Biotransformation capacity.

<p>Benzyloxyresorufin-O-debenzylation [BROD] (A), ethoxyresorufin-O-deethylation [EROD] (B), ethylmorphine-N-demethylation [EMND] (C) and glutathione-S-transferase (D, as 1-chloro-2,4-dinitrobenzene conjugation) activities in 9000g supernatants are shown exemplarily. LPS impaired BROD activities to approximately 40% of the control values, whereas enzyme activities of CTCE-0214D plus LPS treated mice were elevated by more than 90% when compared to endotoxin administration alone. Moreover, no significant difference was detectable between CTCE-0214D plus LPS or saline treated mice. Furthermore, CTCE-0214D plus LPS treated mice revealed significantly elevated EROD activities when compared to LPS treatment, whereas no significance to the control group was detectable. Co-administration of CTCE-0214D and LPS ameliorated endotoxins effects on EMND activities (elevation by about 70%), the activity, however, still being significantly decreased when compared to control. Furthermore, increased glutathione-S-transferase activities were observed when comparing the CTCE-0214D plus LPS to the LPS group. Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001. In (E), the results of all performed model reactions are presented in pmol/(mg protein x min), except PNPH and GST, which are given in nmol/(mg protein x min). Statistical significance was determined and marked as mentioned, with the addition that significance to the LPS group was marked separately; ⁺, p≤0.05; ⁺⁺, p≤0.01; ⁺⁺⁺, p≤0.001 vs. LPS.</p

Immunohistochemical staining of TLR4, NF-κB and cleaved caspase-3 in the spleens.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: (A-I) 200x, (J) 630x). For reasons of clarity, the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control. TLR4 occurred primarily in the white pulp, whereas cells of the red pulp displayed almost no membrane-bound staining. Especially after LPS treatment (B), spleno- and lymphocytes were identified to show an intensive TLR4 expression. Co-administration of CTCE-0214D to LPS clearly decreased the staining intensity of NF-κB, as lympho—and splenocytes in lymphoid follicles of the white pulp displayed diminished NF-κB levels (F). Nevertheless, the nuclear factor occurred ubiquitously in the spleens of all groups. Besides a few spleno- and lymphocytes, especially tingible body macrophages showed advanced cleaved caspase-3 activity and were found unambiguously more often in LPS-challenged mice (H,J). Co-administration of CTCE-0214D plus LPS reduced the appearance of cleaved caspase-3 to a minimum (I).</p

Administration of a CXCL12 Analog in Endotoxemia Is Associated with Anti-Inflammatory, Anti-Oxidative and Cytoprotective Effects <i>In Vivo</i>

<div><p>Background</p><p>The chemokine receptor CXCR4 is a multifunctional receptor which is activated by its natural ligand C-X-C motif chemokine 12 (CXCL12). As CXCR4 is part of the lipopolysaccharide sensing complex and CXCL12 analogs are not well characterized in inflammation, we aimed to uncover the systemic effects of a CXCL12 analog in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by using a sublethal LPS dose.</p><p>Methods</p><p>The plasma stable CXCL12 analog CTCE-0214D was synthesized and administered subcutaneously shortly before LPS treatment. After 24 hours, mice were sacrificed and blood was obtained for TNF alpha, IFN gamma and blood glucose evaluation. Oxidative stress in the liver and spleen was assessed and liver biotransformation capacity was determined. Finally, CXCR4, CXCL12 and TLR4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for apoptosis and oxidative stress were determined by means of immunohistochemistry.</p><p>Results</p><p>CTCE-0214D distinctly reduced the LPS mediated effects on TNF alpha, IFN gamma, ALAT and blood glucose levels. It attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. Furthermore, in all three organs investigated, CTCE-0214D diminished the LPS induced expression of CXCR4, CXCL12, TLR4, NF-κB, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity was induced above average. Additionally, TUNEL staining revealed anti-apoptotic effects of CTCE-0214D.</p><p>Conclusions</p><p>In summary, CTCE-0214D displayed anti-inflammatory, anti-oxidative and cytoprotective features. It attenuated reactive oxygen species, induced heme oxygenase 1 activity and mitigated apoptosis. Thus, the CXCR4/CXCL12 axis seems to be a promising target in the treatment of acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals.</p></div

