Based on the interleukin-17(IL-17) signaling pathway, this study explores the effect and mechanism of Bufei Decoction on Klebsiella pneumoniae pneumonia in rats. SD rats were randomly divided into the control group, model group, Bufei Decoction low-dose group(6.68 g·kg~(-1)·d~(-1)), Bufei Decoction high-dose group(13.36 g·kg~(-1)·d~(-1)), and dexamethasone group(1.04 mg·kg~(-1)·d~(-1)), with 10 rats in each group. A pneumonia model was established by tracheal drip injection of K. pneumoniae. After successful model establishment, the improvement in lung tissue damage was observed following drug administration. Core targets and signaling pathways were screened using transcriptomics techniques. Real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA expression of core targets interleukin-6(IL-6), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and chemokine CXC ligand 6(CXCL6). Western blot was used to assess key proteins in the IL-17 signaling pathway, including interleukin-17A(IL-17A), nuclear transcription factor-κB activator 1(Act1), tumor necrosis factor receptor-associated factor 6(TRAF6), and downstream phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK), and phosphorylated nuclear factor-κB p65(p-NF-κB p65). Apoptosis of lung tissue cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL). The results showed that, compared with the control group, the model group exhibited significant pathological damage in lung tissue. The mRNA expression of IL-6, IL-1β, TNF-α, and CXCL6, as well as the protein levels of IL-17A, Act1, TRAF6, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65, were significantly increased, and the number of apoptotic cells was notably higher, indicating successful model establishment. Compared with the model group, both low-and high-dose groups of Bufei Decoction showed reduced pathological damage in lung tissue. The mRNA expression levels of IL-6, IL-1β, TNF-α, and CXCL6, and the protein levels of IL-17A, Act1, TRAF6, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65, were significantly decreased, with a significant reduction in apoptotic cells in the high-dose group. In conclusion, Bufei Decoction can effectively improve lung tissue damage and reduce inflammation in rats with K. pneumoniae. The mechanism may involve the regulation of the IL-17 signaling pathway and the reduction of apoptosis.
Animals ; Interleukin-17/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Rats ; Male ; Klebsiella pneumoniae/physiology* ; Klebsiella Infections/immunology* ; Humans ; Lung/drug effects*

The study aims to explore the herb-drug interaction between Xuefu Zhuyu Decoction(XFZY) and atorvastatin(AT). Reverse transcription polymerase chain reaction(RT-PCR) was used to analyze the transcription levels of proteins related to drug metabolism and transport in LS174T cells, detect the intracellular drug uptake under various substrate concentrations and incubation time, and optimize the model reaction conditions of transporter multidrug resistance protein 1(MDR1)-specific probe Rhodamine 123 and AT to establish a cell model for investigating the human intestinal drug interaction. The cell counting kit-8(CCK-8) method was adopted to evaluate the cytotoxicity of XFZY on LS174T cells. After a single and continuous 48 h culture with XFZY, AT or Rhodamine 123 was added for co-incubation. The effect and mechanism of XFZY on human intestinal absorption of AT were analyzed by measuring the intracellular drug concentrations and transcription levels of related transporters and metabolic enzymes. The results of in vitro experiments show that a single co-culture with a high concentration of XFZY significantly increases the intracellular concentrations of Rhodamine 123 and AT. A high concentration of XFZY co-culture for 48 h increases the AT uptake level, significantly induces the CYP3A4 and UGT1A1 gene expression levels, and inhibits the OATP2B1 gene expression level. To compare with the evaluation results of the in vitro human cell model, the pharmacokinetic experiment of XFZY combined with AT was carried out in rats. Sprague-Dawley(SD) rats were randomly divided into a blank control group and an XFZY group. After 14 days of continuous intragastric administration, AT was given in combination. The liquid chromatography-mass spectrometry(LC-MS)/MS method was used to detect the concentrations of AT and metabolites 2-hydroxyatorvastatin acid(2-HAT), 4-hydroxyatorvastatin acid(4-HAT), atorvastatin lactone(ATL), 2-hydroxyatorvastatin lactone(2-HATL), and 4-hydroxyatorvastatin lactone(4-HATL) in plasma samples, and the pharmacokinetic parameters were calculated. Pharmacokinetic analysis in rats shows that continuous administration of XFZY does not significantly change the pharmacokinetic characteristics of AT in rats, but the AUC_(0-6 h) values of AT and metabolites 2-HAT, 4-HAT, and 2-HATL increase by 21.37%, 14.94%, 12.42%, and 6.68%, respectively. The metabolic rate of the main metabolites shows a downward trend. The study indicates that administration combined with XFZY can significantly increase the uptake level of AT in human intestinal cells and increase the exposure level of AT and main metabolites in rats to varying degrees. The mechanism may be mainly due to the inhibition of intestinal MDR1 transport activity.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Atorvastatin/administration & dosage* ; Humans ; Rats ; Rats, Sprague-Dawley ; Male ; Intestines/cytology* ; Intestinal Mucosa/metabolism* ; Herb-Drug Interactions ; Cytochrome P-450 CYP3A/metabolism* ; Intestinal Absorption/drug effects*

