This study aimed to explore the mechanisms and molecular targets of total flavones of Abelmoschus manihot(TFA) plus empagliflozin(EM) in attenuating diabetic tubulopathy(DT) by targeting mitochondrial homeostasis and pyroptosis-apoptosis-necroptosis(PANoptosis). In the in vivo study, the authors established the DT rat models through a combination of uninephrectomy, administration of streptozotocin via intraperitoneal injections, and exposure to a high-fat diet. Following modeling successfully, the DT rat models received either TFA, EM, TFA+EM, or saline(as a vehicle) by gavage for eight weeks, respectively. In the in vitro study, the authors subjected the NRK52E cells with or without knock-down Z-DNA binding protein 1(ZBP1) to a high-glucose(HG) environment and various treatments including TFA, EM, and TFA+EM. In the in vivo and in vitro studies, The authors investigated the relative characteristics of renal tubular injury and renal tubular epithelial cells damage induced by reactive oxygen species(ROS), analyzed the relative characteristics of renal tubular PANoptosis and ZBP1-mediatted PANoptosis in renal tubular epithelial cells, and compared the relative characteristics of the protein expression levels of marked molecules of mitochondrial fission in the kidneys and mitochondrial homeostasis in renal tubular epithelial cells, respectively. Furthermore, in the network pharmacology study, the authors predicted and screened targets of TFA and EM using HERB and SwissTargetPrediction databases; The screened chemical constituents and targets of TFA and EM were constructed the relative network using Cytoscape 3.7.2 network graphics software; The relative targets of DT were integrated using OMIM and GeneCards databases; The intersecting targets of TFA, EM, and DT were enriched and analyzed signaling pathways by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG) software using DAVID database. In vivo study results showed that TFA+EM could improve renal tubular injury, the protein expression levels and characteristics of key signaling molecules in PANoptosis pathway in the kidneys, and the protein expression levels of marked molecules of mitochondrial fission in the kidneys. And that, the ameliorative effects in vivo of TFA+EM were both superior to TFA or EM. Network pharmacology study results showed that TFA+EM treated DT by regulating the PANoptosis signaling pathway. In vitro study results showed that TFA+EM could improve ROS-induced cell injury, ZBP1-mediatted PANoptosis, and mitochondrial homeostasis in renal tubular epithelial cells under a state of HG, including the protein expression levels of marked molecules of mitochondrial fission, mitochondrial ultrastructure, and membrane potential level. And that, the ameliorative effects in vitro of TFA+EM were both superior to TFA or EM. More importantly, using the NRK52E cells with knock-down ZBP1, the authors found that, indeed, ZBP1 was mediated PANoptosis in renal tubular epithelial cells as an upstream factor. In addition, TFA+EM could regulate the protein expression levels of marked signaling molecules of PANoptosis by targeting ZBP1. In summary, this study clarified that TFA+EM, different from TFA or EM, could attenuate DT with multiple targets by ameliorating mitochondrial homeostasis and inhibiting ZBP1-mediated PANoptosis. These findings provide the clear pharmacological evidence for the clinical treatment of DT with a novel strategy of TFA+EM, which is named "coordinated traditional Chinese and western medicine".
Animals ; Rats ; Mitochondria/metabolism* ; Benzhydryl Compounds/administration & dosage* ; Glucosides/administration & dosage* ; Abelmoschus/chemistry* ; Male ; Homeostasis/drug effects* ; Flavones/administration & dosage* ; Rats, Sprague-Dawley ; Diabetic Nephropathies/physiopathology* ; Drugs, Chinese Herbal/administration & dosage* ; DNA-Binding Proteins/genetics* ; Humans ; Apoptosis/drug effects*

