OBJECTIVE: To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of Plastrum Testudinis (PT) in the treatment of osteoporosis (OP). METHODS: Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method, including sham group, ovariectomized group (OVX), ovariectomized groups treated with high-, medium-, and low-dose PT (160, 80, 40 mg/kg per day, respectively), with 6 rats in each group. Except for the sham group, the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment, bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry, Western blot, and quantitative polymerase chain reaction. In addition, Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently, PT extract was used to rescue the effects of Dkk-1 and miR-214, and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated. RESULTS: PT-M and PT-L significantly reduced the weight gain in OVX rats (P<0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats, and increased the expressions of bone formation-related factors including alkaline phosphatase, bone morphogenetic protein type 2, collagen type I alpha 1, and runt-related transcription factor 2 when compared with the OVX group (P<0.05 or P<0.01). Meanwhile, different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats, and increased the mRNA or protein expressions of Wnt3a, β-catenin, glycogen synthase kinase-3β, and low-density lipoprotein receptor-related protein 5 (P<0.05 or P<0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition, the expression of miR-214 was decreased in OVX rats (P<0.01), and it was negatively correlated with the osteogenic differentiation of BMSCs (P<0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (P<0.05 or P<0.01). Conversely, PT effectively counteracted the effect of miR-214 mimic, thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (P<0.05 or P<0.01). CONCLUSION PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway, which may be related to inhibiting miR-214 in BMSCs.
Animals ; MicroRNAs/genetics* ; Female ; Rats, Sprague-Dawley ; Wnt Signaling Pathway/genetics* ; Osteogenesis/genetics* ; Mesenchymal Stem Cells/cytology* ; Cell Differentiation/drug effects* ; Bone Density/drug effects* ; Ovariectomy ; Osteoporosis/drug therapy* ; beta Catenin/metabolism* ; Rats ; Intercellular Signaling Peptides and Proteins/metabolism* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE: To explore the main components and potential mechanisms of Sangqi Qingxuan Liquid in the treatment of arterial vascular endothelial cells (AVECs) injury in hypertension through network pharmacology. METHODS: Traditional Chinese Medicine Systems Pharmacology and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID) were used to screen the active components of Sangqi Qingxuan Liquid (SQQX), which met the oral utilization rate and drug similarity criteria. An active component-target network was constructed using Cytoscape 3.6 software. A protein-protein interaction (PPI) network of targets associated with SQQX treatment for hypertension was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The Metascape database was used to perform enrichment analysis of gene ontology biological functions and MSigDB pathway enrichment analysis of proteins in the PPI network. Further analysis of the main components of SQQX was performed using UPLC-MS. Based on the results of network pharmacology, the mechanism of SQQX to improve the injury of AVECs in hypertension was verified through lentiviral transfection by Wnt/ β -catenin signaling pathway. AVECs induced by angiotensin II (Ang II ) was used to establish a model of endothelial function injury in hypertension. Cell viability, intracellular nitric oxide content, malonaldehyde content, and superoxide dismutase activity were measured to determine the optimal induction conditions. The optimal intervention conditions for SQQX were determined based on cell viability, cellular DNA activity, and the gradient method. The cells were further divided into blank, model, overexpression lentivirus negative control, overexpression lentivirus, overexpression lentivirus + SQQX intervention (2.47 mg/mL, 12 h), inhibition lentivirus negative control, inhibition lentivirus, and inhibition lentivirus + SQQX intervention (2.47 mg/mL, 12 h) groups. Finally, quantitative real-time PCR and Western blotting were performed to analyze the molecular mechanisms of SQQX in the Wnt/ β -catenin signaling pathway. RESULTS: The main SQQX components were betaine, buddleoside, and chlorogenic acid, in descending order. Network pharmacology analysis screened 12 pathways associated with the hypertensive vascular endothelium. The results showed that 1 µ mol/L for 12 h was the optimal condition for Ang II to induce AVECs injury, and 2.47 mg/mL SQQX intervention for 12 h was the optimal condition for treating AVECs injury. In the experimental validation based on the interaction network of the Wnt/ β -catenin signaling pathway, SQQX significantly decreased the expressions of β -catenin, Smad2, peroxisome proliferator-activated receptors (PPARs), endothelial nitric oxide synthase (eNOS), and endothelin-1 (ET-1) caused by the β -catenin overexpression lentivirus (P<0.05 or P<0.01). The function of vascular endothelial cells can be improved by the β -catenin inhibition lentivirus, and no obvious changes were observed after further intervention with SQQX. CONCLUSION SQQX may protect against AVECs injury by regulating the Wnt/β -catenin signaling pathway.
