Objective To understand the epidemic characteristics and genotype distribution of acute hepatitis B in Tianjin, and to find out the relationship between genotype and epidemic characteristics. Methods The information of acute hepatitis B cases with a local address in Tianjin was collected through the National Infectious Disease Surveillance System in Tianjin from 2018 to 2022. The patient outcomes were followed up through hospital system records and telephone survey, and hepatitis B virus (HBV) genotypes were detected by fluorescent PCR. Results From 2018 to 2022, there were 387 cases of acute hepatitis B with local address reported in Tianjin, with an average annual reported incidence rate of 0.52/100 000, showing a downward trend in general (χ²=28.553，P<0.001). The reported male to female incidence ratio was 1.68. The age distribution was mainly concentrated in the 30-65 age group, with the highest incidence rate (1.22/100 000) reported in the 35-39 age group. 72.87% of cases showed negative HBsAg after 6 months of follow-up following diagnosis. The proportion of cadres and staff who turned negative (92.16%) was significantly higher than that of those who did not turn negative (0%). The median ALT (1508.00 U/L) in the turning negative group was significantly higher than that in the non-turning negative group (976.00 U/L). Among 315 cases with successful genotyping, genotype C accounted for 81.27%, and genotype B accounted for 14.92%, with 47 cases. The median ALT of genotype B patients with acute hepatitis B (1585.00 U/L) was significantly higher than that of genotype C patients (988.00 U/L). Conclusion The reported incidence rate of acute hepatitis B in Tianjin is relatively low, and shows a downward trend. Young and middle-aged men are prone to infect HBV. Genotype C is the main genotype, and genotype B HBV causes more serious liver damage in patients with acute hepatitis B.

ObjectiveTo preliminarily explore the active components and target pathways of Angelicae Sinensis Radix-Polygonati Rhizoma （ASR-PR） herb pair in the treatment of simple obesity through network pharmacology and molecular docking， and to verify and investigate its mechanism of action via animal experiments. MethodsThe chemical constituents and targets of ASR and PR were predicted using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. Targets related to simple obesity were identified by retrieving the GeneCards， Online Mendelian Inheritance in Man （OMIM）， Pharmacogenomics Knowledgebase （PharmGKB）， and DisGeNET databases. The intersection of drug and disease targets was used to construct an active component-target network using Cytoscape software. This network was imported into the STRING database to construct a protein-protein interaction （PPI） network， and topological analysis was conducted to identify core genes. Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis and mapping were performed using the DAVID database and the Microbioinformatics platform. AutoDock 1.5.7 software was used to perform molecular docking between the top five active components and core targets. An animal model of simple obesity was established by feeding C57BL/6J mice a high-fat diet. The mice were administered ASR （2.06 g·kg^-1）， PR （2.06 g·kg^-1）， or ASR-PR （4.11 g·kg^-1） for 10 weeks， while the model group received an equal volume of purified water by gavage. After the administration period， the mice were sacrificed to measure body fat weight and serum levels of total cholesterol （TC）， triglycerides （TG）， high-density lipoprotein （HDL）， and low-density lipoprotein （LDL）. Hematoxylin-eosin （HE） staining was used to observe histopathological sections of liver and adipose tissue. Serum levels of leptin， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA expression levels of epidermal growth factor receptor （EGFR） and signal transducer and activator of transcription 3 （STAT3） in liver tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsNetwork pharmacology and molecular docking results indicated that the treatment of simple obesity by ASR-PR may involve the regulation of protein expression of core targets EGFR and STAT3 by its main components MOL009760 （Siberian glycoside A_qt）， MOL003889 （methyl protodioscin_qt）， MOL009766 （resveratrol）， MOL006331 （4′，5-dihydroxyflavone）， and MOL004941 （baicalin）， thereby modulating the PI3K/Akt and JAK/STAT signaling pathways. The animal experiment results showed that compared with the normal group， the model group had significantly increased body weight， body fat weight， and serum levels of TG， TC， TNF-α， IL-6， and leptin （P<0.01）. EGFR mRNA expression was significantly elevated （P<0.05）， while STAT3 mRNA expression was significantly decreased （P<0.01）. Histological analysis revealed disordered hepatic architecture in the model group， with pronounced lipid vacuoles， cytoplasmic loosening， lipid accumulation， and steatosis. Adipocytes in white adipose tissue （WAT） and brown adipose tissue （BAT） of the model group exhibited markedly increased diameters， reduced cell counts per unit area， and irregular morphology. Compared with the model group， the ASR-PR group significantly reduced body weight， body fat weight， serum TC， IL-6， TNF-α， leptin levels， and EGFR mRNA expression （P<0.01）. TG levels were also significantly decreased （P<0.05）， while STAT3 mRNA expression was significantly increased （P<0.01）. Histopathological improvements included reduced size and number of hepatic lipid vacuoles and restoration of liver cell morphology toward that of the normal group. The diameter of adipocytes significantly decreased， and the number of adipocytes per unit area increased. ConclusionASR-PR may regulate the expression of key target proteins such as EGFR and STAT3 via its core active components， modulate the PI3K/Akt and JAK/STAT signaling pathways， repair damaged liver and adipose tissues， and thereby alleviate the progression of obesity in mice.

