Background: Influenza A virus (IAV) is a negative-sense RNA virus of the Orthomyxoviridae family and is the etiological agent of a highly contagious acute respiratory disease that can lead to acute lung injury. Objective: To elucidate the molecular mechanisms of IAV infection, an integrative research approach combining gene expression profiling, multinetwork analysis, and in vivo experimental validations was employed. Methods: First, a series of network-based analyses were performed, including protein-protein interaction network construction, weighted gene co-expression network analysis, and subsequent gene set enrichment analysis, to identify the major underlying mechanisms of IAV infection. Following gene expression analysis, core targets, both direct and indirect regulators, were screened. An IAV (H1N1) strain A/PR/8/34-induced acute lung injury mouse model was constructed for in vivo validations. Batch one included two groups to evaluate findings from the multi-network analysis: Mock (n = 10; 5 males and 5 females) and IAV (n = 10; 5 males and 5 females). Batch two included three groups to assess the role of toll-like receptor 3 (TLR3) in IAV infection: Mock (n = 6; 3 males and 3 females), IAV (n = 6; 3 males and 3 females), and TLR3 inhibitor (n = 6; 3 males and 3 females). Body weight was measured on days 0, 3, and 5 after infection. On day 5, lung tissues were collected to assess viral load and histopathological changes. Key targets were examined using enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining, both in sera and lung tissues. Results: IAV infection was significantly associated with dysregulation of the immune-inflammation system, such as the LTR, nucle-otide-binding oligomerization domain-(NOD) like receptor, retinoic acid-inducible gene I-like receptor, and nuclear factor kappa-B signaling pathways. Gene set enrichment analysis further indicated that the TLR and necroptosis signaling pathways played crucial roles in the progression of IAV infection (TLR signaling pathway normalized enrichment score = 2.3941, P = 1.00 × 10 ⁻¹⁰; necroptosis normalized enrichment score = 1.9421, P = 6.21 × 10 ⁻⁷). Among the core targets, TLR3 and mixed lineage kinase domain-like protein (MLKL) may regulate gene expression at the transcriptional level (all P < 0.05). In vivo validation using an IAV (PR8) infected acute lung injury mouse model demonstrated increased viral load and lung index, alveolar structural damage, and inflammatory cell infiltration. Immunofluorescence staining exhibited large gaps in Lamin B1 staining and breaches in Emerin signals following IAV-PR8 infection. Expression levels of TLR3, p-receptor-interacting serine/threonine-protein kinase 3 (RIPK3)/RIPK3, and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in lung tissues, as well as proinflammatory factors and mediators in sera, were significantly elevated after IAV infection. Moreover, enhanced neutrophil infiltration (myeloperoxidase) and citrullinated histone H3 (a neutrophil extracellular trap-specific marker), both established indicators of neutrophil extracellular trap formation, were observed. Notably, treatment with a TLR3 inhibitor significantly ameliorated IAV-induced acute lung injury by regulating necroptosis-related targets. Conclusion: Our study provides network-based in vivo evidence that TLR3-receptor-interacting serine/threonine-protein kinase 3-MLKL-mediated necroptosis may underlie IAV-induced acute lung injury and could serve as a potential therapeutic target in severe influenza cases.

