The effects of ursodeoxycholic acid in the management of sepsis-induced cholestasis in rodents

Background: Cholestasis is a condition in which there is impairment of bile flow from the liver to the small bowel. It is a common complication of bacterial infection and sepsis. Treatment is usually directed towards the eradication of bacterial infection and consequences of sepsis. Ursodeoxycholic acid (UDCA) has been under investigation as a possible therapeutic option for the treatments of sepsis-associated cholestasis. Methods: Sixty male albino rats (weighing 100–150g) were subjected to daily doses of UDCA (100 mg/kg, p.o.) for 10 days before or after lipopolysaccharides (LPS) induction of cholestasis. Then, the following liver enzyme activity was assessed: plasma aspartate transferase (AST), plasma alkaline transferase (ALT), plasma alkaline phosphatase (ALP), total bilirubin (TBIL). Hepatocyte apoptosis and immunomodulatory activity were assessed by flow cytometric analysis. Plasma pro-inflammatory cytokines (TNF-, IL-1 and IL-4) were measured by ELISA. Liver histology changes were assessed by hematoxylin and eosin (H&E) staining. Results: Our results showed that LPS-induced cholestasis resulted in a significant rise in the TBIL and liver enzymes including GGT, ALP, AST, and hepatocytes death. UDCA improves serum liver chemistries and halts bile acid cytotoxicity when it was used either as a treatment or prevention, compared to the LPS group. Moreover, UDCA has immunomodulatory properties: the effect of UDCA on the percentage of natural killer (NK) cells did not change in either the treatment or prevention group when compared to LPS induced cholestasis. However, significant decrease in the CD3 has been found in the treatment group as compared to the LPS group, and an unexpected increase in the prevention group compared to the LPS treated group. UDCA failed to ameliorate the increase in plasma TNF-α concentration in the treatment group. On the other hand, UDCA caused reduction in plasma TNF-α in the prevention group.

reduction in the liver tissue apoptosis in the UDCA treated groups.
Conclusion: Prophylactic treatment and treatment with UDCA appear to exert a beneficial effect against the damaging effect of hydrophobic bile acids by LPS-induced secretary failure. This involved multiple mechanisms of action.

Background
Cholestasis is considered a clinical syndrome that occurs because of disruption of bile formation due to impaired secretion by hepatocytes or blockage in the flow of bile through the intrahepatic or extrahepatic bile ducts (1). Infections may induce cholestasis via bacterial endotoxins such as lipopolysaccharides (LPS), which are released by gramnegative bacteria from the sites of bacterial infection into the circulation; such endotoxins provoke hepatic Kupffer cells to release proinflammatory cytokines, such as tumor necrosis factor-α, (TNF-α), and interleukin-6 (IL-6), which leads to failure of bile secretion (2,3). Sepsis-induced cholestasis may be an early complication in 20% of hospitalized patients with sepsis (4). Severe sepsis may result in life-threatening organ failure due to dysregulated host immune response against infection (5). Sepsis-associated hepatic damage may result from cholestasis induced by the impairment of hepatocellular and bile duct formation due to circulating pro-inflammatory cytokines (2). Apart from the control of sepsis with appropriate antibiotics and supportive care, there is no specific treatment for cholestasis (6).
Ursodeoxycholic acid (UDCA) is the first effective modifying therapy for patients with primary biliary cholangitis (PBC)-previously known as primary biliary cirrhosis-which is a chronic immune-mediated liver disease characterized by progressive cholestasis, biliary fibrosis, and eventually cirrhosis (7). UDCA is very effective in about two thirds of treated patients with an expected survival similar to the general population (8). Furthermore, UDCA is the preferred choice for empiric therapy of most cholestatic liver disease, including cholelithiasis (9). UDCA (3, 7-dihy-droxy-5-cholanic acid) is a hydrophilic bile acid that is more commonly becoming used for the treatment of various cholestatic disorders (10,11). It is normally present in human bile acids in low concentration. UDCA is a major component of bile acid in black bear's bile, which has historically been used in Chinese traditional medicine for the treatment of liver disease; however, the Japanese were the first to formally report its effectiveness on patients with liver disease (12). The mechanisms behind the beneficial effects of UDCA in cholestasis are still unclear.
Evidence from experimental studies suggests three major mechanisms of action: cytoprotection of cholangiocytes from the detergent effect of bile acids, enhancement of hepatobiliary secretion, and hepatocytes protection from apoptosis-induced by bile acids.
One or all of these mechanisms may be relevant to some cholestatic disorders (13). The aims of this study are to investigate the effect of UDCA in the treatment and prevention of LPS-induced cholestasis and to examine the underlying mechanisms, including liver tissue apoptosis, inflammatory mediators, and any possible immunomodulatory mechanisms. GmbH, in Freiburg, Germany). Alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) kits were obtained from ''Wako Pure Chemical Industries", in Osaka, Japan. Annexin V-FITC apoptosis detection kits were purchased from Aldrich Chem. Corp., in the USA. TNF-alpha kits and Interleukin Kits (IL1, IL4) were purchased from "ASSAYPRO" Co. (Universal Biologicals (Cambridge) Ltd., UK). Enzyme-Linked Immunosorbent Assay (ELISA) Kits and T/B/NK Kits were purchased from the "BD Biosciences" Co., in Franklin Lakes, New Jersey.