Patients suffering from nerve injury often experience exacerbated pain responses and complain of memory deficits. The dorsal hippocampus (dHPC), a well-defined region responsible for learning and memory, displays maladaptive plasticity upon injury, which is assumed to underlie pain hypersensitivity and cognitive deficits. However, much attention has thus far been paid to intracellular mechanisms of plasticity rather than extracellular alterations that might trigger and facilitate intracellular changes. Emerging evidence has shown that nerve injury alters the microarchitecture of the extracellular matrix (ECM) and decreases ECM rigidity in the dHPC. Despite this, it remains elusive which element of the ECM in the dHPC is affected and how it contributes to neuropathic pain and comorbid cognitive deficits. Laminin, a key element of the ECM, consists of α-, β-, and γ-chains and has been implicated in several pathophysiological processes. Here, we showed that peripheral nerve injury downregulates laminin β1 (LAMB1) in the dHPC. Silencing of hippocampal LAMB1 exacerbates pain sensitivity and induces cognitive dysfunction. Further mechanistic analysis revealed that loss of hippocampal LAMB1 causes dysregulated Src/NR2A signaling cascades via interaction with integrin β1, leading to decreased Ca²⁺ levels in pyramidal neurons, which in turn orchestrates structural and functional plasticity and eventually results in exaggerated pain responses and cognitive deficits. In this study, we shed new light on the functional capability of hippocampal ECM LAMB1 in the modulation of neuropathic pain and comorbid cognitive deficits, and reveal a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified hippocampal LAMB1/integrin β1 signaling as a potential therapeutic target for the treatment of neuropathic pain and related memory loss.
Animals ; Laminin/genetics* ; Hippocampus/metabolism* ; Neuralgia/metabolism* ; Cognitive Dysfunction/etiology* ; Male ; Peripheral Nerve Injuries/metabolism* ; Extracellular Matrix/metabolism* ; Integrin beta1/metabolism* ; Pyramidal Cells/metabolism* ; Signal Transduction

OBJECTIVE: EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen, an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome. Its composition, anticoagulant and fibrinolytic activities, and relevant mechanisms have been confirmed through in vitro experiments. However, whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown. This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro. METHODS: The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl₃. Then, the protein bands of EPF3 incubated with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and homogenate of Caco-2 cells (HC2C) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability. Finally, the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer. RESULTS: EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid. It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl₃. EPF3 was stable in SIF and HC2C and unstable in SGF. The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent. The efflux ratio was less than 1.2 during transport, and the transport behavior was not affected by inhibitors. EPF3 could reversibly reduce the expression of tight junction-related proteins, including zonula occludens-1, occludin, and claudin-1 in Caco-2 cells. CONCLUSION EPF3 could play a thrombolytic and antithrombotic role in zebrafish. It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction. This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation. Please cite this article as: Zhong WL, Yang JQ, Liu H, Wu YL, Shen HJ, Li PY, Du SY. Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong (Pheretima vulgaris Chen) and its transport mechanism in Caco-2 monolayer through cell bypass pathway. J Integr Med. 2025; 23(4): 415-428.
Animals ; Zebrafish ; Humans ; Caco-2 Cells ; Fibrinolytic Agents/pharmacology* ; Thrombosis/drug therapy* ; Intestinal Absorption

Objective:To analyze the clinical value of delayed solidification of cement at freezing point combined with establishment of cement channels in the treatment of further leakage of cement in percutaneous kyphoplasty (PKP).Methods:A retrospective analysis was performed for the medical records of 261 patients with osteoporotic vertebral fracture in the thoracolumbar segment who underwent PKP treatment in Beijing Puren Hospital from April 2019 to April 2023. According to the method of dealing with PKP cement leakage, it was divided into freezing point group (using bone cement combined with cement channel reconstruction treatment at freezing point temperature) and temperature gradient group (using temperature gradient method). There were 128 cases in the freezing point group, including 37 males and 91 females, aged 75.57±4.60 years (range, 65-85 years), and fracture locations were 18 cases in T 10, 30 cases in T 11, 44 cases in T 12, 23 cases in L 1 and 13 cases in L 2. There were 133 cases in the temperature gradient group, including 36 males and 97 females, aged 75.66±4.51 years (range, 65-85 years), and fracture locations were 17 cases in T 10, 32 cases in T 11, 51 cases in T 12, 22 cases in L 1, and 11 cases in L 2. The intraoperative blood loss, operation time, intravertebral cement area, cement leakage area, cement leakage area increase, cement bolus time and incidence of injection difficulty, as well as the pain visual analogue scale (VAS), Oswestry disability index (ODI), kyphosis angle, the height of the anterior edge of the injured vertebral body and the difference between it before and after surgery were compared. Results:All patients were followed up for 3 consecutive months. The intraoperative blood loss and initial cement leakage area were 9.48±2.64 ml and 32.56±7.05 mm 2 in the freezing point group and 9.04±2.25 ml and 32.86±7.00 mm 2 in the temperature gradient group, respectively, and the difference was not statistically significant ( P>0.05) ; The operation time, the area of bone cement in the vertebral body, the final leakage area of bone cement, and the increase of bone cement leakage in the freezing point group were 55.08±4.13 min, 1 175.45±117.11 mm 2, 35.84±8.67 mm 2, and 0.00(0.00, 13.32) mm 2, respectively, and the temperature gradient group were 53.02±3.96 min, 823.70±144.79 mm 2, and 73.38±29.16 mm 2 and 44.39(20.13, 56.61) mm 2, the differences were statistically significant ( P<0.05). The height of the anterior edge of the vertebral body was 21.54±2.06 mm and 21.24±2.33 mm immediately after surgery and 3 months after surgery in the freezing point group, which were higher than those in the temperature gradient group 21.10±1.60 mm and 18.92±1.51 mm, respectively, and the difference was statistically significant ( P<0.05). The VAS scores of the freezing point group were 2.29±0.62 and 1.03±0.66 points, ODI were 23.20%±3.97%, 10.43%±4.33%, and the kyphosis angles were 9.09°±2.80° and 9.44°±2.93°, respectively, which were lower than those of the temperature gradient group (4.11±0.79 and 2.79±0.65 points), ODI (35.97%±6.42%, and 23.73%±5.72%), and the kyphosis angles (10.24°±2.33° and 13.22°±2.56°), the differences were statistically significant ( P<0.05). The operating time of bone cement in the freezing point group was 10.89±2.35 min, which was longer than that in the temperature gradient group 5.77±0.52 min, and the difference was statistically significant ( t=24.021, P<0.001). The incidence of cement injection difficulty was 0 in the freezing point group and 27.1% (36/133) in the temperature gradient group. Conclusion:The establishment of bone cement combined with bone cement channel at freezing point temperature can effectively prolong the bolus time of bone cement and reduce the re-leakage of bone cement, which is conducive to increasing the injection volume and distribution area of bone cement in the vertebral body, effectively reducing the amount of bone cement leakage and obtaining better clinical efficacy.

