Fibrotic Mice Research Articles

Janus kinase 2 inhibition by pacritinib as potential therapeutic target for liver fibrosis.

Janus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo . Transcriptome analyses of JAK2 in human livers and other targets of pacritinib have been shown to correlate with profibrotic factors. Although transcription of JAK2 correlated significantly with type I collagen expression and other profibrotic genes, no correlation was observed for interleukin-1 receptor-associated kinase and colony-stimulating factor 1 receptor. Pacritinib decreased gene expression of fibrosis markers in mouse primary and human-derived HSCs in vitro . Moreover, pacritinib decreased the proliferation, contraction, and migration of HSCs. C 57 BL/6J mice received ethanol in drinking water (16%) or Western diet in combination with carbon tetrachloride intoxication for 7 weeks to induce alcoholic or nonalcoholic fatty liver disease. Pacritinib significantly reduced liver fibrosis assessed by gene expression and Sirius red staining, as well as HSC activation assessed by alpha-smooth muscle actin immunostaining in fibrotic mice. Furthermore, pacritinib decreased the gene expression of hepatic steatosis markers in experimental alcoholic liver disease. Additionally, pacritinib protected against liver injury as assessed by aminotransferase levels. This study demonstrates that the JAK2 inhibitor pacritinib may be promising for the treatment of alcoholic and nonalcoholic liver fibrosis and may be therefore relevant for human pathology.

Is hepatitis-B immunization effective during chronic liver fibrosis? Investigation of secretory and cellular immune responses on an experimental model

Objective Adults with end-stage of chronic liver diseases have lower antibody titers after hepatitis-B vaccination. We have less amount of knowledge about the effect of non-viral cause chronic liver fibrosis on vaccination. In this study, we investigated the effect of non-viral chronic liver fibrosis on hepatitis B vaccine and the effect of tetanous toxoid co-administration at the level of humoral and cellular immune responses in an experimental model. Methods Hepatitis B vaccine was administered either alone or in combination with tetanus toxoid in thioacetamide-induced fibrotic BALB/c mice. Fibrosis level was determined by Knodell scoring. Anti-HBsAg, biochemical parameters, inflammatory (IL-1β, TNF-α), and anti-inflammatory (IL-10) cytokine levels were investigated in serum samples by automated systems and ELISA; respectively. Frequencies of activated lymphocytes were determined in flow cytometer. Results Antibody titers significantly decreased after immunization of fibrotic mice. However, co-administration of toxoid significantly elevated antibody titer. The percentage of CD19+CD69+ B lymphocytes was found to be lower in vaccinated fibrotic group compared to vaccinated naive group. Simultaneous administration of toxoid significantly increased the frequencies of CD4+ and CD8+ T cells expressing CD69 and CD127. Interestingly, CD19+CD5+CD1high Breg cells were significantly reduced in the group vaccinated with hepatitis B vaccine and toxoid, simultaneously. The reduction in Breg percentage did not expose a significant decrease in the level of IL-10. Conclusion Non-viral chronic liver fibrosis causes a reduction on specific antibody level after vaccination. Reduction on Breg cell frequency may have an effect on elevation of antibody level after co-administration of tetanus toxoid.

Macrophage-mediated PDGF Activation Correlates With Regenerative Outcomes Following Musculoskeletal Trauma.

Our objective was to identify macrophage subpopulations and gene signatures associated with regenerative or fibrotic healing across different musculoskeletal injury types. Subpopulations of macrophages are hypothesized to fine tune the immune response after damage, promoting either normal regenerative, or aberrant fibrotic healing. Mouse single-cell RNA sequencing data before and after injury were assembled from models of musculoskeletal injury, including regenerative and fibrotic mouse volumetric muscle loss (VML), regenerative digit tip amputation, and fibrotic heterotopic ossification. R packages Harmony , MacSpectrum , and Seurat were used for data integration, analysis, and visualizations. There was a substantial overlap between macrophages from the regenerative VML (2mm injury) and regenerative bone models, as well as a separate overlap between the fibrotic VML (3mm injury) and fibrotic bone (heterotopic ossification) models. We identified 2 fibrotic-like (FL 1 and FL 2) along with 3 regenerative-like (RL 1, RL 2, and RL 3) subpopulations of macrophages, each of which was transcriptionally distinct. We found that regenerative and fibrotic conditions had similar compositions of proinflammatory and anti-inflammatory macrophages, suggesting that macrophage polarization state did not correlate with healing outcomes. Receptor/ligand analysis of macrophage-to-mesenchymal progenitor cell crosstalk showed enhanced transforming growth factor β in fibrotic conditions and enhanced platelet-derived growth factor signaling in regenerative conditions. Characterization of macrophage subtypes could be used to predict fibrotic responses following injury and provide a therapeutic target to tune the healing microenvironment towards more regenerative conditions.

