TNF-α Mediated the Disruption of Hepatic Tight Junction Expression in Blood–Biliary Barrier of Colitis via Downregulating PI3K/AKT Signaling Pathway

Abstract

Hepatocyte tight junctions (TJ) constituted blood–biliary barrier is the most important hepatic barrier for separating bile from the bloodstream, disruption or dysfunction of TJ barrier is involved in hepatobiliary manifestations of colitis, but the underlying mechanism is still not clear. This study aims to investigate the effect and underlying mechanism of tumor necrosis factor alpha (TNF-α) on hepatic TJ protein expression in blood–biliary barrier and identify its role in the pathogenesis of acute colitis-related cholestasis. Acute colitis rat model was induced by trinitrobenzene sulfonic acid (TNBS) intra-colonic administration. TJs expression of blood–biliary barrier was tested in colitis rats, the serum TNF-α level was also determined in order to elucidate the correlation of TNF-α and TJs. HepaRG cells were used to investigate the effect of TNF-α on TJs, and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway were also evaluated in rats and TNF-α treated HepaRG cells. Acute colitis was induced in rats at 5 d post TNBS, which is accompanied with cholestasis-like alteration. Serum TNF-α level was increased in colitis rats and positively correlated with the alteration of total bile acids and bilirubin, marked decrease in TJs was found in TNF-α treated HepaRG cells and the rats, down-regulated PI3K/AKT signaling pathway were also identified in TNF-α treated HepaRG cells and the rats. The study concluded that serum TNF-α mediated the down-regulation of PI3K/AKT signaling pathway, which contributed to the reduction of TJ protein expression in acute colitis-related intrahepatic cholestasis. These findings suggest that TNF-α plays an important role in the pathogenesis of intrahepatic cholestasis of colitis.

INTRODUCTION

Ulcerative colitis (UC) is a digestive disease characterized by non-specific chronic inflammation of the colon. It is reported that about 40% of colitis patients have extraintestinal manifestations, particularly in the liver, pancreas, and gallbladder system.¹⁾ Liver manifestations are among the most common extra-intestinal symptoms of colitis,²⁾ it is found that the degree of liver lesions usually correlates positively with the severity of intestinal inflammation.³⁾ It is notable that the incidence of liver lesions in UC patients is significantly higher than in the general population, nearly one-third of colitis patients experience liver lesions, hepatitis, cholestasis, fatty liver, cirrhosis, and even cancer, which are all the common liver diseases that related to UC.⁴⁾ Nowadays, the pathogenesis mechanism of colitis-related liver disease is still not very clear, evidence from previous studies indicated that it may have close correlation with auto-immune activation, intestinal bacterial hepatic translocation, bacterial endotoxin-induced damage, and metabolic impairment, etc.⁵⁾

Liver barrier plays a crucial role in protecting against viruses, bacteria, endotoxin, and other damage to the liver, which is essential for maintaining homeostasis of metabolic and storage functions of liver.⁶⁾ Once the hepatic barrier is destructed, gut-derived pathogens, bacteria and its metabolites may translocate to the liver through portal vein,⁷⁾ which may induce pathological reactions and participate in the pathogenesis of liver diseases.⁸⁾ Blood–biliary barrier is the most important hepatic barrier that separates bile from the bloodstream.⁹⁾ Tight junctions (TJ) is the peculiar architecture that constitute the blood–biliary barrier. TJ, a kind of protein complexes that mainly composed of claudin, occludin, and Zonula occludens (ZO) on hepatocytes, plays an essential role in separating basolateral membranes and intercellular apical membranes.¹⁰⁾ Previous studies have shown that disruption or loss-of-function of TJ in hepatocytes can increase the permeability of liver barrier, and thus cause bile components to flow back into the blood and reduce the biliary osmotic gradient-related bile secretion.^11,12) Moreover, the alteration of hepatic TJ proteins enabled the translocation of the toxic products enter into the liver through systemic circulation, which may be associated with the pathogenesis of chronic liver disease.¹³⁾ All these evidences indicated the vital role of TJ in liver barrier function and liver diseases.

