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  Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 79  |  Page : 641-650  

Salvianolic Acid B delays hepatolithiasis through inhibiting the fibrosis of intrahepatic biliary epithelial cells


1 Department of Hepatobiliary Surgery, Harrison International Peace Hospital, Hengshui, Hebei, China
2 Department of Geriatric Medicine, Harrison International Peace Hospital, Hengshui, Hebei, China

Date of Submission10-Apr-2021
Date of Decision07-Dec-2021
Date of Acceptance27-Apr-2022
Date of Web Publication19-Sep-2022

Correspondence Address:
Hongbin Bao
Department of Hepatobiliary Surgery, Harrison International Peace Hospital, Hengshui, Hebei - 053000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_162_21

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   Abstract 


Background: Hepatolithiasis (HL) is one of the common diseases in hepatobiliary surgery, and bile duct fibrosis is the key step in the pathogenesis of HL. Salvia miltiorrhiza (S. miltiorrhiza) has an inhibitory effect on bile duct fibrosis, but its molecular mechanism remains unclear. Objectives: Our research aimed to clarify the molecular mechanism of S. miltiorrhiza on HL. Materials and Methods: The rats were fed with salvianolic acid B and salvianolic acid B-free lithogenic diet (LD) for 4 weeks to observe the degree of bile duct fibrosis. The expression levels of TNF, IL1B, IL6, and MRP2 in HIBEC stimulated by taurodeoxycholic acid (TDCA) were measured by RT-qPCR and ELISA. E-cadherin and Vimentin expression in HIBEC was detected using immunofluorescence and Western blot. Cell proliferation was detected by CCK8. Results: The fibrosis of the bile duct was the key step of HL. The NF-κB pathway was activated and MRP2 was expressed low in intrahepatic biliary epithelial cells surrounding bile duct stones. Through experiments, salvianolic acid B (Sal B) delays HL via the NF-κB/MRP2 axis. Conclusion: In this research, we confirmed that salvianolic acid B inhibited the HL via the NF-κB/MRP2 axis.

Keywords: Epithelial-mesenchymal transition, hepatolithiasis, MRP2, NF-κB, salvianolic acid B


How to cite this article:
Yao H, Xu W, Liu Z, Ma D, Bao H. Salvianolic Acid B delays hepatolithiasis through inhibiting the fibrosis of intrahepatic biliary epithelial cells. Phcog Mag 2022;18:641-50

How to cite this URL:
Yao H, Xu W, Liu Z, Ma D, Bao H. Salvianolic Acid B delays hepatolithiasis through inhibiting the fibrosis of intrahepatic biliary epithelial cells. Phcog Mag [serial online] 2022 [cited 2022 Sep 26];18:641-50. Available from: http://www.phcog.com/text.asp?2022/18/79/641/356387



SUMMARY

  • Hepatolithiasis is a common disease of intrahepatic calculi in Asia. Salvia miltiorrhiza is a traditional Chinese herb. In this experiment, we found that as a major ingredient in Salvia miltiorrhiza, Salvianolic acid B can delay the development of hepatolithiasis by regulating the NF-κB/MRP2 axis.




Abbreviations used: ABC: ATP-binding cassette; CTS: Cryptotanshinone; EMT: Epithelial-mesenchymal transition; HIBEC: Human intrahepatic biliary epithelial cells; HL: Hepatolithiasis; IHC: Immunohistochemical; LD: Lithogenic diet; Sal A: Salvianolic acid A; Sal B: Salvianolic acid B; Sal C: Salvianolic acid C; S. miltiorrhiza: Salvia miltiorrhiza; Tan I: Tanshinone I; Tan IIA: Tanshinone IIA; TCM: Traditional Chinese medicine; TDCA: Taurodeoxycholic acid.


   Introduction Top


Hepatolithiasis (HL) is the presence of gallstones in the biliary ducts of the liver, which is rare in western countries but is prevalent and common in East Asia.[1],[2] As a type of gallstone disease, HL is characterized by the presence of gallstones in the left and right hepatic ducts proximal to the intrahepatic bile ducts.[3] The deterioration of HL complications includes biliary tract infection, biliary cirrhosis, and even cholangiocarcinoma.[4],[5] Genetic, diet, and environmental factors are suspected for the pathogenesis of HL, but the specific mechanism of HL remains unclear. Although the principles and techniques of surgical treatment of HL continue to develop, residual and recurrence of stones are still the main factors affecting long-term prognosis.[6] The current studies indicate that bile duct stenosis, cholestasis, and bile duct infection have been recognized as the main factors for HL. Among them, bile duct fibrosis has been reported as a key step in the pathogenesis of HL.[7],[8]

