ObjectiveThis study aims to construct a reconstituted high-density lipoprotein (rHDL) delivery system loaded with kinsenoside (KD@rHDL), and to systematically evaluate its function in enhancing the phagocytosis of amyloid β-protein (Aβ) by microglia and improving the inflammatory state of microglia, as well as to preliminarily explore its potential application value in the treatment of Alzheimer’s disease (AD). MethodsKD@rHDL was prepared by the film hydration method combined with probe sonication and co-incubation. Its morphology was observed by transmission electron microscopy, and the particle size and Zeta potential were measured by dynamic light scattering. The encapsulation efficiency and drug loading were determined by high-performance liquid chromatography. The affinity between KD@rHDL and Aβ was analyzed by surface plasmon resonance (SPR) to assess its feasibility as a medium for Aβ clearance. At the cellular level, after treating mouse microglial cells (BV-2 cells) with KD@rHDL and adding fluorescently labeled Aβ, the phagocytic efficiency of microglia for Aβ was detected by confocal microscopy. Meanwhile, the CCK-8 method was used to evaluate the effect of KD@rHDL on cell viability to determine its safety. The trans-barrier transport ability of KD@rHDL was detected by Transwell assay. The expression levels of NLRP3 inflammasome and downstream inflammatory factor IL-1β in LPS-induced microglia were detected by Western blot to evaluate the regulatory effect of KD@rHDL on the inflammatory state of cells. ResultsCharacterization results showed that the successfully prepared KD@rHDL presented a typical discoid structure under transmission electron microscopy, with a uniform particle size distribution, an average particle size of approximately (14.4±0.24) nm, and a suitable negative Zeta potential, demonstrating good colloidal stability. The drug content determination results indicated that the encapsulation efficiency of KD@rHDL for kinsenoside was (42.24±1.30)%, and the drug loading was (6.03±0.19)%, indicating a good drug loading capacity. The CCK-8 assay results showed that in the set concentration range, the survival rates of BV2 and HT22 cells in the KD@rHDL treatment group were all above 90%, with no significant difference from the control group, indicating good cell safety of the formulation. The results of the Aβ phagocytosis experiment indicated that, compared with the Aβ oligomers (Aβo) group alone, the fluorescence signal intensity within microglia in the KD@rHDL treatment group was significantly enhanced, and a large amount of fluorescence-labeled Aβ was observed to accumulate intracellularly under a fluorescence microscope. The SPR assay results showed that rHDL had a strong affinity for Aβ, with an affinity constant reaching the nanomolar level. Transwell assay results indicated that KD@rHDL could effectively cross the bEnd.3 cell monolayer barrier and be taken up by BV2 and HT22 cells. Western blot assay results showed that high-dose KD@rHDL treatment could significantly reduce the expression level of NLRP3 protein in LPS-induced microglia and simultaneously down-regulate the maturation and secretion of IL-1β, indicating that KD@rHDL can effectively inhibit the activation of the NLRP3 inflammasome pathway and improve the neuroinflammatory state mediated by microglia. ConclusionThis study successfully constructed a reconstituted high-density lipoprotein delivery system loaded with kinsenoside (KD@rHDL). This nano-delivery system not only significantly enhances the phagocytic clearance ability of microglia towards Aβ, but also effectively inhibits the NLRP3/IL-1β-mediated inflammatory pathway, improving the inflammatory state of microglia. The above results indicate that KD@rHDL has a synergistic effect in promoting Aβ clearance and alleviating neuroinflammation, demonstrating potential therapeutic value for AD and providing new ideas and experimental basis for the development of subsequent AD treatment strategies.

