Influenza A virus infection activates STAT3 to enhance SREBP2 expression, cholesterol biosynthesis, and virus replication.

Cellular cholesterol plays an important role in influenza A virus (IAV) endocytosis and replication. However, how IAV infection regulates cholesterol biosynthesis remains poorly understood. Here, we report that IAV infection activates SREBP2 and induces the expression of HMGCR, a rate-limiting enzyme in cholesterol synthesis pathway. SREBP2 deficiency suppresses IAV-induced HMGCR expression and virus replication. Mechanistically, IAV infection activates JAK2 and STAT3, inhibition of JAK2 and STAT3 activity by their inhibitors or by gene knockout downregulates IAV-induced SREBP2 and HMGCR expression and IAV replication, reduces the content of cellular cholesterol and virus binding to host cells. Exogenous cholesterol reverses the inhibitory effect of S3I-201 and STAT3 deficiency on virus replication. STAT3 or JAK2 overexpression increases the expression of SREBP2 and its downstream target genes, leading to increased IAV replication. These observations collectively suggest that STAT3 activation facilitates IAV replication by inducing SREBP2 expression and increasing cholesterol biosynthesis.

Unlocking the potential of phenolated kraft lignin as a versatile feed additive.

Lignin, a renewable natural antioxidant and bacteriostat, holds promise as a versatile, cost-effective feed additive. However, traditional industrial lignin faces limitations, including low reactivity, poor uniformity, and unstable properties, necessitating chemical modification. Complex modification methods pose economic and toxicity challenges, so this study adopted a relatively simple alkali-catalyzed phenolization approach, using phenol, catechol, and pyrogallol to modify kraft lignin, and characterized the resulting products using various techniques. Subsequently, their antioxidant, antibacterial, adsorption properties for heavy metal ions and mycotoxins, growth-promoting properties, and antiviral abilities were assessed. The phenolation process led to lignin depolymerization and a notable increase in phenolic hydroxyl content, particularly in pyrogallol-phenolated lignin (Py-L), rising from 3.08 to 4.68 mmol/g. These modified lignins exhibited enhanced antioxidant activity, with over 99 % inhibition against E. coli and S. aureus, and remarkable adsorption capacities for heavy metal ions and mycotoxins. Importantly, Py-L improved the growth performance of mice and reduced influenza mortality. Furthermore, density functional theory calculations elucidated the mechanism behind the enhanced antioxidant properties. This study presents a promising avenue for developing versatile feed additives to address challenges related to animal feed antioxidant supplementation, bacterial control, and growth promotion.

Mesenchymal stem cells prevent H7N9 virus infection via rejuvenating immune environment to inhibit immune-overactivity.

BACKGROUND: Influenza is a clinically important infectious disease with a high fatality rate, which always results in severe pneumonia. Mesenchymal stem cells (MSCs) exhibit promising therapeutic effects on severe viral pneumonia, but whether MSCs prevent virus infection and contribute to the prevention of influenza remains unknown. METHODS: ICR mice were pretreated with human umbilical cord (hUC) MSCs and then infected with the influenza H7N9 virus. Weight, survival days, and lung index of mice were recorded. Serum antibody against influenza H7N9 virus was detected according to the hemagglutination inhibition method. Before and after virus infection, T cell and B cell subtypes in the peripheral blood of mice were evaluated by flow cytometry. Cytokines in the supernatants of MSCs, innate immune cells, and mouse broncho alveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA) or Luminex Assay. RESULTS: Pretreatment with MSCs protected mice against influenza H7N9 virus infection. Weight loss, survival rate, and structural and functional damage to the lungs of infected mice were significantly improved. Mechanistically, MSCs modulated T lymphocyte response in virus-infected mice and inhibited the cGAS/STING pathway. Importantly, the protective effect of MSCs was mediated by cell-to-cell communications and attenuation of cytokine storm caused by immune overactivation.

