Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation, joint destruction, and functional impairment. Angiogenesis plays a key role in the pathological progression of RA with dysfunction of endothelial cells to promote synovial inflammation, sustain pannus formation, subsequently leading to joint damage. Colquhounia Root Tablets (CRT), a Chinese patent drug, has shown a satisfying clinical efficacy in treating RA, while the underlying mechanism by which CRT inhibits RA-associated angiogenesis remains unclear. In this study, we applied a research approach combining transcriptomic data analysis, bio-network mapping, and in vivo and in vitro experiments to explore the molecular mechanisms of CRT in suppressing angiogenesis in RA. Animal welfare and experimental procedures follow the regulations of the Animal Ethics Committee of Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences (ratification number: IBTCMCACMS21-2307-06). Network analysis identified that key genes such as nucleotide-binding oligomerization domain-containing protein 2 (NOD2), SMAD family member 3 (SMAD3), and vascular endothelial growth factor A (VEGFA) significantly enriched in pathways related to NOD-like receptor signaling and VEGF signaling, indicating that CRT may inhibit angiogenesis by regulating vascular endothelial cell function with modulating angiogenesis-related pathways. In vivo data showed that CRT significantly reduced the positive expression of CD31 and VEGF in the ankle joint of adjuvant-induced arthritis (AIA) rats. In vitro data further confirmed that CRT effectively inhibited VEGF-induced migration, invasion, and tube formation in HUVECs, while significantly reduced the expression of angiogenesis-related factors VEGF/CD31/Ang-1, as well as the positive expression of VEGF and CD31 in HUVECs. Furthermore, CRT markedly decreased the protein expression of NOD2, VEGFA, and SMAD3. In conclusion, these findings indicate that CRT may inhibit the RA-related angiogenesis by targeting the NOD2/SMAD3/VEGF signaling axis to improve endothelial cell function, enriching the scientific connotation of CRT in inhibiting pathological angiogenesis in RA and also offer new insights for clinical prevention and treatment of RA.

BACKGROUND: Regulatory dendritic cell (DCreg) subset exhibits a unique capacity for inducing immune tolerance among the variety subsets of dendritic cells (DCs) within gut-associated lymphoid tissues (GALTs). Fms-like tyrosine kinase 3 ligand (FLT3L) is involved in the differentiation of DCregs and the subsequent expansion of regulatory T-cells (Tregs) mediated by DCregs, though the precise mechanism remains poorly understood. This study aimed to explore the expansion mechanism of Treg induced by DCreg and the role of FLT3L in this process. METHODS: DCregs were distinguished from other DC subsets isolated from GALTs of BALB/c mice through a mixed lymphocyte reaction assay. The functions and mechanisms by which FLT3L promoted Treg expansion via DCregs were investigated in vitro through co-culture experiments involving DCregs and either CD4 + CD25 - T-cells or CD4 + CD25 + T-cells. Additionally, an in vivo experiment was conducted using a dextran sulfate sodium (DSS)-induced colitis model in mice. RESULTS: CD103 + CD11b + DC exhibited DCreg-like functionality and was identified as DCreg for subsequent investigation. Analysis of Foxp3 + Treg percentages within a co-culture system of CD4 + CD25 - T-cells and DCregs, with or without FLT3L, demonstrated the involvement of the FLT3/FLT3L axis in driving the differentiation of precursor T-cells into Foxp3 + Tregs induced by DCregs. Cell migration and co-culture assays revealed that the FLT3/FLT3L axis enhanced DCreg migration toward Tregs via the Rho pathway. Additionally, it was observed that DCregs could promote Treg proliferation through the Notch pathway, as inhibition of Notch signaling by DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) suppressed Treg expansion within the co-culture system of DCregs and CD4 + T-cells or CD4 + CD25 + T-cells. Furthermore, the FLT3/FLT3L axis influenced JAG1 expression in DCregs, indirectly modulating Treg expansion. In vivo experiments further established that FLT3L promoted DCreg expansion and restored Treg balance in DSS-induced colitis models, thereby ameliorating colitis symptoms in mice. CONCLUSION The FLT3/FLT3L axis is integral to the maintenance of DCreg function in Treg expansion.
Animals ; T-Lymphocytes, Regulatory/immunology* ; Dendritic Cells/immunology* ; Mice ; Mice, Inbred BALB C ; Membrane Proteins/metabolism* ; Receptors, Notch/metabolism* ; Lymphoid Tissue/metabolism* ; Signal Transduction/physiology* ; Coculture Techniques ; Flow Cytometry

