The Effect of Pomegranate Extract on Survival and Peritoneal Bacterial Load in Cecal Ligation and Perforation Model of Sepsis in Rats Shahryar Eghtesadi,Sanaz Tavasoli ,Amir Hassan Zarnani, Mohamadreza Vafa, Maziar Moradi-Lakeh, Hamidreza Pazoki-Toroudi Azad University, Science and Research Branch, School of Medical Sciences, Tehran, Iran

Sepsis is one of the major causes of death in intensive care units Sepsis is one of the major causes of death in intensive care units. Oxidative stress and hyper-inflammation have been shown to be major causes of mortality and morbidity in septic cases. Pomegranate is a fruit considered for its antioxidant and anti-inflammatory properties.

Obiectives The aims of this study are to evaluate the effect of a standard pomegranate fruit liquid extract (POMx), on mortality and peritoneal bacterial load in cecal ligation and perforation (CLP) sepsis model,and also to evaluate the effect of pomegranate polyphenol liquid extract (POMx) on inflammatory and antioxidant status in cecal ligation and perforation (CLP) rat sepsis model, which is an acute condition.

Materials and Methods In a contrlled experimental study adult male wistar rats were divided into four groups of 24 each: sham; CLP; prevention (consumed POMx [250 mg of polyphenols/kg/day] for 4 weeks before CLP); treatment (received a single drink of POMx [250 mg of polyphenols/kg] after CLP). Each group was divided into three subgroups, each containing eight animals, for bacterial load and survival (with and without antibiotics) studies. Sepsis was induced by CLP surgery. Ten days survival rate was recorded. Peritoneal bacterial load was also assessed. Blood samples (before and 24 hours after surgery) and liver and lung samples (24 hours after surgery) were collected. Data were analyzed using Log-rank and Kruskal-Wallis tests

Statistical analysis Survival rate was analyzed with log-rank test. The difference between bacterial load of groups were analyzed with Kruskal-Wallis test. The level of significance was set at 5%. All analyses were performed with the SPSS statistical package 15.0 (SPSS Inc., Chicago, IL, USA). The graphs were created using GraphPad Prism version 5.04 for Windows (GraphPad Software Inc., San Diego California USA).

Results There was no significant difference in survival rate of CLP, prevention and treatment groups, in subgroups without antibiotics. However, in subgroups with antibiotics, the prevention group had significantly lower survival rate than sham group (P < 0.05). Conversely, the bacterial load of prevention and treatment groups were significantly higher than sham group (P < 0.01). Although antioxidant enzymes were higher in prevention group after sepsis induction, lower Serum total antioxidant status (TAS) and higher liver thiobarbituric acid reactive species (TBARS) levels were observed in this group compared to CLP and Sham groups. Peritoneal neutrophil myeloperoxidase activity was significantly lower in prevention compared with sham and CLP groups.

Figure 1: Effect of pomegranate fruit liquid extract (POMx) on 10 day survival rate of rats with cecal ligation and perforation (CLP)‑induced sepsis. Four subgroups of rats (sham, CLP, prevention and treatment) were subdivided to two subgroups, receiving Imipenem/cilastatin 25 mg/kg every 12 h for 3 days (b), or no antibiotic therapy (a). Ten day survival rate was assessed after CLP surgery. The prevention group consumed POMx (250 mg of polyphenols/kg/day) in their drinking water for 4 weeks before CLP surgery; and treatment group received a single drink of POMx (250 mg of polyphenols/kg) after CLP surgery. *P < 0.05 and **P < 0.01, comparing to sham (log‑rank test)

Figure 2: Peritoneal bacterial load of rats with cecal ligation and perforation (CLP)‑induced sepsis. The prevention group consumed pomegranate fruit liquid extract (POMx) (250 mg of polyphenols/kg/day) in their drinking water for 4 weeks before CLP surgery; and treatment group received a single drink of POMx (250 mg of polyphenols/kg) after CLP surgery. **P < 0.01, comparing to sham (Kruskal‑Wallis test)

† Kruskal–Wallis one-way analysis of variance a N.S. not significant Table 1- The effect of of pomegranate polyphenol liquid extract on the severity of organ failure, studied by biochemical analyses. Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed of pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery) N.S.a 45.00 (31.25) 57.50 (49.75) 79.50 (134.05) 67.00 (58.25) BUN (mg/dl) N.S. 0.60 (0.33) 0.65 (0.15) 0.65 (0.40) 0.65 (0.38) Creatinine (mg/dl)   302.5 (145.2) 347.5 (315.8) 298.0 (137.0) 392.5 (304.3) SGOT (U/L) 65.5 (42.5) 121.5 (76.3) 89.5 (41.8) 106.0 (88.3) SGPT (U/L) 535 (659) 1318 (1177) 935 (1014) 1528 (1544) LDH (U/L) 0.15 (0.10) 0.20 (0.02) 0.20 (0.15) 0.20 (0.08) Bilirubin (mg/dl) † Kruskal–Wallis one-way analysis of variance a N.S. not significant