Serum extracellular vesicle proteome differentiate early stage liver fibrosis

The staging of liver fibrosis is crucial for its early detection and treatment. However, there is still lacking of effective method for the diagnosis of early liver fibrosis. Extracellular vesicles have shown great potential in disease diagnosis due to its specific role in cell-cell communication. Here we analyzed the serum extracellular vesicles proteome from fibrotic mice in a data independent acquisition manner. Totally, we identified 869 proteins commonly present in the serum samples. After systematical data filtering for differentially expressed proteins, the potential markers for the diagnosis of early liver fibrosis were obtained. Six vital differentially expressed proteins (HSPA8, EEF2, EEF1A1, PLG, SERPINC1, HRG) were further verified as a potential candidate biomarker panel, which could discriminate early-stage fibrosis mice from normal mice. This is the first time to differentiate early-stage liver fibrosis with serum extracellular vesicles proteome and its derived marker molecules.

Galectin-3 critically mediates the hepatoprotection conferred by M2-like macrophages in ACLF by inhibiting pyroptosis but not necroptosis signalling

We previously documented that M2-like macrophages exert a hepatoprotective effect in acute-on-chronic liver failure (ACLF) by inhibiting necroptosis signalling. Nevertheless, the molecular mechanism behind this hepatoprotection still needs to be further dissected. Galectin-3 (GAL3) has been reported to be critically involved in the pathogenesis of multiple liver diseases, whereas the potential role of GAL3 in ACLF remains to be explored. Herein, we hypothesised that GAL3 plays a pivotal role in the hepatoprotection conferred by M2-like macrophages in ACLF by inhibiting necroptosis. To test this hypothesis, we first assessed the expression of GAL3 in control and fibrotic mice with or without acute insult. Second, loss- and gain-of-function experiments of GAL3 were performed. Third, the correlation between GAL3 and M2-like macrophage activation was analysed, and the potential role of GAL3 in M2-like macrophage-conferred hepatoprotection was confirmed. Finally, the molecular mechanism underlying GAL3-mediated hepatoprotection was dissected. GAL3 was found to be obviously upregulated in fibrotic mice with or without acute insult but not in acutely injured mice. Depletion of GAL3 aggravated hepatic damage in fibrotic mice upon insult. Conversely, adoptive transfer of GAL3 provided normal mice enhanced resistance against acute insult. The expression of GAL3 is closely correlated with M2-like macrophage activation. Through adoptive transfer and depletion experiments, M2-like macrophages were verified to act as a major source of GAL3. Importantly, GAL3 was confirmed to hold a pivotal place in the hepatoprotection conferred by M2-like macrophages through loss- and gain-of-function experiments. Unexpectedly, the depletion and adoptive transfer of GAL3 resulted in significant differences in the expression levels of pyroptosis but not necroptosis signalling molecules. Taken together, GAL3 plays a pivotal role in the hepatoprotection conferred by M2-like macrophages in ACLF by inhibiting pyroptosis but not necroptosis signalling. Our findings provide novel insights into the pathogenesis and therapy of ACLF.

Caveolin-1 and Its Functional Peptide CSP7 Affect Silica-Induced Pulmonary Fibrosis by Regulating Fibroblast Glutaminolysis.

