Network Pharmacology, Molecular Docking Analysis and Molecular Dynamics Simulation of Scutellaria baicalensis in the Treatment of Liver Fibrosis.

BACKGROUND: Traditional Chinese medicine Scutellaria Baicalensis (SB), one of the clinical firstline heat-clearing drugs, has obvious symptomatic advantages for hepatic fibrosis with dampness-heat stasis as its syndrome. We aim to predict and validate the potential mechanism of Scutellaria baicalensis active ingredients against liver fibrosis more scientifically and effectively. METHODS: The underlying mechanism of Scutellaria baicalensis in inhibiting hepatic fibrosis was studied by applying network pharmacology, molecular docking and molecular dynamics simulation. Expression levels of markers in activated Hepatic Stellate Cells (HSC) after administration of three Scutellaria baicalensis extracts were determined by Western blot and Real-time PCR, respectively, in order to verify the anti-fibrosis effect of the active ingredients Results: There are 164 common targets of drugs and diseases screened and 115 signaling pathways obtained, which were mainly associated with protein phosphorylation, senescence and negative regulation of the apoptotic process. Western blot and Real-time PCR showed that Scutellaria baicalensis extracts could reduce the expression of HSC activation markers, and Oroxylin A had the strongest inhibitory effect on it. Molecular docking results showed that Oroxylin A had high binding activity to target proteins. Molecular dynamics simulation demonstrates promising stability of the Oroxylin A-AKT1 complex over the simulated MD time of 200 ns. CONCLUSION: Scutellaria baicalensis active ingredients may inhibit HSC proliferation, reduce the generation of pro-inflammatory factors and block the anti-inflammatory effect of inflammatory signal transduction by inducing HSC apoptosis and senescence, thus achieving the effect of anti-fibrosis.

New horizons for the role of RNA N6-methyladenosine modification in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancy, presenting a formidable challenge to the medical community owing to its intricate pathogenic mechanisms. Although current prevention, surveillance, early detection, diagnosis, and treatment have achieved some success in preventing HCC and controlling overall disease mortality, the imperative to explore novel treatment modalities for HCC remains increasingly urgent. Epigenetic modification has emerged as pivotal factors in the etiology of cancer. Among these, RNA N6-methyladenosine (m6A) modification stands out as one of the most prevalent, abundant, and evolutionarily conserved post-transcriptional alterations in eukaryotes. The literature underscores that the dynamic and reversible nature of m6A modifications orchestrates the intricate regulation of gene expression, thereby exerting a profound influence on cell destinies. Increasing evidence has substantiated conspicuous fluctuations in m6A modification levels throughout the progression of HCC. The deliberate modulation of m6A modification levels through molecular biology and pharmacological interventions has been demonstrated to exert a discernible impact on the pathogenesis of HCC. In this review, we elucidate the multifaceted biological functions of m6A modifications in HCC, and concurrently advancing novel therapeutic strategies for the management of this malignancy.

Artesunate Induces Ferroptosis in Hepatic Stellate Cells and Alleviates Liver Fibrosis via the ROCK1/ATF3 Axis.

Background and Aims: Development of fibrosis in chronic liver disease requires activation of hepatic stellate cells (HSCs) and leads to a poor outcome. Artesunate (Art) is an ester derivative of artemisinin that can induce ferroptosis in HSCs, and activated transcriptional factor 3 (ATF3) is an ATF/CREB transcription factor that is induced in response to stress. In this study, we examined the role of the Rho-associated protein kinase 1 (ROCK1)/ATF3 axis in Art-induced ferroptosis in HSCs. Methods: HSC activation and ferroptosis were studied in vitro by western blotting, polymerase chain reaction, immunofluorescence, and other assays. ATF3 electrophoretic mobility and ROCK1 protein stability were assayed by western blotting. Immunoprecipitation was used to detect the interaction of ROCK1 and ATF3, as well as ATF3 phosphorylation. A ubiquitination assay was used to verify ROCK1 degradation. Atf3-interfering and Rock1-overexpressing mice were constructed to validate the anti-hepatic fibrosis activity of Art in vivo. Results: Art induced ferroptosis in HSCs following glutathione-dependent antioxidant system inactivation resulting from nuclear accumulation of unphosphorylated ATF3 mediated by ROCK1-ubiquitination in vitro. Art also decreased carbon tetrachloride-induced liver fibrosis in mice, which was reversed by interfering with Atf3 or overexpressing Rock1. Conclusions: The ROCK1/ATF3 axis was involved in liver fibrosis and regulation of ferroptosis, which provides an experimental basis for further study of Art for the treatment of liver fibrosis.

