Role of bile in intestinal barrier function and its inhibitory effect on bacterial translocation in obstructive jaundice in rats1

Journal of Surgical Research 115, 18 –23 (2003) doi:10.1016/S0022-4804(03)00308-1

Role of Bile in Intestinal Barrier Function and its Inhibitory Effect on Bacterial Translocation in Obstructive Jaundice in Rats 1 Yorihiko Ogata, M.D.,* ,† Masaharu Nishi, M.D., Ph.D.,* ,2 Haruyuki Nakayama,† Tomomi Kuwahara, M.D., Ph.D.,† Yoshinari Ohnishi, M.D., Ph.D.,† and Seiki Tashiro, M.D., Ph.D.* *Department of Digestive Surgery and †Department of Molecular Bacteriology, Graduate School of Medicine, University of Tokushima, Tokushima, Japan Submitted for publication August 12, 2002

mesenteric lymph nodes in CBDL rats although the inhibitory effect was weak. The integrity and permeability of the intestinal mucosa were kept at normal levels by bile administration in CBDL rats whereas the morphological changes, such as villous atrophy, villous edema, and lacteal canal dilatation, were observed in other CBDL rats. Conclusion. Bile plays an important role in maintaining the intestinal barrier function to prevent the invasion of enteric bacteria to the underlying tissues, suggesting that the intestinal administration of bile to patients with obstructive jaundice is a useful way to reduce infectious complications by inhibiting bacterial translocation from the intestine to other organs. © 2003 Elsevier Inc. All rights reserved. Key Words: bile; bile acid; JNW14; bacterial translocation; obstructive jaundice.

Background. Our previous study using genetically labeled Escherichia coli strain JNW14 revealed that obstructive jaundice promotes bacterial translocation in rats and that the absence of bile in the intestinal tract is considered to be a factor inducing bacterial translocation. The aim of this study was to investigate the role of bile and bile acids in intestinal barrier function against bacterial translocation. Materials and methods. Eight-week-old male specificpathogen-free Wistar rats were subjected to ligation of their common bile ducts (CBDL). The CBDL rats were treated with bacitracin, neomycin sulfate, and streptomycin sulfate, and the intestinal tract was colonized with E. coli strain JNW14, which was genetically labeled with resistant markers against the above three antibiotics, to monitor the bacterial translocation. The rats were then administered saline, cholic acid (20 mg/100 g BW), taurocholic acid (TCA: 5–50 mg/100 BW), or bile (1.5– 6 mL/day) via a duodenal catheter. The degree of bacterial translocation of E. coli strain JNW14 to the mesenteric lymph nodes was compared. Histopathological examination of the terminal ileum and intestinal permeability test using phenolsulfonphthalein was also performed. Results. Both cholic acid and TCA showed no inhibitory effect on bacterial translocation at any of the doses tested in CBDL rats, although TCA significantly decreased the numbers of E. coli strain JNW14 in the cecum. However, bile administration reduced the numbers of E. coli strain JNW14 in the cecum and

INTRODUCTION

Despite the improvement in operative procedures and the development of powerful antibiotics, septic complication after surgical operation is still a major cause of the high mortality rate in patients with obstructive jaundice [1, 2]. Several reports on model rats with obstructive jaundice have suggested that the absence of bile in the intestine induces the bacterial translocation of enteric bacteria from the intestinal tract to the systemic organs [3–5]. Endotoxin absorption is one of the factors promoting bacterial translocation by increasing the permeability of the intestinal mucosa [6]. However, bile acids and secretory IgA in the intestine inactivate the biological action of endotoxin by chemical and immunological mechanisms. On the basis of these facts, several attempts have been made to prevent the bacterial translocation in obstructive jaundice by administration of bile acid [5, 7, 8].

1

Supported by a Grant-in Aid for Scientific Research C from the Ministry of Education, Science, Sports, and Culture of Japan. 2 To whom correspondence and reprint requests should be addressed to at Department of Digestive Surgery, Graduate School of Medicine, University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima, 770-8503, Japan. E-mail: [email protected].

0022-4804/03 $35.00 © 2003 Elsevier Inc. All rights reserved.