OBJECTIVE: To investigate the regulatory effects of two traditional mineral medicines (TMMs), Gypsum Fibrosum (Shigao, GF) and Terra Flava Usta (Zaoxintu, TFU), on gut-beneficial bacteria in mice, and preliminarily explore their mechanisms of action. METHODS: Mice were randomly divided into 3 groups (n=10 per group): the control group (standard diet), the GF group (diet supplemented with 2% GF), and the TFU group (diet supplemented with 2% TFU). After 4-week intervention, 16S rRNA gene sequencing was used to analyze the changes in the gut microbiota (GM). Scanning electron microscopy, in combination with coumarin A tetramethyl rhodamine conjugate and Hoechst stainings, was used to observe the bacteria and biofilm formation. RESULTS: Principal coordinate analysis revealed that GF and TFU significantly altered the GM composition in mice. Further analysis revealed that GF and TFU affected different types of gut bacteria, suggesting that different TMMs may selectively modulate specific bacterial populations. For certain bacteria, such as Faecalibaculum and Ileibacterium, both GF and TFU exhibited growth-promoting effects, implying that they may be sensitive to TMMs and that different TMMs can increase their abundance through their respective mechanisms. Notably, Lactobacillus reuteri, a widely recognized and used probiotic, was significantly enriched in the GF group. Random forest analysis identified Ileibacterium valens as a potential indicator bacterium for TMMs' impact on GM. Further mechanistic studies showed that gut bacteria formed biofilm structures on the TFU surface. CONCLUSIONS This study provides new insights into the interaction between TMMs and GM. As safe and effective natural clays, GF and TFU hold promise as potential candidates for prebiotic development.
Animals ; Gastrointestinal Microbiome/drug effects* ; Bacteria/growth & development* ; Mice ; Biofilms/drug effects* ; Male ; RNA, Ribosomal, 16S/genetics*

Objective:To establish a Plaque-reduction Neutralization Test (PRNT) for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus (HRSV) and optimize the conditions for preliminary application.Methods:The CHO expression system was used to produce palivizumab monoclonal antibody (palivizumab) and the influencing factors such as cell type, cell culture duration, fixation and permeabilization protocols, and blocking agents. The reproducibility of the method was verified and its correlation was verified with conventional PRNT. Finally, the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum (immunized by intramuscular injection of HRSV fusion proteins).Results:Palivizumab was expressed at approximately 50 mg/L. The optimal working conditions for PRNT were as follows: culturing HEp-2 cells for 2 days, fixing with 4% (V/V) paraformaldehyde at room temperature for 15 min followed by 0.2% (V/V) Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step. The overall coefficient of variation (CV) for the reproducibility validation of this method was <15%, showing a good linear relationship with the conventional PRNT. The Spearman correlation coefficient r s was 0.983. This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320, and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice. Conclusion:The HRSV neutralizing antibody assay established in this study is rapid, reproducible, high-throughput, and can be used to detect neutralizing antibodies to HRSV subtypes A and B.

