Protective Effect of Silymarin on Liver in Experimental in the Sepsis Model of Rats

Abstract

This study, it was investigated whether silymarin has a protective effect by performing histological, immunohistochemical, and biochemical evaluations on the liver damage induced by cecal ligation perforation (CLP). CLP model was established and silymarin was treated at a dose of 50 mg/kg, 100 mg/kg, and 200 mg/kg, by oral one hour before the CLP. As an effect of the histological evaluations of the liver tissues, venous congestion, inflammation, and necrosis in the hepatocytes were observed in the CLP group. A situation close to the control group was observed in the Silymarin (SM)100 and SM200 groups. As a result of the immunohistochemical evaluations, inducible nitric oxide synthase (iNOS), cytokeratine (CK)18, Tumor necrosis factor-alpha (TNF-α), and interleukine (IL)-6 immunoreactivities were intense in the CLP group. In the biochemical analysis, Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST), and Alanine Aminotransferase (ALT) levels were significantly increased in the CLP group, while a significant decrease was observed in the treatment groups. TNFα, IL-1β, and IL-6 concentrations were in parallel with histopathological evaluations. In the biochemical analysis, Malondialdehyte (MDA) level increased significantly in the CLP group, but there was a significant decrease in the SM100 and SM200 groups. Glutathione (GSH), Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione Peroxidase (GSH-Px) activities were relatively low in the CLP group. According to these data, it was concluded that using silymarin reduces the existing liver damage in sepsis.

I.  Introduction

Sepsis represents a clinical syndrome accompanied by biological, physiologic, and biochemical changes caused by the inflammatory response to infection in the body [67]. Septic shock which occurs in sepsis and afterward is a high mortality disease that requires intensive care treatment [11]. The sepsis incidence was detected in 437 in 100,000 individuals and 270 in 100,000 for septic shock according to retrospective research covering the years 1995 and 2015 [21]. Gram-positive bacteria, gram-negative bacteria, viruses, fungi, and parasites are responsible for sepsis etiology [70]. With the entry of microorganisms into the body that causes sepsis; coagulation factors, inflammatory cytokines, and substances that suppress the myocardium, adhesion molecules begin to be released. The response created versus bacterial toxins protects the body against infection under normal conditions, while excessive response results in many harmful effects [20]. In sepsis, the amount of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) increases with the increase in the enzyme nitric oxide synthase (iNOS) that can be stimulated. The increase of these cytokines causes damage to the tissues and the endothelium [55]. The cause of irreversible damage and organ dysfunctions in sepsis are changed in balances such as inflammatory-anti-inflammatory, oxidant-antioxidant, apoptotic-antiapoptotic, and coagulant-anticoagulant [11, 15]. The respiratory system, liver, coagulation system, gastrointestinal tract damage, and kidney impairment are common and are even problems that increase the death rate [62]. Experimental sepsis models are performed by applying live bacteria to the body via intraperitoneal or intravenous, by giving intraperitoneal lipopolysaccharide, or by the cecal ligation and perforation (CLP) method [18, 28]. The clinical results seen in the sepsis model created by the CLP method are quite similar to the results of sepsis in humans. In addition, it is a frequently preferred method in terms of its cheap and easy application and allowing for monitoring the polymicrobial sepsis table. This method is based on the logic of connecting the cecum with a standard-sized thread and perforating it with a standard-sized injector tip without disturbing the intestinal passage of animals under anesthesia [17, 18, 28, 53].

Silymarin (SM) is a polyphenolic flavonoid with antioxidant, and antiapoptotic properties extracted from the Silybum marianum plant [22]. The chemical formula of silymarin is C₂₅H₂₂O₁₀ [24]. The antioxidant property of silymarin is due to the increase in the activity of antioxidant enzymes such as glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT). Besides, it has antioxidant properties which increase the expression of antioxidant enzymes at the mRNA level [59, 77]. Silymarin improves the hyperglycemia caused by alloxan and increases the blood glutathione level [60].

In the literature, it was reported that silymarin has strong effects on inflammation and oxidative stress. For this purpose, it was aimed to examine the anti-inflammatory and antioxidant effects of silymarin applied at various doses by histochemical, immunohistochemical, and biochemical methods in the prevention of liver damage caused by CLP application to rats in the study.

II.  Materials and Methods

Animal procedure

The study was determined to be by the ethical rules by the decision of the Atatürk University Animal Experiments Local Ethics Committee (Decision No: 2019–26). Rats were obtained from Ataturk University Animal Laboratory and Research Center (ATADEM). A total of 50 male Sprague-Dawley rats weighing an average of 230–270 grams were used. Rats were kept in cages where temperature (21–23°C) and moisture (45–50%) were measured up to the day of the experiment, in rooms with half a day of dark and half-day light. The animals were given unlimited pellet feed and water (ad libitum) until the day of the experiment. The animals were starved for 12 hr before the experiment.

Experimental groups

The experimental groups were divided into 5 groups and 10 rats in each group. The characteristics of the groups are as follows: Group 1: Control Group, Group 2: has cecal ligation and perforation (CLP), Group 3: has 50 mg/kg of silymarin and CLP (SM50+CLP), Group 4: has 100 mg/kg of silymarin and CLP (SM100+CLP) and Group 5: has 200 mg/kg of silymarin and CLP (SM200+CLP).

Experimental sepsis model

Except for the control and CLP groups, silymarin (SIGMA S0292-10G) was given to the other groups in 50 mg/kg, 100 mg/kg, and 200 mg/kg doses with a gavage needle and orally. Silymarin serum was applied after being suspended in physiological water. An hour later, CLP was applied to the third, fourth, and fifth groups (Groups 3, 4, and 5). Oral-applied silymarin reaches the highest level in plasma within approximately 2–4 hr and it has a half-life of 6 hr [72]. The application of anesthetic material for the operation was given an hour before the silymarin operation because it slowed down the absorption of the drug. Only the CLP model was applied to the second group.

CLP is a method used to create septicemia in polymicrobial properties. The animals in the operation groups were anesthetized with xylazine (5 mg/kg) and ketamine hydrochloride (40 mg/kg). The rats were secured on the back, and the infiltration line of the abdomen was wiped twice with povidone-iodine solution, providing the antisepsis. The midline of the abdomen was opened with a 3 cm incision and the cecum was explored. The ligation was applied under the ileocecal valve with silk floss (3/0). The connected section was drilled twice with the standard injector tip measuring 18 gauge and tightened to allow some of the fecal matter to come out. Then, the cecum was placed in the abdomen and the abdomen was closed with the stapler in normal anatomical form.

In the CLP rat model, the perforation was constituted with 13, 16, 18, and 22 caliber injector tips and a 0.5 cm scalpel, and the mortality of the experimental subjects was assessed in the literature [45]. Therefore, we used an 18-gauge injector to obtain blood and liver tissue in this study before the rats died.

