Antioxidant Effects of Probiotics in Experimentally Induced Peritonitis

Abstract

Aim:

An experimental study was performed to evaluate the protective effects of probiotics on gut mucosa in peritonitis through antioxidant mechanisms.

Methods:

Thirty-two male Wistar albino rats were divided equally into four groups. The rats in Group 1 (control group) underwent laparotomy only. In group 2 (peritonitis group), peritonitis was induced in the rats by the cecal ligation and puncture (CLP) model. In group 3, the rats were treated with probiotics for five days after CLP-induced peritonitis. The last group of rats (group 4) were fed probiotics for five days before the CLP procedure and five days after the surgery. On the fifth day after surgery, all rats were killed, and tissue samples from the terminal ileum were obtained to evaluate the activities of myeloperoxidase (MPO), malondialdehyde (MDA), and glutathione (GSH). Histopathologic examinations were also performed to evaluate the grade of intestinal injury.

Results:

Myeloperoxidase and MDA activities were increased, GSH concentrations were decreased in group 2, compared with group 1. Intestinal MPO activities in group 4 were decreased compared with group 1 and group 2, indicating a reduction in oxidant activity. Malondialdehyde decreased in group 3 and decreased even more in group 4, compared with the peritonitis group (group 2). Glutathione concentrations were increased in group 4 compared with group 2 and group 3 (p < 0.05). The Chiu scores of the probiotics groups, groups 3 and 4, were lower than those in group 2, indicating reduced mucosal damage in the probiotically fed groups.

Conclusion:

Probiotics have protective effects in peritonitis, which may be related to antioxidant mechanisms. This antioxidant effect of probiotics might occur when pre-conditioning with probiotics before peritonitis because there is sufficient time to prepare the tissues for oxidative damage.

Secondary peritonitis is a life-threatening condition and occurs primarily after the disruption of the integrity of the gastrointestinal tract. Upon induction of experimental peritonitis by cecal ligation and perforation-puncture (CLP), multiple signs of gut barrier failure appear, including bacterial translocation and internalization of tight junction-associated proteins [1]. In critically ill patients, the gut mucosal barrier and immunologic functions may be impaired, leading to dissemination of pathogenic substances into systemic circulation; this process can trigger pathophysiologic changes and an inflammatory response that causes sepsis and multiple organ failure [2]. Reactive oxygen species play an important role in this inflammatory process. A reduction in oxygen, the production of reactive oxygen species by white blood cells, and lipid peroxidation by these reactive oxygen species causes oxidative damage in peritonitis. This oxidant-mediated injury also worsens the progression of peritonitis [3]. The inflammation severity in the intestinal mucosa is an important factor affecting the severity of peritonitis. The intestine plays a central role in sepsis. Current studies regarding symbiotic therapy with probiotics have investigated the ability of probiotics to reduce infection, systemic inflammatory response syndrome, severe sepsis, and mortality rates [4]. We hypothesize that oxidative stress, which is one of the most important components of peritonitis, can be prevented by the anti-inflammatory features of probiotics. In our study, we aimed to evaluate the effects of probiotics as anti-inflammatory and antioxidant agents in a rat model of peritonitis by examining the activity of inflammatory biochemical markers (malondialdehyde [MDA] and myeloperoxidase [MPO]) to reveal oxidative stress and the concentrations of glutathione (GSH) to measure the anti-inflammatory response.

