Shenfu Injection Attenuates Bile Duct Injury in Rats with Acute Obstructive Cholangitis

Abstract

Background:

We investigated the effect of Shenfu injection (SFI) in Wistar rats with acute obstructive cholangitis (AOC) and considered the possible molecular mechanisms of the effects.

Methods:

The 96 rats were divided randomly into three groups. In one group, the common bile duct was subjected to ligation (BDL), and 0.2 mL of saline was injected into the proximal bile ducts. To create AOC, again, the common bile duct was ligated, and 0.2 mL of lipopolysaccharide (LPS)) (2 mg/mL) was injected into the proximal ducts. In the Shenfu injection (SFI) group, the material (10 mg/kg) was injected into the tail vein 2 hours before induction of AOC. The hepatic histopathologic changes were observed under a light microscope. The endotoxin, tumor necrosis factor-α (TNF-α), alanine transaminase (ALT), and total bilirubin (TB) concentrations in the serum were measured at different time points (0, 4, 8, and 16 hours) after ligation. The expression of nuclear transcription factor-κB (NF-κB) and CD14 in Kupffer cells also was analyzed at different times by Western blotting.

Results:

The TNF-α, ALT, and TB concentrations in the serum and the expression of CD14 and NF-κB in Kupffer cells were significantly higher in the SFI group than in the BDL group, but all were significantly lower than in the AOC group. Compared with the AOC group, the edema of cholangiocytes was alleviated in the SFI group, and the infiltration of inflammatory cells around cholangiocytes was reduced.

Conclusion:

Shenfu injection significantly alleviated bile duct injury. The potential mechanism may be associated with inhibition of CD14 expression and prevention of NF-κB activation in Kupffer cells.

Biliary tract infection, especially acute obstructive cholangitis (AOC), is a common disease around the world. Despite the multitude of advances in the surgical treatment of AOC, it remains a significant cause of morbidity and death because it easily leads to sepsis, septic shock, and multiple organ dysfunction syndrome (MODS) [1]. Endotoxin-induced bile duct injury often is the most important contributor to death in AOC.

Liver, as a main immune system organ, participates in the occurrence and development of various diseases [2, 3]. Previous studies have shown that Kupffer cells (KCs) play an important role in endotoxin-induced bile duct injury by secreting a variety of cytokines [4, 5]. In particular, CD14 is an important surface activator during KC activation [6]. It recognizes lipopolysaccharide (LPS) and activates a nuclear transcription factor kappa B (NF-κB) signaling pathway that leads to an excessive inflammatory reaction [7, 8].

Shenfu injection (SFI) using a material derived from Shenfu decoction, which is a preparation of an extract from red ginseng and black aconiti, with the active ingredients of ginseng saponin and aconitum alkaloids. It is used mainly to treat all kinds of shock (especially septic shock), heart failure, myocardial ischemia, and dysrhythmias. It has the effects of increasing coronary blood flow, protecting ischemic myocardium, and relieving inflammation injury [9 –11]. However, any protective effect on endotoxin-induced bile duct injury in AOC has not been reported yet. The aim of this study was to observe the effect of SFI on endotoxin-induced bile duct injury in AOC and the activation of the CD14 signaling pathway.

Materials and Methods

Materials

Ninety-six adult Wistar rats (8 to 10 weeks old, males and females, weighing 200–220 g), were purchased from the Experimental Animal Center of Chongqing Medical University. Lipopolysaccharide from Escherichia coli O₁₁₁B4 was obtained from Sigma Chemical Company (St Louis, MO, USA). The limulus kit was bought from Shanghai Yihua Clinical Medical Science and Technology Company. An ELISA kit was acquired from Boster Biological Technology (Wuhan) Co., Ltd. Antibodies against CD14 and NF-κB p65 were purchased from Santa Cruz Biotechnology (USA) Co., Ltd. Shenfu injection mix was obtained from Sichuan Ya'an SANJIU Pharmaceutical Company.

Animals

All animals received humane care in accordance with institutional guidelines. For the procedure, the rats were given intra-peritoneal anesthesia with 1% pentobarbital (30 mg/kg) after fasting for 12 hours. The rats were divided into three groups by the random number table method. For the bile duct ligation (BDL) group, the common bile duct was ligated and injected with 0.2 mL of normal saline (NS) as a control. In the AOC group, the common bile duct was ligated and injected with 0.2 mL of E. coli LPS 2 mg/mL [12]. The SFI group, SFI (10 mg/kg) was injected in the tail vein 2 hours before induction of AOC. Then, the rats were executed to collect their liver and blood at 0, 4, 8. and 16 h after ligation of the common bile drug (eight rats from each group were sacrificed per time). Primary KCs were isolated from mouse liver using a three-step procedure as described previously [13].

Endotoxin detection

The endotoxin concentration in the serum was determined by Chromogenic End-point Tachypleus Amebocyte Lysate.

Enzyme-linked immunosorbent assay (ELISA)

The TNF-α concentration in the serum was measured using rat ELISA kits according to the manufacturer's protocol.