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OGATA ET AL.: ROLE OF BILE IN BACTERIAL TRANSLOCATION

TABLE 1 Bacterial Numbers in the Cecum

Group Sham CBDL

Treatment

Saline 20 mg/100 g BW: CA 5 mg/100 g BW: TCA 10 mg/100 g BW: TCA 20 mg/100 g BW: TCA 30 mg/100 g BW: TCA 40 mg/100 g BW: TCA 50 mg/100 g BW: TCA 1.5 mL: bile 3.0 mL: bile 6.0 mL: bile

Rats tested

Number of viable E. coli JNW14 (log 10 CFU/g cecal content)

10 10 6 5 5 8 5 5 5 6 9 10

8.61 ⫾ 0.43† 9.50 ⫾ 0.09 9.19 ⫾ 0.41 9.43 ⫾ 0.11 9.19 ⫾ 0.40 9.24 ⫾ 0.16 8.99 ⫾ 0.02* 9.26 ⫾ 0.10 8.92 ⫾ 0.36* 9.02 ⫾ 0.12* 8.99 ⫾ 0.18† 8.95 ⫾ 0.26†

Note. Values are means ⫾ SD. * Significantly different from the saline group; P ⬍ 0.05. † Significantly different from the saline group; P ⬍ 0.001.

subgroups and administered 3 mL of sterilized saline (n ⫽ 10), 20 mg/100 g BW of cholic acid (CA, n ⫽ 6), 5–50 mg/100 g BW of taurocholic acid (TCA, n ⫽ 33), or 1.5– 6 mL/day of bile (n ⫽ 25) through a duodenostomy tube.

Experimental Design The rats were given drinking water containing 2 mg/mL each of streptomycin sulfate, neomycin sulfate and bacitracin for 4 days after the operation. The rats were then administered the culture of E. coli strain JNW14 (2 ⫻ 10 11 CFU/day) three times a day for 3 days through the duodenal catheter. The CBDL rats were also administered 3.0 mL/day of sterilized saline, 20 mg/100 g BW of CA, 5–50 mg/100 g BW of TCA, or 1.5– 6.0 mL/day of bile through the duodenal catheter three times a day for 2 days before the evaluation. The bile was collected by canulation to the common bile duct of normal Wistar rats. The viable cell number of E. coli strain JNW14 in the cecum, mesenteric lymph nodes (MLNs), lungs, liver, and spleen was determined from the duplicated plates as described previously [9]. All animal procedures complied with animal care guidelines of the Institute of Animal Experimentation, School of Medicine, The University of Tokushima.

Phenolsulfonphthalein Test

Seen-Chen et al. have demonstrated that the administration of sodium taurocholic acid to model rats with obstructive jaundice inhibited bacterial multiplication in the cecum, and prevented bacterial translocation [7]. On the contrary, several studies have shown that administration of bile acids provided no beneficial effects on the structure and function of the intestinal mucosa in rats with obstructive jaundice [5, 8]. The aim of this study was to elucidate the role of bile and bile acids on intestinal barrier function by evaluating the intestinal structure, intestinal permeability and the degree of bacterial translocation in obstructive jaundice in rats.

Seven days after the start of the experiment, 10 mg of phenolsulfonphthalein (PSP) was administered to the rats through the duodenal catheter, and total urine for 24 h was collected. The PSP excretion in 24-h urine was measured, and the urinary PSP excretion rate was calculated as an index to represent the intestinal permeability [10, 11]. Urinary PSP was measured by the colorimetric method. The concentration of PSP in the urine was determined as follows. The urine collected was alkalized with 10 mL of 10% NaOH, the volume was adjusted to 1000 mL with distilled water, and the PSP concentration in the urine was then determined using a spectrophotometer (Shimadzu Co., Ltd., Japan) at 559 nm. The urinary PSP excretion ratio was expressed as a percentage of the total PSP amount administered.

MATERIALS AND METHODS

Number of E. coli Strain JNW14 Cells in Mesenteric Lymph Nodes

TABLE 2

Bacterial Strain Escherichia coli strain JNW14, which was labeled with resistant genetic markers against bacitracin, neomycin sulfate, and streptomycin sulfate in the previous study [9], was used for monitoring the bacterial translocation.

Experimental Animals Eight-week-old male SPF Wistar rats (Charles River Japan Inc.) weighing from 200 to 250 g were used in this study. The rats were fed a standard pellet diet (MF, Oriental Yeast, Tokyo) and allowed access to tap water ad libitum throughout the experiment. They were housed in solid plastic cages under controlled temperature (25 ⫾ 2°C), humidity (60 –70%), and lighting (12-h light-dark cycle). The rats were randomized in two groups. One group of rats (shamoperated group, n ⫽ 10) underwent laparotomy without ligation of their common bile ducts. Another group of rats (CBDL group, n ⫽ 74) were subjected to common bile duct ligation (CBDL) as reported in the previous study [9]. All the rats underwent catheter duodenostomy. The rats in the CBDL group were further divided into four

Group Sham CBDL

Treatment

Saline 20 mg/100 g BW: CA 5 mg/100 g BW: TCA 10 mg/100 g BW: TCA 20 mg/100 g BW: TCA 30 mg/100 g BW: TCA 40 mg/100 g BW: TCA 50 mg/100 g BW: TCA 1.5 mL: bile 3.0 mL: bile 6.0 mL: bile

Rats tested

Number of viable E. coli JNW14 (log 10 CFU/g MLN)

10 10 6 5 5 8 5 5 5 6 9 10

2.99 ⫾ 0.35† 3.59 ⫾ 0.14 3.75 ⫾ 0.25 3.67 ⫾ 0.21 3.58 ⫾ 0.22 3.65 ⫾ 0.11 3.55 ⫾ 0.15 3.81 ⫾ 0.27 3.34 ⫾ 0.39 3.55 ⫾ 0.29 3.29 ⫾ 0.25* 3.33 ⫾ 0.22*

Note. Values are means ⫾ SD. * Significantly different from the saline group; P ⬍ 0.05. † Significantly different from the saline group; P ⬍ 0.001.