NF-κB is a crucial transcription factor controlling inflammation, immunity, and apoptosis.[9] The current studies showed that the activation of NF-κB is generally accompanied by the massive production of inflammatory factors.[10],[11],[12],[13] Furthermore, the NF-κB signaling pathway accelerates the epithelial-mesenchymal transition (EMT) process through a variety of pathways including up-regulation of the expression of pro-inflammatory factors.[14] Transmembrane transporters from the ATP-binding cassette (ABC) family mediate the entry and elimination of endogenous compounds and exogenous substances from liver cells.[15] MRP2 is one of the superfamilies of ABC transporters located on the side of the hepatocellular biliary canaliculi and functions in biliary transport.[16] The abnormal expression of MRP2 in epithelial cells leads to cholestasis and fibrosis.[17],[18] The activation of NF-κB has been reported to significantly inhibits the FXR/RXR transactivation of the MRP2 promoter.[19]

Salvia miltiorrhiza (S. miltiorrhiza) as a common traditional Chinese medicine (TCM) has now attracted interest in the therapy of various diseases.[20] S. miltiorrhiza has been reported to promote blood circulation, eliminate blood stasis, relieve pain, dilate coronary arteries, lower blood sugar, and is an anti-inflammatory.[21],[22] Besides, S. miltiorrhiza is a compound preparation for treating gallstones, choledocholithiasis, and cholecystolithiasis, but its specific mechanism remains unclear.[23]

Hence, our study revealed the effect of S. miltiorrhiza on the development of HL. Salvianolic acid B (Sal B) showed an inhibition effect in taurodeoxycholic acid (TDCA)-induced HIBEC and Sal B-rich diet showed a protective effect against HL. These findings lead us to further understand the regulation mechanism of Sal B on the progress of HL and provide new sights for the therapy of HL.


   Materials and Methods Top


Chemicals

S. miltiorrhiza and its ingredients (Salvianolic acid A (Sal A), Salvianolic acid B (Sal B), Salvianolic acid C (Sal C), Tanshinone I (Tan I), Tanshinone IIA (Tan IIA), Cryptotanshinone (CTS)) were purchased from Chengdu PUSH Biotechnology Co. Ltd. (China). The concentrations used in the corresponding experiments were 0, 100, 200, 500, 1,000, 10,000 μM and were freshly diluted using the basal medium.

Animals

All experimental procedures were authorized by the Harrison International Peace Hospital and were conducted based on the instructions of care and use for laboratory animals. Healthy male SD rats aged 8 weeks were obtained from the Beijing HFK Biotechnology Company Limited (Beijing, China) and placed in environmentally controlled plastic cages (22 ± 2°C, a 12 h light cycle). We arbitrarily divided the rats into two groups after partial ligation of the common bile duct: lithogenic diet (LD) in rats (n = 5) and LD with Sal B in rats (n = 5). The concentration of each component was calibrated, and the rats were weighed every day before administration. After 4 weeks of treatment with a different component diet, rats were euthanatized according to the AVMA Guidelines for the Euthanasia of Animals 1 day after the last dose. The liver tissues of rats were harvested, and the fibrotic degree of the intrahepatic bile duct was evaluated using histological analysis.

Cell culture

All cell lines were acquired from ATCC (USA). We cultured HEK293 and HIBEC in DMEM (USA) and added 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, USA) and nurtured them in a 5% CO2 incubator at 37°C.

CCK-8 assays

Cell Counting Kit-8 (CCK-8) was acquired from Dojindo. HEK293 cells and HIBEC were cultured (2 × 103 per well) in 96-well plates overnight, respectively. We treated cells with S. miltiorrhiza for 24 or 72 h. According to the manufacturers' instructions, CCK-8 was used to perform cell viability at 24 and 72 h. Then 96-well plate reader at 450 nm was used to measure the absorbency of cells to evaluate the cell viability.

RT-qPCR assays

Taking out the culture plates, the cells were washed with PBS. After treatment, we extracted total RNA from glioma cells by Trizol reagent (Life Technologies, NY, USA). The reverse transcription of cDNA was performed by the PrimeScript™RT kit (Takara, Japan). PCR primers were synthesized by GenePharma (ShangHai Gene Pharma, Shanghai, China) and the sequences were listed. The expression detected by SYBR Premix Ex Taq II (TaKaRa, Japan).

ELISA

After ventilation, the right lung tissue was collected and ground, followed by centrifugation to collect the supernatant. Concentrations of TNF-α (RTA00), IL-1β (RLB00), IL-6 (R6000B), and MRP2 were detected by ELISA kits (R&D Co., Ltd., Minneapolis, MN, USA). All operations were strictly as per kit instructions.

