Ethanol (EtOH) is a common trigger for gastric mucosal diseases, and mitigating oxidative stress is essential for attenuating gastric mucosal damage. Capsaicin (CAP) has been identified as a potential agent to counteract oxidative damage in the gastric mucosa; however, its precise mechanism remains unclear. This study demonstrates that CAP alleviates EtOH-induced gastric mucosal injuries through two primary pathways: by suppressing the chemokine receptor 4 (CCR4)/Src/p47phox axis, thereby reducing oxidative stress, and by inhibiting the phosphorylation and nuclear translocation of nuclear factor-κB p65 (NF-κB) p65, resulting in diminished inflammatory responses. These findings elucidate the mechanistic pathways of CAP and provide a theoretical foundation for its potential therapeutic application in the treatment of gastric mucosal injuries.
Ethanol/toxicity* ; Animals ; Gastric Mucosa/metabolism* ; Signal Transduction/drug effects* ; Oxidative Stress/drug effects* ; Capsaicin/pharmacology* ; Male ; NADPH Oxidases/genetics* ; Mice ; Humans ; src-Family Kinases/genetics*

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

ObjectiveTo reveal the molecular mechanism of Angong Niuhuangwan（AGNH） in improving traumatic brain injury（TBI） based on single cell sequencing. MethodSeventy-five male SD rats were randomly divided into the sham group， model group， piracetam group（3.6 g·kg^-1）， AGNH low- and high-dose groups（0.09， 0.27 g·kg^-1）， with 15 rats in each group. In addition to the sham group， the other 4 groups used the modified Feeney free-fall impact method to prepare TBI model， and the drugs were administered by gavage immediately after modeling， 24 hours later， the modified neurological deficit score（mNSS） was performed， and brain tissue was isolated to determine the degree of cerebral edema. Hematoxylin-eosin（HE） staining was used to observe the injury degree in the cortex， CA1 region and CA3 region of brain tissue. The expression levels of cyclooxygenase-2（COX-2）， interferon regulatory factor 1（IRF1）， Janus kinase 2（JAK2） and suppressor of cytokine signaling 3（SOCS3） were observed by immunofluorescence（IF） staining. The levels of interleukin（IL）-6， IL-18， IL-1β， IL-17A， tumor necrosis factor-α（TNF-α）， Caspase-1 and nucleotide binding oligomerization domain（NOD）-like receptor heat protein domain associated protein 3（NLRP3） inflammasome were determined by enzyme-linked immunosorbent assay（ELISA）. The regulation of AGNH on each cell population was analyzed by single cell sequencing， and differentially expressed genes were analyzed by Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG）， which led to construct microglia differentially expressed gene network to search for the key targets， and validated by ELISA and IF. ResultCompared with the sham group， the mNSS and brain water content were significantly increased in the model group（P<0.01）. Compared with the model group， mNSS and brain water content in the low and high dose AGNH groups were decreased（P<0.05，P<0.01）. HE staining results showed that compared with the sham group， the cells in the cerebral cortex and hippocampus of rats in the model group were seriously lost， and the cells were arranged loosely（P<0.01）. Compared with the model group， AGNH could significantly increase the density of neurons in the CA1 and CA3 regions of the cerebral cortex and hippocampus， making the arrangement more compact， as well as improved cell morphology（P<0.05，P<0.01）. ELISA and IF staining showed that AGNH could reduce the levels of Caspase-1， IL-17A， TNF-α， NLRP3 and COX-2 in brain tissue of TBI rats（P<0.05， P<0.01）. A total of 13 cell subsets were identified by single cell sequencing， among which microglia played an important role in neuroimmunity. The results of GO enrichment analysis of differentially expressed genes in microglia showed that AGNH improved TBI in response to inflammation and TNF-α. KEGG enriched IL-17 signaling pathway， TNF signaling pathway， Toll-like receptor signaling pathway， etc. The results of network analysis showed that the key targets of AGNH in regulating TBI might be IL-6， IL-1β， JAK2， SOCS3， IRF1. IF and ELISA verification results showed that compared with the sham group， SOCS3 expression in microglia was decreased in the model group， and the expressions of IL-6， IL-1β， JAK2 and IRF1 were increased（P<0.01）. Compared with the model group， AGNH could increase the expression of SOCS3， decrease the expression of IL-6， IL-1β， JAK2， IRF1 （P<0.05， P<0.01）. ConclusionAGNH can reduce the degree of brain edema and brain injury， decrease the expression of inflammatory factors， and inhibit the expression of NLRP3 and its downstream Caspase-1 in TBI rats， which may act on the targets of IL-6， IL-1β， JAK2， IRF1 and SOCS3 in microglia.