Autophagy activation is required for N6-methyladenosine modification to regulate ferroptosis in hepatocellular carcinoma.

BACKGROUND & AIMS: Although ferroptosis holds promise as a new strategy for treating hepatocellular carcinoma (HCC), there are several obstacles that need to be overcome. One major challenge is the lack of understanding about the mechanisms underlying ferroptosis. Additionally, while the m6A modification has been shown to regulate various forms of cell death, its role in regulating ferroptosis in HCC has been largely overlooked. Bridging this knowledge gap, our study aimed to elucidate the regulatory influence of m6A modification on HCC ferroptosis. MATERIALS: Dot blot and EpiQuik m6A RNA Methylation Quantitative kit detected changes in overall m6A modification level during ferroptosis in HCC. MeRIP-qPCR and RIP-qPCR identified that the m6A modification of ATG5 mRNA was significant changed. BALB/c nude mice were used to construct xenograft tumor models to verify the phenotypes upon YTHDC2 silencing. In addition, patient-derived organoid models were used to demonstrate that induction of ferroptosis was an effective strategy against HCC. RESULTS: Our study has shown that inducing ferroptosis is a promising strategy for combatting HCC. Specifically, we have found a significant correlation between ferroptosis and high levels of m6A modification in HCC. Notably, we discovered that the elevation of ATG5 mRNA m6A modification mediated by WTAP is dependent on the reading protein YTHDC2. Importantly, inhibition of either WTAP or YTHDC2 effectively prevented ferroptosis and suppressed HCC development in both in vitro and in vivo models. CONCLUSION: Our study revealed that WTAP upregulates ATG5 expression post-transcriptionally in an m6A-YTHDC2-dependent manner, thereby promoting the translation of ATG5 mRNA during ferroptosis in HCC. These findings have significant implications for the development of innovative and effective therapeutic approaches for HCC treatment.

Canonical Wnt signaling promotes HSC glycolysis and liver fibrosis through an LDH-A/HIF-1α transcriptional complex.

BACKGROUND AND AIMS: Aerobic glycolysis reprogramming occurs during HSC activation, but how it is initiated and sustained remains unknown. We investigated the mechanisms by which canonical Wnt signaling regulated HSC glycolysis and the therapeutic implication for liver fibrosis. APPROACH AND RESULTS: Glycolysis was examined in HSC-LX2 cells upon manipulation of Wnt/ß-catenin signaling. Nuclear translocation of lactate dehydrogenase A (LDH-A) and its interaction with hypoxia-inducible factor-1α (HIF-1α) were investigated using molecular simulation and site-directed mutation assays. The pharmacological relevance of molecular discoveries was intensified in primary cultures, rodent models, and human samples. HSC glycolysis was enhanced by Wnt3a but reduced by ß-catenin inhibitor or small interfering RNA (siRNA). Wnt3a-induced rapid transactivation and high expression of LDH-A dependent on TCF4. Wnt/ß-catenin signaling also stimulated LDH-A nuclear translocation through importin ß2 interplay with a noncanonical nuclear location signal of LDH-A. Mechanically, LDH-A bound to HIF-1α and enhanced its stability by obstructing hydroxylation-mediated proteasome degradation, leading to increased transactivation of glycolytic genes. The Gly28 residue of LDH-A was identified to be responsible for the formation of the LDH-A/HIF-1α transcription complex and stabilization of HIF-1α. Furthermore, LDH-A-mediated glycolysis was required for HSC activation in the presence of Wnt3a. Results in vivo showed that HSC activation and liver fibrosis were alleviated by HSC-specific knockdown of LDH-A in mice. ß-catenin inhibitor XAV-939 mitigated HSC activation and liver fibrosis, which were abrogated by HSC-specific LDH-A overexpression in mice with fibrosis. CONCLUSIONS: Inhibition of HSC glycolysis by targeting Wnt/ß-catenin signaling and LDH-A had therapeutic promise for liver fibrosis.