OBJECTIVE: To evaluate the effects of posterior cruciate ligament(PCL) resection on soft tissue elasticity and knee stability in total knee arthroplasty(TKA). METHODS: Six adult cadaveric knee specimens (involving 10 knees) were included in the study. With the assistance of the robotic system(TiRobot Recon, TINAVI, Beijing), total knee arthroplasty (TKA) was performed sequentially using cruciate retaining (CR) prostheses and posterior stabilizing (PS) prostheses. Between the two surgical procedures, the femoral and tibial osteotomy surfaces were not altered;only the posterior cruciate ligament (PCL) was resected and the intercondylar fossa was treated. After installing the femoral trial component, a soft tissue balance solver was used to apply tension ranging from 30 N to 90 N in 5 N increments at 0°, 10°, and 90° of knee flexion. Meanwhile, the medial and lateral joint gaps were measured synchronously. Based on the tension-gap coupling data, the equivalent elastic coefficients of the medial and lateral soft tissue sleeves at different knee flexion angles, as well as the range of the joint line convergence angle (JLCA) under fixed varus-valgus stress, were calculated. Additionally, the gap balance status under 80 N of tension was analyzed. Self-control comparisons of each indicator were conducted before and after PCL resection to analyze the change patterns. RESULTS: After PCL resection, in the fully extended position (knee flexion 0°). The medial equivalent elastic coefficient was 32.2 (25.7, 63.3) N·mm^-1 for the CR prosthesis and 27.7 (22.0, 51.9) N·mm^-1 for the PS prosthesis, and the statistically significant difference (P=0.013). The range of JLCA was 0.41°(0.26, 0.55)° for the CR prosthesis, which was smaller than 0.75° (0.40, 0.98)° for the PS prosthesis, and the difference was statistically significant(P=0.041). At 90° of knee flexion, the medial joint gap was 10.7(10.1, 11.7) mm for the CR prosthesis, which was smaller than 12.1(10.9, 15.1) mm for the PS prosthesis, with a statistically significant difference(P=0.011). No statistically significant differences were observed in other joint gaps. CONCLUSION PCL resection reduces the rigidity of the medial soft tissues in the fully extended knee and increases the medial joint gap in the flexed position, thereby affecting knee stability and balance. This finding suggests that PS and CR prostheses may require different morphological designs, and there should be differences in indications and osteotomy strategies between CR-TKA and PS-TKA. CR-TKA is more suitable for patients with preoperative medial soft tissue laxity.
Humans ; Posterior Cruciate Ligament/physiopathology* ; Knee Joint/physiopathology* ; Arthroplasty, Replacement, Knee ; Elasticity ; Male ; Female ; Middle Aged ; Aged ; Biomechanical Phenomena ; Adult