Western blot assays

The protein samples were extracted and separated by 10% SDS-PAGE gel, and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skim milk and cultured overnight at 4° with the following primary detection antibodies (Abcam, UK). We washed three times with TBS-T and the membranes were cultured with the secondary antibody at 24°C for 1 h. Western blots were pictured using an ECL reagent (Pierce, USA) and the density was verified using ImageJ software (NIH, USA).

Immunofluorescence

We detect the expression of E-cadherin and Vimentin in HIBEC by using immunofluorescence. The slides were washed with PBS three times in the culture plate for 3 min each time. Then, it was fixed with 4% paraformaldehyde for 35 min and permeabilized with 0.5% Triton X-100 for 10 min. Overnight staining was done with E-cadherin rabbit antibody (Novus) (1:100) or Vimentin rabbit antibody (Abcam) at 4°C, followed by 1 h staining at normal temperature with a fluorescence secondary antibody (Bitianyun Company, China). We washed the cells with PBS among antibody staining. Finally, DAPI (Bitianyun Company, China) staining solution was added at room temperature for more than 15 min and was installed on the slides with an anti-fading patch (Bi Tianyun Company, China). The slides were stored at −20°C. The images were taken from a fluorescence microscope (Leica).

Histopathological and Immunohistochemical (IHC) studies

The experiment rats were euthanized and then the needed tissues were removed, put into washing, and fixed (4% paraformaldehyde). We fixed the tissue in paraffin and sectioned it after fixation. Before IHC, we dewaxed and heated all sections in sodium citrate buffer (pH 6.0) diluted 100 times. We treated all sections with 3% H2O2 at room temperature (15 min). After inactivation, we cultured the serum at room temperature and blocked it for 10 min. IHC staining was performed with primary antibody E-cadherin and primary antibody Vimentin. After incubation with the secondary antibody, it was inoculated with the SABC kit (ZSGB-Bio, Beijing, China) and diaminobenzidine (DAB; Sigma, St. Louis, Mo., USA) to examine the expression of these markers in cells. The sections were stained with hematoxylin. Pictures were taken by Baumer digital camera with Olympus BX51 microscope (Olympus, Tokyo, Japan). The German semiquantitative method was used to evaluate the IHC scores.

Statistical analysis

The data were analyzed and visualized using GraphPad Prism 8.0 (USA). Comparisons between the two groups were performed using the Student's t-test. Comparisons between multiple groups were performed using one-way ANOVA. Each experiment was repeated at least three times independently. The data were expressed as average ± SD, (** P < 0.01).


   Results Top


EMT is closely involved in TDCA-induced HIBEC fibrosis

The previous studies have shown that the fibrosis of the bile duct is a critical event during the occurrence and progression of HL.[8] Here, we used HIBEC as a research cell model, and logarithmic growth phase cells were chosen for the experiment. TDCA was used to induce the fibrosis of HIBEC to mimic the early stage of HL. The expressions of E-cadherin and Vimentin in HIBEC after TDCA stimulation were evaluated by immunofluorescence staining. The fluorescence intensity of E-cadherin was significantly reduced in TDCA-induced HIBEC, while that of Vimentin was significantly enhanced, suggesting the EMT process was accelerated in TDCA-induced cells [Figure 1]a and [Figure 1]b. Western blot also revealed a substantial reduction expression in E-cadherin, whereas Vimentin was significantly increased in TDCA-induced HIBEC [Figure 1]c. The results showed that EMT is integrally involved in the TDCA-induced HIBEC fibrosis. Fibrotic HIBEC lost epithelial features and acquired a mesenchymal phenotype.
Figure 1: EMT is closely involved in TDCA-induced HIBEC fibrosis. The expression of (a) E-cadherin and (b) Vimentin in HIBEC specimens was detected by immunofluorescence assay. (c) Western blot analysis of E-cadherin and Vimentin in TDCA-induced HIBEC. The data are expressed as average ± SD, (** P < 0.01)

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S. miltiorrhiza inhibits the fibrosis of HIBEC in a dose-dependent manner

