Fibrosis In Murine Model Research Articles

Profibrotic monocyte-derived alveolar macrophages are expanded in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy.

Cepharanthine attenuates pulmonary fibrosis via modulating macrophage M2 polarization

BackgroundIdiopathic pulmonary fibrosis (IPF) is a group of chronic interstitial pulmonary diseases characterized by myofibroblast proliferation and extracellular matrix (ECM) deposition. However, current treatments are not satisfactory. Therefore, more effective therapies need to be explored. Cepharanthine (CEP) is a naturally occurring alkaloid that has recently been reported to have multiple pharmacological effects, particularly in chronic inflammation.MethodsFor in vivo experiments, first, a pulmonary fibrosis murine model was generated via tracheal injection of bleomycin (BLM). Second, the clinical manifestations and histopathological changes of the mice were used to verify that treatment with CEP might significantly reduce BLM-induced fibrosis. Furthermore, flow cytometric analysis was used to analyze the changes in the number of M2 macrophages in the lung tissues before and after treatment with CEP to explore the relationship between macrophage M2 polarization and pulmonary fibrosis. In vitro, we constructed two co-culture systems (THP-1 and MRC5 cells, RAW264.7 and NIH 3T3 cells), and measured the expression of fibrosis-related proteins to explore whether CEP could reduce pulmonary fibrosis by regulating macrophage M2 polarization and fibroblast activation.ResultsThe results showed that the intranasal treatment of CEP significantly attenuated the symptoms of pulmonary fibrosis induced by BLM in a murine model. Our findings also indicated that CEP treatment markedly reduced the expression of fibrosis markers, including TGF-β1, collagen I, fibronectin and α-SMA, in the mouse lung. Furthermore, in vitro studies demonstrated that CEP attenuated pulmonary fibrosis by inhibiting fibroblast activation through modulating macrophage M2 polarization and reducing TGF-β1 expression.ConclusionsThis study demonstrated the potential and efficacy of CEP in the treatment of pulmonary fibrosis. In particular, this study revealed a novel mechanism of CEP in inhibiting fibroblast activation by regulating macrophage M2 polarization and reducing the expression of fibrosis-associated factors. Our findings open a new direction for future research into the treatment of pulmonary fibrosis.

PAI-1 Interaction with Sortilin Related Receptor-1 is Required for Lung Fibrosis.

Plasminogen activator inhibitor-1 (PAI-1) has been previously shown to promote lung fibrosis via a mechanism that requires an intact vitronectin (VTN) binding site. In the present study, employing two distinct murine fibrosis models, we find that VTN is not required for PAI-1 to drive lung scarring. This result suggested the existence of a previously unrecognized profibrotic PAI-1-protein interaction involving the VTN-binding site for PAI-1. Using an unbiased proteomic approach, we identified sortilin related receptor 1 (SorlA) as the most highly enriched PAI-1 interactor in the fibrosing lung. We next investigated the role of SorlA in pulmonary fibrosis and found that SorlA deficiency protected against lung scarring in a murine model. We further show that, while VTN deficiency does not influence fibrogenesis in the presence or absence of PAI-1, SorlA is required for PAI-1 to promote scarring. These results, together with data showing increased SorlA levels in human IPF lung tissue, support a novel mechanism through which the potent profibrotic mediator PAI-1 drives lung fibrosis and implicate SorlA as a new therapeutic target in IPF treatment.

The Role of a Cholecystokinin Receptor Antagonist in the Management of Chronic Pancreatitis: A Phase 1 Trial.

Chronic pancreatitis (CP) is a rare but debilitating condition with an 8-fold increased risk of developing pancreatic cancer. In addition to the symptoms that come from the loss of endocrine and exocrine function in CP, the management of chronic pain is problematic. We previously showed that the CCK-receptor antagonist called proglumide could decrease inflammation, acinar-ductal metaplasia, and fibrosis in murine models of CP. We hypothesized that proglumide would be safe and diminish pain caused by CP. A Phase 1 open-labeled safety study was performed in subjects with clinical and radiographic evidence of CP with moderate to severe pain. After a 4-week observation period, the subjects were treated with proglumide in 400 mg capsules three times daily (1200 mg per day) by mouth for 12 weeks, and then subjects returned for a safety visit 4 weeks after the discontinuation of the study medication. The results of three pain surveys (Numeric Rating Scale, COMPAT-SF, and NIH PROMIS) showed that the patients had significantly less pain after 12 weeks of proglumide compared to the pre-treatment observation phase. Of the eight subjects in this study, two experienced nausea and diarrhea with proglumide. These side effects resolved in one subject with doses reduced to 800 mg per day. No abnormalities were noted in the blood chemistries. A blood microRNA blood biomarker panel that corresponded to pancreatic inflammation and fibrosis showed significant improvement. We conclude that proglumide is safe and well tolerated in most subjects with CP at a dose of 1200 mg per day. Furthermore, proglumide therapy may have a beneficial effect by decreasing pain associated with CP.

