BACKGROUND:Spleen has the functions of blood storage,hematopoiesis,and immunity.With the increase of age,the structural degeneration and functional decline of spleen lead to the impairment of immune system function,thus accelerating the aging process of the body.The treatment of spleen aging in tree shrews with highly active umbilical cord mesenchymal stem cells has not been reported. OBJECTIVE:To explore the intervention effect and mechanism of highly active umbilical cord mesenchymal stem cells on spleen aging in tree shrews. METHODS:Highly active umbilical cord mesenchymal stem cells were isolated,cultured,and obtained from the umbilical cord tissue of newborn tree shrews by caesarean section.The differentiation abilities of adipogenesis,osteogenesis,and chondrogenesis were detected by three-line differentiation kit.Cell cycle and surface markers were detected by flow cytometry.The second generation of highly active umbilical cord mesenchymal stem cells were transfected with Genechem Green Fluorescent Protein with infection complex values of 100,120,140,160,180,and 200,respectively,to screen the best transfection conditions.After transfection,the fourth generation of highly active umbilical cord mesenchymal stem cells was injected into the tail vein of tree shrews in the elderly treatment group.The young control group and the aged model group were not given special treatment.After 4 months of treatment,the spleen tissue was taken and the structure of the spleen was observed by hematoxylin-eosin staining.β-Galactosidase staining was used to detect the activity of aging-related galactosidase.Immunohistochemical staining was used to detect the expression levels of p21 and p53 proteins.Ki67 and PCNA immunofluorescence staining was used to detect cell proliferation activity.Immunofluorescence staining was used to detect the expression levels of spleen autophagy protein molecules Beclin 1 and APG5L/ATG5.Reactive oxygen species fluorescence staining was used to detect the content of reactive oxygen species in spleen tissue.CD3 immunofluorescence staining was used to detect the change of the proportion of total T lymphocytes.The secretion levels of interleukin 1β and transforming growth factor β1 in spleen were detected by enzyme linked immunosorbent assay.The distribution of highly active umbilical cord mesenchymal stem cells labeled with green fluorescent protein in spleen tissue was observed by DAPI double staining of nucleus. RESULTS AND CONCLUSION:(1)Highly active umbilical cord mesenchymal stem cells grew in a short spindle shape with fish-like growth,with a large proportion of G0/G1 phase,and had the potential to differentiate into adipogenesis,osteogenesis,and chondrogenesis.(2)Multiplicity of infection=140 and transfection for 72 hours were the best conditions for labeling tree shrews highly active umbilical cord mesenchymal stem cells with Genechem Green Fluorescent Protein.(3)Compared with the aged model group,in the aged treatment group,the spleen tissue cells of tree shrews were arranged closely,and the area of white pulp was increased(P<0.01);the boundary between red pulp and white pulp was clear;the proportion of germinal centers did not show statistically significant difference(P>0.05).The activity level of galactosidase related to spleen tissue aging was decreased(P<0.001),and the expression levels of aging protein molecules p21 and p53 were down-regulated(P<0.001).The expression levels of proliferation-related molecules Ki67 and PCNA were up-regulated(P<0.001,P<0.05);expression levels of autophagy-related molecules Beclin 1 and APG5L/ATG5 were up-regulated(P<0.001),and the content of reactive oxygen species decreased(P<0.001),and the proportion of CD3+T cells increased(P<0.05).The secretion level of interleukin 1β in the aging-related secretion phenotype decreased(P<0.001);no significant difference was found in transforming growth factor β1 level(P>0.05).Compared with the young control group,the above indexes were significantly different in the elderly treatment group(P<0.05).(4)Green fluorescent cells labeled with green fluorescent protein were observed in spleen tissue of tree shrews the elderly treatment group by frozen tissue section observation.The results show that intravenous infusion of highly active umbilical cord mesenchymal stem cells can migrate to spleen tissue,inhibit the production of reactive oxygen species,down-regulate the expression of aging-related proteins,induce autophagy,promote cell proliferation,reduce chronic inflammation,and then improve the structure and function of spleen tissue.