To explore the effect of S. miltiorrhiza on HIBEC fibrosis, we first performed CCK-8 assay to evaluate the effect of S. miltiorrhiza on HIBEC cell viability at different concentrations. The results showed that S. miltiorrhiza were non-toxic to HIBEC within the concentration of 10,000 μM for 1 day [Figure 2]a. Similarly, this non-toxicity did not alter when the treatment was extended for 3 days [Figure 2]b. Next, TDCA-stimulated HIBEC was treated with different concentrations of S. miltiorrhiza for 24 h. Six different groups were set according to the screening concentration of S. miltiorrhiza. Western blot analysis showed that the expression of E-cadherin in TDCA-stimulated HIBEC was significantly increased in the range of 0 to 500 μM S. miltiorrhiza, while it stopped in the range of increasing from 500 to 10,000 μM S. miltiorrhiza. Notably, the expression of Vimentin showed the opposite trend to that of E-cadherin [Figure 2]c. Together, our data suggested that within a concentration of 500 μM, S. miltiorrhiza inhibited the fibrosis of HIBEC in a dose-dependent manner. This response reached saturation after the concentration of S. miltiorrhiza was greater than 500 μM.
Figure 2: S. miltiorrhiza inhibits the fibrosis of HIBEC in a dose-dependent manner. CCK-8 analysis of cell viability at (a) 24 h or (b) 72 h of HIBEC treated with six different dosages of S. miltiorrhiza (0, 100, 200, 500, 1,000, and 10,000 μM). (c) Western blot analysis of E-cadherin and Vimentin in HIBEC induced by TDCA treated with different concentrations of S. miltiorrhiza. The data are expressed as average ± SD, (** P < 0.01)

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S. miltiorrhiza alleviates the fibrosis of HIBEC by inhibiting NF-κB/MRP2 axis

The previous studies demonstrated that MRP2 is an important factor regulated by NF-κB pathway.[24] NF-κB pathway-activating is generally accompanied by overexpression of TNF, IL1B, and IL6.[11] To determine whether TDCA-induced HIBEC affects the NF-κB/MRP2 axis, we first performed RT-qPCR to detect the mRNA expression of NF-κB-related inflammatory factors, and Mrp2 of TDCA-treated HIBEC. The results indicated that the expression of TNF, IL1B, and IL6 was upregulated, while Mrp2 was downregulated in the TDCA group [Figure 3]a. Similarly, ELISA indicated that TDCA treatment significantly aggravated the levels of TNF-α, IL-1β, and IL-6, whereas the levels of MRP2 were alleviated, suggesting the NF-κB/MRP2 axis was activated in HIBEC after TDCA treatment [Figure 3]b. To explore the effect of S. miltiorrhiza on the activation of NF-κB/MRP2 axis, six different concentrations of S. miltiorrhiza were performed to treat TDCA-induced HIBEC. The RT-qPCR analysis indicated S. miltiorrhiza significantly downregulated the mRNA expression of TNF, IL1B, and IL6, while Mrp2 was upregulated [Figure 3]c. We also found downregulated NF-κB-related inflammatory factors levels and upregulated MRP2 levels in the supernatant after S. miltiorrhiza treatment using ELISA [Figure 3]d. Notably, S. miltiorrhiza inhibited the NF-κB/MRP2 axis under a certain dose within a concentration of 500 μM. Nevertheless, the inhibitory effect reached saturation when the concentration of S. miltiorrhiza was over 500 μM. These findings indicated that S. miltiorrhiza alleviates HIBEC fibrosis progression through the NF-κB/MRP2 axis.
Figure 3: S. miltiorrhiza alleviates the fibrosis of HIBEC by inhibiting the NF-κB/MRP2 axis. (a) RT-qPCR analysis of TNF, IL1B, IL6, and Mrp2 in HIBEC and HIBEC induced by TDCA. (b) TNF-α, IL-1β, IL-6, and MRP2 levels in HIBEC and HIBEC induced by TDCA were evaluated by ELISA. (c) RT-qPCR analysis of TNF, IL1B, IL6, and MRP2 in HIBEC induced by TDCA treated with different dosages of S. miltiorrhiza. (d) TNF-α, IL-1β, IL-6, and MRP2 levels in HIBEC induced by TDCA treated with different dosages of S. miltiorrhiza were evaluated by ELISA. The data are expressed as average ± SD, (** P < 0.01)

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Regulation mechanism of Sal B on HIBEC fibrosis