Histologic analysis

Liver tissues were fixed in 10% neutral Formalin, dehydrated, embedded in paraffin, and cut into 5-μm sections. The sections were stained with hematoxylin and eosin (H&E).

Liver function analysis

The alanine transaminase (ALT) and total bilirubin (TB) concentrations in the serum were measured using commercial kits and an automatic biochemical analyzer (Olympus-AU5400, Japan) provided by the Laboratory of the Second Affiliated Hospital of Chongqing Medical University.

Western blotting

Expression of CD14 and NF-κB proteins in the KCs were determined by the Western blot method with β-actin as a control. The protein was extracted, and the concentration was determined spectrophotometrically. After successful polyacrylamide gel electrophoresis, the gel was removed; and electrophoretic printing was carried out. The blots were incubated with primary antibody and secondary antibody according to the manufacturer's instructions. The film was exposed and scanned at the same time to calculate the gray value of the strip to obtain the IA (absorbance × area). The relative expression of protein was represented by the ratio of target strip IA to the β-actin strip IA.

Statistical analysis

Experimental data are expressed as the mean ± standard deviation (SD), and analysis was performed with SPSS 17.0 software (SPSS Inc., Chicago, IL). Statistical significance between subgroups of parametric data was analyzed by a single-factor analysis of variance (ANOVA). A p value <0.05 was considered significant, and a value<0.01 was considered highly significant.

Results

Endotoxin and TNF-α concentrations

Plasma endotoxin concentrations of the AOC and SFI groups increased significantly by four hours after surgery and increased gradually with time. There was a significance difference in the groups in the corresponding time points (p < 0.01), although there was no significant difference between the AOC and SFI groups. The plasma endotoxin concentration was almost unchanged in the BDL group (Fig. 1A).

FIG. 1.

Serum protein concentrations at different time points. (A) Endotoxin. (B) Tumor necrosis factor-α. *P < 0.05 vs. BDL group. ^#P < 0.05 vs. AOC group.

Serum TNF-α in the AOC group was significantly higher at four hours and rose gradually as time went on. Compared with the BDL group at the corresponding time points, the difference was highly significant (p < 0.01). The serum TNF-α concentration in the SFI group also was significantly higher than in the BDL group (p < 0.01) but significantly lower than in the AOC group at the corresponding time points (p < 0.01) (Fig. 1B).

ALT and TB concentrations

Serum ALT and TB was significantly higher in the AOC group at four hours and continued to rise as time passed. Compared with the BDL group at the corresponding time points, the difference was highly significant. Serum ALT began to increase by eight hours in the BDL group, and the TB concentration began to increase at four hours. The serum ALT and TB concentrations of the SFI group were significantly higher than in the BDL group but significantly lower than in the AOC group at the corresponding times (p < 0.01) (Fig. 2).

FIG. 2.

Serum concentrations of proteins at three times. (A) Alanine transaminase. (B) Total bilirubin. *P < 0.05 vs. BDL group. ^#P < 0.05 vs. AOC group.

Shenfu injection inhibited expression of CD14 protein and activation of NF-κB in KCs

The expression of CD14 protein in KCs of the AOC group was significantly higher at four hours after modeling and rose gradually thereafter. Compared with the BDL group at the corresponding time points, the difference was highly significant (p < 0.01). The expression in the SFI group was significantly higher than in the BDL group but significantly lower than in AOC group at the corresponding times.

The expression of NF-κB in KCs of the AOC group increased by four hours after creation of the model, which was highly significant at eight hours. Compared with the BDL group at the corresponding times, the difference was highly significant (p < 0.01). The expression in the SFI group was significantly higher than in the BDL group but significantly lower than in the AOC group (Fig. 3).

FIG. 3.

Expression of proteins. (A) Western blotting electrophoretogram of CD14 and NF-κB expressions in Kupffer cells. (B) Amount of CD14 at different time points. (C) Amount of NF-κB at different times. *P < 0.05 vs. BDL group. ^#P < 0.05 vs. AOC group.

Shenfu injection protects against bile duct injury

In the AOC group, the degree of injury in liver tissues was aggravated gradually with the passage of time. There was central venous dilation, swollen or exfoliated endothelial cells, and neutrophil infiltration in the portal area at four hours. The liver parenchyma was infiltrated focally by neutrophils at eight hours. At 16 hours, the portal area was obviously expanded and infiltrated by a large number of inflammatory cells of various types but especially neutrophils. The inflammation involved the interlobular biliary ductules and liver parenchyma, and degeneration and necrosis of a large area of hepatocytes was observed. The necrotic hepatocytes and infiltrated inflammatory cells were much fewer in the SFI than that in the AOC group, suggesting that the inflammation of liver tissues was alleviated by Shenfu. In the BDL group, the portal area was infiltrated by only a small number of inflammatory cells, and hepatocytes were of normal size and shape (Fig. 4).

FIG. 4.

Pathological changes of liver tissue at 16 h (H&E; original magnification × 400). (A) In BDL group, a few inflammatory cells have infiltrated. (B) Portal area in AOC group is obviously expanded and infiltrated widely by inflammatory cells. Hepatocytes are necrotic. (C) Portal area in SFI group is slightly expanded and infiltrated by a small number of inflammatory cells.