COVID-19-associated liver injury: Adding fuel to the flame.

COVID-19 is mainly characterized by respiratory disorders and progresses to multiple organ involvement in severe cases. With expansion of COVID-19 and SARS-CoV-2 research, correlative liver injury has been revealed. It is speculated that COVID-19 patients exhibited abnormal liver function, as previously observed in the SARS and MERS pandemics. Furthermore, patients with underlying diseases such as chronic liver disease are more susceptible to SARS-CoV-2 and indicate a poor prognosis accompanied by respiratory symptoms, systemic inflammation, or metabolic diseases. Therefore, COVID-19 has the potential to impair liver function, while individuals with preexisting liver disease suffer from much worse infected conditions. COVID-19 related liver injury may be owing to direct cytopathic effect, immune dysfunction, gut-liver axis interaction, and inappropriate medication use. However, discussions on these issues are infancy. Expanding research have revealed that angiotensin converting enzyme 2 (ACE2) expression mediated the combination of virus and target cells, iron metabolism participated in the virus life cycle and the fate of target cells, and amino acid metabolism regulated immune response in the host cells, which are all closely related to liver health. Further exploration holds great significance in elucidating the pathogenesis, facilitating drug development, and advancing clinical treatment of COVID-19-related liver injury. This article provides a review of the clinical and laboratory hepatic characteristics in COVID-19 patients, describes the etiology and impact of liver injury, and discusses potential pathophysiological mechanisms.

Emodin promotes hepatic stellate cell senescence and alleviates liver fibrosis via a nuclear receptor (Nur77)-mediated epigenetic regulation of glutaminase 1.

BACKGROUND AND PURPOSE: Senescence in hepatic stellate cells (HSCs) limits liver fibrosis. Glutaminolysis promotes HSC activation. Here, we investigated how emodin affected HSC senescence involving glutaminolysis. EXPERIMENTAL APPROACH: Senescence, glutaminolysis metabolites, Nur77 nuclear translocation, glutaminase 1 (GLS1) promoter methylation and related signalling pathways were examined in human HSC-LX2 cells using multiple cellular and molecular approaches. Fibrotic mice with shRNA-mediated knockdown of Nur77 were treated with emodin-vitamin A liposome for investigating the mechanisms in vivo. Human fibrotic liver samples were examined to verify the clinical relevance. KEY RESULTS: Emodin upregulated several key markers of senescence and inhibited glutaminolysis cascade in HSCs. Emodin promoted Nur77 nuclear translocation, and knockdown of Nur77 abolished emodin blockade of glutaminolysis and induction of HSC senescence. Mechanistically, emodin facilitated Nur77/DNMT3b interaction and increased GLS1 promoter methylation, leading to inhibited GLS1 expression and blockade of glutaminolysis. Moreover, the glutaminolysis intermediate α-ketoglutarate promoted extracellular signal-regulated kinase (ERK) phosphorylation, which in turn phosphorylated Nur77 and reduced its interaction with DNMT3b. This led to decreased GLS1 promoter methylation and increased GLS1 expression, forming an ERK/Nur77/glutaminolysis positive feedback loop. However, emodin repressed ERK phosphorylation and interrupted the feedback cascade, stimulating senescence in HSCs. Studies in mice showed that emodin-vitamin A liposome inhibited glutaminolysis and induced senescence in HSCs, and consequently alleviated liver fibrosis; but knockdown of Nur77 abrogated these beneficial effects. Similar alterations were validated in human fibrotic liver tissues. CONCLUSIONS AND IMPLICATIONS: Emodin stimulated HSC senescence through interruption of glutaminolysis. HSC-targeted delivery of emodin represented a therapeutic option for liver fibrosis.