Objective:To establish a Plaque-reduction Neutralization Test (PRNT) for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus (HRSV) and optimize the conditions for preliminary application.Methods:The CHO expression system was used to produce palivizumab monoclonal antibody (palivizumab) and the influencing factors such as cell type, cell culture duration, fixation and permeabilization protocols, and blocking agents. The reproducibility of the method was verified and its correlation was verified with conventional PRNT. Finally, the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum (immunized by intramuscular injection of HRSV fusion proteins).Results:Palivizumab was expressed at approximately 50 mg/L. The optimal working conditions for PRNT were as follows: culturing HEp-2 cells for 2 days, fixing with 4% (V/V) paraformaldehyde at room temperature for 15 min followed by 0.2% (V/V) Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step. The overall coefficient of variation (CV) for the reproducibility validation of this method was <15%, showing a good linear relationship with the conventional PRNT. The Spearman correlation coefficient r s was 0.983. This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320, and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice. Conclusion:The HRSV neutralizing antibody assay established in this study is rapid, reproducible, high-throughput, and can be used to detect neutralizing antibodies to HRSV subtypes A and B.

Objective To explore whether dexmedetomidine(DEX)can alleviate exertional heatstroke(EHS)-induced rhabdomyolysis(RM)in rats by activating adrenergic α2 receptors,and to explore its potential mechanism based on the reactive oxygen species(ROS)/NOD-like receptor protein 3(NLRP3)/interleukin-1β(IL-1β)pathway.Methods Thirty-six male Sprague-Dawley(SD)rats,after a 7-day acclimatization training,were randomly divided into six groups:control group(CN group),EHS group,low-dose DEX group(EHS+low DEX group),high-dose DEX group(EHS+high DEX group),DEX combined with yohimbine(YOH)group(EHS+high DEX+YOH group),and YOH group(EHS+YOH),with six rats in each group.Before modeling,EHS+high DEX+YOH group and EHS+YOH group were intraperitoneally injected with YOH at 1 mg/kg,while the other four groups were injected intraperitoneally with an equal dose of physiological saline(0.9%NS).During modeling,except for CN group,the other 5 groups of rats were subjected to heat exercise in a high-temperature and high-humidity chamber to construct an EHS rat model.After successful modeling,EHS+low DEX group was intraperitoneally injected with DEX at 10 μg/kg,EHS+high DEX group and EHS+high DEX+YOH group were intraperitoneally injected with DEX at 30 μg/kg,and CN group,EHS group and DEX+YOH group were intraperitoneally injected with equal doses of saline.After 6 h of observation,all rats were anesthetized,and their blood from the abdominal aorta and gastrocnemius muscle tissue were taken.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of serum tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β and myoglobin(MB)in rats;biochemical assay kit was used to measure the level of creatine kinase(CK)in rat serum;HE staining was used to observe pathological changes in rat gastrocnemius muscle tissues;transmission electron microscopy was used to observe ultrastructural changes in gastrocnemius muscle;2′,7′-dichlorofluorescent yellow diacetate(DCFH-DA)fluorescent probe was used to detect the level of reactive oxygen species(ROS);and Western blotting was performed to detect the expression levels of NOD-like receptors 3(NLRP3),aspartic protease-1(caspase-1)and adrenergic α2A receptor(ADRA2A).Results Compared with CN group,the levels of serum IL-6,IL-1β,TNF-α,CK and MB in EHS group rats were significantly elevated(P<0.01).HE staining results revealed that the gastrocnemius muscle tissues of rats in EHS group had these pathological manifestations such as disarray of muscle fibrous structure,hemorrhage,edema,and infiltration of inflammatory cells.Transmission electron microscopy results showed that the ultrastructure of the gastrocnemius muscle in EHS group exhibited myofibroblasts with swelling and enlarging in size,cytoplasmic vacuolization,and mitochondria with obvious swelling,degranulation,and disappearance of double cristae.Compared with CN group,the expression levels of ROS,NLRP3,and caspase-1 in gastrocnemius of rats in EHS group significantly increased(P<0.01);Compared with EHS group,the levels of TNF-α,IL-6,IL-1β,CK,MB and the expression levels of ROS,NLRP3,caspase-1 in gastrocnemius tissue of rats in EHS+low DEX group and EHS+high DEX group decreased in a dose-dependent manner(P<0.05),and the pathological damage observed with HE staining and transmission electron microscopy was alleviated by DEX.After YOH pretreatment,compared with the EHS+high DEX group,the serum levels of TNF-α,IL-6,IL-1β,CK,MB and ROS,NLRP3 and caspase-1 in the gastrocnemius muscle tissue of rats in EHS+high DEX+YOH group relatively increased(P<0.05),and the pathological damage observed with HE staining and transmission electron microscopy was exacerbated.The expression of ADRA2A in gastrocnemius muscle of EHS group significantly decreased compared with CN group(P<0.01),and the expression of ADRA2A in muscle of rats in DES+low DEX group and EHS+high DEX group was higher than that in EHS group(P<0.05).Conclusions DEX can alleviate EHS-induced RM by activating ADRA2A,potentially through inhibiting the ROS-dependent NLRP3/IL-1β inflammatory pathway.

