Type 2 diabetes (T2D) impairs antiviral immunity; however, the causal link between T2D and interferon-α2 (IFN-α2) deficiency remains unclear. This study used genome-wide association study-based Mendelian randomization (MR) to investigate this relationship and validated the findings in an H1N1-infected diabetic mouse model. MR analysis of 26 single nucleotide polymorphisms showed a significant negative association between T2D and IFN-α2 levels (inverse variance weighted odds ratio 0.667; P = 0.000116) without heterogeneity or pleiotropy. In vivo experiments confirmed that db/db mice exhibited more severe H1N1-induced lung injury, higher viral loads, and lower survival rates compared with nondiabetic controls. However, exogenous IFN-α2 treatment significantly reversed these pathologic outcomes. Inflammatory cytokine profiling showed that IFN-α2 downregulated 21 elevated cytokines and restored Fas ligand levels in lung tissue. Mechanistically, Western blotting demonstrated that IFN-α2 inhibited the phosphorylation of JAK1/2 and STAT3, thereby suppressing excessive inflammation. In conclusion, our findings indicate that T2D leads to IFN-α2 deficiency, contributing to susceptibility to viral infection. Supplementation with IFN-α2 effectively attenuated virus-induced lung injury by inhibiting JAK/STAT3 signaling and cytokine storms, positioning IFN-α2 supplementation as a promising therapeutic strategy for managing influenza complications in patients with diabetes. ARTICLE HIGHLIGHTS:Type 2 diabetes (T2D) is linked to impaired antiviral immunity, but whether it drives interferon-α2 (IFN-α2) deficiency remains unknown. We asked whether T2D causally suppresses IFN-α2 levels and whether exogenous supplementation could rescue host defense mechanisms against influenza infection. By integrating genetic analysis with an H1N1-infected diabetic mouse model, we show that T2D genetically lowers IFN-α2 and that treatment reverses lung injury by inhibiting JAK/STAT3-mediated hyperinflammation. Our study positions IFN-α2 supplementation as a promising therapeutic strategy to prevent severe viral pneumonia in patients with T2D.

Background: Corylin is a bioactive extract of Psoralea corylifolia L. with reported antioxidant, anti-tumor, and anti-inflammatory effects. Its anti-tumor effects have been investigated, but mechanistic explanations are lacking. This study mainly focuses on the role and mechanism of Corylin in inducing liver cancer cell death. Methods: The effects of Corylin on liver cancer cell proliferation were analyzed using crystal violet staining and cell counting kit-8 (CCK-8) assays. Nuclear changes were detected via Hoechst staining, while apoptosis levels were assessed using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Changes in mitochondrial membrane potential and reactive oxygen species (ROS) levels were analyzed using JC-1 and tetramethylrhodamine methyl ester (TMRM) staining combined with flow cytometry. Immunofluorescence staining and Western blot (WB) experiments assessed alterations in apoptosis-and autophagy-related proteins. Finally, the effects of Corylin on liver cancer cells in vivo were validated by establishing a subcutaneous tumor model. Results: In this study, we found that Corylin had a significant anti-liver cancer effect and significantly increased the apoptosis level of liver cancer cells. Additionally, Corylin was found to significantly reduce mitochondrial membrane potential and significantly increase ROS release. It was also observed that Corylin significantly influenced autophagy levels in liver cancer cells. Further analysis revealed that Corylin induced mitophagy, with the increase in autophagy levels being directly proportional to the rise in apoptosis levels. Finally, the establishment of a subcutaneous tumor-bearing model showed that Corylin also had a significant anti-liver cancer effect in vivo. Conclusions: The above results demonstrated that Corylin mainly caused the death of liver cancer cells through the endogenous apoptosis and mitophagy pathway and had great potential as an anti-liver cancer drug.