Studies using experimental colitis model revealed that destroyed TJ function and increased permeability of hepatic barrier were considered as the key pathogenic factors that responsible for the hepatobiliary changes in UC.^14,15) Results from previous study showed that gut-derived endotoxin lead to intrahepatic cholestasis via increasing hepatocyte TJ permeability and altering canalicular transporters function in trinitrobenzene sulfonic acid (TNBS)-induced colitis rats.¹⁶⁾ All these above evidences indicated that impaired hepatocyte barrier presents a pathogenetic mechanism for hepatobiliary injury of UC, but the underlying mechanism is still not fully established. Moreover, a variety of cytokines, such as tumor necrosis factor α (TNF-α),^17,18) interferon-gamma,¹⁹⁾ interleukin 10,²⁰⁾ as well as inducible nitric oxide,²¹⁾ were reported as the important factors that disrupting barrier permeability of the intestine, it is not clear whether these factors are involved in the disrupted TJ barrier function in hepatocytes, and thus responsible for the hepatobiliary changes of UC.

In this present study, TNBS-induced acute colitis rat model was used to investigate the colitis-related alteration in hepatic function and TJ protein expression, serum TNF-α level was also determined dynamically in the progression of experimental colitis in order to elucidate the correlation of TNF-α level and hepatic alteration. Moreover, HepaRG cells were used to investigate the effect of TNF-α on the expression of TJ protein and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway. The aim of this study is to explore the effect and underlying mechanism of TNF-α on TJ protein expression in blood–biliary barrier so as to investigate its role in the pathogenesis of acute colitis-related cholestasis.

MATERIALS AND METHODS

Animals

Male Wistar rats weighing 220–240 g were obtained from the Animal Center of Gansu University of Traditional Chinese Medicine. The rats were housed in the animal room of the Pharmacology Department of Lanzhou University. The rats were kept in a controlled temperature and humidity, and ad libitum access to food and water. Prior to the experiment, approval was obtained from the Ethics Committee to ensure compliance with relevant regulations and guidelines (SCYK2015-0005).

Colitis Induction and Evaluation

All rats were fasted for 12 h prior the experiment. Rats were weighed and anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg), then TNBS solution (2.5 mg/mL, 50% ethanol, 0.8 mL per rat) was administered into the colon (8 cm proximal to the anus) through a thin catheter.²²⁾ Rats in the normal control group were given 0.8 mL saline using the same method. After TNBS solution administration, the rats were placed on a warm pad until recovering from anesthesia. Disease activity index (DAI) and myeloperoxidase (MPO) activity assay were conducted in order to evaluate colonic inflammation. The body weight change and stool consistency were recorded daily to calculate the DAI score,²³⁾ and MPO activity of colon tissue was determined using MPO activity assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instruction.²⁴⁾

Hematoxylin–Eosin (H&E) Staining

Colon tissue were embedded in paraffin and subjected to paraffin sectioning. After sectioning, the slides were dewaxed by sequential immersion in xylene I and II, anhydrous ethanol I and II, and 75% alcohol, followed by rinsing with tap water. Hematoxylin staining, differentiation, bluing, and rinsing with running water were then conducted. Eosin staining was performed by first dehydrating the slides, followed by sequential immersion in 85 and 95% graded ethanol and staining with eosin staining solution. Finally, the slides were dehydrated and mounted with neutral gum.

In Vivo Study Design

Rats were randomly divided into four groups, i.e., normal control as well as colitis model at 1, 3, and 5 d after TNBS administration. Rats were fasted overnight before samples collection. After pentobarbital sodium (30 mg/kg, intraperitoneally (i.p.)) administration, blood was collected from the abdominal aorta of rats, then the blood samples were centrifuged at 1500 G for 10 min to obtain serum. Liver and colon tissues were obtained simultaneously. The collected tissues were divided into two parts, one part was fixed in 4% paraformaldehyde for the preparation of pathological sections, while the other part was stored at −80 °C for Western-bolt or MPO activity analysis.

