Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC.

BACKGROUND AND AIMS: Sorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC. MATERIALS AND METHODS: A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array. RESULTS: CRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC. CONCLUSION: Our study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.

CRISPR/Cas9 screens unravel miR-3689a-3p regulating sorafenib resistance in hepatocellular carcinoma via suppressing CCS/SOD1-dependent mitochondrial oxidative stress.

AIMS: Therapeutic outcome of sorafenib in hepatocellular carcinoma (HCC) is undermined by the development of drug resistance. This study aimed to identify the critical microRNA (miRNA) which is responsible for sorafenib resistance at the genomic level. METHODS: CRISPR/Cas9 screen followed by gain- and loss-of-function assays both in vitro and in vivo were applied to identify the role of miR-3689a-3p in mediating sorafenib response in HCC. The upstream and downstream molecules of miR-3689a-3p and their mechanism of action were investigated. RESULTS: CRISPR/Cas9 screening identified miR-3689a-3p was the most up-regulated miRNA in sorafenib sensitive HCC. Knockdown of miR-3689a-3p significantly increased sorafenib resistance, while its overexpression sensitized HCC response to sorafenib treatment. Proteomic analysis revealed that the effect of miR-3689a-3p was related to the copper-dependent mitochondrial superoxide dismutase type 1 (SOD1) activity. Mechanistically, miR-3689a-3p targeted the 3'UTR of the intracellular copper chaperone for superoxide dismutase (CCS) and suppressed its expression. As a result, miR-3689a-3p disrupted the intracellular copper trafficking and reduced SOD1-mediated scavenge of mitochondrial oxidative stress that eventually caused HCC cell death in response to sorafenib treatment. CCS overexpression blunted sorafenib response in HCC. Clinically, miR-3689a-3p was down-regulated in HCC and predicted favorable prognosis for HCC patients. CONCLUSION: Our findings provide comprehensive evidence for miR-3689a-3p as a positive regulator and potential druggable target for improving sorafenib treatment in HCC.

Immunomodulatory potential of natural products from herbal medicines as immune checkpoints inhibitors: Helping to fight against cancer via multiple targets.

Immunotherapy sheds new light to cancer treatment and is satisfied by cancer patients. However, immunotoxicity, single-source antibodies, and single-targeting stratege are potential challenges to the success of cancer immunotherapy. A huge number of promising lead compounds for cancer treatment are of natural origin from herbal medicines. The application of natural products from herbal medicines that have immunomodulatory properties could alter the landscape of immunotherapy drastically. The present study summarizes current medication for cancer immunotherapy and discusses the potential chemicals from herbal medicines as immune checkpoint inhibitors that have a broad range of immunomodulatory effects. Therefore, this review provides valuable insights into the efficacy and mechanism of actions of cancer immunotherapies, including natural products and combined treatment with immune checkpoint inhibitors, which could confer an improved clinical outcome for cancer treatment.

Lysyl Oxidase-Like 4 Fosters an Immunosuppressive Microenvironment During Hepatocarcinogenesis.

BACKGROUND AND AIMS: Lysyl oxidase-like 4 (LOXL4) is an amine oxidase that is primarily involved in extracellular matrix remodeling and is highly expressed in HCC tissues, but its functional role in mediating liver carcinogenesis is poorly understood. Therefore, we aimed to investigate the role of LOXL4 in hepatocarcinogenesis. APPROACH AND RESULTS: Here, we demonstrate that hepatic LOXL4 expression was increased during the liver carcinogenesis in mice concomitantly fed a choline-deficient, l-amino acid-defined diet. LOXL4 was secreted by the neoplastic cells and primarily localized within hepatic macrophages through exosome internalization. Supplementation of LOXL4 had minimal effect on neoplastic cells. In vitro exposure of macrophages to LOXL4 invoked an immunosuppressive phenotype and activated programmed death ligand 1 (PD-L1) expression, which further suppressed the function of CD8+ T cells. Injection of LOXL4 promoted macrophages infiltration into the liver and accelerated tumor growth, which was further abolished by adoptive T-cell transfer or PD-L1 neutralization. Label-free proteomics analysis revealed that the immunosuppressive function of LOXL4 on macrophages primarily relied on interferon (IFN)-mediated signal transducer and activator of transcription-dependent PD-L1 activation. Hydrogen peroxide scavenger or copper chelation on macrophages abolished the IFN-mediated PD-L1 presentation by LOXL4. In human HCC tissue, expression of LOXL4 in CD68+ cells was positively correlated with PD-L1 level. High expression of LOXL4 in CD68+ cells and low expression of CD8A in tumor tissue cooperatively predict poor survival of patients with HCC. CONCLUSIONS: LOXL4 facilitates immune evasion by tumor cells and leads to hepatocarcinogenesis. Our study unveils the role of LOXL4 in fostering an immunosuppressive microenvironment during hepatocarcinogenesis.

