Upregulation of granzyme B and C-X3-C motif receptor 1 in circulating plasmablasts was negatively regulated by Notch signal in patients with systemic lupus erythematosus.

As one molecule related to cytotoxicity, surface expression of C-X3-C motif receptor 1 (CX3CR1) was highly correlated with intracellular granzyme B (GZMB) in NK and cytolytic T cells. However, the expression of CX3CR1 and GZMB in B cells has not been clarified, and their clinical significance in systemic lupus erythematosus (SLE) remains unclear. This study aimed to clarify the changes and clinical significance of peripheral blood B cells expressing GZMB and/or CX3CR1 in SLE. Peripheral blood was collected from 39 SLE patients and 48 healthy controls. We found that GZMB and CX3CR1 expression varied in different B-cell subsets, with plasmablasts possessing the highest positive percentages, consistent with bioinformatics prediction. GZMB+ and CX3CR1+ percentages in circulating B cells and plasmablasts were increased in SLE patients. CX3CR1 was upregulated on B cells after in vitro stimulation. Notch intracellular domain (NICD) expression was significantly decreased in plasmablasts of SLE patients and CX3CR1 in plasmablasts was downregulated with the addition of JAG1. In conclusion, GZMB and CX3CR1 were increased in B cells and in plasmablasts of SLE patients and CX3CR1 was negatively regulated by Notch signal in plasmablasts, which may be involved in SLE pathogenesis.

SIT1 identifies circulating hypoactive T cells with elevated cytotoxic molecule secretion in systemic lupus erythematosus patients.

This study aims to elucidate the expression and functionality of SIT1 in circulating CD8/CD4 + T cells in humans and to delineate its significance in systemic lupus erythematosus (SLE) patients. We employed multiparametric flow cytometry to investigate the expression of SIT1 in circulating CD8/CD4 + T cells and their respective subsets, comparing healthy controls (HCs) with SLE patients. Furthermore, we assessed the levels of granzyme B, perforin, IL-17, and IFN-γ in SIT1-related CD8/CD4 + T cells from both HCs and SLE patients, both before and after PMA stimulation. Clinically, we conducted receiver operating characteristic curve analysis and correlation analysis to evaluate the clinical relevance of SIT1-related CD8/CD4 + T cells in SLE patients. SIT1 exhibited higher expression in CD4 + T cells, with SIT1 - T cells demonstrating elevated levels of granzyme B, perforin, and IFN-γ compared to SIT1 + T cells. PMA-stimulated T cells exhibited reduced SIT1 expression compared to unstimulated T cells. SLE patients displayed increased SIT1 + proportions in CD8 + T cells and decreased SIT1 + CD4 + T cell numbers. Additionally, SIT1 + cells in SLE patients exhibited significantly higher levels of granzyme B and perforin compared to HCs. SIT1 + cells demonstrated significant associations with clinical indicators in SLE patients, with indicators related to SIT1 proving valuable in the diagnosis of SLE patients. SIT1 is inversely correlated with T cell activation. In SLE patients, SIT1 expression is altered in T cells concomitant with an augmented secretion of cytotoxic molecules. This upregulation may contribute to the pathogenesis of SLE and enhance its diagnostic potential.

In vitro digestion mimicking conditions in adults and elderly reveals digestive characteristics of pork from different cooking ways.

This study aimed to investigate the impact of three cooking ways (sous vide (SV), frying (FR) and roasting (RO)) on pork protein digestion characteristics under conditions simulating healthy adult (control, C) and elderly individuals with achlorhydria (EA). Changes in degree of hydrolysis (DH), SDS-PAGE profiles, zeta potential, particle size and secondary structure during digestion were evaluated. Our results revealed the EA condition markedly affected the protein digestion process of pork with different cooking ways. The DH values of SV (25.62%), FR (21.38%) and RO (19.40%) under the EA condition were significantly lower than those of under the control condition (38.32%, 33.00% and 30.86%, respectively). Moreover, differences were also observed among three cooking ways under the EA condition. For a given cooking way, the differences between control and EA conditions gradually diminished from the gastric to the intestinal phase. Under a certain digestion condition, SV maintained the highest degree of digestion throughout the process, particularly under the EA condition. Therefore, we conclude that pork cooked by sous vide is more recommendable for the elderly considering protein digestibility.