Serum Biochemical Index Determination

Serum level of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), and total bilirubin (TBil) were measured using Chemistry Analyzer (AU400, Olympus, Tokyo, Japan) in clinical lab of First Hospital of Lanzhou University. Serum level of total bile acids (TBA) was analyzed by a Microplate Reader (Multiskan FC, Thermo Scientific, U.S.A.) according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China).²⁵⁾

In Vitro Study Design

HepaRG cells (HPRGC10, Invitrogen, U.S.A.), the liver cancer cells of human origin, were applied in this study. Cells were cultured in 1640 medium containing 10% fetal bovine serum (Serena, South America) and 1% penicillin and streptomycin (Hyclone, U.S.A.), and cultured at 37 °C with 5% CO₂. When HepaRG cells reached approximately 70–80% confluency, experiments were conducted. The cells were seeded in a 6-well plate and incubated overnight in 1640 medium containing 10% serum. In the next day, TNF-α was added to the medium at a non-cytotoxic concentration of 5 ng/mL,²⁶⁾ and further incubated for 24 h. Finally, the cells were harvested, the protein was extracted for Western blot analysis.

Western Blot Analysis

Protein was extracted from cultured cells or liver tissue using RIPA lysis buffer. Protein quantification was performed using the BCA kit, and the concentrations of all samples were adjusted to ensure consistency. Equal amounts of protein were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% skimmed milk for 1 h, the PVDF membrane was incubated with an appropriate dilution of primary antibody (Table 1) overnight at 4 °C. Next, a suitable dilution of secondary antibody was added to the membrane, incubated at room temperature for 1 h. The PVDF membrane was visualized by exposing it to ECL reagent. Gray values of the bands were analyzed using ImageJ software, and the optical density values were statistically analyzed after normalization to GAPDH.

Table 1. Primary Antibodies Used for Western Blot Analysis

Antigen	Source	Dilution
ZO-1	Thermo (QG215365)	1 : 2000
Occludin	Abcam (ab168986)	1 : 1000
Claudin	PTM BIO (PTM-5134)	1 : 1000
PI3K	Cell Signaling Technology (4292S)	1 : 1000
p-PI3K	Cell Signaling Technology (17366)	1 : 1000
AKT	Cell Signaling Technology (9272S)	1 : 1000
p-AKT	Cell Signaling Technology (9271)	1 : 1000
GAPDH	Santa Cruz (SC-25778)	1 : 1000

Immunofluorescence Staining

The liver tissue sections (5 µm thick) were deparaffinized and rehydrated for immunohistochemistry, sections were heated in incitrate buffer (0.01M, pH 6.0) solution by microwave for antigen retrieval. After incubation with rabbit monoclonal anti-ZO-1 antibody (1 : 500, Thermo Scientific) overnight at 4 °C, the sections were then incubated with Alexa Fluor^® 488-coupled secondary antibody (1 : 500, Abcam, U.K.) at room temperature for 1 h. The nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI) (20 µg/mL, Sigma) and observed under a fluorescence microscope (Olympus) at 400× magnification.

Enzyme-Linked Immunosorbent Assay ELISA Assay

Blood samples were collected from the rat abdominal aorta and centrifuged at 1500 G for 10 min. The serum was isolated, and the serum levels of TNF-α were measured in duplicate using ELISA kit (Wuhan Huamei Biotechnology Company, China) following the manufacturer’s instructions, the results were expressed at pg/mL serum.

Statistical Analysis

The mean and standard deviation (S.D.) of all relevant data were calculated using GraphPad Prism v 8.0 software and presented. Statistical analysis was performed using Student’s t-test or one-way ANOVA to determine the significance of differences between groups. A p-value of less than 0.05 was considered statistically significant.

RESULTS

Acute Colitis Model Induced by TNBS Administration

As shown in Fig. 1A, colon sections with H&E staining showed that there was marked mucosal swelling and inflammatory cells infiltration in the colon tissue of rats at 5 d post TNBS. Compared to the normal rats, the DAI value and MPO activity in rats at 5 d post TNBS were both significantly increased (p < 0.05, Figs. 1B, D). And, compared with the normal group, the body weight of rats at 2, 3, and 4 d post TNBS were also significantly reduced (p < 0.05, Fig. 1C). All these results indicated that acute colitis was induced at 5 d post TNBS administration.