Exposure to silica is a cause of pulmonary fibrosis disease termed silicosis, which leads to respiratory failure and ultimately death. However, what drives fibrosis is not fully elucidated and therapeutic options remain limited. Our previous RNA-sequencing analysis showed that the expression of caveolin-1 (CAV1) was downregulated in silica-inhaled mouse lung tissues. Here, we not only verified that CAV1 was decreased in silica-induced fibrotic mouse lung tissues in both messenger RNA and protein levels, but also found that CSP7, a functional peptide of CAV1, could attenuate pulmonary fibrosis in vivo. Further in vitro experiments revealed that CAV1 reduced the expression of Yes-associated protein 1(YAP1) and affected its nuclear translocation in fibroblasts. In addition, Glutaminase 1 (GLS1), a key regulator of glutaminolysis, was identified to be a downstream effector of YAP1. CAV1 could suppress the activity of YAP1 to decrease the transcription of GLS1, thereby inhibiting fibroblast activation. Taken together, our results demonstrated that CAV1 and its functional peptide CSP7 may be potential molecules or drugs for the prevention and intervention of silicosis.

Olmesartan Improves Hepatic Sinusoidal Remodeling in Mice with Carbon Tetrachloride-Induced Liver Fibrosis.

Aim In mice with liver fibrosis produced by carbon tetrachloride (CCl4), the effects of olmesartan on intrahepatic angiogenesis and sinusoidal remodeling will be evaluated. Methods By injecting CCl4 into the peritoneal cavity, we established a mouse model of liver fibrosis. Using Sirius red and Masson trichrome staining, the extent of liver fibrosis in the animals was determined. Using immunohistochemical labeling and western blotting, the level of α-smooth muscle actin (α-SMA) expression, a characteristic of hepatic stellate cell activation, was assessed. Electron microscopy was used to determine the effect of olmesartan on hepatic sinusoidal capillarization, and immunohistochemical labeling was used to determine the expression levels of endothelial and basement membrane proteins in mouse liver tissues. Platelet-derived growth factor (PDGF), IL-10, vascular endothelial growth factor (VEGF), and angiotensin II levels in mouse serum were measured by Luminex multifactor analysis and ELISA. Olmesartan's effect on the angiotensin II type 1 receptor (AT1R) and the VEGF receptor (VEGFR) was evaluated using western blotting. Results Olmesartan reduced CCl4-induced inflammatory cell infiltration and collagen deposition to alleviate liver fibrosis. α-SMA expression was decreased, and HSC activation was inhibited in mouse liver tissues by olmesartan treatment. In addition, hepatic sinusoidal capillarization was improved under the action of olmesartan. The expression of collagen IV, fibronectin, CD31, and von Willebrand factor (VWF) in the olmesartan group was also markedly downregulated. In fibrotic mice, olmesartan medication decreased the levels of PDGF, VEGF, and angiotensin II, but it increased the level of IL-10. Moreover, olmesartan reduced the expression of VEGFR-1, VEGFR-2, and AT1R relative to CCl4-induced liver fibrosis. Conclusions In mice with CCl4-induced fibrosis, olmesartan lowers angiogenesis and improves hepatic sinusoidal remodeling, according to our findings. By acting on the angiotensin II-AT1R-VEGF axis, this is achieved.

Loss of MEN1 leads to renal fibrosis and decreases HGF-Adamts5 pathway activity via an epigenetic mechanism.