Antifibrotic effects of vitamin D3 on human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. Up to now, no treatment can stop the progression of IPF. Vitamin D3 (VD) reduces experimental lung fibrosis in murine models and depletion of vitamin D3 might be associated with the reduced survival of patients with IPF. In this context, we determined if VD can prevent the pro-fibrotic functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and control HLFs were derived from surgical lung biopsies collected from patients with IPF or with primary lung cancer, respectively. VD (3-100 nM) markedly reduced the basal and PDGF-induced proliferation of HLFs. VD also altered cell cycle by increasing the percentage of IPF HLFs arrested in the G0/G1 phase, and by downregulating the expression of various cell cycle regulatory proteins. In addition, VD barely prevented the TGF-β1-induced differentiation in HLFs. At 100 nM, VD slightly reduced the expression of the pro-fibrotic marker α-smooth muscle actin, and had no effect on fibronectin and collagen-1 expression. In contrast, 100 nM VD strongly inhibited the aerobic glycolytic metabolism induced by TGF- β1. Finally, VD reduced both the secretion of lactate, the levels of lactate deshydrogenase mRNA and the activity of intracellular LDH in IPF HLFs. In conclusion, our study shows that VD reduced pro-fibrotic functions of HLFs. These findings suggest that it might be interesting to assess the potential clinical benefits of vitamin D supplementation in patients with IPF, especially on lung function decline.

FOXS1 is increased in liver fibrosis and regulates TGFβ responsiveness and proliferation pathways in human hepatic stellate cells

Liver fibrosis commences with liver injury stimulating transforming growth factor beta (TGFβ) activation of hepatic stellate cells (HSCs), causing scarring and irreversible damage. TGFβ induces expression of the transcription factor Forkhead box S1 (FOXS1) in hepatocytes and may have a role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no studies have determined how it affects HSCs. We analyzed human livers with cirrhosis, HCC, and a murine fibrosis model and found that FOXS1 expression is significantly higher in fibrotic livers but not in HCC. Next, we treated human LX2 HSC cells with TGFβ to activate fibrotic pathways, and FOXS1 mRNA was significantly increased. To study TGFβ-FOXS1 signaling, we developed human LX2 FOXS1 CRISPR KO and scrambled control HSCs. To determine differentially expressed gene transcripts controlled by TGFβ-FOXS1, we performed RNA-seq in the FOXS1 KO and control cells and over 400 gene responses were attenuated in the FOXS1 KO HSCs with TGFβ-activation. To validate the RNA-seq findings, we used our state-of-the-art PamGene PamStation kinase activity technology that measures hundreds of signaling pathways nonselectively in real time. Using our RNA-seq data, kinase activity data, and descriptive measurements, we found that FOXS1 controls pathways mediating TGFβ responsiveness, protein translation, and proliferation. Our study is the first to identify that FOXS1 may serve as a biomarker for liver fibrosis and HSC activation, which may help with early detection of hepatic fibrosis or treatment options for end-stage liver disease.

The miR-3074/BMP7 axis regulates TGF-β-caused activation of hepatic stellate cells in vitro and CCl4-caused murine liver fibrosis in vivo.