Blockade of KLF5/LDH-A feedback loop contributes to Curcumol inhibition of sinusoidal endothelial cell glycolysis and mitigation of liver fibrosis.

BACKGROUND: LSECs (Liver sinusoidal endothelial cells) are the portal of liver, their pathological angiogenesis plays a constructive role in etiopathogenesis of liver fibrosis by affecting liver tissue repair and inflammatory drive. Although intervention in angiogenesis can effectively inhibit abnormal activation of LSEC, no effective drugs have been found to treat liver fibrosis. PURPOSE: We investigated the effect of the natural compound Curcumol on LSEC angiogenesis and elucidated the novel underlying mechanism, expecting to provide a scientific basis for exploring potential therapeutic drugs for liver fibrosis. METHODS: Various cellular and molecular assays, as well as genetic assays, were used to detect pathological angiogenesis and changes in glycolysis levels in cultured rat LSECs and mouse liver fibrosis models. RESULTS: Transcription factor KLF5 is able to influence the angiogenic properties of LSEC by regulating the glycolytic process, and affect the expression of LDH-A by transcriptionally binding to its promoter. In our study, we were surprised to find that LDH-A (the final step of glycolysis) has a strong regulatory effect on the glycolytic process of LSEC. Through in-depth study, we found that LDH-A could affect the transcriptional activity of KLF5, thus forming a positive feedback loop. Curcumol could break this positive feedback loop and inhibit the glycolysis-dependent angiogenic nature of LSEC, thus alleviating liver fibrosis. Curcumol reduced extracellular matrix (ECM) deposition, attenuated pathological angiogenesis in LSEC, and decreased the level of CCl4-induced liver fibrosis in mice. CONCLUSION: Our results demonstrated the great utilization potentiality of KLF5 in liver fibrosis, and the innovative discovery that LDH-A regulates the glycolytic process and forms a malignant feedback loop by exerting non-enzymatic effects. It also reveals the prospect of Curcumol-regulated KLF5/LDH-A feedback loop in the treatment of liver fibrosis, providing a new option for the future medicine of liver fibrosis.

Oroxylin A activates ferritinophagy to induce hepatic stellate cell senescence against hepatic fibrosis by regulating cGAS-STING pathway.

In recent study, the pathological mechanism of liver fibrosis has been associated with hepatic stellate cell (HSC) senescence. Targeted induction of HSC senescence is considered as a new strategy to remove activated HSC. Nevertheless, little is known about the role of ferritinophagy in cell senescence. In this study, we reported that Oroxylin A from Scutellaria baicalensis Georgi can regulate HSC senescence induced by ferritinophagy through the cGAS-STING pathway to reduce liver fibrosis. We first found that Oroxylin A treatment alleviated the pathological changes of liver fibrosis, reduced collagen deposition, and significantly inhibited liver fibrosis. Interestingly, Oroxylin A treatment can activate HSC ferritinophagy and further induce HSC senescence. It is noteworthy that ferritinophagy is mediated by nuclear receptor coactivator 4 (NCOA4), an important selective mediator for ferritin degradation. NCOA4 siRNA causes Oroxylin A to reduce the degree of telomerase activity in HSCs and induce the expression of senescence markers, such as SA-ß-Gal and related marker proteins. Importantly, the cGAS-STING pathway is crucial to the activation of HSC ferritinophagy by Oroxylin A. Specifically, Oroxylin A can promote the secretion of cytokines like IFN-ß by the cGAS-STING pathway to regulate ferritinophagy. cGAS siRNA resulted in a dose-dependent decrease in the expression of NCOA4, a significant reduction in the expression level of autophagy-related phenotype, and a decrease in the content of ROS and iron ions in HSCs. In conclusion, we identified the new role of ferritinophagy and the GAS-STING pathway in Oroxylin A -mediated anti-hepatic fibrosis.

m6A methylation-induced NR1D1 ablation disrupts the HSC circadian clock and promotes hepatic fibrosis.