Differential GPR56 Expression in T Cell Subpopulations for Early-Stage Lung Adenocarcinoma Patient Identification.

OBJECTIVE: This study aimed to investigate the expression of GPR56 in the T cells of early-stage lung adenocarcinoma (LUAD) patients and clarify its diagnostic significance. METHODS: Blood samples were collected from 32 patients with stage IA LUAD and 31 healthy controls. GPR56 and perforin were analysed in circulating T-cell subsets by flow cytometry. In addition, a correlation between perforin and GPR56 expression was detected. Changes in GPR56+ cells in early LUAD patients were analysed, and the diagnostic significance of GPR56+ T cells for early LUAD was studied by receiver operating characteristic (ROC) curve analysis. RESULTS: The expression of GPR56 in CD8+ T cells from early-stage LUAD patients was significantly greater than that in CD4+ T cells. The percentage of perforin-positive GPR56+ cells in early-stage LUAD patients was high. GPR56 levels in the T cells of LUAD patients were significantly lower than those in healthy controls. ROC analysis revealed that the area under the curve for the percentage of GPR56-positive CD8+ TEMRA cells to distinguish early-stage LUAD patients from healthy individuals- reached 0.7978. CONCLUSION: The decreased expression of GPR56 in the peripheral blood of early-stage LUAD patients correlated with perforin levels, reflecting compromised antitumor immunity and aiding early-stage LUAD screening.

Zirconium-Catalyzed Synthesis of 6-(Trifluoromethyl)-2H-pyran-2-ones via C-C Bond Cleavage.

A strategy for the annulation reaction of alkynones with ethyl 4,4,4-trifluoro-3-oxobutanoate through C-C bond cleavage is described. The zirconium-catalyzed transformation provides access to a wide range of structurally diverse 6-(trifluoromethyl)-2H-pyran-2-ones in moderate to good yields, utilizing Na2CO3 as a base. Further transformations into trifluoromethylated arene derivatives have been demonstrated as well. Furthermore, plausible reaction pathways are proposed by conducting various control experiments and isolating a ß-diketone intermediate (X-ray) containing an intramolecular hydrogen bond.

FCRL3 expression is upregulated and closely correlates with TIGIT expression in regulatory T cells of patients with systemic lupus erythematosus.

Using data from single-cell RNA sequencing and flow cytometry, we initially examined the expression of FCRL3, finding it to be elevated and positively associated with TIGIT expression in the regulatory T cells of patients with systemic lupus erythematosus. This also suggests that the co-expression of FCRL3 and TIGIT warrants further attention.

Group 1 innate lymphoid cell activation via recognition of NKG2D and liver resident macrophage MULT-1: Collaborated roles in triptolide induced hepatic immunotoxicity in mice.

Triptolide (TP) is the major bioactive component of traditional Chinese medicine Tripterygium wilfordii Hook. F., a traditional Chinese medicinal plant categorized within the Tripterygium genus of the Celastraceae family. It is recognized for its therapeutic potential in addressing a multitude of diseases. Nonetheless, TP is known to exhibit multi-organ toxicity, notably hepatotoxicity, which poses a significant concern for the well-being of patients undergoing treatment. The precise mechanisms responsible for TP-induced hepatotoxicity remain unresolved. In our previous investigation, it was determined that TP induces heightened hepatic responsiveness to exogenous lipopolysaccharide (LPS). Additionally, natural killer (NK) cells were identified as a crucial effector responsible for mediating hepatocellular damage in this context. However, associated activating receptors and the underlying mechanisms governing NK cell represented innate lymphoid cell (ILC) activation remained subjects of inquiry and were not yet investigated. Herein, activating receptor Killer cell lectin like receptor K1 (NKG2D) of group 1 ILCs was specifically upregulated in TP- and LPS-induced acute liver failure (ALF), and in vivo blockade of NKG2D significantly reduced group 1 ILC mediated cytotoxicity and mitigated TP- and LPS-induced ALF. NKG2D ligand UL16-binding protein-like transcript 1 (MULT-1) was found upregulated in liver resident macrophages (LRMs) after TP administration, and LRMs did exhibit NK cell activating effect. Furthermore, M1 polarization of LRMs cells was observed, along with an elevation in intracellular tumor necrosis factor (TNF)-α levels. In vivo neutralization of TNF-α significantly alleviated TP- and LPS-induced ALF. In conclusion, the collaborative role of group 1 ILCs and LRMs in mediating hepatotoxicity was confirmed in TP- and LPS-induced ALF. TP-induced MULT-1 expression in LRMs was the crucial mechanism in the activation of group 1 ILCs via MULT-1-NKG2D signal upon LPS stimulation, emphasizing the importance of infection control after TP administration.