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FIG. 1. Intestinal permeability test evaluated by 24-h urinary PSP excretion ratio in sham-operated control rats (Sham) and common bile duct-ligated rats administered saline (CBDL), 30 mg/100 g BW of taurocholic acid (CBDL ⫹ TCA), and 3.0 mL/day of bile (CBDL ⫹ bile). *Significantly different from the CBDL ⫹ saline group (P ⬍ 0.01).

Histopathological Analysis The terminal ileum of the rats in each group was used for histopathological examination. The samples were fixed immediately in neutral-buffered 10% formalin, and embedded in paraffin wax. The 5-mm sections were stained with hematoxylin and eosin and then subjected to examination by light microscopy. For each sample, five randomly chosen mucosal regions were traced, and morphometric variations in villous height, mucosal width, and lacteal diameter were measured using an ocular micrometer. Histological evaluation was performed by an independent pathologist who had no knowledge of the experimental groups from which the specimens were derived.

Statistical Analysis The data were expressed as mean ⫾ SD. Multiple comparisons were made using analysis of variance followed by the Fisher’s PLSD test for the comparisons among groups. A probability value less than 0.05 was considered to be significant.

RESULTS

To determine the role of bile and bile acids on the bacterial translocation, bile, CA or TCA was administered to the CBDL rats, and their effects on viable cell numbers of colonized E. coli strain JNW14 in the cecum and the MLNs, intestinal permeability, and morphological change in the ileum were examined. Table 1 shows the number of E. coli strain JNW14 in

the cecal contents. In the CBDL rats administered saline, the cecal number of E. coli strain JNW14 was significantly higher (P ⬍ 0.001) than that in the shamoperated rats. The administration of TCA (30 and 50 mg/100 g BW) or bile (1.5– 6.0 mL/day) to the CBDL rats reduced the number of E. coli strain JNW14 in the cecum. However, no significant difference was observed in the cecal numbers of E. coli strain JNW14 between the CA (20 mg/100 g BW)-administered rats and CBDL rats. The numbers of E. coli strain JNW14 in the MLNs of rats in each group are shown in Table 2. The viable cell number of E. coli strain JNW14 in the MLNs in the CBDL rat administered saline was significantly higher (P ⬍ 0.001) than that in the shamoperated group. The number of translocated E. coli strain JNW14 to the MLNs was slightly reduced by bile administration (3.0 – 6.0 mL/day). However, neither CA nor TCA gave difference on the number of E. coli strain JNW14 cells in the MLNs despite the cecal population levels of the bacterium being decreased by the administration of TCA (30 and 50 mg/100 g BW). These results suggest that the inhibitory effect of bile on bacterial overgrowth in the cecum is weak; however, the some components in bile other than TCA and CA have protective effect on the intestinal barrier function.

OGATA ET AL.: ROLE OF BILE IN BACTERIAL TRANSLOCATION

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FIG. 2. Histopathological examination of the terminal ileum stained with hematoxylin– eosin. (A), sham-operated control rats illustrating a normal mucosal architecture; (B), CBDL rats administered saline; (C), CBDL rats administered taurocholic acid (TCA); and (D), bile. Arrows indicate dilated lacteal canals. Bar expresses 10 ␮m.

The result of an intestinal permeability test is shown in Fig. 1. The 24-h urinary PSP excretion rate in the CBDL group (7.32 ⫾ 1.87%) was significantly increased in comparison with that in the sham-operated group (4.44 ⫾ 0.31%). The administration of bile at 3.0 mL/day to the CDBL rats normalized the intestinal permeability (4.61 ⫾ 0.90%). However, no significant difference was observed between the 30 mg/100 g BW of TCA-administered group (5.98 ⫾ 1.91%) and the CBDL group (Fig. 1). The PSP excretion rates of shamoperated rats treated with 20 mg/100 g BW of CA (4.52 ⫾ 0.98%) or 30 mg/100 g BW of TCA (4.50 ⫾ 0.76%) were equal levels to that of untreated sham-operated rats. The histopathological examination of the terminal ileum showed submucosal edema in the CBDL and CBDL ⫹ TCA groups, whereas no pathological change was observed in the sham-operated and CBDL ⫹ Bile groups (Fig. 2). The villous height of the terminal ileum

in the CBDL and 30 mg/100 g BW of TCA-administered groups was significantly lower than that in the shamoperated group, whereas the villous height in the group administered with 3.0 mL of bile was almost the same as that in the sham-operated group (Table 3). The villous width in the CBDL and CBDL ⫹ TCA groups was significantly wider than that in the sham-operated group, and apparent lacteal canal dilatation was observed in the CBDL and CBDL ⫹ TCA groups (Fig. 2 and Table 3). These results suggest that certain components in the bile other than major bile acids play an important role in the reduction of bacterial translocation by maintaining the structure and barrier function of the intestinal mucosa. DISCUSSION

