{'fraction_non_alphanumeric': 0.034822419533851276, 'fraction_numerical': 0.0177118017018128, 'mean_word_length': 5.284510316768381, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Triptolide increases resistance to bile duct ligation-induced liver injury and fibrosis in mice by inhibiting RELB

Cholestasis is a common, chronic liver disease that may cause fibrosis and cirrhosis. Tripterygium wilfordii Hook.f (TWHF) is a species in the Euonymus family that is commonly used as a source of medicine and food in Eastern and Southern China. Triptolide (TP) is an epoxy diterpene lactone of TWHF, as well as the main active ingredient in TWHF. Here, we used a mouse model of common bile duct ligation (BDL) cholestasis, along with cultured human intrahepatic biliary epithelial cells, to explore whether TP can relieve cholestasis. Compared with the control treatment, TP at a dose of 70 or 140 μg/kg reduced the serum levels of the liver enzymes alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in mice; hematoxylin and eosin staining also showed that TP reduced necrosis in tissues. Both in vitro and in vivo analyses revealed that TP inhibited cholangiocyte proliferation by reducing the expression of RelB. Immunohistochemical staining of CK19 and Ki67, as well as measurement of Ck19 mRNA levels in hepatic tissue, revealed that TP inhibited the BDL-induced ductular reaction. Masson 3 and Sirius Red staining for hepatic hydroxyproline showed that TP alleviated BDL-induced hepatic fibrosis. Additionally, TP substantially inhibited BDL-induced hepatic inflammation. In summary, TP inhibited the BDL-induced ductular reaction by reducing the expression of RelB in cholangiocytes, thereby alleviating liver injury, fibrosis, and inflammation.


Introduction
Cholestasis can reflect either a functional defect in bile formation at the hepatocyte level or impairment in bile secretion/flow at the bile duct level (1). Cholestasis manifests as the excessive accumulation of biliary components (e.g., bile acid, cholesterol, and bilirubin) in the liver and systemic circulation. The clinical symptoms include liver injury, severe pruritus, jaundice, and fatigue; severe cases can cause acute liver failure (2). Chronic cholestasis may eventually lead to liver fibrosis and cirrhosis (3).
Ursodeoxycholic acid and chenodeoxycholic acid are the preferred drugs for treatment of cholestasis (4). However, tolerance may develop (5). Recently, obeticholic acid (a 6ethyl derivative of chenodeoxycholic acid) was approved for the treatment of primary biliary cirrhosis-in conjunction with ursodeoxycholic acid-in patients with inadequate responses to ursodeoxycholic acid; it was approved as monotherapy for patients who are unable to tolerate ursodeoxycholic acid (6)(7)(8). However, hepatic decompensation, liver failure, and death have been reported in patients with Child-Pugh B or C cirrhosis who receive doses of obeticholic acid above the recommended level. Thus, the Food and Drug Administration placed a black box warning on the obeticholic acid label for patients with decompensated liver disease. New, inexpensive therapeutic agents are needed for effective relief of cholestasis symptoms.
Traditional Chinese medicine dietary supplements alleviate various forms of liver injury including cholestasis (9)(10)(11)(12). Tripterygium wilfordii Hook.f (TWHF) is a species of Tripterygium in the Euonymus family (13); the dried root ("thunder god vine") serves as a "bitter and cold" traditional Chinese medicine (14) in Eastern and Southern China. The root is also cooked in southern China. Triptolide (TP) is an epoxy diterpene lactone of TWHF (15), as well as the principal active ingredient in TWHF (13). TP exhibits potent immunosuppressive and antiproliferative activities (16); it effectively treats rheumatoid arthritis (17), diabetic kidney disease (18), and prostate cancer (19,20). A TP dietary supplement reportedly alleviates senile osteoporosis (21), reduces stress, and increases longevity (22). We previously showed that TP was active against colon cancer (23,24). The NF-κB protein complex regulates cell survival (25), aging (26), cytokine production (27), and obesity (28); NF-κB is the principal target of TP. NF-κB transcriptional inhibition by TP can suppress inflammation (29) and tumor growth (30). Thus far, the effects of TP on cholestasis remain unknown. In this study, we used a mouse model of common bile duct ligation (BDL) to explore whether TP can effectively treat cholestasis. Our findings provide a rationale for TP as complementary medicine of the preferred drugs or alternative medicine for cholestasis.