BackgroundRenal fibrosis is a serious condition that results in the development of chronic kidney diseases. The MEN1 gene is an epigenetic regulator that encodes the menin protein and its role in kidney tissue remains unclear.MethodsKidney histology was examined on paraffin sections stained with hematoxylin‐eosin staining. Masson’s trichrome staining and Sirius red staining were used to analyze renal fibrosis. Gene and protein expression were determined by quantitative real‐time PCR (qPCR) and Western blot, respectively. Immunohistochemistry staining in the kidney tissues from mice or patients was used to evaluate protein levels. Flow cytometry was used to analyze the cell cycle distributions and apoptosis. RNA‐sequencing was performed for differential expression genes in the kidney tissues of the Men1f/f and Men1∆/∆ mice. Chromatin immunoprecipitation sequencing (ChIP‐seq) was carried out for identification of menin‐ and H3K4me3‐enriched regions within the whole genome in the mouse kidney tissue. ChIP‐qPCR assays were performed for occupancy of menin and H3K4me3 at the gene promoter regions. Luciferase reporter assay was used to detect the promoter activity. The exacerbated unilateral ureteral obstruction (UUO) models in the Men1f/f and Men1∆/∆ mice were used to assess the pharmacological effects of rh‐HGF on renal fibrosis.ResultsThe expression of MEN1 is reduce in kidney tissues of fibrotic mouse and human diabetic patients and treatment with fibrotic factor results in the downregulation of MEN1 expression in renal tubular epithelial cells (RTECs). Disruption of MEN1 in RTECs leads to high expression of α‐SMA and Collagen 1, whereas MEN1 overexpression restrains epithelial‐to‐mesenchymal transition (EMT) induced by TGF‐β treatment. Conditional knockout of MEN1 resulted in chronic renal fibrosis and UUO‐induced tubulointerstitial fibrosis (TIF), which is associated with an increased induction of EMT, G2/M arrest and JNK signaling. Mechanistically, menin recruits and increases H3K4me3 at the promoter regions of hepatocyte growth factor (HGF) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (Adamts5) genes and enhances their transcriptional activation. In the UUO mice model, exogenous HGF restored the expression of Adamts5 and ameliorated renal fibrosis induced by Men1 deficiency.ConclusionsThese findings demonstrate that MEN1 is an essential antifibrotic factor in renal fibrogenesis and could be a potential target for antifibrotic therapy.

Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.

Measurement of Liver Stiffness using Atomic Force Microscopy Coupled with Polarization Microscopy.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level. To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice. A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level. To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice. A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

FRI205 - Perihepatic implantation of matrix-embedded endothelial cells reduce inflammation and collagen deposition in fibrotic mice

FRI205 - Perihepatic implantation of matrix-embedded endothelial cells reduce inflammation and collagen deposition in fibrotic mice

SAT170 - Selective induction of macrophage RNF41 stimulates liver regeneration in hepatectomized healthy and fibrotic mice

SAT170 - Selective induction of macrophage RNF41 stimulates liver regeneration in hepatectomized healthy and fibrotic mice

Hyaluronan synthase 2, a target of miR-200c, promotes carbon tetrachloride-induced acute and chronic liver inflammation via regulation of CCL3 and CCL4

Liver fibrosis occurs during wound healing after repeated liver injury and is characterized by extensive extracellular matrix deposition. We previously identified hyaluronan synthase 2 (HAS2) as a driver of liver fibrosis and hepatic stellate cell (HSC) activation. Developing strategies to suppress HSC activation is key to alleviating liver fibrosis, and HAS2 is an attractive candidate for intervention. To gain insight into the molecular function of HAS2, we investigated its posttranscriptional regulation. We found that miR-200c directly targets the 3’ untranslated regions of HAS2. Moreover, miR-200c and HAS2 were inversely expressed in fibrotic human and mouse livers. After establishing the direct interaction between miR-200c and HAS2, we investigated the functional outcome of regulating HAS2 expression in three murine models: CCl4-induced acute liver injury, CCl4-induced chronic liver fibrosis, and bile duct ligation-induced liver fibrosis. Hepatic Has2 expression was induced by acute and chronic CCl4 treatment. In contrast, miR-200c expression was decreased after CCl4 treatment. HSC-specific Has2 deletion reduced the expression of inflammatory markers and infiltration of macrophages in the models. Importantly, hyaluronidase-2 (HYAL2) but not HYAL1 was overexpressed in fibrotic human and murine livers. HYAL2 is an enzyme that can cleave the extracellular matrix component hyaluronan. We found that low-molecular-weight hyaluronan stimulated the expression of inflammatory genes. Treatment with the HA synthesis inhibitor 4-methylumbelliferone alleviated bile duct ligation-induced expression of these inflammatory markers. Collectively, our results suggest that HAS2 is negatively regulated by miR-200c and contributes to the development of acute liver injury and chronic liver inflammation via hyaluronan-mediated immune signaling.

