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 observe the efficacy and safety of Morinda officinalis oligosaccharides in the continuation treatment of mild and moderate depression.Methods An open,single-arm,multi-center design was adopted in our study.Adult patients with mild and moderate depression who had received acute treatment of Morinda officinalis oligosaccharides were enrolled and continue to receive Morinda officinalis oligosaccharides capsules for 24 weeks,the dose remained unchanged during continuation treatment.The remission rate,recurrence rate,recurrence time,and the change from baseline to endpoint of Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA),Clinical Global Impression-Severity(CGI-S)and Arizona Sexual Experience Scale(ASEX)were evaluated.The incidence of treatment-related adverse events was reported.Results The scores of HAMD-17 at baseline and after treatment were 6.60±1.87 and 5.85±4.18,scores of HAMA were 6.36±3.02 and 4.93±3.09,scores of CGI-S were 1.49±0.56 and 1.29±0.81,scores of ASEX were 15.92±4.72 and 15.57±5.26,with significant difference(P＜0.05).After continuation treatment,the remission rate was 54.59％(202 cases/370 cases),and the recurrence rate was 6.49％(24 cases/370 cases),the recurrence time was(64.67±42.47)days.The incidence of treatment-related adverse events was 15.35％(64 cases/417 cases).Conclusion Morinda officinalis oligosaccharides capsules can be effectively used for the continuation treatment of mild and moderate depression,and are well tolerated and safe.

Objective:To provide reference for hospital drug selection and clinical rational drug selection,through evaluating the nutritional value of six commonly used balanced compound amino acid injection (BCAA) in clinical practice,including 18AA (250 mL:12.5 g),18AA-I (250 mL:17.5 g),18AA-Ⅱ(250 mL:21.25 g),18AA-IV (250 mL:8.7 g),18AA-V (250 mL:8.06 g),and 18AA-V-SF (250 mL:8.06 g). Methods:Based on the whole egg protein model,the nutritional value of six varieties of BCAA from two aspects were evaluated,including the first limiting amino acid chemical score (CS),value of essential amino acid (EAA) and the comprehensive quality of total EAA (both essential amino acid index and closeness to standard protein). Results:The first limiting amino acid CS value from high to low was 18AA-Ⅱ>18AA>18AA-V=18AA-V-SF>18AA-I=18AA-Ⅳ. Total EAA comprehensive quality:the essential amino acid index from high to low was 18AA-Ⅱ>18AA>18AA-I>18AA-Ⅳ>18AA-V=18AA-V-SF. The closeness to whole egg protein from high to low was 18AA-Ⅱ=18AA=18AA-I>18AA-Ⅳ>18AA-V=18AA-V-SF. Ultimately,the nutritional value of the 6 varieties of BCAA decreased from high to low:18AA-Ⅱ>18AA>18AA-I>18AA-Ⅳ>18AA-V=18AA-V-SF. Conclusions:Among the six varieties of BCAA,18AA-Ⅱ has the highest nutritional value and the highest amino acid content in the same liquid volume,making it the preferred drug for patients with normal liver and kidney function.

Objective To investigate the mechanism of Chonghe soft extract on ulcer wound healing in diabetic rats through protein kinase B(Akt)/mammalian Sirolimus target protein(mTOR)-mediated nucleotides binding oligomeric acid domain-like receptor protein 3(NLRP3)inflammasome inactivation.Methods Thirty six SD rats with diabetic ulcer,which were established by feeding with high glucose and high fat diet and injecting intraperitoneally with streptozocin(STZ)combined with skin defect,were randomly divided into model group,Chonghe soft extract group and growth factor group,with twelve rats in each group.Another twelve SD rats were injected an equal dose of citric acid-sodium citrate buffer solution and used as blank group.The blank group and the model group were not received drug intervention,but the Chonghe soft extract group and the growth factor group were externally applied Chonghe soft extract and growth factor gel,respectively.The wound healing of each group was observed and recorded.After 7 days and 14 days of treatment,the histopathology of wound were observed by HE staining and the number of fibroblasts were counted.The levels of IL-1β,IL-18 and TNF-α in serum were detected by ELISA.The expression of autophagy-related protein Beclin-1 and LC3Ⅱ in granulation tissue was detected by immunohistochemistry.The expression of NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),Caspase1,Pro-Caspase1 and Akt/mTOR autophagy pathway-related proteins Akt,p-Akt,mTOR and p-mTOR were detected by Western Blot.Results Compared with the blank control group,the pathological wound repair of the model group was delayed on the 7th day and 14th day,the number of fibroblasts per unit area was decreased(P<0.01).The levels of IL-1β,IL-18 and TNF-α were increased(P<0.01).The expression levels of ASC,Pro-Caspase1,Caspase1,and NLRP3 were increased in the wound tissues(P<0.01),while the expression levels of Beclin-1,LC3-Ⅱ,mTOR,p-mTOR,Akt and p-Akt were decreased in the wound tissues(P<0.01).Compared with the model group,the pathological injury in Chonghe soft extract group and growth factor group was significantly improved on the 7th day and 14th day.The number of fibroblasts per unit area was significantly increased(P<0.01).The levels of IL-1β,IL-18 and TNF-α were significantly decreased(P<0.01).The expression levels of ASC,Pro-Caspase1,Caspase1,and NLRP3 in the wound tissues were decreased(P<0.01),while the expression levels of Beclin-1,LC3-Ⅱ,mTOR,p-mTOR,Akt and p-Akt were increased(P<0.01,P<0.05).Conclusion Chonghe soft extract can reduce inflammatory reaction,promote the generation of fibro,regulate the Akt/mTOR-mediated NLRP3 inflammasome inactivation,improve the level of autophagy in wound,and promote ulcer wound healing in diabetic rats.