PURPOSE: To investigate the protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on ischemic hypoxic injury of yorkshire brain tissue caused by traumatic blood loss. METHODS: This article performed a random controlled trial. Brain tissue of 7 yorkshire was selected and divided into the sub-low temperature anterograde machine perfusion group (n = 4) and the blank control group (n = 3) using the random number table method. A yorkshire model of brain tissue injury induced by traumatic blood loss was established. Firstly, the perfusion temperature and blood oxygen saturation were monitored in real-time during the perfusion process. The number of red blood cells, hemoglobin content, NA⁺, K⁺, and Ca²⁺ ions concentrations and pH of the perfusate were detected. Following perfusion, we specifically examined the parietal lobe to assess its water content. The prefrontal cortex and hippocampus were then dissected for histological evaluation, allowing us to investigate potential regional differences in tissue injury. The blank control group was sampled directly before perfusion. All statistical analyses and graphs were performed using GraphPad Prism 8.0 Student t-test. All tests were two-sided, and p value of less than 0.05 was considered to indicate statistical significance. RESULTS: The contents of red blood cells and hemoglobin during perfusion were maintained at normal levels but more red blood cells were destroyed 3 h after the perfusion. The blood oxygen saturation of the perfusion group was maintained at 95% - 98%. NA⁺ and K⁺ concentrations were normal most of the time during perfusion but increased significantly at about 4 h. The Ca²⁺ concentration remained within the normal range at each period. Glucose levels were slightly higher than the baseline level. The pH of the perfusion solution was slightly lower at the beginning of perfusion, and then gradually increased to the normal level. The water content of brain tissue in the sub-low and docile perfusion group was 78.95% ± 0.39%, which was significantly higher than that in the control group (75.27% ± 0.55%, t = 10.49, p < 0.001), and the difference was statistically significant. Compared with the blank control group, the structure and morphology of pyramidal neurons in the prefrontal cortex and CA1 region of the hippocampal gyrus were similar, and their integrity was better. The structural integrity of granulosa neurons was destroyed and cell edema increased in the perfusion group compared with the blank control group. Immunofluorescence staining for glail fibrillary acidic protein and Iba1, markers of glial cells, revealed well-preserved cell structures in the perfusion group. While there were indications of abnormal cellular activity, the analysis showed no significant difference in axon thickness or integrity compared to the 1-h blank control group. CONCLUSIONS Mild hypothermic machine perfusion can improve ischemia and hypoxia injury of yorkshire brain tissue caused by traumatic blood loss and delay the necrosis and apoptosis of yorkshire brain tissue by continuous oxygen supply, maintaining ion homeostasis and reducing tissue metabolism level.
Animals ; Perfusion/methods* ; Disease Models, Animal ; Brain Injuries/etiology* ; Swine ; Male ; Hypothermia, Induced/methods*

OBJECTIVE: To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells (MSCs). METHODS: Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with ⁶⁰Co, and the radiation dose rate was 0.98 Gy/min. Bulk RNA-seq was performed on control and irradiated MSCs. The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis. Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro, and real-time fluorescence quantitative PCR (qPCR) was used to detect the expression differences of key regulatory factors Cebpa, Lpl and Pparg after radiation treatment. At the same time, qPCR and Western blot were used to detect the effect of inhibition of Nrf2, a key factor of antioxidant stress pathway, on the expression of key regulatory factors of adipogenesis. Moreover, the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR. RESULTS: Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways. The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs, with high expression of Cebpa, Lpl and Pparg, as well as oxidative stress-related gene Nrf2. Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation. Notably, the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs. In addition, irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs. CONCLUSION Ionizing radiation promotes adipogenesis of MSCs in mice, and oxidative stress pathway participates in this effect, blocking Nrf2 further promotes the adipogenesis of MSCs. Additionally, irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.
Mesenchymal Stem Cells/cytology* ; Oxidative Stress/radiation effects* ; Animals ; Adipogenesis/radiation effects* ; Mice ; Radiation, Ionizing ; Cell Differentiation/radiation effects* ; Humans ; NF-E2-Related Factor 2/metabolism* ; PPAR gamma ; Cells, Cultured

OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

OBJECTIVE: To explore the efficacy and safety of Juan Bi Pill (JBP) in treatment of active rheumatoid arthritis (RA). METHODS: From February 2017 to May 2018, 115 participants from 4 centers were randomly divided into JBP group (57 cases) and placebo group (58 cases) in a 1:1 ratio using a random number table method. Participants received a dose of JBP (4 g, twice a day, orally) combined with methotrexate (MTX, 10 mg per week) or placebo (4 g, twice a day, orally) combined with MTX for 12 weeks. Participants were required with follow-up visits at 24 and 48 weeks, attending 7 assessment visits. Participants were undergo disease activity assessment 7 times (at baseline and 2, 4, 8, 12, 24, 48 weeks) and safety assessments 6 times (at baseline and 4, 8, 12, 24, 48 weeks). The primary endpoint was 28-joint Disease Activity Score (DAS28-ESR and DAS28-CRP). The secondary endpoints included American College of Rheumatology (ACR) criteria for 20% and 50% improvement (ACR20/50), Health Assessment Questionnaire Disability Index (HAQ-DI), clinical disease activity index (CDAI), visual analog scale (VAS), Short Form-36 (SF-36) score, Medial Outcomes Study (MOS) sleep scale score, serum erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tender joint count, swollen joint count, and morning stiffness. The adverse reactions were observed during the treatment. RESULTS: After 12 weeks of treatment, DAS28-ESR and DAS28-CRP scores in both groups were lower than before treatment (both P<0.01), while the remission rate of DAS28-ESR and DAS28-CRP and low disease activity of JBP group were higher than those in the placebo group (both P<0.01). JBP demonstrated better efficacy on ACR20 and ACR50 compliance rate at 12 and 48 weeks comparing to placebo (all P<0.05). The CDAI and HAQ-DI score, pain VAS and global VAS change of RA patients and physicians, the serum ESR and CRP levels, and the number of tenderness and swelling joints were lower than before treatment at 4, 8, 12, 24, 48 weeks in both groups (P<0.05 or P<0.01), while the reduction of above indices in the JBP group was more obvious than those in the placebo group at 12 weeks (ESR and CRP, both P<0.05) or at 12 and 48 weeks (all P<0.01). There was no difference in adverse reactions between the 2 groups during treatment (P=0.75). CONCLUSION JBP combined with MTX could effectively reduce disease activity in patients with RA in active stage, reduce the symptoms of arthritis, and improve the quality of life, while ensuring safety, reliability, and fewer adverse effects. (Trial Registration: ClinicalTrials.gov, No. NCT02885597).
Humans ; Arthritis, Rheumatoid/drug therapy* ; Methotrexate/adverse effects* ; Female ; Double-Blind Method ; Male ; Middle Aged ; Treatment Outcome ; Drugs, Chinese Herbal/adverse effects* ; Drug Therapy, Combination ; Adult ; Antirheumatic Agents/adverse effects* ; Aged