Continuously progressive hepatic fibrosis might cause chronic liver diseases, resulting in hepatic failure. The activation of hepatic stellate cells (HSCs) residing in the liver might induce and influence hepatic fibrosis. In the present study, microRNA 3074 (miR-3074) was found increased within transforming growth factor-β (TGF-β)-activated HSCs and enriched within the TGF-β signaling. In activated HSCs by TGF-β, miR-3074 overexpression aggravated TGF-β-induced fibrotic changes, whereas miR-3074 inhibition exerted opposite effects. miR-3074 directly targeted bone morphogenetic protein 7 (BMP7) and inhibited BMP7 expression. Under TGF-β induction, overexpressed BMP7 notably attenuated the promotive roles of miR-3074 overexpression in TGF-β-activated HSCs. Within carbon tetrachloride (CCl4)-caused liver fibrosis murine model, miR-3074 agomir administration promoted, while LV-BMP7 administration alleviated CCl4-induced fibrotic changes; LV-BMP7 significantly attenuated the effects of miR-3074 agomir. Lastly, mmu-miR-3074 also targeted mouse BMP7 and inhibited mouse BMP7 expression. In conclusion, the miR-3074/BMP7 axis regulates TGF-β-caused activation of HSCs in vitro and CCl4-caused murine liver fibrosis in vivo. BMP7-mediated Smad1/5/8 activation might be involved.

Clinical validation of the suppressive impact of letrozole on liver fibrosis in patients with breast cancer undergoing continuous letrozole administration: A retrospective study.

Treatment strategies for preventing liver fibrosis have not yet been established. Letrozole, widely used for breast cancer, has recently been reported to suppress liver fibrosis in murine models. Therefore, we aimed to validate the suppressive effects of letrozole on liver fibrosis in the clinical setting. From 2006 to 2020, 23 consecutive patients who received continuous letrozole treatment for 24 months or more and had a liver fibrosis marker FIB-4 index of ≥ 2.30, were included. Forty-three patients who underwent anastrozole treatment for 24 months or more and had a liver fibrosis marker FIB-4 index of ≥ 2.30, were also included as controls. The Fisher exact, chi-square, unpaired Student t, and paired Student t test were used to analyze the data. The patient characteristics were similar between the letrozole- and anastrozole-treated patient groups. Among the letrozole-treated patients, the mean FIB-4 index tended to decline during letrozole treatment; a significant decrease was observed at 18 and 24 months compared with the baseline values (p = 0.044 and p = 0.013). In addition, the mean aspartate aminotransferase-to-platelet ratio index (APRI) decreased during letrozole treatment; the values at 18 and 24 months were significantly lower than those at baseline (p = 0.024 and p = 0.026). In contrast, among anastrozole-treated patients, the mean FIB-4 index and APRI did not change during anastrozole treatment. When changes in the FIB-4 index were further examined in a limited number of patients with a FIB-4 index ≥ 2.67, a significant reduction in the FIB-4 index at 24 months compared with baseline was also observed in letrozole-treated patients (p = 0.023), but not in anastrozole-treated patients. In conclusion, our findings support a possible suppressive effect of letrozole on liver fibrosis in the clinical setting. Further studies are required to better understand the pharmacological effects of letrozole.

Chemical characterization and effects of volatile oil of Alpinia zerumbet on the quality of collagen deposition and caveolin-1 expression in a muscular fibrosis murine model.

This study evaluated the effect of the volatile oil of Alpinia zerumbet (VOAz) on caveolin-1 gene expression and muscular fibrosis. The rats were immobilized to induce fibrosis of the gastrocnemius muscle, and they were treated with VOAz. Collagen quality was assessed by histology and the expression of the caveolin-1 (CAV-1) gene was evaluated using qPCR. Histomorphological analysis indicated a significant reduction in the perimeter, width, and intensity of collagen in the treated groups, thus showing that the oil was effective in regulating the quality of collagen at the three concentrations. The results of expression levels suggested a decrease in the lesioned group and in two treatment groups (0.0115 µg/g and 0.009 µg/g). However, with the lowest concentration (0.0065 µg/g), no significant difference was observed, with levels similar to those found in healthy tissue. Therefore, the results showed that VOAz has the potential to be a non-invasive and low-cost alternative to aid in the treatment of muscular fibrosis.

