The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Bioindicator responses to extreme conditions: Insights into pH and bioavailable metals under acidic metal environments.

Acid mine drainage (AMD) caused environmental risks from heavy metal pollution, requiring treatment methods such as chemical precipitation and biological treatment. Monitoring and adapting treatment processes was crucial for success, but cost-effective pollution monitoring methods were lacking. Using bioindicators measured through 16S rRNA was a promising method to assess environmental pollution. This study evaluated the effects of AMD on ecological health using the ecological risk index (RI) and the Risk Assessment Code (RAC) indices. Additionally, we also examined how acidic metal stress affected the diversity of bacteria and fungi, as well as their networks. Bioindicators were identified using linear discriminant analysis effect size (LEfSe), Partial least squares regression (PLS-R), and Spearman analyses. The study found that Cd, Cu, Pb, and As pose potential ecological risks in that order. Fungal diversity decreased by 44.88% in AMD-affected areas, more than the 33.61% decrease in bacterial diversity. Microbial diversity was positively correlated with pH (r = 0.88, p = 0.04) and negatively correlated with bioavailable metal concentrations (r = -0.59, p = 0.05). Similarly, microbial diversity was negatively correlated with bioavailable metal concentrations (bio_Cu, bio_Pb, bio_Cd) (r = 0.79, p = 0.03). Acidiferrobacter and Thermoplasmataceae were prevalent in acidic metal environments, while Puia and Chitinophagaceae were identified as biomarker species in the control area (LDA>4). Acidiferrobacter and Thermoplasmataceae were found to be pH-tolerant bioindicators with high reliability (r = 1, P < 0.05, BW > 0.1) through PLS-R and Spearman analysis. Conversely, Puia and Chitinophagaceae were pH-sensitive bioindicators, while Teratosphaeriaceae was a potential bioindicator for Cu-Zn-Cd metal pollution. This study identified bioindicator species for acid and metal pollution in AMD habitats. This study outlined the focus of biological monitoring in AMD acidic stress environments, including extreme pH, heavy metal pollutants, and indicator species. It also provided essential information for heavy metal bioremediation, such as the role of omics and the effects of organic matter on metal bioavailability.

Liver ACSM3 deficiency mediates metabolic syndrome via a lauric acid-HNF4α-p38 MAPK axis.

Metabolic syndrome combines major risk factors for cardiovascular disease, making deeper insight into its pathogenesis important. We here explore the mechanistic basis of metabolic syndrome by recruiting an essential patient cohort and performing extensive gene expression profiling. The mitochondrial fatty acid metabolism enzyme acyl-CoA synthetase medium-chain family member 3 (ACSM3) was identified to be significantly lower expressed in the peripheral blood of metabolic syndrome patients. In line, hepatic ACSM3 expression was decreased in mice with metabolic syndrome. Furthermore, Acsm3 knockout mice showed glucose and lipid metabolic abnormalities, and hepatic accumulation of the ACSM3 fatty acid substrate lauric acid. Acsm3 depletion markedly decreased mitochondrial function and stimulated signaling via the p38 MAPK pathway cascade. Consistently, Acsm3 knockout mouse exhibited abnormal mitochondrial morphology, decreased ATP contents, and enhanced ROS levels in their livers. Mechanistically, Acsm3 deficiency, and lauric acid accumulation activated nuclear receptor Hnf4α-p38 MAPK signaling. In line, the p38 inhibitor Adezmapimod effectively rescued the Acsm3 depletion phenotype. Together, these findings show that disease-associated loss of ACSM3 facilitates mitochondrial dysfunction via a lauric acid-HNF4a-p38 MAPK axis, suggesting a novel therapeutic vulnerability in systemic metabolic dysfunction.

Unraveling assemblage of microbial community dwelling in Dabaoshan As/Pb/Zn mine-impacted area: A typical mountain mining area of South China.