To explore the main components of S. miltiorrhiza that attenuated the fibrosis of HIBEC, we investigated several major tanshinones (Tan I, Tan IIA, CTS) and salvianolic acids (Sal A, Sal B, Sal C) ingredients of S. miltiorrhiza. Each of the main components was used to treat HIBEC in the presence of TDCA for 24 h, then the expression levels of inflammatory factors and MRP2 in HIBEC were detected using the RT-qPCR analysis [Figure 4] and [Figure 5]. The results showed that treatment of Tan I and Sal C had little effect on the TNF, IL1B, IL6, and Mrp2 expression [Figure 4]a and [Figure 5]c. Treatment of Tan IIA and CTS merely suppressed the expression of TNF [Figure 4]b and [Figure 4]c. Treatment of Sal A downregulated the expression of TNF and IL1B but had little effect on IL6 and Mrp2 [Figure 5]a. Interestingly, Sal B significantly impaired TNF, IL1B, and IL6 expression levels and elevated Mrp2 expression levels. Sal B significantly increased the Mrp2 expression level and decreased the expression levels of TNF, IL1B, and IL6 [Figure 5]b. Besides, the influence of Sal B on Mrp2 and its downstream molecules showed a dose-dependent manner within 500 μM and reached saturation when the concentration exceeded 500 μM. We also performed ELISA to detect the expression concentrations of inflammatory factors and MRP2 in cell culture supernatant after treatment with each of the main components. The results were consistent with the RT-qPCR analysis, suggesting that Sal B significantly upregulated the levels of the Mrp2 via inhibited the activation NF-κB pathway [Figure 6] and [Figure 7]. Together, these results confirmed that Sal B acted as a main functional component of S. miltiorrhiza in delaying fibrosis through NF-KB/MRP2 axis under a certain dose.
Figure 4: Effects of main tanshinones of S. miltiorrhiza on the NF-κB/MRP2 axis. RT-qPCR analysis of TNF, IL1B, IL6, and Mrp2 in HIBEC and HIBEC induced by TDCA treated with different dosages of (a) Tan I, (b) Tan IIA, (c) CTS. The data are expressed as average ± SD, (** P < 0.01)

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Figure 5: Effects of main salvianolic acids of S. miltiorrhiza on the NF-κB/MRP2 axis. RT-qPCR analysis of TNF, IL1B, IL6, and Mrp2 in HIBEC and HIBEC induced by TDCA treated with different dosages of (a) Sal A, (b) Sal B, (c) Sal C. The data are expressed as average ± SD, (** P < 0.01)

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Figure 6: Effects of main tanshinones of S. miltiorrhiza on NF-κB/MRP2 axis. TNF-α, IL-1β, IL-6, and MRP2 levels in HIBEC induced by TDCA treated with different dosages of (a) Tan I, (b) Tan IIA, (c) CTS were evaluated by ELISA. The data are expressed as average ± SD, (** P < 0.01)

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Figure 7: Effects of main salvianolic acids of S. miltiorrhiza on the NF-κB/MRP2 axis. TNF-α, IL-1β, IL-6, and MRP2 levels in HIBEC induced by TDCA treated with different dosages of (a) Sal A, (b) Sal B, (c) Sal C were evaluated by ELISA. The data are expressed as average ± SD, (** P < 0.01)

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Sal B-rich diet alleviates the fibrosis of hepatic bile ducts in SD rats

To further confirm the role of Sal B on bile duct cells in vivo, 10 male rats were randomly arranged with LD or LD with Sal B (LD + Sal B) after partial ligation of the common bile ducts. After 4 weeks of treatment with a different component diet, we performed histological analysis to assess the fibrotic degree of intrahepatic bile ducts. The findings revealed that Sal B markedly reduces the infiltration of inflammatory cells in the portal vein and the proliferation of fibrous tissue. In addition, the biliary dilation and necrosis were alleviated after treatment with Sal B [Figure 8]a. Furthermore, IHC staining and semiquantitative analysis revealed that treatment of Sal B significantly increased E-cadherin levels and decreased Vimentin levels, suggesting the decreased fibrotic degree of the intrahepatic bile duct [Figure 8]b. Collectively, these findings suggested that Sal B prevented HL by inhibiting intrahepatic bile duct fibrosis.
Figure 8: Sal B-rich diet alleviates the fibrosis of hepatic bile ducts in SD rats. Histopathological evaluation of HL in SD rats. (a) The histopathological assessment (hematoxylin-eosin staining) was performed in both the control group (LD) and the Sal B group (LD + Sal B). (b) Immunofluorescence assays evaluated the levels of E-cadherin and Vimentin expression in the control group (LD) and Sal B group (LD + Sal B). The data are expressed as average ± SD, (** P < 0.01)

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   Discussion Top


Biliary tract obstruction, biliary tract infection, and hepatic parenchyma destruction are the basic pathological changes of HL, and bile duct fibrosis is the key step in causing these basic pathological changes.[25],[26] EMT is the transformation of epithelial cells to mesenchymal cells, manifested by the loss of epithelial marker E-cadherin and the acquisition of mesenchymal marker Vimentin.[27] As a transient and dynamic procedure regulated by NF-κB, Wnt, ErbB, and other cellular signaling pathways, EMT is closely attached to chronic inflammation, cancer metastasis, and fibrotic diseases.[28],[29] Besides, cholestasis has also been revealed to stimulate EMT processes in biliary epithelial cells.[30] As one of the ABC transporters, MRP2 contributes to the clearance of both endogenous and exogenous toxic compounds in bile. Furthermore, current studies suggest that low MRP2 expression, which is associated with NF-κB activation, contributes to cholestasis.[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31] However, our research provided novel evidence to support the low expression of MRP2 in fibrotic intrahepatic biliary epithelial cells, which is also associated with NF-κB activation.