Helios characterized circulating follicular helper T cells with enhanced functional phenotypes and was increased in patients with systemic lupus erythematosus.

Helios was related to the immunosuppressive capacity and stability of regulatory T cells. However, the significance of Helios in follicular help T (TFH) and follicular regulatory T (TFR) cells is unclear. This research aimed to clarify the significance of Helios (IKZF2) in TFH and TFR cells and its clinical value in systemic lupus erythematosus (SLE). IKZF2 mRNA in different cell subsets was analyzed. Helios+ percentages in TFH and TFR cells were identified in the peripheral blood of 75 SLE patients and 62 HCs (healthy controls). PD-1 and ICOS expression were compared between Helios+ and Helios- cells. The capacity of TFH cells to secrete IL-21 and TFR cells to secrete IL-10 was measured. Correlation analysis and receiver operating characteristic (ROC) curve analysis were conducted to assess the clinical significance of Helios-related TFH and TFR cell subsets in SLE. There was Helios expression in TFH and TFR cells. PD-1 and ICOS were lower in Helios+ TFR than in Helios- TFR. ICOS was increased in Helios+ TFH cells compared with Helios- TFH cells, and ICOS in Helios+ TFH cells was downregulated in SLE. Helios+ TFH cells secreted more IL-21 than Helios- TFH cells, and Helios+ TFH cells from SLE patients had a stronger IL-21 secretion than HCs. Helios+ TFH percentages were negatively correlated with C3 and C4 and positively related to CRP and SLEDAI, and the AUC of Helios+ TFH to distinguish SLE from HC was 0.7959. Helios characterizes circulating TFH cells with enhanced function. Increased Helios+ TFH cells could reflect the autoimmune status of SLE.

Upregulation of CX3CR1 expression in circulating T cells of systemic lupus erythematosus patients as a reflection of autoimmune status through characterization of cytotoxic capacity.

OBJECTIVE: This study investigated CX3CR1 expression in human peripheral blood T lymphocytes and their subsets, exploring changes in SLE patients and its diagnostic potential. METHODS: Peripheral blood samples from 31 healthy controls and 50 SLE patients were collected. RNA-Seq data from SLE patient PBMCs were used to analyze CX3CR1 expression in T cells. Flow cytometry determined CX3CR1-expressing T lymphocyte subset proportions in SLE patients and healthy controls. Subset composition and presence of GZMB, GPR56, and perforin in CX3CR1+ T lymphocytes were analyzed. T cell-clinical indicator correlations were assessed. ROC curves explored CX3CR1's diagnostic potential for SLE. RESULTS: CX3CR1+CD8+ T cells exhibited higher GPR56, perforin, and GZMB expression than other T cell subsets. The proportion of CX3CR1+ was higher in TEMRA and lower in Tn and TCM. PMA activation reduced CX3CR1+ T cell proportions. Both RNA-Seq and flow cytometry revealed elevated CX3CR1+ T cell proportions in SLE patients. Significantly lower perforin+ and GPR56+ proportions were observed in CX3CR1+CD8+ T cells in SLE patients. CX3CR1+ T cells correlated with clinical indicators. CONCLUSION: CX3CR1+ T cells display cytotoxic features, with heightened expression in CD8+ T cells, particularly in adult SLE patients. Increased CX3CR1 expression in SLE patient T cells suggests its potential as an adjunctive diagnostic marker for SLE.