Microbial community assemblage includes microorganisms from the three domains including Bacteria, Archaea, and Eukarya (Fungi), which play a crucial role in geochemical cycles of metal(loid)s in mine tailings. Mine tailings harbor vast proportions of metal(loid)s, representing a unique source of co-contamination of metal(loid)s that threaten the environment. The elucidation of the assembly patterns of microbial communities in mining-impacted ecospheres has received little attention. To decipher the microbial community assembly processes, the microbial communities from the five sites of the Dabaoshan mine-impacted area were profiled by the MiSeq sequencing of 16S rRNA (Bacteria and Archaea) genes and internal transcribed spacers (Fungi). Results indicated that the coexistence of 31 bacterial, 10 fungal, and 3 archaeal phyla, were mainly dominated by Mucilaginibacter, Cladophialophora, and Candidatus Nitrosotalea, respectively. The distribution of microorganisms was controlled by deterministic processes. The combination of Cu, Pb, and Sb was the main factor explaining the structure of microbial communities. Functional predicting analysis of bacteria and archaea based on the phylogenetic investigation of communities by reconstruction of unobserved states analyses revealed that the metabolic pathways related to arsenite transporter, arsenate reductase, and FeS cluster were important for metal detoxification. Furthermore, the ecological guilds (pathogens, symbiotrophs, and saprotrophs) of fungal communities explained 44.5 % of functional prediction. In addition, metal-induced oxidative stress may be alleviated by antioxidant enzymes of fungi communities, such as catalase. Such information provides new insights into the microbial assembly rules in co-contaminated sites.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader.

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability. The telomeric repeat-containing RNA (TERRA) that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop. In tumor cells, R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres (ALT) pathway. Dysregulated R-loops can cause stalled replication forks and telomere instability. However, how R-loops are recognized and regulated, particularly at telomeres, is not well understood. We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination. Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses (DDR). In addition, ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway. Using the proximity-dependent biotin identification (BioID) technology, we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases, including DHX9. Importantly, ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9. Our findings suggest that ILF3 may function as a reader of telomeric R-loops, helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

GPRASP1 loss-of-function links to arteriovenous malformations by endothelial activating GPR4 signals.

Arteriovenous malformations (AVMs) are fast-flow vascular malformations and refer to important causes of intracerebral hemorrhage in young adults. Getting deep insight into the genetic pathogenesis of AVMs is necessary. Herein, we identified two vital missense variants of G protein-coupled receptor (GPCR) associated sorting protein 1 (GPRASP1) in AVM patients for the first time and congruously determined to be loss-of-function (LoF) variants in endothelial cells. GPRASP1 LoF caused endothelial dysfunction in vitro and in vivo. Endothelial Gprasp1 knockout mice suffered a high probability of cerebral hemorrhage, AVMs, and exhibited vascular anomalies in multiple organs. GPR4 was identified to be an effective GPCR binding with GPRASP1 to develop endothelial disorders. GPRASP1 deletion activated GPR4/cAMP/MAPK signaling to disturb endothelial functions, thus contributing to vascular anomalies. Mechanistically, GPRASP1 promoted GPR4 degradation. GPRASP1 enabled GPR4 K63-linked ubiquitination, enhancing the binding of GPR4 and RABGEF1 to activate RAB5 for conversions from endocytic vesicles to endosomes, and subsequently increasing the interactions of GPR4 and ESCRT members to package GPR4 into multivesicular bodies or late endosomes for lysosome degradation. Notably, the GPR4 antagonist NE 52-QQ57 and JNK inhibitor SP600125 effectively rescued the vascular phenotype caused by endothelial Gprasp1 deletion. Our findings provided novel insights into the roles of GPRASP1 in AVMs and hinted at new therapeutic strategies.

Ilexchinene, a new seco-ursane triterpenoid from the leaves of Ilex chinensis with therapeutic effect on neuroinflammation by attenuating the MAPK/NF-κB signaling pathway.