S. miltiorrhiza is considered a kind of TCM with high medicinal value for the reason of its ability involved in the treatment of multiple diseases. Its main components are composed of tanshinone and salvianolic acid. Recently, S. miltiorrhiza has been reported to treat a variety of cardiovascular diseases, cirrhosis, and modulate the EMT process.[32],[33],[34],[35] Meanwhile, S. miltiorrhiza is also an ingredient of compound preparations used in the therapy of intrahepatic bile duct stones and participates in the treatment of gallstones and HL. S. miltiorrhiza has the function of anti-bile duct cell fibrosis and inhibiting primary bile duct gallstones.[36] Further studies have shown that different components of S. miltiorrhiza are involved in treating multiple diseases. Tan IIA has been revealed to improve myocardial function by activating AMPK-mTOR signaling pathway and Sal A strengthens the transfer of anti-tumor drugs to brain tumor tissues via PKB/Src/Cav-1 axis.[37],[38] The previous studies have demonstrated that Sal B can attenuate EMT progression by activating Sirt1-mediated autophagy or modulating TGF-β1/Smads pathway.[39],[40] In our study, we found that Sal B reduces HL by inhibiting bile duct fibrosis caused by cholestasis.


   Conclusion Top


In our research, we first found that the NF-κB signaling pathway was activated in fibrotic HIBEC, while MRP2 was inhibited. We demonstrated that compared with other ingredients of S. miltiorrhiza, Sal B has the effect of inhibiting TDCA-induced fibrotic HIBEC. Sal B was found to have a significant effect on the inhibition of HL via the NF-κB/MRP2 axis. Interestingly, Sal B upregulated MRP2 via inhibiting the NF-κB signaling pathway. Moreover, a diet rich in Sal B could significantly prevent HL in SD rats. In summary, our results suggested that supplementary Sal B may have potential therapeutic value for HL.

Acknowledgements

We greatly appreciate the support from the Harrison International Peace Hospital.

Ethics approval and consent to participate

The present study was approved by the Institutional Ethics Committee of China Harrison International Peace Hospital. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki.

Authors' contribution

Hao Yao designed study, analyzed data, wrote the initial draft of manuscript and revised manuscript. Wenpin Xu performed experiments. Zhaoming Liu performed experiments and contributed to the revision of manuscript. Dawei Ma prepared the experiment resources. Hongbin Bao provided supervised the progress of study, provided experimental platform and financial support.

Financial support and sponsorship

This study was supported by Hebei Province 2020 Medical Science Research Project (No. 20200401).

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Catena M, Aldrighetti L, Finazzi R, Arzu G, Arru M, Pulitano C, et al. Treatment of non-endemic hepatolithiasis in a Western country. The role of hepatic resection. Ann R Coll Surg Engl 2006;88:383-9.  Back to cited text no. 1
    
2.
Jarufe N, Figueroa E, Munoz C, Moisan F, Varas J, Valbuena JR, et al. Anatomic hepatectomy as a definitive treatment for hepatolithiasis: A cohort study. HPB (Oxford) 2012;14:604-10.  Back to cited text no. 2
    
3.
Kim HJ, Kim JS, Joo MK, Lee BJ, Kim JH, Yeon JE, et al. Hepatolithiasis and intrahepatic cholangiocarcinoma: A review. World J Gastroenterol 2015;21:13418-31.  Back to cited text no. 3
    
4.
Jiang H, Jiang O, Xia X, Su S, Li B. Laparoscopic versus open hepatectomy approach for regional hepatolithiasis: A meta-analysis. J Chin Med Assoc 2018;81:429-36.  Back to cited text no. 4
    
5.
Nakayama F, Koga A, Ichimiya H, Todo S, Shen K, Guo RX, et al. Hepatolithiasis in East Asia: Comparison between Japan and China. J Gastroenterol Hepatol 1991;6:155-8.  Back to cited text no. 5
    