Animal surgery procedure
Male C57BL/6J mice (6-8 weeks) were supplied by Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). The animal study was reviewed and approved by the China Pharmaceutical University Experimental Animal Ethics Committee. Mice were housed in conditions with controlled light (12 h light/dark cycle), temperature (24 ± 2 • C), and humidity (50-60%) and had adequate food and tap water. Cholestasis was induced by common bile duct ligation (BDL). Mice were anesthetized using 3% isoflurane and kept under anesthesia using 2-3% isoflurane during the entire infection procedure, where the abdominal cavity was opened from the abdominal midline. All experiments on mice were performed under the guidelines of Ethical Committee of China Pharmaceutical University. Triptolide in powder was suspended in 0.5% CMC-Na and administered to mice by gavage. The doses selected for TP in animal experiments were 70 µg/kg and 140 µg/kg (31). The common bile duct was ligated twice with a 7-0 nylon suture. The sham operation group involved the same operation, but the common bile duct was not ligated. After one-week acclimatization, the mice were then randomly separated into four groups (n = 6 per group): (1) Control mice (sham operated); (2) BDL mice; (3) BDL with TP at 70 µg/kg administration; (4) BDL with TP at 140 µg/kg administration. BDL performed at three days after TP treatment. Mice in sham and BDL group were given corresponding vehicle. After BDL the mice still were treated with TP once a day. Seven days after BDL surgery, mice were sacrificed ( Figure 1A).
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using kits from Whitman Biotech (Nanjing, China). Hepatic hydroxyproline was measured using kits from Nanjing   observe pathological changes. All slides were scanned with a NanoZoomer S60 (Hamamatsu, Japan).
Quantitative real-time polymerase chain reaction RNA from tissues and cells was extracted with TRIzol (vazyme, China). The RNA concentration was determined using Nanodrop2000 Spectrophotometers (Thermo Scientific, USA). cDNA was generated using BIO-RAD MyCyclerThermal Cycler (BIO-RAD, USA) and the HighCapacity cDNA Reverse Kit. qPCR was performed using StepOnePlus (Applied Biosystems, USA) with specific primers ( Table 1). Primers were purchased from Genescipt (China). Results were normalized using GAPDH as an internal control.

Immunoblot analysis
Protein content was analyzed by lysing tissues and cells with RIPA buffer containing protease inhibitors and the Bradford Protein Assay Kit. Western blot analysis was performed following a previously described method (32). Protein bands were detected with a Tanon 5200Muti (Tanon, China) using ECL reagents. The gray density of the protein bands was determined using ImageJ. All quantitative comparisons between samples were on the same gels/blots.   Frontiers in Nutrition 07 frontiersin.org Immunofluorescence Cells on coverslips were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized in PBS with 1% Triton for 10 min. Cells were then incubated with 5% goat serum in PBS for 1 h at room temperature before being incubated with antibodies overnight at 4 • C. The next day, cells on a round coverslip were washed three times with PBS and incubated for 1 h at room temperature with secondary Alexa antibodies and DAPI. Fluorescence images were scanned using a FV3000 (Olympus, Japan).

Statistical analysis
All data were shown as mean ± SD and at least three replicate experiments were performed in vitro and in vivo. The necrotic, Masson3 positive and Sirius Red positive area were analyzed using Image J software. Statistical significance was determined using one-way analysis of variance as appropriate (GraphPad Prism 9, GraphPad Software Inc., CA).

