Effects of LPS-induced cholangitis on the cytoskeleton morphology of bile duct epithelium and the intervention mechanism of Dahuang Lingxian formula for these changes

Cheng-Ji Li, Yuan Yu, Yi-Rong Gan, Jiao-An Pang, Wen Yang

1. Guangxi University of Chinese Medicine, Nanning 530001, China

2. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China

Keywords:Bile duct epithelial cells Dahuang Lingxian formula Lipopolysaccharide Cytoskeleton Bile duct inflammation

ABSTRACT Objective: To observe the effect of lipopolysaccharide (LPS) on cytoskeleton and the effect of Dahuang Lingxianfang on nF-KB /MAPK signaling pathway. Methods: Biliary epithelial cells of each group were stained with photolipin fluorescent staining, and the arrangement of cytoskeleton was observed under laser confocal microscope. Western blotting was used to detect the expression of f-actin. Results: After LPS intervention, the biliary epithelial cells showed nuclear shrinkage or damage, and the skeleton was broken or lumped. The cytoskeleton was partially repaired after the intervention of pathway blocking preparation combined with RHUbarb Lingxianfang. All the other groups had different degree of cytoskeleton fracture.Compared with normal group, the expression of F-actin protein in LPS group was decreased(P < 0.05); Compared with LPS group, the expression of F-actin in LPS+ TCM group,LPS+PDTC+ TCM group, LPS+SB203580+ TCM group and LPS+PDTC+SB203580+TCM group was significantly increased (P < 0.05); Compared with traditional Chinese medicine group, the expression of F-actin in LPS+PDTC+ traditional Chinese medicine group, LPS+SB203580+ traditional Chinese medicine group and LPS+PDTC+SB203580+traditional Chinese medicine group had no significant difference (P > 0.05). Conclusion:RHUbarb Lingxianfang can restore the sequence of biliary epithelial cytoskeleton and protect its microfilament structure under inflammation, and its mechanism may be related to the regulation of NF-KB /MAPK signaling pathway.

1. Introduction

Intrahepatic cholangiolithiasis, known as the “incurable disease”in benign bile duct diseases, is an important factor causing bile duct suppurative inflammation and cholangiocarcinoma, which are frequently seen in China, Japan, South Korea and other Asia-Pacific regions[1]. The pathogenesis of intrahepatic cholangiolithiasis is complicated and related to many factors, but the correlation between the formation of intrahepatic cholangiolithiasis and biliary tract infection has been confirmed in animal experiments and clinical studies[2]. Biliary tract infection is mainly caused by Gram-negative bacteria, and gram-negative bacteria endotoxin lipopolysaccharide is an important part of the cell wall of gram-negative bacteria.LPS can activate downstream NF-kB (nuclear factor kappa-lightchain-enhancer of activated B cells) and MAPK (mitogen-activated protein kinase) signaling pathways to participate in inflammatory responses through the signaling pathway Toll-like receptor 4 (TLR4)[3]. Persistent inflammatory injury can cause the proliferation of bile duct epithelial cells (BDECs), change of the bile flow in the bile duct tree, and the formation and secretion of lithogenic bile[4].

Rhubarb Lingxianfang is Professor Tang Ganli’s empirical prescription for the prevention and treatment of intrahepatic bile duct stones. Long-term clinical and basic studies have found that rHUbarb can reduce the formation of lithogenic bile and relieve biliary tract inflammation by regulating key downstream factors of NF-kB /MAPK signaling pathway[5][6]. F-actin, as an important structure for maintaining cell homeostasis, is involved in the destruction of epithelial junctions and formation of permeability barriers in inflammatory responses[7]. Therefore, whether regulation of NF-kB/MAPK signaling pathway can cause changes in F-actin and abnormal changes in the structure and function of bile duct epithelial cells, thus affecting the formation of calculi, arouses people’s thinking. Therefore, this study started from the level of cytoskeleton (F-actin), used LPS-induced biliary inflammatory cell model, combined with western blot and laser confocal detection technology, to observe the changes of biliary epithelial cytoskeleton protein F-actin under different signal pathway treatment and intervention of RHUbarb Lingxianfang. To improve the rHUbarb lingxianfang to relieve bile duct inflammation and treat the mechanism of intrahepatic bile duct stones.