Lactiplantibacillus pentoses CCFM1227 Produces Desaminotyrosine to Protect against Influenza Virus H1N1 Infection through the Type I Interferon in Mice.

Microbiota-derived desaminotyrosine (DAT) protects the host from influenza by modulating the type I interferon (IFN) response. The aim of this study was to investigate the antivirus effects of a DAT-producing bacteria strain. A comparative genomics analysis and UHPLC Q-Exactive MS were used to search for potential strains and confirm their ability to produce DAT, respectively. The anti-influenza functions of the DAT producer were evaluated using an antibiotic-treated mouse model by orally administering the specific strain before viral infection. The results showed the Lactiplantibacillus pentosus CCFM1227 contained the phy gene and produced DAT by degrading phloretin. In vivo, L. pentosus CCFM1227 re-inoculation increased the DAT level in feces, and protected from influenza through inhibiting viral replication and alleviating lung immunopathology. Furthermore, CCFM1227-derived DAT was positively correlated with the IFN-ß level in the lung. The transcriptome results showed that CCFM1227 activated gene expression in the context of the defense response to the virus, and the response to interferon-beta. Moreover, CCFM1227 treatment upregulated the expression of MHC-I family genes, which regulate the adaptive immune response. In conclusion, L. pentosus CCFM1227 exerted antiviral effects by producing DAT in the gut, and this may provide a potential solution for creating effective antiviral probiotics.

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K.

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P â€‹+ â€‹M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P â€‹+ â€‹M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.

Clinical features of the first critical case of acute encephalitis caused by the avian influenza A (H5N6) virus.

Highly pathogenic avian influenza viruses (HPAIV), such as H5N1, H5N6, and H7N9, have been reported to frequently infect humans, but acute encephalitis caused by HPAIV in humans has been rarely reported. We report the first critical case of acute encephalitis with mild pneumonia caused by the H5N6 virus. On January 25 of 2022, a 6-year-old girl with severe neurological symptoms was admitted to our hospital and rapidly developed into seizures and coma. Brain imaging showed abnormalities. Electroencephalogram (EEG) presented abnormal slow waves. Cerebrospinal fluid (CSF) contained elevated protein (1.64 g/L) and white cells (546 × 106/L). Laboratory investigations revealed abnormally elevated transaminases, lactate dehydrogenase, and cytokines in serum. A novel reassortant H5N6 virus was identified from the patient's serum, CSF, and tracheal aspirate specimens. Phylogenic analysis indicated that this virus was a novel reassortant avian-origin influenza A (H5N6) virus that belonged to clade 2.3.4.4b. This patient was diagnosed with acute encephalitis and discharged from the hospital accompanied by a language barrier. An epidemiological investigation confirmed that wild waterfowls were the direct source of infection in this case. Our study highlights the urgent need to pay attention to acute encephalitis caused by HPAIV.

Lactobacillus mucosae exerted different antiviral effects on respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) infection is a constant threat to the health of young children, and this is mainly attributed to the lack of effective prevention strategies. This study aimed to determine whether Lactobacillus (L.) mucosae, a potential probiotic, could protect against respiratory viral infection in a mouse model. Naive 3-4-week-old BALB/c mice were orally administered with three L. mucosae strains (2.5 × 108 CFU/mouse) 7 days before RSV infection (105 TCID50/mouse). Results showed that all three strains inhibited RSV replication and reduced the proportions of inflammatory cells, including granulocytes and monocytes in the blood. The L. mucosae M104R01L3 treatment maintained stable weight in mice and increased interferon (IFN)-ß and tumor necrosis factor (TNF)-α levels. The L. mucosae DCC1HL5 treatment increased interleukin (IL)-1ß and IL-10 levels. Moreover, the M104R01L3 and DCC1HL5 strains increased the proportions of Akkermansia, Alistipes, and Anaeroplasma which contributed to the advantageous modulation of the gut microbiota. Besides, L. mucosae affected the gut levels of short-chain fatty acids (SCFAs) that are important for the antiviral response. L. mucosae 1,025 increased acetate, propionate, and butyrate levels, whereas L. mucosae M104R01L3 increased the level of acetate in the gut. L. mucosae M104R01L3 may protect against viral infection by upregulating the IFN-ß levels in the lungs and its antiviral effect may be related to the increase of acetate levels in the gut. In conclusion, the three L. mucosae strains exerted antiviral effects against RSV infection by differentially regulating immune responses and intestinal micro-ecological balance. This study can provide a reference for studying the mechanisms underlying the antiviral effects of L. mucosae.

