Molecular and Cellular Biochemistry https://doi.org/10.1007/s11010-017-3234-3

Lipopolysaccharide stimulates endogenous β-glucuronidase via PKC/ NF-κB/c-myc signaling cascade: a possible factor in hepatolithiasis formation Dianbo Yao1 · Qianze Dong2 · Yu Tian1 · Chaoliu Dai1 · Shuodong Wu1,3  Received: 21 August 2017 / Accepted: 24 November 2017 © Springer Science+Business Media, LLC, part of Springer Nature 2017

Abstract Hepatolithiasis is commonly encountered in Southeastern and Eastern Asian countries, but the pathogenesis mechanism of stone formation is still not well understood. Now, the role of endogenous β-glucuronidase in pigment stones formation is being gradually recognized. In this study, the mechanism of increased expression and secretion of endogenous β-glucuronidase during hepatolithiasis formation was investigated. We assessed the endogenous β-glucuronidase, c-myc, p-p65, and p-PKC expression in liver specimens with hepatolithiasis by immunohistochemical staining, and found that compared with that in normal liver samples, the expression of endogenous β-glucuronidase, c-myc, p-p65, and p-PKC in liver specimens with hepatolithiasis significantly increased, and their expressions were positively correlated with each other. Lipopolysaccharide (LPS) induced increased expression of endogenous β-glucuronidase and c-myc in hepatocytes and intrahepatic biliary epithelial cells in a dose- and time-dependent manner, and endogenous β-glucuronidase secretion increased, correspondingly. C-myc siRNA transfection effectively inhibited the LPS-induced expression of endogenous β-glucuronidase. Furthermore, NF-κB inhibitor pyrrolidine dithiocarbamate or PKC inhibitor chelerythrine could effectively inhibit the LPS-induced expression of c-myc and endogenous β-glucuronidase, and the expression of p-p65 was also partly inhibited by chelerythrine. Our clinical observations and experimental data indicate that LPS could induce the increased expression and secretion of endogenous β-glucuronidase via a signaling cascade of PKC/NF-κB/c-myc in hepatocytes and intrahepatic biliary epithelial cells, and endogenous β-glucuronidase might play a possible role in the formation of hepatolithiasis. Keywords  Hepatolithiasis · β-Glucuronidase · c-Myc · NF-κB Abbreviations LPS Lipopolysaccharide NF-κB Nuclear factor kappa B PKC Protein kinase C Electronic supplementary material  The online version of this article (https://doi.org/10.1007/s11010-017-3234-3) contains supplementary material, which is available to authorized users. * Shuodong Wu [email protected] 1



Department of General Surgery, Shengjing Hospital, China Medical University, Shenyang, China

2



Department of Pathology, The First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, Liaoning, China

3

Department of General Surgery, Shengjing Hospital, China Medical University, No. 36, San Hao Street, Heping District, Shenyang 110004, Liaoning, China



PI3K Phosphoinositide-3-kinase IKK IκB-α kinase

Introduction Hepatolithiasis, or primary intrahepatic stones, are commonly encountered in Southeastern and Eastern Asian countries [1–3]. However, increasing immigration from the Asian-Pacific region might make this disease gradually become a therapeutic challenge to clinicians in the West as well. At present, hepatolithiasis is still recognized as an intractable hepatobiliary disease, with high recurrence rate. Patients with recurrent stones would have to suffer from repeated surgery or endoscopic lithotomy, which would gradually increase the treatment risks, difficulties, and pains of patients [1, 2]. Until now, the pathogenetic mechanism of stone formation is still not yet well understood, and much more efforts should be made to further reveal the related