Epigenetic regulation in human cancer: the potential role of epi-drug in cancer therapy.

Epigenetics is dynamic and heritable modifications to the genome that occur independently of DNA sequence. It requires interactions cohesively with various enzymes and other molecular components. Aberrant epigenetic alterations can lead to inappropriate onset of genetic expressions and promote tumorigenesis. As the epigenetic modifiers are susceptible to extrinsic factors and reversible, they are becoming promising targets in multiple cancer therapies. Recently, various epi-drugs have been developed and implicated in clinical use. The use of epi-drugs alone, or in combination with chemotherapy or immunotherapy, has shown compelling outcomes, including augmentation of anti-tumoral effects, overcoming drug resistance, and activation of host immune response.

Combination of Gentiana rhodantha and Gerbera anandria in the BL02 formula as therapeutics to non-small cell lung carcinoma acting via Rap1/cdc42 signaling: A transcriptomics/ bio-informatics biological validation approach.

BACKGROUND: Non-small cell lung cancer (NSCLC) ranks the most commonly diagnosed and highest mortality-leading cancer worldwide despite a variety of treatment strategies are available. The highly heterogeneous and aggressive property of NSCLC as well as its poor prognosis indicates the need for novel therapeutic targets identification. The objective of this study is to identify potential targets from the adjuvant herbal formula BL02 using a combined approach of high throughput transcriptomics and network pharmacology. METHODS: The quality and stability of BL02 were assessed by UHPLC analysis. The inhibitory effect of BL02 on NSCLC was measured by in vivo orthotopic intrathoracic mouse model and in vitro cellular models. EGFR-mutant HCC827 and wild type A549 cell lines were employed. Transcriptomics analysis was introduced to profile the gene expression of NSCLC cells treated with BL02; Network pharmacology and molecular docking analyses predicted the interaction of compounds and NSCLC targets. Immuno-blotting and pull-down assays verified the putative targets. RESULTS: The UHPLC analysis revealed that BL02 was relatively stable between batches of production and for 24 months of storage. Orally administration of BL02 was safe and effective to inhibit pulmonary NSCLC growth in mice implanted with A549 and HCC827-generated tumors. BL02 exhibited relatively low cytotoxicity to NSCLC cells in vitro, but potently suppressed NSCLC cell motility. The transcriptomic analysis illustrated that EGFR and cellular adhesion-related signaling is involved in BL02 action. Further bioinformatics analysis validated BL02 activity is mediated by cdc42-regulated signaling. BL02 depolymerized the actin cytoskeleton through suppressing cdc42 and deactivating its upstream molecule Rap1. These effects may be primarily mediated by the direct binding of 5-methylcoumarin-4-cellobioside and mangiferin from BL02 to Rap1 protein. CONCLUSION: Our study proposes an integration model of experimental, transcriptomic and bioinformatics analyses in the identification of novel therapeutic target of NSCLC from an adjuvant herbal formula BL02. Our findings revealed that inhibition of Rap1/cdc42 signaling by active compounds 5-methylcoumarin-4-cellobioside and mangiferin from BL02 might be potentially effective therapy for NSCLC.

Tumor microenvironment-driven non-cell-autonomous resistance to antineoplastic treatment.

Drug resistance is of great concern in cancer treatment because most effective drugs are limited by the development of resistance following some periods of therapeutic administration. The tumor microenvironment (TME), which includes various types of cells and extracellular components, mediates tumor progression and affects treatment efficacy. TME-mediated drug resistance is associated with tumor cells and their pericellular matrix. Noninherent-adaptive drug resistance refers to a non-cell-autonomous mechanism in which the resistance lies in the treatment process rather than genetic or epigenetic changes, and this mechanism is closely related to the TME. A new concept is therefore proposed in which tumor cell resistance to targeted therapy may be due to non-cell-autonomous mechanisms. However, knowledge of non-cell-autonomous mechanisms of resistance to different treatments is not comprehensive. In this review, we outlined TME factors and molecular events involved in the regulation of non-cell-autonomous resistance of cancer, summarized how the TME contributes to non-cell-autonomous drug resistance in different types of antineoplastic treatment, and discussed the novel strategies to investigate and overcome the non-cell-autonomous mechanism of cancer non-cell-autonomous resistance.