BACKGROUND: Neuroinflammation is a vital factor participating in the whole pathogenetic process of diverse neurodegenerative disorders, but accessible clinical drugs are still insufficient due to their inefficacy and side effects. Triterpenoids are reported to possess potential anti-neuroinflammatory activities, and the leaves of Ilex chinensis are a commonly used herbal medicine containing many ursane-type and oleanane-type triterpenoids. However, the novel triterpenoids from I. chinensis and their underlying mechanisms are still elusive. PURPOSE: To isolate novel seco-ursane triterpenoids with anti-neuroinflammatory effects from the leaves of I. chinensis and reveal their underlying mechanisms. STUDY DESIGN AND METHODS: The novel compound was purified by column chromatography and identified by comprehensive spectroscopic experiments. The LPS-induced BV-2 cell model and LPS-induced acute murine brain inflammation model were used to assess the anti-neuroinflammatory effect of the structure and further understand its underlying mechanisms by cell viability, ELISA, Western blot analysis, qRT‒PCR analysis, behavior analysis, H&E staining, and immunofluorescence staining experiments. RESULTS: Ilexchinene is a novel ursane-type triterpenoid with a rare 18,19-seco-ring skeleton that was first isolated and identified from I. chinensis. Ilexchinene evidently reduced the overexpression of inflammatory substances in vitro. A mechanistic study suggested that ilexchinene could decrease NF-κB activation to prevent the formation of the NLRP3 inflammasome in the early neuroinflammatory response; in addition, it could prevent the phosphorylation of ERK and JNK. In vivo, ilexchinene remarkably improved LPS-induced mouse behavioral deficits and diminished the number of overactivated microglial cells. Furthermore, ilexchinene evidently diminished the overexpression of inflammatory substances in mouse brains. A mechanistic study confirmed that ilexchinene markedly suppressed the MAPK/NF-κB pathway to relieve the neuroinflammatory response. CONCLUSION: We identified a novel 18,19-seco-ursane triterpenoid from the leaves of I. chinensis and revealed its underlying mechanism of neuroinflammation for the first time. These findings suggest that ilexchinene might possess promising therapeutic effects in neuroinflammation.

Microbial metabolic activity in metal(loid)s contaminated sites impacted by different non-ferrous metal activities.

Many non-ferrous metal mining and smelting activities have caused severe metal(loid) contamination in the local soil environment. The metabolic activity of soil microorganisms in four areas affected by different metallurgical activities (production vs. waste disposal) was characterized using a contamination gradient from the contaminated site to the surrounding soils. Results indicated that the soil microcalorimetric and enzyme activities were correlated with the fractionated metal(loid) properties (p < 0.05). All four areas had high total As, Cd, Pb, Sb, and Zn concentrations, of which mobile As, Cu, Ni, Pb, Sb, and Zn were higher in the contaminated sites than the surrounding sites, reflecting an elevated environmental risk. Three contaminated site areas had lower microbial activities than their surrounding sites suggesting that high metal(loid) concentrations inhibited soil microbial communities. Interestingly, the fourth area (tailing pond) showed an opposite trend (i.e., increased microbial activity in contaminated vs. surrounding areas). The microbial thermodynamic parameters of this contaminated site were higher than its surrounding sites, suggesting that the selected microbial communities can develop a functional resistance to metal(loid)s stress. This study provides a theoretical basis for ecological prevention and control of metal-polluted areas.

Transmembrane nuclease NUMEN/ENDOD1 regulates DNA repair pathway choice at the nuclear periphery.

Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP-inhibitor resistance using an inducible CRISPR-Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and non-homologous end joining-dependent repair of double-stranded DNA breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5' overhangs through its endonuclease and 3'→5' exonuclease activities, promotes the repair of DNA lesions-including heterochromatic lamina-associated domain breaks as well as deprotected telomeres-and functions as a downstream effector of DNA-dependent protein kinase catalytic subunit. These findings underline the role of NUMEN as a key player in DNA repair pathway choice and genome-stability maintenance, and have implications for ongoing research into the development and treatment of genome instability disorders.

mTORC1-Induced Bone Marrow-Derived Mesenchymal Stem Cell Exhaustion Contributes to the Bone Abnormalities in Klotho-Deficient Mice of Premature Aging.

Stem cell exhaustion is a hallmark of aging. Klotho-deficient mice (kl/kl mice) is a murine model that mimics human aging with significant bone abnormalities. The aim of this study is using kl/kl mice to investigate the functional change of bone marrow-derived mesenchymal stem cells (BMSCs) and explore the underlying mechanism. We found that klotho deficiency leads to bone abnormalities. In addition, kl/kl BMSCs manifested hyperactive proliferation but functionally declined both in vivo and in vitro. Mammalian target of rapamycin complex 1 (mTORC1) activity was higher in freshly isolated kl/kl BMSCs, and autophagy in kl/kl BMSCs was significantly decreased, possibly through mTORC1 activation. Conditional medium containing soluble Klotho protein (sKL) rescued hyperproliferation of kl/kl BMSCs by inhibiting mTORC1 activity and restoring autophagy. Finally, intraperitoneal injection of mTORC1 inhibitor rapamycin restored BMSC quiescence, ameliorated bone phenotype, and increased life span of kl/kl mice in vivo. This research highlights a therapeutic strategy to maintain the homeostasis of adult stem cell pool for healthy bone aging.