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mechanisms, to explore more effective methods to prevent stone formation or recurrence. Major type of hepatolithiasis is brown pigment stone, and bacterial infection is thought to be crucial for its formation [4]. The well-known and widely accepted Maki’s theory suggests that bacterial β-glucuronidase (exogenous β-glucuronidase) could hydrolyze bilirubin glucuronides to unconjugated bilirubin, and the latter could combine with calcium ions to produce calcium bilirubinate stones [5]. In addition, some studies found that in the bile of patients with hepatolithiasis, besides bacterial β-glucuronidase, endogenous β-glucuronidase activity and concentration in patients with pigment stones also increased significantly [6, 7]. Furthermore, it was found that with the emptying of infected bile, exogenous β-glucuronidase gradually declined, while endogenous β-glucuronidase could maintain at a high level for a long time. As endogenous β-glucuronidase could also catalyze the formation of calcium bilirubinate, these suggested that endogenous β-glucuronidase might play a role for a long time in pigment stones formation [8, 9]. Nowadays, the role of endogenous β-glucuronidase in pigment stones formation is being gradually recognized, and increasingly attended. Revealing the related mechanisms of endogenous β-glucuronidase expression and secretion might help us control the lithogenic potentials, to prevent stone recurrence. C-myc, a basic helix-loop-helix leucine zipper (HLHZIP) transcription factor, is involved in multiple biological processes, including cell proliferation, growth, energy metabolism, and apoptosis [10, 11]. In liver diseases, it is not only related with the proliferation and metastasis of cancer cells, but also increasingly expressed in some chronic liver diseases [12, 13]. In the Li’s study for chronic proliferative cholangitis, specific blockage of c-myc expression by c-myc shRNA inhibited the secretion of endogenous β-glucuronidase, revealing a possible role of c-myc in the regulation of endogenous β-glucuronidase secretion [13]. In addition, some studies found that lipopolysaccharide (LPS), a major endotoxin from Gram-negative bacteria, could increase the expression of c-myc [14], and nuclear factor kappa B (NF-κB), protein kinase C (PKC), or phosphoinositide-3-kinase (PI3K) may play an important role in the regulation process [15, 16]. In this study, we aimed to elucidate the role of endogenous β-glucuronidase in the formation of hepatolithiasis, and its possible regulation mechanism.

Materials and methods Cell culture Intrahepatic biliary epithelial cell line (HiBEpiC) was purchased from Sciencell, while human liver cell line (L02) was provided by the department of Pathology of China Medical

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Molecular and Cellular Biochemistry

University. Cells were cultured in epithelial cell medium with 10% fetal bovine serum in a humidified atmosphere of 5% ­CO2 at 37 °C. The cells were stimulated with different concentrations of LPS (0, 1, 5, 10, 50, 100, 250, 500 μg/ml) for different time (0, 0.5, 1, 3, 6, 12, 24, 48, 72 h), and the expression change of c-myc and endogenous β-glucuronidase was detected. C-myc low-expression cells were generated by transient transfection (Human MYC (4609) siRNA, M-00328207-0005, siGENOME), to reveal the role of c-myc in the regulation of endogenous β-glucuronidase expression. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC), PKC inhibitor chelerythrine, or PI3K inhibitor LY294002 was applied to reveal the roles of NF-κB, PKC, PI3K in LPS-induced expression of c-myc and endogenous β-glucuronidase.

Immunohistochemistry Paraffin-embedded samples (41 liver specimens with hepatolithiasis and 10 normal liver samples obtained from patients with haemangioma) were obtained from Shengjing Hospital of China Medical University. The staining procedures were performed on 4 μm paraffin-embedded tissue sections. The slides were deparaffinized in xylene and dehydrated in a graded ethanol series, and the sections underwent antigen retrieval in citrate solution. Endogenous peroxidase was blocked with 3% hydrogen peroxide, and the sections were washed with phosphate-buffered saline. After blocking, they were incubated overnight with β-glucuronidase (1:150, ab166904, Abcam), c-myc (1:100, ab32072, Abcam), p-Akt (1:40, 13038, Cell Signaling Technology), p-PKC (1:40, 9921, Cell Signaling Technology), p-p65 (1:100, 3033, Cell Signaling Technology) primary antibodies, respectively. Antigen staining was performed using DAB horseradish peroxidase color development kit, and then counter was stained with hematoxylin. The immunoreactivity of proteins was assessed independently by two experienced pathologists for staining intensity (0 absent or weak, 1 intermediate or strong staining).