Contribution of common and rare variants to Asian neovascular age-related macular degeneration subtypes

Neovascular age-related macular degeneration (nAMD), along with its clinical subtype known as polypoidal choroidal vasculopathy (PCV), are among the leading causes of vision loss in elderly Asians. In a genome-wide association study (GWAS) comprising 3,128 nAMD (1,555 PCV and 1,573 typical nAMD), and 5,493 controls of East Asian ancestry, we identify twelve loci, of which four are novel (P , < , 1.19times {10}^{-8}). Substantial genetic sharing between PCV and typical nAMD is noted (rg = 0.666), whereas collagen extracellular matrix and fibrosis-related pathways are more pronounced for PCV. Whole-exome sequencing in 259 PCV patients revealed functional rare variants burden in collagen type I alpha 1 chain gene (COL1A1; P=1.05times {10}^{-6}) and potential enrichment of functional rare mutations at AMD-associated loci. At the GATA binding protein 5 (GATA5) locus, the most significant GWAS novel loci, the expressions of genes including laminin subunit alpha 5 (Lama5), mitochondrial ribosome associated GTPase 2 (Mtg2), and collagen type IX alpha 3 chain (Col9A3), are significantly induced during retinal angiogenesis and subretinal fibrosis in murine models. Furthermore, retinoic acid increased the expression of LAMA5 and MTG2 in vitro. Taken together, our data provide insights into the genetic basis of AMD pathogenesis in the Asian population.

Abstract P2011: Reduced Activity Of Activin Like Kinase 1 (ALK1) Increases Mortality And Myocardial Rupture After Acute Myocardial Infarction

Introduction: Acute myocardial infarction (AMI) is a primary cause of heart failure (HF). Transforming growth factor beta (TGFβ) plays a central role in cardiac remodeling after AMI. Reduced activity of the TGFβ Type I receptor, activin like kinase (ALK)-1 promotes cardiac fibrosis in murine models of heart failure. A functional role for ALK1 in post-infarct cardiac remodeling remains poorly understood. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after AMI. Methods: AMI was induced by left coronary ligation in wild type (WT), and ALK+/- mice. After 14 days, LV function was assessed using conductance catheters and myocardial ALK1 signaling was quantified. Using siRNAs targeting ALK1 in in human cardiac fibroblasts (H-CFs), we further tested the role of ALK1 in cardiac fibrosis. Results: Reduced ALK1 activity significantly reduced survival compared to WT (p= 0.008 respectively) after AMI. ALK1 mice demonstrated a higher rate of cardiac rupture compared to WT after AMI (0% vs 85.7%). Both WT ad ALK1+/- mice demonstrated reduced LV ejection fraction and stroke work compared to sham controls (p=0.001 and p=0.0001 respectively). Profibrotic SMAD3 signaling was increased and antifibrotic SMAD1 signaling decreased in ALK1-/+ (pSMAD3, p=0.03, pSMAD1, p=0.003) mice 14 days after AMI. Reduced SMAD1 signaling was directly associated with increased myocardial Type 1 collagen protein levels in ALK1-/+ (R2=0.42, p=0.01) mice compared to WT. Myocardial levels of the TGFb1 co-receptor, endoglin, were increased significantly in ALK+/- as compared to WT (p=0.001) after AMI. Activity of MMP9 was increased in ALK1+/- mice, but protein levels were not affected 2 weeks after MI. Conclusion: We identify for the first time that loss of ALK1 activity increases cardiac rupture and mortality after AMI. These findings identify a novel functional role for ALK1 as a determinant of post-infarct survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after AMI.

Innovative Pre-Clinical Data Using Peptides to Intervene in the Evolution of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive, relentless, and deadly disease. Little is known about its pathogenetic mechanisms; therefore, developing efficient pharmacological therapies is challenging. This work aimed to apply a therapeutic alternative using immunomodulatory peptides in a chronic pulmonary fibrosis murine model. BALB/c mice were intratracheally instilled with bleomycin (BLM) and followed for 30 days. The mice were treated with the immune modulatory peptides ToAP3 and ToAP4 every three days, starting on the 5th day post-BLM instillation. ELISA, qPCR, morphology, and respiratory function analyses were performed. The treatment with both peptides delayed the inflammatory process observed in the non-treated group, which showed a fibrotic process with alterations in the production of collagen I, III, and IV that were associated with significant alterations in their ventilatory mechanics. The ToAP3 and ToAP4 treatments, by lung gene modulation patterns, indicated that distinct mechanisms determine the action of peptides. Both peptides controlled the experimental IPF, maintaining the tissue characteristics and standard function properties and regulating fibrotic-associated cytokine production. Data obtained in this work show that the immune response regulation by ToAP3 and ToAP4 can control the alterations that cause the fibrotic process after BLM instillation, making both peptides potential therapeutic alternatives and/or adjuvants for IPF.