Long-term excessive alcohol intake can directly injure the gastroduodenal mucosa, causing gastric erosions, gastric ulcers, and gastrorrhagia. Fritillaria ussuriensis Maxim is a famous traditional Chinese medicine and health food produced in China. Sipeimine is an alkaloidal component of Fritillaria ussuriensis Maxim. This research aimed to investigate the protective effects of sipeimine on ethanol-induced gastric ulcers in mice. The results displayed that sipeimine could alleviate gastric tissue damage and decrease the levels of SOD, MDA, IL-6, IFN-γ, TNF-α, and IL-1β. Sipeimine treatment also adjusted macrophage polarization and the balance of Th17/Treg cell by reducing the expression of Jak1/2, p-Jak1/2, Stat1/3, and p-Stat1/3. Moreover, sipeimine could increase the abundance of Lactobacillus_johnsonii and decrease the abundance of Bacteroides_vulgatus in the gut microbiota. Meanwhile, sipeimine treatment significantly decreased the abundance of Rodentibacter_heylii and Streptococcus_cuniculi in the gastric microbiota. In conclusion, sipeimine can improve gastric ulcers by suppressing the Jak-Stat pathway, reversing gut-gastro microbiota dysbiosis, inhibiting macrophage M1 polarization, maintaining the balance of Th17/Treg cell, and lessening sustained inflammatory injury.

Abstract Monkeypox virus (MPXV) is an important zoonotic pathogenic virus, which poses serious threats to public health. MPXV infection can be prevented by immunization against the variola virus. Because of the safety risks and side effects of vaccination with live vaccinia virus (VACV) strain Tian Tan (VTT), we constructed two gene-deleted VTT recombinants (TTVAC7 and TTVC5). The immunogenicity and protective effects of the gene-deleted VTT vaccine were assessed using BALB/C mice challenged with VTT and New Zealand rabbits challenged with MPXV. The results demonstrated strong humoral and cellular immune responses. The VTT-specific and neutralizing antibody titers, specific T cell levels, and degree of dendritic cell maturation of the mice, in addition to the MPXV neutralizing antibody titers and IFN-γ, IL-6, and TNF-α levels of the rabbits were markedly higher in the groups immunized with TTVAC7 and TTVC5 than the control groups (p < 0.05). Moreover, immunization with TTVAC7 and TTVC5 reduced morbidities caused by VACV and MPXV infection. The weight change, lung histological score, and residual virus of the mouse model (p < 0.05). Similarly, the temperature change, pock number, lung histological score, and residual virus of the rabbit model were significantly reduced in the groups immunized with TTVAC7 and TTVC5 (p < 0.05). Collectively, these results demonstrate that TTVAC7 and TTVC5 may be used as potential live attenuated vaccines against MPXV infection.

Purpose: The study was aimed at identifying the pathogenic gene responsible for X-linked nonsyndromic hearing loss (NSHL) in a five-generation Chinese family and at elucidating the gene's function both in vivo using a zebrafish model and in vitro using PRPS1 knockdown HEI-OC1 cells. Methods: Exome sequencing (ES) and Sanger sequencing were used to identify the pathogenic variants. A transgenic zebrafish model overexpressing the novel PRPS1 variant (c.494G>A: p.Cys165Tyr) was constructed, and PRPS1 was knocked down in HEI-OC1 cells using siRNA to explore the underlying mechanisms. Hair cell development and behavior were assessed in zebrafish, and mitochondrial function and cell viability were analyzed in HEI-OC1 cells. Results: A novel missense variant (c.494G>A: p.Cys165Tyr) in the PRPS1 gene was identified as the pathogenic variant causing progressive X-linked deafness-1 (DFNX1). The variant led to hair cell death in zebrafish, with disrupted swimming behavior. In HEI-OC1 cells, PRPS1 knockdown resulted in downregulation of the nicotinamide adenine dinucleotide (NAD+)/sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) pathway, increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and apoptosis, which were partially rescued by pretreatment with nicotinamide mononucleotide (NMN), a precursor of NAD+. Conclusion: The study reports a novel PRPS1 variant contributing to the variant spectrum of PRPS1 and highlights the role of PRPS1 deficiency in increasing oxidative stress-induced hair cell apoptosis via the NAD+/SIRT3/SOD2 pathway. These findings provide new insights into the molecular mechanisms of PRPS1-related hearing loss and potential therapeutic targets.

Recombinant antigens have been used for vaccine development, which need suitable adjuvants to boost the immunogenicity. To this end, we report here our discovery on rationally designed lipopeptide amphiphiles could be applied for adjuvants. After accompanying with lipopeptidic adjuvants, multi-antigens subunit vaccines consisting of mature virus and enveloped virus surface proteins exhibited good immunogenicity and effectively elicited humoral immune response. Both structural screening and in vitro studies demonstrated that among these adjuvants, C16-GCV2E3, one of self-assembling lipopeptides with the palmitic acid capped on N terminus, could significantly enhance the antigen uptake, DC maturation, and T cell responses with Th2 skewing. Notably, in vivo results further confirmed that C16-GCV2E3 augmented the titer of antigen-specific antibodies, enhanced the neutralizing activity of immune serum to MPXV, and boosted efficacy of the subunit vaccine against VACV compared to several counterparts in mice. We believe our discovery opens a new avenue for the development of effective adjuvants.