OBJECTIVES: To observe the evolution of intrahepatic macrophage polarization in mice with liver fibrosis induced by iron overload. METHODS: Thirty-two C57BL/6 mice (6-8 weeks) were randomized into control group (n=8) and liver fibrosis model group (n=24) induced by aidly intraperitoneal injection of iron dextran. At the 3rd, 5th, and 7th weeks of modeling, 8 mice in the model group were sacrificed for observing liver fibrosis using Masson, Sirius Red and immunohistochemical staining and detecting serum levels of ALT, AST and the levels of serum iron, ferritin, liver total Fe and ferrous Fe. iNOS⁺/F4/80⁺ cells and CD206⁺/F4/80⁺ cells were detected by double immunofluorescence assay to observe the proportion and distribution of M1 and M2 macrophages. The hepatic expressions of Arg-1, iNOS, IL-6, IL-10, and TNF‑α proteins were detected using Western blotting or ELISA, and the expression of CD206 mRNA was detected using RT-PCR. RESULTS: The mice in the model group showed gradual increase of fibrous tissue hyperplasia in the portal area over time, structural destruction of the hepatic lobules and formation of pseudolobules. With the passage of time during modeling, the rat models showed significantly increased hepatic expressions of α-SMA and COL-1, elevated serum levels of ALT, AST, Fe, ferritin, and increased liver total Fe and ferrous Fe levels. The expressions of M1 polarization markers IL-6, TNF‑α, and iNOS all increased with time and reached their peak levels at the 3rd week; The expressions of M2 polarization markers (IL-10 and Arg-1 proteins and CD206 mRNA) significantly increased in the 3rd week and but decreased in the 5th and 7th weeks. CONCLUSIONS Iron overload promotes M1 polarization of macrophages in mice. Liver fibrosis in the early stage promotes M2 polarization of macrophages but negatively regulate M2 polarization at later stages.
Animals ; Mice ; Mice, Inbred C57BL ; Iron Overload/pathology* ; Macrophages/metabolism* ; Male ; Liver Cirrhosis/etiology* ; Nitric Oxide Synthase Type II/metabolism* ; Interleukin-10/metabolism* ; Liver/pathology* ; Interleukin-6/metabolism* ; Mannose Receptor ; Tumor Necrosis Factor-alpha/metabolism* ; Mannose-Binding Lectins/metabolism* ; Arginase

In analytical techniques such as ion mobility spectrometry(IMS)and mass spectrometry(MS),the ionization efficiency of target analytes is primarily constrained by the type of ionization source and factors such as the species and number density of the reactant ions.Systematic investigation into the reactivity differences of various reactant ions under varying conditions can not only significantly enhance the detection sensitivity of target compound product ions but also provide a theoretical foundation for establishing efficient detection methods based on ion-molecule reaction mechanisms.In this study,the pressure of a pressure-tunable photoionization tandem ion mobility spectrometry(PI-tandem-IMS)was reduced from ambient pressure(100 kPa)to low pressure(20 kPa)to systematically examine the reactivity differences between two negative reactant ions,CO3-and CO4-,and methyl salicylate(MeSA)under varying pressures.When the pressure decreased,the increased relative signal intensity of CO4-significantly influenced the detection sensitivity of the characteristic product ion[MeSA·O2]-.Based on differences in ion mobility(k0),the delay time for the opening of TPG2 was adjusted to selectively inject CO-3 and CO-4 in the drift region 2.Independent characterization of the reactivity of these reactant ions with MeSA in the reaction region confirmed that CO4-exhibited superior reactivity toward MeSA.The theoretical model revealed an Arrhenius plot for the ion-molecule reaction between CO4-and MeSA,showing a positive correlation between the reaction rate coefficient(k)and temperature,the activation energy Ea was 62.45 kJ/mol.Furthermore,controlling parameters such as pressure or temperature significantly influenced the progression of this ion-molecule reaction,demonstrating the technical advantages of PI-tandem-IMS in mechanistic studies and regulation of ion-molecule reactions.

The resolving power and sensitivity are critical for on-site detection of hazardous chemicals using stand-alone ion mobility spectrometry(IMS).However,improving the sensitivity and resolving power of IMS has long been a prominent research hot spot.In the commonly used IMS based on the Bradbury-Nielsen gate(BNG),the gating voltage difference(GVD)applied between the two sets of grid wires affects the electric field distribution in the ionization region and the drift region.This,in turn,influences the spatial distribution and temporal width of the injected ion swarm,and has an impact on the ion mobility discrimination,sensitivity,and resolving power of the instrument.This study showed that increasing the GVD could induce an ion converging effect,boosting the ion number density in front of the BNG by nearly 300％.To simultaneously utilize temporal compression and ion converging effects,a novel BNG controlling mode was proposed by adding a chopping state to the conventional controlling mode.This chopping state reduced the mobility discrimination effects between ions with mobility differences up to about 0.90 cm2/(V·s)to 1/22 of their original value.When analyzing hazardous chemical mixtures using the novel BNG controlling mode,compared with conventional mode,the signal intensity of low-mobility methyl salicylate ions(MS·O2)-increased by 18-fold while the resolving power maintained around 100,and the detection limit for MS was improved from 3.75 μg/L to 97 ng/L.This novel BNG controlling mode only added a potential wave to the low voltage wires,with no requirement of changing the structure of the drift tube,and was easy to apply to existing commercial instruments.

