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Human ghrelin mitigates intestinal injury and mortality after whole body irradiation in rats - PubMed

  • ️Thu Jan 01 2015

Human ghrelin mitigates intestinal injury and mortality after whole body irradiation in rats

Zhimin Wang et al. PLoS One. 2015.

Abstract

Widespread use of ionizing radiation has led to the realization of the danger associated with radiation exposure. Although studies in radiation countermeasures were initiated a half century ago, an effective therapy for a radiomitigator has not been identified. Ghrelin is a gastrointestinal hormone, and administration of ghrelin is protective in animal models of injuries including radiation combined injury. To test whether ghrelin can be protective in whole body irradiaton (WBI) alone, male Sprague Dawley (SD) rats were treated with human ghrelin (20 nmol/rat) daily for 6 days starting at either 24 h or 48 h after 10 Gray (Gy) WBI and survival outcome was examined. The 10 Gy WBI produced a LD70/30 model in SD rats (30% survival in 30 days). The survival rate in rats treated with ghrelin starting at 24 h was significantly improved to 63% and when treatment was initiated at 48 h, the survival remained at 61%. At 7 days post WBI, plasma ghrelin was significantly reduced from the control value. Ghrelin treatment starting at 24 h after WBI daily for 6 days improved histological appearance of the intestine, reduced gut permeability, serum endotoxin levels and bacterial translocation to the liver by 38%, 42% and 61%, respectively at day 7 post WBI. Serum glucose and albumin were restored to near control levels with treatment. Ghrelin treatment also attenuated WBI-induced intestinal apoptosis by 62% as evidenced by TUNEL staining. The expression of anti-apoptotic cell regulator Bcl-xl was decreased by 38% in the vehicle and restored to 75% of the control with ghrelin treatment. Increased expression of intestinal CD73 and pAkt were observed with ghrelin treatment, indicating protection of the intestinal epithelium after WBI. These results indicate that human ghrelin attenuates intestinal injury and mortality after WBI. Thus, human ghrelin can be developed as a novel mitigator for radiation injury.

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Conflict of interest statement

Competing Interests: PW and WL are inventors of US provisional application #62/047,729: “Treatment of radiation injury using ghrelin”. The patent application covers the fundamental concept of using ghrelin for the treatment of radiation injury. TheraSource LLC holds the exclusive option to license the technology from the Feinstein Institute for Medical Research. PW is a co-founder of TheraSource LLC., and other authors (Z.W., W.L.Y., A.J.) are employed by TheraSource LLC. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Human ghrelin improves survival after WBI.

Male Sprague-Dawley rats were exposed to 10 Gy WBI and treated with vehicle or human ghrelin (20 nmol/rat) for 6 days starting at 24 h (A) or 48 h (B) and observed for 30 days. The survival rate was estimated by the Kaplan-Meir method and compared by Log Rank test. *P = 0.04 vs. Vehicle. The percent body weight change was calculated for each animal from Vehicle (C) and human ghrelin treatment starting at 24 h (D) and plotted. Those animals in both groups that were dead as designated as such.

Fig 2
Fig 2. Plasma ghrelin levels after WBI.

Ghrelin levels from plasma of non-irradiated (Control) and 10 Gy WBI were measured using an Enzyme Immunoassay Kit. Ghrelin levels (ng/ml) were calculated against a standard curve ranging from 0 to 100 ng/ml. Data are presented as mean ± SE (n = 6–7) and compared with Student’s t-test. *P = 0.008 vs. Control.

Fig 3
Fig 3. Histology of rat jejunum after WBI.

Histological sections of the rat jejunum from Control (A), Vehicle (B) and human ghrelin (C) were stained with hematoxylin and eosin stain (100 × magnification). Villus length (D) and crypt depth (E) were measured using Nikon software and indicated by vertical and horizontal dotted lines, respectively. Data are presented as mean ± SE (n = 3) and compared by Student Neuman Keul’s test by ANOVA. *P<0.001 vs. Control; #P<0.003 vs. Vehicle.

