Sepsis, caused primarily by gram-negative bacterial infection, is part of a spectrum of conditions ranging from the systemic inflammatory response syndrome (SIRS) to septic shock and multiple organ dysfunction syndrome (MODS) (1, 2). It is estimated that 215,000 of the 751,000 patients who develop sepsis in the United States every year die of the condition (3). Despite significant advances in managing sepsis, it continues to be the main cause of mortality in intensive care units (2). The mortality associated with sepsis ranges from around 26% for SIRS to around 82% for septic shock (4).
Lipid peroxidation is one of the most investigated consequences of reactive oxygen species acting on cell membrane structures and functions. Recent studies (5-7) have confirmed that lipid peroxidation plays an important role in the development of multiple organ failure during sepsis. Nitric oxide overproduced by inducible nitric oxide synthase (iNOS) is involved in oxidative stress during sepsis (8). Nitric oxide, with its highly spontaneous chemical reactivities, interacts with superoxide to generate peroxynitrite and hydroxyl radical (9), both of which are the important mediators of lipid peroxidation in many pathological states (10,11).
One study (12) reported that a sesame oil-rich diet increased the survival rate in mice during the first 4 days after cecal ligation and puncture (CLP) and suggested that sesamol (3,4-methylenedioxyphenol), a constituent of sesame oil, may be responsible for the protective action of sesame oil against sepsis.
In addition, to manage the critical situation in endotoxemia, sesamol might be more beneficial than sesame oil. First, water-soluble sesamol can easily be prepared and administered to mitigate liver-damaging oxidative stress. Second, during sepsis, impaired gastric mucosa and acid secretion may decrease the digestion and absorption of drugs (13, 14). Although sesamol protects against endotoxin-induced oxidative stress and organ injury (15), the effect of sesamol on rats with CLP-induced sepsis in a clinically relevant model of sepsis has never been investigated. The aims of this study were (a) to investigate the effect of sesamol on mortality and hepatic injury, and (b) to examine the involvement of the antioxidative mechanism of sesamol against hepatic injury in rats with CLP-induced sepsis.
MATERIALS AND METHODS
Sesamol was obtained from Sigma (St. Louis, Mo).
Male SPF Wistar rats, weighing 200 to 300 g, were obtained from and housed in our institution's Laboratory Animal Center. Rats were housed individually in a room with a 12-h dark/light cycle and central air conditioning (25°C, 70% humidity). Rats were allowed free access to tap water and were fed a rodent diet from Richmond Standard, PMI Feeds, Inc. (St. Louis, Mo). The animal care and experimental protocols were in accordance with nationally approved guidelines.
In the mortality study, the rats were divided into 2 groups of 16: group 3 (CLP group), rats received CLP only; and group 4 (CSM group), rats were given sesamol (10 mg/kg, s.c.) 0, 6, 12, 18, 24, 30, 36, and 42 h after CLP. Animal mortality was recorded during the 48 h after CLP. In other studies, rats were divided into 4 groups of 6: group 1 (SO group), rats received a sham operation only; group 2 (SM group), rats were given sesamol (10 mg/kg, s.c.) 0 and 6 h after the sham operation; group 3 (CLP group), rats received CLP only; and group 4 (CSM group), rats were given sesamol (10 mg/kg, s.c.) 0 and 6 h after CLP. Parameters were determined 12 h after CLP. Hepatic injury was assessed using blood biochemistry and histological examination. Hepatic oxidative stress was assessed by determining lipid peroxidation, hydroxyl radical, superoxide anion, and nitrite levels in the liver. iNOS expression in the liver was also determined.
Cecal ligation and puncture
Rats were anesthetized using light diethylether and then shaved over the anterior abdominal wall. A 2-cm-long midline incision, sufficient to expose the cecum and the adjacent intestine, was made. The ligated cecum was punctured twice with an 18-gauge needle, after which the cecum was gently squeezed to exude fecal matter. The abdominal incision was then closed, and 1 mL of saline was administered subcutaneously for fluid resuscitation (16, 17).
Rat blood samples were collected from the femoral vein under light ethylether anesthesia. Blood was drawn via venipuncture into serum separation tubes, allowed to clot for 30 min at room temperature, and then centrifuged at 1000× g for 10 min at 4°C.