This study aims to investigate the effects of aerobic exercise on neuroinflammation in AD mice and explore the mechanisms of neuroinflammation regulated by the blood-brain barrier,lipopolysaccharide(LPS)displacement,and glial cell activation.Twenty 3-month-old male APP/PS1 double transgenic mice were used,which were randomly divided into a sedentary group(SE-AD)and an aerobic exercise group(Run-AD),and 10 3-month-old male C57BL/6 mice were used as the control group(WT).The Run-AD group underwent 12 weeks of aerobic training.The results of the water maze showed that aerobic exercise improved the learning and memory capacity of AD mice(P＜0.05).The results of H&E stai-ning and Nissl staining showed that aerobic exercise reduced necrotic cells and inflammatory cell infiltra-tion in the cerebral cortex,as well as nuclear condensation in the CA1 and GD regions of the hippocam-pus(P＜0.05,P＜0.01),and increased the area of Nissl bodies in the cerebral cortex and hippocam-pal CA3 and DG regions.Western blotting and ELISA results showed that aerobic exercise increased the expression of Occludin,ZO-1 and Claudin-5 proteins in the brain(P＜0.01),and decreased the levels of LPS in the brain(P＜0.01).The qRT-PCR results exhibited that aerobic exercise decreased the ex-pression of TLR4,MyD88,NF-κB,IL-1β,and TNF-α mRNA(P＜0.05,P＜0.01).The results of immunofluorescence staining revealed that aerobic exercise reduced the fluorescence area of brain IL-1βand TNF-α proteins(P＜0.05,P＜0.01),as well as the fluorescence area of Iba-1,GFAP,and TLR4 proteins in the cerebral cortex and hippocampus(P＜0.05,P＜0.01).There was a high degree of overlap between Iba-1 and TLR4 fluorescence in the cerebral cortex,and GFAP was localized around Iba-1.In summary,aerobic exercise attenuates neuroinflammation in AD mice by protecting the blood-brain barrier,reducing the displacement of LPS,and subsequently weakening the immune activation of microglia to regulate the TLR4/MyD88/NF-κB signaling pathway to alleviate neuroinflammation.

Dipsaci Radix is a commonly used Chinese herbal medicine in China, with triterpenoid saponins as the main active components. β-Amyrin synthase, a member of the oxidosqualene cyclase superfamily, plays a crucial role in the biosynthesis of oleanane-type triterpenoid saponins. Asperosaponin Ⅵ is an oleanane-type triterpenoid saponin. To explore the β-amyrin synthase genes involved in the biosynthesis of asperosaponin Ⅵ in Dipsacus asper, this study screened the candidate genes from the transcriptome data of D. asper. Two β-amyrin synthase genes, Da OSC1 and Da OSC2, were identified by phylogenetic analysis and correlation analysis. The coding sequences of Da OSC1 and Da OSC2 were 2 286 bp and 2 295 bp in length, encoding 761 and 764 amino acids,respectively. Multiple sequence alignments showed that Da OSC1 and Da OSC2 had three conserved motifs( DCTAE, QW, and MWCYCR) unique to the oxidosqualene cyclase family. Real-time quantitative PCR results showed that Da OSC1 and Da OSC2 had the highest expression levels in the roots. Compared with normal growth conditions, the low-temperature treatment significantly upregulated the expression of Da OSC1 and Da OSC2. Agrobacterium-mediated transient expression of Da OSC1 and Da OSC2 in Nicotiana benthamiana resulted in the production of β-amyrin, which suggested that Da OSC1 and Da OSC2 were able to catalyze the synthesis of β-amyrin. This study clarified the catalytic functions of two β-amyrin synthases in D. asper, analyzed their expression patterns in different tissue and at low temperatures. The findings provide a foundation for further studying the biosynthetic pathway and regulatory mechanism of asperosaponin Ⅵ in D. asper.
Intramolecular Transferases/chemistry* ; Phylogeny ; Plant Proteins/chemistry* ; Gene Expression Regulation, Plant ; Dipsacaceae/classification* ; Saponins/metabolism* ; Oleanolic Acid/metabolism*

To clarify the species, pathogenicity, and distribution of the pathogens causing the root rot of Atractylodes lancea in Hubei province, the tissue separation method was used to isolate the pathogens from root rot samples in the main planting areas of A. lancea in Hubei. Based on the preliminary identification of the Fusarium genus by the internal transcribed spacer(ITS) sequence, three housekeeping genes, EF1/EF2, Btu-F-FO1/Btu-F-RO1, and FF1/FR1, were amplified and sequenced. Subsequently, a phylogenetic tree was constructed based on these TEF gene sequences to classify the pathogens. The pathogenicity of these strains was determined using the root irrigation method. A total of 194 pathogen strains were isolated using the tissue separation method. Molecular identification using the three housekeeping genes identified the pathogens as F. solani, F. oxysporum, F. commune, F. equiseti, F. tricinctum, F. redolens, F. fujikuroi, F. avenaceum, F. acuminatum, and F. incarnatum. Among them, F. solani and F. oxysporum were the dominant strains, widely distributed in multiple regions, with F. solani accounting for approximately 54% of the total isolated strains and F. oxysporum accounting for approximately 34%. Other strains accounted for a relatively small proportion, totaling approximately 12%. The results of pathogenicity determination showed that there were certain differences in pathogenicity among strains. The analysis of the pathogenicity differentiation of the widely distributed F. solani and F. oxysporum strains revealed that these dominant strains in Hubei were mainly highly pathogenic. This study determined the species, pathogenicity, and distribution of the pathogens causing the root rot of A. lancea in Hubei province. The results provide a scientific basis for further understanding the root rot of A. lancea and its epidemic occurrence and scientifically preventing and controlling this disease.