ObjectiveTo preliminarily explore the active components and target pathways of Angelicae Sinensis Radix-Polygonati Rhizoma （ASR-PR） herb pair in the treatment of simple obesity through network pharmacology and molecular docking， and to verify and investigate its mechanism of action via animal experiments. MethodsThe chemical constituents and targets of ASR and PR were predicted using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. Targets related to simple obesity were identified by retrieving the GeneCards， Online Mendelian Inheritance in Man （OMIM）， Pharmacogenomics Knowledgebase （PharmGKB）， and DisGeNET databases. The intersection of drug and disease targets was used to construct an active component-target network using Cytoscape software. This network was imported into the STRING database to construct a protein-protein interaction （PPI） network， and topological analysis was conducted to identify core genes. Gene Ontology （GO） analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis and mapping were performed using the DAVID database and the Microbioinformatics platform. AutoDock 1.5.7 software was used to perform molecular docking between the top five active components and core targets. An animal model of simple obesity was established by feeding C57BL/6J mice a high-fat diet. The mice were administered ASR （2.06 g·kg^-1）， PR （2.06 g·kg^-1）， or ASR-PR （4.11 g·kg^-1） for 10 weeks， while the model group received an equal volume of purified water by gavage. After the administration period， the mice were sacrificed to measure body fat weight and serum levels of total cholesterol （TC）， triglycerides （TG）， high-density lipoprotein （HDL）， and low-density lipoprotein （LDL）. Hematoxylin-eosin （HE） staining was used to observe histopathological sections of liver and adipose tissue. Serum levels of leptin， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA expression levels of epidermal growth factor receptor （EGFR） and signal transducer and activator of transcription 3 （STAT3） in liver tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsNetwork pharmacology and molecular docking results indicated that the treatment of simple obesity by ASR-PR may involve the regulation of protein expression of core targets EGFR and STAT3 by its main components MOL009760 （Siberian glycoside A_qt）， MOL003889 （methyl protodioscin_qt）， MOL009766 （resveratrol）， MOL006331 （4′，5-dihydroxyflavone）， and MOL004941 （baicalin）， thereby modulating the PI3K/Akt and JAK/STAT signaling pathways. The animal experiment results showed that compared with the normal group， the model group had significantly increased body weight， body fat weight， and serum levels of TG， TC， TNF-α， IL-6， and leptin （P<0.01）. EGFR mRNA expression was significantly elevated （P<0.05）， while STAT3 mRNA expression was significantly decreased （P<0.01）. Histological analysis revealed disordered hepatic architecture in the model group， with pronounced lipid vacuoles， cytoplasmic loosening， lipid accumulation， and steatosis. Adipocytes in white adipose tissue （WAT） and brown adipose tissue （BAT） of the model group exhibited markedly increased diameters， reduced cell counts per unit area， and irregular morphology. Compared with the model group， the ASR-PR group significantly reduced body weight， body fat weight， serum TC， IL-6， TNF-α， leptin levels， and EGFR mRNA expression （P<0.01）. TG levels were also significantly decreased （P<0.05）， while STAT3 mRNA expression was significantly increased （P<0.01）. Histopathological improvements included reduced size and number of hepatic lipid vacuoles and restoration of liver cell morphology toward that of the normal group. The diameter of adipocytes significantly decreased， and the number of adipocytes per unit area increased. ConclusionASR-PR may regulate the expression of key target proteins such as EGFR and STAT3 via its core active components， modulate the PI3K/Akt and JAK/STAT signaling pathways， repair damaged liver and adipose tissues， and thereby alleviate the progression of obesity in mice.

OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

BACKGROUND:The definitive cause of adolescent idiopathic scoliosis is not yet known.The search for a clinical approach to address adolescent idiopathic scoliosis is imminent.OBJECTIVE:To investigate the effect of Schroth therapy combined with core strength training on mild adolescent idiopathic scoliosis and to provide more bases for the clinical treatment of mild adolescent idiopathic scoliosis.METHODS:110 patients with mild adolescent idiopathic scoliosis attending the Department of Rehabilitation Medicine and Department of Spine Surgery of Affiliated Hospital of Nantong University from July 2022 to January 2024 were selected as the study subjects.They were divided into the trial group and the control group according to the wishes of the patients and their parents,with 55 cases in each group.The control group was observed and followed up,and the trial group underwent Schroth therapy combined with core strength training for 45 minutes a day for 24 weeks.The differences in imaging parameters,body surface indexes,three-dimensional ultrasound imaging angle,and quality of life were compared between the two groups before and after treatment.RESULTS AND CONCLUSION:(1)At 24 weeks after treatment,major curve Cobb,apical vertebral translation,and cervical lordosis were significantly improved in the trial group(P＜0.05),while there was no significant difference in the control group(P＞0.05).Major curve Cobb and apical vertebral translation in the trial group were significantly better than those in the control group(P＜0.05).(2)At 24 weeks after treatment,angle of trunk rotation in the trial group was significantly lower than that before treatment(P＜0.05),while there was no significant difference between before and after treatment in the control group(P＞0.05),and angle of trunk rotation in the trial group was significantly lower than that of the control group(P＜0.05).(3)At 24 weeks after treatment,the center of laminae angle of three-dimensional ultrasound imaging was significantly reduced in the trial group(P＜0.05),while there was no significant difference in the control group before and after treatment(P＞0.05).The center of laminae angle of three-dimensional ultrasound imaging was smaller in the trial group than that in the control group(P＜0.05).(4)At 24 weeks after treatment,in terms of the quality of life,pain dimension score in the trial group was significantly increased(P＜0.05).Both trial and control groups showed significantly higher scores in the self-image dimension compared with that before treatment(P＜0.05).Both groups had significantly lower scores in the mental health dimension compared with that before treatment(P＜0.05).In the dimensions of pain,self-image,mental health,and satisfaction,the trial group was significantly higher than the control group(P＜0.05).(5)It is indicated that Schroth therapy combined with core strength training can improve the major curve Cobb,apical vertebral translation,and cervical lordosis angle,reduce the angle of trunk rotation,decrease the center of laminae angle of three-dimensional ultrasound imaging,and improve the quality of life,and it is effective in the treatment of mild adolescent idiopathic scoliosis.

Objective:To investigate the impact of inserting an exogenous gene, NanoLuc (Nluc), at different sites in the influenza virus genome on viral properties and analyze the expression stability of the exogenous gene both in vitro and in vivo. Methods:Using molecular cloning techniques and reverse genetics, eight recombinant influenza viruses were constructed by inserting the exogenous Nluc gene into the gene segments encoding hemagglutinin (HA), neuraminidase (NA), non-structural protein (NS), and polymerase basic protein 1 (PB1). Viral replication capacity was evaluated by hemagglutination and plaque assays. Nluc expression in infected cells was monitored by fluorescence imaging. The potential impact of the exogenous gene insertion on viral infectivity was examined in a mouse infection model. Independent samples t-test were used for statistical analysis. Results:The recombinant viruses with insertions in the HA, NA, and NS gene segments generated fluorescent signals in the first generation of rescued viruses and demonstrated replication capabilities in plaque and hemagglutination assays. The recombinant viruses based on the NA and NS genes were capable of stably expressing Nluc across different generations, and exhibited correct fluorescent distribution patterns in mouse infection experiments. Meanwhile, the NS gene-based recombinant virus demonstrated superior stability in the mouse model.Conclusions:This study demonstrates that the NS gene segment of influenza virus can serve as an effective insertion site for exogenous genes without impairing the viral replication or infectivity, and the recombinant virus constructed based on it exhibits high integration stability and substantial application potential.