TUNEL assay in the spleens and immunohistochemical staining of the cleaved caspase-3 in the thymi.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: (A-C) 400x, (D-F) 200x, (G,H) 630x). For reasons of clarity, the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control group. Whereas endotoxin induced cell death in both the white and red pulp (B), CTCE-0214D was able to significantly diminish the amount of cells undergoing apoptosis (C), which is particularly evident in the white pulp. In the thymi of the LPS group, endotoxin caused a massive appearance of cleaved caspase-3 in the cortex (E), whereas the medulla did not significantly differ from the other treatment groups. CTCE-0214D decreased the cleaved caspase-3 expression in the cortical regions to control level and caused a raise in the amount of lymphocytes (F), especially when compared to the LPS group. The magnifications in (G) and (H) reveal the tingible body macrophages in the white pulp to be the main locus of apoptotic cell death.</p

Oxidative stress in the liver and spleen.

<p>Tissue content of lipid peroxidation products as determined by thiobarbituric acid reactive substances (TBARS) in the livers of all animal groups 24 hours after treatment (A). CTCE-0214D reduced TBARS elevated due to endotoxemia to almost the half. As further parameters, total glutathione content (B), reduced glutathione levels (GSH, C) and the GSH/GSSG ratio were evaluated (D). In comparison to the control group, endotoxin caused a decrease in the total as well as reduced glutathione content and minimized the GSH/GSSG ratio, while co-administration of CTCE-0214D was able to mitigate all LPS effects significantly. Endotoxin caused a significantly reduced GSH/GSSG ratio in the spleen, whereas CTCE-0214D was able to increase the ratio which was attributable mainly to reduced GSSG levels. Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001.</p

Immunohistochemical staining of CXCR4 and CXCL12 in the spleens.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: 200x). For reasons of clarity, the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control. After saline treatment (A), CXCR4 appeared on macrophages and on lymphocytes in the red and in the white pulp, wherein in lymphoid follicles its presence was predominantly limited to lymphocytes in the marginal zone. Several cells in the red pulp are stained non-specifically (clearly distinguishable from the specific staining because of a granular appearance of the staining). LPS challenge (B) caused various CXCR4 positive cells to appear in the white pulp. The additional administration of CTCE-0214D diminished the amount of CXCR4 positive cells in the tissue to a minimum (C). CXCL12 staining in the spleen of saline treated animals (D) revealed that particularly splenocytes in the white and in the red pulp as well as plasma cells in the red pulp produced CXCL12. LPS treatment caused an increased CXCL12 expression in plasma cells, splenocytes and endothelial vascular cells in the red pulp, whereas in the white pulp predominantly splenocytes and tingible body macrophages became positive. Co-administration of CTCE-0214D to the endotoxin (F) evidently reduced the appearance of CXCL12. In the photomicrographs of (G) and (H) the tingible body macrophages (CXCL12 positive) and the engulfed lymphocytes in their cell bodies (CXCR4 positive) are depicted at a higher magnification (630x).</p

Clinical Severity Score, body temperatures, blood glucose, serum TNF-α and IFN-γ levels.

