Antibiotic adjuvants offer a promising strategy for restoring antibiotic sensitivity, expanding antibacterial spectra, and reducing required dosages. Previously, compound 15 was identified as a potential adjuvant for Polymyxin B (PB) against multidrug-resistant (MDR) Pseudomonas aeruginosa DK2; however, its clinical utility was hindered by high cytotoxicity, uncertain in vivo efficacy, and an unclear synergetic mechanism. To address these challenges, we synthesized and evaluated a series of novel benzamide derivatives, with A22 emerging as a particularly promising candidate. A22 demonstrated potent synergistic activity to PB, minimal cytotoxicity, improved water solubility, and broad-spectrum synergism of polymyxins against various clinically isolated MDR Gram-negative strains. In vivo studies using Caenorhabditis elegans and mouse models further confirmed the efficacy of A22. Moreover, A22 effectively suppressed the development of PB resistance in Pseudomonas aeruginosa DK2. Mechanistic investigations revealed that A22 enhances polymyxins activity by inducing reactive oxygen species production, reducing ATP levels, increasing NOX activity, and inhibiting biofilm formation, leading to bacterial death. These findings position A22 as a highly promising candidate for the development of polymyxin adjuvants, offering a robust approach to combating MDR Gram-negative bacterial infections.

OBJECTIVES: To investigate the effects of quercetin on cuproptosis and L-type calcium currents in the myocardium of diabetic rats. METHODS: Forty SD rats were randomized into control group and diabetic model groups. The rat models of diabetes mellitus (DM) induced by high-fat and high-sugar diet combined with streptozotocin (STZ) injection were further divided into DM model group, quercetin treatment group, and empagliflozin treatment group (n=10). Blood glucose and body weight were measured every other week, and cardiac function of the rats was evaluated using echocardiography. HE staining, Sirius red staining, and wheat germ agglutinin (WGA) analysis were used to observe the changes in myocardial histomorphology, and serum copper levels and myocardial FDX1 expression were detected. In cultured rat cardiomyocyte H9c2 cells with high-glucose exposure, the effects of quercetin and elesclomol, alone or in combination, on intracellular CK-MB and LDH levels and FDX1 expression were assessed, and the changes in L-type calcium currents were analyzed using patch-clamp technique. RESULTS: The diabetic rats exhibited elevated blood glucose, reduced body weight, impaired left ventricular function, increased serum copper levels and myocardial FDX1 expression, decreased L-type calcium currents, and prolonged action potential duration. Quercetin and empagliflozin treatment significantly lowered blood glucose, improved body weight, and restored cardiac function of the diabetic rats, and compared with empagliflozin, quercetin more effectively reduced serum copper levels, downregulated FDX1 expression, and enhanced myocardial L-type calcium currents in diabetic rats. In H9c2 cells, high glucose exposure significantly increased myocardial expressions of FDX1, CK-MB and LDH, which were effectively lowered by quercetin treatment; Elesclomol further elevated FDX1, CK-MB and LDH levels in the exposed cells, and these changes were not significantly affected by the application of quercetin. CONCLUSIONS Quercetin ameliorates myocardial injury in diabetic rats possibly by suppressing myocardial cuproptosis signaling and restoring L-type calcium channel activity.
Animals ; Quercetin/pharmacology* ; Calcium Channels, L-Type/metabolism* ; Diabetes Mellitus, Experimental/metabolism* ; Rats, Sprague-Dawley ; Rats ; Myocytes, Cardiac/drug effects* ; Myocardium/pathology* ; Male

Given the increasing concern regarding antibacterial resistance, the antimicrobial properties of naphthoquinones have recently attracted significant attention. While 1,4-naphthoquinone and its derivatives have been extensively studied, the antibacterial properties of 5,8-dihydroxy-1,4-naphthoquinone derivatives remain relatively unexplored. This study presents a comprehensive in vitro and in vivo analysis of the antibacterial activity of 35 naturally sourced and chemically synthesized derivatives of 5,8-dihydroxy-1,4-naphthoquinone. Kirby-Bauer antibiotic testing identified three compounds with activity against methicillin-resistant Staphylococcus aureus (MRSA), with one compound (PNP-02) demonstrating activity comparable to vancomycin in minimum inhibitory concentration, minimum bactericidal concentration (MBC), and time-kill assays. Microscopic and biochemical analyses revealed that PNP-02 adversely affects the cell wall and cell membrane of MRSA. Mechanistic investigations, including proteomic sequencing analyses, Western blotting, and RT-qPCR assays, indicated that PNP-02 compromises cell membrane integrity by inhibiting arginine biosynthesis and pyrimidine metabolism pathways, thereby increasing membrane permeability and inducing bacterial death. In an in vivo mouse model of skin wound healing, PNP-02 exhibited antibacterial efficacy similar to vancomycin. The compound demonstrated low toxicity to cultured human cells and in hemolysis assays and remained stable during serum incubation. These findings suggest that PNP-02 possesses promising bioactivity against MRSA and represents a potential novel antibacterial agent.
Methicillin-Resistant Staphylococcus aureus/genetics* ; Anti-Bacterial Agents/chemistry* ; Naphthoquinones/administration & dosage* ; Animals ; Microbial Sensitivity Tests ; Mice ; Humans ; Staphylococcal Infections/microbiology* ; Molecular Structure