An O-Benzyl Phosphonamidate Prodrug of Tenofovir for the Treatment of Hepatitis B Virus Infection.

A series of new O-(substituted benzyl) phosphoramidate prodrugs of tenofovir for the treatment of hepatitis B virus (HBV) infections have been designed and synthesized. An investigation of structure-activity relationships revealed that the compound bearing an o-methylbenzyl group (1a) has the most potent in vitro anti-HBV activity. This prodrug (1a) was well-tolerated in KM mice via intragastric administration at a dosage of up to 1.5 g/kg. In DHBV-infected ducks, prodrug 1a displayed a good inhibitory effect on the viral DNA replication in both the serum and the liver in a time- and dose-dependent manner and did not cause any necrosis, hemorrhage, or inflammatory response in the animal livers. Further investigation demonstrated that prodrug 1a achieved a higher exposure of the bioactive metabolite (tenofovir diphosphate, TFV-DP) in the liver, the target organ for the treatment of HBV infection, than tenofovir alafenamide fumarate (TAF) did at an equimolar dose.

Efficacy of Dexamethasone versus Dexmedetomidine Combined with Local Anaesthetics in Brachial Plexus Block: A Meta-Analysis and Systematic Review.

Background: Both dexamethasone and dexmedetomidine are commonly used local anaesthetic adjuvants in brachial plexus block to enhance the blocking effect. However, it is unclear which of the two drugs is more effective in a brachial plexus block. This article compares the effects of dexamethasone and dexmedetomidine combined with local anaesthetics in brachial plexus block through meta-analysis, availing information for current practice and future research. Methods: We conducted a search of the PubMed, Embase, Cochrane Library, and Web of Science databases to identify studies investigating the effects of dexamethasone and dexmedetomidine combined with local anaesthetics on brachial plexus block. The databases were searched from their inception to October 2021. Clinical randomized controlled trials were included. Two researchers independently conducted literature screening. The Cochrane System Review Manual was adopted for literature quality evaluation, whereas Stata 14.0 software aided in the meta-analysis. The duration of analgesia was the primary outcome indicator; whereas, the secondary outcome indicators included the duration of sensory block and motor block. Results: Seven articles were analysed, including 465 patients. Compared to the dexmedetomidine group, the dexamethasone group exhibited longer durations of analgesia (WMD = 111.29, 95% CI: 16.49-206.10, P = 0.021), sensory block (WMD = 173.20, 95% CI: 86.69-259.71, P < 0.0001), and motor block (WMD = 121.03, 95% CI: 12.87-229.20, P = 0.028). Conclusion: The present meta-analysis results affirm that dexamethasone is a better local anaesthetic adjuvant in brachial plexus block that enhances the blocking effect. Nevertheless, the existing heterogeneity warrants additional large-scale, multicentre, high-quality randomized controlled trials in the future for further verification and to provide more reliable clinical evidence.

Inoscavin A, a pyrone compound isolated from a Sanghuangporus vaninii extract, inhibits colon cancer cell growth and induces cell apoptosis via the hedgehog signaling pathway.