The 2022 monkeypox outbreak involved rapid global dissemination, prompting research into animal models for the monkeypox virus (MPXV), including non-human primates and mice. However, studies utilizing rabbits as models remain limited. In this study, we established three rabbit models using the current epidemic MPXV strain. Following intravenous MPXV injection, adult rabbits exhibited characteristic clinical manifestations, including widespread rash and fever, with viral replication in the skin, lungs, and testes, resulting in severe pathological damage by 6 days post-infection (dpi). Intradermal injection of MPXV into the dorsal skin of adult rabbits produced red lesions with central necrosis and hemorrhage accompanied by dense inflammatory infiltrates. Abundant viral particles were observed in epidermal cells at 6 dpi. Additionally, a fatal MPXV model was developed in 10-day-old rabbits using intranasal virus administration. These young rabbits exhibited lethargy and diarrhea beginning at 2 dpi, significant weight loss, and a 50% mortality rate by 15 dpi. Viral dissemination was detected in multiple organs, leading to extensive multi-organ damage. This study highlights the utility of rabbit models for MPXV, displaying typical clinical features and pathogenic mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE:Licorice is one of the most commonly used Chinese medicines. Licochalcone A (Lico A) is a flavonoid, which is one of the main components extracted from licorice. It has a variety of pharmacological activities such as improving glucose and lipid metabolism. However, the therapeutic efficacy of Lico A against non-alcoholic fatty liver disease (NAFLD) and the underlying mechanism of this action remains unclear. AIM OF THE STUDY:The aim of this study was to investigate the interventional effect and mechanism of action of Licochalcone A in NAFLD by combining in vivo and in vitro experiments. MATERIALS AND METHODS:NAFLD mouse model was established with MCD feed, and an in vitro cell model was established with free fatty acid (FFA) formulated with palmitic acid (PA) and oleic acid (OA). After stimulation with Licochalcone A, the changes in the in vivo and in vitro models were observed to clarify whether Licochalcone A has an ameliorative effect on NAFLD. The mechanism of action of Licochalcone A in ameliorating NAFLD was further verified by transcriptomics, mTOR-related protein detection, agonist and inhibitor experiments. RESULTS:Compared with the model group, the number of intracellular lipid vacuoles and red fat droplets was reduced in the Lico A group, and the size of adipocytes was reduced. Meanwhile, the liver function and lipid indexes such as AST, ALT, TG, CHO, HDL-C and LDL-C were reduced. mTOR expression level was reduced, and the expression levels of ULK1, Beclin-1, VPS34, Atg5 and Atg12 were increased. CONCLUSION:Licochalcone A alleviates NAFLD, reduces hepatic lipid deposition and enhances liver function. Its mechanism of action is related to the inhibition of mTOR expression and regulation of ULK1/Beclin-1/VPS34 pathway thereby promoting autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE:Zhichuanling oral liquid (ZCLOL) is a multi-component traditional Chinese medicine formulation that has been widely used in the clinical management of asthma. However, the molecular mechanisms underlying its clinical efficacy remain largely undefined, particularly regarding calcium homeostasis and endoplasmic reticulum stress (ERS) regulation. AIM OF THE STUDY:This study aimed to elucidate the mechanisms by which ZCLOL mitigates asthma-related airway inflammation through the integration of in vivo and in vitro models with proteomic profiling. METHODS:In this study, an OVA-induced asthma model was established to evaluate the therapeutic effects of ZCLOL by assessing pulmonary function, serum Th2 cytokine levels, and macrophage polarization. To further elucidate the underlying mechanisms of ZCLOL in asthma treatment, we conducted proteomic analysis, measured calcium levels in lung tissue, and performed Western blotting. Additionally, an inflammatory BEAS-2B cell model induced by LPS and IL-13, combined with BAPTA-AM treatment, was used to explore the molecular pathways involved. RESULTS:ZCLOL effectively alleviated airway hyperresponsiveness, reduced lung tissue injury, inhibited M1 macrophage polarization and Th2 cytokine secretion, decreased intracellular calcium ion levels, mitigated endoplasmic reticulum stress, and downregulated the expression of STIM1, Bip, Ero1-Lα, PDI, CHOP, and phosphorylated JNK (p-JNK). CONCLUSION:ZCLOL alleviates airway inflammation and hyperresponsiveness in asthma by restoring Ca2+ homeostasis and suppressing ERS through inhibition of the STIM1/CHOP/JNK axis. These findings provide mechanistic evidence supporting ZCLOL as a potential multi-target therapeutic strategy for asthma.