Targeting pathogenic macrophages by the application of SHP-1 agonists reduces inflammation and alleviates pulmonary fibrosis

Idiopathic pulmonary fibrosis is a progressive fibrotic disorder with no cure that is characterized by deterioration of lung function. Current FDA-approved drugs for IPF delay the decline in lung function, but neither reverse fibrosis nor significantly improve overall survival. SHP-1 deficiency results in hyperactive alveolar macrophages accumulating in the lung, which contribute to the induction of pulmonary fibrosis. Herein, we investigated whether employing a SHP-1 agonist ameliorates pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis murine model. Histological examination and micro-computed tomography images showed that SHP-1 agonist treatment alleviates bleomycin-induced pulmonary fibrosis. Reduced alveolar hemorrhage, lung inflammation, and collagen deposition, as well as enhanced alveolar space, lung capacity, and improved overall survival were observed in mice administered the SHP-1 agonist. The percentage of macrophages collected from bronchoalveolar lavage fluid and circulating monocytes in bleomycin-instilled mice were also significantly reduced by SHP-1 agonist treatment, suggesting that the SHP-1 agonist may alleviate pulmonary fibrosis by targeting macrophages and reshaping the immunofibrotic niche. In human monocyte-derived macrophages, SHP-1 agonist treatment downregulated CSF1R expression and inactivated STAT3/NFκB signaling, culminating in inhibited macrophage survival and perturbed macrophage polarization. The expression of pro-fibrotic markers (e.g., MRC1, CD200R1, and FN1) by IL4/IL13-induced M2 macrophages that rely on CSF1R signaling for their fate-determination was restricted by SHP-1 agonist treatment. While M2-derived medium promoted the expression of fibroblast-to-myofibroblast transition markers (e.g., ACTA2 and COL3A1), the application of SHP-1 agonist reversed the transition in a dose-dependent manner. Our report indicates that pharmacological activation of SHP-1 ameliorates pulmonary fibrosis via suppression of CSF1R signaling in macrophages, reduction of pathogenic macrophages, and the inhibition of fibroblast-to-myofibroblast transition. Our study thus identifies SHP-1 as a druggable target for the treatment of IPF, and suggests that the SHP-1 agonist may be developed as an anti-pulmonary fibrosis medication that both suppresses inflammation and restrains fibroblast-to-myofibroblast transition.

WED-216 - TLC-3595, a selective acetyl-CoA carboxylase 2 (ACC2) inhibitor, improves steatosis and fibrosis in murine models of NASH via pleiotropic mechanisms

WED-216 - TLC-3595, a selective acetyl-CoA carboxylase 2 (ACC2) inhibitor, improves steatosis and fibrosis in murine models of NASH via pleiotropic mechanisms

OA28 CCL24 serum concentration predicts both vascular and fibrotic complications in systemic sclerosis: rationale for a biological target driven basket trial across disease subsets