Assessment of liver injury
Hepatic dysfunction was assessed by measuring rises in serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), and total bilirubin (TBIL) (18, 19). Serum samples were spotted to slides (Vitros DT; Johnson & Johnson Co., Rochester, NY) and evaluated for all the above indicators using a blood biochemical analyzer (Ektachem DTSCII; Eastman Kodak, Rochester, NY) (20). For the histological studies, we used 4 livers from rats in each of the 4 groups. These tissues were fixed in 4% formaldehyde buffered with a phosphate solution (phosphate-buffered saline (PBS), 0.1 mol; pH 7.4) at room temperature. Liver fragments were washed in phosphate buffer, dehydrated in graded concentrations of ethanol, and then embedded in paraffin. From each liver, 4-μm-thin sections were obtained and stained with hematoxylin and eosin to evaluate hepatic morphology.
Measurement of serum lipid peroxidation level in liver
Liver tissue was homogenized in Tris-HCl (20 mmol, pH 7.4). Tissue homogenate (500 μL) was centrifuged at 2500× g for 10 min at 4°C. Supernatant (200 μL) was taken for lipid peroxidation measurement using a commercial assay kit (Lipid Peroxidase Assay Kit; Merck Biosciences GmbH, Darmstadt, Germany) and the spectrophotometer (DU 640B; Beckman, Fullerton, Calif) was read at 586 nm (21, 22).
Determination of hydroxyl radical and superoxide anion in liver tissue
Briefly, tissue was homogenized in Tris-sucrose buffer (0.24 mol sucrose in 20 mmol Tris-HCl buffer containing 1 mmol EDTA, pH 7.4) (1:10, wt/vol). The homogenate was centrifuged at 400× g at 4°C for 30 min. Superoxide anion and hydroxyl radical were measured using a high-performance chemiluminescence (CL) analyzer (CLA-2100; Tohoku Electronic Industrial Co. Ltd., Rifu, Japan). Briefly, 400 μL of whole blood sample was mixed with 200 μL of PBS in a stainless dish and then the background CL count was read for 60 s. One hundred microliters of lucigenin, indoxyl β-D-glucuronide, or luminol (17 mmol dissolved in PBS, for determination of superoxide anion, hydroxyl radical, and peroxynitrite, respectively) was injected into the machine, and CL was counted for another 1200 s at 10-s intervals. The data were analyzed using Chemiluminescence Analyzer Data Acquisition Software (Tohoku Electronic Industrial Co.) (23).
Measurement of nitrite production in liver tissue
Briefly, the amounts of nitrite in liver tissue were measured following the Griess reaction. Liver tissue was homogenized in deionized water (1:10, wt/vol). Tissue homogenate (500 μL) was centrifuged at 2500× g for 10 min at 4°C. Supernatant (100 μL) was incubated with 100 μL of Griess reagent at room temperature for 20 min. The absorbance was measured at 550 nm using a spectrophotometer (24). Nitrite concentration was calculated by comparing it with a standard solution of known sodium nitrite concentration (25).
Determination of iNOS expression in liver tissue
Liver tissue was homogenized in ice-cold lysis buffer (1:10, wt/vol) containing 20 mmol Hepes (pH 7.2), 1% Triton X-100, 10% glycerol, 1 mmol phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and 10 μg/mL aprotinin. We centrifuged this solution at 12,000 rpm for 30 min and then determined the protein concentration in the supernatant using protein assay dye (Bio-Rad Laboratories, Hercules, Calif) with bovine serum albumin as the standard. We loaded 50 μg of protein on 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred it to nitrocellulose sheets (NEN Life Science Products, Inc, Boston, Mass) in a transfer apparatus (Bio-Rad) run at 1.2 A for 3 h. After we blocked the blots in 5% nonfat skim milk in TBST, we incubated the blots with primary iNOS polyclonal antibody (dilution, 1:1000) (BD Biosciences, San Diego, Calif) against target protein in 5% nonfat skim milk, and then with antirabbit IgG conjugated with alkaline phosphatase (dilution, 1:3000) (Jackson ImmunoResearch Laboratories, Inc., Philadelphia, Pa). Immunoblots were developed using alkaline phosphatase substate (5-bromo-4-chloro-3-indoyl-phosphate/nitroblue tetrazolium) solution (Kirkegaard & Perry Laboratories, Inc., Baltimore, Md) (26). Relative quantification of iNOS protein was carried out using densitometry with the ImageJ computer program (National Institutes of Health), available at http://rsb.info.nih.gov/ij/.
Data are expressed as mean ± SD. The log-rank test was used to compare mortality data in different groups. One-way analysis of variance and then the Tukey Honestly Significant Difference method were used to make pairwise comparisons between the treatments in most experiments. Statistical significance was set at P < 0.05.