Fig. 3-The effect of pomegranate polyphenol liquid extract on serum total antioxidant status before (a) and after (b) cecal ligation and perforation surgery and thiobarbituric acid reactive species(TBARS) level in liver (c) and lung (d) tissue after cecal ligation and perforation surgery. The Consuming POMx group consumed pomegranate polyphenol liquid extract (250 mg of polyphenols/kg/day) in their drinking water for four weeks and Not consuming POMx group were normal rats before randomization in to groups and having cecal ligation and perforation surgery (a). Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery) (b,c,d). Values are expressed as median ± Interquartile range (* p<0.05, ** p<0.01, *** p<0.001)

Fig4- The effect of pomegranate polyphenol liquid extract on activity of antioxidant enzymes; superoxide dismutasev (SOD) in liver (a) and lung (b), catalase (CAT) in liver (c) and lung (d) and glutathione peroxidase (GluPx) in liver (e) and lung (f). Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract ,250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery). Values are expressed as median ± Interquartile range (* p<0.05, ** p<0.01)

Fig 5-The effect of pomegranate polyphenol liquid extract on activity of antioxidant enzymes; superoxide dismutasev (SOD) in liver (a) and lung (b), catalase (CAT) in liver (c) and lung (d) and glutathione peroxidase (GluPx) in liver (e) and lung (f). Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract ,250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery). Values are expressed as median ± Interquartile range (* p<0.05, ** p<0.01)

Fig 6-The effect of pomegranate polyphenol liquid extract on serum Nitrate/nitrite level before (a) and after (b) CLP surgery. The Consuming POMx group consumed pomegranate polyphenol liquid extract (250 mg of polyphenols/kg/day) in their drinking water for four weeks and Not consuming POMx group were normal rats before randomization in to groups and having cecal ligation and perforation surgery (a). Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery) (b). Values are expressed as median ± Interquartile range (* p<0.05, *** p<0.001

Fig7-The effect of pomegranate polyphenol liquid extract on peritoneal neutrophil Myeloperoxidase (MPO) activity after cecal ligation and perforation surgery. Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery). Values are expressed as median ± Interquartile range (* p<0.05, ** p<0.01)

Fig 8-The effect of pomegranate polyphenol liquid extract on serum cytokines after cecal ligation and perforation surgery, tumor necrosis factor (TNF)-α (a), interleukin (IL)-6 (b), IL-10 (c). Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery). Values are expressed as median ± Interquartile range (* p<0.05, ** p<0.01, *** p<0.001)

Fig9-The effect of pomegranate polyphenol liquid extract on immunohistochemical analysis of inducible nitric oxide synthase (iNOS) (a-h, q) and cyclooxygenase 2 (COX-2) (i-p, r) in liver tissue of experimental groups. Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation17

Fig 10-The effect of pomegranate polyphenol liquid extract on western blot analysis of cyclooxygenase 2 (COX-2) in liver tissue of experimental groups. Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery). Values are expressed as median ± Interquartile range (b

Fig 11- The effect of pomegranate polyphenol liquid extract on histological examination (H&E staining, X40) of liver, lung and kidney tissues of experimental groups after cecal ligation and perforation surgery. Experimental groups included: Sham (animals underwent the sham surgery without any other intervention), CLP (animals underwent cecal ligation and perforation surgery without any other intervention), prevention (animals consumed pomegranate polyphenol liquid extract, 250 mg of polyphenols/kg/day, in their drinking water four weeks before cecal ligation and perforation surgery) and treatment (animals received a single drink of pomegranate polyphenol liquid extract, 250mg of polyphenols/kg, after cecal ligation and perforation surgery)

Conclusions: Our study demonstrates that pomegranate extract could increase mortality rate via increasing peritoneal cavity bacterial load, in CLP sepsis model. POMx consumption prior to sepsis induction, suppressed the vital function of neutrophils in early hours of sepsis induction, thus resulted in higher oxidative stress observed in prevention group after CLP induction. The results may suggest that despite its anti-inflammatory action in chronic conditions, pomegranate may not work properly in rapid progressing conditions like sepsis.