Western blot Cell lysates were resolved on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies against β-glucuronidase (1:1000, ab166904, Abcam), c-myc (1:1000, ab32072, Abcam), p-Akt (1:1000, 13038, Cell Signaling Technology), p-PKC (1:1000, 9921, Cell Signaling Technology), p-p65 (1:1000, 3033, Cell Signaling Technology), GAPDH (1:1000, G5262, Sigma), β-actin (1:3000, 60008-1-Ig, Proteintech), respectively, followed by incubation with peroxidase-conjugated secondary antibodies and chemiluminescence detection.

Molecular and Cellular Biochemistry

Enzyme‑linked immunosorbent assay L02 or HiBEpiC cells were seeded as 1 × 104 cells per well of 24-well plates, with each well containing 200 μl epithelial cell medium. After about 8 h, cells were treated with LPS (0, 10, 50, or 100 μg/ml) for different time (0, 3, 6, or 24 h). The β-glucuronidase of supernatants was determined using enzyme-linked immunosorbent assay (ELISA) kit for human β-glucuronidase (CSB-E09469h, CUSABIO) according to the manufacturer’s instructions.

Transient transfection L02 or HiBEpiC cells were seeded as 6 × 105 cells per well of 6-well plate. After about 16 h, cells were transfected with 4 μg of Human MYC (4609) siRNA (siGENOME,

Fig. 1  Relationship between c-myc and endogenous β-glucuronidase in the liver samples with hepatolithiasis. a Endogenous β-glucuronidase expression in the liver specimens with hepatolithiasis significantly increased. b C-myc expression in the liver specimens with hepatolithiasis significantly increased. c The example of the

M-003282-07-0005), and 10 μl of Lipofectamine2000 (Invitrogen, CA) according to the manufacturer’s protocol. After 48 h transfection, the cells were collected for detection.

Statistical analysis Rank-sum test was used to test the differences of β-glucuronidase, c-myc, p-p65, p-PKC, or p-Akt expression between liver specimens with hepatolithiasis and normal liver samples. Spearman correlation analysis and multiple regression analysis were used to test the correlation of β-glucuronidase and c-myc, with p-p65, p-PKC and p-Akt expression in liver specimens with hepatolithiasis. All p values reported are two-sided, and the significance level was set at less than 0.05. The analyses were performed with the SPSS 19.0 statistical software program.

cases with increased β-glucuronidase expression in the liver specimens with hepatolithiasis compared with the normal liver tissues. d The example of the cases with increased c-myc expression in the liver specimens with hepatolithiasis compared with the normal liver tissues

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Molecular and Cellular Biochemistry

Results C‑myc and endogenous β‑glucuronidase expressions in hepatolithiasis formation To conduct our survey focusing on the mechanism of hepatolithiasis formation, liver specimens with hepatolithiasis and normal liver samples were examined by immunohistochemistry stain. It was found that compared with that in normal liver samples, endogenous β-glucuronidase expression in the liver specimens with hepatolithiasis significantly increased (Fig. 1a, c), suggesting that increased expression of endogenous β-glucuronidase in hepatocytes and intrahepatic biliary epithelial cells might take part in the formation of hepatolithiasis. Similarly, the expression of c-myc in the liver specimens with hepatolithiasis also increased significantly (Fig. 1b, d), and spearman correlation analysis showed that the expression of c-myc in the liver samples with hepatolithiasis was positively correlated with that of endogenous β-glucuronidase (Table 1). As Li’s study found that c-myc shRNA could inhibit the secretion of endogenous β-glucuronidase [13], what we found could suggest that in the formation of hepatolithiasis, c-myc might take part in the regulation of endogenous β-glucuronidase secretion, by regulating endogenous β-glucuronidase expression.