Plant Diseases/microbiology* ; Atractylodes/microbiology* ; Phylogeny ; Plant Roots/microbiology* ; Fusarium/classification* ; China ; Virulence ; Fungal Proteins/genetics*

OBJECTIVE: To explore clinical efficacy of autologous platelet-rich plasma(PRP) in treating Rockwood type Ⅲ acromioclavicular dislocation. METHODS: From January 2019 to July 2021, 32 patients with Rockwood type Ⅲ acromioclavicular dislocation were treated with minimally invasive adjustable titanium plate internal fixation, and were divided into PRP group and control group according to whether PRP treatment was performed, with 16 patients in each group. In PRP group, there were 10 males and 6 females, aged from 28 to 47 years old with an average of (36.75±7.14) years old;the time from injury to surgery ranged from 1 to 31 h with an average of (26.13±3.98) h;5 patients on the left side and 11 patients on the right side;PRP was injected once during operation and the 4th and 8th weeks after operation respectively. In control group, there were 8 males and 8 females, aged from 30 to 52 years old with an average of (38.50±5.48) years old; the time from injury to surgery ranged from 1 to 29 h with an average of (25.48±3.11) h;7 patients on the left side and 9 patients on the right side; minimally invasive surgical treatment was performed. Visual analogue scale(VAS) was used to evaluate pain and Constant-Murley score for shoulder joint function was used to evaluate the recovery of shoulder joint movement function before operation and 1, 3, 6, and 12 months after operation respectively. RESULTS: All patients were followed up for 12 to 28 months with an average of (18.3±5.2) months. All incisions patients healed well without adverse events such as infection. Postoperative VAS of PRP group at 1, 3, and 6 months were (5.5±1.2), (3.7±1.6), and (2.4±1.2), respectively, while were lower than those of control group (6.6±1.4), (4.9±1.1), and (3.7±1.3), respectively;and had statistical differences between two groups (P<0.05). There was no statistically significant difference in VAS between two groups before operation and 12 months after operation (P>0.05). Postoperative Constant-Murley scores of PRP group at 1, 3, and 6 months were (64.09±11.61), (73.19±12.89), and (82.61±14.81) points, respectively, which were higher than those of control group were (52.32±17.42), (61.65±14.43), and (72.52±11.04) respectively;and the differences were statistically significant (P<0.05). There was no statistically significant difference in Constant-Murley scores at 12 months after operation between two groups (P>0.05). In PRP group, there was no statistically significant difference at 6 months and 12 months after operation (P>0.05), while there were statistically significant differences at the other time points (1 month after operation compared with before operation, 3 months after operation compared with 6 months after operation, and 3 months after operation compared with 1 month after operation) (P<0.05). In control group, there was no statistically significant difference when comparing 1 month and 3 months after operation (P>0.05), while at the other time points (1 month after operation with before operation, 3 months after operation with 6 months after operation, and 6 months after operation with 12 months after operation), the differences were all statistically significant (P<0.05). CONCLUSION Adjustable titanium plate fixation combined with postoperative injection of PRP for the treatment of Rockwood type III acromioclavicular joint dislocation has effect of promoting the recovery of shoulder joint function and reducing pain.