BACKGROUND: Sanghuangporus vaninii, a large precious medicinal fungus called Sanghuang in China, has significant antitumor activity. We previously reported that a Sanghuangporus vaninii extract could lead to apoptosis in HT-29 cells through the intrinsic apoptotic pathway. We further found that Inoscavin A exhibited anti-colon cancer activity, but its specific mechanisms have not been fully elucidated. METHODS: Inoscavin A was obtained from Sanghuangporus vaninii by the classic phytochemical separation technology. The male BALB/c nude mice were injected with HT-29 colon cancer cells as animal model. In order to observe the pathological changes of tumor section, the hematoxylin-eosin(H&E) staining was applied in the histological analysis. Metabolomics was utilized for the investigation of the overall changes of serum metabolites in animal model, and the potential targets of Inoscavin A were analyzed by Ingenuity Pathway Analysis (IPA). We further employed a molecular docking approach to predict the degree of combination of Inoscavin A and Smo. Then we further performed Western blotting and immunofluorescence analysis to investigate the expression of proteins involved in Hh-related pathways in tumor tissues. In addition, the colony formation assay, scratch-wound assay and transwell migration and invasion assay were conducted to evaluate the anti-colon-cancer activity of Inoscavin A. Concurrently, the mitochondrial membrane potential assay and TUNEL apoptosis assay were detected to demonstrate the effect of Inoscavin A on promoting HT-29 cells apoptosis. Western blot experiments verified the anti-tumor effects of Inoscavin A were modulated the protein expression of Shh, Ptch1, Smo and Gli1 in HT-29 cells. RESULTS: We showed that Inoscavin A, a pyrone compound isolated from the Sanghuangporus vaninii extract, exerted its antitumor activity in an HT-29 colon cancer cell xenograft mouse model. Subsequently, we first time prove that the antitumor effects of Inoscavin A were related to the hedgehog (Hh) signaling pathway. Furthermore, we demonstrated that Smo, the core receptor of the Hh pathway, was critical for the induction of apoptosis of Inoscavin A and that overexpression of this target could significantly rescue cell apoptosis induced by Inoscavin A treatment. CONCLUSION: Thus, our studies first propose that the natural outgrowth Inoscavin A exerted its anti-cancer effects by inhibiting Smo to suppress the activity of the Hh pathway though inhibiting cell proliferation and promoting apoptosis. These findings further indicate that Inoscavin A will be expected to be a prospective remedical compound for the treatment of colon cancer.

Emergence of a novel reassortant avian influenza virus (H10N3) in Eastern China with high pathogenicity and respiratory droplet transmissibility to mammals.

Decades have passed since the first discovery of H10-subtype avian influenza virus (AIV) in chickens in 1949, and it has been detected in many species including mammals such as minks, pigs, seals and humans. Cases of human infections with H10N8 viruses identified in China in 2013 have raised widespread attention. Two novel reassortant H10N3 viruses were isolated from chickens in December 2019 in eastern China during routine surveillance for AIVs. The internal genes of these viruses were derived from genotype S (G57) H9N2 and were consistent with H5N6, H7N9 and H10N8, which cause fatal infections in humans. Their viral pathogenicity and transmissibility were further studied in different animal models. The two H10N3 isolates had low pathogenicity in chickens and were transmitted between chickens via direct contact. These viruses were highly pathogenic in mice and could be transmitted between guinea pigs via direct contact and respiratory droplets. More importantly, these viruses can bind to both human-type SAα-2,6-Gal receptors and avian-type SAα-2,3-Gal receptors. Asymptomatic shedding in chickens and good adaptability to mammals of these H10N3 isolates would make it easier to transmit to humans and pose a threat to public health.

The Potential Role of Probiotics in Protection against Influenza a Virus Infection in Mice.