Fig 4
Fig 4. Periodic Acid Schiff (PAS) staining of rat jejunum after WBI.

Histological sections from Control (A), Vehicle (B) and human ghrelin (C) were stained with PAS (200 × magnification). Goblet cells/villus (D) was counted using Nikon software. Data are presented as mean ± SE (n = 3) and compared by Student Neuman Keul’s test by ANOVA. *P<0.001 vs. Control; #P<0.001 vs. Vehicle.

Fig 5
Fig 5. Gut permeability, serum endotoxin levels, and bacterial translocation after WBI.

Gut permeability of the rat ileum (A) was assessed. Data are presented as mean ± SE (n = 5–6) and compared by Student Neuman Keul’s test by ANOVA. *P<0.001 vs. Control; #P<0.004 vs. Vehicle. Serum endotoxin levels (B) were determined by the endpoint chromogenic Limulus Amebocyte Lysate (LAL) assay and expressed in EU/ml. *P<0.025 vs. Control; #P<0.05 vs. Vehicle. Liver 16S rRNA was performed using real time PCR. Bacterial DNA was used as standards and the counts are expressed as ng/mg tissue. *P<0.04 vs. Control; #P<0.04 vs. Vehicle.

Fig 6
Fig 6. Serum glucose and albumin levels after WBI.

Serum glucose (A) and albumin (B) were measured using Liquid Glucose Hexokinase Reagent Set and Albumin Reagent Set, respectively. Data are presented as mean ± SE (n-6–7) and compared by Student Neuman Keul’s test by ANOVA. *P<0.01 vs. Control; #P<0.03 vs. Vehicle.

Fig 7
Fig 7. Intestinal TUNEL staining after WBI.

Jejunal sections from Control (A, D), Vehicle (B, E) and human ghrelin treatment (C, F) were stained with terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL) staining kit. The sections were counterstained with DAPI and images were merged with the TUNEL stain (MERGED). TUNEL positive cells/crypt (D) was counted. Data are presented as mean ± SE (n = 3) and compared by Student Neuman Keul’s test by ANOVA. *P<0.001 vs. Control; #P<0.001 vs. Vehicle.

Fig 8
Fig 8. Bcl-xl and Bax protein levels after WBI.

Protein lysates from Jejunal segments were examined by Western blotting for Bcl-xl (A) and Bax (B) protein levels. Bcl-xl/Bax ratio (C) was calculated and plotted. Data are presented as mean ± SE (n = 6–7) and compared by Student Neuman Keul’s test by ANOVA. *P<0.001 vs. Control; #P<0.011 vs. Vehicle.

Fig 9
Fig 9. Leukocyte trafficking and intestinal epithelial cell survival after WBI.

Total RNA from jejunal segments were examined for ICAM-1 (A), CD73 (B) mRNA expressions by real time PCR. Protein lysates from jejunal segments were examined for pAkt expression (C) by Western blotting. Data are presented as mean ± SE (n = 6–7) and compared by Student Neuman Keul’s test by ANOVA. *P<0.05 vs. Control; #P<0.05 vs. Vehicle.

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References

    1. Hauer-Jensen M, Wang J, Boerma M, Fu Q, Denham JW (2007) Radiation damage to the gastrointestinal tract: mechanisms, diagnosis, and management. Curr Opin Support Palliat Care 1: 23–29. 10.1097/SPC.0b013e3281108014 - DOI - PubMed
    1. Mettler FA Jr, Voelz GL (2002) Major radiation exposure—what to expect and how to respond. N Engl J Med 346: 1554–1561. - PubMed
    1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656–660. - PubMed
    1. van der Lely AJ, Tschop M, Heiman ML, Ghigo E (2004) Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 25: 426–457. - PubMed
    1. Wang G, Lee HM, Englander E, Greeley GH Jr (2002) Ghrelin—not just another stomach hormone. Regul Pept 105: 75–81. - PubMed

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