Single-cell sequencing reveals that endothelial cells, EndMT cells and mural cells contribute to the pathogenesis of cavernous malformations.

Cavernous malformations (CMs) invading the central nervous system occur in ~0.16-0.4% of the general population, often resulting in hemorrhages and focal neurological deficits. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of CMs in humans. Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from twelve human CM samples and three control samples. A total of 112,670 high-quality cells were clustered into 11 major cell types, which shared a number of common features in CMs harboring different genetic mutations. A new EC subpopulation marked with PLVAP was uniquely identified in lesions. The cellular ligand‒receptor network revealed that the PLVAP-positive EC subcluster was the strongest contributor to the ANGPT and VEGF signaling pathways in all cell types. The PI3K/AKT/mTOR pathway was strongly activated in the PLVAP-positive subcluster even in non-PIK3CA mutation carriers. Moreover, endothelial-to-mesenchymal transition (EndMT) cells were identified for the first time in CMs at the single-cell level, which was accompanied by strong immune activation. The transcription factor SPI1 was predicted to be a novel key driver of EndMT, which was confirmed by in vitro and in vivo studies. A specific fibroblast-like phenotype was more prevalent in lesion smooth muscle cells, hinting at the role of vessel reconstructions and repairs in CMs, and we also confirmed that TWIST1 could induce SMC phenotypic switching in vitro and in vivo. Our results provide novel insights into the pathomechanism decryption and further precise therapy of CMs.

An inducible CRISPR/Cas9 screen identifies DTX2 as a transcriptional regulator of human telomerase.

Most tumor cells reactivate telomerase to ensure unlimited proliferation, whereas the expression of human telomerase reverse transcriptase (hTERT) is tightly regulated and rate-limiting for telomerase activity maintenance. Several general transcription factors (TFs) have been found in regulating hTERT transcription; however, a systematic study is lacking. Here we performed an inducible CRISPR/Cas9 KO screen using an hTERT core promoter-driven reporter. We identified numerous positive regulators including an E3 ligase DTX2. In telomerase-positive cancer cells, DTX2 depletion downregulated hTERT transcription and telomerase activity, contributing to progressive telomere shortening, growth arrest, and increased apoptosis. Utilizing BioID, we characterized multiple TFs as DTX2 proximal proteins, among which NFIC functioned corporately with DTX2 in promoting hTERT transcription. Further analysis demonstrated that DTX2 mediated K63-linked ubiquitination of NFIC, which facilitated NFIC binding to the hTERT promoter and enhanced hTERT expression. These findings highlight a new hTERT regulatory pathway that may be exploited for potential cancer therapeutics.

S100A10 regulates tumor necrosis factor alpha-induced apoptosis in chondrocytes via the reactive oxygen species/nuclear factor-kappa B pathway.

Aberrant chondrocyte apoptosis and inflammation are the most critical causes of osteoarthritis (OA) development. This study was designed to demonstrate the relationship between S100A10 and OA. In this study, S100A10 was overexpressed or silenced in rat chondrocytes. Cell viability, apoptosis, reactive oxidative species (ROS), and calcium ion detection were assessed using Cell Counting Kit-8 assay and flow cytometry. The levels of key oxidation-related enzymes and tumor necrosis factor-alpha (TNF-α) were quantified using enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and Western blotting. S100A10 was highly expressed in patients with OA and positively correlated with TNF-α level. Knockdown of S100A10 effectively counteracted TNF-α-induced ROS level, apoptosis, and calcium level and associated with decreased inflammation-related metalloproteinase 1 (MMP1), MMP13, and nuclear necrosis factor-kappa B (NF-κB)-p65 and increased survivin and cytoplasmic NF-κB-p65. Overexpression of S100A10 had an effect similar to TNF-α, which was significantly counteracted by pyrrolidine dithiocarbamate, an NF-κB inhibitor, or verapamil, a calcium-channel blocker. S100A10 contributed to chondrocyte apoptosis through the ROS/NF-κB pathway. This study has established the relationship between S100A10 and the NF-κB pathway, thus providing novel perspectives for exploring S100A10 functions.

