic sections. n = 5. J) mRNA expression of HSP22 in the aorta. *P < 0.05 vs. Control; #P < 0.05 vs. HFD-control.
Fig 3: HSP22 reduces hyperglycemia-induced endothelial injuryin vitro. HUVECs were transfected with plasmids containing HSP22 or a dominant-negative form for 48 h and then stimulated with high glucose for 24 h. A) HSP22 protein expression was determined by Western blotting. GAPDH was used as a loading control. n = 3. B) Cells in 96-well plates were stained with a Cell Counting Kit-8 (CCK8) to evaluate cell viability. n = 3. C) Cytotoxicity was measured by LDH release in the cell media. n = 3. D) Adhesion of nonstimulated primary human peripheral mononuclear cells (PBMCs) to stimulated HUVECs was quantified. n = 3. E) mRNA expression of ICAM-1 and VCAM-1 was determined by RT-PCR. n = 3. F) Endothelial cell activation-related cytokines were examined by RT-PCR. n = 3. *P < 0.05 vs. NC-NG; #P < 0.05 vs. NC-HG.
Fig 4: Hyperglycemia induces HSP22 upregulation and endothelial activationin vitro. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal medium (EBM) containing normal glucose (NG, 5 mM D-Glucose), osmotic control (OC, 30 mM D-mannitol), or high glucose (HG, 30 mM D-Glucose) for 24 h. A) Cell viability was evaluated in cells in 96-well plates by a Cell Counting Kit-8 (CCK8) after treatment with NG, OC or HG for 24 h. n = 3. B) HUVECs were cultured in EBM containing NG, OC or HG for 24 h, and the cell media were collected for the measurement of lactate dehydrogenase (LDH) release as an indicator of cytotoxicity. n = 3. C) HUVECs were cultured in EBM containing NG, OC or HG for 24 h, and the adhesion of nonstimulated primary human peripheral mononuclear cells (PBMCs) to stimulated HUVECs was quantified. n = 3. D) mRNA expression of ICAM-1 and VCAM-1 was determined by RT-PCR. n = 3. E) Endothelial cell activation-related cytokines were examined by RT-PCR. n = 3. F) Representative immunofluorescence staining images of HSP22 (green), the endothelial cell marker CD31 (red), and nuclei (blue) (original magnification ×63). G) Quantification of the fluorescence intensity of HSP22 and CD31 in HUVECs. n = 3. H) mRNA expression of HSP22 was determined by RT-PCR. n = 3. I) HSP22 protein expression was determined by Western blotting. J) Quantification of HSP22 protein expression. GAPDH was used as a loading control. n = 3. *P < 0.05 vs. NG; #P < 0.05 vs. OC.
Fig 5: In vivo near-infrared fluorescence imaging of Xenolight 750 probes in the rat AVM model.Rats with an AVM creation were sham treated or irradiated with a 15 Gy marginal dose to the AVM region by Gamma Knife and imaging performed 12 h after conjugate dye injection (25 µg/kg). Representative montages of x-ray (left), fluorescent (centre) and merged (right) images after injection of Xenolight 750 probes: (A) Xenolight-750 isotype control in irradiated animal; (B) Xenolight 750-ICAM-1 probe and; (C) Xenolight 750-VCAM-1 probe, at day 21 after sham (top panels) or radiation (bottom panels). Image J quantitation of fluorescence at day 21 post-irradiation or sham with Xenolight 750-ICAM-1 (D) or Xenolight 750-VCAM-1 (E) probes and Xenolight-750 isotype control probe.
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