Discussion

Acute obstructive cholangitis is a serious hepatobiliary disease, with a high mortality rate. It can easily cause a series of serious complications such as sepsis and MODS at any time during the infection [14, 15]. Bacterial endotoxin and monocyte macrophages, especially KCs, produce and release excessive inflammatory mediators, leading to an uncontrolled inflammatory response and immune dysfunction, which are the main causes of sepsis and MODS [16]. Previous research indicated that bile duct obstruction alone does not induce acute cholangitis, but infection of the bile by enteric bacteria can trigger fatal septicemia [17]. Septicemia and endotoxin-mediated liver injury often are the direct causes of death.

The liver is not only a place for removal of endotoxin, but also one of the most severely damaged organs in the process of severe biliary tract infection; and the extent of functional recovery of the liver will determine the prognosis of the patient [18]. As a repository of macrophages, it is rich in KCs, which play an important role in immune defense and inflammatory reaction, as well as alcoholic liver disease and ischemia–reperfusion injury [19]. Many LPS receptors on the liver cell membranes are involved in the inflammatory response, especially an important molecule named CD14. The activation of liver cells induced by combining LPS with CD14 can promote LPS signal transduction by connecting with TLR4 on receptors on cell membranes and forming TLR4/MD-2 [20 –22].

When activated, KCs release numerous mediators, including cytokines (TNF-α, interleukin [IL]-1, and IL-6) and reactive oxygen species [23]. As a major inflammatory cytokine, TNF-α promotes liver injury in several ways. In addition, TNF-α can prime the accumulation of neutrophils by activating endothelial cells to release toxic products (i.e., reactive oxygen species and proteases) that can damage nearby cells, which leads to liver injury [24,25]. As a member of a class of key nuclear transcription factors present in the cytoplasm of almost all cells, NF-κB is involved in a variety of physiological or pathological processes, including cell proliferation, differentiation, apoptosis, stress responses, and immune inflammatory reactions [26]. The activation of the NF-κB-mediated signal pathway is related to hepatic inflammatory injury [27]. Therefore, how to inhibit a series of activation reactions became the key to inhibiting hepatic inflammatory injury in AOC. This study was designed to examine the relation between endotoxin-mediated bile duct injury and changes in the expression of CD14 and NF-κB in KCs, and the potential mechanism by which SFI can protect against hepatic inflammation injury in rats with AOC.

Our results indicated that endotoxin concentrations in the serum of the animals with AOC increased gradually after ligation. There was no difference between them. The expressions of CD14 and NF-κB in KCs and TNF-α in the serum were up-regulated. There was a significant positive correlation between them and concentration of endotoxin. Both serum ALT and TB increased significantly in the AOC group in parallel with the increase in endotoxin. At the same time, bile duct injury was aggravated histopathologically. Many neutrophil granulocytes infiltrated the interlobular biliary ductules and liver parenchyma, and hepatocytes were necrotic in the later period. This response also was correlated positively with the concentration of TNF-α, indicating that the increase in CD14 expression and the activation of NF-κB can induce or aggravate endotoxin-mediated bile duct injury during the development of AOC. However, clearly the expression of CD14 and NF-κB was down-regulated and the concentrations of TNF-α, ALT, and TB in the serum were decreased after SFI treatment compared with the AOC group. The bile duct injury was less severe than in the AOC group. These results indicated that SFI can down-regulate CD14 expression, inhibit NF-kB activation, and decrease TNF-α production to protect against endotoxin-mediated bile duct injury during the development of AOC.

In this experiment, we also noticed that the expression of NF-κB in KCs of the BDL group increased gradually as time went on, but the expression of NF-κB in the KCs of both the AOC and the SFI group dropped suddenly at 16 hours. Clearly, there is an unknown mechanism that regulates the expression of NF-κB in AOC. This is worth our attention in further research.

Conclusion

Shenfu injection significantly alleviated bile duct injury in rats with AOC. The potential mechanism may be associated with inhibition of CD14 expression and prevention of NF-κB activation in KCs.

Footnotes

Author Disclosure Statement

The authors thank Pro. Zuo-jin Liu for reviewing an early draft of this article and giving valuable comments.

This study was supported by the National Natural Science Foundation of China (Nos. 30500473 and 30972888) and the Postdoctoral Fellow Science Foundation of the Ministry of Personnel of China (No. 20070411152).

Procedures involving animals and their care were conducted in conformity with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023; revised 1978) and were approved by the Animal Care and Use Committee of the Chongqing Medical University.

All authors declare that they have no competing interests.

Please contact the corresponding author for data requests.

Mr. Hao-yang Tan contributed to the conception of the study and wrote the manuscript. Dr. Pei-zhi Li contributed significantly to the analysis and to manuscript preparation. Prof. Jian-ping Gong helped perform the analysis with constructive discussions. Prof. Kang Yang designed the experiments and performed the data analyses.

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