Over-expression of SRD5A3 and its prognostic significance in breast cancer.

OBJECTIVE: The study aimed to compare the Steroid 5 alpha-reductase 3 (SRD5A3) expression levels in breast cancer (BC) and normal tissues, to investigate the prognostic value of SRD5A3 mRNA expression in BC patients and to identify the SRD5A3-related signaling pathways using bioinformatics approaches. METHODS: We evaluated the expression levels of SRD5A3 and survival data in BC patients using different bioinformatic databases. Further, Cox regression analysis was conducted to predict the independent prognostic factors for BC. Moreover, the association of SRD5A3 with clinicopathological factors was measured through LinkedOmics database. And the potential role of SRD5A3 was determined by Gene Ontology and KEGG pathway enrichment analysis. Finally, protein network of SRD5A3 was constructed and genetic alterations were analyzed. RESULTS: Bioinformatic data indicated that both mRNA and protein expression levels of SRD5A3 were higher in BC group than those in the normal group (P < 0.05). Besides, BC patients with higher SRD5A3 mRNA expression levels had a lower overall survival (all P < 0.05). Cox regression analysis further demonstrated the independent prognostic value of SRD5A3 in BC (P = 0.015). SRD5A3 mRNA expression was significantly associated with N stage (P < 0.001), age (P < 0.05), and histologic subtype (P < 0.001) but had no significant relationship with other clinical characteristics (all P > 0.05). Moreover, the functional enrichment analysis revealed that the SRD5A3 was involved in metabolism-related pathways (all P < 0.05). CONCLUSIONS: SRD5A3 was highly expressed in BC tissues and high SRD5A3 expression was related to poorer prognosis. SRD5A3 serves as an oncogene and might function as a potential biomarker for prognosis and a therapeutic target for BC.

New triterpenoid saponins from the leaves of Ilex chinensis and their hepatoprotective activity.

Seven new triterpenoid saponins, including five ursane-type saponins, ilexchinenosides R-V (1-5), and two oleanane-type saponins, ilexchinenosides W-X (6-7), with four known triterpenoid saponins (8-11) were isolated from the leaves of Ilex chinensis. Their structures were elucidated by comprehensive spectroscopic 1D and 2D NMR and HR-ESI-MS data. Their sugar moieties were determined by HPLC analysis compared with standards after hydrolysis and derivatization. Compounds 1, 2, 4, 9 and 10 exhibited potential hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced HepG2 cell injury in vitro.

Minor alkaloids from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Seven new monoterpene alkaloids (1-7), along with 18 known analogues, were isolated from an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). Their structures were determined by spectroscopic data analysis and electronic circular dichroism (ECD) calculations. Compound 1 is the first monoterpene 22-norindoloquinolizidine alkaloid with a ketene unit, while 2 and 3 are unusual indoloquinolizidine alkaloids having an oxazinane ring.[Formula: see text].

Minor triterpenes from an aqueous extract of the hook-bearing stem of Uncaria rhynchophylla.

Six new triterpenes, uncarinic acids KP (1-6), along with 24 known analogues, were isolated as minor constituents of an aqueous decoction of the hook-bearing stems of Uncaria rhynchophylla (Gou-teng). By comprehensive spectroscopic data analysis, their structures were elucidated as derivatives of olean-12-en-28-oic acid and urs-12-en-28-oic acid with different oxidized forms at C-3, C-6, and/or C-23, respectively. Cell-based preliminary bioassay showed that the (E)-/(Z)-coumaroyloxy and (E)-/(Z)-feruloyloxy units at C-27 of olean-12-en-28-oic acid and urs-12-en-28-oic acid played roles in their bioactivities.[Formula: see text].