Animals
Sixty male albino rats (weighing 100-150g) were obtained from King Fahd Medical Research Center, King Abdulaziz University, in Jeddah, Saudi Arabia. Animals were housed in plastic cages with free access to water and standard chow. They were placed in an adjusted temperature of 20-25oC and controlled humidity of 50 ± 5% on a 12 h dark/light cycle. They were kept for acclimation one week before the start of experimental work.

Experimental Design
Rats were randomly classified into five groups (12 rats each).Thecontrol (untreated) group received saline (0.5 ml/kg, i.p.) and water (0.5 ml/kg, p.o), both used as a vehicle for delivering LPS and UDCA respectively for 10 days. The UDCA-treated group received a daily dose of UDCA (100 mg/kg, p.o) for 10 days (14). In the LPS-induced cholestasis group, rats were injected with a single sublethal dose of LPS (5 mg/kg, i.p.) (3,15). In the treatment group, rats were injected with LPS; 24 hours later, these rats received UDCA in a dose of 100 mg/kg, p.o. for 10 days (16). In the preventive group, rats received a daily dose of UDCA for 10 days before LPS injection and then 24 hours later the rats were euthanized. At the end of the experiment, rats were exsanguinated by severing the subclavian artery and vein under lethal anesthesia using pentobarbital sodium, in a dose of 50 mg/kg, i.p. Blood samples were collected from the retro-orbital venous plexus with heparinized sterile capillary tubes for further analysis.

Cytokine Quantification
Cytokines (TNF-, IL-1 and IL-4) in the sera or the culture supernatants were measured by ELISA according to the manufacturer's instructions.

Histopathology
Liver tissue was obtained from UDCA-fed or control rats 24 hours after or before LPS injection. Sections were prepared from formalin-fixed (5-μm-thick slides) paraffinembedded tissue samples stained with hematoxylin and eosin (H&E) by routine procedure.
Stained sections were examined using a microscope (SPI Supplies®) connected to a digital camera (camera fits eyepiece sizes 23mm to 30mm).
Statistical analysis SPSS version 20 (SPSS, Chicago, IL, USA) was used for data processing. Data are expressed as means ± S. D. A one-way ANOVA and Tukey's post hoc test were used for multiple comparisons. The number of animals per group was (n = 12): A P-value <0.05 was considered statistically significant.

Results
The following variables indicated the effect of UDCA on prevention and treatment groups of LPS-induced cholestasis.

Effect of UDCA on biochemical parameters of liver function
To determine the efficacy of the UDCA, the liver function parameters in the serum were measured as indicators of liver-injury status, and the results for each group are listed in Table 1. The rats on the LPS group had significantly higher values for GGT, ALP, TBIL, and AST compared with that of the control group or UDCA group (p<0.05). The treatment group with UDCA had only significantly lower values for GGT, ALP, and AST (P<0.05).
However, in UDCA pretreatment, the values for GGT, ALP, TBIL, and AST were significantly lower than the corresponding values in the LPS group (p<0.05). Moreover, the ALP and TBIL values were significantly lower than those in the treatment group (p<0.05).