Patients and Methods

Animals and study design

After receiving approval from the Animal Ethics Committee, 32 male Wistar albino rats (Institute of Experimental Medical Research and Application, Istanbul University) weighing 300–350 g were divided equally into four groups. They were housed under controlled temperature (21°C ± 2°C), lighting (12-h light/dark cycle), and humidity conditions. Each group contained 8 rats. Rats in group 1 underwent laparotomy only. In group 2, peritonitis was induced in the rats using the CLP model. A median laparotomy was performed in which the cecum was first ligated with a silk suture and then punctured with a 20-gauge needle, as described in the literature [5]. Rats in group 3 were treated with probiotics for five days after peritonitis had been induced using CLP. The probiotic solution containing 2 × 10⁹ colony-forming units per milliliter (CFU/mL) Bifidobacterium bifidum, Lactobacillus acidophilus, and Lactobacillus bulgaricus (Probiyotik Pur, Ella Farma, Turkey) was administered through an orogastric tube twice daily. The last group of rats, group 4 was fed probiotics for five days before the CLP procedure and probiotic feeding continued after surgery for five days. On the fifth day after surgery, all rats were killed and tissue samples from the terminal ileum were obtained to evaluate MPO activity, as well as tissue MDA and GSH content for histopathologic analysis.

Biochemical analysis

Myeloperoxidase

Myeloperoxidase is a heme protein synthesized during myeloid differentiation that constitutes the major component of neutrophil azurophilic granules. Its concentration is correlated with the severity of inflammation. To estimate MPO concentrations, 200 mg of tissue samples were homogenized in 3 mL of 0.05 M K-phosphate buffer (pH = 6.0) containing 0.5% hexadecyltrimethylammonium bromide. Homogenates were sonicated for 20 sec, freeze-thawed three times, and centrifuged at 40,000g for 15 min. Then, 0.1 mL of supernatants was added to 2.9 mL 0.05 M K-phosphate buffer (pH = 6.0) containing 0.53 mM o-dianisidine and 0.15 mM H₂O₂ and mixed. Changes in absorbance at 460 nm were recorded for five minutes. The results are expressed as nmol/min/mg protein [6].

Malondialdehyde

Reactive oxygen species degrade polyunsaturated lipids to form malondialdehyde; therefore, MDA, which results from this lipid peroxidation, is used as a marker of oxidative stress. The colored complex of MDA and thiobarbituric acid was measured spectrophotometrically at 532 nm [7].

Glutathione

Glutathione is an important antioxidant that prevents damage to important cellular components caused by reactive oxygen species. The GSH concentrations were measured using Ellman reagent (5,5′-dithiobis-2-nitro benzoic acid) [8].

Histopathologic examination

After fixation with formol, the ileal segments were embedded in paraffin blocks. The paraffin blocks were cut into 4-mcm–wide sections, and these tissue sections were stained with hematoxylin and eosin. Histopathologic scoring was performed according to the Chiu scoring scale (Table 1) [9].

Table 1.

Histopathologic Grades of Intestinal Tissue (Chiu Scoring System [9])

Grade	Histology
0	Normal mucosal villi
I	Development of a sub-epithelial space, usually at the tip of the villus, with capillary congestion.
II	Extension of the sub-epithelial space with moderate lifting of the epithelial layer.
III	Massive epithelial lifting down the sides of the villi.
IV	Denuded villi with lamina propria, dilated capillaries exposed, and increased cellularity of the lamina propria.
V	Digestion and disintegration of the lamina propria, haemorrhage and ulceration.

Statistical analysis

Descriptive statistical methods were used to analyze the results in the groups. Kolmogorov-Smirnov and Shapiro-Wilk tests were used for the normality analysis. Analysis of variance (ANOVA) and t-test methods were used to compare the groups and to ensure normal distributions. The Kruskal-Wallis test was used for the variance analysis, and the Mann-Whitney U test was used to compare the groups that did not have a normal distribution. The confidence interval was 95% and a p value ≤0.05 was considered significant.

Results

Anorexia, piloerection, and immobilization occurred in all rats after the CLP procedure. In all groups, the rats' weights decreased, particularly in the first 24 h after the induction of peritonitis. The loss of body weight (10%) was similar among the groups.

Biochemical results

Myeloperoxidase concentrations

Myeloperoxidase activities were increased in group 2, compared with group 1. Group 3 demonstrated increased MPO concentrations compared with group 1 and group 2. Intestinal MPO activities in group 4 were decreased compared with group 1 and group 2, indicating a reduction in oxidant activity.

Malondialdehyde concentrations

The MDA concentrations were slightly increased in group 2 compared with group 1. The concentrations decreased in group 3 and decreased even more in group 4 compared with group 1 and group 2.