Abstract Background/Aims CCL24 is a novel target that was found to play a dual role in advancing pro-inflammatory and pro-fibrotic processes in systemic sclerosis (SSc). Previous studies reported that skin and serum CCL24 levels are elevated in SSc and that blocking of CCL24 was effective in preventing and attenuating experimental-induced fibrosis in murine models as well as interfering with endothelial cell activation. Here we tested the concentration of CCL24 in an observational cohort of patients with SSc and available clinical follow-up to determine its clinical value in predicting fibrotic and vascular complications of the disease. Methods Consecutive patients from a single-centre observational cohort were enrolled in this analysis. Clinical data were collected according to the EUSTAR minimal essential data set. Serum Concentration of CCL24 was measured by Luminex assay (Myriad RBM) and defined as “high” for patients over the upper quartile of the healthy control population (110 ng/ml). Interstitial lung disease (ILD) was defined by at least 10% parenchymal involvement at a high-resolution CT scan of the chest, while digital ulcer (DU) disease was defined by the presence of current or history of DU in the previous 6 months. Progressive fibrotic ILD was defined as previously described, according to Erice's criteria within 24 months. Clinical features were compared between CCL24 high vs low by Chi-Square and T-student tests. R software was employed for statistical analysis. Results 189 SSc patients (13% male) fulfilling 2013 ACR/EULAR classification criteria were enrolled in this study. Median (IQR) disease duration was 4 (IQR 2-10) years. 37 (27%) had diffuse cutaneous SSc, 60 (32%) had ILD, and 90 (48%) had DU disease. 52 patients (26%) were CCL24 High. These patients had a higher prevalence of DU (60%, p = 0.042), a higher median (IQR) mRSS [4(2-7) vs 2(0-5), p = 0.036) and showed a higher prevalence of ILD (42% vs 28%, p = 0.055); this was more significant among patients with limited cutaneous SSc (7% vs 12%, p = 0.037). Most importantly, among patients with PF-ILD (19/43 with available 24-month follow-up), 44% had significantly higher CCL24 serum concentration at baseline, compared to stable patients (1150 [817-1593] vs 725 [460-955]; p = 0.017). Consistent with these findings, in patients with ILD, having high CCL24 gave double the chance of progressing within 24 months (66% vs 32% of CCL24 low; p = 0.03). Conclusion We show here that within the context of an observational cohort of unselected patients, CCL24 serum concentration is associated with the presence of DU as well as worse skin and lung fibrotic involvement, suggesting that this molecule may be involved in both vascular and fibrotic manifestations of SSc. These results informed the rationale for a target-enriched basket randomized controlled trial to test the biological and clinical effect of CCL24 inhibition in SSc across cutaneous disease subsets. Disclosure E. De Lorenzis: None. A. Mor: None. R. Ross: None. S. Di Donato: None. R. Aricha: None. H. Levi: None. I. Vaknin: None. F. Del Galdo: Other; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

Berberine attenuates liver fibrosis by autophagy inhibition triggering apoptosis via the miR-30a-5p/ATG5 axis

Berberine (BBR) is an effective drug against liver fibrosis (LF). Autophagy is involved in the pathogenesis of LF; however, the mechanism linking BBR to autophagy in LF remains unresolved. To explore the underlying mechanism, we assessed the effects of BBR on autophagy and apoptosis of activated hepatic stellate cells (HSCs) in vitro and in a murine model of fibrosis. The decreased expression of the autophagy activation marker ATG5, autophagosome formation, and autophagy flux in the HSC model confirmed that BBR inhibited autophagy in activated HSCs and in mice with liver fibrosis. Moreover, ATG5 was necessary for inducing autophagy and HSC activation. BBR suppressed ATG5 expression by upregulating miR-30a-5p expression, which affected the stability of ATG5 mRNA by binding to its 3′-untranslated region, an effect that was attenuated by treatment with a miR-30a-5p inhibitor. BBR also markedly induced HSC apoptosis, as indicated by the upregulated expression of the pro-apoptosis markers p53, BAX, and cleaved PARP and the downregulated expression of the anti-apoptosis marker BCL-2, effects that were reversed by ATG5 overexpression. In vivo, BBR improved mouse LF by decreasing collagen deposition, inflammatory cell infiltration, and expression of fibrosis markers hydroxyproline, α-smooth muscle actin, and collagen type 1-A1 and the autophagy marker LC3. BBR had a protective effect on mouse fibrotic livers and reduced serum aspartate aminotransferase and alanine aminotransferase levels. Collectively, these results reveal a novel mechanism of BBR-induced autophagy inhibition triggering apoptosis in HSCs, providing a reliable experimental and theoretical basis for developing BBR-based candidate drugs for LF.

Taurocholic acid promotes hepatic stellate cell activation via S1PR2/p38 MAPK/YAP signaling under cholestatic conditions.