Objective:To investigate the mechanism of Polygonati Rhizoma-Angelicae Sinensis Radix in the treatment of prediabetes based on network pharmacology and animal experiments.Methods:The active components of Polygonati Rhizoma and Angelicae Sinensis Radix were retrieved from the TCMSP database. The potential targets of the active components were predicted using the Swiss Target Prediction database. The "drug-active component-target" network was constructed by Cytoscape 3.10 software. The disease targets were obtained from OMIM, Genecards, TTD and the U.S. National Library of Medicine. The common targets of drugs and diseases were screened by Venny 2.1.0 platform and imported into STRING database to construct the PPI network. The DAVID database was employed to perform Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on the common targets of drugs and diseases. The prediabetes model was established by feeding C57BL/6J mice with high-fat diet. 24 high-fat fed mice were divided into four groups using a random number table: the model group, Polygonati Rhizoma group, Angelicae Sinensis Radix group and Polygonati Rhizoma-Angelicae Sinensis Radix group (herb pair group), with 6 mice in each group. Another 6 normal mice were set as the normal group. Polygonati Rhizoma group was intragastrically administered with Polygonati Rhizoma granule solution at 2.055 g/kg, the Angelicae Sinensis Radix group was intragastrically administered with Angelicae Sinensis Radix granule solution at 2.055 g/kg, and the herb pair group was intragastrically administered with Polygonati Rhizoma-Angelicae Sinensis Radix herb pair at 4.11 g/kg, once daily. The model group and the normal group were intragastrically administered with an equal volume of deionized water for 10 weeks. Fasting blood glucose (FBG) and oral glucose tolerance tests (OGTT) were regularly performed. After 10 weeks of intragastric administration, the levels of serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and fasting insulin (FINS) were measured; glycogen deposition in liver tissues was observed using periodic acid-Schiff (PAS); the mRNA expression levels of FGF1, FGF2, PGF, KDR, IGF1R, INSR, PI3Kca, PI3Kcb, PI3Kcg, Akt1, Akt2 and GSK-3β in liver tissues were detected by Real-time PCR; the protein expression levels of PI3K, phosphorylated (p)-Akt, Akt, p-GSK-3β and GSK-3β in liver tissues of mice were detected by Western blot.Results:205 core targets of Polygonati Rhizoma-Angelicae Sinensis Radix in the treatment of prediabetes were identified; KEGG enrichment analysis revealed that the herb pair may exert hypoglycemic effects by activating the PI3K-Akt signaling pathway. Compared with the model group, the FBG level and AUC values in the herb pair group decreased ( P<0.05), the levels of LDL-C and HDL-C decreased ( P<0.01), the FINS and HOMA-IR levels decreased ( P<0.05), the mRNA expression levels of FGF1, FGF2, PGF, KDR, IGF1R, INSR, PI3Kca, PI3Kcb, PI3Kcg, Akt1, Akt2 and GSK-3β in liver tissues were elevated significantly ( P<0.01), and the protein expression levels of PI3K/β-actin, p-Akt/Akt and p-GSK-3β/GSK-3β in liver tissues of the herb pair group significantly increased ( P<0.01). Conclusion:Polygonati Rhizoma-Angelicae Sinensis Radix may activate PI3K/Akt/GSK-3β signaling pathway by up-regulating FGF1, FGF2, PGF, etc., and affect insulin resistance, glycogen synthesis and other processes, so as to treat prediabetes.

ObjectiveTo evaluate the effects of Wuhua herbal tea on chronic unpredictable mild stress (CUMS)-induced depression and explore its mechanism of action in combating depression.MethodsWe tested the antidepressant effects of Wuhua herbal tea in a rat model of CUMS-induced depression using fluoxetine as a positive control. The rats were divided into four groups: control group, model group, fluoxetine group, and Wuhua herbal tea group. The rats underwent body weight measurements, sucrose preference test, and open-field test. Enzyme-linked immunosorbent assay kits were used to detect the serum levels of serotonin, dopamine, adrenocorticotropic hormone (ACTH), corticosterone, norepinephrine, and interleukin-6. Intergroup comparisons and detection of brain-derived neurotrophic factor (BDNF), cAMP-response element binding protein (CREB), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3) mRNA expression in the hippocampus were performed using RT-PCR. Immunohistochemistry was used to identify the expression of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) proteins in hippocampal paraffin sections of CUMS rats.ResultsCompared with the control group, the model group rats had depressive tendencies, exhibiting low vitality and interest in various behavioral indicators which were signs of despair. The Wuhua herbal tea group statistically increased the levels of serotonin and dopamine in the serum of CUMS rats to varying degrees (P = .015 and P = .002); reduced serum levels of ACTH, corticosterone, norepinephrine, and interleukin-6 (all P .05); and decreased mRNA expression of BDNF, CREB, JAK2, and STAT3 in the hippocampus (all P .05); and decreased p-STAT3 protein levels (P = .006).ConclusionWuhua herbal tea shows antidepressant potential in CUMS rats by modulating the HPA axis and inhibiting JAK2-STAT3 overactivation, alleviating neuroinflammation. It also restores BDNF-CREB pathway function, reducing depressive symptoms.