OMICs approaches-assisted identification of macrophages-derived MIP-1γ as the therapeutic target of botanical products TNTL in diabetic retinopathy.

BACKGROUND: Inflammatory reaction in the dysfunction of retinal endotheliocytes has been considered to play a vital role in diabetic retinopathy (DR). Anti-inflammatory therapy so far gains poor outcome as DR treatment. This study aims to identify a novel therapeutic target of DR from the OMICs studies of a traditional anti-DR botanical products TNTL. METHODS: Hyperglycemic mice were treated with TNTL. The anti-hyperglycemic effect of TNTL was validated to confirm the biological consistency of the herbal products from batches. Improvement of DR by TNTL was examined by various assays on the retina. Next-generation transcriptome sequencing and cytokine array was used to identify the therapeutic targets. In vitro study was performed to validate the target. RESULTS: We observed that TNTL at its high doses possessed anti-hyperglycemic effect in murine type I diabetic model, while at its doses without reducing blood glucose, it suppressed DR incidence. TNTL restored the blood-retina barrier integrity, suppressed retinal neovascularization, and attenuated the retinal ganglion cell degeneration. Transcriptomic analysis on the retina tissue of hyperglycemic mice with or without TNTL revealed that the inflammatory retina microenvironment was significantly repressed. TNTL treatment suppressed pro-inflammatory macrophages in the retina, which resulted in the inactivation of endothelial cell migration, restoration of endothelial cell monolayer integrity, and prevention of leakage. Cytokine array analysis suggested that TNTL could significantly inhibit the secretion of MIP1γ from pro-inflammatory macrophages. Prevention of endothelial dysfunction by TNTL may be mediated by the inhibition of MIP1γ/CCR1 axis. More specifically, TNTL suppressed MIP1γ release from pro-inflammatory macrophages, which in turn inhibited the activation of CCR1-associated signaling pathways in endothelial cells. CONCLUSION: Our findings demonstrated that TNTL might be an alternative treatment to DR, and the primary source of potential drug candidates against DR targeting MIP1γ/CCR1 axis in the retinal microenvironment.

Targeting tumour microenvironment by tyrosine kinase inhibitor.

Tumour microenvironment (TME) is a key determinant of tumour growth and metastasis. TME could be very different for each type and location of tumour and TME may change constantly during tumour growth. Multiple counterparts in surrounding microenvironment including mesenchymal-, hematopoietic-originated cells as well as non-cellular components affect TME. Thus, therapeutics that can disrupt the tumour-favouring microenvironment should be further explored for cancer therapy. Previous efforts in unravelling the dysregulated mechanisms of TME components has identified numerous protein tyrosine kinases, while its corresponding inhibitors have demonstrated potent modulatory effect on TME. Recent works have demonstrated that beyond the direct action on cancer cells, tyrosine kinase inhibitors (TKIs) have been implicated in inactivation or normalization of dysregulated TME components leading to cancer regression. Either through re-sensitizing the tumour cells or reversing the immunological tolerance microenvironment, the emergence of these TME modulatory mechanism of TKIs supports the combinatory use of TKIs with current chemotherapy or immunotherapy for cancer therapy. Therefore, an appropriate understanding on TME modulation by TKIs may offer another mode of action of TKIs for cancer treatment. This review highlights mode of kinase activation or paracrine ligand production from TME components and summarises the findings on the potential use of various TKIs on regulating TME components. At last, the combination use of current TKIs with immunotherapy in the perspectives of efficacy and safety are discussed.

Repression of WT1-Mediated LEF1 Transcription by Mangiferin Governs ß-Catenin-Independent Wnt Signalling Inactivation in Hepatocellular Carcinoma.