Objective To determine the effects of cardiac resynchronization therapy at different pacing sites on electrocardiogram(ECG),left ventricular(LV)function and adverse events in elderly patients with heart failure(HF).Methods A total of 214 elderly HF patients admitted to our department between July 2021 and July 2024 were retrospectively recruited.According to different pacing sites in cardiac resynchronization therapy,they were divided into His bundle group(102 cases)and left bundle branch group(112 cases).Their grades of New York Heart Association(NYHA)cardiac function,duration of QRS complex,pacing parameters(pacing threshold,pacing perception,pacing resistance),cardiac function indicators,LV function,LV systolic synchrony[standard deviation of time to peak longitudinal strain,to peak radial strain and to peak circumferential strain(Tls-SD,Trs-SD and Tcs-SD)],and incidence of adverse events were compared between the two groups before and at 6 months after cardiac pacemaker implantation.Results In 6 months after surgery the left bundle branch group had significantly lower N-terminal pro-B-type natriuretic peptide level,smaller LV end-diastolic diameter and LV end-systolic diameter,decreased Tls-SD,Trs-SD and Tcs-SD,and shorter duration of QRS complex,but higher LV ejection fraction,cardiac index and cardiac output when compared with the His bundle group(P＜0.01).The incidence of adverse events was obviously lower in the left bundle branch group than the His bundle group(6.25％vs 15.69％,P＜0.05).Conclusion Left bundle branch pacing shows significant improvement for cardiac function in elderly HF patients,and can effectively maintain ECG stability and improve LV function.It is a safe and effective cardiac resynchronization therapy.

Objective To investigate the value of peripheral blood circRNA9226 combined with tumor markers for the diagnosis of lung adenocarcinoma.Methods A total of 30 patients with lung adenocarcinoma who were admitted to the Affiliated Lihuili Hospital of Ningbo University from December 2022 to January 2023 were selected as case group.Additionally,twenty healthy individuals who underwent physical examinations during the same period were selected as control group.The expression levels of circRNA9226,carcinoembryonic antigen(CEA),carbohydrate antigen(CA)199,and albumin(Alb)were compared between two groups.The diagnostic efficacy of circRNA9226 and CEA for lung adenocarcinoma was evaluated by receiver operating characteristic(ROC)curve,and the correlation between circRNA9226 and other indicators was analyzed.Results The peripheral blood circRNA9226 expression levels in case group was higher than that in control group(P＜0.05).The sensitivity of circRNA9226 and CEA for diagnosing lung adenocarcinoma was 83.3％and 46.7％,respectively,with specificity of 90.0％and 100.0％.Area under the curve(AUC)was 0.897 and 0.776,respectively.When both tests were combined,the sensitivity was 93.3％,accuracy was 92.0％,and AUC was 0.918,showing a significant improvement in both sensitivity and accuracy compared to single tests.The correlation analysis results showed that circRNA9226 was positively correlated with CEA concentration(r=0.371,P＜0.05)and negatively correlated with Alb concentration(r=-0.548,P＜0.05).Conclusion Peripheral blood circRNA9226 has advantages in distinguishing lung adenocarcinoma from healthy controls,and combined with CEA can further improve the diagnostic efficacy.

This study explored whether Astragalus polysaccharide(APS)can regulate the VEGF/VEGFR signaling pathway to affect the proliferative activity of rat intestinal mucosal microvascu-lar endothelial cells(RIMMVECs).RIMMVECs were isolated from newborn rats,then purified and treated with APS at concentrations of 0.1,1.0,10.0,100.0,1 000.0,and 10 000.0 mg/L.MTT was used to determine the effect of APS on RIMMVECs proliferation and screen for the optimal concentration of APS.Subsequently,flow cytometry was used to detect the changes in cell cycle to evaluate the stage of action of APS on the cell cycle in RIMMVECs.Then,the ELISA was used to detect the changes of VEGFA in cell supernatant to evaluate the potential of cell proliferation and angiogenesis.The changes in fluorescence intensity of Fluo-8AM was observed using fluorescence microscopy to evaluate intracellular Ca2+levels.Finally,Western blot was used to detect the ex-pression of PERK in RIMMVECs to analyze the possible mechanism of APS.The results showed that 100 mg/L APS significantly enhanced the proliferative activity of RIMMVECs,increased the content of VEGFA in the cell supernatant,the intracellular Ca2+levels,and the expression of PERK protein,indicating that APS promotes the proliferation of RIMMVECs,which may be a-chieved by promoting the expression of VEGFA and activating the ERK pathway.