Objective To investigate the role of circular RNAs(circRNA)in Alzheimer's disease(AD)and its potential mechanism.Methods Six-month-old APP/PS1 mouse model of AD and wild type(WT)mice were subjected and then randomly divided into WT group,WT+circ-Olfm1 knockout group,AD group(transgenic APP/PS1 mice),AD+circ-Olfm1 knockout group,AD+FOXO3a knockout group,with 3 mice in each group.① The total RNA of mouse brain was extracted,and the differential expression of circRNAs and mRNAs between the AD mice and WT mice was detected,and the obtained circRNAs and mRNAs were analyzed with gene ontology(GO)analysis.② RT-qPCR was used to detect the expression of the top 10 up-regulated and down-regulated circRNAs,as well as the expression of circ-Olfm1 and miR-330-5p.③ Lentiviral vectors were prepared and stereotaxically injected into the cortex or hippocampus of WT and AD mice to knock out circ-Olfm1 gene.Water maze test was used to evaluate the effect of circ-Olfm1 knockout on cognitive function,and immunofluorescence assay was employed to observe the deposition of amyloid β(Aβ)plaque in the brain.④ The interaction between circ-Olfm1 and miR-330-5p was verified by double luciferase reporter gene analysis.⑤ The protein levels of AMPK and FOXO3a were detected by Western blotting.⑥ Transmission electron microscopy was utilized to observe the mitochondria of the hippocampus.⑦ The levels of inflammatory factors IL-6,IL-1β and TNF-α were detected by ELISA.Results There were totally 52 differentially expressed circRNAs identified between the AD and WT mice,including 28 up-regulated and 24 down-regulated(fold change>1.5,P<0.05).These differentially expressed genes are mainly involved in signal transduction,learning and memory and other functions.circ-Olfm1 was identified as the most significantly differentially expressed circRNA,which is highly expressed in the neurons and up-regulated in the cerebral cortex and hippocampus of the AD mice.Knockout of circ-Olfm1 reduced the number of Aβ plaques in the cerebral cortex and hippocampus of AD mice(P<0.01).In starBase database,there are complementary sequences observed between circ-Olfm1 and miR-330-5p.Western blotting showed that the addition of Aβ42 significantly increased the expression of AMPK and FOXO3a in the neuronal cells(P<0.01).And silencing circ-Olfm1 led to decreased expression of AMPK and FOXO3a in neuronal cells+Aβ42(P<0.01).ELISA revealed that knockout of FOXO3a significantly increased the levels of inflammatory factors IL-6,IL-1β,and TNF-α(P<0.01).Transmission electron microscopy displayed that knocking FOXO3a out significantly aggravated mitochondrial damage(P<0.01).Conclusion circ-Olfm1 is up-regulated in the brain tissue and neurons+Aβ42 of AD rats,and the mechanism of cognitive impairment in AD rats may be through its regulating FOXO3a protein.