Fig. 1. Acute Colitis at 5 d after Intro-Colonic TNBS Administration in Rats

A: Representative histological changes in the colon of TNBS-treated rats (H&E staining, scale bar:100 µm), B: Statistical analysis of the disease activity index, C: Statistical analysis of body weight, and D: Statistical analysis of MPO activity. Data are presented as mean ± standard error of mean, n = 6 per group. * p < 0.05, ** p < 0.01 vs. normal group.

Alteration of Hepatic Function Index in Acute Colitis Model

As shown in Fig. 2A, compared to the normal rats, the serum level of ALP and GGT were markedly increased in rats at 5 d post TNBS administration (p < 0.05), while the serum level of ALT and AST did not show markedly differences between these two groups (p > 0.05). It is notable that the serum level of TBil and TBA were also significantly increased in TNBS-treated rats when compared to that of the normal rats (p < 0.05, Fig. 2B). As shown in Fig. 2C, histopathological observation on liver tissue did not showed marked alterations between normal rats and TNBS-treated rats. All these results indicated that there is a cholestasis-like alteration in acute colitis rats.

Fig. 2. Alteration of Hepatic Function Index in Rats at 5 d Post TNBS Administration

A: Statistical analysis of the serum level of ALP, GGT, ALT, and AST in TNBS-treated rats, B: Statistical analysis of the serum level of TBil and TBA in TNBS-treated rats, C: Representative histological changes of liver tissues from normal and TNBS-treated rats (H&E staining, scale bar:100 µm). Data are presented as mean ± standard error of the mean, n = 6 per group. * p < 0.05 vs. normal group.

Serum TNF-α Level Corelated with Elevated TBA and TBil in Colitis Model

As shown in Fig. 3A, compared with the normal rats, the serum level of TNF-α is significantly increased in colitis rats at 5 d post TNBS administration (p < 0.001). Results from correlation analysis showed that the alteration of serum TNF-α level was positively correlated with elevated serum level of TBA (r = 0.798, p = 0.002) and TBil (r = 0.683, p = 0.014) (Fig. 3B), indicating that serum TNF-α may play an important role in cholestasis-like alteration in acute colitis rats.

Fig. 3. Serum Alteration of TNF-α Correlated with Elevated Serum Level of TBA and TBil in Colitis Rats

A: Statistical analysis of serum level of TNF-α between normal and colitis rats; B: Correlation analysis of serum TNF-α, TBA and TBil in colitis rats. Data are presented as mean ± standard error of the mean, n = 6 per group. *** p < 0.001 vs. normal rats.

Decreased TJ Protein Expression in TNF-α Treated HepaRG Cells and in the Liver of Colitis Model

As shown in Fig. 4A, compared to the control, the expression of TJ protein, i.e., ZO-1, occludin, and claudin, all significantly decreased in HepaRG cells incubated with TNF-α (5 ng/mL) (p < 0.05), indicating the down-regulatory effect of TNF-α on TJ protein expression in HepaRG cells. As shown in Fig. 4B, compared to the normal control, the serum levels of TNF-α in colitis rats at 1, 3, and 5 d post TNBS was gradually increased, significant difference was found in colitis rats at 3 d (p < 0.05) and 5 d (p < 0.001) post TNBS, respectively. Immunofluorescence staining also showed that the hepatic ZO-1 expression was reduced in acute colitis rats when compared to the normal control (Fig. 4C). Consistent with the findings from TNF-α treated HepaRG cells, ZO-1 protein expression was markedly decreased in colitis rats at 1, 3, and 5 d post TNBS (p < 0.05); claudin expression was significantly decreased in colitis rats at 5 d post TNBS (p < 0.05); while occludin expression was initially markedly increased at one day post TNBS (p < 0.05), and then showed gradually decrease trend at 3 and 5 d post TNBS (Fig. 4D). All these data indicated that serum TNF-α contribute to the decreased hepatic expression of TJ protein in acute colitis rats.