N-Acetyl-l-tryptophan inhibits CCl4-induced hepatic fibrogenesis via regulating TGF-β1/SMAD and Hippo/YAP1 signal

BackgroundAlthough liver fibrosis is a key pathologic process in many liver diseases, therapeutic approaches for inhibiting liver fibrosis are still very limited. N-Acetyl-l-tryptophan (l-NAT) has a hepatoprotective effect via inhibiting the destruction of liver cells, enhancing cell viability and reducing the inflammation. However, the effect of l-NAT on liver fibrosis is not determined. PurposeThe present study investigated the effect of l-NAT on liver fibrosis and explored it potential molecular mechanism. MethodsTo address this concern, this study was carried out via fibrotic mice model induced by CCl4 and many approaches such as various histological staining methods, western blot assay, etc. Resultl-NAT decreased the levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in fibrotic mice model induced by carbon tetrachloride (CCl4). Histological staining showed that l-NAT ameliorated liver injury and fibrosis, and reduced the expression of α-smooth muscle actin (α-SMA) and Collagen I protein. l-NAT also attenuated apoptosis by down-regulating the level of pro-apoptotic protein Bax and up-regulating that of anti-apoptotic protein Bcl-2. Moreover, l-NAT inhibited the expressions of TGF-β1/SMAD and matrix metalloproteinase 9 (MMP9) proteins, and reversed the expression of YAP1 protein in CCl4-induced liver fibrosis. ConclusionThese results clearly demonstrated that l-NAT attenuated CCl4-induced liver fibrosis in mice, and this protective mechanism might relate to TGF-β1/SMAD and Hippo/YAP1 signaling pathway. Thus, this study provided data basis for the prevention and treatment of liver fibrosis.

Forsythiaside A alleviated carbon tetrachloride-induced liver fibrosis by modulating gut microbiota composition to increase short-chain fatty acids and restoring bile acids metabolism disorder

Liver fibrosis is a chronic and progressive disease with complex pathogenesis related to bile acids (BAs) and gut microbiota. Forsythiaside A (FTA), isolated from the traditional Chinese medicine Forsythiae Fructus (Lian Qiao), is a natural hepatoprotective agent. The purpose of this study was to investigate the protective effect of FTA on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Liver fibrosis was induced in mice by intraperitoneal injection of 2 mL/kg CCl4 three times a week for 4 weeks. FTA attenuated CCl4-induced liver fibrosis in mice, which was proved by the results of Masson and Sirius red staining, liver hydroxyproline, hyaluronic acid, laminin, type III procollagen, and type IV collagen assays. FTA inhibited hepatic stellate cell activation, and reduced hepatic inflammation and oxidative stress in mice treated with CCl4. What’s more, FTA ameliorated CCl4-induced gut dysbiosis, maintained intestinal barrier function, increased the production of short-chain fatty acids (SCFAs), and improved endotoxemia, as manifested by decreased serum lipopolysaccharide levels and increased expression of ileal tight junction proteins. Besides, FTA can modulate the genes related to bile acid metabolism to alter the distribution of fecal BAs in fibrotic mice. In a word, FTA can improve liver fibrosis by inhibiting inflammation and oxidative stress, regulating gut microbiota and BA metabolism, and increasing the content of SCFAs. The results of this study provided an important reference for the study on the mechanisms by which natural products prevent liver fibrosis.