Alterations in Helios+ T cell subsets in peripheral blood of early-stage lung adenocarcinoma patients: Implications for early diagnosis.

OBJECTIVE: This study aimed to investigate the changes and significance of circulating Helios-associated T cell subsets in patients with early-stage lung adenocarcinoma (LUAD). METHODS: Blood samples were collected from 35 healthy controls and 34 patients with early-stage LUAD. Flow cytometry was used to analyze various CD4+ T cell subsets, including regulatory T(Treg) cells, follicular regulatory T(Tfr) cells, follicular helper T (Tfh) cells, and conventional T (con-T) cells. Correlation analysis was conducted to investigate the association of Helios-related subsets with clinical indicators. The ROC curve was used to explore the potential clinical value of Helios+ T cell subsets in the screening of patients with early LUAD. Fifteen of these patients were tracked after lung cancer resection and changes in Helios+ T cell subsets before and after treatment were analyzed. RESULTS: The percentage and absolute number of Tregs were up-regulated in LUAD patients while Tfh and con-T cells expressing Helios were down-regulated. Absolute counts of Tfr and con-T cells and Helios expression in Tfr and Treg decreased significantly after resection. Helios+ Tfh and con-T were negatively correlated with certain tumor markers. Areas under the curve (AUCs) of percentages and absolute counts of Helios+ Tfh, Treg, Tfr and con-T cells to distinguish early LUAD from healthy individuals were 0.7277, 0.5697, 0.5718, 0.7210 (percentages), 0.7336, 0.7378, 0.5908 and 0.7445(absolute numbers), respectively. CONCLUSION: Helios+ T cell subsets in peripheral blood of early-stage LUAD patients has changed significantly, which may be related to the pathogenesis of LUAD and could help for early diagnosis of LUAD.

Elevated Layilin-Positive Monocyte Levels in the Peripheral Blood of Patients with Systemic Lupus Erythematosus Reflect Their Autoimmune Status.

OBJECTIVE: To investigate the expression of layilin (LAYN) in human circulating monocytes and lymphocytes and its clinical significance in systemic lupus erythematosus (SLE). METHODS: Blood samples were collected from 51 SLE patients and 50 healthy controls. Flow cytometry was used to analyze LAYN in lymphocytes and monocyte subsets. Functionally characterized molecules including human HLA, CD74 and CD62L were studied in LAYN+ monocytes. A correlation analysis was conducted between LAYN-related subsets and clinical indicators of SLE such as anti-double-stranded DNA and complements levels. ROC curves were used to explore the potential clinical diagnostic value of LAYN in SLE. RESULTS: LAYN was significantly higher in monocytes than in lymphocytes and higher in CD14+CD16+ monocytes than in CD14-CD16+ and CD14+CD16- monocytes. CD74 was upregulated and CD62L was downregulated in LAYN+ monocytes compared with LAYN- monocytes. The absolute number of LAYN+ monocytes was increased in SLE patients, and the median fluorescence intensity of HLA was decreased. LAYN+ monocytes were positively correlated with complement C4, while decreased CD62L+ percentages in LAYN+ monocytes were negatively correlated with C4. The ROC analysis revealed that the area under the curve (AUCs) for CD62L+ percentages in LAYN+ monocytes, LAYN+ lymphocyte numbers, and LAYN+ monocyte numbers to distinguish SLE from healthy individuals were 0.6245, 0.6196 and 0.6173, respectively. CONCLUSION: LAYN is differentially expressed in monocytes and their subpopulations and has corresponding functional differences. Changes in LAYN expression on monocytes are associated with complement C4 levels in SLE patients. These suggest that LAYN may be involved in the pathogenesis of SLE. ABBREVIATION: ANOVA: analysis of variance; anti-dsDNA: anti-double-stranded DNA; anti-ENA: anti-extractable nuclear antigen; anti-SSA: anti-Sjogren syndrome A; anti-SSB: anti-Sjogren syndrome B; anti-U1RNP: anti-U1 ribonucleoprotein; AUC: area under the ROC curve; CBC: complete blood count; CD62L: L-selectin; CD74/Ii: MHC class II invariant chain; CD44/HCAM: homing cell adhesion molecule; cMos: classical monocytes; CRP: C-reactive protein; CXCR2: C-X-C motif chemokine receptor 2; CXCR4: C-X-C motif chemokine receptor 4; ESR: erythrocyte sedimentation rate; HCs: healthy controls; HA: hyaluronan; HLA: human leukocyte antigen; Ig: immunoglobulin; iMos: intermediate monocytes; LAYN: layilin; MFI: median fluorescence intensity; MIF: migration inhibitory factor; ncMos: nonclassical monocytes; PBMCs: peripheral blood mononuclear cells; ROC: receiver operating characteristic curve; SLE: systemic lupus erythematosus; SLEDAI, SLE disease activity index; Treg: regulatory T cells; WBCs: white blood cells.