Drugs, Chinese Herbal/therapeutic use* ; beta Catenin/metabolism* ; Hypertension/metabolism* ; Endothelial Cells/metabolism* ; Protein Interaction Maps/drug effects* ; Humans ; Wnt Signaling Pathway/drug effects* ; Network Pharmacology ; Endothelium, Vascular/injuries* ; Cell Survival/drug effects* ; Angiotensin II/pharmacology* ; Nitric Oxide/metabolism*

Ribosome is an intracellular ribonucleoprotein particle that serves as the site of protein biosynthesis. Ribosomal dysfunction caused by mutations in genes encoding ribosomal proteins (RPs) and ribosome biogenesis factors (RBFs) can lead to a spectrum of diseases, collectively known as ribosomopathy. Phase separation is a thermodynamic process that produces multiple phases from a homogeneous mixture. The formation of membraneless organelles and intracellular structures, including ribosomes and nucleoli, cannot occur without the involvement of phase separation. Here, ribosome structure, biogenesis, and their relationship with ribosomopathy are systematically reviewed. The tissue specificity of ribosomopathy and the role of phase separation in ribosomopathy are particularly discussed, which may offer some clues for understanding the mechanisms of ribosomopathy. Then, some new ideas for the prevention, diagnosis, and treatment of ribosomopathy are provided.
Humans ; Ribosomes/physiology* ; Ribosomal Proteins/metabolism* ; Mutation ; Animals ; Cell Nucleolus/metabolism* ; Protein Biosynthesis ; Phase Separation

INTRODUCTION: The profile of patients referred from primary to tertiary nephrology care is unclear. Ethnic Malay patients have the highest incidence and prevalence of kidney failure in Singapore. We hypothesised that there is a Malay predominance among patients referred to nephrology due to a higher burden of metabolic disease in this ethnic group. METHODS: This is a retrospective observational cohort study. From 2014 to 2018, a coordinator and physician triaged patients referred from primary care, and determined co-management and assignment to nephrology clinics. Key disease parameters were collated on triage and analysed. RESULTS: A total of 6,017 patients were studied. The mean age of patients was 64 ± 16 years. They comprised 57% men; 67% were Chinese and 22% were Malay. The proportion of Malay patients is higher than the proportion of Malays in the general population (13.4%) and they were more likely than other ethnicities to have ≥3 comorbidities, including diabetes mellitus, hypertension, hyperlipidaemia, coronary artery disease and stroke (70% vs. 57%, P < 0.001). Malay and Indian patients had poorer control of diabetes mellitus compared to other ethnicities (glycated haemoglobin 7.8% vs. 7.4%, P < 0.001). Higher proportion of Malay patients compared to other ethnicities had worse kidney function with estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m 2 on presentation (28% vs. 24%, P = 0.003). More ethnic Malay, Indian and younger patients missed appointments. CONCLUSION A disproportionately large number of Malay patients are referred for kidney disease. These patients have higher metabolic disease burden, tend to miss appointments and are referred at lower eGFR. Reasons underpinning these associations should be identified to facilitate efforts for targeting this at-risk population, ensuring kidney health for all.