Influenza A virus induces severe respiratory tract infection and results in a serious global health problem. Influenza infection disturbs the cross-talk connection between lung and gut. Probiotic treatment can inhibit influenza virus infection; however, the mechanism remains to be explored. The mice received Lactobacillus mucosae 1025, Bifidobacterium breve CCFM1026, and their mixture MIX for 19 days. Effects of probiotics on clinical symptoms, immune responses, and gut microbial alteration were evaluated. L. mucosae 1025 and MIX significantly reduced the loss of body weight, pathological symptoms, and viral loading. B. breve CCFM1026 significantly reduced the proportion of neutrophils and increased lymphocytes, the expressions of TLR7, MyD88, TRAF6, and TNF-α to restore the immune disorders. MIX increased the antiviral protein MxA expression, the relative abundances of Lactobacillus, Mucispirillum, Adlercreutzia, Bifidobacterium, and further regulated SCFA metabolism resulting in an enhancement of butyrate. The correlation analysis revealed that the butyrate was positively related to MxA expression (p < 0.001) but was negatively related to viral loading (p < 0.05). The results implied the possible antiviral mechanisms that MIX decreased viral loading and increased the antiviral protein MxA expression, which was closely associated with the increased butyrate production resulting from gut microbial alteration.

Antiviral effect of fufang yinhua jiedu (FFYH) granules against influenza A virus through regulating the inflammatory responses by TLR7/MyD88 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-Yinhua-Jiedu Granules (FFYH) optimized from a Yin-Qiao-San, as traditional Chinese medicine (TCM), was used to treat influenza and upper respiratory tract infection and was recommended for the prevention and treatment of SARS in 2003 and current COVID-19 in Anhui Province in 2020. AIM OF STUDY: In the clinical studies, FFYH was very effective for the treatment of influenza, but the mechanism of action against influenza A virus remains unclear. In the present study, we investigated the antiviral effect of FFYH against influenza A virus in vitro and vivo. Moreover, the potential mechanism of FFYH against influenza A virus in vivo was investigated for the first time. MATERIALS AND METHODS: CPE inhibition assay and HA assay were used to evaluate the in vitro antiviral effects of FFYH against influenza A virus H1N1, H3N2, H5N1, H7N9 and H9N2. Mice were used to evaluate the antiviral effect of FFYH in vivo with ribavirin and lianhuaqingwen as positive controls. RT-PCR was used to quantify the mRNA transcription of TNF-α, IL-6, IFN-γ, IP10, and IL-1ß mRNA. ELISA was used to examine the expression of inflammatory factors such as TNF-α, IL-6, IFN-γ, IP10, and IL-1ß in sera. The blood parameters were analyzed with auto hematology analyzer. Moreover, the potential mechanism of FFYH against influenza A virus in vivo was also investigated. RESULTS: FFYH showed a broad-spectrum of antiviral activity against H1N1, H3N2, H5N1, H7N9, and H9N2 influenza A viruses. Furthermore, FFYH dose-dependently increased the survival rate, significantly prolonged the median survival time of mice, and markedly reduced lung injury caused by influenza A virus. Also, FFYH significantly improve the sick signs, food taken, weight loss, blood parameters, lung index, and lung pathological changes. Moreover, FFYH could markedly inhibit the inflammatory cytokine expression of TNF-α, IL-6, IFN-γ, IP10, IL-10, and IL-1ß mRNA or protein via inhibition of the TLR7/MyD88/NF-κB signaling pathway in vivo. CONCLUSION: FFYH not only showed a broad-spectrum of anti-influenza virus activity in vitro, but also exhibited a significant protective effect against lethal influenza virus infection in vivo. Furthermore, our results indicated that the in vivo antiviral effect of FFYH against influenza virus may be attributed to suppressing the expression of inflammatory cytokines via regulating the TLR7/MyD88/NF-κB signaling pathway. These findings provide evidence for the clinical treatment of influenza A virus infection with FFYH.

ATG7-dependent and independent autophagy determine the type of treatment in lung cancer.