The operated animals were taken into postoperative care for 16 hr. At the end of 16 hr, 15 mg/kg xylazine and 100 mg/kg ketamine were administered intraperitoneally to all subjects. Thus, the reflexes of the anesthetized rats were controlled and an incision was made at the level of the abdominal diaphragm. The animals whose blood samples were taken from their hearts under deep anesthesia were sacrificed by cervical dislocation. Then, the liver tissues of all animals were taken to a solution containing 10% formaldehyde for histopathological and immunohistochemical studies. The blood samples taken were used for biochemical analysis.

Histological analyses

Hepatic tissues fixed in a 10% formaldehyde solution were passed through a series of alcohol and xylol and embedded in paraffin blocks. Serial sections of 5-micrometer thickness were taken from the blocks (Leica RM2125 RTS). The sections were used in histopathological and immunohistochemical stains. Mallory’s Triple Stain, modified by Crossman, was conducted in histopathologic studies.

Immunohistochemical analyses

Primary antibodies used for immunohistochemical staining (Avidin-Biotin Method): iNOS (Abcam, Anti-iNOS antibody, 1/50 dilution, ab3523), CK18 (Abcam, Anti-Cytokeratin antibody, 1/50 dilution, ab203233), TNFα (Santa Cruz, TNFα antibody, 1/50 dilution, sc-52746), IL-6 (Santa Cruz, IL-6 antibody, 1/50 dilution, sc-57315). After the tissues taken on adhesive slides were passed through xylol and graded alcohol series, 3% hydrogen peroxide was dropped and left for 10 min to prevent methylation. Then the slides were washed with phosphate-buffered saline (PBS). The slides were then taken to containing citrate buffer solution (Citrate buffer; 1 cc was dropped into 100 ml distilled water) and kept in the microwave at 500 Watt for 15 min, and the antigen mask was removed. To prevent nonspecific binding, protein blocks were dripped onto the slides and waited for 10 min. Antibodies were prepared by diluting 1/50 with PBS. After protein blocking, 100 microliters of the primary antibody were dropped onto each slide and kept in a dark and humid environment for approximately 1 hr. Then washing was done with PBS. After treatment with complement (Thermo Fisher, DAB Substrate System) for 10 min, washing was done with PBS. Conjugate (Thermo Fisher, DAB Substrate System) was added and left for 25 min. After washing with PBS, a solution of DAB (3,3'diaminobenzidine) (Thermo Fisher, Catalog no: 34002) was added and browning was observed in the tissues. Again, after washing with PBS, counterstaining was performed with Mayer’s hematoxylin solution. After washing the slides with PBS, they were kept in 96% alcohol for 1 min, 2 times in absolute alcohol for 5 min, 2 times in xylol for 5 min, and covered with entellan.

The semiquantitative scoring method was used in the immunohistochemical evaluation of this study. Based on this method, the average dyeing density of at least 5 different regions from each liver tissue was observed and scoring was performed. Immune positive and immune negative coloring scores were detected according to the assessments: There is little or no coloring − (0%), slightly dense + (0–30%), medium density ++ (30–60%) and intensive coloring +++ (60–100%).

After all, a computer (Zeiss AXIO Scope-A1) and a microscope with a camera add-on were used for microscopic imaging and assessments.

Biochemical analyses

At the end of the experiment, the blood was taken with the injector from the left ventricle of the heart for performing biochemical analyses. The blood was collected into gel serum tubes, centrifuged for 10 min at 3000 rpm, serums were removed, transferred to eppendorf tubes, and placed at +4°C. Then, the serums were frozen at −20°C and stored in the freezer at −80°C until biochemical analyses were performed. ALT (Cat. No: AL 8304, Randox Laboratories Limited, 55 Diamond Road, Crumlin, Country Antrim, BT29 4QY, United Kingdom), AST (Cat. No: AS 8306, Randox Laboratories Limited, 55 Diamond Road, Crumlin, Country Antrim, BT29 4QY, United Kingdom) and ALP (Cat. No: AL 8304, Randox Laboratories Limited, 55 Diamond Road, Crumlin, Country Antrim, BT29 4QY, United Kingdom) serum concentrations were prepared using commercial kits. The serum TNF-α (SunRed, Cat. No: 201-11-0765), IL-1β (SunRed, Cat. No: 201-11-0120), IL-6 (SunRed, Cat. No: 201-11-0136), and GSH-Px (SunRed, Cat. No: 201-11-5104) concentrations were measured with ELISA Reader (Bio Tek μQuant MQX200 Elisa reader/USA) by the protocol specified in rat ELISA kits.

The MDA (Malondialdehyde), GSH (Glutathione), CAT (Catalase), and SOD (Superoxide dismutase) determinations of serum samples were performed according to the methods reported by the references Yoshioka et al. [74], Tietze et al. [64], Goth et al. [26] and Sun et al. [61] respectively.

Statistical analyses

The findings of the biochemical analysis were evaluated statistically in this study. To this end, One-Way ANOVA variance analysis was performed using the SPSS 22.0 package program to reveal the importance of diversity between groups. The Tukey test was performed for multiple comparisons in the data which has a normal distribution. Statistical significance was considered p < 0.05.

III.  Results

Histopathologic findings

Triple staining findings in liver tissues

Histopathologic examination of the liver sections of the control group revealed remark cords around both the central vena and portal area. Remark cords consisted of the hepatocytes which had regular sequences, and cell boundaries are smooth and polygonal-shaped (Fig. 1A, B).

Fig. 1.

Light microscopic sections of the probability of the results of Control (A, B), CLP (C, D), SM50+CLP (E, F), SM1000+CLP (G, H), SM200+CLP (I, J). arrow: hepatocytes with hydropic hydropic degeneration with pycnotic nuclei, white arrow: necrotic hepatocytes with eosinophilic cytoplasm, arrowhead: sinusoidal dilatation, star: mononuclear cell infiltration, c: congestion. Stain: Crosman modified Mallory’s triple staining, ×400. No, we didn’t use eosin. We used hematoxylen, acid fuchsin and aniline blue.

There was liver damage which was seen clearly in the liver sections of the CLP group. Many hepatocytes were detected in hepatic acinus; the hepatocytes had pyknotic nuclei, eosinophilic cytoplasm, and hydropic degenerative image. In the portal area, sinusoidal dilatation, venous congestion, and mononuclear cell infiltration were observed in addition to necrotic hepatocytes (Fig. 1C, D). Similar to the CLP group, numerous hepatocytes were detected in the sections of the SM50+CLP group; hepatocytes were hydropically degenerated, had pyknotic nuclei and eosinophilic cytoplasm, and were located around the central. In addition, sinusoidal dilatation, venous congestion, the hepatocytes which hydropic degenerated, mononuclear cell infiltration, and an increase in the connective tissue around the vessel were detected in the portal area (Fig. 1E, F). Compared to the CLP group, the SM100+ CLP group sections determined that the damage was very low. Decreased number of cells that had hydropic degeneration and necrosis were around the central vena and portal area. Also, sinusoidal dilatation and venous congestion were found to regress (Fig. 1G, H). It was determined that the healing in the SM200+ CLP group was at the highest level. In addition, it was detected that this group had the closest image to the control group in the sections (Fig. 1I, J).