Humans ; Male ; Female ; Retrospective Studies ; Middle Aged ; Singapore/epidemiology* ; Referral and Consultation/statistics & numerical data* ; Aged ; Nephrology ; Glomerular Filtration Rate ; Metabolic Diseases/epidemiology* ; Ethnicity ; Diabetes Mellitus/epidemiology* ; Malaysia/ethnology* ; Adult

OBJECTIVE: To observe the effects of ultrasound-guided foraminal electroacupuncture on neuronal apoptosis and motor function in rats with spinal cord injury (SCI), and to explore the potential underlying mechanisms. METHODS: Thirty-six SPF-grade Sprague-Dawley rats were randomly assigned to a sham operation group, a model group, and an ultrasound-guilded electroacupuncture group (electroacupuncture group), with 12 rats in each group. In the sham operation group, the spinal cord was exposed and then the incision was sutured without contusion. In the other two groups, SCI models were established using a modified Allen's impact method. On days 1, 3, 7, and 14 after modeling, the electroacupuncture group received electroacupuncture intervention at the T₉/T₁₀ and T₁₀/T₁₁ intervertebral foramen under ultrasound guidance, avoiding spinal cord injury. Stimulation parameters were dense-disperse wave at 2 Hz/100 Hz and 1-2 mA for each session. Following interventions on days 1, 3, 7, and 14, the Basso-Beattie-Bresnahan (BBB) score was assessed; the inclined plane test was used to assess hindlimb grip strength in rats. After the intervention, HE staining was used to observe spinal cord morphology; TUNEL staining was used to detect neuronal apoptosis; ELISA was used to measure the serum levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α); Western blot was used to analyze the protein expression of Wnt-4, β-catenin, c-Myc, Bax, Bcl-2, and NeuN in spinal tissue; quantitative real-time PCR was used to detect the mRNA expression of Wnt-4, β-catenin, c-Myc, Bax, Bcl-2, and NeuN. RESULTS: Compared with the sham operation group, the model group showed significantly reduced BBB scores (P<0.05), and reduced inclined plane angles (P<0.05) at all time points. Compared with the model group, the electroacupuncture group exhibited increased BBB scores on days 3, 7, and 14 (P<0.05), and higher inclined plane angles on days 1, 3, 7, and 14 (P<0.05). Compared with the sham operation group, the model group showed disorganized spinal cord structure with increased inflammatory cells and necrotic neurons, higher number of apoptotic neurons in spinal tissue (P<0.05), elevated serum IL-6, IL-1β, and TNF-α levels (P<0.05), increased protein and mRNA expression of Wnt-4, β-catenin, c-Myc, and Bax (P<0.05), and decreased protein and mRNA expression of Bcl-2 and NeuN in spinal tissue (P<0.05). Compared with the model group, the electroacupuncture group had fewer inflammatory cells and apoptotic neurons in spinal tissue (P<0.05), reduced serum IL-6, IL-1β, and TNF-α levels (P<0.05), increased protein and mRNA expression of Wnt-4, β-catenin, Bcl-2, and NeuN (P<0.05), and decreased protein and mRNA expression of c-Myc and Bax in spinal tissue (P<0.05). CONCLUSION Ultrasound-guided foraminal electroacupuncture could improve motor function in rats with SCI, potentially by regulating the expression of molecules related to the Wnt-4/β-catenin signaling pathway to inhibit neuronal apoptosis and inflammatory responses.
Animals ; Electroacupuncture/methods* ; Spinal Cord Injuries/physiopathology* ; Rats, Sprague-Dawley ; Rats ; Wnt Signaling Pathway ; Male ; Humans ; Female ; beta Catenin/metabolism* ; Apoptosis ; Ultrasonography ; Tumor Necrosis Factor-alpha/genetics* ; Spinal Cord/metabolism*

INTRODUCTION: Conservative kidney management (CKM) is a recognised treatment option for selected patients with chronic kidney disease stage 5 (CKD G5), but prognostic indicators for mortality and optimal timing for palliative care transition remain uncertain. METHOD: This is a single-centre, prospective cohort study of CKD G5 patients who opted for CKM, conducted between April 2021 and September 2024, with longitudinal monitoring of Edmonton Symptom Assessment System Revised: Renal; Palliative Perfor-mance Scale (PPS); Resources Utilisation Group.Activities of Daily Living (RUG-ADL) scale; Clinical Frailty Score; Karnofsky Performance Score; and clinical and laboratory data. Primary outcomes included identifying baseline mortality predictors and validating the PPS for survival estimation. Cox proportional hazards models were used to identify independent predictors of mortality. RESULTS: Among 109 patients (mean age 79.8±7.3 years, 64.2% female), 62 (56.9%) died during follow-up. Multivariate analysis identified baseline estimated glomerular filtration rate (eGFR) (hazard ratio [HR] 1.32, 95% confidence interval [CI] 1.08.1.68, P<0.01) and serum albumin (HR 1.24, 95% CI 1.08.1.43, P<0.01) as predictors of 1-year mortality. Median survival varied by eGFR: 3.0 months (95% CI 0.6.2) for eGFR .5 mL/min/1.73 m², 13.0 months (95% CI 9.1.16.9) for eGFR 6.10 mL/ min/1.73 m², and 20.0 months (95% CI 16.5.23.5) for eGFR >10 mL/min/1.73 m² (P<0.01). Subsequent PPS correlated strongly with survival, with median survival of 1.8 months for PPS <50, 5.3 months for PPS 50.60, and 7.9 months for PPS 70.80 (P=0.03). CONCLUSION Baseline eGFR and serum albumin predict 1-year mortality in CKM patients. PPS offers a practical tool for identifying patients requiring palliative care transition, supporting personalised care pathways and timely integration of palliative care.
