Objective To express nucleoprotein(NP) of influenza A virus in Bac-to-Bac TOPO system and evaluate its immunogenicity,so as to lay a foundation for the research of influenza virus NP protein vaccine.Methods The NP gene of influenza A H1N1(A/Guangdong-Maonan/SWL1536/2019_CNIC-1909) was amplified by PCR and connected to the vector pFastBac~(TM)/CT-TOPO~(TM),which was transformed into competent E.coli One Shot~(TM) Mach1~(TM) T1~R to prepare donor plasmid,and identified by PCR and sequencing.The donor plasmid was transformed into competent E.coli MAX Efficiency~(TM) DH10Bac~(TM),the positive monoclone was screened by blue-white spot screening,and the recombinant bacmid was extracted and identified by PCR.The recombinant bacmid was transfected into Sf9 insect cells to prepare the recombinant baculovirus,and the virus titer was detected by flow cytometry.After multiple rounds of amplification,the recombinant baculovirus infected Sf9 insect cells for the expression of the target protein NP,and the recombinant protein was purified by nickel ion affinity chromatography.Female BALB/c mice were subcutaneously inoculated with recombinant NP protein and PBS respectively,with six mice in each group.The serum specific antibody levels of mice were detected by ELISA,the IFN-γ secreted by spleen cells were detected by flow cytometry,and the CD4~+ and CD8~+ lymphocytes produced by spleen cells were detected by ELISpot.Two weeks after the last immunization,the mice were challenged with A/Guangdong Maonan/SWL 1536/2019_CNIC-1909 influenza virus through nasal cavity,and the body mass changes and survival rate of mice within 14 days after challenge were monitored.Results The results of PCR and sequencing showed that the donor plasmid was constructed correctly.PCR identification results confirmed that the recombinant bacmid was successfully constructed.The titer of recombinant baculovirus was 2.875 × 10~8ivp/mL.The recombinant NP protein showed specific binding to mouse anti-influenza A virus NP monoclonal antibody,with the purity of 91.3% after purification.Two weeks after the last immunization,compared with the PBS group,the serum specific antibody titer(t=0.288,P <0.000 1),the average number of IFNγ spots produced by spleen cells(t=9.235,P <0.000 1),and the number of CD4~+ and CD8~+T lymphocytes produced(t=10.870 and 6.200,P=0.008 4and 0.025 0,respectively) in mice of NA group increased significantly.The mice in PBS group all died within 5 days after infection,while in NP group,one mouse died 5 days after infection,and the other five mice survived.Conclusion The NP protein of influenza A virus expressed by Bac-to-Bac TOPO system has high immunogenicity and can produce strong humoral and cellular immune responses,which can provide certain protection for mice against influenza virus infection.

Acute-on-chronic liver failure （ACLF） is an acute and critical illness with a high short-term mortality rate， and current therapies mainly focus on elimination of causes， organ support， and prevention of complications. Although liver transplantation is the most effective treatment modality， its clinical application is limited， and traditional Chinese medicine has shown significant advantages and characteristics in the treatment of ACLF. In traditional Chinese medicine， ACLF is classified into the same category as diseases such as “jaundice”， and unlike traditional jaundice which is mostly characterized by excess and heat syndromes， the syndrome of ACLF has gradually transformed from Yang jaundice to Yin jaundice due to the changing disease spectrum of ACLF. With reference to the pathogenesis of ACLF in Western medicine and traditional Chinese medicine theories， this article discusses the essential pathogenesis of ACLF in traditional Chinese medicine， explores the evolution of ACLF syndromes， and reviews the research advances in the clinical efficacy and mechanisms of traditional Chinese medicine based on the three-factor differentiation-based treatment of damp-heat， blood stasis-heat， and spleen deficiency， as well as the safety of spleen-strengthening and Yang-warming drugs in the clinical treatment of ACLF， in order to provide ideas， methods， and evidence for the application of traditional Chinese medicine in ACLF.