The roles of nuclear receptor subfamily 1 group d member 1 (NR1D1) and the circadian clock in liver fibrosis remain unclear. Here, we showed that liver clock genes, especially NR1D1, were dysregulated in mice with carbon tetrachloride (CCl4)-induced liver fibrosis. In turn, disruption of the circadian clock exacerbated experimental liver fibrosis. NR1D1-deficient mice were more sensitive to CCl4-induced liver fibrosis, supporting a critical role of NR1D1 in liver fibrosis development. Validation at the tissue and cellular levels showed that NR1D1 was primarily degraded by N6-methyladenosine (m6A) methylation in a CCl4-induced liver fibrosis model, and this result was also validated in rhythm-disordered mouse models. In addition, the degradation of NR1D1 further inhibited the phosphorylation of dynein-related protein 1-serine site 616 (DRP1S616), resulting in weakened mitochondrial fission function and increased mitochondrial DNA (mtDNA) release in hepatic stellate cell (HSC), which in turn activated the cGMP-AMP synthase (cGAS) pathway. Activation of the cGAS pathway induced a local inflammatory microenvironment that further stimulated liver fibrosis progression. Interestingly, in the NR1D1 overexpression model, we observed that DRP1S616 phosphorylation was restored, and cGAS pathway was also inhibited in HSCs, resulting in improved liver fibrosis. Taken together, our results suggest that targeting NR1D1 may be an effective approach to liver fibrosis prevention and management.

Naringenin is a Potential Immunomodulator for Inhibiting Liver Fibrosis by Inhibiting the cGAS-STING Pathway.

Background and Aims: Naringenin is an anti-inflammatory flavonoid that has been studied in chronic liver disease. The mechanism specific to its antifibrosis activity needs further investigation This study was to focused on the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) pathway in hepatic stellate cells and clarified the antifibrosis mechanism of naringenin. Methods: The relationship between the cGAS-stimulator of interferon genes (STING) pathway and liver fibrosis was analyzed using the Gene Expression Omnibus database. Histopathology, immunohistochemistry, fluorescence staining, Western blotting and polymerase chain reaction were performed to assess gene and protein expression levels associated with the cGAS pathway in clinical liver tissue samples and mouse livers. Molecular docking was performed to evaluate the relationship between naringenin and cGAS, and western blotting was performed to study the expression of inflammatory factors downstream of cGAS in vitro. Results: Clinical database analyses showed that the cGAS-STING pathway is involved in the occurrence of chronic liver disease. Naringenin ameliorated liver injury and liver fibrosis, decreased collagen deposition and cGAS expression, and inhibited inflammation in carbon tetrachloride (CCl4)-treated mice. Molecular docking found that cGAS may be a direct target of naringenin. Consistent with the in vivo results, we verified the inhibitory effect of naringenin on activated hepatic stellate cells (HSCs). By using the cGAS-specific agonist double-stranded (ds)DNA, we showed that naringenin attenuated the activation of cGAS and its inflammatory factors affected by dsDNA. We verified that naringenin inhibited the cGAS-STING pathway, thereby reducing the secretion of inflammatory factors by HSCs to ameliorate liver fibrosis. Conclusions: Interrupting the cGAS-STING pathway helped reverse the fibrosis process. Naringenin has potential as an antihepatic fibrosis drug.

Oroxylin A regulates cGAS DNA hypermethylation induced by methionine metabolism to promote HSC senescence.

Relevant studies have recognized the important role of hepatic stellate cell (HSC) senescence in anti-liver fibrosis. Cellular senescence is believed to be regulated by the cGAS-STING signaling pathway. However, underlying exact mechanisms of cGAS-STING pathway in hepatic stellate cell senescence are still unclear. Here, we found that Oroxylin A could promote senescence in HSC by activating the cGAS-STING pathway. Moreover, activation of the cGAS-STING pathway was dependent on DNMT3A downregulation, which suppressed cGAS gene DNA methylation. Interestingly, the attenuation of DNMT activity relied on the reduction of methyl donor SAM level. Noteworthy, the downregulation of SAM levels implied the imbalance of methionine cycle metabolism, and MAT2A was considered to be an important regulatory enzyme in metabolic processes. In vivo experiments also indicated that Oroxylin A induced senescence of HSCs in mice with liver fibrosis, and DNMT3A overexpression partly offset this effect. In conclusion, we discovered that Oroxylin A prevented the methylation of the cGAS gene by preventing the production of methionine metabolites, which promoted the senescence of HSCs. This finding offers a fresh hypothesis for further research into the anti-liver fibrosis mechanism of natural medicines.