Humans ; Female ; Male ; Aged ; Prospective Studies ; Glomerular Filtration Rate ; Palliative Care/methods* ; Conservative Treatment/methods* ; Aged, 80 and over ; Prognosis ; Serum Albumin/analysis* ; Proportional Hazards Models ; Activities of Daily Living ; Singapore/epidemiology*

BACKGROUND: The benefits of ideal cardiovascular-health metrics (ICVHMs) in patients with renal insufficiency remain unclear. This study aimed to investigate the associations between ICVHM and prognosis in a renal insufficiency population. METHODS: The trial enrolled 29,682 participants from the US National Health and Nutrition Examination Survey (NHANES), 2007-2018, with mortality follow-up through December 31, 2019. Participants were divided into three groups based on estimated glomerular filtration rates. Cardiovascular health was assessed using new "Life's Essential 8" metrics. Cox regression analyses based on NHANES data were used to determine the associations between ICVHMs and cardiovascular mortality in patients with renal insufficiency. RESULTS: During a mean follow-up of 6.58 years, ideal cardiovascular health (hazard ratio [HR] = 0.42; 95% confidence interval [CI]; 0.25-0.70) and ideal health behavior (HR = 0.53; 95% CI; 0.39-0.73) reduced cardiovascular mortality in participants with renal insufficiency. For each one ICVHM increment, a 25% reduction in cardiovascular mortality was recorded (95% CI; 0.69-0.82). When compared with participants with normal renal function, for those with mild renal insufficiency, the HR for cardiovascular mortality gradually decreased from 1.47 (95% CI; 0.85-2.52) in those who had ≤1 ICVHMs to 0.30 (95% CI; 0.12-0.77) in participants who had >6 ICVHMs. CONCLUSIONS From an ICVHM perspective, enhanced cardiovascular benefits were observed in individuals with renal insufficiency, coupled with a reduced risk of all-cause mortality. Furthermore, when compared with individuals with normal renal function, increased ICVHMs can mitigate adverse risks associated with renal impairment.
Humans ; Male ; Female ; Nutrition Surveys ; Middle Aged ; Renal Insufficiency/physiopathology* ; Aged ; Prognosis ; Adult ; Cardiovascular Diseases/mortality* ; Glomerular Filtration Rate/physiology* ; Proportional Hazards Models

BACKGROUND: Enzalutamide, a second-generation androgen receptor (AR) pathway inhibitor, is widely used in the treatment of castration-resistant prostate cancer. However, after a period of enzalutamide treatment, patients inevitably develop drug resistance. In this study, we characterized leucine-rich repeated G-protein-coupled receptor 5 (LGR5) and explored its potential therapeutic value in prostate cancer. METHODS: A total of 142 pairs of tumor and adjacent formalin-fixed paraf-fin-embedded tissue samples from patients with prostate cancer were collected from the Pathology Department at Sun Yat-sen Memorial Hos-pital. LGR5 was screened by sequencing data of enzalutamide-resistant cell lines combined with sequencing data of lesions with different Gleason scores from the same patients. The biological function of LGR5 and its effect on enzalutamide resistance were investigated in vitro and in vivo . Glutathione-S-transferase (GST) pull-down, coimmunoprecipitation, Western blotting, and immunofluorescence assays were used to explore the specific binding mechanism of LGR5 and related pathway changes. RESULTS: LGR5 was significantly upregulated in prostate cancer and negatively correlated with poor patient prognosis. Overexpression of LGR5 promoted the malignant progression of prostate cancer and reduced sensitivity to enzalutamide in vitro and in vivo . LGR5 promoted the phosphorylation of glycogen synthase kinase-3β (GSK-3β) by binding heat shock protein 90,000 alpha B1 (HSP90AB1) and mediated the activation of the Wingless/integrated (WNT)/β-catenin signaling pathway. The increased β-catenin in the cytoplasm entered the nucleus and bound to the nuclear AR, promoting the transcription level of AR, which led to the enhanced tolerance of prostate cancer to enzalutamide. Reducing HSP90AB1 binding to LGR5 significantly enhanced sensitivity to enzalutamide. CONCLUSIONS LGR5 directly binds to HSP90AB1 and mediates GSK-3β phosphorylation, promoting AR expression by regulating the WNT/β-catenin signaling pathway, thereby conferring resistance to enzalutamide treatment in prostate cancer.