Objective To investigate the effect of pramlintide,a pancreatic amyloid peptide analog,on learning and memory of Alzheimer's disease(AD)mice through antioxidant stress,and to determine the expression of phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Methods The APP/PS1 mice were divided into a pramlintide treatment group(intraperitoneal injection of 0.5 μmol/L per day for 10 weeks)and an AD group(same dose of PBS),with 5 mice in each group.The learning and memory abilities were detected with water maze test,the pathological changes of the hippocampus were observed with HE staining and immunohistochemistry,the morphological characteristics of dendritic spines in hippocampus were observed after Golgi staining,and the ultrastructure of hippocampal neurons was observed through transmission electron microscopy(TEM).The content of malondialdehyde(MDA)and the level of superoxide dismutase(SOD)in the hippocampal tissue were detected by biochemical assay,and the levels of inflammatory factors IL-6,TNF-α and IL-1β were determined with ELISA.Western blotting was applied to measure the expression of PI3K/Akt signaling pathway related proteins in the hippocampus.In the cell experiment,SH-SY5Y cells were added with Aβ 1-42 to establish a cell model of AD.After the cells were treated with pramlintide,the levels of oxidative stress and inflammatory response were detected,and cell apoptosis was detected by immunofluorescence.Results The animal experiments showed that pramlintide treatment resulted in significantly shortened escape latency(P<0.01),increased platform crossings(P<0.01),and prolonged time to exploring hidden platform(P<0.01).In the hippocampal tissue of the pramlintide treatment group,HE staining displayed hippocampal neurons in high density and neat arrangement(P<0.05),immunohistochemical results showed significantly reduced Aβ protein(P<0.01),Golgi staining results demonstrated more dendritic spines(P<0.05),TEM revealed almost intact neuronal mitochondrial structure,with reduced vacuolization and clear and identifiable morphology.When compared with the AD group,the levels of oxidative stress and inflammatory response were decreased(P<0.01),and the relative expression of p-PI3K/PI3K and p-Akt/Akt proteins was increased(P<0.01)in the treatment group.In cell experiments,the levels of oxidative stress and inflammatory response were decreased in AD cell model after pramlintide treatment(P<0.01),and the results of immunofluorescence showed that cell apoptosis was declined(P<0.01).Conclusion Pramlintide can improve the cognitive function,reduce the hippocampal deposition of Aβ,reduce oxidative stress and inflammatory response,alleviate the pathological changes of neuronal ultrastructure,and enhance the expression of PI3K/Akt signaling pathway in AD mice.

Objective To investigate the expression level of circular RNA circ-Tns3 in Alzheimer's disease(AD)mice and its role in Aβ-induced neuronal damage.Methods Five APP/PS1 transgenic AD mice and 5 wild-type(WT)mice,weighting of 23～26 g and aged 6 months were subjected in the study.Morris water maze test was used to assess learning and memory abilities,and immunohistochemical staining was performed to observe the number of Aβ plaques in the hippocampal tissue.Subsequently,total RNA was extracted from the brains to detect the differential expression of circRNAs between AD and WT mice,and the results were further analyzed with Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.The top 6 differentially expressed circRNAs were validated by real-time quantitative PCR(RT-qPCR).In in vitro experiments,Aβ1-42 was used to treat neuronal cells to establish AD cell model,and si-circ-Tns3 was transfected into Aβ1-42-treated neuronal cells to knock down circ-Tns3.RT-qPCR was used to determine the expression levels of circ-Tns3 and miR-671-5p.Cell viability and apoptotic rate were detected by CCK-8 assay and TUNEL staining,respectively.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were measured using corresponding kits,and the levels of inflammatory factors IL-6,IL-1β,and TNF-α were detected with ELISA.The interaction between circ-Tns3 and miR-671-5p was verified by dual-luciferase reporter assay and RNA-binding protein immunoprecipitation(RIP)assay.The expression level of Sirt1 protein was detected by Western blotting.Results The 6-month-old AD mice exhibited significant cognitive impairment and Aβ deposition(P<0.01).There were 269 differentially expressed circRNAs identified between AD and WT mice,of which 159 were up-regulated and 110 down-regulated.GO and KEGG enrichment analyses showed that these differentially expressed circRNAs were mainly involved in synaptic transmission,memory,and cholinergic synapse signaling pathways.The expression of circ-Tns3 was significantly increased not only in the brain tissue of AD mice but also in neuronal cells after Aβ1-42 treatment.In cellular experiments,knockdown of circ-Tns3 significantly reduced cell viability and number of apoptotic cells in Aβ1-42-treated neuronal cells,decreased MDA content,increased SOD activity,and reduced the levels of IL-6,IL-1β,and TNF-α(P<0.01).The starBase database predicted that circ-Tns3 and miR-671-5p have complementary sequences,and dual-luciferase reporter and RIP assays confirmed their interaction.The bioinformatics database predicted that miR-671-5p and sirt1 have complementary sequences.Western blotting indicated that in neuronal cells treated with Aβ1-42,the expression of sirt1 was increased after knockdown of circ-Tns3(P<0.01).In Aβ1-42-treated neuronal cells,after knockdown of circ-Tns3,addition of miR-671-5p inhibitor significantly decreased the expression level of sirt1 protein(P<0.01).Conclusions circ-Tns3 is highly expressed in AD mice and cell model of AD.Knocking circ-Tns3 down improves neuronal damage.circ-Tns3 may be involved in the neuronal damage through regulating sirt 1 protein by binding to miR-671-5p.