2. Materials and Methods

2.1 Experimental Materials

Bile duct epithelial cells from SD rats (Shanghai Cell Bank);DMEM culture medium, Fetal bovine serum (FBS) (Gibco,USA, Batch No. C11885500BT; A3161002); Rhodamine labeled Gholopeptide (Solarbio, A1610-300T); LPS (LPS, Sigma); 0.25%trypsin -0.02% ethylenediamine tetraacetic acid (EDTA) solution(Lot no. 25200056 Company: Gibco); Phosphate buffer PBS (Batch Number: P1022-500), the water is ultrapure.

2.2 Experimental Instrument

BSC-1300II B2 biosafety cabinet (Shanghai Boxun Medical BioInstrument Co., LTD.); 5410 low temperature high speed centrifuge (Eppendorf, Germany); 371 CO2 incubator, Multiskan FC enzyme reader (Thermo Fisher Scientific, USA); Confocal laser scanning microscope (Olympus Corporation, Japan, FV3100); IX71 inverted fluorescence microscope (Olympus Corporation, Japan).

2.3 Culture of Bile Duct Epithelial Cells

DMEM medium containing 10% fetal bovine serum and 1% double antibody (penicillin and streptomycin) was used for routine culture at 37℃, 5%CO2, saturated temperature.

2.4 Cell Grouping and Intervention

Cultured bile duct epithelial cells were inoculated in 9 laser confocal petri dishes respectively, 3×3cm per dish. After the cells were 80% full, intervention was performed with drugs respectively:

2.5 Observing the Biliary Epithelial Cytoskeleton Rearrangement in Each Group

10μl of the original solution was prepared and diluted with 20ulBSA and 2mlPBS. The cells were washed twice with PBS,fixed with 2.5% glutaraldehyde fixative for 15min, washed three times with PBS for 3min each; 0.5% Trito-X-100 permeable at room temperature for 20min. Wash by PBS 3 times, 3min each. Take 300μ1 TRIT-labeled gitopeptide working solution, and incubate for 30min at room temperature, avoiding light, then wash with PBS twice. Nucleate with 200μl DAPI for 3min and wash with PBS twice, the results were analyzed by confocal laser scanning. The fluorescence intensity was analyzed by ImageJ 6 software.

2.6 Detecting the Expression of Cytoskeleton Protein F-actin with Western Blot

The total protein of the cells is extracted by the total protein reagent extraction box, and the protein concentration is detected by the BCA protein content detection kit. After SDS-PAGE electrophoresis, the proteins on the gel are transferred to PVDF membrane. After sealing with 5% skim milk, the cells are incubated with F-Actin primary antibody at 4 ℃ overnight, and rinsed with 1×TBST for 3 times.Anti-rabbit/mouse IgG secondary antibody (HRP-labeled) is added,and incubated for 1h at room temperature. After 1× washing, ECL luminescence agent is used for development. Finally, the bands are analyzed by Image J software.

2.7 Statistical Analysis

All data are input into SPSS20.0 for statistical analysis. Oneway anOVA is used for comparison between groups. Measurement data are expressed as mean ± standard deviation, and P<0.05 is considered statistically significant.

3. Results

3.1 Morphological Changes of Biliary Epithelial Cells in Each group

Under light microscope, normal bile duct epithelial cells are observed to grow in adherent monolayer, with irregular appearance,large cell body and dark brown nucleus. After LPS intervention,compared with Figure A, cell morphology changes. Cell bodies become significantly round and smaller, and A large number of cells fall off and float on the liquid level. After the intervention of RHUbarb Lingxianfang, compared with Figure B, cells adhere to the wall and grow well, with irregular appearance, enlarged cell body and obvious nucleus. After the intervention of pathway blocking agents, cell death increases. After the intervention of RHUbarb Lingxianfang, the growth of cells in each group recovers and the detachment of walls is less. The results are shown in Figure 1.