Glutathione concentrations

Glutathione concentrations were lower in group 2 compared with group 1. Glutathione concentrations were lower in group 3 compared with group 1, yet were increased compared with group 2. Also, GSH concentrations were increased in group 4 compared with both group 1 and group 2. This increase is significant in group 4 and group 2 as an indicator of restoration of antioxidant activity. There was also a significant increase in the GSH concentrations in group 4 compared with group 3 (p < 0.05; Table 2).

Table 2.

Mean Values and Standard Deviations of Myeloperoxidase, Malondialdehyde, and Glutathione Levels

Groups		MPO (nmol/mg)	MDA (nmol/mg)	GSH (nmol/mg)
Group 1	Mean	6.760	1.278	0.672
	SD	4.315	0.691	0.360
Group 2	Mean	7.760	1.282	0.380^a
	SD	4.375	1.669	0.129
Group 3	Mean	11.040	1.116	0.626^a
	SD	3.103	1.435	0.160
Group 4	Mean	4.240	0.700	0.888^a
	SD	1.803	0.182	0.120

Significance among the groups (p < 0.05).

Group 1 = laparatomy only; group 2 = peritonitis only; group 3 = treated with probiotics for five days after CLP-induced peritonitis; group 4 = were fed probiotics for five days before the CLP procedure and five days after the surgery; MDA = malondialdehyde, nmol/mg protein; GSH = glutathione, nmol/mg protein; MPO = myeloperoxidase, nmol/mg; SD = standard deviation; CLP = cecal ligation and puncture.

Histopathologic results

The median concentrations of the groups' Chiu scores are listed in Table 3. The Chiu score was lowest in the group 1. Although the Chiu scores of the probiotic groups (group 3 and 4) were lower than group 2, the difference was not significant.

Table 3.

Median Levels of the Chiu Score According to Histologic Examination

Groups	Chiu score
Group 1	0.5 (0–1)
Group 2	1.5 (0–4)
Group 3	1 (0–3)
Group 4	0.75 (0–3)

Discussion

The present study demonstrated that probiotics may alter intestinal mucosal damage during peritonitis via antioxidant mechanisms and pre-treatment with probiotics reduces the effects of mucosal damage more efficiently.

Peritonitis continues to be a life-threatening condition related to surgery despite modern broad-spectrum antibiotics and advanced intensive care units. Peritonitis is especially fatal in infants and newborns, therefore, new treatments are being investigated. However, these treatments are still not enough to cure patients, and antibiotics alone cannot be the definitive solution. Alternative treatments are also being investigated. Among these new experimental drugs, probiotics show promising results. Probiotics are living micro-organisms that survive in the gastrointestinal tract after ingestion and have beneficial effects on the host. Probiotics that contain lactobacillus and bifidobacterium have been recommended for use in several diseases [10]. These probiotics have shown positive effects on bacterial translocation during laparoscopic surgery [11] and in experimental short-bowel syndrome [12]. In the study by Carlisie et al. [13], intra-luminal chloramine-T caused full-thickness necrosis in mice and was characterized by a loss of probiotic bacteria such as lactobacillus, showing that oxidative stress and colonization by pathogenic bacteria may play important roles in intestinal necrosis. Recently, the importance of the gut microbiome in critically ill patients has been studied by many authors [14,15].

Various animal models of intra-abdominal sepsis have been defined, and CLP is frequently used [16,17]. In this model, peritonitis is experimentally induced by cecal ischemia and leakage of cecal contents into the abdominal cavity [18,19]. We used this model to induce experimental peritonitis. In all rats, anorexia, piloerection, and immobilization occurred after the CLP procedure. In all groups, the rats' weight decreased, especially in the first 24 h after peritonitis induction. Throughout the experiment, the weight loss ratios among the groups were approximately the same. Weight loss did not increase in either of the probiotic-treated groups. Our results showed that the rats were seriously affected by peritonitis.