Aim To explore how Danzhi Jiangtang cap-sules(DJC)safeguard the mitochondrial activity of vascular smooth muscle cells(VSMCs)by controlling the LncRNA TUG1/β-catenin signaling pathway to de-crease vascular calcification(VC).Methods Vascu-lar smooth muscle cell calcification models were in-duced with β-glycerin and diabetic vascular calcifica-tion rat models were induced with vitamin D3+high-fat diet.Von Kossa staining was applied to detect cal-cification of cells and vascular tissue.Colorimetric method of phthalein complex was used to determine calcium content.P-nitrobenzene phosphate colorimetry was employed to assess alkaline phosphatase(ALP)activity.RT-qPCR was used to analyze the expression of VSMCs'osteoblast transformation related genes bone morphogenetic protein2(BMP2),smooth muscle actin alpha(α-SMA),taurine up-regulated1,LncRNA Tug1(Lnc-RNA TUG1),and β-catenin.Western blotting was utilized to detect the protein expression of BMP2,α-SMA and β-catenin.The mitochondrial membrane potential was detected by JC-1 fluorescence probe.Mitochondrial structure was observed by trans-mission electron microscope.Results DJC reduced LncRNA TUG1 expression,down-regulated β-catenin expression,decreased ALP activity and calcium depo-sition,protected mitochondrial function,restored mem-brane potential,and decreased osteoblastic transforma-tion of VSMCs induced by glycerin phosphate.Impor-tantly,DJC attenuated diabetic lower limb VC by down-regulating the expression of LncRNA TUG1,β-catenin,and elevating the expression of α-SMA.Con-clusions DJC capsules significantly improved VSMCs by protecting mitochondrial function by LncRNA TUG1/β-catenin signaling to reduce VSMCs'osteo-blast transformation.

Objective:To understand the serological characteristics of irregular antibodies in pregnant women and explore their clinical significance.Methods:From January 2017 to March 2022,151 471 pregnant women in Women and Children's Hospital of Chongqing Medical University were enrolled in this study,microcolumn gel card test was used for irregular antibody screening,and antibody specificity identification was further performed in some antibody-positive subjects.Results:The positive rate of irregular antibody screening in the enrolled pregnant women was 0.91％(1 375/151 471),0.23％(355/151 471)was detected in the first trimester,0.05％(71/151 471)in the second trimester,and 0.63％(949/151 471)in the third trimester.The positive rate of irregular antibody screening in the third trimester was significantly higher than that in the first and second trimester,and a significant increase in the number of positive cases was found in the third trimester than that in the second trimester.The analysis of agglutination intensity of 1 375 irregular antibody screening positive results showed that the weakly positive agglutination intensity accounted for 50.11％(689/1 375),which was the highest,the suspicious positive was 18.69％(257/1 375),and the positive was 31.20％(429/1 375).The significant difference in distribution of agglutination intensity was not observed between the first trimester group and the second trimester group,however,in the third trimester,the proportion of suspicious positive and weakly positive was lower than the first trimester,while,the proportion of positive was higher than the first trimester,and the difference was statistically significant(P＜0.001).Among the irregular antibody screening positive pregnant women,the proportion of pregnant women with pregnancy number ≥ 2 was significantly higher than that with pregnancy≤1.Among 60 pregnant women who underwent antibody identification,the distributions of the antibodies were as follows:Rh blood group system accounted for 23.33％(14/60),Lewis system 43.33％(26/60),Kidd system 3.33％(2/60),MNS system 16.67％(10/60),P1PK system 1.67％(1/60),autoantibodies 1.67％(1/60),and 4 cases was unable to identify(6.67％,4/60).Among specific antibodies,the anti-Lea was the most common(30.00％),followed by anti-E(16.67％)and anti-M(16.67％).Conclusion:The differences of irregular antibody serological characteristics exist in pregnant women from different regions with different genetic backgrounds,understanding the characteristics of irregular antibody in local pregnant women is of great significance for ensuring transfusion safety in pregnant women and preventing hemolytic disease of newborn.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

Objective To investigate the expression of CD123 in children with acute lymphoblastic leukemia(ALL)and its effect on the clinical characteristics and prognosis of children with B-lineage acute lymphoblastic leukemia(B-ALL).Methods A retrospective analysis was conducted on the clinical data of 251 children with ALL who were admitted to the Department of Hematology and Oncology,Children's Hospital of Kunming Medical University,from December 2019 to June 2022.According to the expression of CD123 at initial diagnosis,the children were divided into CD123+group and CD123-group,and the two groups were compared in terms of clinical characteristics and treatment outcome.The factors influencing the prognosis were analyzed.Results Among the 251 children with ALL,there were 146 children(58.2%)in the CD123+group.The B-ALL group had a significantly higher positive expression rate of CD123 than the acute T lymphocyte leukemia group(P<0.05).Compared with the CD123-group,the CD123+group had significantly lower peripheral blood leukocyte count and percentage of juvenile cells and a significantly higher proportion of children with high hyperdiploid karyotype or an age of 1-10 years,with a relatively low proportion of children with E2A-PBX1 fusion gene(P<0.05).The multivariate Cox proportional-hazards regression model analysis showed that compared with the>10 years group,the 1-10 years group had a significantly higher overall survival rate(P<0.05),and compared with the high risk group,the moderate risk group had a significantly higher event-free survival rate in children with B-ALL(P<0.05).Conclusions CD123 is widely expressed in children with B-ALL,and positive expression of CD123 might be an indicator for good prognosis in children with B-ALL,which is of great significance for evaluating the efficacy of remission induction therapy and survival prognosis of children with B-ALL.