Figure1 Cell morphology of each group after different intervention(observed under 4×20 microscope)

3.2 Cytoskeleton Image

Confocal microscopy shows that the nucleus (blue) of the normal group is intact, the capsule is clearly visible, and the cytoskeleton(red fluorescence) is filiform. After LPS intervention, the nucleus is shrunken or damaged, and the skeleton is broken or clumped.After the intervention of RHUbarb Lingxianfang, nuclear wrinkling is reduced less and cytoskeleton is clumped. After the intervention of pathway blocking agents, the nucleus is obviously shrunked, the cytoskeleton is broken to varying degrees, and the microfilaments are disordered. After the intervention of RHUbarb Lingxianfang, the nuclear shrinkage is not obvious and the microfilaments are arranged neatly. The results are shown in Figures 2, 3 and 4.

FIgure 2 Cytoskeleton diagram

Figure 3 Cytoskeleton after intervention with pathway blocking agents

Figure 4 Cytoskeleton diagram of RHUbarb Lingxianfang + pathway blocking preparation after intervention

3.3 Fluorescence Intensity

① Compared with the normal group, the fluorescence intensity of model group is significantly decreased (t =5.816, P=0.004<0.05);

② Compared with model group, the fluorescence intensity of LPS+TCM group is increased (t=5.381, P=0.006<0.05);

The fluorescence intensity of LPS+PDTC group is increased(t=9.021, P=0.001<0.05).The fluorescence intensity of LPS+SB203580 group is increased(t=13.798, P=0.000<0.05);

The fluorescence intensity of LPS+PDTC+SB203580 group is increased (t=15.520, P=0.000<0.05);

The fluorescence intensity of LPS+PDTC+TCM group is increased(t =9.643, P=0.000<0.05);

The fluorescence intensity of LPS+SB203580+TCM group is increased (t=9.160, P=0.001<0.05);

The fluorescence intensity of LPS+PDTC+SB203580+TCM group is increased (t=3.613, P=0.023<0.05);

③ Compared with LPS+ TCM group, the fluorescence intensity of LPS+SB203580+TCM group is increased, but there is no statistical significance (t =2.438, P=0.071>0.05).

The fluorescence intensity of LPS+PDTC+SB203580+TRADITIONAL Chinese medicine group increased, but there is no statistical significance(t=0.403, P=0.708>0.05). Compared with the model group, the fluorescence intensity of the remaining groups is enhanced to varying degrees.

Table 2 Fluorescence intensity of F-actin in each group(x±s)

3.4 Expression of F-actin Skeleton Protein was Detected by Western Blot

① Compared with the normal group, the expression of F-actin in LPS group is significantly decreased (t=6.473, P=0.003<0.05);

② Compared with LPS group, the expression of F-actin in LPS+TCM group is significantly increased (t=10.624,P=0.000<0.05); The expression of F-actin in LPS+PDTC+TCM group is significantly increased (t=7.639, P=0.002<0.05).

The expression of F-actin protein in LPS+SB203580+TCM group is significantly increased (t=7.243, P=0.002<0.05); The expression of F-actin in LPS+PDTC+SB203580+TCM group is significantly increased (t=3.685, P=0.021<0.05);

There is no significant difference in F-actin protein expression in other groups (P>0.05).

③ Compared with traditional Chinese medicine group,the expression of F-actin in LPS+PDTC+TCM group,LPS+SB203580+TCM group and LPS+PDTC+SB203580+TCM group has no significant difference (P>0.05).

Table 3 Expression of f-actin skeleton protein(x±s)

Figure 5 Expression of F-actin protein in each group

4. Discussion

Made up of microtubule, microfilament and intermediate filaments of the cytoskeleton is the main structure of the cell, including the principal compositions of the cytoskeleton protein is cytoskeleton fibrous actin (F-actin), start and regulating intracellular and extracellular signal when the primary target protein and its stability in cells, motion, contraction, adhesion, proliferation and apoptosis, and other ACTS, Important mechanisms of ganglion cell permeability[8].The cytoskeleton is able to change shape or move in response to the environment. When cells suffered inflammation such as malignant stimulation, F-actin reorganization happens and redistribution, cells surrounding F-actin ring fracture, cells in the central of a dense array of beam shaped fiber stress, increased tension cause cells to center,can accelerate cell shrinkage, cause apoptosis necrosis of the final ending, further aggravate the inflammatory response [9][10].