Based on the role of ATG7 in the initiation of autophagy, autophagy can be divided into ATG7-dependent selective autophagy and ATG7-independent alternative autophagy. However, the detailed roles of two different types of autophagy in antitumor therapy have not been fully elucidated so far. Here, we for the first time demonstrated an investigational inducer, w09, could induce both selective autophagy and alternative autophagy in NSCLC, but the phenotypes of these two kinds of autophagy are different：(1) w09-induced selective autophagy mainly promoted cell apoptosis, while w09-triggered alternative autophagy markedly induced autophagic cell death in NSCLC；(2) w09-induced ATG7 dependent autophagy mainly promoted the accumulation of SQSTM1/p62, while w09-triggered ATG7 independent autophagy markedly accelerated the degradation of SQSTM1/p62. These above results were further confirmed by knockout ATG7 gene in A549 cells or restoration of ATG7 function in H1650 cells. Deletion of ATG7 gene markedly attenuated the effect of w09-induced autophagy or apoptosis on A549 cells, while restoration of functional ATG7 markedly enhanced the effect of w09-induced autophagy and apoptosis on H1650 cells. Mechanistically, we further revealed that w09 induced two different types of autophagy through inhibiting PI3K/AKT/mTOR signaling pathway. Notably, compared with A549WT xenograft model, the in vivo antitumor effect of w09 or Taxel on the ATG7-deficient A549 xenograft model was significantly attenuated. Therefore, a special attention must be paid to distinguish which kinds of autophagy have been induced by autophagy inducers with antitumor agents by targeting PI3K/AKT/mTOR signaling pathway.

Discovery of novel 2-aminonicotinonitrile derivatives with new potential autophagy activity.

Background: To clarify the molecular mechanism of novel 2-aminonicotinonitrile autophagy enhancers, two series of novel 2-aminonicotinonitrile derivatives are synthesized and their structure-activity relationship and biological activity were analyzed. Results & methodology: Structure-activity relationship analysis revealed that substituents at C-4 and C-6 position of 7a contribute to enhance their autophagy-inducing activity, while C-5 position substituents have the opposite effect. The most promising compound 7g showed the strongest autophagy-inducing activity and better antiproliferative activity by inducing cell apoptosis and blocking cell cycle G1 arrest in SGC-7901 cells. Conclusion: The novel 2-aminonicotinonitrile autophagy enhancers were for the first time discovered and 7g might be a promising new autophagy enhancer with potential anticancer activity.

Metabolomics study of the hepatoprotective effect of Phellinus igniarius in chronic ethanol-induced liver injury mice using UPLC-Q/TOF-MS combined with ingenuity pathway analysis.

BACKGROUND: Phellinus igniarius (L.) Quèl as a potential medicinal mushroom possesses multiple biological activities including hepatoprotection, but the hepatoprotective mechanism is not clear. PURPOSE: To elucidate the hepatoprotective effect and potential target of P. igniarius. METHODS: The male C57BL/6 mice were fed with the Lieber-DeCarli diet containing alcohol or isocaloric maltose dextrin as control diet with or without P. igniarius decoction (PID) in the dosage of 0.65 g/kg and 2.6 g/kg. The levels of serum biomarkers were detected by an automatic biochemistry analyser. The histopathological changes of liver were observed by hematoxylin and eosin (H&E) staining. Ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was applied for investigating the dynamic changes of serum metabolites in chronic ethanol-induced liver injury mice and after treatment with PID. Ingenuity pathway analysis (IPA) was employed to identify the potential target of PID. RESULTS: PID could significantly reduce the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and total bile acid (TBA) in serum and improved hepatic steatosis and inflammation. In terms of metabolism, a total of 36 serum differential metabolites were identified, and PID intervention regulated 24 of them, involving the key metabolic pathways such as the biosynthesis of unsaturated fatty acids, primary bile acid biosynthesis, glycerophospholipid metabolism, fatty acids biosynthesis, ether lipid metabolism and arachidonic acid metabolism. On the mechanism, IPA showed that farnesol X receptor (FXR) was the major potential target for PID, and PID could improve chronic alcohol intake induced by the inhibition of mRNA expression of FXR in the liver and the activation of mRNA expression of FXR in the intestine in mice. CONCLUSION: The present study for the first time systematically illustrated the hepatoprotective effect of P. igniarius and preliminarily explored its potential target FXR. P. igniarius might be exploited as a promising therapeutic option for alcoholic liver injury.