Effect of UDCA on hepatocyte apoptosis
Apoptosis is one of the mechanisms underlying UDCA growth inhibition in vitro. To determine whether UDCA elicits growth inhibition in vivo through apoptosis, liver tissue was isolated and examined using flow cytometric analysis, as demonstrated in Figures   1&2. The results indicated that the LPS injected group had significantly increased apoptotic activity as compared to the control group (p = 0.003). Both treatment and prevention groups demonstrated reduced apoptosis when compared to the LPS group (p = 0.013 and p = 0.002, respectively). There was no significant difference found between the control group and the UDCA (p = 0.63), treatment (p = 0.44), and prevention groups (p = 0.9) ( Table 2). To further investigate the immunomodulatory properties of UDCA, liver tissues were isolated in five rat groups. Table 3 shows a decrease in the activity of NK cells in the LPS group compared to the control group, but reduction was not statistically significant (P = 0.14). Similarly, no statistical significance was detected in NK cells activity between LPS and either the UDCA treatment group or the pretreatment group (p = 0.12, p = 0.74, respectively). The effect of UDCA on T-lymphocytes (CD3) activity in the different study groups was examined. The percentage of CD3 when UDCA was used as treatment increased compared to the LPS group (p = 0.000), however, the pretreatment group showed that the percentage of CD3 cells had decreased significantly as compared to either LPS group or the control group (p<0.05). No significant difference was found between the treatment and pretreatment groups (p = 0.17).

Effect of UDCA on proinflammatory cytokines
The effect of UDCA on the inflammatory mediators in treatment and prevention groups were also investigated, as shown in Table 4 (12,17).
In this study, the intra-peritoneal injection of LPS induces cholestasis in rats that resulted in significant rise in the TBIL and liver enzymes, comprising GGT, ALP, and AST (Table 1), in agreement with a number of other investigators (15,18). Liver enzyme ALP is a cellular membrane-bound enzyme that is induced and released into the circulation in response to cellular membrane damage by the endogenous bile acids, and causes hepatocytes damage and lysis (19). The UDCA had anti-cholestatic effects on the LPS-treated rats in our experiment that was manifested by reduction in the mean level of liver enzymes: GGT, ALP, and AST. UDCA had also a preventive role when administered to experimental rats before LPS induction of cholestasis, as evidenced by the comparable liver enzymes and TBIL to the control, and significantly lowered the level of liver enzymes GGT, ALP, and AST compared to LPS-induced rats. This response may be explained partly by its membrane protective effects (20).
The accumulation of endogenous bile acids in the hepatocytes results in cellular damage and hepatocytes death. We found that LPS induced hepatocytes death was evident by higher percentage of apoptosis as compared to the control group; UDCA resulted in a lower percentage of an apoptosis comparable to the control group, which favors the positive effect of UDCA in minimizing or preventing hepatocellular damage and death. This finding was in agreement with one previous study (21). The improvement in the liver enzymes in the groups who received UDCA, either as treatment or prevention, in comparison with the LPS group, coincides with significant improvement in the liver tissue when examining the hepatic histopathology. These findings were consistent with some other reports (16). In addition to the histopathological improvement, we also found significant reduction in the liver tissue apoptosis in the UDCA treated groups. Rodrigues et al. (27) showed that co-administration of UDCA with the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes in all cell types. A recent study (24) has suggested that UDCA can reverse the cytokineinduced impairment in gene expression, and improves localization of the canalicular membrane transporters of bile acids, the bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), by counteracting LPS-induced endocytic internalization. Future efforts will be focused on optimized dosage regimens, as well as on further elucidation of the mechanisms of action of UDCA at the molecular level.

Conclusion
In conclusion, our study showed that UDCA is beneficial for the treatment and prevention of LPS-induced cholestasis in animals; an effect that can be manifested by improvement in liver enzymes and histopathological abnormalities.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.         Effect of UDCA on the nuclear area length in liver tissue in the prevention and treatment of cholestasis induced by LPS in rats.

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