Male ; Humans ; Phenylthiohydantoin/pharmacology* ; Benzamides ; Receptors, G-Protein-Coupled/genetics* ; Nitriles ; Cell Line, Tumor ; HSP90 Heat-Shock Proteins/metabolism* ; Drug Resistance, Neoplasm/genetics* ; Prostatic Neoplasms/drug therapy* ; beta Catenin/metabolism* ; Receptors, Androgen/genetics* ; Animals ; Mice ; Wnt Signaling Pathway/physiology*

Persistent inflammation plays a pivotal role in the initiation and progression of renal fibrosis. Activation of the pattern recognition receptor retinoic acid-inducible gene-I (RIG-I) is implicated in the initiation of inflammation. This study aimed to investigate the upstream mechanisms that regulates the activation of RIG-I and its downstream signaling pathway. Eight-week-old male C57BL/6 mice were used to establish unilateral ureteral obstruction (UUO)-induced renal fibrosis model, and the renal tissue samples were collected 14 days later for analysis. Transforming growth factor-β (TGF-β)-treated mouse renal tubular epithelial cells were used in in vitro studies. The results demonstrated that, compared to the control group, UUO kidney exhibited significant fibrosis, which was accompanied by the increases of RIG-I, p-NF-κB p65 and inflammatory cytokines, such as TNF-α and IL-1β. Additionally, the protein level of the E3 ubiquitin ligase RNF125 was significantly downregulated and predominantly localized in the renal tubular epithelial cells. Similarly, the treatment of tubular cells with TGF-β induced the increases in RIG-I, p-NF-κB p65 and inflammatory cytokines while decreasing RNF125. Co-immunoprecipitation (Co-IP) assays confirmed that RNF125 was able to interact with RIG-I. Overexpression of RNF125 promoted the ubiquitination of RIG-I, and accelerated its degradation via the ubiquitin-proteasome pathway. Overexpression of RNF125 in UUO kidneys and in vitro tubular cells effectively mitigated the inflammatory response and renal fibrosis. In summary, our results demonstrated that the decrease in RNF125 under pathological conditions led to reduction in RIG-I ubiquitination and degradation, activation of the downstream NF-κB signaling pathway and increase in inflammatory cytokine production, which promoted the progression of renal fibrosis.
Animals ; Fibrosis ; Male ; Ubiquitination ; Mice ; Mice, Inbred C57BL ; DEAD Box Protein 58 ; Ubiquitin-Protein Ligases/physiology* ; Inflammation/metabolism* ; Ureteral Obstruction/complications* ; Kidney/pathology* ; Signal Transduction ; Transforming Growth Factor beta/pharmacology*

The circadian clock plays a critical role in regulating various physiological processes, including gene expression, metabolic regulation, immune response, and the sleep-wake cycle in living organisms. Post-translational modifications (PTMs) are crucial regulatory mechanisms to maintain the precise oscillation of the circadian clock. By modulating the stability, activity, cell localization and protein-protein interactions of core clock proteins, PTMs enable these proteins to respond dynamically to environmental and intracellular changes, thereby sustaining the periodic oscillations of the circadian clock. Different types of PTMs exert their effects through distincting molecular mechanisms, collectively ensuring the proper function of the circadian system. This review systematically summarized several major types of PTMs, including phosphorylation, acetylation, ubiquitination, SUMOylation and oxidative modification, and overviewed their roles in regulating the core clock proteins and the associated pathways, with the goals of providing a theoretical foundation for the deeper understanding of clock mechanisms and the treatment of diseases associated with circadian disruption.
Protein Processing, Post-Translational/physiology* ; Circadian Rhythm/physiology* ; Humans ; Animals ; CLOCK Proteins/physiology* ; Circadian Clocks/physiology* ; Phosphorylation ; Acetylation ; Ubiquitination ; Sumoylation