Disrupted bile acid regulation and accumulation in the liver can contribute to progressive liver damage and fibrosis. However, the effects of bile acids on the activation of hepatic stellate cells (HSCs) remain unclear. This study investigated the effects of bile acids on HSC activation during liver fibrosis, and examined the underlying mechanisms. The immortalized HSCs, LX-2 and JS-1cells were used for the in vitro study. in vitro, the adeno-associated viruses adeno-associated virus-sh-S1PR2 and JTE-013 were used to pharmacologically inhibit the activity of S1PR2 in a murine model of fibrosis induced by a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Histological and biochemical analyses were performed to study the involvement of S1PR2 in the regulation of fibrogenic factors as well as the activation properties of HSCs. S1PR2 was the predominant S1PR expressed in HSCs and was upregulated during taurocholic acid (TCA) stimulation and in cholestatic liver fibrosis mice. TCA-induced HSC proliferation, migration and contraction and extracellular matrix protein secretion were inhibited by JTE-013 and a specific shRNA targeting S1PR2 in LX-2 and JS-1 cells. Meanwhile, treatment with JTE-013 or S1PR2 deficiency significantly attenuated liver histopathological injury, collagen accumulation, and the expression of fibrogenesis-associated genes in mice fed a DDC diet. Furthermore, TCAmediated activation of HSCs through S1PR2 was closely related to the yes-associated protein (YAP) signaling pathway via p38 mitogen-activated protein kinase (p38 MAPK). TCA-induced activation of the S1PR2/p38 MAPK/YAP signaling pathways plays a vital role in regulating HSC activation, which might be therapeutically relevant for targeting cholestatic liver fibrosis.

TREM2 Regulates the Removal of Apoptotic Cells and Inflammatory Processes during the Progression of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver pathology worldwide. In mice and humans, NAFLD progression is characterized by the appearance of TREM2-expressing macrophages in the liver. However, their mechanistic contributions to disease progression have not been completely elucidated. Here, we show that TREM2+ macrophages prevent the generation of a pro-inflammatory response elicited by LPS-laden lipoproteins in vitro. Further, Trem2 expression regulates bone-marrow-derived macrophages (BMDMs) and Kupffer cell capacity to phagocyte apoptotic cells in vitro, which is dependent on CD14 activation. In line with this, loss of Trem2 resulted in an increased pro-inflammatory response, which ultimately aggravated liver fibrosis in murine models of NAFLD. Similarly, in a human NAFLD cohort, plasma levels of TREM2 were increased and hepatic TREM2 expression was correlated with higher levels of liver triglycerides and the acquisition of a fibrotic gene signature. Altogether, our results suggest that TREM2+ macrophages have a protective function during the progression of NAFLD, as they are involved in the processing of pro-inflammatory lipoproteins and phagocytosis of apoptotic cells and, thereby, are critical contributors for the re-establishment of liver homeostasis.

Synergistic Effects of Forskolin and Rutin in the Treatment of Pulmonary Fibrosis in Murine Model

Synergistic Effects of Forskolin and Rutin in the Treatment of Pulmonary Fibrosis in Murine Model

Tanreqing injection protects against bleomycin-induced pulmonary fibrosis via inhibiting STING-mediated endoplasmic reticulum stress signaling pathway

Ethnopharmacological relevanceIdiopathic pulmonary fibrosis (IPF), characterized by excessive collagen deposition, is a progressive and typically fatal lung disease without effective therapeutic methods. Tanreqing injection (TRQ), a Traditional Chinese Patent Medicine, has been widely used to treat inflammatory respiratory diseases clinically. Aim of the studyThe present work aims to elucidate the therapeutic effects and the possible mechanism of TRQ against pulmonary fibrosis. MethodsThe pulmonary fibrosis murine model were constructed by the intratracheal injection of bleomycin (BLM). 7 days later, TRQ-L (2.6 ml/kg) and TRQ-H (5.2 ml/kg) were administered via intraperitoneal injection respectively for 21 days. The efficacy and underlying molecular mechanism of TRQ were investigated. ResultsHere, we showed that TRQ significantly inhibited BLM-induced lung edema and pulmonary function. TRQ markedly reduced BLM-promoted inflammatory cell infiltration in BALF and inflammatory cytokines release (TNF-α, IL-6, and IL-1β) in serum and lung tissues. Meanwhile, TRQ also alleviated BLM-induced collagen synthesis and deposition. Simultaneously, TRQ attenuated BLM-induced pulmonary fibrosis through regulating the expression of fibrotic hallmarks, manifested by down-regulated α-SMA and up-regulated E-cadherin. Moreover, we found that TRQ significantly prevented STING, p-P65, BIP, p-PERK, p-eIF2α, and ATF4 expression in lung fibrosis mice. ConclusionsTaken together, our results indicated that TRQ positively affects inflammatory responses and lung fibrosis by regulating STING-mediated endoplasmic reticulum stress (ERS) signal pathway.