C‑myc takes part in the regulation of LPS‑induced expression and secretion of endogenous β‑glucuronidase HiBEpiC cells or L02 cells were treated with different concentrations of LPS for different time. When HiBEpiC cells Table 1  Correlation between p-Akt, p-PKC, p-P65, c-myc, and endogenous β-glucuronidase expression in the samples with hepatolithiasis

c-myc  Spearman correlation coefficient  p value β-Glucuronidase  Spearman correlation coefficient  p value p-p65  Spearman correlation coefficient  p value p-PKC  Spearman correlation coefficient  p value p-Akt  Spearman correlation coefficient  p value

were treated with LPS for 24 h, c-myc and endogenous β-glucuronidase expression were markedly elevated in a dose-dependent manner, and when LPS was at 100 μg/ml, c-myc and endogenous β-glucuronidase expression were near the maximum, showing no significant increase with the increase of LPS concentration (Fig. 2a). When the concentration of LPS was maintained at 100 μg/ml, it was found that c-myc and endogenous β-glucuronidase expression were markedly elevated in a time-dependent manner, and at 24 h, their expressions were near the maximum (Fig. 2b). In the L02 cells, the result was similar (Fig. 2c, d). These results confirmed that LPS could induce increased expression of endogenous β-glucuronidase and c-myc in hepatocytes and intrahepatic biliary epithelial cells in a dose- and timedependent manner. As bacterial infection is believed to be crucial for intrahepatic bile duct stones formation [4], and during the formation of hepatolithiasis, endotoxemia were observed [17], it could be inferred that in the formation of hepatolithiasis, the increased expression of endogenous β-glucuronidase and c-myc might be induced by LPS. To determine whether c-myc take part in the regulation of endogenous β-glucuronidase expression, we assessed the effect of altered c-myc expression in LPS-stimulated HIBEpiC or L02 cells. Cells were transfected with c-myc siRNA to inhibit the LPS-induced expression of c-myc. It was confirmed that c-myc expression was effectively inhibited, and it was found that the LPS-induced expression of endogenous β-glucuronidase in the HIBEpiC or L02 cells with c-myc siRNA transfection was also effectively inhibited (Fig. 3a, b), confirming that it was via c-myc that LPS induced the increased expression of endogenous β-glucuronidase.

c-myc

β-Glucuronidase

p-p65

p-PKC

p-Akt

1.000

0.824** 0.000

0.511** 0.001

0.585** 0.000

0.245 0.123

0.824** 0.000

1.000

0.481** 0.001

0.452** 0.003

0.232 0.145

0.511** 0.001

0.481** 0.001

1.000

0.459** 0.003

0.188 0.238

0.585** 0.000

0.452** 0.003

0.459** 0.003

1.000

0.428** 0.005

0.245 0.123

0.232 0.145

0.188 0.238

0.428** 0.005

1.000

*Correlation is significant at the 0.05 level (2-tailed) **Correlation is significant at the 0.01 level (2-tailed)

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Molecular and Cellular Biochemistry

Fig. 2  LPS induce increased expression of endogenous β-glucuronidase and c-myc in a dose- and time-dependent manner. a In HiBEpiC cells, LPS induced increased expression of endogenous β-glucuronidase and c-myc in a dose-dependent manner. b In HiBEpiC cells, LPS  induced increased expression of endogenous β-glucuronidase and c-myc in a time-dependent manner. c, d In L02 cells, LPS  induced increased expression of endogenous β-glucuronidase and c-myc in a dose- and time-dependent manner

In addition, the endogenous β-glucuronidase secretion of LPS-stimulated HIBEpiC or L02 cells was also detected by ELISA analysis. It was found that the secretion of endogenous β-glucuronidase also increased, similarly with the expression of endogenous β-glucuronidase, in a dose- and time-dependent manner (Fig. 4a, b), suggesting that the increased activity of endogenous β-glucuronidase in the bile with pigment gallstones [6] may come from the secretion of LPS-stimulated hepatocytes and intrahepatic biliary epithelial cells with increased expression of endogenous β-glucuronidase.