BACKGROUND/AIMS: The development of hepatocellular carcinoma (HCC) is a complex process which involves deregulation of multiple signalling pathways. The hyper-activation of Wnt signalling promotes sustained expansion, invasion, and neovascularization of HCC. Mangiferin, a natural small molecule present in Mangifera indica L. has been shown to inactivate ß-catenin, which is an indispensable regulator in Wnt pathway. Our study aimed to determine whether mangiferin has any inhibitory effect on HCC and examine how it modulates Wnt signalling. METHODS: The tumour inhibitory effect of mangiferin was examined by in vitro cellular models and an in vivo orthotopic HCC implantation model. The genes responsible for mangiferin-mediated anti-HCC were delineated by polymerase chain reaction (PCR) microarray. The expression of target genes was further determined by quantitative PCR and immuno-blotting assays. The binding capacity of Wilms' tumour 1 (WT1) to the lymphoid enhancer-binding factor 1 (LEF1) promoter was confirmed by chromatin immunoprecipitation-qPCR. RESULTS: Oral administration of mangiferin inhibited orthotopic tumour growth. Cellular investigations confirmed the dose-dependent inhibition of mangiferin on HCC expansion and invasion. PCR array combined with Gene Ontology analysis revealed that the Wnt pathway was the predominant target of mangiferin and LEF1 was the most reduced gene in the Wnt pathway. Overexpression of LEF1 diminished repression of Wnt signalling and reduced proliferation activity in mangiferin-treated HCC cells. The mangiferin-mediated down-regulation of LEF1 was independent of ß-catenin but associated with WT1 protein. WT1 knock-in in HCC cells further enhanced LEF1 expression. Chromatin immunoprecipitation assays revealed that the mangiferin induced repression of LEF1 was associated with decreased occupancy of WT1 on the LEF1 promoter. CONCLUSION: Our study identifies a novel mechanism of hepatocellular carcinoma inhibition through ß-catenin-independent Wnt signalling, which is regulated by WT1-associated LEF1 repression. The study also highlights mangiferin as a promising Wnt inhibitor for HCC treatment.

Molecular Mechanisms Involved in Oxidative Stress-Associated Liver Injury Induced by Chinese Herbal Medicine: An Experimental Evidence-Based Literature Review and Network Pharmacology Study.

Oxidative stress, defined as a disequilibrium between pro-oxidants and antioxidants, can result in histopathological lesions with a broad spectrum, ranging from asymptomatic hepatitis to hepatocellular carcinoma in an orchestrated manner. Although cells are equipped with sophisticated strategies to maintain the redox biology under normal conditions, the abundance of redox-sensitive xenobiotics, such as medicinal ingredients originated from herbs or animals, can dramatically invoke oxidative stress. Growing evidence has documented that the hepatotoxicity can be triggered by traditional Chinese medicine (TCM) during treating various diseases. Meanwhile, TCM-dependent hepatic disorder represents a strong correlation with oxidative stress, especially the persistent accumulation of intracellular reactive oxygen species. Of note, since TCM-derived compounds with their modulated targets are greatly diversified among themselves, it is complicated to elaborate the potential pathological mechanism. In this regard, data mining approaches, including network pharmacology and bioinformatics enrichment analysis have been utilized to scientifically disclose the underlying pathogenesis. Herein, top 10 principal TCM-modulated targets for oxidative hepatotoxicity including superoxide dismutases (SOD), malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), glutathione peroxidase (GPx), Bax, caspase-3, Bcl-2, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and nitric oxide (NO) have been identified. Furthermore, hepatic metabolic dysregulation may be the predominant pathological mechanism involved in TCM-induced hepatotoxic impairment.

Hepatoprotective Effects of a Functional Formula of Three Chinese Medicinal Herbs: Experimental Evidence and Network Pharmacology-Based Identification of Mechanism of Action and Potential Bioactive Components.