Immunohistochemical stain findings in liver tissues

The scoring results obtained in the assessment of the immunopositive density of the liver sections are given in Table 1.

Table 1.  Immunohistochemical staining scores

Groups	iNOS	CK18	TNF-α	IL-6
Control	—	—	—	—
CLP	+++	+++	+++	+++
SM50+CLP	++	+++	++	++
SM100+CLP	++	+	+	+
SM200+CLP	+	++	+	+

As a result of immunohistochemical staining with iNOS antibody; it was observed that there were intense (+++) iNOS positive cells around the central vein in the liver sections of the CLP group while iNOS positive cells were not visible on the liver slices of the control group. The medium-intensity (++) immunopositive staining was observed in the liver tissues of the SM50+CLP and SM100+CLP groups while slightly (+) immunopositive staining was detected in the sections in the SM200+CLP group. The reaction was observed to be intense in endothelial cells (Fig. 2).

Fig. 2.

Immunreactivity of iNOS, CK18, TNF-α and IL-6 antibodies from liver sections in all groups. Streptavidin-biotin peroxidase staining ×100.

As a result of immunohistochemical staining with CK18 antibody; it was detected that there were dense (+++) CK18 positive cells in the liver sections of the CLP and SM50+CLP groups while CK18 positive cells were not seen in the liver sections of the control group. Slight intensity (+) immune positive staining was detected in the liver sections of the SM100+CLP group. It was observed that there was a medium-density (++) painting in the sections of the SM200+CLP group. It was determined that the reaction was especially in perinuclear areas in the cell cytoplasm (Fig. 2).

As a result of immunohistochemical staining with TNF-α antibody; it was observed that there were dense (+++) TNF-α positive cells in the liver sections of the CLP group while TNF-α positive cells were not seen in the liver sections of the control group. Moderate-density (++) immune positivity was detected in the sections belonging to the SM50+CLP group. Slightly intensity (+) immune positivity staining was observed in the SM100+CLP and SM200+CLP groups. The reaction was found to be common in hepatocyte cell membranes (Fig. 2).

As a result of immunohistochemical staining with IL-6 antibody; it was determined that there were dense (+++) IL-6 positive cells in the liver sections of the CLP group while IL-6 positive cells were not seen in the liver sections of the control group. Moderate-density (++) immune positivity was detected in sections belonging to the SM50+CLP group, while slightly dense (+) immune positivity was detected in the SM100+CLP and SM200+CLP groups. The reaction intensity was determined to be around the central vein and in the cell (Fig. 2).

Biochemical results

The serums were taken from rats that created experimental sepsis by CLP method: The changing levels of ALT, AST, ALP, TNF-α, IL-1β, IL-6; the levels of MDA, GSH, and the change activities of GSH-Px, CAT, SOD found in serums were shown in Table 2.

Table 2.  Biochemical parameters measured in the serum of all groups

GROUPS	CONTROL	CLP	SM50+CLP	SM100+CLP	SM200+CLP	p
ALT (U/L)	37.10 ± 0.43d	153.30 ± 0.47a	143.60 ± 0.37b	120.90 ± 1.96c	139.10 ± 1.46b	***
AST (U/L)	37.36 ± 0.40e	257.95 ± 0.85a	252.62 ± 0.43b	228.04 ± 2.61d	243.56 ± 0.46c	***
ALP (U/L)	92.30 ± 0.47e	252.6 ± 0.50a	242.7 ± 0.54b	203.5 ± 0.37d	221.6 ± 1.07c	***
TNF-α (ng/L)	86.66 ± 0.32e	176.51 ± 0.31a	161.98 ± 0.30b	132.02 ± 0.37d	144.25 ± 1.02c	***
IL-1β (pg/L)	524.27 ± 1.18e	1335.41 ± 2.56a	1236.42 ± 1.87b	934.55 ± 2.27d	1032.02 ± 2.05c	***
IL-6 (pg/mL)	33.04 ± 0.42e	112.96 ± 0.50a	108.79 ± 0.29b	102.53 ± 0.50d	106.18 ± 0.39c	***
MDA (mmol/L)	13.40 ± 0.21d	32.74 ± 0.40a	31.65 ± 0.29a	26.85 ± 0.46c	28.21 ± 0.21b	***
GSH (mmol/L)	2.06 ± 0.01a	1.51 ± 0.09c	1.70 ± 0.03b	2.01 ± 0.00a	1.78 ± 0.02b	***
GSH-Px (ng/mL)	10.65 ± 0.44a	5.15 ± 0.21d	6.89 ± 0.16c	8.78 ± 0.17b	6.11 ± 0.15c,d	***
CAT (kU/L)	273.12 ± 0.36a	152.79 ± 0.46e	157.42 ± 0.35d	181.01 ± 1.72b	163.21 ± 0.54c	***
SOD (EU/mL protein)	15.93 ± 0.10a	9.13 ± 0.20e	10.73 ± 0.15d	12.56 ± 0.10b	11.68 ± 0.10c	***

The values are expressed as mean ± standart deviaiton. Significance accepted as 0.05. **: p < 0.01, ***: p < 0.001. The letters (a, b, c, d, e) indicates the statsical differences between the same line.

Serum liver function test

The ALT, AST, and ALP activities of serum samples were highest in the CLP group while the values were significantly decreased in the control and silymarin applied CLP groups (p < 0.001). In addition, these values were found to be significantly lower in the SM100+CLP group than in the CLP group (p < 0.001) (Table 2).

Serum cytokines concentrations

In our study, immunohistochemical staining of TNF-α and IL-6 antibodies; we found intensive (+++) staining in the liver sections of the CLP group while TNF-α and IL-6 positive cells were not seen in the liver sections of the control group. We detected slightly (+) immunopositive staining in the SM100+CLP and SM200+CLP groups while moderate intensity (++) immune positivity was observed in the sections belonging to the SM50+CLP group. Similarly, there was a significant increase in serum TNF-α, IL-1β, and IL-6 levels in the CLP group compared with all groups in the biochemical findings while it was determined that there was a decrease in these values in the treatment groups compared to the CLP group (p < 0.001) (Table 2).

Serum oxidative stress parameters

MDA levels of serum samples were statistically different (p < 0.001) among all groups. The lowest MDA level was detected in the control group, followed by the SM100+CLP, SM200+CLP, CLP, and SM50+CLP groups, respectively. MDA levels were found to be similar in CLP and SM50+CLP groups.