Objective To investigate the effect of asiaticoside on blood pressure and relaxation of thoracic aorta in rats and explore the underlying mechanism.Methods SD rats treated with 50 and 100 mg/kg asiaticoside by daily gavage for 2 weeks were monitored for systolic blood pressure changes,and histological changes of the thoracic aorta were evaluated using HE staining.In isolated rat endothelium-intact and endothelium-denuded thoracic aorta rings,the effects of asiaticoside on relaxation of the aortic rings were tested at baseline and following norepinephrine(NE)-and KCl-induced constriction.The vascular relaxation effect of asiaticoside was further observed in NE-stimulated endothelium-intact rat aortic rings pretreated with L-nitroarginine methyl ester,indomethacin,zinc protoporphyrin Ⅸ,tetraethyl ammonium chloride,glibenclamide,barium chloride,Iberiotoxin,4-aminopyridine,or TASK-1-IN-1.The aortic rings were treated with KCl and NE followed by increasing concentrations of CaCl2 to investigate the effect of asiaticoside on vasoconstriction induced by external calcium influx and internal calcium release.Results Asiaticoside at 50 and 100 mg/kg significantly lowered systolic blood pressure in rats without affecting the thoracic aorta histomorphology.While not obviously affecting resting aortic rings with intact endothelium,asiaticoside at 100 mg/kg induced significant relaxation of the rings constricted by KCl and NE,but its effects differed between endothelium-intact and endothelium-denuded rings.In endothelium-intact aortic rings pretreated with indomethacin,ZnPP Ⅸ,barium chloride,glyburide,TASK-1-IN-1 and 4-aminopyridine,asiaticoside did not produce significant effect on NE-induced vasoconstriction,and tetraethylammonium,Iberiotoxin and L-nitroarginine methyl ester all inhibited the relaxation effect of asiaticoside.In KCl-and NE-treated rings,asiaticoside obviously inhibited CaCl2-induced vascular contraction.Conclusion Asiaticoside induces thoracic aorta relaxation by mediating high-conductance calcium-activated potassium channel opening,promoting nitric oxide release from endothelial cells and regulating Ca2+ influx and outflow,thereby reducing systolic blood pressure in rats.

Objective To investigate whether activation of mitochondrial acetal dehydrogenase 2(ALDH2)alleviates hypoxic pulmonary hypertension by regulating the SIRT1/PGC-1α signaling pathway.Methods Thirty 8-week-old C57 BL/6 mice were randomized into control,hypoxia,and hypoxia+Alda-1(an ALDH2 activator)group(n=10),and the mice in the latter two groups,along with 10 ALDH2 knockout(ALDH2-/-)mice,were exposed to hypoxia(10%O2,90%N2)with or without daily intraperitoneal injection of Alda-1 for 4 weeks.The changes in right ventricular function and pressure(RVSP)of the mice were evaluated by echocardiography and right ventricular catheter test,and pulmonary artery pressure was estimated based on RVSP.Pulmonary vascular remodeling,right ventricular injury,myocardial α-SMA expression,distal pulmonary arteriole muscle normalization,right ventricular cross-sectional area,myocardial cell hypertrophy,and right cardiac hypertrophy index were assessed with HE staining,immunofluorescence staining and WGA staining,and the expressions of ALDH2,SIRT1,PGC-1α,P16INK4A and P21CIP1 were detected.In pulmonary artery smooth muscle cells with hypoxic exposure,the effect of Alda-1 and EX527 on cell senescence and protein expressions was evaluated using β-galactose staining and Western blotting.Results The wild-type mice with hypoxic exposure showed significantly increased RVSP,right ventricular free wall thickness and myocardial expressions of P16INK4A and P21CIP1,which were effectively lowered by treatment with Alda-1 but further increased in ALDH2-/-mice.In cultured pulmonary artery smooth muscle cells,hypoxic exposure significantly increased senescent cell percentage and cellular expressions of P16INK4A and P21CIP1,which were all lowered by treatment with Alda-1,but its effect was obviously attenuated by EX527 treatment.Conclusion ALDH2 alleviates hypoxia-induced senescence of pulmonary artery smooth muscle cells by upregulating the SIRT1/PGC-1α signaling pathway to alleviate pulmonary hypertension in mice.