O-GlcNAcylation Coordinates Glutaminolysis by Regulating the Stability and Membrane Trafficking of ASCT2 in Hepatic Stellate Cells.

Background and Aims: Recognition of excessive activation of hepatic stellate cells (HSCs) in liver fibrosis prompted us to investigate the regulatory mechanisms of HSCs. We aimed to examine the role of O-GlcNAcylation modification of alanine, serine, cysteine transporter 2 (ASCT2) in HSCs and liver fibrosis. Methods: The expression of O-GlcNAcylation modification in fibrotic mice livers and activated HSCs was analyzed by western blotting. Immunoprecipitation was used to assess the interaction of ASCT2 and O-GlcNAc transferase (OGT). In addition, ASCT2 protein stability was assayed after cycloheximide (CHX) treatment. The O-GlcNAcylation site of ASCT2 was predicted and mutated by site-directed mutagenesis. Real-time PCR, immunofluorescence, kit determinations and Seahorse assays were used to clarify the effect of ASCT2 O-GlcNAcylation on HSC glutaminolysis and HSC activation. Western blotting, immunochemistry, and immunohistofluorescence were used to analyze the effect of ASCT2 O-GlcNAcylation in vivo. Results: We observed significantly increased O-GlcNAcylation modification of ASCT2. ASCT2 was found to interact with OGT to regulate ASCT2 stability. We predicted and confirmed that O-GlcNAcylation of ASCT2 at Thr122 site resulted in HSCs activation. We found Thr122 O-GlcNAcylation of ASCT2 mediated membrane trafficking of glutamine transport and attenuated HSC glutaminolysis. Finally, we validated the expression and function of ASCT2 O-GlcNAcylation after injection of AAV8-ASCT2 shRNA in CCl4-induced liver fibrosis mice in vivo. Conclusions: Thr122 O-GlcNAcylation regulation of ASCT2 resulted in stability and membrane trafficking-mediated glutaminolysis in HSCs and liver fibrosis. Further studies are required to assess its role as a putative therapeutic target.

Qingchang Wenzhong Decoction reduce ulcerative colitis in mice by inhibiting Th17 lymphocyte differentiation.

BACKGROUND: Qingchang Wenzhong Decoction (QCWZD), a chinese herbal prescription, is widely used for ulcerative colitis (UC). Nevertheless, the active ingredients and mechanism of QCWZD in UC have not yet been explained clearly. PURPOSE: This research focuses on the identification of the effective ingredients of QCWZD and the prediction and verification of their potential targets. METHODS: The UC mice were established by adding 3.0% dextran sulfate sodium (DSS) to sterile water for one week. Concurrently, mice in the treatment group were gavage QCWZD or mesalazine. LC-MS analyzed the main components absorbed after QCWZD treatment, and network pharmacology predicted their possible targets. ELISA, qPCR, immunohistochemistry and immunofluorescence experiments were used to evaluate the colonic inflammation level and the intestinal barrier completeness. The percentage of Th17 and Treg lymphocytes was detected by flow cytometry. RESULTS: After QCWZD treatment, twenty-seven compounds were identified from the serum. In addition, QCWZD treatment significantly reduced the increased myeloperoxidase (MPO) and inflammatory cell infiltration caused by DSS in the colonic. In addition, QCWZD can reduce the secretion of inflammatory factors in serum and promote the expression of mRNAs and proteins of occludin and ZO-1. Network pharmacology analysis indicated that inhibiting IL-6-STAT3 pathway may be necessary for QCWZD to treat UC. Flow cytometry analysis showed that QCWZD can restore the normal proportion of Th17 lymphocytes in UC mice. Mechanistically, QCWZD inhibited the phosphorylation of JAK2-STAT3 pathway, reducing the transcriptional activation of RORγT and IL-17A. CONCLUSIONS: Overall, for the first time, our work revealed the components of QCWZD absorbed into blood, indicated that the effective ingredients of QCWZD may inhibit IL-6-STAT3 pathway and inhibit the differentiation of Th17 lymphocytes to reduce colon inflammation.