Lipid metabolism,as the basis of life maintenance,is a prerequisite for cell survival,and lipid homeostasis can rapidly respond to metabolic changes in a coordinated manner.In cancers,there is an increase in lipid metabolism in cancer cells to meet the requirements for plasma membrane synthesis and energy production.Abnormal lipid metabolism plays an important role in the progression of primary liver cancer.This article reviews the association between abnormal lipid metabolism and primary liver cancer,in order to find targets for the prevention and treatment of primary liver cancer.

Objective To analyze the status of the bacteria in the operative area of transnasal skull base surgery and its correlation with postoperative intracranial infection.Methods The procedure of transnasal skull base surgery was divided into three stages:nasal passage preparation(stage 1),tumor resection(stage 2),and skull base reconstruction(stage 3).Bacterial sampling was taken from the mucosa of the anterior wall of sphenoid sinus or clival recess of sellar floor at the beginning of each stage;and the positive rate of bacterial culture in different stages of operation and its correlation with postoperative intracranial infection were analyzed.Results A total of 105 patients were enrolled in this study,and 315 samples were taken.The average time point of sampling in the three stages was 20.3,45.1 and 131.3 min after the beginning of operation,respectively.The positive results were 9 cases(2.9％)in the stage 1,8 cases(2.5％)in the stage 2,and 23 cases(7.3％)in the stage 3,which were 24 cases of Staphylococcus epidermidis,7 cases of Staphylococcus aureus,3 cases of hemolytic streptococcus,2 cases of Klebsiella pneumonia,and 4 cases of Escherichia coli.There was no significant difference in the positive cases between stage 1 and stage 2(P=0.955),but there were significant differences between stage 1 or 2 and stage 3(P=0.013;P=0.007).There were 36(11.4％)patients with at least one positive result in the three stages,17(16.2％)with cerebrospinal fluid leakage,and 12(11.4％)with intracranial infection.The risk of intracranial infection was 3.1 times higher in patients with positive bacterial culture than patients with negative bacterial culture(OR=3.1,95％CI:0.9-10.6),which was not statistically significant;patients with CSF leakage were 61.4 times higher than those without CSF leakage(OR=61.4,95％CI:11.2-337.1),which was statistically significant(P＜0.001).The consistency rate of bacteria in nasal cavity and postoperative cerebrospinal fluid culture was 57.1％.Conclusion The positive rate of bacterial culture in the operative area of transnasal skull base surgery increases significantly with the extension of operation time,which is a potential risk index of postoperative intracranial infection.