Fig. 4. Decreased TJ Protein Expression in TNF-α (5 ng/mL) Treated HepaRG Cells and in the Liver of Colitis Rats

A: Western immunoblots of ZO-1, occludin, and claudin as well as the statistical analysis of protein level in HepaRG cells treated with TNF-α (n = 3 per group); B: Statistical analysis of serum TNF-α level in colitis rats at 1, 3, and 5 d post TNBS (n = 5 per group); C: Representative immunofluorescent micrographs indicating ZO-1 (green; FITC, scale bar:50 µm) and cell nuclei (blue, DAPI) in the liver of normal rats and colitis rats; D: Western immunoblots of ZO-1, occludin and claudin and statistical analysis of the protein level in the liver of colitis rats. Data are presented as mean ± standard error of the mean, n = 5 per group in animal study. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. normal control group.

Down-Regulated PI3K/AKT Signaling Pathway in TNF-α Treated HepaRG Cells and in the Liver of Acute Colitis

Concerning the important role of PI3K/AKT signaling in regulating TJ protein expression in the intestine and blood-brain barrier, we further investigate the expression of PI3K/AKT signaling pathway proteins in TNF-α treated HepaRG cells and in the liver of acute colitis rats. As shown in Fig. 5A, the protein expression of p-PI3K and p-AKT were markedly decreased after TNF-α treatment (p < 0.05), while no significant differences were found in the total protein expression of PI3K and AKT (p > 0.05). Consistent well with the results from HepaRG cells, results from animal study showed that the expression of p-PI3K decreased significantly in the colitis rats at 3 and 5 d post TNBS (p < 0.05), the expression of p-AKT decreased markedly at 5 d post TNBS (p < 0.05), while no significant differences were found in the protein expression of PI3K and AKT in the liver of colitis rats (p > 0.05, Fig. 5B).

Fig. 5. Down-Regulated PI3K/AKT Signaling Pathway in TNF-α (5 ng/mL) Treated HepaRG Cells and in the Liver of Acute Colitis Rats

A: Western immunoblots of PI3K, p-PI3K, AKT, and p-AKT expression and statistical analysis of the protein level in HepaRG cells treated with TNF-α (n = 3 per group); B: Western immunoblots of PI3K, p-PI3K, AKT, and p-AKT expression and statistical analysis of the protein level in the liver of colitis rats. Data are presented as mean ± standard error of mean, n = 6 per group in animal study. * p < 0.05, ** p < 0.01 vs. control group.

DISCUSSION

The liver diseases, including cholangitis, cholestasis, and liver damage, are the most common reported manifestations in UC patients, which pose a serious threat to the life and health of UC patients beside the primary intestinal disease. Accumulated evidences showed that hepatic translocation of intestinal flora, inflammatory factors, liver barrier dysfunction, and cholestasis may play a vital role in the development of colitis-related liver diseases. Currently, the pathogenesis mechanism of UC-related liver injury is not yet fully understood. Nowadays, to investigate the pathogenesis of UC-related liver diseases and discover the strategies for its prevention and treatment are urgently needed. In this present study, we demonstrated that gut-derived TNF-α mediated the down-regulation of PI3K/AKT signaling pathway is involved in the impaired hepatic TJ protein expression in blood–biliary barrier, which may contribute to acute colitis-related intrahepatic cholestasis (Fig. 6).

Fig. 6. A Proposed Effect of TNF-α Mediated the Down-Regulation of PI3K/AKT Signaling Pathway Is Involved in the Impaired Hepatic TJ Protein Expression in Blood–Biliary Barrier, Which May Contribute to Acute Colitis-Related Intrahepatic Cholestasis

Schematic depiction of the blood–bile barrier in the liver. The blood–bile barrier is composed of the tight junction proteins (i.e., ZO-1, occludin, and claudin) present at the apical membrane of the hepatocytes, which restricts the mixing of sinusoidal blood and bile (left schematic diagram). In the colitis, gut-derived TNF-α entered into the liver via the portal vein and down-regulated the expression of tight junction proteins through the PI3K/AKT signaling pathway, and further resulted in blood–bile barrier dysfunction and bile acids leaking into the blood, which contribute to the pathogenesis of acute colitis-related intrahepatic cholestasis (right schematic diagram).