Inhibition of ASCT2 induces hepatic stellate cell senescence with modified proinflammatory secretome through an IL-1α/NF-κB feedback pathway to inhibit liver fibrosis.

Senescence of activated hepatic stellate cells (aHSCs) is a stable growth arrest that is implicated in liver fibrosis regression. Senescent cells often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). But little is known about how alanine-serine-cysteine transporter type-2 (ASCT2), a high affinity glutamine transporter, affects HSC senescence and SASP during liver fibrosis. Here, we identified ASCT2 is mainly elevated in aHSCs and positively correlated with liver fibrosis in human and mouse fibrotic livers. We first discovered ASCT2 inhibition induced HSCs to senescence in vitro and in vivo. The proinflammatory SASP were restricted by ASCT2 inhibition at senescence initiation to prevent paracrine migration. Mechanically, ASCT2 was a direct target of glutaminolysis-dependent proinflammatory SASP, interfering IL-1α/NF-κB feedback loop via interacting with precursor IL-1α at Lys82. From a translational perspective, atractylenolide III is identified as ASCT2 inhibitor through directly bound to Asn230 of ASCT2. The presence of -OH group in atractylenolide III is suggested to be favorable for the inhibition of ASCT2. Importantly, atractylenolide III could be utilized to treat liver fibrosis mice. Taken together, ASCT2 controlled HSC senescence while modifying the proinflammatory SASP. Targeting ASCT2 by atractylenolide III could be a therapeutic candidate for liver fibrosis.

Erratum: Ligand Activation of PPARγ by Ligustrazine Suppresses Pericyte Functions of Hepatic Stellate Cells via SMRT-Mediated Transrepression of HIF-1α: Erratum.

[This corrects the article DOI: 10.7150/thno.22237.].

Curcumol alleviates liver fibrosis by inducing endoplasmic reticulum stress-mediated necroptosis of hepatic stellate cells through Sirt1/NICD pathway.

Liver fibrosis is a repair response process after chronic liver injury. During this process, activated hepatic stellate cells (HSCs) will migrate to the injury site and secrete extracellular matrix (ECM) to produce fibrous scars. Clearing activated HSCs may be a major strategy for the treatment of liver fibrosis. Curcumol isolated from plants of the genus Curcuma can effectively induce apoptosis of many cancer cells, but whether it can clear activated HSCs remains to be clarified. In the present study, we found that the effect of curcumol in treating liver fibrosis was to clear activated HSCs by inducing necroptosis of HSCs. Receptor-interacting protein kinase 3 (RIP3) silencing could impair necroptosis induced by curcumol. Interestingly, endoplasmic reticulum (ER) stress-induced cellular dysfunction was associated with curcumol-induced cell death. The ER stress inhibitor 4-PBA prevented curcumol-induced ER stress and necroptosis. We proved that ER stress regulated curcumol-induced necroptosis in HSCs via Sirtuin-1(Sirt1)/Notch signaling pathway. Sirt1-mediated deacetylation of the intracellular domain of Notch (NICD) led to degradation of NICD, thereby inhibiting Notch signalling pathway to alleviate liver fibrosis. Specific knockdown of Sirt1 by HSCs in male ICR mice further exacerbated CCl4-induced liver fibrosis. Overall, our study elucidates the anti-fibrotic effect of curcumol and reveals the underlying mechanism between ER stress and necroptosis.

Modification of lysine deacetylation regulates curcumol-induced necroptosis through autophagy in hepatic stellate cells.