In our study, serum SOD activity decreased significantly in the CLP group, while silymarin, which is used as an antioxidant substance, significantly increased serum SOD activity compared to the CLP group. It was determined that silymarin, which is used as an antioxidant substance, significantly increased serum SOD activity compared to the CLP group while serum SOD activity decreased significantly in the CLP group. In our study, it was determined that there was a very significant difference (p < 0.001) between all groups when serum samples were examined in terms of CAT. The lowest CAT activity belonged to the CLP group, followed by SM50+CLP, SM200+CLP, SM100+CLP, and the control group. GSH and GSH-Px activity of serum samples differed statistically in all groups (p < 0.001). The lowest GSH-Px activity belonged to the CLP group, while it was observed in the SM200+CLP, SM50+CLP, SM100+CLP, and control groups, respectively. The GSH level was the lowest in the CLP group, followed by the SM50+CLP, SM200+CLP, SM100+CLP, and control groups, respectively (Table 2).

IV.  Discussion

Septic Peritonitis Model which was created with the CLP method in mice, was developed to investigate septic shock and sepsis data encountered in humans [57]. The cecal contents leaking into the peritoneum upon puncture of the cecum cause fecal contamination and bacteremia. As a result of this situation, the body’s natural response develops and septicemia is shaped. Septic shock and sepsis are severe diseases that can cause organ failure along with dysfunction even in organs far from the damage zone [51]. The most important role at the organ level in sepsis is the liver. The liver provides detoxification and clearance of endotoxins, the bacteria responsible for septicemia and vasoactive substances formed during sepsis, and regulates the activation of cells involved in host defense. The liver is both the target organ affected by inflammatory mediators and the source of these mediators [35]. Despite the latest technologies and antibiotics in intensive care for sepsis treatment today, sepsis remains a major problem. The search for solutions for the treatment of sepsis is still ongoing [1, 27]. This study was trying to determine the protective effect of different doses of silymarin against sepsis-induced liver damage in rats.

When the liver with septic is investigated histopathologically, it is often reported that pathological findings such as Kupffer cell hyperplasia, focal hepatocyte changes, intrahepatic alcohol, portal tract mononuclear cell infiltrates and steatosis emerge [41]. Al-Kadi et al. [4] reported that there are degenerative hepatocytes, vacuolization, apoptotic and acidophilous cells in the liver tissues in the CLP group as a result of a study in which they created sepsis and treated it with silymarin. Song et al. [58] determined that hepatocyte necrosis, cytoplasmic vacuolization, lysosome proliferation, and neutrophil cell infiltration occurred in the liver morphology of the rats that had sepsis by CLP method in their study. Gong et al. [25] also found that rats had significant interstitial edema and congestion in liver tissues in a study in which they created septicemia using CLP. Within the scope of our work; similar to the CLP group, many hepatocytes with pycnotic nuclei, hydropic degeneration, and eosinophilic cytoplasm were detected around the central vein in sections belonging to the SM50+CLP group. The venous congestion, sinusoidal dilatation, the hepatocytes which hydropic degenerated, mononuclear cell infiltration, and increased connective tissue around the vessels were observed in the portal area. Compared to the CLP group, in the sections belonging to the SM100+CLP group, it was determined that the damage was considerably less. Necrosis and hydropic degeneration around both the central vein and portal area and a decrease in the number of cells undergoing necrosis and hydropic degeneration were detected. In addition, the regressed sinusoidal dilatation and venous congestion were recorded. It was determined that the improvement in the SM200+ CLP group was close to the control group.

In sepsis, pro-inflammatory cytokines such as TNF-α and IL-1β are increasing with the increase of iNOS expression. As a result of this situation, cytotoxic substances such as reactive oxygen species are released. These substances could deform the surrounding tissues with endothelial cells [55]. When iNOS is stimulated, it causes NO production in large quantities and over a long period. NO produced in this way is related to various pathophysiological processes and is associated with various events such as inhibition of mitochondrial respiration [16], stimulation of tissue damages and protein nitration [12, 34], antiviral [31] and antibacterial [40] activity. LPS has been shown to activate the Kupffer cells to overexpress iNOS mRNA and to generate an abundance of NO [13]. The expression of iNOS leads to overproduction of NO, resulting in damage to endothelial, neuronal, and epithelial cells and it has been shown to be an important event in various models of liver injury [23, 30]. Liver damage was induced by doxorubicin (DOX) and the therapeutic effect of thymoquinone (THQ) was investigated. iNOS, eNOS, Cas-3 expressions and the number of apoptotic cells were found to be decreased in the DOX+THQ group compared to the DOX group [3]. In our study, iNOS positive cells were not observed in the liver sections of the control group, whereas there were intense (+++) iNOS positive cells around the central vein in the liver sections of the CLP group; there was moderate density (++) in liver tissues of SM50+CLP and SM100+CLP groups, and slight (+) immunopositive staining was detected in the sections of SM200+CLP group. Cytokeratin is used as an indicator of liver damage [71]. The expression of cytokeratin has not been observed in hepatic echinococcosis disease. Intermediate filaments are an important component of the three major mammalian cytoskeletal proteins, and cytokeratin is the largest subgroup in the intermediate filament protein family, mainly expressed in epithelial cells [39]. CK8/CK18 is expressed in large amounts in the liver, and the imbalance of the ratio can cause liver damage and participate in processes such as apoptosis [29]. The serological concentrations of CK18 were reported to increase significantly compared to healthy controls in patients with severe sepsis [43]. Similarly, studies are showing that CK18 is significantly higher in patients with severe sepsis than in the healthy control group [52]. In a study in which liver injury was induced by carbon tetrachloride, CK8 and CK18 protein levels were found to increase significantly as liver fibrosis increased [56]. Similar to the literature, in our study, CK18 positive cells were not seen in the liver sections of the control group; intensive (+++) CK18 positive cells in liver sections belonging to CLP and SM50+CLP groups; slight (+) in liver sections belonging to the SM100+CLP group and it was determined that there was a medium density in the SM200+CLP group.

In the septic process, some studies report that IL-6 and TNF-α levels increase in endotoxic shock situations and a negative correlation with survival time occurs in this process [36]. In another study, it was stated that TNF-α and IL-6 from inflammatory cytokines decreased in serum one hour after treatment of septic animals induced with CLP with silymarin [4]. Immunohistochemical stains with TNF-α and IL-6 antibodies; we found that there was intensive (+++) dyeing in the liver sections of the CLP group while TNF-α and IL-6 positive cells were not seen in the liver sections of the control group in our study. Moderate-density (++) immune positivity was detected in sections belonging to the SM50+CLP group, while slight intensity (+) immune positive stain was detected in the SM100+CLP and SM200+CLP groups. Similarly, in the biochemical findings, serum TNF-α, IL-1β, and IL-6 levels were significantly increased in the CLP group compared to all groups, while these values decreased according to the CLP group in the treatment groups (p < 0.001). According to Toklu et al. [65] in a sepsis study conducted by the CLP method, they reported that IL-1β, IL-6 and TNF-α levels of sepsis groups increased in serum samples, and there was a significant decrease after silymarin therapy. TNF-α and IL-6 levels were examined in rats treated with CLP, and it was reported that TNF-α and IL-6 levels increased at the sixth hour in rats with severe sepsis in literature [57]. It was reported that serum IL-6 and TNF-α levels were significantly increased in the CLP-induced sepsis groups of rats compared to the control groups [9, 68, 73, 75]. Cihan et al. [14] examined the effects of silymarin on the liver in the experimental sepsis model they created with CLP: they found that TNF-a, IL-1β and IL-6 levels increased significantly in the sepsis group in serum samples, which was significantly eliminated by silymarin treatment.