Triptolide alleviates bile duct ligation-induced liver injury
To explore the effects of TP on cholestasis-induced liver injury, we established a mouse model of BDL and administered two TP doses by oral gavage; such doses were previously reported to attenuate chronic kidney disease (31). A schematic of the mouse model is depicted in Figure 1A. Figure 1B shows that TP at a dose of 70 or 140 µg/kg attenuated the BDLinduced weight loss. BDL increased the serum levels of the liver enzymes alanine transaminase, aspartate aminotransferase, and alkaline phosphatase. Either dose of TP substantially reduced the levels of these enzymes (Figure 1C). Histopathological staining revealed less necrosis around the portal tract when BDL mice were treated with TP ( Figure 1D). Thus, TP effectively treated BDL-induced liver injury.

Triptolide inhibits proliferation and RelB expression in human intrahepatic biliary epithelial cells
Bile duct hyperplasia is common in patients with cholestasis; cholangiocyte proliferation and a ductular reaction contribute to the onset and progression of liver disease (32)(33)(34). Members of the NF-κB family of transcription factors act through a canonical pathway and a non-canonical pathway. Noncanonical NF-κB signaling activates predominantly p100sequestered NF-kB proteins, the most important of which is RelB (35). This protein is involved in the ductular reaction; the bile ducts of patients with primary sclerosing cholangitis and primary biliary cirrhosis exhibit increased levels of RelB. RelB and its downstream target lymphotoxin β (LTβ) affect the proliferation of bile duct epithelial cells (36). TP inhibits the expression of NF-κB proteins (37). Here, we analyzed HiBECs in vitro. We hypothesized that TP would reduce cholangiocyte proliferation by inhibiting the expression of RelB.
Growth curve analyses showed that siRNA-mediated RelB knockdown inhibited the growth of HiBECs (Figure 2A). Western blotting revealed that TP (100 nM) significantly inhibited the expression of RelB in HiBECs (Figure 2B). TP at 50 and 100 nM inhibited the growth of HiBECs ( Figure 2C); this was confirmed (for TP at 100 nM) by immunofluorescence staining of CK19 and Ki67 (proliferation markers) ( Figure 2D). Quantitative polymerase chain reaction (qPCR) analysis demonstrated that TP significantly reduced the mRNA expression levels of Relb and the downstream genes Tnfsf14 and Ltβ (Figure 2E).

Triptolide inhibits bile duct ligation-induced expression of RelB and downstream genes
Western blotting revealed that the protein level of RelB increased after BDL. Both TP doses substantially reduced the level of RelB (Figure 3A). Enzyme-linked immunosorbent assay analysis showed that BDL increased the expression of serum tumor necrosis factor superfamily member 14 (TNFSF14), whereas TP inhibited this increase ( Figure 3B). qPCR analysis of hepatic tissue showed that BDL upregulated the mRNA expression levels of Relb, Tnfsf14, and Ltβ, but these increases were inhibited by TP at a dose of 70 or 140 µg/kg ( Figure 3C).

Triptolide relieves bile duct ligation-induced bile duct hyperplasia
The above results indicated that TP inhibited the BDLinduced upregulation of Relb and downstream genes (Tnfsf14 and Ltβ) in hepatic tissue. Increased levels of RelB lead to a ductular reaction. Cytokeratin-19 (CK19) is solely expressed by cholangiocytes. Immunohistochemical analysis of CK19 revealed that BDL induced prominent bile duct hyperplasia; TP inhibited this process (Figures 4A,B). Immunohistochemical analysis of Ki67 revealed many positive cells (black arrows) in bile ducts after BDL; TP significantly reduced the numbers of these cells (Figures 4A,B), indicating that TP alleviated bile duct Frontiers in Nutrition 08 frontiersin.org hyperplasia. qPCR analysis showed that TP significantly reduced the BDL-induced upregulation of Ck19 (Figure 4C).

