Combined Administration of Human Ghrelin and Human Growth Hormone Attenuates Organ Injury and Improves Survival in Aged Septic Rats

Abstract

Sepsis is a major healthcare concern, especially in the elderly population. The use of an animal model closely resembling clinical conditions in this population may provide a better prediction in translating bench studies to the bedside. Ghrelin inhibits sympathetic nerve activity and inflammation in young septic animals; however, aged animals become hyporesponsive to ghrelin. In this study, we evaluated the efficacy of combined human ghrelin and growth hormone (GH) for sepsis treatment in the elderly utilizing a clinically relevant animal model of sepsis. Male Fischer 344 rats 22 to 24 months old were subjected to cecal ligation and puncture (CLP). Human ghrelin plus GH or vehicle (normal saline) was administered subcutaneously at 5 h after CLP. At 20 h after CLP, blood and tissue samples were collected for various analyses. Combined treatment attenuated serum levels of lactate, lactate dehydrogenase, creatinine, blood urea nitrogen, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in aged septic rats. The integrity of the microscopic structure in the lungs, liver and kidneys was well preserved after treatment. Expression of IL-6, TNF-α, macrophage inflammatory protein-2 and keratinocyte-derived chemokine as well as myeloperoxidase activity and caspase-3 activation were significantly reduced in the lungs and liver of treated rats. Moreover, treated rats showed an improvement in cardiovascular function and increased expression of ghrelin receptor and c-fos in the brainstem. Finally, the 10-d survival of aged septic rats was increased from 29% to 64% after combined treatment and was associated with less body weight loss. Our findings warrant the development of combined human ghrelin and GH for sepsis treatment in the geriatric population.

Figure 1.

Effect of the combined treatment on organ injury makers and systemic inflammation in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or human ghrelin plus GH at two different doses, GG-1 (40 nmol/kg of ghrelin and 25 μg/kg GH) and GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. Blood samples were collected at 20 h after CLP for measuring (A) lactate, (B) LDH, (C) BUN, (D) creatinine, (E) IL-6 and (F) TNF-α. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 2.

Effect of the combined treatment on the morphological structure of lung tissues in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The lung tissues were harvested at 20 h after CLP. Sections of lung tissues were stained with H&E and examined under light microscopy. (A) Representative images of the stained lung tissues are shown. Original magnification 200×. (B) Histologic injury scores of the lungs in different groups were quantified as described in Materials and Methods. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 3.

Effect of the combined treatment on lung inflammation in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The lung tissues were harvested at 20 h after CLP for measuring (A) protein levels of IL-6 by ELISA, mRNA levels of (B) MIP-2 and (C) KC by qPCR, and (D) MPO activity by colorimetric assay. mRNA levels in sham group are designated 1 for comparison. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 4.

Effect of the combined treatment on the morphological structure of liver tissues in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The liver tissues were harvested at 20 h after CLP. Sections of liver tissues were stained with H&E and examined under light microscopy. (A) Representative images of the stained liver tissues are shown. Sham tissues have normal architecture, whereas the vehicle group shows hepatocytes vacuolization, cell necrosis and inflammatory cell infiltration (arrows). The treatment of GG-2 markedly maintains the integrity of the liver structure. Original magnification 200×. (B) Histologic injury scores of the liver in different groups were quantified as described in Materials and Methods. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 5.

Effect of the combined treatment on liver inflammation in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The liver tissues were harvested at 20 h after CLP for measuring (A) protein levels of TNF-α by ELISA, mRNA levels of (B) MIP-2 and (C) KC by qPCR, and (D) MPO activity by colorimetric assay. mRNA levels in sham group are designated 1 for comparison. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 6.

Effect of the combined treatment on the morphological structure of kidney tissues in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The kidney tissues were harvested at 20 h after CLP. Sections of kidney tissues were stained with H&E and examined under light microscopy. (A) Representative images of the stained kidney tissues are shown. The kidneys obtained from the vehicle group demonstrated a significant renal injury with glomerular destruction (black arrow), tubular proteinaceous casts (red arrow), loss of tubular brush borders and epithelial cell injury (blue arrows). The integrity of renal morphology in the GG-2 treatment group is significantly improved. Original magnification 200×. (B) Histologic injury scores of the kidney in different groups were quantified as described in Materials and Methods. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 7.

Effect of the combined treatment on caspase-3 activation in the organs of aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The tissues were harvested at 20 h after CLP for the protein extraction. Levels of cleaved caspase-3 in the (A) lung and (B) liver tissues were determined by Western blotting. Representative blots against cleaved caspase-3 and β-actin. Blots were scanned and quantified with densitometry. The levels of protein expression in sham group are designated 1 for comparison. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 8.

Effect of the combined treatment on cardiac contractile function in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. At 20 h after CLP, rats were anesthetized, and left ventricular pressure measurements and heart rate were recorded using a Millar pressure transducer. (A) LVDP, (B) maximal rate of ventricular pressure development (+dP/dt_max) and (C) work product were calculated. Data are expressed as mean ± SE (n = 5–7 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 9.

Effect of the combined treatment on brain activity in aged septic rats. Rats were sham operated or subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. The brainstem was harvested at 20 h after CLP for protein extraction. (A) Representative Western blots against GHSR-1a, c-fos and β-actin. Quantitative levels of (B) GHSR-1a and (C) c-fos in different groups. Blots were scanned and quantified with densitometry. The levels of protein expression in sham group are designated 1 for comparison. Data are expressed as mean ± SE (n = 5–7 per group). ^#P < 0.05 versus vehicle.

Figure 10.

Effect of the combined treatment on survival and body weight change in aged septic rats. Rats were subjected to CLP with injection of vehicle (normal saline) or GG-2 (80 nmol/kg of ghrelin and 50 μg/kg of GH) at 5 h after CLP. (A) Rats were monitored daily for 10 d. The survival rate was analyzed by Kaplan-Meier survival analysis and compared by the log-rank test (n = 14 per group). *P < 0.05 versus vehicle. (B) The daily body weight change of the surviving rats in a 10-d survival study. The percentage of body weight change is calculated by the difference from d 0. Data are expressed as mean ± SE.