The ALT, AST, and ALP activity of serum samples were increased significantly in the CLP group compared to control and other groups while these values were decreased in the SM100+CLP group than CLP group. The increase in ALP activity is generally known as an indicator of hepatosellüler liver disease, viral hepatitis, cholestatic liver disease, and tumors [46, 66]. AST and ALT are enzymes of the aminotransferase group that causes hepatocellular damage [19]. AST is found in both mitochondria and the cytosol, while ALT is found only in the cytosol [66]. Therefore, ALT is a more accurate indication of the detection of hepatocellular damage [7, 63]. In a study of the literature, in parallel with our findings, in the blood samples of rats with sepsis caused by LPS; it has been reported that ALT, AST, and ALP enzymes were found to be significantly higher in the sepsis group [42]. In addition, it was reported that ALT, AST, and ALP enzymes were found to be higher in the sepsis group compared to the control group in a study that created sepsis by giving mice intraperitoneal Escherichia coli [44]. Many studies have been carried out to assess the hepatic protective effects of silymarin in terms of ALP, AST, and ALT enzymes. In groups with hepatotoxicity using CCl₄ and DEN; ALP [2, 50, 54], AST [2, 47, 50, 54], and ALT [2, 47, 50, 54] enzyme levels increased in the sepsis group compared to the control group and it was reported that the enzyme levels decreased in the silymarin applied groups. In the pathogenesis of sepsis; oxidative damage caused by free oxygen radicals produced in large quantities by activated immune system cells is thought to be involved. Many methods are applied in the body as an indicator of oxidative stress. Measuring the amount of MDA, one of the final products of lipid peroxidation, is one of the most preferred methods of current experimental and clinical research [10]. Serum MDA levels were lowest in the control group, followed by SM100+CLP, SM200+CLP, CLP, and SM50+CLP groups, respectively. MDA levels were found to be similar in CLP and SM50+CLP groups (p > 0.05). A study in which they investigated the role of oxidative stress in the model of CLP sepsis; found that MDA levels increased in tissue and plasma and as a result, free oxygen radicals played a role in sepsis pathophysiology in the CLP model. Koksal et al. [32] investigated the role of oxidative stress in CLP sepsis; it found that MDA levels increased in tissue and plasma and as a result, free oxygen radicals played a role in sepsis pathophysiology in the CLP model. In addition, [48] reported that MDA levels in the CLP group’s liver tissue rose significantly compared to the control group.

In our study, it was detected that while serum SOD activity was significantly reduced in the CLP group; silymarin used as an antioxidant substance significantly increased serum SOD activity compared to the CLP group. Zheng et al. [76] reported that in the experimental sepsis model created by the CLP method, the SOD activity in the renal tissue of rats was significantly reduced compared to the control group. Wang et al. [69] found that serum SOD activity increased in sepsis animals in rats where they formed sepsis using the CLP method and that the application of exogenous carbon monoxide significantly increased the SOD activity. In a study investigating the effects of silymarin, it was found that the application of silymarin increased SOD activity in the formation of benzoyl peroxide-induced tumors in mouse (or rat) skin [38]. In addition, the lowest activity of CAT, an enzymatic antioxidant with high activity in the liver, was measured in the CLP group and was followed by SM50+CLP, SM200+CLP, SM100+CLP, and the control group, respectively. In a study that created experimental sepsis with CLP, CAT enzyme activity was researched in the liver tissues of rats and it was reported that cat activity of the sepsis group was suppressed compared to the control group [48]. Allameh et al. [6] reported that CAT levels were lower in liver tissues of rats that they created experimental sepsis with CLP than in the control group. In the experimental sepsis model created by applying LPS, CAT levels were measured in renal tissue; it was found that there was a significant decrease in the sepsis group and edaravone application corrected this situation [38].

One of the most commonly found tripeptides in the liver is GSH. It is tasked with eliminating superoxide radicals and free radicals such as hydrogen peroxide. GSH is also a substrate for GSH-Px [8, 33, 37, 49]. GSH-Px is an antioxidant enzyme that prevents oxidative stress and neutralizes the harmful effects of free radicals. GSH and GSH-Px levels were lowest in the CLP group (p < 0.001). The GSH-Px activity was the lowest in the SM200+CLP, SM50+CLP, SM100+CLP, and control groups after the CLP group while the GSH level was the lowest in the SM50+CLP, SM200+CLP, SM100+CLP and control groups after the CLP group. In the literature, a study investigating the role of the oxidative system in sepsis, Koksal et al. [32] detected that GSH levels decreased in the liver, kidney, heart, lung, and plasma. In CLP studies in rats, it was reported that GSH levels decreased in the liver tissues of sepsis groups compared to the control group [5, 9]. Aydın et al. [9] researched the effects of ferulic acid in the sepsis model they created with the CLP method in rats; they found that GSH level and GSH-Px activity decreased in the sepsis group and significant increases were observed in the groups that applied ferulic acid.

Our study findings revealed that the application of silymarin reduced liver damage in septic rats by dose-dependent. In addition, it was determined that there was some improvement in the oxidant-antioxidant balance, which was impaired in septic rats, and that silymarin had a hepatoprotective effect in the liver in histological evaluation. However, more comprehensive experimental and clinical studies are needed to better understand the protective effects of silymarin on the liver.

V.  Disclosure Statement

No potential conflict of interest was reported by the author(s).

VI.  Funding

This study was supported by Atatürk University Scientific Research Projects Coordination Unit. Project Number: TDK-2019-7285.

VII.  Acknowledgments

This study was produced from Nevra AYDEMİR CELEP’s doctoral thesis titled “Investigation of the Protective Effects of Silymarin in Rats with Liver Damage by the Sepsis Model Induced by the Cecal Ligation-Perforation Method”.