Good endometrial receptivity is an essential factor for embryo implantation,and gene expression in endometrial tissue during the window of implantation(WOI)is closely related to receptivity.Transcriptome sequencing technology enables the identification of gene expression profiles of endometrium during different menstrual phases,as well as microRNAs and long-chain non-coding RNA sequences involved in regulating gene expression.Combining this technology with bioinformatics analysis provides a better understanding of specific gene expression during the receptive period and offers technical support for studying its regulatory mechanism.Moreover,gene expression profiles of the endometrium during different menstrual phases hold significant clinical application value for accurately assessing endometrium receptivity in infertility patients and those with repeated implantation failure,thereby guiding individualized embryo transfer strategies.This review summarizes the progress of transcriptome sequencing in evaluating human endometrial receptivity and discusses future research directions.This review aims to understand the complex molecular mechanisms of endometrial receptivity formation and regulation from the transcriptional level,in order to improve the implantation rate of embryos in assisted reproductive technology and reduce the abortion rate.

Objective:To explore the relationship between mechanical ventilation based on volumetric guaranteed(VG)ventilation mode and weaning outcomes of neonatal respiratory distress syndrome(RDS).Methods:This study was a single-center prospective study,according to random number table method,129 neonatal with RDS who were admitted to our hospital from January 2022 to October 2024 were divided into control group(received pressure-controlled ventilation,n=64)and observation group(received pressure-controlled ventilation combined with VG,n=65).The duration of invasive ventilation,total duration of respiratory support,length of hospital stay,arterial blood gas parameters[partial pressure of carbon dioxide(PaCO2),pH value,partial pressure of oxygen(PaO2),PaO2/fraction of inspired oxygen(FiO2)],weaning time,extubation failure rate and incidence of complications were compared between the two groups.Results:The duration of invasive ventilation,the total duration of respiratory support,the weaning time and the length of hospital stay in the observation group were shorter than those in the control group,and the extubation failure rate and the incidence of complications were lower than those in the control group(P＜0.05).24 h,48 h after intervention,PaCO2 decreased,while pH value,PaO2 and PaO2/FiO2 increased.The PaCO2 in the observation group was lower than that in the control group,while the pH value,PaO2 and PaO2/FiO2 were higher than those in the control group(P＜0.05).Conclusion:Mechanical ventilation based on VG can shorten the mechanical ventilation time of neonatal with RDS,improve blood gas analysis indicators,and reduce weaning time,extubation failure rate and the risk of complications.