Triptolide relieves bile duct ligation-induced liver fibrosis
TNFSF14, which acts downstream of RelB, promotes hepatic stellate cell activation and exacerbates liver fibrosis (38). Staining with Masson 3 and Sirius Red confirmed that TP decreased collagen deposition around the portal fields in BDL mice (Figures 5A,B). α-Smooth muscle actin [also known as actin alpha 2 (ACTA2)] is a marker of hepatic stellate cell activation; immunohistochemical staining of α-smooth muscle actin decreased around the portal area ( Figure 5A). Hydroxyproline is a characteristic component of collagen; in BDL mice, the hepatic levels of hydroxyproline were substantially lower after treatment with TP at a dose of 70 or 140 µg/kg, compared with those levels in the control group ( Figure 5C). Next, we examined the expression of liver fibrosis-related genes. Col1a1 is an important collagen component, and its expression significantly increases in fibrotic tissues. Connective tissue growth factor [also known as cellular communication network factor 2 (CTGF/CCN2)] and transforming growth factor beta-1 (TGF-β1) are markers of liver fibrosis, and they both directly activate hepatic stellate cells and promote collagen deposition; BDL elevates the levels of both proteins (39,40). The mRNA expression levels of Acta2, Col1a1, Ccn2, and Tgf-β1 were TP inhibited the BDL-induced ductular reaction by reducing the expression of RelB in cholangiocytes, thereby alleviating liver injury, fibrosis, and inflammation. Created with BioRender.com. downregulated when BDL mice were treated with TP at a dose of 70 or 140 µg/kg (Figure 5D).

Triptolide relieves bile duct ligation-induced hepatic inflammation
Ductular reactions are often accompanied by inflammatory infiltrates (33,41). Therefore, we examined the effect of TP on hepatic inflammation. For this purpose, we conducted immunohistochemical staining of F4/80 (also known as mouse EGF-like module-containing mucin-like hormone receptor-like 1), which is expressed by various mature macrophages including Kupffer cells; we also performed immunohistochemical staining of the lymphocyte antigen 6 complex locus G6D (LY6G), a neutrophil-specific marker. BDL-induced enhancement of F4/80 and LY6G staining was decreased by TP at a dose of 70 or 140 µg/kg; thus, TP reduced hepatic inflammatory infiltration ( Figure 6A). The mRNA expression levels of genes encoding the inflammatory factors F4/80, interleukin-1β, and tumor necrosis factor-α were significantly reduced when BDL mice received TP at a dose of 70 or 140 µg/kg (Figure 6B). These findings indicated that TP attenuated hepatic inflammatory infiltration in BDL mice.