The excessive deposition of extracellular matrix (ECM) is the main characteristic of liver fibrosis, and hepatic stellate cells (HSCs) are the main source of ECM. The removal of activated HSCs has a reversal effect on liver fibrosis. Western blot and MTT analysis indicated that curcumol could relieve hepatic fibrosis by promoting HSCs receptor-interacting protein kinase 1/3 (RIP1/RIP3)-dependent necroptosis. Importantly, autophagy flow was monitored by constructing the mRFP-GFP-LC3 plasmid, and it was found that curcumol cleared activated HSCs in a necroptosis manner that was dependent on autophagy. Our study suggested that the activation of necrosome formed by RIP1 and RIP3 depended on Atg5, and that autophagosomes were also necessary for curcumol-induced necroptosis. Furthermore, microscale thermophoresis and co-immunoprecipitation assay results proved that curcumol could target Sirt1 to regulate autophagy by reducing the acetylation level of Atg5. The HSCs-specific silencing of Sirt1 exacerbated CCl4 -induced liver fibrosis in mice. The deacetylation of Atg5 not only accelerated the accumulation of autophagosomes but also enhanced the interaction between Atg5 and RIP1/RIP3 to induce necroptosis. Overall, our study indicated that curcumol could activate Sirt1 to promote Atg5 deacetylation and enhanced its protein-protein interaction function, thereby inducing autophagy and promoting the necroptosis of HSCs to reduce liver fibrosis.

m6A methylation is required for dihydroartemisinin to alleviate liver fibrosis by inducing ferroptosis in hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) is a central event in the development of liver fibrosis, and the elimination of activated HSCs is considered to be an effective anti-fibrotic strategy. Here, we report that dihydroartemisinin (DHA) prevented the activation of HSCs via ferroptosis pathway. Importantly, DHA treatment increased the level of autophagy in HSCs. The inhibition of autophagy by 3-MA dramatically abolished the DHA-induced ferroptosis in HSCs. Mechanistically, the up-regulated m6A modification is essential for the activation of autophagy by DHA through the reduction of fat mass and obesity-associated gene (FTO). Down-regulation of m6A modification by FTO overexpression could impair autophagy and the classical ferroptotic events. Interestingly, the m6A modification of BECN1 mRNA was evidently up-regulated compared with other autophagy-related genes. More importantly, YTHDF1 was identified as a key m6A reader protein for BECN1 mRNA stability, and knockdown of YTHDF1 could prevent DHA-induced HSC ferroptosis. Noteworthy, YTH domain was essential for YTHDF1 to prolong the half-life of BECN1 mRNA in DHA-induced HSC ferroptosis. In mice, DHA treatment alleviated liver fibrosis by triggering HSC ferroptosis. HSC-specific inhibition of m6A modification and autophagy could impair DHA-induced HSC ferroptosis in murine liver fibrosis. Overall, these results provided novel implications to reveal the molecular mechanism of DHA-induced ferroptosis, by which pointed to m6A modification-dependent ferroptosis as a potential target for the treatment of liver fibrosis.

Dihydroartemisinin Induces Ferroptosis in HCC by Promoting the Formation of PEBP1/15-LO.

Relevant researches have recognized the vital role of inducing ferroptosis in the treatment of tumor. The latest findings indicate that PEBP1/15-LO can play an essential role in the process of cell death. However, its role in regulating ferroptosis in hepatocellular carcinoma (simplified by HCC) remains unclear. The previous research of our team has proved that DHA can induce ferroptosis of hepatic stellate cells. In this study, we found that DHA could also induce ferroptosis in HCC cells. Interestingly, DHA induced ferroptosis by promoting the formation of PEBP1/15-LO and promoting cell membrane lipid peroxidation. In addition, we also found that DHA had no obvious regulatory effect on 15-LO, but it could promote PEBP1 protein expression. Importantly, we discovered the upregulation of PEBP1 induced by DHA was related to the inhibition of its ubiquitination degradation. In vivo experiments have also obtained consistent results that DHA can inhibit tumor growth and affect the expression of ferroptosis markers in tumor tissues, which would be partially offset by interference with PEBP1.