Myeloperoxidase activity is used widely in experimental studies as an indicator of neutrophil infiltration to determine the severity of inflammation [19]. During peritonitis, leukocyte infiltration occurs, abdominal vascular permeability increases, and MPO activity increases in peritoneal fluid [20]. The concentration of MPO was increased in group 2 compared with group 1, which was expected because inflammation is aggravated in the peritonitis process. Group 3 demonstrated increased MPO concentrations, indicating that only administering probiotics after peritonitis was not sufficient to prevent inflammation. The reason that we could not detect the antioxidant effect of probiotics in group 3 might be insufficient time to prepare the tissues for peritonitis damage with probiotics. Although not significant, in our study, the group that was fed probiotics before and after peritonitis induction (group 4) had the lowest MPO concentrations, which indicates a reduction in inflammation. We concluded that to prevent oxidative injury in peritonitis probiotics should be given pre-operatively in addition to post-operative usage. Oxidative tissue damage is a result of surgery and intra-abdominal sepsis, therefore, the usage of probiotics only prior to peritonitis may not be adequate and should be continued post-operatively. Prophylaxis with probiotics was more efficient than administering only probiotics after peritonitis. Tok et al. [21] also demonstrated that pre-treatment with probiotics is efficient in reducing acute lung injury in rats.

Malondialdehyde, a product of lipid peroxidation, is a measure of oxidative injury [18,19]. In our study, MDA concentrations were greater in the peritonitis group than in the laparotomy group, as expected. Administering probiotics reduced MDA concentrations in the mice of the probiotic groups (group 3 and 4) compared with the peritonitis and laparotomy groups. Our study confirms that MDA concentrations decrease in the probiotic-treated groups, which indicates a reduction in the inflammatory process.

Glutathione is an important endogenous antioxidant that shows protective effects against free oxygen radicals. Glutathione peroxidase and GSH reductase are involved in the main reactions in which GSH is consumed to eliminate hydrogen peroxide and other reactive oxygen species. Therefore, reduced GSH content can be used as an indicator of oxidative stress induced by reactive oxygen species [20 –22]. When our peritonitis group is compared with the laparotomy group, as a result of oxidative injury caused by peritonitis there is an increase in oxidants such as MPO and MDA, and a decrease in antioxidants such as GSH as expected. In our study, in contrast to the expectations, there was a decrease in the GSH concentrations of group 3 compared with group 1, indicating a lower antioxidant effect despite probiotics. This may be caused by insufficient time for the tissues to be prepared for peritonitis damage. Glutathione concentrations increased statistically in group 4 compared with group 1 and group 2 (p < 0.05), indicating an increase in the anti-inflammatory pathways. There was also a significant increase in the GSH concentrations in group 4 compared with group 3, suggesting that pre-treatment with probiotics can result in this antioxidant effect. Similar to our study, Teke et al. [23] studied the effects of caffeic acid phenethyl ester (CAPE) on anastomotic healing in secondary peritonitis. They observed restoration in reduced GSH concentrations and superoxide dismutase (SOD) activity after CAPE administration in peritonitis, which were explained by the anti-inflammatory and anti-oxidative effects of CAPE [23].

Our histologic analysis showed that probiotics administered before and after peritonitis reversed intestinal tissue damage. Probiotics easily provide live bacteria and are beneficial for health. In surgical clinics, human beings can benefit from their anti-inflammatory and antioxidant effects. For example, these bacteria can be used for acute or perforated appendicitis. In patients with abdominal pain with suspicion of appendicitis, probiotics can be used for prophylaxis and continued for therapy in addition to antibiotics. These treatments may be possible with further clinical studies on the dosage and timing of use.

Conclusion

Our results showed antioxidant and anti-inflammatory effects of probiotics in experimental peritonitis. These findings suggest that probiotics are possible therapeutic agents in the prevention and treatment of peritonitis.

Footnotes

Acknowledgment

This study was presented at the 15th Congress of the European Paediatric Surgeons Association in Dublin.

Author Disclosure Statement

The authors have no conflicts of interest.

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