Sesamol increased the short-term survival rate in septic rats
To assess the protective effect of sesamol against sepsis, we assessed the animal survival rate after CLP. The survival rate was significantly higher (P = 0.0005) in the CSM group than in the CLP group (Fig. 1).
Sesamol attenuated hepatic dysfunction in septic rats
To examine the effects of sesamol on CLP-induced hepatic injury, we assessed AST, ALT, ALKP, and TBIL levels. Serum AST, ALT, ALKP, and TBIL were significantly higher in the CLP (all P < 0.01) than in SO or SM group, and significantly (P < 0.05) lower in the CSM group than in the CLP group (Fig. 2). Microscopic examination of livers from the CLP group showed severe hepatic necrosis. The magnitude of the necrosis was significantly attenuated in the CSM group. Livers from the SO and SM groups had a normal histological appearance (Fig. 2).
Sesamol decreased lipid peroxidation levels in liver tissue
We next assessed the involvement of lipid peroxidation in CLP-induced organ injury. After CLP, lipid peroxidation levels in liver tissue were significantly (P < 0.005) higher in the CLP group than in the other 3 groups. There was no significant difference in lipid peroxidation levels between the SO, SM, or CSM groups, which indicates that sesamol reduced or prevented any increases in the CSM group (Fig. 3).
Sesamol decreased hydroxyl radical and superoxide anion generation in liver tissue
To examine the role of free radicals in the development of lipid peroxidation, we assessed hydroxyl radical and superoxide anion counts in liver tissue. Both hydroxyl radical (Fig. 4) and superoxide anion (Fig. 5) were significantly (both P < 0.001) higher in the CLP group than in the other 3 groups. There were no significant differences in hydroxyl radical and superoxide anion counts between the SO, SM, or CSM groups, which indicates that sesamol significantly reduced or prevented any increases in the CSM group (Figs. 4 and 5).
Sesamol decreased nitric oxide production and iNOS expression in liver tissue
To further assess the involvement of nitric oxide in the generation of free radicals, we determined nitrite production and iNOS expression in liver tissue. Both nitrite production and iNOS expression were significantly (P < 0.01 and P < 0.0001, respectively) higher in the CLP group than in the other 3 groups (Fig. 6). There were no significant differences in nitrite production and iNOS expression between the SO, SM, or CSM groups, which indicates that sesamol potently reduced or prevented nitrite production and iNOS expression in the CSM group (Fig. 6).
We demonstrated that sesamol delayed mortality and attenuated hepatic dysfunction in rats with CLP-induced sepsis. Inhibition of hepatic lipid peroxidation and nitric oxide may be at least partially involved in sesamol's protection against liver injury in septic rats. Although 1 or more components of sesame oil may contribute to the antioxidative effect (27), sesamol might be very important in sesame oil's protection against sepsis and might be appropriate for treating sepsis in rats.
Because the animal mortality rate during sepsis is related to end-organ (hepatic or renal) damage (22, 28), it is suggested that sesamol increases animal survival via amelioration of LPS-induced multiple organ failure. Recent studies have confirmed that patients with SIRS and MODS also show increased lipid peroxidation (7). The plasma level of lipid peroxidation is higher in patients with MODS than without MODS, and this level is correlated with the Sequential Organ Failure Assessment score (5, 6). Because sesamol reduced lipid peroxidation levels and attenuated hepatic injury, we suggest that the reduction of lipid peroxidation in tissue might contribute to delayed mortality and the attenuation of organ dysfunction after CLP in rats.
Inhibiting hydroxyl radical, superoxide anion, and nitric oxide production might contribute to the antilipid peroxidation effect of sesamol in septic rats. Both reactive oxygen intermediates, such as hydroxyl radical and superoxide anion, and nitric oxide have been shown to play a potential role in organ failure (29, 30) during sepsis. Nitric oxide and superoxide anion interact to form hydroxyl radical, which is the most important free radical in the development of lipid peroxidation and multiple organ failure during sepsis (9-11). Because sesamol reduced serum nitric oxide and lipid peroxidation levels after CLP in rats, sesamol might exert its protection by inhibiting nitric oxide-mediated lipid peroxidation. However, its mechanism is still unknown. More investigations will be needed.
In summary, it is likely that sesamol is the important component in sesame oil that accounts for its protective effect against sepsis in rats. We hypothesize that it inhibits the production of nitric oxide, thereby attenuating lipid peroxidation-associated hepatic injury and delaying mortality in septic rats.
The authors thank Bill Franke for the editorial assistance.
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hepatic injury; lipid peroxidation; nitric oxide; sepsis; sesamol