Objective:To analyze the clinical value of delayed solidification of cement at freezing point combined with establishment of cement channels in the treatment of further leakage of cement in percutaneous kyphoplasty (PKP).Methods:A retrospective analysis was performed for the medical records of 261 patients with osteoporotic vertebral fracture in the thoracolumbar segment who underwent PKP treatment in Beijing Puren Hospital from April 2019 to April 2023. According to the method of dealing with PKP cement leakage, it was divided into freezing point group (using bone cement combined with cement channel reconstruction treatment at freezing point temperature) and temperature gradient group (using temperature gradient method). There were 128 cases in the freezing point group, including 37 males and 91 females, aged 75.57±4.60 years (range, 65-85 years), and fracture locations were 18 cases in T 10, 30 cases in T 11, 44 cases in T 12, 23 cases in L 1 and 13 cases in L 2. There were 133 cases in the temperature gradient group, including 36 males and 97 females, aged 75.66±4.51 years (range, 65-85 years), and fracture locations were 17 cases in T 10, 32 cases in T 11, 51 cases in T 12, 22 cases in L 1, and 11 cases in L 2. The intraoperative blood loss, operation time, intravertebral cement area, cement leakage area, cement leakage area increase, cement bolus time and incidence of injection difficulty, as well as the pain visual analogue scale (VAS), Oswestry disability index (ODI), kyphosis angle, the height of the anterior edge of the injured vertebral body and the difference between it before and after surgery were compared. Results:All patients were followed up for 3 consecutive months. The intraoperative blood loss and initial cement leakage area were 9.48±2.64 ml and 32.56±7.05 mm 2 in the freezing point group and 9.04±2.25 ml and 32.86±7.00 mm 2 in the temperature gradient group, respectively, and the difference was not statistically significant ( P>0.05) ; The operation time, the area of bone cement in the vertebral body, the final leakage area of bone cement, and the increase of bone cement leakage in the freezing point group were 55.08±4.13 min, 1 175.45±117.11 mm 2, 35.84±8.67 mm 2, and 0.00(0.00, 13.32) mm 2, respectively, and the temperature gradient group were 53.02±3.96 min, 823.70±144.79 mm 2, and 73.38±29.16 mm 2 and 44.39(20.13, 56.61) mm 2, the differences were statistically significant ( P<0.05). The height of the anterior edge of the vertebral body was 21.54±2.06 mm and 21.24±2.33 mm immediately after surgery and 3 months after surgery in the freezing point group, which were higher than those in the temperature gradient group 21.10±1.60 mm and 18.92±1.51 mm, respectively, and the difference was statistically significant ( P<0.05). The VAS scores of the freezing point group were 2.29±0.62 and 1.03±0.66 points, ODI were 23.20%±3.97%, 10.43%±4.33%, and the kyphosis angles were 9.09°±2.80° and 9.44°±2.93°, respectively, which were lower than those of the temperature gradient group (4.11±0.79 and 2.79±0.65 points), ODI (35.97%±6.42%, and 23.73%±5.72%), and the kyphosis angles (10.24°±2.33° and 13.22°±2.56°), the differences were statistically significant ( P<0.05). The operating time of bone cement in the freezing point group was 10.89±2.35 min, which was longer than that in the temperature gradient group 5.77±0.52 min, and the difference was statistically significant ( t=24.021, P<0.001). The incidence of cement injection difficulty was 0 in the freezing point group and 27.1% (36/133) in the temperature gradient group. Conclusion:The establishment of bone cement combined with bone cement channel at freezing point temperature can effectively prolong the bolus time of bone cement and reduce the re-leakage of bone cement, which is conducive to increasing the injection volume and distribution area of bone cement in the vertebral body, effectively reducing the amount of bone cement leakage and obtaining better clinical efficacy.

AIM To investigate the effects of Gan Jiang-Huang Qin-Huang Lian-Ren Shen Decoction(GJHQHLRSD)on the pyroptosis,pathway of colonic epithelial cells in mouse models of ulcerative colitis(UC).METHODS Among the 63 C57BL/6J mice,13 were randomly selected and assigned to the model group,and the others were divided into the control group,the positive Sulfasalazine Enteric-Coated Tablets group(0.6 g/kg),and low,medium,and high dose GJHQHLRSD groups(3.9,7.8,15.6 g/kg),with 10 mice in each group.The UC mouse model was established using DSS,and the corresponding drugs were administered by gavage.The mice had their general condition observed;their disease activity index(DAI)score assessed;their colon length measured;their histopathological damage of the colon analyzed using HE staining;their colonic IL-1β,IL-8,and TNF-α levels measured by ELISA method;their colonic NLRP3,GSDMD,pro-IL-1β,pro-caspase-1,and IL-1βprotein expression detected by Western blot method;and their cell pyroptosis detected by TUNEL and GSDMD fluorescence double staining.RESULTS Compared with the control group,the model group exhibited significant decrease in body weight and a shortened colon length(P＜0.01);increases in DAI score,levels of IL-1β,IL-8,TNF-α,as well as the protein expressions of NLRP3,GSDMD,and active-caspase-1(P＜0.05,P＜0.01);significant increase of colonic GSDMD and TUNEL positivity;indicating increased tissue damage and inflammatory response.Compared with the model group,the groups intervened with GJHQHLRSD showed a significant increase in body weight and colonic elongation(P＜0.05,P＜0.01);decreases in DAI score,levels of IL-1β,IL-8,TNF-α,as well as the protein expressions of NLRP3,GSDMD,and active-caspase-1(P＜0.05,P＜0.01);a gradient decrease in positivity of GSDMD and TUNEL;indicating a significantly reduced colonic pathological damage.CONCLUSION GJHQHLRSD can improve the DSS-induced inflammatory reaction of colonic mucosa in UC mice,and its mechanism mainly involves the NLRP3/caspase-1,thereby the regulation of the cell pyroptosis process.