Humans ; Male ; Female ; Adult ; Middle Aged ; Platelet-Rich Plasma ; Acromioclavicular Joint/surgery* ; Bone Plates ; Titanium ; Joint Dislocations/therapy* ; Fracture Fixation, Internal/methods*

Peri-implant keratinized mucosa (PIKM) augmentation refers to surgical procedures aimed at increasing the width of PIKM. Consensus reports emphasize the necessity of maintaining a minimum width of PIKM to ensure long-term peri-implant health. Currently, several surgical techniques have been validated for their effectiveness in increasing PIKM. However, the selection and application of PIKM augmentation methods may present challenges for dental practitioners due to heterogeneity in surgical techniques, variations in clinical scenarios, and anatomical differences. Therefore, clear guidelines and considerations for PIKM augmentation are needed. This expert consensus focuses on the commonly employed surgical techniques for PIKM augmentation and the factors influencing their selection at second-stage surgery. It aims to establish a standardized framework for assessing, planning, and executing PIKM augmentation procedures, with the goal of offering evidence-based guidance to enhance the predictability and success of PIKM augmentation.
Humans ; Consensus ; Dental Implants ; Mouth Mucosa/surgery* ; Keratins

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This study aims to investigate the effects of aerobic exercise on neuroinflammation in AD mice and explore the mechanisms of neuroinflammation regulated by the blood-brain barrier,lipopolysaccharide(LPS)displacement,and glial cell activation.Twenty 3-month-old male APP/PS1 double transgenic mice were used,which were randomly divided into a sedentary group(SE-AD)and an aerobic exercise group(Run-AD),and 10 3-month-old male C57BL/6 mice were used as the control group(WT).The Run-AD group underwent 12 weeks of aerobic training.The results of the water maze showed that aerobic exercise improved the learning and memory capacity of AD mice(P＜0.05).The results of H&E stai-ning and Nissl staining showed that aerobic exercise reduced necrotic cells and inflammatory cell infiltra-tion in the cerebral cortex,as well as nuclear condensation in the CA1 and GD regions of the hippocam-pus(P＜0.05,P＜0.01),and increased the area of Nissl bodies in the cerebral cortex and hippocam-pal CA3 and DG regions.Western blotting and ELISA results showed that aerobic exercise increased the expression of Occludin,ZO-1 and Claudin-5 proteins in the brain(P＜0.01),and decreased the levels of LPS in the brain(P＜0.01).The qRT-PCR results exhibited that aerobic exercise decreased the ex-pression of TLR4,MyD88,NF-κB,IL-1β,and TNF-α mRNA(P＜0.05,P＜0.01).The results of immunofluorescence staining revealed that aerobic exercise reduced the fluorescence area of brain IL-1βand TNF-α proteins(P＜0.05,P＜0.01),as well as the fluorescence area of Iba-1,GFAP,and TLR4 proteins in the cerebral cortex and hippocampus(P＜0.05,P＜0.01).There was a high degree of overlap between Iba-1 and TLR4 fluorescence in the cerebral cortex,and GFAP was localized around Iba-1.In summary,aerobic exercise attenuates neuroinflammation in AD mice by protecting the blood-brain barrier,reducing the displacement of LPS,and subsequently weakening the immune activation of microglia to regulate the TLR4/MyD88/NF-κB signaling pathway to alleviate neuroinflammation.