EGFR-mediated autophagy in tumourigenesis and therapeutic resistance.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that links extracellular signals to the control of cell survival, growth, proliferation and differentiation. Due to its frequent overexpression and hyperactivation, EGFR has been a therapeutic target for human malignancies. Unfortunately, the specialized inhibitors of EGFR or EGFR-mediated pathways have not yet achieved the desired clinical effects. Therefore, it is necessary to elucidate the EGFR-mediated molecular basis of tumourigenesis, development and therapeutic resistance and to identify potential therapeutic targets. Interestingly, emerging research has indicated that autophagy is closely related to tumourigenesis, tumour progression and chemoresistance. Both autophagy upregulation and downregulation have been observed in cancers, suggesting its dual oncogenic and tumour suppressor properties during malignant transformation. Importantly, EGFR has been demonstrated to be a critical determinant of whether autophagy has a cytoprotective or cytotoxic effect. Therefore, here, we mainly focus on the function of EGFR in autophagy, especially the potential mechanism. The EGFR-mediated pathways or proteins involved in autophagy regulation include (1) the EGFR-mTOR pathway; (2) the EGFR-RAS pathway; (3) EGFR-Beclin1; (4) the EGFR-STAT3 pathway and (5) EGFR-LAPTM4B (oncoprotein lysosomal-associated transmembrane protein 4B). In addition, we also describe the role of EGFR-mediated autophagy in chemoresistance and tumour therapy. We attempt to summarized the mechanism by which EGFR-mediated signalling pathways participate in regulating autophagy and to investigate how to use the existing knowledge to identify potential cancer therapeutic targets.

A Combined Phytochemistry and Network Pharmacology Approach to Reveal the Potential Antitumor Effective Substances and Mechanism of Phellinus igniarius.

Phellinus igniarius (P. igniarius) is a medicinal fungus that is widely used in East Asia for the adjuvant treatment of cancer. To elucidate the antitumor effective substances and mechanism of P. igniarius, we designed an approach incorporating cytotoxicity screening, phytochemical analysis, network pharmacology construction, and cellular and molecular experiments. The dichloromethane extract of P. igniarius (DCMPI) was identified as the active portion in HT-29 cells. Nineteen constituents were identified, and 5 were quantified by UPLC-ESI-Q/TOF-MS. Eight ingredients were obtained in the network pharmacology study. In total, 473 putative targets associated with DCMPI and 350 putative targets related to colon cancer were derived from online databases and target prediction tools. Protein-protein interaction networks of drug and disease putative targets were constructed, and 84 candidate targets were identified based on topological features. Pathway enrichment analysis showed that the candidate targets were mostly related to reactive oxygen species (ROS) metabolic processes and intrinsic apoptotic pathways. Then, a cellular experiment was used to validate the drug-target mechanisms predicted by the system pharmacology analysis. Experimental results showed that DCMPI increased intracellular ROS levels and induced HT-29 cell apoptosis. Molecular biology experiments indicated that DCMPI not only increased Bax and Bad protein expression and promoted PARP and caspase-3/9 cleavage but also down-regulated Bcl-2 and Bcl-xl protein levels to induce apoptosis in HT-29 cells. In conclusion, our study provides knowledge on the chemical composition and antitumor mechanism of P. igniarius, which may be exploited as a promising therapeutic option for colon cancer.

In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine.

2',3'-dideoxyguanosine (DoG) has been demonstrated to inhibit duck hepatitis B virus (DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus (WHV) replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations (EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 µmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors, DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181V, and adefovir-resistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.