Divergent Construction of Azaheterocycles via Alkoxyl Radical-Triggered C-C Bond Cleavage/Cyclization of N-Functionalized Acrylamides.

An array of redox-neutral alkylation/cyclization cascade reactions of N-functionalized acrylamides with cycloalkyl hydroperoxides were achieved via the alkoxyl radical-triggered C-C bond cleavage. Through adjusting the radical acceptors on the N atom, a variety of keto-alkylated chain-containing azaheterocycles, including indolo[2,1-a]isoquinolin-6(5H)-ones, quinoline-2,4-diones, and pyrido[4,3,2-gh]phenanthridines were constructed by a one-pot procedure with good yields and excellent functional group tolerance.

Optimal velocity loss threshold for inducing post activation potentiation in track and field athletes.

The aim of this study was to determine the optimal velocity loss (VL) threshold that maximises the post activation potentiation (PAP) stimulus for achieving larger and more consistent performance gains in track and field athletes. Twenty-two athletes from athletics participated in four back squat PAP tests with four different VL threshold (5%, 10%, 15% and 20% VL) at an intensity of 85%1RM. Countermovement jump (CMJ) height, power, and momentum were assessed before, and 10 s, 4, 8, 12, 16 minutes after the PAP condition. Repetitions of the squat in all the PAP conditions were also recorded. Only the 5% VL condition produced significant improvements in height (ES = 0.73, P = 0.038), peak power output (ES = 0.73, P = 0.038) and momentum (ES = 0.72, P = 0.041) of CMJ, and these changes appeared 8 minutes after the condition. The total number of repetitions during the 5% VL condition was significantly lower than that observed in the 15% (P = 0.003) and 20% VL (P < 0.001) trials. The results from this study indicate that 5%VL during the 2 sets preconditioning squat at 85%1RM was optimal for eliciting PAP in a CMJ exercise, and resulted in significant increases at the 8-min recovery period. The same squat condition also had the least number of repetitions. However, considering the efficiency in practice, athletes can also choose the rest time of 4-min, which can also achieve similar results.

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules.

BACKGROUND: Tripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PURPOSE: To identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. STUDY DESIGN: In vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. METHODS: Flow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. RESULTS: Interferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 µg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. CONCLUSION: The TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects.

Glycocholic acid aggravates liver fibrosis by promoting the up-regulation of connective tissue growth factor in hepatocytes.