AIM:To investigate the function of mitochondrial reactive oxygen species(mtROS)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway in modulating macrophage polarization in liver fibrosis resulting from iron overload.METHODS:Thirty-two male C57BL/6 mice were randomly allocated into four groups:control group,model group(iron dextran,50 mg/kg),MitoTEMPO(3 mg/kg)group,and MCC950(10 mg/kg)group,comprising eight mice per group.All mice,with the exception of the control group,were administered daily in-traperitoneal injections of iron dextran for a duration of seven consecutive weeks,whereas the control group received equiv-alent volumes of normal saline.Starting in week four,the MitoTEMPO and MCC950 cohorts received their designated treatments through intraperitoneal injection three times weekly.Serum alanine aminotransferase(ALT)and aspartate ami-notransferase(AST)concentrations were assessed through biochemical analysis.Liver tissues were analyzed utilizing HE,Masson,Sirius red and immunohistochemical staining.The concentrations of mtROS were evaluated utilizing the MitoSOX Red probe.Cytokines and polarization markers,such as interleukin-1β(IL-1β),IL-18,IL-6,tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS),IL-10,and arginase-1(Arg-1),were quantified via ELISA.Western blot analysis was performed to quantify the protein expression levels of Arg-1,iNOS,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),and caspase-1.The mRNA expression of NLRP3,ASC,and caspase-1 was assessed using RT-qPCR.Immunofluorescence double labeling was employed to identify M1 and M2 macrophages.RESULTS:(1)In comparison to the control group,the model group demonstrated notable inflammatory cell infiltration,pronounced fibrous tissue hyperplasia,significant disruption of hepatic lobular architecture,and the develop-ment of pseudo-lobules in certain areas.Serum ALT and AST levels were markedly elevated(P<0.01),as were the mRNA and protein expression levels of NLRP3,ASC,caspase-1,and mtROS(P<0.01).Iron overload resulted in markedly ele-vated serum iron,ferritin,total liver iron,and ferrous iron concentrations(P<0.01).Markers indicative of M1 macrophage polarization,including IL-6,TNF-α,and iNOS,exhibited upregulation(P<0.01),whereas M2 markers such as IL-10,Arg-1,and CD206 were significantly downregulated(P<0.01).(2)Compared with model group,inhibiting mtROS or NL-RP3 substantially reduced inflammation and fibrous tissue hyperplasia.ALT and AST levels were markedly diminished(P<0.01),as were the areas of positive staining for α-smooth muscle actin and collagen type Ⅰ(P<0.01).Markers of iron over-load,such as serum iron,ferritin,total liver iron,and ferrous iron,were significantly ameliorated(P<0.01).M1 polariza-tion markers were significantly downregulated(P<0.01).CONCLUSION:The mtROS/NLRP3 signaling pathway facili-tates liver fibrosis caused by iron overload by enhancing macrophage polarization to the M1 phenotype.

Objective To explore the efficacy of auricular plaster therapy with press needles combined with eszopiclone tablets in the treatment of patients with post-stroke insomnia(PSI)and its effect on the serum levels of brain-derived neurotrophic factor(BDNF)and interleukin-1β(IL-1β).Methods A total of 120 PSI patients were selected as the study subjects and randomly divided into group A(60 cases)and group B(60 cases).Group A was treated with eszopiclone tablets,while group B was treated with auricular plaster therapy with press needles combined with eszopiclone tab-lets.Both groups received continuous treatment for 4 weeks.The clinical efficacy,sleep status,in-flammatory factors,neurotrophic factors,psychological state,quality of life,and adverse reactions were compared between the two groups after treatment.Results After treatment,the total clinical effective rate in the group B was 93.33％,which was higher than 80.00％in the group A(P＜0.05).The levels of tumor necrosis factor-α(TNF-α),IL-1β,IL-6,norepinephrine(NE),the scores of Pittsburgh Sleep Quality Index(PSQI),Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA),and Self-rating Scale of Sleep(SRSS)in both groups were lower than those before treatment(P＜0.05),and the above indicators in the group B were lower than those in group A,with statistically significant differences(P＜0.05).The levels of 5-hydroxytryptamine(5-HT),BDNF,and the scores of Stroke-Specific Quality of Life Scale(SS-QOL)were higher than those be-fore treatment,and the levels in the group B were higher than those in the group A,with statistically significant differences(P＜0.05).There was no statistically significant difference in the total inci-dence of adverse reactions between the two groups(P＞0.05).Conclusion Auricular plaster therapy with press needles combined with eszopiclone tablets can improve the clinical efficacy,sleep status,and quality of life in PSI patients,reduce the levels of BDNF and IL-1β,alleviate anxiety and depression,and have high safety.