Relevant studies have shown that the activation of NF-kB/MAPK inflammatory signaling pathway mainly affects the cytoskeleton from the following two aspects. On the one hand, MAPK signaling pathway can not only reduce the stability of microtubules by over-phosphorylation of MAPs, but also affect the stability of microtubules by regulating the phosphorylation of other proteins,such as DOC1R/MRP14[11]. On the other hand, when cells are stimulated by external factors such as inflammation, oxidative stress occurs, and the activation of nuclear transcription factor NF-κB is further promoted through MAPK signal transduction pathway. The activated NF-κB enters the nucleus, and the release of inflammatory factors such as TNF-a and IL-6 is accelerated, leading to massive depolymerization of F-actin[12]. In addition, pharmacological regulation of inflammatory signaling pathways has been reported in the literature on the changes of different cytoskeletons, for example,icariin protects lPS-induced osteoblast F-actin injury by inhibiting the mRNA expression of cytoskeleton related factors RhoA and Cofilin[13]. Bergenin inhibits the NF-κB and MAPKs pathways by reducing the production of pro-inflammatory factors NO, TNF-α,IL-1β and IL-6 to maintain the homeostasis of macrophage cytoskeleton[14].

Based on the “inflammation hypothesis”, the research group conducted a long-term study on the mechanism of intrahepatic bile duct stones, and explored the influence of RHUbarb Lingxianfang on the formation of intrahepatic bile duct stones by regulating bile duct inflammation. Previous studies have shown that DHUANG Lingxianfang can regulate the expression of Myd88, P-P38, TLR4,NF-κB and other proteins and mRNA in TLR4/NF-κB/MAPK signaling pathway, thus reversing the inflammatory response of bile duct cells[15][16]. This prescription is based on the principle of “soothing the liver and promoting gallbladder, attacking and removing stones”. Rhubarb[17] and Mirabilite[18], which are composed of rhubarb[17] and Mirabilite[18], have the pharmacological effects of purging and anti-inflammatory, and the two drugs together play the role of King medicine in attacking stagnation, promoting gallbladder and removing stones. Pennilaria[19] and turmeric[20]can protect bile duct epithelial cells through anti-inflammatory effect. Saponins A and D contained in Bupleurum[21] can inhibit lipopolysaccharide (LPS) activity. Fructus aurantii and Z. aurantii can protect the liver and reduce the secretion of stone-induced bile[22]. It can be seen that rHUbarb lingxianfang has the functions of protecting liver, regulating biliary tract and anti-inflammation.

In this study, the research group observed the changes of cytoskeleton proteins under the intervention of inflammatory states and different pathway blocking agents, so as to improve the molecular mechanism of DHUANGlingxianfang for alleviating bile duct inflammation and treating intrahepatic bile duct stones.The results showed that after LPS intervention, the content of F-actin skeleton protein in biliary epithelial cells was reduced,and the myofilament was disordered and broken. However, after DHUANGlingxianfang intervention, the content of F-actin protein was improved, especially after combined with pathway blocking preparation, and the myofilament was more orderly. It can be seen that RHUbarb Lingxianfang can restore the sequence of biliary epithelial cytoskeleton and protect its microfilament structure under inflammation. These results indicate that RHUbarb Lingxianfang may regulate NF-kB /MAPK inflammatory pathway, relieve bile duct cell inflammation, repair and maintain the integrity of cytoskeleton. The change of F-actin skeleton protein is closely related to epithelial-mesenchymal transformation, and the molecular mechanism between the occurrence of biliary inflammation and epithelial-mesenchymal transformation will be the next research direction of our research group.