AIMS: The precise role of bile acid in the progression of liver fibrosis has yet to be elucidated. In this study, common bile duct ligation was used as an in vivo mouse model for the evaluation of bile acids that promote liver connective tissue growth factor expression. MAIN METHODS: Primary rat and mice hepatocytes, as well as primary rat hepatic stellate and HepaRG cells were evaluated as in vitro models for promoting the expression of connective tissue growth factor by bile acids. KEY FINDINGS: Compared with taurochenodeoxycholic acid, glycochenodeoxycholic acid, and taurocholic acid, glycocholic acid (GCA) most strongly promoted the secretion of connective tissue growth factor in mouse primary hepatocytes, rat primary hepatocytes and HepaRGs. GCA did not directly promote the activation of hepatic stellate cells. The administration of GCA in mice with ligated bile ducts promotes the progression of liver fibrosis, which may promote the yes-associated protein of hepatocytes into the nucleus, resulting in the hepatocytes secreting more connective tissue growth factor for hepatic stellate cell activation. In conclusion, our data showed that GCA can induce the expression of connective tissue growth factor in hepatocytes by promoting the nuclear translocation of yes-associated protein, thereby activating hepatic stellate cells. SIGNIFICANCE: Our findings help to elucidate the contribution of GCA to the progression of hepatic fibrosis in cholestatic disease and aid the clinical monitoring of cholestatic liver fibrosis development.

Proteomics analysis reveals novel insights into the mechanism of hepatotoxicity induced by Tripterygium wilfordii multiglycoside in mice.

Tripterygium wilfordii multiglycoside (GTW), extracted and purified from the peeled roots of T. wilfordii Hook.f. (TwHF), is a well-known traditional Chinese medicine and applied to various autoimmune diseases clinically. However, it has been reported to cause severe liver injury. At present, the mechanism underlying GTW-induced hepatotoxicity remain poorly defined. Here, we evaluated the effects of GTW on mouse liver and elucidated the associated mechanisms via label-free proteomics combined with bioinformatics analysis. Male C57BL/6J mice were randomly divided into normal group, a low-dose GTW (70 mg/kg) group and a high-dose GTW (140 mg/kg) group. After 1-week administration, GTW dose-dependently induced hepatotoxicity. Further analysis showed that GTW could act on the intestinal immune network for IgA production pathway, which plays an important role in maintaining intestinal homeostasis and influences the crosstalk between gut and liver. Western blots confirmed that GTW could decrease pIgR protein expression in the liver and ileum, and, as a result, the secretion of IgA into gut lumen was reduced. Further validation showed that intestinal barrier integrity was impaired in GTW-treated mice, promoting bacteria transferring to the liver and triggering proinflammatory response. Our study demonstrated that gut-liver axis may play a vital part in the progression of GTW-induced hepatotoxicity, which provides guidance for basic research and clinical application of GTW.

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.

Obeticholic acid aggravates liver injury by up-regulating the liver expression of osteopontin in obstructive cholestasis.

AIMS: Obeticholic acid (OCA) was approved for the treatment of primary biliary cholangitis (PBC) patients, as it can significantly improve the level of serum alkaline phosphatase. However, OCA-induced liver injury in PBC patients puts them at risk of acute chronic liver failure, thus limiting the clinical application of OCA. Osteopontin (OPN), an extracellular cell matrix molecule, is highly induced in many cholestatic liver diseases. Herein we explored whether liver injury exacerbation by OCA was related to OPN. MAIN METHODS: Bile duct ligation (BDL) mice were treated with OCA (40 mg/kg) to evaluate its effect on liver injury and OPN involvement. Enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and other assays were used to detect OPN levels in serum and liver. Immunohistochemistry, and immunofluorescence, among other assays, were used to evaluate the extent of ductular reaction. The extent of fibrosis was also determined using various assays, such as immunohistochemistry, quantitative real-time PCR (qPCR), and hydroxyproline assays. KEY FINDINGS: OPN was overexpressed in the liver of BDL mice treated with OCA. OCA induced overexpression of OPN exacerbated ductular reaction, fibrosis, and liver inflammation, and reduced hepatocyte proliferation. SIGNIFICANCE: Upon liver injury, OCA upregulates the expression of OPN in the liver and accelerates disease progression. This mechanism helps explain the risk of liver damage associated with OCA.