Objective To investigate the relationship between the levels of serum circular RNA protein tyrosine phosphatase 4A2(circ_PTP4A2),circular RNA precocious dissociation of sisters 5 homolog B(circ_PDS5B)and the volume of cerebral infarction and the degree of neurological deficits in patients with acute is-chemic stroke(AIS).Methods Ninety patients with AIS who visited the hospital from January 2021 to De-cember 2023 were selected as the AIS group,and 90 healthy individuals who underwent physical examinations during the same period were selected as the control group.The levels of serum circ_PTP4A2 and circ_PDS5B were detected by real-time fluorescence quantitative PCR,the volume of cerebral infarction was measured by magnetic resonance diffusion tensor imaging,and the degree of neurological deficit was evaluated by the Na-tional Institutes of Health Stroke Scale(NIHSS)score.According to the volume of cerebral infarction,they were divided into large-volume group(volume of cerebral infarction≥ 20 cm3,29 cases),medium-volume group(1 cm3＜volume of cerebral infarction＜20 cm3,34 cases),and small-volume group(volume of cerebral infarction≤ 1 cm3,27 cases),and according to the NIHSS scores,patients with AIS were classified into the se-vere group(NIHSS scores≥21 points,27 cases),the moderate group(NIHSS scores 5-20 points,32 cases)and the mild group(NIHSS score≤ 4 points,31 cases).Factors contributing to the increased volume of cere-bral infarction and the increased degree of neurological deficit in patients with AIS were analyzed by ordered multi-categorical Logistic regression.Results Compared with the control group,serum circ_PTP4A2 and circ_PDS5B levels were elevated in the AIS group(P＜0.05).Serum circ_PTP4A2 and circ_PDS5B levels were sequentially increased in the small-volume group,medium-volume group,and large-volume group(P＜0.05).Serum circ_PTP4A2 and circ_PDS5B levels were sequentially increased in the mild group,moderate group,and severe group(P＜0.05).Ordered multi-categorical Logistic regression showed that high NIHSS score,high circ_PTP4A2,and high circ_PDS5B were independent risk factors for increased cerebral infarction volume in AIS patients(P＜0.05),and high cerebral infarction volume,high circ_PTP4A2,and high circ_PDS5B were independent risk factors(P＜0.05).Conclusion The elevated levels of serum circ_PTP4A2 and circ_PDS5B in AIS patients are related to the increase in cerebral infarction volume and the aggravation of neurological deficits.Early detection of serum circ_PTP4A2 and circ_PDS5B levels is helpful for risk stratification in AIS patients.

Objective To observe the effects of asiaticoside on renal fibrosis in diabetic nephropathy(DN)rats by regulating the Hippo-yes-associated protein(YAP)/transcriptional co-activator with PDZ-binding motif(TAZ)signaling pathway.Methods A DN rat model was established by feeding a high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin(STZ).The successfully modeled rats were randomly divided into model group,low-and high-dose asiaticoside groups,and high-dose asiaticoside+XMU-MP-1(Hippo-YAP/TAZ pathway activator)group,with a normal group set as control.After group intervention,blood urea nitrogen(BUN),serum creatinine(SCr),fasting blood glucose(FBG),and 24-hour urinary protein levels were measured.Hematoxylin-eosin(HE)staining was used to observe renal histopathological changes,Masson staining was used to assess renal fibrosis,and Western Blot was used to detect the protein expressions of YAP and TAZ in renal tissue.Results Compared with the normal group,the model group showed disordered renal tissue structure,enlarged and irregular cystic cavities,enlarged renal tubules with thickened basement membranes,inflammatory cell infiltration,and significant collagen deposition in the glomeruli,renal tubules,and vascular walls.The levels of FBG,BUN,SCr,24-hour urinary protein,and the protein expressions of YAP and TAZ in renal tissue were significantly increased(P＜0.05).Compared with the model group,the low-and high-dose asiaticoside groups showed significant improvement in renal tissue damage and fibrosis,with reduced levels of FBG,BUN,SCr,24-hour urinary protein,and decreased protein expressions of YAP and TAZ in renal tissue(P＜0.05).Compared with the high-dose asiaticoside group,the high-dose asiaticoside+XMU-MP-1 group showed a reversal in all the above indicators.Conclusion Asiaticoside may alleviate renal fibrosis and improve renal injury in DN rats by inhibiting the Hippo-YAP/TAZ pathway.