The normal and fibrotic mouse lung classified by spatial proteomic analysis

Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

TRPM8 deficiency attenuates liver fibrosis through S100A9-HNF4α signaling

BackgroundLiver fibrosis represent a major global health care burden. Data emerging from recent advances suggest TRPM8, a member of the transient receptor potential (TRP) family of ion channels, plays an essential role in various chronic inflammatory diseases. However, its role in liver fibrosis remains unknown. Herein, we assessed the potential effect of TRPM8 in liver fibrosis.MethodsThe effect of TRPM8 was evaluated using specimens obtained from classic murine models of liver fibrosis, namely wild-type (WT) and TRPM8−/− (KO) fibrotic mice after carbon tetrachloride (CCl4) or bile duct ligation (BDL) treatment. The role of TRPM8 was systematically evaluated using specimens obtained from the aforementioned animal models after various in vivo and in vitro experiments.ResultsClinicopathological analysis showed that TRPM8 expression was upregulated in tissue samples from cirrhosis patients and fibrotic mice. TRPM8 deficiency not only attenuated inflammation and fibrosis progression in mice but also helped to alleviate symptoms of cholangiopathies. Moreover, reduction in S100A9 and increase in HNF4α expressions were observed in liver of CCl4- and BDL- treated TRPM8−/− mice. A strong regulatory linkage between S100A9 and HNF4α was also noticed in L02 cells that underwent siRNA-mediated S100A9 knockdown and S100A9 overexpressing plasmid transfection. Lastly, the alleviative effect of a selective TRPM8 antagonist was confirmed in vivo.ConclusionsThese findings suggest TRPM8 deficiency may exert protective effects against inflammation, cholangiopathies, and fibrosis through S100A9-HNF4α signaling. M8-B might be a promising therapeutic candidate for liver fibrosis.Graphical

Resveratrol ameliorates liver fibrosis induced by nonpathogenic Staphylococcus in BALB/c mice through inhibiting its growth

BackgroundThe altered gut microbiota is implicated in the pathogenesis of liver fibrosis. Resveratrol is a candidate for the treatment of liver fibrosis, which could ameliorate the dysregulation of gut microbiota in mice. This study aimed to clarify the role and mechanism of resveratrol in gut microbiota during liver fibrosis.MethodsA mouse model of liver fibrosis induced by CCl4 was conducted to assess the effect of resveratrol on liver fibrosis. The changes of gut microbiota in liver fibrotic mice after resveratrol intervention were assessed using 16S ribosomal RNA sequencing. The mechanism of the gut microbiota dysregulation in liver fibrosis was investigated by Sirius red staining, immunohistochemical assay, bacterial translocation (BT), EUB338 fluorescence in situ hybridization, immunofluorescence, trans-epithelial electrical resistance analysis and paracellular permeability analysis.ResultsResveratrol relieved CCl4-induced liver fibrosis. Besides, resveratrol restrained the gut microbiota Staphylococcus_lentus and Staphylococcus_xylosus in the liver fibrotic mice, and the Staphylococcus_xylosus and Staphylococcus_lentus facilitated the occurrence of BT and the cultures of them enhanced the permeability of intestine. The in vivo assay corroborated that the excessive Staphylococcus_xylosus and Staphylococcus_lentus canceled the protecting effect of resveratrol on liver fibrosis, and Staphylococcus_xylosus or Staphylococcus_lentus alone had a limited impact on the liver injury of normal mice.ConclusionResveratrol ameliorated liver fibrosis by restraining the growth of Staphylococcus_xylosus and Staphylococcus_lentus.

Cholesterol-Mediated Seeding of Protein Corona on DNA Nanostructures for Targeted Delivery of Oligonucleotide Therapeutics to Treat Liver Fibrosis.

The protein corona is a protein layer formed on the surface of nanoparticles administered in vivo and considerably affects the in vivo fate of nanoparticles. Although it is challenging to control protein adsorption on nanoparticles precisely, the protein corona may be harnessed to develop a targeted drug delivery system if the nanoparticles are decorated with a ligand with enhanced affinity to target tissue- and cell-homing proteins. Here, we prepared a DNA tetrahedron with trivalent cholesterol conjugation (Chol3-Td) that can induce enhanced interaction with lipoproteins in serum, which in situ generates the lipoprotein-associated protein corona on a DNA nanostructure favorable for cells abundantly expressing lipoprotein receptors in the liver, such as hepatocytes in healthy mice and myofibroblasts in fibrotic mice. Chol3-Td was further adopted for liver delivery of antisense oligonucleotide (ASO) targeting TGF-β1 mRNA to treat liver fibrosis in a mouse model. The potency of ASO@Chol3-Td was comparable to that of ASO conjugated with the clinically approved liver-targeting ligand, trivalent N-acetylgalactosamine (GalNAc3), demonstrating the potential of Chol3-Td as a targeted delivery system for oligonucleotide therapeutics. This study suggests that controlled seeding of the protein corona on nanomaterials can provide a way to steer nanoparticles into the target area.