BACKGROUND: It has been reported that human adipose-derived mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells with the function of albumin synthesis and urea secretion in vitro.OBJECTIVE: To investigate the potential of adipose-derived mesenchymal stem cells differentiating into hepatocyte-like cells in vitro.METHODS: Adipose-derived mesenchymal stem cells were isolated from the subcutaneous fat of hepatitis B virus infection patients by collagenase digestion and adherent method. Adipose-derived mesenchymal stem cells were induced by three-phase induction method and observed morphologically. The expression levels of alpha-fetoprotein, albumin and cytokeratin 18 were detected by immunohistochemical staining and glycogen synthesis function was detected by glycogen staining method.RESULTS AND CONCLUSION: Most of adipose-derived mesenchymal stem cells induced by three-phase induction method were differentiated into hepatocyte-like cells with polygonal morphology. Immunohistochemistry staining results showed that hepatocyte-like cells expressed alpha-fetoprotein, albumin and cytokeratin 18, and the expression levels of albumin and cytokeratin 18 increased with the culture time. The induced cells had the function of glycogen synthesis and were positive for periodic acid Schiff staining. These results showed that the subcutaneous adipose-derived mesenchymal stem cells could be induced into functional hepatocyte-like cells in hepatitis B virus infection patients.

Objective To observe the clinical efficacy of Zhishanghan Zhufeng Formula in treating knee osteoarthritis.Methods Totally 90 patients were randomly divided into treatment group and control group, 45 cases in each group. The treatment group was treated with Zhishanghan Zhufeng Formula for 30 days. The control group was treated with routine medicine (diclofenac sodium sustained release tablets and glucosamine sulfate tablets) for 30 days. The clinical efficacy and the changes of VAS, WOMAC score and knee temperature before and after treatment of two groups were observed.Results TCM clinical symptoms were significantly improved, and the effects in the treatment group were more evident compared with the control group (P<0.05). After treatment, VAS and WOMAC score of the two groups decreased obviously, while the knee temperature increased, and the treatment group was superior to the control group, with significant significance (P<0.05).Conclusion Zhishanghan Zhufeng Formula has effective clinical efficacy for knee osteoarthritis.

Objective:To highly express HIV-1p17 in E.coli and purify the protein.Methods:①Recombinant plasmid was constructed by inserting HIV-1p17 gene amplified by PCR into plasmid vector,pET28c;②The recombinant plasmid was expressed in BL21,BL21(DE3),BL21(DE3) plysS and HMS174(DE3) of E.coli separately;③The target protein were purified with Ni-NTA resin;④The purified protein was detected by western blot and ELISA.Results:The expression of the P17 protein in BL21(DE3) represented up to 32% of total protein in E.coli,which was the most amounts compared with other kinds of E.coli.The purity of the purified protein reached 95%.The purified protein was recognized by HIV-1P17McAb as well as by HIV-1 positive serum.Conclusion:The recombinant plasmid is highly expressed in BL21(DE3) of E.coli that can be proceeded to the immunocompetence and the bioactivity research.The method of Ni-NTA resin is simple with low protein losing and high purity.And the purified p17 can be employed in early detection of HIV-1 infection and prediction of the clinical progression.