VIII. References

• 1   Abd El,  A. A. E.-A.,  Sayed,  A. A.,  Soliman,  A. M. and  Fahmy,  S. R. (2016) Exploration of the therapeutic potential effect of Sepia officinalis in animal model of sepsis induced by cecal ligation and puncture. Injury 47; 2709–2717.
• 2   Abdel-Moneim,  A. M.,  Al-Kahtani,  M. A.,  El-Kersh,  M. A. and  Al-Omair,  M. A. (2015) Free radical-scavenging, anti-inflammatory/anti-fibrotic and hepatoprotective actions of taurine and silymarin against CCl4 induced rat liver damage. PLoS One 10; e0144509.
• 3   Akin,  A. T.,  Öztürk,  E.,  Kaymak,  E.,  Karabulut,  D. and  Yakan,  B. (2021) Therapeutic effects of thymoquinone in doxorubicin‐induced hepatotoxicity via oxidative stress, inflammation and apoptosis. Anat. Histol. Embryol. 50; 908–917.
• 4   Al-Kadi,  A.,  Ahmed,  A.-S.,  El-Tahawy,  N. F. G.,  Khalifa,  M. M. A. and  El-Daly,  M. (2020) Silymarin protects against sepsis-induced acute liver and kidney injury via anti-inflammatory and antioxidant mechanisms in the rat. J. Adv. Biomed. Pharm. Sci. 3; 190–197.
• 5   Aliomrani,  M.,  Sepand,  M. R.,  Mirzaei,  H. R.,  Nekonam,  S. and  Sabzevari,  O. (2016) Effects of phloretin on oxidative and inflammatory reaction in rat model of cecal ligation and puncture induced sepsis. DARU J. Pharm. Sci. 24; 1–8.
• 6   Allameh,  A.,  Razavi-Azarkhiavi,  K.,  Mohsenifar,  A. and  Jamali-Zavarei,  M. (2012) Effect of acute ethanol treatment on biochemical and histopathological factors in rat liver in an experimental sepsis model. Pathol. Res. Pract. 208; 331–337.
• 7   Ambade,  A.,  Satishchandran,  A.,  Gyongyosi,  B.,  Lowe,  P. and  Szabo,  G. (2016) Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease. World J. Gastroenterol. 22; 4091–4108.
• 8   Atkuri,  K. R.,  Mantovani,  J. J.,  Herzenberg,  L. A. and  Herzenberg,  L. A. (2007) N-Acetylcysteine—a safe antidote for cysteine/glutathione deficiency. Curr. Opin. Pharmacol. 7; 355–359.
• 9   Aydin,  S.,  Şahin,  T. T.,  Bacanli,  M.,  Taner,  G.,  Başaran,  A. A.,  Aydin,  M., et al. (2016) Resveratrol protects sepsis-induced oxidative DNA damage in liver and kidney of rats. Balkan Med. J. 33; 594–601.
• 10  Bayraktar, O. (2013) Deneysel sepsis modeli oluşturulan ratlarda s-allilsistein’in transglutaminaz ii üzerine etkileri, ESOGÜ, Sağlık Bilimleri Enstitüsü.
• 11   Bilgili,  B.,  Haliloğlu,  M. and  Cinel,  İ. (2014) Sepsis ve akut böbrek hasarı. Turk. J. Anaesthesiol. Reanim. 42; 294–301.
• 12   Boughton-Smith,  N.,  Evans,  S.,  Whittle,  B.,  Moncada,  S.,  Hawkey,  C.,  Cole,  A., et al. (1993) Nitric oxide synthase activity in ulcerative colitis and Crohn’s disease. Lancet 342; 338–340.
• 13   Chen,  J. X.,  Berry,  L. C.,  Tanner,  M.,  Chang,  M.,  Myers,  R. P. and  Meyrick,  B. (2001) Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium. J. Cell. Physiol. 186; 116–123.
• 14   Cihan,  B.,  Yarat,  A.,  Akbay,  T. T. and  Şener,  G. (2015) Investigation of the effect of silymarin on the liver in experimental sepsis. Marmara Pharm. J. 19; 52–59.
• 15   Cinel,  I. and  Dellinger,  R. P. (2007) Advances in pathogenesis and management of sepsis. Curr. Opin. Infect. Dis. 20; 345–352.
• 16   Clementi,  E.,  Brown,  G. C.,  Feelisch,  M. and  Moncada,  S. (1998) Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc. Natl. Acad. Sci. U S A 95; 7631–7636.
• 17   Coşkunfırat,  N.,  Cengiz,  M. and  Yılmaz,  M. (2010) Akut böbrek yetersizliği üzerine hayvan modelleri. Türk Yoğun Bakım Derneği Dergisi 8; 38–45.
• 18   Doi,  K.,  Leelahavanichkul,  A.,  Yuen,  P. S. and  Star,  R. A. (2009) Animal models of sepsis and sepsis-induced kidney injury. J. Clin. Invest. 119; 2868–2878.
• 19   Feng,  L.,  Mao,  W.,  Zhang,  J.,  Liu,  X.,  Jiao,  Y.,  Zhao,  X., et al. (2014) Pharmacokinetic variations of tetramethylpyrazine phosphate after oral administration in hepatic precancerous mice and its hepatoprotective effects. Drug Dev. Ind. Pharm. 40; 1–8.
• 20   Fidan,  H.,  Koca,  U.,  Ergür,  B. U.,  Ahmedova,  A. and  Kaymakçi,  F. (2012) N-Asetilsisteinin Rat Intraabdominal Sepsis Modelinde Böbrek Apoptozisi Üzerine Olan Etkileri/The Effects of N-Acetylcysteine on Kidney Apoptosis in a Rat Intraabdominal Sepsis Model. Türkiye Klinikleri Tip Bilimleri Dergisi 32; 1653.
• 21   Fleischmann,  C.,  Scherag,  A.,  Adhikari,  N. K.,  Hartog,  C. S.,  Tsaganos,  T.,  Schlattmann,  P., et al. (2016) Assessment of global incidence and mortality of hospital-treated sepsis. Current estimates and limitations. Am. J. Respir. Crit. Care Med. 193; 259–272.
• 22   Fraschini,  F.,  Demartini,  G. and  Esposti,  D. (2002) Pharmacology of silymarin. Clin. Drug Investig. 22; 51–65.
• 23   Gardner,  C. R.,  Laskin,  J. D.,  Dambach,  D. M.,  Sacco,  M.,  Durham,  S. K.,  Bruno,  M. K., et al. (2002) Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-α and interleukin-10. Toxicol. Appl. Pharmacol. 184; 27–36.
• 24   Gillessen,  A. and  Schmidt,  H. H.-J. (2020) Silymarin as supportive treatment in liver diseases: A narrative review. Adv. Ther. 37; 1279–1301.
• 25  Gong, W. and Wen, H. (2019) Sepsis induced by cecal ligation and puncture. In “Mouse Models of Innate Immunity”, Springer, pp. 249–255.
• 26   Goth,  L. (1991) A simple method for determination of serum catalase activity and revision of reference range. Clin. Chim. Acta 196; 143–151.