PKC/NF‑κB take part in the regulation of c‑myc and endogenous β‑glucuronidase expression To clarify whether PI3K, PKC, or NF-κB take part in the LPS-induced expression of c-myc and endogenous

β-glucuronidase, the relative proteins were also detected in this study. Immunohistochemistry analyses revealed that compared with that in normal liver samples, p-p65 and p-PKC expressions in the liver samples with hepatolithiasis also increased significantly (Fig. 5a–d). Spearman correlation analysis showed that the expression of p-p65 and p-PKC in the liver samples with hepatolithiasis was positively correlated with that of c-myc or endogenous β-glucuronidase (Table 1), suggesting that in the formation of hepatolithiasis, these proteins might take part in the regulation of c-myc and endogenous β-glucuronidase expression. Multiple logistic regression analysis suggested that p-p65 and p-PKC expressions in the liver samples with hepatolithiasis were positively associated with the expression of c-myc (Table 2), and partial least squares regression model suggested that the expression of c-myc was positively associated with the expression of endogenous β-glucuronidase (Table 3). In addition, in the LPS-stimulated HiBEpiC or L02 cells, NF-κB and PKC were also activated, as p-p65 and p-PKC were increasingly expressed (Fig. 6a, b). Furthermore, NF-κB inhibitor PDTC, or PKC inhibitor chelerythrine could effectively inhibit the LPS-induced expression of c-myc and endogenous β-glucuronidase, and chelerythrine could also partly inhibit the expression of p-p65 (Fig. 6a, b), providing the proof that NF-κB and PKC were critical regulators in the LPS-stimulated expression of c-myc and endogenous β-glucuronidase, and PKC was a regulator of NF-κB, at least partially in the process. PI3K signal proteins were also detected in our study. However, the expression of p-Akt in liver samples with hepatolithiasis did not significantly increase (Fig. 5e, f), and spearman correlation analysis showed that the expression of p-Akt in the liver samples with hepatolithiasis was not correlated with that of c-myc or endogenous β-glucuronidase, either (Table 1). Furthermore, it was found that in LPSstimulated HiBEpiC or L02 cells, the expression of p-Akt did not increase, though PI3K inhibitor LY294002 could partly inhibit the LPS-stimulated expression of p-PKC, p-p65, c-myc, and endogenous β-glucuronidase (Fig. 6a, b). These results demonstrated that PI3K should not be the main regulator for the endogenous β-glucuronidase in the process of hepatolithiasis formation.

Discussion The mechanism of pigment gallstones formation is being gradually explored [18]. Since Maki’s description, the important role of exogenous β-glucuronidase in the formation of pigment gallstone is well known and widely accepted [18, 19]. Recently, the role of endogenous β-glucuronidase in the pathogenesis of pigment gallstone formation is also being gradually revealed [7, 17, 18, 20]. It was found that

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Molecular and Cellular Biochemistry

Fig. 3  LPS induce increased expression of endogenous β-glucuronidase via c-myc. a In HiBEpiC cells, when c-myc expression was effectively inhibited via c-myc siRNA, the LPS-induced expression of endogenous β-glucuronidase in the transfected cells was also inhibited. b In L02 cells, the result was the same

Fig. 4  LPS induce increased secretion of endogenous β-glucuronidase in a dose- and time-dependent manner. a LPS stimulated increased endogenous β-glucuronidase secretion of HIBEpiC cells, in a dose- and time-dependent manner. b In L02 cells, the result was the same

in the infected bile, besides large amounts of exogenous β-glucuronidase that were released by bacteria, endogenous β-glucuronidase expression and secretion also increased. Correspondingly, the activity of exogenous and endogenous β-glucuronidase would increase [6, 7]. Because both exogenous and endogenous β-glucuronidase could hydrolyze bilirubin glucuronides to free bilirubinate, which combines with calcium to yield water-insoluble calcium bilirubinate, it could be speculated that besides exogenous β-glucuronidase, endogenous β-glucuronidase may also take part in the formation of hepatolithiasis. It was revealed that bacterial infection and bile duct obstruction were important factors for increased activity of human endogenous β-glucuronidase [21]. With the emptying of infected bile, exogenous β-glucuronidase decreased quickly, but endogenous β-glucuronidase could maintain at a high level for a long time, suggesting that endogenous β-glucuronidase may play a role for a long time in pigment stones formation [8, 22]. Now, it was revealed that the expression of endogenous β-glucuronidase in the liver