OBJECTIVE: Lipid oxidation is involved in the pathogenesis of atherosclerosis and may be contribute to the development of Ischemic stroke (IS). However, the lipid profiles associated with IS have been poorly studied. We conducted a pilot study to identify potential IS-related lipid molecules and pathways using lipidomic profiling. METHODS: Serum lipidomic profiling was performed using LC-MS in 20 patients with IS and 20 age- and sex-matched healthy controls. Univariate and multivariate analyses were simultaneously performed to identify the differential lipids. Multiple testing was controlled for using a false discovery rate (FDR) approach. Enrichment analysis was performed using MetaboAnalyst software. RESULTS: Based on the 294 lipids assayed, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) models were used to distinguish patients with IS from healthy controls. Fifty-six differential lipids were identified with an FDR-adjusted P less than 0.05 and variable influences in projection (VIP) greater than 1.0. These lipids were significantly enriched in glycerophospholipid metabolism (FDR-adjusted P = 0.009, impact score = 0.216). CONCLUSIONS Serum lipid profiles differed significantly between patients with IS and healthy controls. Thus, glycerophospholipid metabolism may be involved in the development of IS. These results provide initial evidence that lipid molecules and their related metabolites may serve as new biomarkers and potential therapeutic targets for IS.
Humans ; Pilot Projects ; Lipidomics ; Male ; Female ; Biomarkers/blood* ; Middle Aged ; Ischemic Stroke/blood* ; Aged ; China ; Lipids/blood* ; Adult ; Case-Control Studies ; East Asian People

Objective To investigate the clinical effect of anterior cervical discectomy and fusion(ACDF)in the treatment of cervical spondylosis of vertebral artery type(CSA).Methods The clinical data of 42 patients with CSA from January 2020 to January 2022 were retrospectively analyzed.There were 25 males and 17 females,aged from 30 to 74 years old with an average of(53.9±11.0)years old.There were 18 cases with single-segment lesions,17 cases with two-segment lesions,and 7 cases with three-segment lesions.The American Academy of Otolaryngology-Head and Neck Surgery's Hearing and Balance Committee score(CHE),the Neck Disability Index(NDI)and the cervical curvature Cobb angle were recorded before surgery and after surgery at 6 months.Results All 42 ACDF patients were followed up for 6 to 30 months with an average of(14.0±5.2)months.The operative time ranged from 95 to 220 min with an average of(160.38±36.77)min,the intraoperative blood loss ranged from 30 to 85 ml with an average of(53.60±18.98)ml.Tow patients had mild postoperative dysphagia,which improved with symptomatic treatment such as nebulized inhalation.CHE score decreased from(4.05±0.96)preoperatively to(2.40±0.70)at 6 months postoperatively(t=12.97,P＜0.05).The number of improved vertigo at 6 months postoperatively was 38,with an im-provement rate of 90.5％.NDI score was reduced from(34.43±8.04)preoperatively to(20.76±3.91)at 6 months postopera-tively(t=1 1.83,P＜0.05).The cervical curvature Cobb angle improved from(8.04±6.70)° preoperatively to(12.42±5.23)° at 6 months postoperatively(t=-15.96,P＜0.05).Conclusion The ACDF procedure has outstanding clinical efficacy in treating CSA.The operation can rapidly relieve patients'episodic vertigo symptoms by relieving bony compression and reconstructing cervical curvature.However,it is necessary to strictly grasp the indications for surgery and clarify the causes of vertigo in pa-tients,and ACDF surgery is recommended for CSA patients for whom conservative treatment is ineffective.