• 27   Gül,  F.,  Arslantaş,  M. K.,  Cinel,  İ. and  Kumar,  A. (2017) Changing definitions of sepsis. Turk. J. Anaesthesiol. Reanim. 45; 129.
• 28   İskit,  A. B. (2005) Sepsiste deneysel modeller. Yoğun Bakım Dergisi 5; 133–136.
• 29   Jacob,  J. T.,  Coulombe,  P. A.,  Kwan,  R. and  Omary,  M. B. (2018) Types I and II keratin intermediate filaments. Cold Spring Harb. Perspect. Biol. 10; a018275.
• 30   Kamanaka,  Y.,  Kawabata,  A.,  Matsuya,  H.,  Taga,  C.,  Sekiguchi,  F. and  Kawao,  N. (2003) Effect of a potent iNOS inhibitor (ONO-1714) on acetaminophen-induced hepatotoxicity in the rat. Life Sci. 74; 793–802.
• 31   Karupiah,  G.,  Xie,  Q.-W.,  Buller,  R. M. L.,  Nathan,  C.,  Duarte,  C. and  MacMicking,  J. D. (1993) Inhibition of viral replication by interferon-γ-induced nitric oxide synthase. Science 261; 1445–1448.
• 32   Koksal,  G.,  Sayilgan,  C.,  Aydin,  S.,  Oz,  H. and  Uzun,  H. (2004) Correlation of plasma and tissue oxidative stresses in intra-abdominal sepsis. J. Surg. Res. 122; 180–183.
• 33   Konukoğlu,  D. and  Akçay,  T. (1995) Glutatyon metabolizması ve klinik önemi. Turkiye Klinikleri J. Med. Sci. 15; 214–218.
• 34   Kooy,  N. W.,  Lewis,  S. J.,  Royall,  J. A.,  Yao,  Z. Y.,  Kelly,  D. R. and  Beckman,  J. S. (1997) Extensive tyrosine nitration in human myocardial inflammation: evidence for the presence of peroxynitrite. Crit. Care Med. 25; 812–819.
• 35   Kurt,  C. (2006) Sepsis ile ilişkili tanımlar, epidemiyoloji, insidans ve klinik. Güncel Bilgiler Işığında Sepsis”, İÜ Cerrahpaşa Tıp Fakültesi Sürekli Tıp Eğitimi Etkinlikleri Sempozyum Dizisi; 17–26.
• 36   Leon,  L. R.,  White,  A. A. and  Kluger,  M. J. (1998) Role of IL-6 and TNF in thermoregulation and survival during sepsis in mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 275; R269–R277.
• 37   Lima,  E. S.,  Roland,  I. d. A.,  Maroja,  M. d. F. and  Marcon,  J. L. (2007) Vitamin A and lipid peroxidation in patients with different forms of leprosy. Rev. Inst. Med. Trop. Sao Paulo 49; 211–214.
• 38   Liu,  L.,  Song,  Y.,  Zhao,  M.,  Yi,  Z. and  Zeng,  Q. (2015) Protective effects of edaravone, a free radical scavenger, on lipopolysaccharide-induced acute kidney injury in a rat model of sepsis. Int. Urol. Nephrol. 47; 1745–1752.
• 39   Loschke,  F.,  Seltmann,  K.,  Bouameur,  J.-E. and  Magin,  T. M. (2015) Regulation of keratin network organization. Curr. Opin. Cell Biol. 32; 56–64.
• 40   MacMicking,  J. D.,  North,  R. J.,  LaCourse,  R.,  Mudgett,  J. S.,  Shah,  S. K. and  Nathan,  C. F. (1997) Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc. Natl. Acad. Sci. U S A 94; 5243–5248.
• 41   Mete,  B. (2006) Sepsiste böbrek ve karaciğer. İÜ Cerrahpaşa Tıp Fakültesi Sürekli Tıp Eğitimi Etkinlikleri Güncel Bilgiler Işığında Sepsis Sempozyumu Bildirisi 51; 35–43.
• 42   Mohamadin,  A. M.,  Elberry,  A. A.,  Elkablawy,  M. A.,  Gawad,  H. S. A. and  Al-Abbasi,  F. A. (2011) Montelukast, a leukotriene receptor antagonist abrogates lipopolysaccharide-induced toxicity and oxidative stress in rat liver. Pathophysiology 18; 235–242.
• 43   Moore,  D. J.,  Greystoke,  A.,  Butt,  F.,  Wurthner,  J.,  Growcott,  J.,  Hughes,  A., et al. (2012) A pilot study assessing the prognostic value of CK18 and nDNA biomarkers in severe sepsis patients. Clin. Drug Investig. 32; 179–187.
• 44   Oter,  S.,  Edremitlioglu,  M.,  Korkmaz,  A.,  Coskun,  O.,  Kilic,  D.,  Kisa,  U., et al. (2005) Effects of hyperbaric oxygen treatment on liver functions, oxidative status and histology in septic rats. Intensive Care Med. 31; 1262–1268.
• 45   Otero-Anton,  E.,  Gonzalez-Quintela,  A.,  Lopez-Soto,  A.,  Lopez-Ben,  S.,  Llovo,  J. and  Perez,  L. (2001) Cecal ligation and puncture as a model of sepsis in the rat: influence of the puncture size on mortality, bacteremia, endotoxemia and tumor necrosis factor alpha levels. Eur. Surg. Res. 33; 77–79.
• 46   Parveen,  R.,  Baboota,  S.,  Ali,  J.,  Ahuja,  A.,  Vasudev,  S. S. and  Ahmad,  S. (2011) Effects of silymarin nanoemulsion against carbon tetrachloride-induced hepatic damage. Arch. Pharm. Res. 34; 767–774.
• 47   Pradeep,  K.,  Mohan,  C. V. R.,  Gobianand,  K. and  Karthikeyan,  S. (2007) Silymarin modulates the oxidant–antioxidant imbalance during diethylnitrosamine induced oxidative stress in rats. Eur. J. Pharmacol. 560; 110–116.
• 48   Prauchner,  C. A.,  Prestes,  A. d. S. and  da Rocha,  J. B. (2011) Effects of diphenyl diselenide on oxidative stress induced by sepsis in rats. Pathol. Res. Pract. 207; 554–558.
• 49   Rajkapoor,  B.,  Venugopal,  Y.,  Anbu,  J.,  Harikrishnan,  N.,  Gobinath,  M. and  Ravichandran,  V. (2008) Protective effect of Phyllanthus polyphyllus on acetaminophen induced hepatotoxicity in rats. Pak. J. Pharm. Sci. 21; 57–62.
• 50   Ramakrishnan,  G.,  Augustine,  T.,  Jagan,  S.,  Vinodhkumar,  R. and  Devaki,  T. (2007) Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats. Exp. Oncol. 29; 39–44.
• 51   Rittirsch,  D.,  Huber-Lang,  M. S.,  Flierl,  M. A. and  Ward,  P. A. (2009) Immunodesign of experimental sepsis by cecal ligation and puncture. Nat. Protoc. 4; 31–36.