BACKGROUND: Enzalutamide, a second-generation androgen receptor (AR) pathway inhibitor, is widely used in the treatment of castration-resistant prostate cancer. However, after a period of enzalutamide treatment, patients inevitably develop drug resistance. In this study, we characterized leucine-rich repeated G-protein-coupled receptor 5 (LGR5) and explored its potential therapeutic value in prostate cancer. METHODS: A total of 142 pairs of tumor and adjacent formalin-fixed paraf-fin-embedded tissue samples from patients with prostate cancer were collected from the Pathology Department at Sun Yat-sen Memorial Hos-pital. LGR5 was screened by sequencing data of enzalutamide-resistant cell lines combined with sequencing data of lesions with different Gleason scores from the same patients. The biological function of LGR5 and its effect on enzalutamide resistance were investigated in vitro and in vivo . Glutathione-S-transferase (GST) pull-down, coimmunoprecipitation, Western blotting, and immunofluorescence assays were used to explore the specific binding mechanism of LGR5 and related pathway changes. RESULTS: LGR5 was significantly upregulated in prostate cancer and negatively correlated with poor patient prognosis. Overexpression of LGR5 promoted the malignant progression of prostate cancer and reduced sensitivity to enzalutamide in vitro and in vivo . LGR5 promoted the phosphorylation of glycogen synthase kinase-3β (GSK-3β) by binding heat shock protein 90,000 alpha B1 (HSP90AB1) and mediated the activation of the Wingless/integrated (WNT)/β-catenin signaling pathway. The increased β-catenin in the cytoplasm entered the nucleus and bound to the nuclear AR, promoting the transcription level of AR, which led to the enhanced tolerance of prostate cancer to enzalutamide. Reducing HSP90AB1 binding to LGR5 significantly enhanced sensitivity to enzalutamide. CONCLUSIONS LGR5 directly binds to HSP90AB1 and mediates GSK-3β phosphorylation, promoting AR expression by regulating the WNT/β-catenin signaling pathway, thereby conferring resistance to enzalutamide treatment in prostate cancer.
Male ; Humans ; Phenylthiohydantoin/pharmacology* ; Benzamides ; Receptors, G-Protein-Coupled/genetics* ; Nitriles ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Prostatic Neoplasms/drug therapy* ; beta Catenin/metabolism* ; Receptors, Androgen/genetics* ; Animals ; Mice ; Wnt Signaling Pathway/physiology*

Loss of protein homeostasis is a hallmark of cellular senescence, and ribosome pausing plays a crucial role in the collapse of proteostasis. However, our understanding of ribosome pausing in senescent cells remains limited. In this study, we utilized ribosome profiling and G-quadruplex RNA immunoprecipitation sequencing techniques to explore the impact of RNA G-quadruplex (rG4) on the translation efficiency in senescent cells. Our results revealed a reduction in the translation efficiency of rG4-rich genes in senescent cells and demonstrated that rG4 structures within coding sequence can impede translation both in vivo and in vitro. Moreover, we observed a significant increase in the abundance of rG4 structures in senescent cells, and the stabilization of the rG4 structures further exacerbated cellular senescence. Mechanistically, the RNA helicase DHX9 functions as a key regulator of rG4 abundance, and its reduced expression in senescent cells contributing to increased ribosome pausing. Additionally, we also observed an increased abundance of rG4, an imbalance in protein homeostasis, and reduced DHX9 expression in aged mice. In summary, our findings reveal a novel biological role for rG4 and DHX9 in the regulation of translation and proteostasis, which may have implications for delaying cellular senescence and the aging process.
G-Quadruplexes ; Cellular Senescence ; Ribosomes/genetics* ; Humans ; Animals ; Mice ; DEAD-box RNA Helicases/genetics* ; Protein Biosynthesis ; RNA/chemistry* ; Neoplasm Proteins