Various Chinese herbal medicines (CHMs) have shown beneficial liver protection effects. Jian-Gan-Bao (JGB), a functional herbal formula, consists of three famous CHMs, including Coriolus versicolor, Salvia miltiorrhiza and Schisandra chinensis, which has been used as a folk medicine for several chronic liver diseases. In the present study, we aim systemically to evaluate the effects of JGB on acute and chronic alcoholic liver diseases (ALD) as well as non-alcoholic fatty liver disease (NAFLD) in mouse models, and identify its potential bioactive components and mechanism of action. JGB showed preventive effects for acute and chronic ALD as well as NAFLD, while post-treatment of JGB showed no significant effect, suggesting the nature of JGB as a health supplement rather than a drug. Furthermore, a compound-target network was constructed to identify the potential bioactive compounds and pathways that regulate its hepatoprotective effects. There are 40 bioactive compounds and 15 related targets that have been identified via this network pharmacology study. Among them are miltirone, neocryptotanshinone II and deoxyshikonin, with desirable pharmaceutical properties. Pathways relating to inflammation, fatty acid oxidation, tumor necrosis factor (TNF) production and cell proliferation were predicted as bioactive compounds and potential underlying mechanisms, which should be the focus of study in this field in the future.

A Biomedical Investigation of the Hepatoprotective Effect of Radix salviae miltiorrhizae and Network Pharmacology-Based Prediction of the Active Compounds and Molecular Targets.

Radix salviae miltiorrhizae (Danshen in Chinese), a classic traditional Chinese medicine (TCM) herb, has been used for centuries to treat liver diseases. In this study, the preventive and curative potential of Danshen aqueous extract on acute/chronic alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) was studied. The in vivo results indicated that Danshen could alleviate hepatic inflammation, fatty degeneration, and haptic fibrogenesis in ALD and NAFLD models. In the aspect of mechanism of action, the significant reduction in MDA levels in both ALD and NAFLD models implies the decreased levels of oxidative stress by Danshen. However, Danshen treatment could not activate the internal enzymatic antioxidant system in ALD and NAFLD models. To further explore the hepatoprotective mechanism of Danshen, an in silico-based network pharmacology approach was employed in the present study. The pharmacological network analysis result revealed that six potential active ingredients such as tanshinone iia, salvianolic acid b, and Danshensu may contribute to the hepatoprotective effects of Danshen on ALD and NAFLD. The action mechanism may relate with regulating the intracellular molecular targets such as PPARα, CYP1A2, and MMP2 for regulation of lipid metabolism, antioxidant and anti-fibrogenesis by these potential active ingredients. Our studies suggest that the combination of network pharmacology strategy with in vivo experimental study may provide a forceful tool for exploring the mechanism of action of traditional Chinese medicine (TCM) herb and developing novel bioactive ingredients.

New Natural Pigment Fraction Isolated from Saw Palmetto: Potential for Adjuvant Therapy of Hepatocellular Carcinoma.

For the first time, we discovered a small proportion of aqueous fraction from Saw Palmetto apart from the fatty acid-rich fraction exhibited pharmacological activity. Therefore, this study aims to explore the anti-tumor potential of red pigmented aqueous fraction of Saw Palmetto, NYG on human hepatocellular carcinoma and its possible targets. Subcutaneous xenograft and orthotopic implantation models of HCC were used to evaluate the tumor inhibitory effect of NYG. Human hepatocellular carcinoma (HCC) cell lines and human umbilical vein endothelial cells (HUVEC) were used as in vitro model. The mRNA expression was conducted by qPCR. Protein expression was monitored by immunoblotting and immunohistochemistry. Cell migration and blood vessel formation were determined by chamber assay and tube formation assay, respectively. Significant tumor inhibition of NYG in dose-dependent manner was observed on subcutaneous xenograft and orthotopic HCC model. NYG has no direct action on cell viability or VEGF secretion of HCC cells. However, NYG reduced in vitro migration and vessel formation activities of HUVEC cells, as well as in vivo intratumoral neovascularization. NYG attenuated extracellular signal-regulated kinases (ERK) activation in endothelial cells, which may be associated with the suppression of migration and tube formation of HUVEC. NYG suppressed tumor expansion of HCC via inhibiting neovascularization, and may be potential adjuvant treatment for HCC.

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells.

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-ß-Transducin Repeat Containing Protein (SCFß-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of ß-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine's potential as an anti-tumor agent for clinical cancer therapy.

Berberine-induced tumor suppressor p53 up-regulation gets involved in the regulatory network of MIR-23a in hepatocellular carcinoma.