6.
Al-Sukhni W, Gallinger S, Pratzer A, Wei A, Ho CS, Kortan P, et al. Recurrent pyogenic cholangitis with hepatolithiasis--The role of surgical therapy in North America. J Gastrointest Surg 2008;12:496-503.  Back to cited text no. 6
    
7.
Alvaro D. Gallstones: Bad company for the steatotic liver. Gastroenterology 2017;152:1284-6.  Back to cited text no. 7
    
8.
Zhao L, Yang R, Cheng L, Wang M, Jiang Y, Wang S. Epithelial-mesenchymal transitions of bile duct epithelial cells in primary hepatolithiasis. J Korean Med Sci 2010;25:1066-70.  Back to cited text no. 8
    
9.
Tang S, Jiang X, Wu L, Chen S, Chen L, Jiang J, et al. Toll-like receptor 4 shRNA attenuates lipopolysaccharide-induced epithelial-mesenchymal transition of intrahepatic biliary epithelial cells in rats. Biomed Pharmacother 2018;107:1210-7.  Back to cited text no. 9
    
10.
Liu H, Dong F, Li G, Niu M, Zhang C, Han Y, et al. Liuweiwuling tablets attenuate BDL-induced hepatic fibrosis via modulation of TGF-beta/Smad and NF-kappaB signaling pathways. J Ethnopharmacol 2018;210:232-41.  Back to cited text no. 10
    
11.
Wang B, Zhao KL, Hu WJ, Zuo T, Ding YM, Wang WX. Macrophage migration inhibitor promoted the intrahepatic bile duct injury in rats with severe acute pancreatitis. Dig Dis Sci 2019;64:759-72.  Back to cited text no. 11
    
12.
Pan WZ, Du J, Zhang LY, Ma JH. The roles of NF-kB in the development of lung injury after one-lung ventilation. Eur Rev Med Pharmacol Sci 2018;22:7414-22.  Back to cited text no. 12
    
13.
Yang W, Liu L, Li C, Luo N, Chen R, Li L, et al. TRIM52 plays an oncogenic role in ovarian cancer associated with NF-kB pathway. Cell Death Dis 2018;9:908.  Back to cited text no. 13
    
14.
Yoshimatsu Y, Wakabayashi I, Kimuro S, Takahashi N, Takahashi K, Kobayashi M, et al. TNF-alpha enhances TGF-beta-induced endothelial-to-mesenchymal transition via TGF-beta signal augmentation. Cancer Sci 2020;111:2385-99.  Back to cited text no. 14
    
15.
Jetter A, Kullak-Ublick GA. Drugs and hepatic transporters: A review. Pharmacol Res 2020;154:104234.  Back to cited text no. 15
    
16.
Marrone J, Tocchetti GN, Danielli M, Mottino AD, Marinelli RA. Improved hepatic MRP2/ABCC2 transport activity in LPS-induced cholestasis by aquaporin-1 gene transfer. Biochimie 2019;165:179-82.  Back to cited text no. 16
    
17.
Chai J, Cai SY, Liu X, Lian W, Chen S, Zhang L, et al. Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis. J Hepatol 2015;63:1440-8.  Back to cited text no. 17
    
18.
Forsgren MF, Nasr P, Karlsson M, Dahlstrom N, Noren B, Ignatova S, et al. Biomarkers of liver fibrosis: Prospective comparison of multimodal magnetic resonance, serum algorithms and transient elastography. Scand J Gastroenterol 2020;55:848-59.  Back to cited text no. 18
    
19.
Balasubramaniyan N, Ananthanarayanan M, Suchy FJ. Nuclear factor-kappaB regulates the expression of multiple genes encoding liver transport proteins. Am J Physiol Gastrointest Liver Physiol 2016;310:G618-28.  Back to cited text no. 19
    
20.
Jia Q, Zhu R, Tian Y, Chen B, Li R, Li L, et al. Salvia miltiorrhiza in diabetes: A review of its pharmacology, phytochemistry, and safety. Phytomedicine 2019;58:152871.  Back to cited text no. 20
    
21.
Yang W, Ju JH, Jeon MJ, Han X, Shin I. Danshen (Salvia miltiorrhiza) extract inhibits proliferation of breast cancer cells via modulation of Akt activity and p27 level. Phytother Res 2010;24:198-204.  Back to cited text no. 21
    
22.
Zhang Q, Liu X, Yan L, Zhao R, An J, Liu C, et al. Danshen extract (Salvia miltiorrhiza Bunge) attenuate spinal cord injury in a rat model: A metabolomic approach for the mechanism study. Phytomedicine 2019;62:152966.  Back to cited text no. 22
    