Objective:To explore the relationship between mechanical ventilation based on volumetric guaranteed(VG)ventilation mode and weaning outcomes of neonatal respiratory distress syndrome(RDS).Methods:This study was a single-center prospective study,according to random number table method,129 neonatal with RDS who were admitted to our hospital from January 2022 to October 2024 were divided into control group(received pressure-controlled ventilation,n=64)and observation group(received pressure-controlled ventilation combined with VG,n=65).The duration of invasive ventilation,total duration of respiratory support,length of hospital stay,arterial blood gas parameters[partial pressure of carbon dioxide(PaCO2),pH value,partial pressure of oxygen(PaO2),PaO2/fraction of inspired oxygen(FiO2)],weaning time,extubation failure rate and incidence of complications were compared between the two groups.Results:The duration of invasive ventilation,the total duration of respiratory support,the weaning time and the length of hospital stay in the observation group were shorter than those in the control group,and the extubation failure rate and the incidence of complications were lower than those in the control group(P＜0.05).24 h,48 h after intervention,PaCO2 decreased,while pH value,PaO2 and PaO2/FiO2 increased.The PaCO2 in the observation group was lower than that in the control group,while the pH value,PaO2 and PaO2/FiO2 were higher than those in the control group(P＜0.05).Conclusion:Mechanical ventilation based on VG can shorten the mechanical ventilation time of neonatal with RDS,improve blood gas analysis indicators,and reduce weaning time,extubation failure rate and the risk of complications.

AIM To investigate the effects of Gan Jiang-Huang Qin-Huang Lian-Ren Shen Decoction(GJHQHLRSD)on the pyroptosis,pathway of colonic epithelial cells in mouse models of ulcerative colitis(UC).METHODS Among the 63 C57BL/6J mice,13 were randomly selected and assigned to the model group,and the others were divided into the control group,the positive Sulfasalazine Enteric-Coated Tablets group(0.6 g/kg),and low,medium,and high dose GJHQHLRSD groups(3.9,7.8,15.6 g/kg),with 10 mice in each group.The UC mouse model was established using DSS,and the corresponding drugs were administered by gavage.The mice had their general condition observed;their disease activity index(DAI)score assessed;their colon length measured;their histopathological damage of the colon analyzed using HE staining;their colonic IL-1β,IL-8,and TNF-α levels measured by ELISA method;their colonic NLRP3,GSDMD,pro-IL-1β,pro-caspase-1,and IL-1βprotein expression detected by Western blot method;and their cell pyroptosis detected by TUNEL and GSDMD fluorescence double staining.RESULTS Compared with the control group,the model group exhibited significant decrease in body weight and a shortened colon length(P＜0.01);increases in DAI score,levels of IL-1β,IL-8,TNF-α,as well as the protein expressions of NLRP3,GSDMD,and active-caspase-1(P＜0.05,P＜0.01);significant increase of colonic GSDMD and TUNEL positivity;indicating increased tissue damage and inflammatory response.Compared with the model group,the groups intervened with GJHQHLRSD showed a significant increase in body weight and colonic elongation(P＜0.05,P＜0.01);decreases in DAI score,levels of IL-1β,IL-8,TNF-α,as well as the protein expressions of NLRP3,GSDMD,and active-caspase-1(P＜0.05,P＜0.01);a gradient decrease in positivity of GSDMD and TUNEL;indicating a significantly reduced colonic pathological damage.CONCLUSION GJHQHLRSD can improve the DSS-induced inflammatory reaction of colonic mucosa in UC mice,and its mechanism mainly involves the NLRP3/caspase-1,thereby the regulation of the cell pyroptosis process.