BACKGROUND:The definitive cause of adolescent idiopathic scoliosis is not yet known.The search for a clinical approach to address adolescent idiopathic scoliosis is imminent.OBJECTIVE:To investigate the effect of Schroth therapy combined with core strength training on mild adolescent idiopathic scoliosis and to provide more bases for the clinical treatment of mild adolescent idiopathic scoliosis.METHODS:110 patients with mild adolescent idiopathic scoliosis attending the Department of Rehabilitation Medicine and Department of Spine Surgery of Affiliated Hospital of Nantong University from July 2022 to January 2024 were selected as the study subjects.They were divided into the trial group and the control group according to the wishes of the patients and their parents,with 55 cases in each group.The control group was observed and followed up,and the trial group underwent Schroth therapy combined with core strength training for 45 minutes a day for 24 weeks.The differences in imaging parameters,body surface indexes,three-dimensional ultrasound imaging angle,and quality of life were compared between the two groups before and after treatment.RESULTS AND CONCLUSION:(1)At 24 weeks after treatment,major curve Cobb,apical vertebral translation,and cervical lordosis were significantly improved in the trial group(P＜0.05),while there was no significant difference in the control group(P＞0.05).Major curve Cobb and apical vertebral translation in the trial group were significantly better than those in the control group(P＜0.05).(2)At 24 weeks after treatment,angle of trunk rotation in the trial group was significantly lower than that before treatment(P＜0.05),while there was no significant difference between before and after treatment in the control group(P＞0.05),and angle of trunk rotation in the trial group was significantly lower than that of the control group(P＜0.05).(3)At 24 weeks after treatment,the center of laminae angle of three-dimensional ultrasound imaging was significantly reduced in the trial group(P＜0.05),while there was no significant difference in the control group before and after treatment(P＞0.05).The center of laminae angle of three-dimensional ultrasound imaging was smaller in the trial group than that in the control group(P＜0.05).(4)At 24 weeks after treatment,in terms of the quality of life,pain dimension score in the trial group was significantly increased(P＜0.05).Both trial and control groups showed significantly higher scores in the self-image dimension compared with that before treatment(P＜0.05).Both groups had significantly lower scores in the mental health dimension compared with that before treatment(P＜0.05).In the dimensions of pain,self-image,mental health,and satisfaction,the trial group was significantly higher than the control group(P＜0.05).(5)It is indicated that Schroth therapy combined with core strength training can improve the major curve Cobb,apical vertebral translation,and cervical lordosis angle,reduce the angle of trunk rotation,decrease the center of laminae angle of three-dimensional ultrasound imaging,and improve the quality of life,and it is effective in the treatment of mild adolescent idiopathic scoliosis.

Objective:To investigate the impact of inserting an exogenous gene, NanoLuc (Nluc), at different sites in the influenza virus genome on viral properties and analyze the expression stability of the exogenous gene both in vitro and in vivo. Methods:Using molecular cloning techniques and reverse genetics, eight recombinant influenza viruses were constructed by inserting the exogenous Nluc gene into the gene segments encoding hemagglutinin (HA), neuraminidase (NA), non-structural protein (NS), and polymerase basic protein 1 (PB1). Viral replication capacity was evaluated by hemagglutination and plaque assays. Nluc expression in infected cells was monitored by fluorescence imaging. The potential impact of the exogenous gene insertion on viral infectivity was examined in a mouse infection model. Independent samples t-test were used for statistical analysis. Results:The recombinant viruses with insertions in the HA, NA, and NS gene segments generated fluorescent signals in the first generation of rescued viruses and demonstrated replication capabilities in plaque and hemagglutination assays. The recombinant viruses based on the NA and NS genes were capable of stably expressing Nluc across different generations, and exhibited correct fluorescent distribution patterns in mouse infection experiments. Meanwhile, the NS gene-based recombinant virus demonstrated superior stability in the mouse model.Conclusions:This study demonstrates that the NS gene segment of influenza virus can serve as an effective insertion site for exogenous genes without impairing the viral replication or infectivity, and the recombinant virus constructed based on it exhibits high integration stability and substantial application potential.