Th17/Treg imbalance mediates hepatic intolerance to exogenous lipopolysaccharide and exacerbates liver injury in triptolide induced excessive immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: Triptolide (TP) is a major active ingredient and toxic component of Tripterygium wilfordii Hook F (TWHF), which exhibits multiple activities and remarkable hepatotoxicity, the latter of which limits its clinical application due to the risk of liver injury. Previous research has revealed the hepatotoxicity of TP resulting in liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. However, existing research has not elucidated the potential immune mechanism such as Th17/Treg imbalance in TP-induced hepatic excessive immune response to exogenous LPS. AIM OF THE STUDY: To investigate the role of Th17/Treg imbalance in TP-induced hepatic excessive immune response to exogenous LPS. MATERIALS AND METHODS: Mice were administered with TP, LPS, neutralization antibody and small molecule inhibitor respectively. Serum transaminase level was measured to determine the severity of liver injury. Frequencies of liver Th17 and Treg cells were analyzed by flow cytometry. Serum cytokine levels were performed by ELSIA, and mRNA levels of liver cytokine were performed by qPCR. The status of neutrophil infiltration was performed by myeloperoxidase (MPO) IHC measurement. Morphological observation of liver was performed by hematoxylin and eosin (H&E) staining. RESULTS: Mice given a single intragastric dose of TP (500 µg/kg) developed lethal fulminant hepatitis following intraperitoneal injection of LPS (0.1 mg/kg), characterized by low survival rate, severe liver injury, high levels of inflammation and neutrophil infiltration. Hepatic Th17/Treg imbalance emerged together with liver injury in these mice. Neutralization of IL-17A attenuated the liver injury and ameliorated the neutrophil infiltration. The TP-induced alteration of hepatic Th17/Treg balance was closely related to the outcome of immune-mediated acute liver injury triggered by LPS. Pretreatment with the STAT3 inhibitor AG490 effectively restored Th17/Treg balance, significantly reducing the production of IL-17A and finally attenuating the degree of liver injury. CONCLUSION: Hepatic Th17/Treg imbalance not only exacerbates TP- and LPS-induced liver injury, but also serves as an indispensable part in the mechanisms of TP-induced hepatic intolerance to exogenous endotoxin.

SEW2871 attenuates ANIT-induced hepatotoxicity by protecting liver barrier function via sphingosine 1-phosphate receptor-1-mediated AMPK signaling pathway.

Cholestatic liver injury, a group of diseases characterized with dysregulated bile acid (BA) homeostasis, was partly resulted from BA circulation disorders, which is commonly associated with the damage of hepatocyte barrier function. However, the underlying hepatocyte barrier-protective molecular mechanisms of cholestatic liver injury remain poorly understood. Interestingly, recent studies have shown that sphingosine-1-phosphate (S1P) participated in the process of cholestasis by activating its G protein-coupled receptors S1PRs, regaining the integrity of hepatocyte tight junctions (TJs). Here, we showed that SEW2871, a selective agonist of sphingosine-1-phosphate receptor 1(S1PR1), alleviated ANIT-induced TJs damage in 3D-cultured mice primary hepatocytes. Molecular mechanism studies indicated that AMPK signaling pathways was involved in TJs protection of SEW2871 in ANIT-induced hepatobiliary barrier function deficiency. AMPK antagonist compound C (CC) and agonist AICAR were all used to further identify the important role of AMPK signaling pathway in SEW2871's TJs protection of ANIT-treated mice primary hepatocytes. The in vivo data showed that SEW2871 ameliorated ANIT-induced cholestatic hepatotoxicity. Further protection mechanism research demonstrated that SEW2871 not only regained hepatocyte TJs by the upregulated S1PR1 via AMPK signaling pathway, but also recovered hepatobiliary barrier function deficiency, which was verified by the restored BA homeostasis by using of high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results revealed that the increased expression of S1PR1 induced by SEW2871 could ameliorate ANIT-induced cholestatic liver injury through improving liver barrier function via AMPK signaling and subsequently reversed the disrupted BA homeostasis. Our study provided strong evidence that S1PR1 may be a promising therapeutic approach for treating intrahepatic cholestatic liver injury. Graphical abstract.