Discussion
We explored whether TP protected against liver injury progression in a mouse model of common BDL. TP at a dose of 70 or 140 µg/kg effectively treated BDL-induced liver injury. Liver enzyme measurement and H&E staining revealed that TP at a dose of 70 or 140 µg/kg significantly alleviated liver damage. Analysis of the liver hydroxyproline content, along with Masson 3 and Sirius Red staining, revealed that TP inhibited BDL-induced liver fibrosis. qPCR analysis of Ck19 transcripts, as well as immunohistochemical staining of CK19 and Ki67, showed that TP significantly inhibited the BDL-induced ductular reaction. TP substantially reduced hepatic inflammatory infiltration after BDL, as revealed by immunohistochemical staining of F4/80 and Ly6G, as well as the mRNA expression levels of F4/80, Il-1β, and Tnfα in hepatic tissue. In vitro analysis demonstrated that TP dramatically downregulated the protein and mRNA Frontiers in Nutrition expression levels of RelB, as well as the downstream genes Tnfsf14 and Ltβ, thereby slowing the growth of HiBECs. Assessment of protein and mRNA expression levels in hepatic tissue revealed that TP attenuated the BDL-induced upregulation of RelB and downstream genes. The serum TNFSF14 assay confirmed that TP alleviated the BDLinduced upregulation of RelB. Graphic abstract was shown in Figure 7.
Tripterygium wilfordii Hook.f (TWHF) exhibits antiinflammatory (41), anti-fertility (42), anti-colitis (43), and anti-cancer activities (44). At present, the clinical medication of TWHF is mainly used for rheumatoid arthritis, lupus and purpuric nephritis, psoriasis, erythroderma and allergic diseases. There are no clinical trials linking TWHF with cholestatic disease. However, the therapeutic window for TP is narrow; clinical applications are compromised by severe toxicities, including hepatotoxicity (37). The doses in this study were chosen because TP at a dose of 70 µg/kg substantially alleviated chronic kidney disease (31); we also used a higher dose for comparison. In our previous study, it was found that there was no obvious liver toxicity and cholestasis symptoms when TP 250 µg/kg was administered for 7 days (45). Therefore, we believe that the TP dose used in this study is a safe dose. TP-induced hepatotoxicity cannot be ignored. We previously found that TP was hepatotoxic at a dose of 500 or 600 µg/kg (46,47). Evidently, TP at a dose of 70 or 140 µg/kg significantly alleviated BDL-induced liver injury, liver fibrosis, the ductular reaction, and hepatic inflammatory infiltration. Therefore, the role of TP in cholestasis is dose-dependent. The specific mechanism may be related to the complex immune homeostasis in the liver, and the specific mechanism will be carried out in future studies. The above content shows that TP needs more research before clinical treatment of cholestasis and may require structural modification and more accurate individualized diagnosis and treatment strategies.
Cholestasis can be caused by certain drugs, abnormal hormone levels, hepatitis, and dietary habits, but the underlying mechanism remains unknown (48). Recent studies have shown that the ductular reaction plays an important role in cholestasisinduced liver fibrosis and injury. The ductular reaction, characterized by cholangiocyte hyperproliferation, is commonly observed in patients with biliary disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, or biliary atresia; this reaction is usually associated with liver fibrosis, and the extent of fibrosis is often correlated with mortality (32,34). We presume that the reaction reflects the intense local inflammatory microenvironment present in cholangiocellular cholestasis; damaged cholangiocytes proliferate to compensate for reduced biliary cell function. The ductular reaction exacerbates hepatic inflammation, inhibits liver regeneration, and promotes fibrosis (41,(49)(50)(51)(52). Previous studies showed that inhibition of the ductular reaction alleviated cholestasis-induced liver damage, inflammation, and fibrosis (53,54). Some authors reported that the level of RelB was directly related to the extent of ductular reaction. RelB and LTβ were highly expressed in cholangiocytes from patients with chronic liver diseases (hepatitis C and hepatitis B virus infections, alcoholic liver disease, non-alcoholic fatty liver disease, and autoimmune hepatitis) or cholangiopathies (primary biliary cirrhosis and primary sclerosing cholangitis) (36). The activation of RelB in cholangiocytes and hepatocytes induces the secretion of LTβ, which activates cholangiocyte RelB in both an autocrine and paracrine manner through the LTβ receptor, thereby stimulating bile duct proliferation. Thus, RelB is essential for cholangiocyte proliferation and the ductular reaction. But so far, there are no drugs targeting RelB yet in clinical, therefore, the design of new drugs for RelB has broad prospects. Accordingly, we first examined the effect of TP on cholangiocyte proliferation in vitro. As expected (37), TP (an NF-κB inhibitor) significantly reduced cell proliferation, as well as the expression of RelB and its downstream genes. In vivo analysis showed that TP significantly alleviated the BDL-induced upregulation of RelB. This finding suggested that TP reduces cholangiocyte expression of RelB, thus suppressing cholangiocyte proliferation; alleviating the BDL-induced ductular reaction; and reducing liver damage, inflammation, and fibrosis.
RelB is frequently associated with liver fibrosis. In addition to its presence in cholangiocytes, RelB is expressed in Kupffer cells (55) and hepatocytes (36). RelB-regulated TNFSF14 is presumed to promote hepatic stellate cell activation and the resulting liver fibrosis (38). Our assays of serum TNFSF14 levels indicated that TP reduced the BDL-induced increase in the TNFSF14 level; this may partly explain why TP relieves hepatic inflammation and fibrosis.
Finally, TP is a small-molecule drug and may thus act via several mechanisms. We focused on RelB. More detailed clinical and translational studies are needed to substantiate the potential utility of TP as a cholestasis treatment. Careful dosing studies are also essential.

Conclusion
Triptolide (TP) at certain doses improved cholestasis. TP may be useful in the prevention or treatment of cholestasisinduced liver injuries, fibrosis, and other inflammatory diseases.

Data availability statement
The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by China Pharmaceutical University Experimental Animal Ethics Committee.