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specimens with hepatolithiasis significantly increased, and hepatocytes and intrahepatic biliary epithelial cells with increased expression of endogenous β-glucuronidase could secrete more endogenous β-glucuronidase. Therefore, revealing the regulation mechanism of endogenous β-glucuronidase expression might help us control the lithogenic potentials. LPS, a major endotoxin from gram-negative bacteria, is believed to be crucial for the formation of intrahepatic bile duct stones. In our previous study for the pathogenesis of pigment gallstones, endotoxemia was observed in the pigment gallstones group, and the activity of endogenous β-glucuronidase also increased, suggesting the close relationship between endotoxin and increased activity of endogenous β-glucuronidase in pigment gallstones formation [17]. Now, in this study, it is further confirmed that LPS could induce the increased expression of endogenous β-glucuronidase, in a dose- and time-dependent manner. What is even more important is that the secretion of endogenous β-glucuronidase also increased, correspondingly.

Molecular and Cellular Biochemistry

Fig. 5  The expression of p-p65, p-PKC, and p-Akt in the liver samples with hepatolithiasis. a and b Compared with that in normal liver samples, the expression of p-p65 in the liver samples with hepatolithiasis significantly increased, and the example of the cases was showed. c and d Compared with that in normal liver samples, the expression of p-PKC in the liver samples with hepatolithiasis signifi-

cantly increased, and the example of the cases was showed. e Compared with that in normal liver samples, the expression of p-Akt in liver samples with hepatolithiasis did not significantly increase. f The example of the cases with increased p-Akt expression in the liver specimens with hepatolithiasis compared with the normal liver tissues

In addition, bacterial infection and inflammatory necrosis would decrease the pH of surrounding tissues, and this would further help increase the activity of endogenous β-glucuronidase [21], to catalyze the precipitation of calcium bilirubinate. So, the LPS-stimulated expression and secretion of β-glucuronidase should be important for the formation of calcium bilirubinate (pigment) stones.

In addition, it was found that in the LPS-stimulated L02 and HiBEpiC cells, similarly with that of endogenous β-glucuronidase, the expression of c-myc also increased. In the Li’s study for chronic proliferative cholangitis, it was found that inhibition of c-myc with c-myc shRNA could reduce the secretion of endogenous β-glucuronidase in the bile duct wall, suggesting the possible role of c-myc for the

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Molecular and Cellular Biochemistry

Table 2  Multivariate logistic regression of c-myc and p-PKC, p-p65 expression

Variables

p-PKC p-P65 Constent

B

SE

2.860 1.921 − 0.944

Table 3  Partial least squares regression of β-glucuronidase and p-PKC, p-p65, c-myc expression Model

Constent c-myc

Unstandardized coefficients

Standardized coefficients

B

Std. error

Beta

0.242 0.761

0.098 0.128

0.837

1.169 0.983 0.597

endogenous

t

Sig.

2.473 5.957

0.018 0.000

p-PKC and p-P65 were instrumental variables

regulation of endogenous β-glucuronidase secretion [13]. Now, in our study, it was found that the increased secretion of endogenous β-glucuronidase might come from the LPS-stimulated hepatocytes and intrahepatic biliary epithelial cells with increased expression of endogenous β-glucuronidase, and in the LPS-stimulated L02 and HiBEpiC cells, downregulation of c-myc could effectively suppress the increased expression of endogenous β-glucuronidase, suggesting that in the physiopathologic process of hepatolithiasis formation, c-myc might be the main regulator of the endogenous β-glucuronidase. In addition, the close relationship between

Fig. 6  PKC/NF-κB take part in the regulation of c-myc and endogenous β-glucuronidase expression. a In the LPS-stimulated HiBEpiC cells, p-p65 and p-PKC expression significantly increased, but p-Akt expression did not. PDTC and chelerythrine could effectively inhibit the LPS-induced expression of c-myc and endog-