Objective To establish a rapid,sensitive,and specific multiplex PCR detection method for the simultaneous detection of Cryptosporidium,Eimeria,and bovine parvovirus.Methods Specific primers targeting the SSU rRNA genes of Cryptosporidium and Eimeria,as well as the VP2 gene of bovine parvovirus were designed and the corresponding recombinant plasmid standards were constructed.To establish the multiplex PCR method,the reaction conditions were optimized using temperature gradient PCR and single-variable control methods.The sensitivity,specificity,reproducibility,and clinical application of the protocol were evaluated.Results The optimal annealing temperature was found to be 60.5℃,and the forward and reverse primer concentrations were determined to be 0.2 μmol/L for Eimeria,and 0.4 μmol/L for Cryptosporidium and bovine parvovirus.The assay demonstrated high sensitivity,with detection limits of 243,260,and 3 110 copies for the recombinant plasmid standards of Cryptosporidium,Eimeria,and bovine parvovirus,respectively.Specificity testing showed no cross-reactivity with ten common bovine pathogens,including Salmonella,bovine viral diarrhea virus,and bovine rotavirus.Consistent intra-and inter-batch results confirmed the strong reproducibility of the method.Clinical application to 81 diarrhea samples from various regions in the Ganzi Prefecture,Sichuan,revealed positivity rates of 18.52%(15/81)for Cryptosporidium,34.57%(28/81)for Eimeria,and 18.52%(15/81)forbovineparvovirus,withamixedinfectionrateof3.7%(3/81).Conclusions Themultiplex PCR method established in this study offers a reliable tool for differential diagnosis and epidemiological investigation of the three common diarrheal pathogens in yaks.

Objective To identify the core constitutions and prodromal symptoms of relapse in schizophrenia patients using network analysis,and to analyze their relationships as well as gender differences.Methods Schizophrenia patients hospitalized in the psychiatry department of Hunan Brain Hospital in Hunan Province between October 2022 and December 2023 were selected as survey participants.A general information questionnaire,a Traditional Chinese Medicine Constitution Scale,and a Schizophrenia Prodromal Symptoms of Relapse Scale were used for investigation.Network anal-ysis was conducted using R language.Results The core constitutions of schizophrenia patients were qi deficiency constitution[Expected Influence(EI)=1.08]and dampness-heat constitution(EI=1.00),and the core prodromal symptoms of relapse were depression/withdrawal(EI=0.84)and ini-tial psychotic manifestations(EI=0.81).There were statistically significant differences in constitutions of traditional Chinese medicine and prodromal symptoms of relapse between patients of different genders(P＜0.05).Females mainly exhibited qi deficiency constitution(EI=1.20)with anxiety as the core prodromal symptom(EI=0.98),while males mainly exhibited yin deficiency constitution(EI=1.05)with depression/withdrawal as the core prodromal symptom(EI=1.00).Conclusion Healthcare professionals can early identify core prodromal symptoms of relapse by core imbalanced constitutions of schizophrenia patients,and develop precise intervention strategies,thereby promoting changes in their network structure,preventing disease relapse,and improving patients'quality of life.

AIM To study the chemical constituents from Citri reticulatae Pericarpium Viride and their anti-triple negative breast cancer activities in vitro.METHODS The ethanolic extract of Citri reticulatae Pericarpium Viride was isolated and purified by silica gel,polyamide,MCI,Sephadex LH-20 and semi-preparative HPLC,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The anti-triple negative breast cancer activities were screened by SRB assay,and their effects on the proliferation of triple negative breast cancer cell lines HCC1806,HCC1937 and MDA-MB-231 in vitro were evaluated.RESULTS Twenty compounds were isolated and identified as nobiletin(1),tangeritin(2),5,4'-dihydroxy-7,8-dimethoxy flavonoid(3),naringenin(4),artemetin(5),5-demethynobiletin(6),3,5,6,7,8,3',4'-pentamethoxy flavonoid(7),5,4'-dihydroxy-3,6,7,8,3'-pentamethoxyflavone(8),xanthomicrol(9),p-hydroxycinnamic acid(10),5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavone(11),pectolinarigenin(12),4'-dihydroxy-5,6,7-tetramethoxyflavone(13),hispidulin(14),4',5,6,7-tetramethoxy-flavone(15),1-methyl-4-(prop-1-en-2-yl)cyclohexane-1,2-diol(16),umbelliferone(17),5-hydroxymethyl furfural(18),hydroquinone(19),1H-indole-3-carbaldehyde(20).Compound 8 showed a significant inhibitory effect with the IC50 value of(5.36±0.24)μmol/L on HCC1806 cells.CONCLUSION Compound 20 is isolated from genus Citrus for the first time,8,12-13,16-17 are isolated from this plant for the first time.Compound 8 show inhibitory effects on the proliferation of HCC1806,HCC1937 and MDA-MB-231 cells in vitro and have the strongest activities.Compounds 3-4,11-12,15,17 and 19 show strong inhibitory effect on HCC1806 cells.Compounds 15,19 also inhibit the proliferation of HCC1937 cells in vitro.