AIM OF THE STUDY: To investigate the involvement of p53 in the regulatory network of microRNA-23a (miR-23a) in berberine-treated hepatocellular carcinoma (HCC) cells. METHODS: The biogenesis of miR-23a upon berberine treatment was monitored by detecting the transcript expression of primary precursor, precursor and mature forms of miR-23a. Protein expression was detected with immunoblotting. The binding capacity between p53 and chromatin DNA was determined by chromatin immunoprecipitation. The role of miR-23a in mediating suppression of HCC by berberine was determined both in vitro and in vivo. RESULTS: miR-23a was up-regulated upon berberine treatment in human HCC cells, and berberine could increase the expression of primary precursor, precursor and mature forms of miR-23a. The up-regulation of miR-23a by berberine is p53-dependent. Inhibition of p53 expression and activity could block the up-regulation of miR-23a induced by berberine. Furthermore, berberine-induced miR-23a expression may mediate the transcription activation of p53-related tumor suppressive genes p21 and GADD45α. Inhibition of miR-23a abolishes the binding of p53 onto chromatin and attenuates transcription activation of p21 and GADD45α. Target prediction and experimental validation demonstrate that berberine-induced miR-23a may target to Nek6 to suppress its expression. Berberine-induced G2/M cell cycle arrest in HCC was attenuated when miR-23a was inhibited. Berberine-induced cell death and in vivo tumor growth inhibition are attenuated upon inhibition of miR-23a. CONCLUSION: Our study reveals that miR-23a may be involved in regulating the anti-HCC effect of berberine by mediating the regulation of p53.

The Role of Oxidative Stress and Antioxidants in Liver Diseases.

A complex antioxidant system has been developed in mammals to relieve oxidative stress. However, excessive reactive species derived from oxygen and nitrogen may still lead to oxidative damage to tissue and organs. Oxidative stress has been considered as a conjoint pathological mechanism, and it contributes to initiation and progression of liver injury. A lot of risk factors, including alcohol, drugs, environmental pollutants and irradiation, may induce oxidative stress in liver, which in turn results in severe liver diseases, such as alcoholic liver disease and non-alcoholic steatohepatitis. Application of antioxidants signifies a rational curative strategy to prevent and cure liver diseases involving oxidative stress. Although conclusions drawn from clinical studies remain uncertain, animal studies have revealed the promising in vivo therapeutic effect of antioxidants on liver diseases. Natural antioxidants contained in edible or medicinal plants often possess strong antioxidant and free radical scavenging abilities as well as anti-inflammatory action, which are also supposed to be the basis of other bioactivities and health benefits. In this review, PubMed was extensively searched for literature research. The keywords for searching oxidative stress were free radicals, reactive oxygen, nitrogen species, anti-oxidative therapy, Chinese medicines, natural products, antioxidants and liver diseases. The literature, including ours, with studies on oxidative stress and anti-oxidative therapy in liver diseases were the focus. Various factors that cause oxidative stress in liver and effects of antioxidants in the prevention and treatment of liver diseases were summarized, questioned, and discussed.

The double-edged sword effect of indigo naturalis.

Indigo naturalis (IN) is a dried powder derived from plants such as Baphicacanthus cusia (Neeks) Bremek., Polygonum tinctorium Ait. and Isatis indigotica Fork. It has a historical application as a dye in ancient India, Egypt, Africa and China. Over time, it has been introduced to China and Japan for treatment of various ailments including hemoptysis, epistaxis, chest discomfort, and aphtha. Clinical and pre-clinical studies have widely demonstrated its promising effects on autoimmune diseases like psoriasis and Ulcerative colitis (UC). Despite the documented efficacy of IN in UC patients, concerns have been raised on the development of adverse effects with long term consumption, prompting a closer examination of its safety and tolerability in these contexts. This review aims to comprehensively assess the efficacy of IN in both clinical and pre-clinical settings, with a detailed exploration of the mechanisms of action involved. Additionally, it summarizes the observed potential toxicity of IN in animal and human settings was summarized. This review will deepen our understanding on the beneficial and detrimental effects of IN in UC, providing valuable insights for its future application in patients with this condition.

Modified Zhenwu Decoction suppresses chronic colitis via targeting macrophage CCR2/Fyn/p38 MAPK signaling axis.

