Fig 1.
Influence of ghrelin treatment (0.5 μg/g BW, twice a day, intraperitoneal (ip.)) on survival, body weight and body temperature within 48 hours after cecal ligation and puncture (CLP)-induced sepsis.
Survival of (A) control mice with ghrelin treatment (ip.) (n = 26) or vehicle (volume adapted, ip.) (n = 30) and (B) mice with chronic high fat diet (hfd) and ghrelin treatment (ip.) (n = 24) or vehicle (volume adapted, ip.) (n = 24) within the first 48 hours after CLP. The survival curve combines data from eight independent experiments; * p < 0.05 of log-rank test as compared to the vehicle treated group. (C) Body weight was measured in control mice (ghrelin: n = 14–40/timepoint, vehicle: n = 11–24/timepoint) and hfd mice (ghrelin: n = 19–38/timepoint, vehicle: n = 13–30/timepoint) before, 6h, 24h and 48h after conducting CLP (combined data from several independent experiments); * p < 0.05 of two-way ANOVA for repeated measurements followed by a post hoc Bonferroni test. (D) Body temperature as independent predictor of mortality was measured rectally in control mice (ghrelin: n = 14–40/timepoint, vehicle: n = 11–24/timepoint) and hfd mice (ghrelin: n = 19–38/timepoint, vehicle: n = 13–30/timepoint) before, 6h, 24h and 48h after conducting CLP (combined data from several independent experiments); * p < 0.05 of two-way ANOVA for repeated measurements followed by a post hoc Bonferroni test. All values are expressed as means ± SEM.
Fig 2.
Influence of ghrelin treatment (0.5 μg/g BW, twice a day, intraperitoneal (ip.)) on early cytokine response after cecal ligation and puncture (CLP)-induced sepsis.
Serum was collected from control mice (ghrelin: n = 5–9 / timepoint, untreated: n = 4–5 / timepoint) and high fat diet (hfd) mice (ghrelin: n = 4–10 / timepoint, untreated: n = 4–5 / timepoint) before, 6h and 24h following CLP. Serum Interleukin (IL)-6 levels of (A) control and (B) hfd mice and serum IL-10 levels of (C) control and (D) hfd mice were determined by ELISA (combined data from several independent experiments); * indicates p < 0.05 using two-way ANOVA for unmatched samples followed by a post hoc Bonferroni test for analyses between more than two groups or within groups over time. All values are expressed as means ± SEM.
Fig 3.
Influence of ghrelin treatment (0.5 μg/g BW, twice a day, intraperitoneal (ip.)) on inflammatory mediators after cecal ligation and puncture (CLP)-induced sepsis.
Serum was collected from control mice (ghrelin: n = 5–9/timepoint, untreated: n = 4–5/timepoint) and high fat diet (hfd) mice (ghrelin: n = 4–10/timepoint, untreated: n = 4–5/timepoint) before, 6h, 24h and 48h following CLP. Serum leptin levels of (A) control and (B) hfd mice, Interleukin (IL)-1ß levels of (C) control and (D) hfd mice and tumor necrosis factor (TNF)α levels of (E) control and (F) hfd mice were determined by ELISA (combined data from several independent experiments); * p < 0.05 of two-way ANOVA for unmatched samples followed by a post hoc Bonferroni test for analyses between more than two groups or within groups over time. All values are expressed as means ± SEM.
Fig 4.
Influence of ghrelin treatment (0.5 μg/g BW, twice a day, intraperitoneal (ip.)) on bacterial load after cecal ligation and puncture (CLP)-induced sepsis.
Blood and peritoneal lavage fluid from control mice (ghrelin: n = 5/timepoint, untreated: n = 5/timepoint) and high fat diet (hfd) mice (ghrelin: n = 5/timepoint, untreated: n = 5/timepoint) were collected before, 24h and 48h following CLP. Blood of (A) control and (B) hfd mice and and peritoneal lavage fluid of (C) control and (D) hfd mice were plated on tryptic soy agar pour plates and incubated at 37°C for 48 hours. Bacterial colony forming units (CFU) were determined. All values are expressed as means ± SEM; * p < 0.05 of of two-way ANOVA for unmatched samples followed by a post hoc Bonferroni test for analyses between more than two groups or within groups over time. n.d. = not detected.
Fig 5.
Influence of ghrelin treatment (0.5 μg/g BW, twice a day, intraperitoneal (ip.)) on peritoneal cellular immune response after cecal ligation and puncture (CLP)-induced sepsis.
Peritoneal lavage fluid from control mice (ghrelin: n = 5/timepoint, untreated: n = 5–11/timepoint) and high fat diet (hfd) mice (ghrelin: n = 5/timepoint, untreated: n = 4–10/timepoint) were collected before, 24h and 48h following CLP. Numbers of neutrophils of control (A) and (B) hfd mice, natural killer (NK) cell numbers of (C) control and (D) hfd mice and γδ T cell numbers of (E) control and (F) hfd mice were were enumerated by flow cytometric analysis (combined data from two independent experiments). All values are expressed as means ± SEM; * p < 0.05 of unpaired t-test for comparison of baseline levels between two groups (hfd and control) and of two-way ANOVA for unmatched samples followed by a post hoc Bonferroni test for analyses between more than two groups or within groups over time.
Fig 6.
Influence of ghrelin treatment (0.5 μg/g BW, twice a day, intraperitoneal (ip.)) on oxidative burst capacity of neutrophils after cecal ligation and puncture (CLP)-induced sepsis.
Blood and peritoneal lavage were collected from controls (n = 5/timepoint), controls with ghrelin treatment (n = 5/timepoint), high fat diet (hfd) (n = 5/timepoint) and hfd with ghrelin treatment (n = 5/timepoint) before, 24h and 48h after CLP-induced sepsis. Ghrelin dependent changes of spontaneous oxidative burst capacity of neutrophils and capacity after stimulation with fMLP in serum of (A) control and (B) hfd mice and in peritoneal lavage fluid of (C) control and (D) hfd mice were measured in blood and peritoneal lavage by flow cytometric analysis (combined data from two independent experiments). All values are expressed as means ± SEM; * p < 0.05 of of two-way ANOVA for unmatched samples followed by a post hoc Bonferroni test for analyses between more than two groups or within groups over time.