23.
Su CY, Ming QL, Rahman K, Han T, Qin LP. Salvia miltiorrhiza: Traditional medicinal uses, chemistry, and pharmacology. Chin J Nat Med 2015;13:163-82.  Back to cited text no. 23
    
24.
Wang Z, Sun X, Feng Y, Wang Y, Zhang L, Wang Y, et al. Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-kappaB-Nrf2 signaling in colorectal cancer cell. Phytomedicine 2021;82:153414.  Back to cited text no. 24
    
25.
Lammert F, Gurusamy K, Ko CW, Miquel JF, Mendez-Sanchez N, Portincasa P, et al. Gallstones. Nat Rev Dis Primers 2016;2:16024.  Back to cited text no. 25
    
26.
Banales JM, Huebert RC, Karlsen T, Strazzabosco M, LaRusso NF, Gores GJ. Cholangiocyte pathobiology. Nat Rev Gastroenterol Hepatol 2019;16:269-81.  Back to cited text no. 26
    
27.
Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 2014;15:178-96.  Back to cited text no. 27
    
28.
Wang M, Ren D, Guo W, Huang S, Wang Z, Li Q, et al. N-cadherin promotes epithelial-mesenchymal transition and cancer stem cell-like traits via ErbB signaling in prostate cancer cells. Int J Oncol 2016;48:595-606.  Back to cited text no. 28
    
29.
Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP. Stabilization of snail by NF-kappaB is required for inflammation-induced cell migration and invasion. Cancer Cell 2009;15:416-28.  Back to cited text no. 29
    
30.
Omenetti A, Porrello A, Jung Y, Yang L, Popov Y, Choi SS, et al. Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans. J Clin Invest 2008;118:3331-42.  Back to cited text no. 30
    
31.
Vollrath V, Wielandt AM, Iruretagoyena M, Chianale J. Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene. Biochem J 2006;395:599-609.  Back to cited text no. 31
    
32.
Avila-Carrasco L, Majano P, Sanchez-Tomero JA, Selgas R, Lopez-Cabrera M, Aguilera A, et al. Natural Plants Compounds as Modulators of Epithelial-to-Mesenchymal Transition. Front Pharmacol 2019;10:715.  Back to cited text no. 32
    
33.
Li CL, Liu B, Wang ZY, Xie F, Qiao W, Cheng J, et al. Salvianolic acid B improves myocardial function in diabetic cardiomyopathy by suppressing IGFBP3. J Mol Cell Cardiol 2020;139:98-112.  Back to cited text no. 33
    
34.
Sun C, Su S, Zhu Y, Guo J, Guo S, Qian D, et al. Salvia miltiorrhiza stem-leaf active components of salvianolic acids and flavonoids improved the hemorheological disorder and vascular endothelial function on microcirculation dysfunction rats. Phytother Res 2020;34:1704-20.  Back to cited text no. 34
    
35.
Wang R, Song F, Li S, Wu B, Gu Y, Yuan Y. Salvianolic acid A attenuates CCl4-induced liver fibrosis by regulating the PI3K/AKT/mTOR, Bcl-2/Bax and caspase-3/cleaved caspase-3 signaling pathways. Drug Des Devel Ther. 2019;13:1889-900.  Back to cited text no. 35
    
36.
Xiao Z, Liu W, Mu YP, Zhang H, Wang XN, Zhao CQ, et al. pharmacological effects of salvianolic acid b against oxidative damage. Front Pharmacol 2020;11:572373.  Back to cited text no. 36
    
37.
Zhang C, Pan Y, Cai R, Guo S, Zhang X, Xue Y, et al. Salvianolic acid A increases the accumulation of doxorubicin in brain tumors through Caveolae endocytosis. Neuropharmacology 2020;167:107980.  Back to cited text no. 37
    
38.
Zhang X, Wang Q, Wang X, Chen X, Shao M, Zhang Q, et al. Tanshinone IIA protects against heart failure post-myocardial infarction via AMPKs/mTOR-dependent autophagy pathway. Biomed Pharmacother 2019;112:108599.  Back to cited text no. 38
    
39.
He Y, Lu R, Wu J, Pang Y, Li J, Chen J, et al. Salvianolic acid B attenuates epithelial-mesenchymal transition in renal fibrosis rats through activating Sirt1-mediated autophagy. Biomed Pharmacother 2020;128:110241.  Back to cited text no. 39
    
40.
Wang QL, Tao YY, Yuan JL, Shen L, Liu CH. Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-beta1 signal transduction pathway in vivo and in vitro. BMC Cell Biol 2010;11:31.  Back to cited text no. 40
    


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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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