Chronic, unresolved inflammation correlates with persistent hepatic injury and fibrosis, ultimately progressing to hepatocellular carcinoma (HCC). Bisdemethoxycurcumin (BDMC) demonstrates therapeutic potential against HCC, yet its mechanism in preventing hepatic "inflammation-carcinoma transformation" remains incompletely understood. In the current research, clinical HCC specimens underwent analysis using hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) to evaluate the expression of fibrosis markers, M2 macrophage markers, and CXCL12. In vitro, transforming growth factor-β1 (TGF-β1)-induced LX-2 cells and a co-culture system of LX-2, THP-1, and HCC cells were established. Cell functions underwent assessment through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and Transwell assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blotting and immunofluorescence evaluated the differential expression of molecules. The interaction between β-catenin/TCF4 and CXCL12 was examined using co-immunoprecipitation (Co-IP), dual luciferase, and chromatin immunoprecipitation (ChIP) assays. A DEN-induced rat model was developed to investigate BDMC's role in liver fibrosis-associated HCC (LFAHCC) development in vivo. Our results showed that clinical HCC tissues exhibited elevated fibrosis and enriched M2 macrophages. BDMC delayed liver fibrosis progression to HCC in vivo. BDMC inhibited the inflammatory microenvironment induced by activated hepatic stellate cells (HSCs). Furthermore, BDMC suppressed M2 macrophage-induced fibrosis and HCC cell proliferation and metastasis. Mechanistically, BDMC repressed TCF4/β-catenin complex formation, thereby reducing CXCL12 transcription in LX-2 cells. Moreover, CXCL12 overexpression reversed BDMC's inhibitory effect on macrophage M2 polarization and its mediation of fibrosis, as well as HCC proliferation and metastasis. BDMC significantly suppressed LFAHCC development through CXCL12 in rats. In conclusion, BDMC inhibited LFAHCC progression by reducing M2 macrophage polarization through suppressing β-catenin/TCF4-mediated CXCL12 transcription.
Animals ; Liver Neoplasms/etiology* ; Humans ; Carcinoma, Hepatocellular/immunology* ; Liver Cirrhosis/complications* ; Macrophages/drug effects* ; Male ; Rats ; Chemokine CXCL12/genetics* ; Diarylheptanoids/pharmacology* ; Rats, Sprague-Dawley ; beta Catenin/genetics*

ObjectiveTo investigate the clinical features and traditional Chinese medicine （TCM） syndrome distribution of treatment-naïve patients with hepatitis B virus-related primary liver cancer （HBV-PLC）， and to provide a basis for integrated traditional Chinese and Western medicine in the prevention and treatment of HBV-PLC. MethodsA retrospective analysis was performed for the clinical data of 99 treatment-naïve HBV-PLC patients who were admitted to Department of Hepatology and Infectious Diseases in The First Affiliated Hospital of Hunan University of Chinese Medicine from January 2019 to December 2024. According to whether the patient received standardized antiviral therapy （for ≥3 years）， they were divided into antiviral group and non-antiviral group， and according to the status of HBeAg， they were divided into HBeAg-positive group and HBeAg-negative group. Demographic features， laboratory test results， imaging data， and TCM syndrome data were collected， and neutrophil-to-lymphocyte ratio （NLR）， Child-Pugh score， and CNLC stage were calculated. The independent samples t-test was used for comparison of normally distributed continuous data between two groups， and the chi-square test was used for comparison of categorical data between groups. ResultsThe 99 treatment-naïve HBV-PLC patients had a mean age of 57.12±11.60 years， and the patients aged 50 — 75 years accounted for the highest proportion of 72.7%， with a male/female ratio of 5.2∶1. The patients with liver cirrhosis accounted for 81.8%， and 67.7% of the patients did not receive antiviral therapy in the past. The positive rates of HBV DNA， HBeAg， and alpha-fetoprotein were 80.8%， 18.2%， and 69.7%， respectively， and the patients with Child-Pugh class A/B disease accounted for 89.9%. Compared with the non-antiviral group， the antiviral group had a significantly smaller maximum tumor diameter （t=2.310， P=0.024）， a significantly lower HBV DNA positive rate （χ²=14.006， P<0.001）， and a significantly lower number of tumor thrombi （χ²=7.347， P=0.007）. In addition， there were significant differences between the HBeAg-negative group and the HBeAg-positive group in Child-Pugh class （χ²=6.780， P=0.034） and CNLC stage （χ²=8.746， P=0.033）. Among the 99 treatment-naïve HBV-PLC patients， 41.4% had liver depression and spleen deficiency with blood stasis， 22.2% had Qi deficiency and blood stasis syndrome， and 19.2% had damp-heat accumulation with blood stasis. ConclusionTreatment-naïve HBV-PLC patients are mainly middle-aged and elderly male individuals， and most of the patients are comorbid with liver cirrhosis. Standardized antiviral therapy can significantly reduce tumor burden and improve virologic response， with better hepatic compensation in HBeAg-negative patients， and hypoproteinemia is more common in patients with Qi deficiency and blood stasis syndrome.

In the traditional theoretical system of traditional Chinese medicine， latent pathogenic factors deeply hiding in the liver and gallbladder of the Jueyin meridian are the core pathogenesis of chronic hepatitis B. Interferon can regulate immunity and eliminate viruses， and clinical cure can be achieved by penetrating and removing pathogenic factors and toxins. However， it often causes dizziness and confusion reactions in the initial stage of treatment， and long-term use can damage Qi-blood； furthermore， its therapeutic effect depends on the patient’s own vital Qi. When traditional Chinese medicine is used in combination with interferon， drug compatibility should be adjusted according to the dynamic changes of pathogenesis， which can reduce adverse reactions， enhance the therapeutic outcome， expand the applicable population of interferon， and improve the clinical symptoms of patients.