<p>24 hours after LPS administration, the Clinical severity score was improved after CTCE-0214D plus LPS treatment as compared to mice which had received LPS only (A, 1.9±0.4 vs. 2.5±0, p≤0.001). In addition, endotoxic mice showed reduced body temperatures in comparison to the control (B, 36.2±0.2°C vs. 37.2±0.3°C, p = 0.09) and to the CTCE 0214D plus LPS group (37.1±0.3°C, vs. LPS p = 0.14). Moreover, a decrease of blood glucose levels (C) by about 50% when compared to the control group was seen (4.6±0.2 mmol/L vs. 10.1±0.9 mmol/L, p≤0.001). The CTCE-0214D plus LPS group showed a blood sugar level of 7.8±0.5 mmol/L, which is equivalent to an elevation by more than 65% when compared to endotoxin treatment only (p≤0.01). In addition, no significance was detectable in comparison to the control group. Serum TNF-α concentrations (D) were significantly reduced after co-administration of CTCE-0214D and LPS (27.7±1.4 ng/mL vs. 18.5±3.3 ng/mL, p≤0.01), while LPS caused an increase of more than 330% in comparison to the control group (6.6±0.5 ng/mL, p≤0.001). Similar results were achieved when determining serum IFN-γ levels (E). Compared to the control, the LPS challenge provoked an increase of approximately 160% (107.7±15.6 pg/mL vs. 41.1±3.0 pg/mL, p≤0.001), while CTCE0214D plus LPS co-administration did not result in significantly elevated serum INF-γ concentrations (60.5±13.8 pg/mL vs. 41.1±3.0 pg/mL, p = 0.58). Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001.</p

Immunohistochemical staining of CXCR4, CXCL12, TLR4, NF-κB, cleaved caspase-3, gp91 phox, heme oxygenase 1 and the TUNEL assay.

<p>Presented are the results in the tissues of liver, spleen and thymus of all treatment groups (each n = 7). Occurrence (Occ) was assessed as following: 0: negative; 1: seldom; 2: frequent; 3: diffuse. Additionally, the intensity of staining was evaluated as follows: 0: no staining; 1: mild; 2: moderate; 3: strong.</p><p>Immunohistochemical staining of CXCR4, CXCL12, TLR4, NF-κB, cleaved caspase-3, gp91 phox, heme oxygenase 1 and the TUNEL assay.</p

Primary antibodies used for the immunohistochemical investigations.

<p>Primary antibodies used for the immunohistochemical investigations.</p

Periodic acid-Schiff (PAS) staining and immunohistochemical staining of heme oxygenase 1 in the livers.

<p>Representative photomicrographs from one of seven different tissue samples are shown (magnification: (A-C) 200x, (D-G) 400x, (H) 630x). For reasons of clarity, the PAS staining of the CTCE-0214D group is not shown as there were no differences to be observed when compared to the control. Exposure to LPS (B) evoked an impressive loss of glycogen content in the livers, whereas the livers of CTCE-0214D plus LPS treated mice (C) showed no difference to the control group (A) at all. LPS exposure (E) enhanced HO-1 occurrence in Kupffer and pit cells, whereas CTCE-0214D on its own (F) was able to induce HO-1 above average. Combined CTCE-0214D plus LPS administration (G) resulted in maximum HO-1 levels. The photomicrograph in (H) shows single Kupffer and pit cells, respectively, at a higher magnification (630x). To quantify the HO-1 activity in the liver, we performed an assay in the 9000g supernatants (I). LPS, CTCE-0214D and CTCE-0214D plus LPS administration increased the HO-1 activity by approximately 90%, 120% and 210%, respectively, when compared to the control group. The combined CTCE-0214D plus LPS treatment revealed the highest HO-1 activity levels, which were additionally significantly elevated in comparison to the values of the LPS or CTCE-0214D group, respectively. Statistical significance (p≤0.05) was determined by using the one-way analysis of variance (ANOVA) and the Tukey post hoc test. Data are given as mean ± standard error of the mean (SEM), n = 7; *, p≤0.05; **, p≤0.01; ***, p≤0.001 vs. control; ⁺, p≤0.05; ⁺⁺, p≤0.01; ⁺⁺⁺, p≤0.001 vs. LPS; #, p≤0.05; ##, p≤0.01; ###, p≤0.001 vs. CTCE-0214D.</p