The chemical substance TNBS can induce rodent acute colitis by a single intracolonic administration in ethanol, the acute transmural damage and inflammation in the colon became maximal from 3 to 5 d post TNBS instillation,²⁷⁾ which appears similar to the pathological manifestations of human UC.²⁸⁾ Consistent with previous report, results from our study showed that the DAI value and MPO activity were all significantly increased in rats at 5 d post TNBS, and there is marked mucosal swelling and inflammatory cells infiltration in the colon tissue, indicating the typical features of acute colitis in the model. We also found that the serum level of ALP, GGT, TBil, and TBA were all markedly increased in acute colitis rats, indicating the existence of cholestatic alteration in acute colitis rats. Our finding is coincided with previous studies showing that TNBS-induced colitis is one of the models to obtain insights into the mechanism of intrahepatic cholestasis associated with UC.^29,30) Different from previous reports, this present study used relatively low TNBS dosage to induced acute colitis, and the serum level of ALT and AST in the colitis rats were unchanged at 5 d after TNBS, suggesting the feature of solitary acute colitis-related intrahepatic cholestasis, but not acute colitis-related hepatitis in this model.

TNF-α, a highly pleiotropic cytokine involved in a variety of physiological processes, plays a crucial role in the pathogenic inflammatory process.³¹⁾ It is also reported that TNF-α could shift the predominant cytokine environment towards pro-inflammatory cytokines (i.e., interleukin (IL)-1, IL-6, IL-17, interferon-γ (IFN-γ), etc.), and simultaneously reducing the secretion of anti-inflammatory cytokines (i.e., IL-4, IL-10, etc.) in the inflamed colon.³²⁾ Accumulated evidences showed that the serum TNF-α level as well as colonic TNF-α concentration were all significantly elevated in acute colitis,^33–36) and the elevated TNF-α is positively correlated with the severity of UC,³⁶⁾ indicating the vital role of TNF-α in the pathogenesis of UC.³⁷⁾ Results from our study showed that the serum TNF-α level was markedly increased in colitis rats, and the increased TNF-α level was positively correlated with elevated TBA and TBil, indicating that the increased TNF-α may contribute to the intrahepatic cholestasis of acute colitis. Considering the important roles of TJ protein in blood–biliary barrier and the pathogenesis of intrahepatic cholestasis, human hepatoma HepaRG cells were used in this study to investigate the effect of TNF-α. We found that TJ protein expression, i.e., ZO-1, occludin, and claudin, all significantly decreased in TNF-α treated HepaRG cells. Knowing that the portal vein mainly collects the blood from the intestine (i.e., inflamed colon), we speculated that the liver may be exposed to relative higher TNF-α concentrations than that in the circulating serum, so TNF-α concentration (5 ng/mL) applied to HepaRG cells was higher than that of the serum level (about 25 pg/mL) of colitis rats. Results from animal study also showed that serum TNF-α level was gradually increased, hepatic ZO-1 expression in colitis model was markedly decreased, similar trends were also found in the hepatic expression of claudin. For occludin expression, it is found to be increased at 1 d, and then decreased at 3 and 5 d after TNBS, we proposed that this may be resulted from the compensatory response to the reduced function of occludin, as TNF-α may firstly induce the redistribution of TJ protein from a predominately membranous to a more diffuse cytoplasmic localization,³⁸⁾ and the prolonged TNF-α exposure may finally lead to damage in TJ structure and function.³⁹⁾ It is well known that cholestasis is linked to the disruption of the structural and functional integrity of the TJ protein in hepatocytes,^40,41) decreased expression of hepatocyte TJ protein can increase hepatic barrier permeability, cause bile components to flow back into the plasma, reduce the bile duct permeability gradient, and thus result in intrahepatic cholestasis.⁴²⁾ Taken together, it is suggested that the increased serum TNF-α may contribute to acute colitis-related intrahepatic cholestasis via down-regulating hepatic TJ expression and impairing TJ barrier function.

