Fig 1: Clopidogrel inhibits the expression of tumor necrosis factor-α and interleukin-1β in db/db mice. Data are shown as mean ± SD values of n = 6 per group. aP < 0.05 vs wild-type mice; bP < 0.05 vs db/db mice. A: Tumor necrosis factor (TNF)-α and interleukin (IL)-1β gene expression, determined using real-time polymerase chain reaction; B: Immunohistochemical staining; C: Semi-quantitative analysis of TNF-α and IL-1β protein expression. WT: Wild type; db/db: db/db mice; Clo: Clopidogrel; TNF: Tumor necrosis factor; IL: Interleukin.
Fig 2: CEL-PRNPs reverse bone erosion in rats with advanced arthritis.a Representative micro-CT images of the ankle joints at the endpoint of the experiment from different treatment groups in therapeutic efficacy study (n = 3 independent animals). b Representative micro-CT images of the trabecular in bone and the reconstructed trabecular structure (n = 3 independent animals). c, d Quantitative micro-CT analysis of BMD (c) and BS/BV (d) of the ankle joints at the endpoint of the experiment. Data represent mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test. e–g Quantitative micro-CT analysis of trabecular number (Tb.N) (e), trabecular bone thickness (Tb.Th) (f), and decreasing trabecular separation (Tb.Sp) (g). Data represent mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test. Anti-TNF anti-TNF (tumor necrosis factor) antibody, CEL celastrol, CEL-NPs CEL-loaded poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles, CEL-RNPs CEL-loaded RGD peptide-modified PLGA nanoparticles, CEL-PRNPs CEL-loaded matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles.
Fig 3: Neuroinflammatory markers support the validity of the rotenone model. Increased tumor necrosis factor alpha (TNF-alpha, green in A and B) specific signal density (SSD) in glial fibrillary acidic protein (GFAP; red in A and B)-expressing astrocytes was detected in the substantia nigra pars compacta (SNpc) as shown in graph C also. No TNF-alpha immunoreactivity was observed in ionized calcium binding adaptor molecule 1 (IBA1)-containing microglia (white in A and B). Inducible nitric oxide synthase (iNOS, red in D and E) immunoreactivity was detected only in very few IBA1 (white in A and B)-containing SNpc microglia in control rats (boxed area and red arrowhead in D). Rotenone treatment increased the number of iNOS immunoreactive (-ir) IBA-immunopositive microglia (inset in E) that occasionally formed active cell clusters (yellow arrowheads in E). Some faintly iNOS-ir nerve cell bodies (blue arrowheads in E and graph G) were also found and occasionally, TH-ir neurons also appeared to show weak iNOS positivity (see inset in E). IBA1 (green in H and I) and cluster of differentiation 68 (CD68, red in H and I) double-labeling revealed that upon rotenone treatment (I), the microglial cells in the centrally-projecting Edinger–Westphal nucleus (EWcp) co-express CD68 (J) suggesting their reactivity. Black bars: vehicle (oil) injected rats, red columns: rotenone-treated group. *p < 0.05, **p < 0.01, ***p < 0.001 in Student’s t test, n = 6. Bars: 50 µm. Scatter plot K demonstrates the negative correlation between the TH immunoreactivity in the SNpc and EWcp microglial activity scores. Scatter plot L illustrates the inverse relationship between SNpc/TH immunoreactivity and astroglial activity scores
Fig 4: CEL-PRNPs reduce the number of OCs and inflammatory macrophages in joints of rats with advanced arthritis.a TUNEL immunofluorescence staining in ankle joints from AIA rats receiving the indicated treatment (Scale bar = 200 μm) (n = 5 independent animals). b Immunohistochemical analyses of the TRAP-stained OCs and CD68-stained synovial macrophages in the joint tissues from rats receiving the indicated treatment (Scale bar = 100 μm) (n = 5 independent animals). c RANKL/OPG ratio in arthritic joints, IL-1β secretion in blood, and TNF secretion in blood from rats receiving the indicated treatment. Data represent mean ± SD (n = 5 independent animals). Statistical significance was determined by a two-sided Student’s t test. d Detection of IL-1β, TNF, OCN, and ALP expression levels in arthritic joints in different groups. Arthritic joints in different groups were stained with IL-1β, TNF, and OCN antibodies, respectively. ALP was stained light–dark in arthritic joints from different groups (Scale bar = 100 μm) (n = 5 independent animals). CEL celastrol CEL-NPs CEL-loaded poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, CEL-RNPs CEL-loaded RGD peptide-modified PLGA nanoparticles, CEL-PRNPs CEL-loaded matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles, TUNEL TdT-mediated dUTP nick end labeling, TRAP tartrate-resistant acid phosphatase, RANKL receptor of activator of NF-kB ligand, OPG osteoprotegerin, IL-1β interleukin-1 β, TNF tumor necrosis factor, OCN osteocalcin, ALP alkaline phosphatase.
Fig 5: Therapeutic efficacy of CEL-PRNPs in rats with advanced arthritis.a The schematic illustration of CEL-PRNPs treatment. b, c Ankle diameter (b) and paw thickness (c) of AIA rats were recorded every other day during the treatment period. Data represent mean ± SD (n = 7 independent animals). Statistical significance was determined by a two-sided Student’s t test. d Representative photographs of hindlimbs at the endpoint of the experiment from different treatment groups (Scale bar = 10 mm); histopathology evaluation of ankle joints was identified using H&E (scale bar = 200 µm), safranin-O and toluidine blue staining (scale bar = 100 µm) (n = 5 independent animals). i.v. intravenous, anti-TNF anti-TNF (tumor necrosis factor) antibody, CEL celastrol, CEL-NPs CEL-loaded poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, CEL-RNPs CEL-loaded RGD peptide-modified PLGA nanoparticles, CEL-PRNPs CEL-loaded matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles, H&E hematoxylin-eosin.
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