Background The senescence of alveolar type II epithelial cells is an important driving factor for the progression of silicotic fibrosis, and the regulatory effects of oxamate on the senescence of alveolar type II epithelial cells is still unclear. Objective To explore whether lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting senescence of alveolar type II epithelial cellsMethods This study was divided into two parts: in vivo experiments and in vitro experiments. In the first part, forty SPF C57BL/6J male mice were randomly divided into four groups with 10 in each group: control group, silicosis model group, low-dose oxamate treatment group, and high-dose oxamate treatment group. The silicotic mouse model was established by intratracheal instillation of 50 μL SiO₂ suspension (100 mg·mL⁻¹). The treatment models were prepared by intraperitoneal injection of 100 μL oxamate (225 mmol·L⁻¹ and 1125 mmol·L⁻¹). In the second part, induction of MLE-12 mouse alveolar type II epithelial cells was conducted with SiO₂. The in vitro experimental groups were ① SiO₂ induction groups: control group, 50 μg·mL⁻¹ SiO₂ group, 100 μg·mL⁻¹ SiO₂ group, and 200 μg·mL⁻¹ SiO₂ group, and ② oxamate treatment groups: control group, SiO₂ group (100 μg·mL⁻¹), low-dose oxamate (25 mmol·L⁻¹) treatment group, and high-dose oxamate (50 mmol·L⁻¹) treatment group. Pathological morphology of lung tissues was evaluated after hematoxylin-eosin (HE) staining; deposition of collagen in lung tissues was evaluated after sirius red staining; positive co-expression of prosurfactant protein C (Pro-SPC) and β-galactosidase was detected by immunofluorescence staining; positive expression of β-galactosidase in MLE-12 cells was detected by immunofluorescence staining. The protein expression levels of collagen type I (CoL I), fibronectin1 (FN1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), lactate dehydrogenase A (LDHA), p-ataxia telangiectasia and Rad3-related kinase (ATR), and cyclin-dependent kinase inhibitors p21, and p16 were detected by Western blotting. Results Compared with the control group, the protein expression levels of HK2, PKM2, LDHA, p-ATR, p21, and p16 were significantly upregulated in the silicosis model group and the SiO₂-induced MLE-12 cells (P＜0.05). The in vivo studies showed that, compared with the control group, the silicon nodule area, the collagen deposition area, the proportion of β-galactosidase positive cells, and the protein expression levels of CoL I, FN1, LDHA, p-ATR, p21, and p16 were significantly upregulated in the silicosis model group (P＜0.05). Compared with the silicosis model group, the oxamate treatment groups showed significant downregulation of the silicon nodule area, the collagen deposition area, the proportion of β-galactosidase positive cells, and the the CoL I, FN1, LDHA, p-ATR, p21, and p16 protein expression levels, and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group (P＜0.05). The in vitro studies showed that, compared with the control group, the proportion of β-galactosidase positive cells and the protein expression levels of p-ATR, p21, and p16 were significantly upregulated in the SiO₂-induced group (P＜0.05). Compared with the SiO₂ group, the proportion of β-galactosidase positive cells and the LDHA, p-ATR, p21 and p16 protein expression levels were significantly downregulated in the oxamate treatment groups, and the high-dose oxamate treatment group showed a higher efficacy on these indicators than the low-dose oxamate treatment group (P＜0.05). Conclusion Lactate dehydrogenase inhibitor oxamate can alleviate silicotic fibrosis in mice by inhibiting the senescence of alveolar type II epithelial cells.

Hepatic encephalopathy （HE） is a common severe liver disease syndrome in clinical practice and is one of the critical and severe diseases in internal medicine， and more than half of liver failure patients diagnosed with overt HE have a survival time of less than 1 year. A comprehensive analysis of the complex pathogenesis of HE and the development of diagnosis and treatment regimens based on evidence-based medicine are of great importance for alleviating high medical resource consumption， high medical expenses， and high incidence and mortality rates in clinical practice. The latest studies have shown that the intestinal tract and the central nervous system can perform bidirectional continuous interaction and signal transmission and regulate the function of inflammation signals， molecules， cells， and organs， which is known as neuroimmune communication and is highly consistent with the main pathological features of HE. With a focus on the mechanism of neuroimmune communication in HE， this article reviews the association between inflammation signal transduction via the gut-brain axis and neurotransmitter regulation and its role in neuroimmune communication in HE， which provides new ideas for the clinical diagnosis and treatment of HE and the research and development of related drugs.