Objective To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells(EMSCs-exo)for repairing secondary spinal cord injury.Methods EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa,identified by transmission electron microscope,nanoparticle tracking analysis(NTA),and Western blotting,and quantified using the BCA method.Neonatal rat microglia purified by differential attachment were induced with 100 μg/L lipopolysaccharide(LPS)and treated with 37.5 or 75 mg/L EMSCs-exo.PC12 cells were exposed to 400 μmol/L H2O2 and treated with EMSCs-exo at 37.5 or 75 mg/L.The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT-PCR,and the concentrations of IL-6,IL-10,and IGF-1 in the supernatants were measured with ELISA.The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry.Results The isolated rat EMSCs showed high expressions of nestin,CD44,CD105,and vimentin.The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63,CD81,and TSG101 but not vimentin.In LPS-treated microglia,EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression(P<0.05).EMSCs-exo treatment at 75 mg/L,as compared with the lower concentration at 37.5 mg/L,more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia,and more effectively promoted cell survival and decreased apoptosis rate of H2O2-induced PC12 cells(P<0.05).Conclusion EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival,suggesting its potential in controlling secondary spinal cord injury.

Objective To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells(EMSCs-exo)for repairing secondary spinal cord injury.Methods EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa,identified by transmission electron microscope,nanoparticle tracking analysis(NTA),and Western blotting,and quantified using the BCA method.Neonatal rat microglia purified by differential attachment were induced with 100 μg/L lipopolysaccharide(LPS)and treated with 37.5 or 75 mg/L EMSCs-exo.PC12 cells were exposed to 400 μmol/L H2O2 and treated with EMSCs-exo at 37.5 or 75 mg/L.The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT-PCR,and the concentrations of IL-6,IL-10,and IGF-1 in the supernatants were measured with ELISA.The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry.Results The isolated rat EMSCs showed high expressions of nestin,CD44,CD105,and vimentin.The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63,CD81,and TSG101 but not vimentin.In LPS-treated microglia,EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression(P<0.05).EMSCs-exo treatment at 75 mg/L,as compared with the lower concentration at 37.5 mg/L,more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia,and more effectively promoted cell survival and decreased apoptosis rate of H2O2-induced PC12 cells(P<0.05).Conclusion EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival,suggesting its potential in controlling secondary spinal cord injury.

Objective:To evaluate modified extended trochanteric osteotomy (ETO) applied in the revision of Vancouver B2/B3 periprosthetic femoral fractures (PFF).Methods:A retrospective study was conducted to analyze the 35 patients with Vancouver B2/B3 PFF who had been treated at Joint Disease Department Ⅱ, Zhengzhou Orthopedic Hospital from January 2012 to November 2020. There were 10 males and 15 females with an age of (74.3±7.8) years. The time from their primary replacement to revision was (120.3±28.6) months. By the Vancouver classification, 26 cases were type B2 and 9 ones type B3. The modified ETO was used in the revision surgery for all patients. The clinical efficacy was evaluated using Harris hip score, imaging evaluation was performed using the Beals and Tower criteria at the last follow-up, and complications were recorded.Results:The operation time for this cohort was (148±32) min and intraoperative bleeding (800±150) mL. All patients were followed up for (45.2±15.3) months. The Harris score increased significantly from preoperative (21.3±11.2) points to (86.2±5.2) points at the last follow-up ( P < 0.001). By the Beals and Tower evaluation, 9 cases were rated as excellent, 24 cases as good, and 2 as poor. All the fractures and sites of trochanteric osteotomy got healed after (4.4±2.8) months except for 1 case of nonunion. Prosthesis subsidence occurred in 3 cases, in 2 of which the subsidence stopped 6 months later and in only 1 of which revision was needed due to the subsidence. Upward block displacement of the greater trochanteric fracture occurred in 2 cases, but did not exceed 1 cm. One case of postoperative dislocation responded to manual reduction. Conclusion:In the revision of Vancouver B2/B3 PFF, the modified ETO can improve fracture healing, and reduce postoperative dislocations and complications, leading to satisfactory clinical efficacy.