The incidence of intrahepatic cholangiocarcinoma (ICC) continues to rise, and there are no effective drugs to treat it. The immune microenvironment plays an important role in the development of ICC and is currently a research hotspot. Icaritin (ICA) is an innovative traditional Chinese medicine for the treatment of advanced hepatocellular carcinoma. It is considered to have potential immunoregulatory and anti-tumor effects, which is potentially consistent with the understanding of "Fuzheng" in the treatment of tumor in traditional Chinese medicine. However, whether ICA can be used to treat ICC has not been reported. Therefore, in this study, sgp19/kRas, an in situ ICC mouse model with intact immune system, was selected to evaluate the efficacy of ICA in the treatment of ICC in vivo for the first time, and the effects of ICA on the tumor immune microenvironment of ICC mice were analyzed by flow cytometry. This experiment was approved by the Experimental Animal Ethics Committee of Capital Medical University (approval number: AEEI-2023-138). In this study, sgp19/kRas ICC mouse model was treated with oral gavage of 100 mg·kg^-1 ICA. We found that ICA significantly inhibited tumor growth and tumor cell proliferation in the sgp19/kRas ICC mouse model after 3 weeks of treatment. Flow cytometry analysis indicated that ICA treatment markedly reduced the proportion of M2 macrophages and increased the number of CD3⁺CD4⁺ T cells. In vitro experiments demonstrated that ICA promoted macrophage polarization towards the M1 phenotype while inhibiting polarization towards the M2 phenotype. Furthermore, transcriptomic analysis suggested that ICA enhanced Toll-like receptor 9 (TLR9) expression, influencing macrophage nitric oxide metabolism synthesis pathways and receptor-related activities, thereby regulating macrophage polarization. In summary, this study demonstrates that ICA treatment significantly delays tumor progression in sgp19/kRas ICC mouse models. Mechanistically, ICA may achieve its anti-ICC effects by upregulating TLR9 receptor expression levels, promoting macrophage polarization towards the M1 phenotype, and altering the tumor immune microenvironment.

Objective To observe the efficacy of Chinese medicine in the treatment of pregnancy complicated with thalassemia and to investigate its influence on pregnancy outcomes.Methods A retrospective cohort study was carried out in 175 pregnant women complicated with thalassemia who met the inclusion criteria.According to the treatment methods,the patients were divided into Chinese medicine group(105 cases),iron supplementation group(41 cases)and untreated group(29 cases).The changes of hematological indicators and pregnancy outcomes in the second and third trimesters of pregnancy were compared among the three groups.The Chinese medicine group was further divided into three subgroups:56 cases in the Chinese patent medicine group,20 cases in the single Chinese medicine group and 29 cases in the Chinese herbal compound group.The changes of hematological indexes in the second and third trimesters of pregnancy in the three subgroups were compared.Moreover,the normative management of pregnant women with thalassemia during pregnancy was explored.Results(1)The concentration of hemoglobin(Hb)in the third trimester of the Chinese medicine group was(2.28±7.27)g/L higher than that in the second trimester,and the curative effect of Chinese medicine group was superior to that in the iron supplementation group and the untreated group(P＜0.05).The Hb concentration in the Chinese medicine group before delivery was(12.17±10.81)g/L higher than that in the second trimester,and the increase in the Chinese medicine group was more significantly than that in the other two groups(P＜0.05).(2)The level of serum ferritin(FER)in the third trimester of the three groups was lower than that in the second trimester,and the decrease of FER level in the iron supplementation group was less significantly than that in the other two groups(P＜0.05).(3)The Hb concentration in the third trimester of the single Chinese medicine group and the Chinese herbal compound group increased by(4.50±4.66),(3.62±8.77)g/L respectively compared with that in the second trimester,and the increase in the above two groups was more significantly than that in the Chinese patent medicine group(P＜0.05).(4)There was no significant difference in pregnancy outcomes among the three groups of pregnant women with thalassemia(P＞0.05).(5)Only 52.0％(91/175)of pregnant women with thalassemia underwent three or more blood routine tests after 20-24 weeks of pregnancy,34.3％(60/175)of pregnant women failed in re-examining the FER levels,and 21.2％(37/175)of pregnant women had no standardized iron supplementation.Conclusion Chinese medicine therapy is effective on improving anemia in pregnant women with thalassemia.Chinese herbal compound and single Chinese medicinal are more effective than Chinese patent medicine,and have not increased the risk of adverse pregnancy outcomes.Oral administration of iron can increase the iron reserve of pregnant women with thalassemia,but its effect on improving anemia is not as good as that of Chinese medicine.Attention should be got to the monitoring of anemia and iron metabolism indicators as well as the standardized use of iron supplements and Chinese patent medicines in pregnant women with thalassemia.