BACKGROUND:Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells.OBJECTIVE:The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified.METHODS:In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue.RESULTS:As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway.CONCLUSION:This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

Platycodin D2 (PD2) is a triterpenoid saponin extracted from the root of Platycodon grandiflorum , a common source of medicine and food. Platycodon grandiflorum saponins have anti-inflammatory, antioxidative, antitumor, and immunity-promoting effects. However, the effect of PD2 on breast cancer cells has not been reported. The purpose of this study is to explore the molecular mechanism underlying the effect of PD2 on breast cancer cells. We analyzed the inhibitory effects and pathways of PD2 on breast cancer by CCK-8 assay, WB assay, and immunofluorescence assay. Subsequently, autophagy and ferroptosis were analyzed using different inhibitors. It was found that PD2 caused mitochondrial damage and promoted mitochondrial reactive oxygen species (mtROS) production, leading to autophagy flux inhibition and ferroptosis. Blockage of autophagy flux and ferroptosis promoted each other, resulting in the inhibition of breast cancer cell proliferation. Similar results were obtained in the tumor-bearing model in vivo. PD2 promoted autophagy flux blockage and ferroptosis in breast cancer cells, which induced each other under the action of mtROS, thus inhibiting the proliferation of breast cancer cells. PD2 is a potential new strategy for the treatment of breast cancer.

OBJECTIVES Inflammatory cytokine secretion and gut microbiota dysbiosis play crucial roles in ulcerative colitis. In this research, the protective effects of peimisine on colitis mice were investigated. METHODS The protective effects were evaluated by the disease activity index, colonic length, hematoxylin-eosin, and AB/PAS Staining. The protective mechanisms were analyzed by ELISA, Western-blot, immunohistochemistry staining, immunofluorescence staining, and 16S rRNA gene analysis. KEY FINDINGS The results showed that peimisine treatment could reduce the disease activity index, prevent colonic shortening, and alleviate colon tissue damage. Peimisine treatment also decreased the levels of MCP-1, IL-1β, IL-6, IFN-γ, TNF-α and affected macrophage polarization and Th17/Treg cell balance by downregulating the expression of jak1/2, p-jak1/2, stat1/3, and p-stat1/3. Moreover, peimisine treatment significantly increased the abundances of beneficial microbes (e.g. Ruminococcaceae UCG-014 and Lachnospiraceae_NK4A136_group) and decreased the abundances of harmful microbes (e.g. Bacteroides and Escherichia). CONCLUSIONS Peimisine can ameliorate colitis by inhibiting Jak-Stat signaling pathway, reversing gut microbiota alterations, suppressing macrophage M1 polarization, maintaining the Th17/Treg cell balance, and reducing sustained inflammatory cytokines-related inflammatory injury.

Background Hepatocellular carcinoma (HCC) cells usually show strong resistance to chemotherapy, which not only reduces the efficacy of chemotherapy but also increases the side effects. Regulation of autophagy plays an important role in tumor treatment. Cell senescence is also an important anti-cancer mechanism, which has become an important target for tumor treatment. Therefore, it is of great clinical significance to find anti-HCC drugs that act through this new mechanism. Platycodin D2 (PD2) is a new saponin compound extracted from the traditional Chinese medicine Platycodon grandiflorum. Purpose Our study aimed to explore the effects of PD2 on HCC and identify the underlying mechanisms. Methods First, the CCK8 assay was used to detect the inhibitory effect of PD2 on HCC cells. Then, different pathways of programmed cell death and cell cycle regulators were measured. In addition, we assessed the effects of PD2 on the autophagy and senescence of HCC cells by flow cytometry, immunofluorescence staining, and Western blotting. Finally, we studied the in vivo effect of PD2 on HCC cells by using a mouse tumor-bearing model. Results Studies have shown that PD2 has a good anti-tumor effect, but the specific molecular mechanism has not been clarified. In this study, we found that PD2 has no obvious toxic effect on normal hepatocytes, but it can significantly inhibit the proliferation of HCC cells, induce mitochondrial dysfunction, enhance autophagy and cell senescence, upregulate NIX and P21, and downregulate CyclinA2. Gene silencing and overexpression indicated that PD2 induced mitophagy in HCC cells through NIX, thereby activating the P21/CyclinA2 pathway and promoting cell senescence. Conclusions These results indicate that PD2 induces HCC cell death through autophagy and aging. Our findings provide a new strategy for treating HCC. Graphical Abstract

BackgroundApoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. Herein, we found that Apoptin induced a strong and lasting endoplasmic reticulum (ER) stress response, Ca2+ imbalance, and triggered the mitochondrial apoptotic pathway. The aim of this study was to explore the mechanisms by which Apoptin exhibited anti-tumor effects in HepG-2 cells.MethodsThe intracellular levels of calcium (Ca2+) were induced by ER stress and determined by electron microscopy, flow cytometry, and fluorescence staining. The mitochondrial injury was determined by mitochondrial membrane potential and electron microscopy. Western blotting was used to investigate the levels of key proteins in ER stress and the apoptotic pathway in mitochondria. The relationship between Ca2+ levels and apoptosis in Apoptin-treated cells was analyzed using a Ca2+ chelator (BAPTA-AM), flow cytometry, and fluorescence staining. We also investigated the in vivo effects of Ca2+ imbalance on the mitochondrial apoptotic pathway using tumor tissues xenografted on nude mice.ResultsThis study showed that Apoptin induced a strong and long- lasting ER stress and injury, which subsequently led to an imbalance of cellular Ca2+ levels, a reduction in the mitochondrial membrane potential, a significant extent image in the mitochondrial structure, and an increase in the expression levels of Smac/Diablo and Cyto-C.ConclusionsIn summary, Apoptin induced apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway. This study provided a new direction for antitumor research in Apoptin.

Liver cancer is a common malignant tumor, and its incidence is increasing yearly. Millions of people suffer from liver cancer annually, which has a serious impact on global public health security. Licochalcone A (Lico A), an important component of the traditional Chinese herb licorice, is a natural small molecule drug with multiple pharmacological activities. In this study, we evaluated the inhibitory effects of Lico A on hepatocellular carcinoma cell lines (HepG2 and Huh-7), and explored the inhibitory mechanism of Lico A on hepatocellular carcinoma. First, we evaluated the inhibitory effects of Lico A on hepatocellular carcinoma, and showed that Lico A significantly inhibited and killed HepG2 and Huh-7 cells in vivo and in vitro. Transcriptomic analysis showed that Lico A inhibited the expression of solute carrier family 7 member 11 (SLC7A11), which induced ferroptosis. We confirmed through in vivo and in vitro experiments that Lico A promoted ferroptosis in hepatocellular carcinoma cells by downregulating SLC7A11 expression, thereby inhibiting the glutathione (GSH)-glutathione peroxidase 4 (GPX4) pathway and inducing activation of reactive oxygen species (ROS). In this study, we suggest that Lico A is a potential SLC7A11 inhibitor that induces ferroptotic death in hepatocellular carcinoma cells, thereby providing a theoretical basis for the development of natural small molecule drugs against hepatocellular carcinoma.

Influenza viruses pose a serious threat to human health, infecting hundreds of millions of people worldwide each year, resulting in a significant increase in global morbidity and mortality. Influenza activity has declined at the onset of the COVID-19 pandemic, but the genetic diversity of B/Victoria lineage viruses has increased significantly during this period. Therefore, the prevention and treatment of the influenza B Victoria strain virus should continue to attract research attention. In this study, we found that Atractyloside A (AA), one of the effective components in Atractylodes lancea (Thunb.) DC shows potential antiviral properties. This study shows that AA not only possesses anti-influenza B virus infection effects in vivo and in vitro but also can regulate macrophage polarization to the M2 type, which can effectively attenuate the damage caused by influenza B virus infection. Therefore, Atractyloside A may be an effective natural drug against B/Victoria influenza infection.