Objective To investigate the effect of melanoma associated antigen D1 (Mage-D1)on mouse femoral bone mass and mineralization ability of mouse bone marrow mesenchymal cells (BMSCs)and its potential molecular mechanism.Methods Female Mage-D1 gene knockout heterozygous mice and male wild-type (WT)mice were subjected as parent mice to breed Mage-D1 gene knockout homozygous (Mage-D1 KO)mice.PCR and agarose gel electrophoresis were used to identify male Mage-D1 knockout (Mage-D1 KO)mice and littermate male wild-type (WT)mice.Micro-CT scanning was performed to observe mouse femoral bone mass,and ELISA and chemical assay were employed to detect serum levels of calcium,phosphorus,calcitonin,and parathyroid hormone in mice.After primary cultured BMSCs were identified with flow cytometry,immunofluorescence staining was utilized to detect the expression of Mage-D1 in BMSCs.BMSCs were infected by Mage-D1 silencing lentivirus,and then the cells were divided into negative control group (sh-NC)and silencing group (sh-Mage-D1).Cell scratch assay was conducted to detect the migration ability of BMSCs,and flow cytometry and CCK-8 assay were conducted to detect the cycle change and proliferation ability of BMSCs.After mineralization induction,alkaline phosphatase (ALP) staining and alizarin red staining were performed;RT-qPCR and Western blotting were used to measure the expression levels of ALP,Runx2 and Col1.RT-qPCR was used to detect mineralization-related genes p75NTR and Msx1.Results Compared with the WT mice,the femoral cortical bone thickness,cortical bone mineral content,cancellous bone mineral content,trabecular number,and cancellous bone surface density were decreased,and trabecular separation was increased in the Mage-D1 knockout homozygous mice (P<0.05).There were no significant changes in the serum levels of calcium,phosphorus,calcitonin and parathyroid hormone in mice after Mage-D1 knockout.Mage-D1 was expressed in the whole BMSCs and was highly expressed in the nucleus and perinuclear regions.Compared with the sh-NC BMSCs,the sh-Mage-D1 group had decreased proliferation ability (P<0.01),enhanced migration ability (P<0.01),and decreased expression of ALP,Runx2 and Col1 genes (P<0.05)and protein (P<0.01)after mineralization induction,milder ALP and alizarin red stain,and lower expression levels of p75NTR and Msx1.Conclusion Mage-D1 knockout can significantly reduce femur bone mass in mice.It can promote the proliferation and inhibit migration of BMSCs,and positively regulate their mineralization in vitro,and the p75NTR-Dlx1/Msx1 signaling axis may be involved in the regulation of bone metabolism by Mage-D1.

Objective To investigate the effects of amylin,also known as islet amyloid polypeptide(IAPP),on learning and memory abilities and the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway in APP/PS1 mice.Methods A total of 20 APP/PS1 mice were randomly divided into Alzheimer's disease(AD)group and IAPP group,with 10 mice in each group.The mice in the latter group were given an intraperitoneal injection of 0.5 μmol/L IAPP,and those of the former group received same dose of PBS.Both interventions were given once per day,for 10 weeks.Morris water maze test was used to measure the learning and memory abilities;HE staining was employed to observe the pathological changes in the hippocampus;Transmission electron microscopy was utilized to observe the ultrastructure of hippocampal neurons;Biochemical assay were conducted to detect the contents of glutathione peroxidase(GSH-Px),malondialdehyde(MDA)and superoxide dismutase(SOD)in hippocampal tissues;ELISA was applied to measure the levels of inflammatory factors such as IL-1β,IL-6,and TNF-α as well as content of Aβ42 in hippocampal tissues;And Western blotting was conducted to detect the expression of PI3K/Akt proteins.Results Compared with the AD group,significantly shorter platform latency(P＜0.01),increased number of traversing the platform and longer time to explore the hidden platform(P＜0.01)were observed in the IAPP group,but no such difference was seen in the swimming speed of the mice.HE staining displayed that the IAPP group had more and well-arranged nerve cells in the hippocampal tissue when compared with the AD group(P＜0.05).Lower Aβ protein expression(P＜0.01),reduced oxidative stress and decreased contents of inflammatory factors(P＜0.01)in hippocampal tissue were observed in the IAPP group than the AD group.The IAPP group showed clearer structure of neuronal mitochondria,reduced vacuolization,and better arranged microtubules and microfilaments,and elevated expression of p-PI3K/PI3K and p-Akt/Akt proteins when compared with the AD group(P＜0.01).Conclusion Amylin can reduce oxidative stress and inflammatory responses,improve learning and memory abilities in AD mice,and promote the activity of PI3K/Akt signaling pathway.