BACKGROUND: Ulcerative colitis (UC) is associated with intestinal macrophage infiltration due to disruption of the mucosal barrier and bacterial invasion. Therefore, it is crucial to identify therapeutic agents capable of attenuating the macrophage-induced inflammatory response to preserve mucosal homeostasis and immune tolerance. The modified Zhenwu decoction (CDD-2103) is a novel herbal formulation developed based on the principles of Traditional Chinese medicine. To date, there are no clinically approved herbal formulations for UC with a well-known mechanism of action on macrophages. PURPOSE: The objective of this study was to systematically investigate the inhibitory effect of the active fraction of CDD-2103 in a mouse model of chronic colitis and delineate the mechanisms underlying its inhibitory action. METHODS: CDD-2103 was extracted into four fractions using organic solvents with increasing polarity. A chronic 49-day dextran sulfate sodium (DSS)-induced colitis mice model, closely resembling human clinical conditions, was used to examine the effect of CDD-2103 on chronic colitis. To confirm the effect of CDD-2103 on macrophages in this chronic colitis model, adoptive macrophage transfer and CCL2 supplementation were conducted. The mechanisms of action of CDD-2103 were further elucidated utilizing bone marrow-derived macrophages (BMDMs). Transcriptome analysis was conducted to gain insights into the underlying mechanism of action of CDD-2103 in BMDMs. RESULTS: Our in vitro and in vivo findings demonstrated that the ethanol-enriched fraction of CDD-2103 exhibited significant anti-inflammatory effects, leading to the suppression of colitis severity. This effect was associated with diminished accumulation of colonic macrophages in the lamina propria of CDD-2103-intervened colitis mice. Specifically, CDD-2103 inhibited CCR2/L2-mediated proinflammatory macrophage infiltration into the colon without affecting macrophage proliferation. Mechanistically, CDD-2103 inhibited Fyn expression-mediated p38 MAPK activation and subsequently suppressed CCR2 expression in BMDMs. CONCLUSIONS: Collectively, our study supports the potential use of CDD-2103 to limit macrophage infiltration, thereby reducing inflammation during UC treatment. CDD-2103 and the components in the ethanolic fraction are promising candidates for the development of novel drugs for UC management. Additionally, our study underscores Fyn-mediated CCR2 expression as a potential therapeutic target for the management of UC.

PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer.

BACKGROUND: Par-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of PARD3 in hepatic tumorigenesis. METHODS: The essential role of PARD3 in mediating hepatic tumorigenesis was assessed in diet-induced spontaneous liver tumour and syngeneic tumour models. The mechanism of PARD3 was delineated by bulk and single-cell RNA sequencing. The clinical significance of PARD3 was identified by tissue array analysis. RESULTS: PARD3 was overexpressed in tumour tissues and PARD3 overexpression was positively correlated with high tumour stage as well as the poor prognosis in patients. In models of spontaneous liver cancer induced by choline-deficient, amino acid-defined (CDAA) and methionine-choline-deficient (MCD) diets, upregulation of PARD3 was induced specifically at the tumorigenesis stage rather than other early stages of liver disease progression. Site-directed knockout of PARD3 using an adeno-associated virus 8 (AAV8)-delivered CRISPR/Cas9 single-guide RNA (sgRNA) plasmid blocked hepatic tumorigenesis, while PARD3 overexpression accelerated liver tumour progression. In particular, single-cell sequencing analysis suggested that PARD3 was enriched in primitive tumour cells and its overexpression enhanced tumour-initiating cell (TICs). Overexpression of PARD3 maintained the self-renewal ability of the CD133+ TIC population within hepatocellular carcinoma (HCC) cells and promoted the in vitro and in vivo tumorigenicity of CD133+ TICs. Transcriptome analysis revealed that Sonic Hedgehog (SHH) signalling was activated in PARD3-overexpressing CD133+ TICs. Mechanistically, PARD3 interacted with aPKC to further activate SHH signalling and downstream stemness-related genes. Suppression of SHH signalling and aPKC expression attenuated the in vitro and in vivo tumorigenicity of PARD3-overexpressing CD133+ TICs. Tissue array analysis revealed that PARD3 expression was positively associated with the phosphorylation of aPKC, SOX2 and Gli1 and that the combination of these markers could be used to stratify HCC patients into two clusters with different clinicopathological characteristics and overall survival prognoses. The natural compound berberine was selected as a potent suppressor of PARD3 expression and could be used as a preventive agent for liver cancer that completely blocks diet-induced hepatic tumorigenesis in a PARD3-dependent manner. CONCLUSION: This study revealed PARD3 as a potential preventive target of liver tumorigenesis via TIC regulation.