OBJECTIVE: To investigate the effect and mechanism of Shuangshi Tonglin Capsules (SSTL) in the treatment of prostate fibrosis (PF). METHODS: Human prostate stromal cells (WPMY-1) were used for in vitro experiments to establish PF cell models induced with estradiol (E₂). The cell proliferation, migration and clonogenic capacity were determined by cell counting kit-8, scratch assay, and crystal violet staining, respectively. Sprague-Dawley rats were used for in vivo experiments. The changes in histomorphology and organ index of rat prostate by SSTL were determined. Pathologic changes and collagen deposition changes in rat prostate were observed by haematoxylin and eosin (HE) and Masson staining. Enzyme-linked immunosorbent assay kits were used to determine changes in rat PF markers fibroblast growth factor-23 (FGF-23), E₂ and prostate specific antigen (PSA). Mechanistically, changes in oxidative stress indicators by SSTL were determined in WPMY-1 cells and PF rats. Then the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) and transforming growth factor-β1 (TGF-β1)/Smad pathway-related proteins as well as Nrf2 and TGF-β1 mRNA were further detected by Western blot or quantitative real-time polymerase chain reaction both in vivo and in vitro. RESULTS: In the efficacy study, SSTL significantly reduced the proliferation, migration, and clonogenic ability of cells, improved the morphology of the glandular tissue, significantly reduced the prostate index, reduced glandular fibrous tissue and collagen deposition, and resulted in a significant decrease in the levels of FGF-23, E₂ and PSA (P<0.01 or P<0.05). In the mechanistic study, SSTL ameliorated oxidative stress by significantly increasing superoxide dismutase and glutathione peroxidase levels and decreasing malondialdehyde level in WPMY-1 cells and rats (P<0.01 or P<0.05). SSTL significantly elevated the expressions of Nrf2, HO-1, NAD(P)H quinone oxidoreductase 1 (NQO-1), and Smad7 proteins in both cells and rats, and significantly decreased the expressions of TGF-β1, collagen I, α-smooth muscle actin and Smad4 proteins (P<0.01 or P<0.05). SSTL also elevated the content of Nrf2 mRNA and decreased the content of TGF-β1 mRNA in cells and rats (P<0.01 or P<0.05). The Nrf2 inhibitor ML385 was added in in vitro experiments to further validate the pathway relevance. CONCLUSION SSTL was effective in improving PF in vivo and in vitro, and its mechanism of action may function through the Nrf2/TGF-β1 signaling pathway.
Male ; NF-E2-Related Factor 2/metabolism* ; Animals ; Drugs, Chinese Herbal/therapeutic use* ; Signal Transduction/drug effects* ; Transforming Growth Factor beta1/metabolism* ; Rats, Sprague-Dawley ; Humans ; Fibrosis ; Prostate/drug effects* ; Cell Proliferation/drug effects* ; Capsules ; Cell Movement/drug effects* ; Oxidative Stress/drug effects* ; Rats

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Antibody-drug conjugates (ADCs) are antitumor drugs composed of monoclonal antibodies and cytotoxic payload covalently coupled by a linker. Currently, 15 ADCs have been clinically approved worldwide. More than 100 clinical trials at different phases are underway to investigate the newly developed ADCs. ADCs represent one of the fastest growing classes of targeted antitumor drugs in oncology drug development. It takes advantage of the specific targeting of tumor-specific antigen by antibodies to deliver cytotoxic chemotherapeutic drugs precisely to tumor cells, thereby producing promising antitumor efficacy and favorable adverse effect profiles. However, emergence of drug resistance has severely hindered the clinical efficacy of ADCs. In this review, we introduce the structure and mechanism of ADCs, describe the development of ADCs, summarized the latest research about the mechanisms of ADC resistance, discussed the strategies to overcome ADCs resistance, and predicted biomarkers for treatment response to ADC, aiming to contribute to the development of ADCs in the future.