As an attractive signaling pathway, PI3K/Akt signal controls a variety of cellular events, including proliferation, apoptosis, and synthesis of protein.^43,44) Recent studies have demonstrated that PI3K/Akt signaling was involved in LPS-induced down-regulation of ZO-1 expression in bEND.3 cells,⁴⁵⁾ and was involved in regulating TJ protein expression and function in blood–brain barrier,⁴⁶⁾ indicating the regulatory role of PI3K/Akt signal in TJ expression and TJ barrier function. Results from our study demonstrated that TNF-α treatment induced down-regulatory expression of p-PI3K and p-AKT in HepaRG cells, the protein expression of p-PI3K and p-AKT were also significantly decreased in acute colitis rats, all these evidences indicated that the down-regulatory effect of TNF-α on PI3K/Akt signaling pathway may contribute to hepatic TJ barrier impairment and intrahepatic cholestasis of acute colitis. Consistent with our findings, previous studies also showed that TNF-α induced cells injury and inflammatory response in human umbilical vein endothelial cells via down-regulating the expression of p-PI3K and p-Akt,⁴⁷⁾ and the reduced p-Akt expression was also detected in TNF-α and IFN-γ treated mesenchymal stem cells,⁴⁸⁾ suggesting the important and regulatory roles of TNF-α on PI3K/Akt signal. Based on the results of ZO-1 expression, we speculated that ZO-1 may be more sensitive to TNF-alpha mediated regulatory effect of PI3K/Akt signal than other TJ proteins in colitis rats, because ZO-1 expression was markedly decreased not only at 1 d post TNBS, but also significantly reduced at 3 and 5 d post TNBS, even though the reduced expression ration of p-AKT/AKT and p-PI3K/PI3K did not reach to the statistical significance at this time.

Concerning the extensive inflammatory response in acute colitis, multiple pro-inflammatory cytokines may also participate in hepatic TJ barrier impairment of colitis. For example, anti-inflammatory cytokine IL-10 absence is reported to represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in IBD patients.⁴⁹⁾ The limitation of this study is that we only investigated the effect of TNF-α on hepatic TJ protein expression, more studies are still needed in the future to elucidate the effect of pro-inflammatory cytokines and anti-inflammatory cytokines on the expression and function of TJ proteins during the colitis. Even though, the present results also give a hint that increased serum TNF-α level contributed much to the hepatic TJ barrier damage and intrahepatic cholestasis in acute colitis, for the central role of TNF-α in the pathogenic inflammatory process of colitis. Moreover, in view of the disrupted hepatic TJ barrier in acute colitis, it is possible that the enterotoxins, lipopolysaccharide, as well as the microbial metabolites may also enter the liver and contribute to the liver dysfunction, more study are still needed in the future in order to elucidate the mechanism of colitis-related liver manifestations.

CONCLUSION

This study demonstrated that TNF-α mediated the down-regulation of PI3K/AKT signaling pathway is involved in the reduced hepatic TJ protein expression in blood–biliary barrier of acute colitis, which may contribute to the onset and progression of colitis-related intrahepatic cholestasis. The findings of this present work may give further evidence about the important role of TNF-α in the pathogenesis of intrahepatic cholestasis of colitis, and provide insights for the prevention and treatment of colitis-related cholestasis.

Acknowledgments

This work was supported by the National Science Foundation of China (81770657); Key-area research and development program of Guangdong province (Grant No. 2020B1111110003), Institution of higher education industry support project of Gansu Province (2020C-01), First Hospital of Lanzhou University internal fund (ldyyyn2018-50), and College student innovation and entrepreneurship action plan of Lanzhou University (20220060072).

Conflict of Interest

The authors declare no conflict of interest.

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