Monkeypox was declared a global health emergency by the World Health Organization, and as of March 2023, 86,000 confirmed cases and 111 deaths across 110 countries have been reported. Its causal agent, monkeypox virus (MPV) belongs to a large family of double-stranded DNA viruses, Orthopoxviridae, that also includes vaccinia virus (VACV) and others. MPV produces two distinct forms of viral particles during its replication cycles: the enveloped viron (EV) that is released via exocytosis, and the mature viron (MV) that is discharged through lysis of host cells. This study was designed to develop multi-valent mRNA vaccines against monkeypox EV and MV surface proteins, and examine their efficacy and mechanism of action. Four mRNA vaccines were produced with different combinations of surface proteins from EV (A35R and B6R), MV (A29L, E8L, H3L and M1R), or EV and MV, and were administered in Balb/c mice to assess their immunogenicity potentials. A dynamic immune response was observed as soon as seven days after initial immunization, while a strong IgG response to all immunogens was detected with ELISA after two vaccinations. The higher number of immunogens contributed to a more robust total IgG response and correlating neutralizing activity against VACV, indicating the additive potential of each immunogen in generating immune response and nullifying VACV infection. Further, the mRNA vaccines elicited an antigen-specific CD4+ T cell response that is biased towards Th1. The mRNA vaccines with different combinations of EV and MV surface antigens protected a mouse model from a lethal dose VACV challenge, with the EV and MV antigens-combined vaccine offering the strongest protection. These findings provide insight into the protective mechanism of multi-valent mRNA vaccines against MPV, and also the foundation for further development of effective and safe mRNA vaccines for enhanced protection against monkeypox virus outbreak.

Objective: To explore the effects of Esculetin on liver cancer and explore potential mechanisms of Esculetin-inducing cells death. Methods: Esculetin's effects on the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells were detected by using CCK8, crystal violet staining, wound healing, TranswellTM and Annexin V-FITC/PI. Flow cytometry, fluorescence staining, Western blot, T-AOC, DPPH radical scavenging assay, hydroxyl radical's inhibitory capability and GSH test were used to examine the esculetin's effects on the ROS level, the oxidation-related substances and proteins' expression in hepatoma cells. In vivo experiment was performed by xenograft model. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by esculetin. Live cell probe, Western blot, Fe2+ content, MDA, HE staining, Prussian blue staining and immunohistochemistry were used to examine the ferritinophagy-related phenomenon induced by esculetin in hepatoma cells. The relationship between esculetin and NCOA4-mediated ferritinophagy was confirmed through gene silence and overexpression, immunofluorescence staining and Western blot. Results: Esculetin suppressed the proliferation, migration and apoptosis of HUH7 and HCCLM3 cells significantly, influenced the oxidative stress level, altered the autophagy and iron metabolism levels in cells, and produced a ferritinophagy-related phenomena. Esculetin increased the levels of cellular lipid peroxidation and reactive oxygen species. In vivo, esculetin could decrease tumour volume, promote LC3 and NCOA4 expressions, suppresse hydroxyl radical's inhibiting capacity and GSH, increase Fe2+ and MDA levels, decrease antioxidant proteins expression in tumour tissue. In addition, Esculetin could also increase the iron deposition of tumour tissues, promote ferritinophagy, and induce tumours' ferroptosis. Conclusion: Esculetin has an inhibitory effect on liver cancer in vivo and in vitro through triggering NCOA4 pathway-mediation ferritinophagy.

Betulinic acid is found in the several foods and plants, such as sour jujube fruits, papaya, persimmon, betulinic acid exhibits low toxicity and a wide range of anticancer activities. Ferritinophagy and ferroptosis are related to tumor formation and therapeutic effects, both of which are controlled by iron metabolism and inextricably linked. In this study, we discovered that betulinic acid could suppress proliferation and migration of hepatoma cells, raised ROS level and inhibited antioxidation level in cells. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by betulinic acid. Betulinic acid could cause ferritinophagy-related phenomenon in vivo and in vitro, promote ferritinophagy-protiens expressions, induce ferritinophagy to inhibit tumours finally. In vivo studies revealed the low toxicity of betulinic acid in mice, the dose of human was about 10 times that of mice, the using dose of human could also be achievable. These findings illustrate the potential of betulinic acid for treatment of cancer.