• 52   Roth,  G. A.,  Krenn,  C.,  Brunner,  M.,  Moser,  B.,  Ploder,  M.,  Spittler,  A., et al. (2004) Elevated serum levels of epithelial cell apoptosis-specific cytokeratin 18 neoepitope m30 in critically ill patients. Shock 22; 218–220.
• 53   Savcun,  G.,  Ozkan,  E.,  Dulundu,  E.,  Topaloglu,  U.,  Sehirli,  A.,  Tok,  O., et al. (2013) Antioxidant and anti-inflammatory effects of curcumin against hepatorenal oxidative injury in an experimental sepsis model in rats. Ulus. Travma Acil Cerrahi Derg. 19; 507–515.
• 54   Shaarawy,  S. M.,  Tohamy,  A. A.,  Elgendy,  S. M.,  Abd Elmageed,  Z. Y.,  Bahnasy,  A.,  Mohamed,  M. S., et al. (2009) Protective effects of garlic and silymarin on NDEA-induced rats hepatotoxicity. Int. J. Biol. Sci. 5; 549–557.
• 55   Shafaroodi,  H.,  Hassanipour,  M.,  Mousavi,  Z.,  Rahimi,  N. and  Dehpour,  A. R. (2015) The effects of sub-chronic treatment with pioglitazone on the septic mice mortality in the model of cecal ligation and puncture: Involvement of nitric oxide pathway. Acta Med. Iran. 53; 608–616.
• 56   Shen,  D.,  Tao,  Q.,  Hu,  Y. and  Liu,  P. (2012) Dynamic proteomic analysis of liver tissue in the carbon tetrachloride-induced liver fibrosis rat model. Zhonghua Gan Zang Bing Za Zhi 20; 664–670.
• 57   Singleton,  K. and  Wischmeyer,  P. (2003) Distance of cecum ligated influences mortality, tumor necrosis factor-alpha and interleukin-6 expression following cecal ligation and puncture in the rat. Eur. Surg. Res. 35; 486–491.
• 58   Song,  X.-M.,  Li,  J.-G.,  Wang,  Y.-L.,  Hu,  Z.-F.,  Zhou,  Q.,  Du,  Z.-H., et al. (2008) The protective effect of the cholinergic anti-inflammatory pathway against septic shock in rats. Shock 30; 468–472.
• 59   Soto,  C.,  Recoba,  R.,  Barron,  H.,  Alvarez,  C. and  Favari,  L. (2003) Silymarin increases antioxidant enzymes in alloxan-induced diabetes in rat pancreas. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 136; 205–212.
• 60   Soto,  C. P.,  Perez,  B. L.,  Favari,  L. P. and  Reyes,  J. L. (1998) Prevention of alloxan-induced diabetes mellitus in the rat by silymarin. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 119; 125–129.
• 61   Sun,  Y.,  Oberley,  L. W. and  Li,  Y. (1988) A simple method for clinical assay of superoxide dismutase. Clin. Chem. 34; 497–500.
• 62   Sungur,  M. (2005) Sepsiste organ destek tedavileri. Yoğun Bakım Dergisi 5; 112–121.
• 63   Tekin,  N.,  Ustuner,  M. C.,  Akyuz,  F.,  Ozbayer,  C. S.,  Aydın,  O.,  Benkli,  K., et al. (2014) Apoptotic effects of dipyrido [3, 2-a: 2', 3'-c] phenazine (dppz) Au (III) complex against diethylnitrosamine/phenobarbital induced experimental hepatocarcinogenesis in rats. Mol. Biol. Rep. 41; 5109–5121.
• 64   Tietze,  F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27; 502–522.
• 65   Toklu,  H. Z.,  Akbay,  T. T.,  Velioglu-Ogunc,  A.,  Ercan,  F.,  Gedik,  N.,  Keyer-Uysal,  M., et al. (2008) Silymarin, the antioxidant component of Silybum marianum, prevents sepsis-induced acute lung and brain injury. J. Surg. Res. 145; 214–222.
• 66   Uygun,  A. and  Polat,  Z. (2009) Viral hepatit dışı serum transaminaz düzeyinde artışa neden olan hastalıklar. Güncel Gastroenteroloji 13; 211–224.
• 67   Vincent,  J.-L.,  Opal,  S. M.,  Marshall,  J. C. and  Tracey,  K. J. (2013) Sepsis definitions: time for change. Lancet 381; 774–775.
• 68   Wang,  A.,  Xiao,  Z.,  Zhou,  L.,  Zhang,  J.,  Li,  X. and  He,  Q. (2016) The protective effect of atractylenolide I on systemic inflammation in the mouse model of sepsis created by cecal ligation and puncture. Pharm. Biol. 54; 146–150.
• 69   Wang,  P.,  Huang,  J.,  Li,  Y.,  Chang,  R.,  Wu,  H.,  Lin,  J., et al. (2015) Exogenous carbon monoxide decreases sepsis-induced acute kidney injury and inhibits NLRP3 inflammasome activation in rats. Int. J. Mol. Sci. 16; 20595–20608.
• 70  Wen, H. (2013) Sepsis induced by cecal ligation and puncture. In “Mouse Models of Innate Immunity”, Springer, pp. 117–124.
• 71   Woolbright,  B. L.,  Bridges,  B. W.,  Dunn,  W.,  Olson,  J. C.,  Weinman,  S. A. and  Jaeschke,  H. (2017) Cell death and prognosis of mortality in alcoholic hepatitis patients using plasma keratin-18. Gene Expr. 17; 301–312.
• 72   Wu,  J.-W.,  Lin,  L.-C. and  Tsai,  T.-H. (2009) Drug–drug interactions of silymarin on the perspective of pharmacokinetics. J. Ethnopharmacol. 121; 185–193.
• 73   Xu,  S.,  Gao,  Y.,  Zhang,  Q.,  Wei,  S.,  Chen,  Z.,  Dai,  X., et al. (2016) SIRT1/3 activation by resveratrol attenuates acute kidney injury in a septic rat model. Oxid. Med. Cell. Longev. 2016; 7296092.
• 74   Yoshioka,  T.,  Kawada,  K.,  Shimada,  T. and  Mori,  M. (1979) Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am. J. Obstet. Gynecol. 135; 372–376.
• 75   Yu,  C.,  Li,  P.,  Qi,  D.,  Wang,  L.,  Qu,  H.-l.,  Zhang,  Y.-j., et al. (2017) Osthole protects sepsis-induced acute kidney injury via down-regulating NF-κB signal pathway. Oncotarget 8; 4796–4813.
• 76   Zheng,  Q.,  Liu,  W.,  Liu,  Z.,  Zhao,  H.,  Han,  X. and  Zhao,  M. (2014) Valproic acid protects septic mice from renal injury by reducing the inflammatory response. J. Surg. Res. 192; 163–169.
• 77   Zi,  X.,  Mukhtar,  H. and  Agarwal,  R. (1997) Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: Inhibition of mRNA expression of an endogenous tumor promoter TNFα. Biochem. Biophys. Res. Commun. 239; 334–339.