Berg and Garlington have defined bacterial translocation as “the passage of viable enteric bacteria

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TABLE 3 Morphological Changes of the Ileum

Sham CBDL ⫹ saline CBDL ⫹ TCA† CBDL ⫹ Bile‡

Rat tested

Villous height (␮m)

Villous width (␮m)

Lacteal diameter (␮m)

10 10 5 9

68.8 ⫾ 8.35 52.3 ⫾ 8.81* 50.5 ⫾ 5.29* 64.4 ⫾ 9.00

8.25 ⫾ 0.83 13.3 ⫾ 0.68* 10.7 ⫾ 0.65* 8.50 ⫾ 0.40

0.78 ⫾ 0.31 1.93 ⫾ 0.61* 1.57 ⫾ 0.34* 1.18 ⫾ 0.24

Note. Values are means ⫾ SD. * Significantly different from sham-operated group; P ⬍ 0.01. † 30 mg/100 g BW of TCA was administered to the CBDL rats. ‡ 3.0 mL/day of bile was administered to the CBDL rats.

through the epithelium into the lamina propria and then to the mesenteric lymph nodes, and possibly to other tissues” [12]. Various studies have been performed to clarify the mechanisms of bacterial translocation. Bile is considered to be a protective factor in intestinal barrier function because obstructive jaundice promotes bacterial translocation. However, the role of bile and bile acids in bacterial translocation is still unclear. In the present study, bile acids and bile were administered to CBDL rats to evaluate their inhibitory effects on bacterial translocation in obstructive jaundice. The most important action of bile acids and bile in bacterial translocation is their inhibition of bacterial overgrowth in the intestine because the degree of bacterial translocation depends on the number of intestinal bacteria [3–5]. However, in the overgrowth model of E. coli strain JNW14 used in this study, the inhibitory effect of bile and bile acids on the growth of the strain was weak. The slight reduction of the number of E. coli strain JNW14 in the MLNs by bile administration seems to be derived from the other mechanisms than the inhibition of bacterial overgrowth, although the effect of the bile acids and bile against the other intestinal bacteria must be determined. In the rodent, ligation of the bile duct reduces the amount of s-IgA in the intestine because the majority of s-IgA is supplied through the bile [13]. Together with this fact, a lack of s-IgA is considered to be a factor influencing the degree of bacterial translocation in obstructive jaundice. However, it might be difficult to apply this hypothesis to a human setting because in humans most s-IgA is directly supplied from plasma cells present in the lamina propria and about 1% of s-IgA is supplied through the bile. Another candidate as an inhibitory factor against bacterial translocation in bile is cholesterol. Cholesterol must be continuously supplied for cytopoiesis and for repair of the intestinal mucosa. Quraishy et al. have reported that cholesterol administration recovered the intestinal epithelial cells from the damage caused by obstructive jaundice [14]. In cases of obstructive jaundice or endotoxemia, deterioration of the microcircula-

tion caused by coagulopathy disorder [15, 16], vasoconstriction [17], adhesion of the leukocytes to the vessel wall [18, 19], and portal hypertension [20] are known to disturb the villous function. In the present study, we observed the changes in the intestinal structure such as lowering of villous height, extension of villous width, expansion of the central lacteal cavity, and submucosal edema in the terminal ileum in the CBDL group. Kordzaya and Goderdzishvili reported that expansion of the central lacteal cavity and villous edema caused by obstructive jaundice extends the gap of the lymphatic capillary, and these histopathological changes result in the increased permeability of the intestinal mucosa [21]. Although several reports have shown that administration of bile acid has no effect on histopathological changes in the intestinal mucosa in obstructive jaundice [5, 8], we observed a protective effect on mucosal integrity and permeability with “bile,” not with bile acids. Summing up these results, an intact barrier function in the intestinal mucosa could block the translocation of enteric bacteria even though they overgrew in the intestine, and bile, not major bile acids, plays an important role in maintaining the intact intestinal barrier function. Bile administration is thought to be an effective way to reduce bacterial translocation in patients with obstructive jaundice. Furthermore, identification of the components that protect the intestinal barrier function will provide useful information for establishing novel approaches to prevent bacterial translocation. REFERENCES 1.

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