Inhibitory effects of alkaline extract from the pericarp of Citrus reticulata Blanco on collagen behavior in bleomycin-induced pulmonary fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Peel of Citrus reticulata, a Chinese herbal drug with functions of regulating Qi and expelling phlegm, has been used for the treatment of lung related diseases in Chinese medicine for a long time. Its detailed effects on collagen in anti-idiopathic pulmonary fibrosis (IPF) is still unclear. AIM OF THE STUDY: To explore the effects of citrus alkaline extract (CAE) on collagen synthesis, crosslinking and deposition in pulmonary fibrosis and understand the possible signal pathways involved in the activity. MATERIALS AND METHODS: CAE was prepared from C. reticulata. Bleomycin-induced pulmonary fibrosis mouse model was applied. Pulmonary fibrosis of lung was estimated with histopathology analysis, and collagen deposition was evaluated with immunohistochemistry. Collagen crosslinking related biomarkers and enzymes were analyzed with chemical methods, immunohistochemical and western blot analyses. RESULTS: CAE oral administration lowered hydroxyproline content, inhibited the collagen deposition including expressions of collagen I and III, and relieved bleomycin-induced pulmonary fibrosis in mice model. The productions of a collagen crosslink pyridinoline and crosslinking related enzymes including lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1) in lung were suppressed by CAE treatment. Furthermore, the protein expressions of TGF-ß1 and Smad3 levels in lungs were also downregulated by CAE. CONCLUSIONS: This study demonstrated that CAE inhibited collagen synthesis, crosslinking and deposition, and ameliorated bleomycin-induced pulmonary fibrosis. Preliminary mechanism study revealed that CAE exerted its bioactivity at least via downregulation of TGF-ß1/Smad3 pathway. Our findings provided a great potential in fighting IPF based on CAE.

Baihe Wuyao decoction ameliorates CCl4-induced chronic liver injury and liver fibrosis in mice through blocking TGF-ß1/Smad2/3 signaling, anti-inflammation and anti-oxidation effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Baihe Wuyao decoction (BWD), a prescription of Traditional Chinese Medicines, composed of Lilium brownii var. viridulum Baker.(Lilii Bulbus) and Lindera aggregata (Sims) Kosterm. (Linderae Radix), has been used to treat epigastric pain and superficial gastritis for hundreds of years in China. Recently, some compounds obtained from Lilii Bulbus and Linderae Radix had active effects of hepatic protection or liver fibrosis alleviation. Thus, we aim to evaluate the effects of BWD on treatment of chronic liver injury and liver fibrosis induced by carbon tetrachloride (CCl4) and to elucidate the possible molecular mechanism. MATERIALS AND METHODS: Mice were treated with BWD (low, medium and high dose), diammonium glycyrrhizinate or vehicle by oral gavage once daily, simultaneously intraperitoneal injected with a single dose of CCl4 (1 µl/g body weight) twice a week for consecutive 6 weeks. Next, all mice were sacrificed after fasted 12 h, and serums and liver tissues were harvested for analysis. The hepatic injury was detected by serum biomarker assay, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The hepatic histology and collagen were illustrated by hematoxylin-eosin staining and Sirius red staining respectively. The antioxidant capacity of liver tissues was evaluated by the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenization. The mRNA gene or protein expressions related to fibrosis, oxidative stress and inflammation molecules were performed by real-time quantitative PCR (RT-PCR) or Western-blot. RESULTS: BWD exhibited a good hepatic protection with ameliorating liver histological changes, decreasing serum AST and ALT contents, and reducing hepatic fibrosis with stimulation ECMs (such as Collagen1 and Collagen3) degradation. BWD inhibited hepatic stellate cells (HSCs) activation, promoted matrix metalloproteinase-2 (MMP2), MMP9, and MMP12 while suppressing tissue inhibitors of matrix metalloproteinase-1 (TIMP1) expression, and blocked traditional fibrosis TGF-ß1/Smad2/3 signal pathway. Moreover, BWD exhibited anti-inflammation effect proved by the reduction of liver Interleukin-1ß (IL-1ß), TNF-α, IL-11 mRNA levels and promoted anti-oxidation effects determined by inhibition of liver MDA and iNOS levels while promoting liver SOD and Mn-SOD. CONCLUSION: BWD ameliorates CCl4-induced CLI and liver fibrosis which is correlated to its blocking TGF-ß1/Smad2/3 signaling, anti-inflammation, and anti-oxidation effects. BWD, as a small traditional prescription, is a promising treatment for CLI and liver fibrosis through multiple pharmacological targets.