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Wald

5.989 3.819 2.499

df

1 1 1

Sig

0.014 0.051 0.114

OR

17.469 6.826 0.389

95% CI of OR Lower limit

Upper limit

1.768 0.994

172.640 46.852

c-myc and endogenous β-glucuronidase was also demonstrated in the liver samples with hepatolithiasis in our study. However, until now, the related mechanism by which LPS induces c-myc and endogenous β-glucuronidase expressions in the process of hepatolithiasis formation has not yet been revealed in the literature. In our study, the role of PKC and NF-κB in LPS-induced expression of c-myc and endogenous β-glucuronidase was detected. In LPS-stimulated HiBEpiC or L02 cells, NF-κB was activated, and NF-κB inhibitor PDTC could effectively inhibit LPS-induced expression of c-myc and endogenous β-glucuronidase. In addition, LPS could also induce increased expression of phosphorylated PKC, and PKC inhibitor chelerythrine could inhibit LPS-induced expression of p-p65, c-myc and endogenous β-glucuronidase. Our results demonstrated that LPS induced c-myc and endogenous β-glucuronidase expressions via PKC/NF-κB signaling pathway. Combined with the immunohistochemical findings of increased expression of p-p65 and p-PKC in the liver samples with hepatolithiasis, and close relationship of p-p65 and p-PKC with the c-myc or endogenous β-glucuronidase expression, our results suggested that LPS

enous β-glucuronidase, and chelerythrine could also partly inhibit the expression of p-p65. LY294002 could partly inhibit the LPSinduced expression of p-PKC, p-p65, c-myc, and endogenous β-glucuronidase. b In L02 cells, the result was the same

Molecular and Cellular Biochemistry

may play a certain role in the formation of hepatolithiasis by stimulating c-myc and endogenous β-glucuronidase expression in hepatocytes and intrahepatic biliary epithelial cells via a signaling cascade of PKC/NF-κB. PKC and NF-κB are common targets for lipopolysaccharide (LPS). Many studies have reported that LPSinduced PKC-α was responsible for the activation of the redox-sensitive transcription factor, NF-κB [23–25]. LPS can activate PKC isoenzymes in many kinds of cells [24, 25]. However, which kinds of PKC isoenzymes are responsible for NF-κB activation is not well understood in different cells, including in hepatocytes and intrahepatic biliary epithelial cells. It was demonstrated that the PKC family could activate the IκB-α kinase (IKK) complex, modulate degradation of IκB-α, and further result in nuclear translocation of NF-κB, to activate its target genes [23, 26]. Furthermore, C-myc has been found to be one of the molecular targets of NF-κB [22, 27], and can be modulated by NF-κB-dependent transcriptional activity [28]. It has been even demonstrated that the murine c-myc gene contains two functional NF-κB binding sites, which were located at 1101–1081  bp upstream of the P1 promoter (URE), and + 440 to + 459 bp (IRE) of internal within exon 1 [29–31]. Multiple agents that were found to be able to activate NF-κB could also induce c-myc gene transcription [32–34], and LPS, as an important factor in the physiopathologic process of hepatolithiasis formation, is also the main inducer of NF-κB activation, in multiple kinds of cells [35, 36]. Now, in our study, LPS, the possible activating factor of hepatolithiasis formation, was found to be the inducer of PKC, NF-κB, c-myc, and endogenous β-glucuronidase, and their ordinal relationships were also demonstrated in hepatocytes and intrahepatic biliary epithelial cells. Further revealing the related mechanism may help us regulate the expression and secretion of endogenous β-glucuronidase, and further explore more effective methods to prevent stone formation or recurrence. In conclusion, our study demonstrated that in hepatocytes and intrahepatic biliary epithelial cells LPS could induce the increased expression and secretion of endogenous β-glucuronidase via a signaling cascade of PKC/ NF-κB/c-myc, and the increased expression of endogenous β-glucuronidase might play a possible role in the formation of hepatolithiasis. Our findings provide a potential target of preventing hepatolithiasis formation or recurrence. Acknowledgements  This work was funded by National Natural Science Foundation of China (81400659). Author Contributions  YD and DQ performed experiments; YD and TY analyzed data, interpreted results of experiments, and prepared figures; YD, DC, and WS designed the overall study; YD drafted manuscript; DC and WS approved final version of manuscript.

Compliance with ethical standards  Conflict of interest  No conflicts of interest, financial or otherwise, are declared by the authors. Ethical approval  The study was reviewed and approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University (reference number: 2012PS34K) and written informed consent was obtained from all patients, and performed in accordance with the principles outlined in the Declaration of Helsinki.

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