We pharmacologically analyzed clonidine-induced tension changes in endothelium-denuded guinea pig thoracic aorta (GP-TA). Clonidine alone produced little change in basal tension; however, in the presence of tetraethylamm...We pharmacologically analyzed clonidine-induced tension changes in endothelium-denuded guinea pig thoracic aorta (GP-TA). Clonidine alone produced little change in basal tension; however, in the presence of tetraethylammonium (TEA) or Ba, it elicited a pronounced contraction that was almost completely abolished by verapamil. In contrast, phenylephrine alone evoked a robust contraction, which was also further enhanced in the presence of Ba; this Ba-potentiated component was nearly abolished by verapamil. The Ba-enhanced clonidine-evoked contraction was insensitive to the α-adrenoceptor (AR) antagonist idazoxan but was competitively inhibited by the α-AR antagonists prazosin and tamsulosin, yielding pA values of 8.26 and 9.92, respectively. Clonidine competitively inhibited phenylephrine-induced contraction, with a pA value of 5.67 in the presence of verapamil. Moreover, clonidine suppressed the sustained component of the phenylephrine response; this inhibition was attenuated by Ba but remained unchanged in the presence of verapamil. These findings indicate that, in GP-TA, clonidine acts as a biased and partial agonist primarily at α-ARs, promoting Ca influx through L-type voltage-dependent Ca channels (VDCCs). These L-type VDCC-mediated responses are negatively regulated by Ba-sensitive K channels. In addition, clonidine itself appears to directly or indirectly activate Ba-sensitive K channels, thereby suppressing the α-AR-L-type VDCC functional coupling.
The orexinergic system is dysregulated in patients with Alzheimer's disease (AD). In the present study, we evaluated the effects of chronic administration of dual orexin receptor antagonists, suvorexant (Suv) and lembore...The orexinergic system is dysregulated in patients with Alzheimer's disease (AD). In the present study, we evaluated the effects of chronic administration of dual orexin receptor antagonists, suvorexant (Suv) and lemborexant (Lem), on long-term potentiation (LTP) in the hippocampal CA1 region of wild-type (WT) and APP knock-in (APP-KI) mice. LTP was enhanced in APP-KI mice compared with WT mice. Chronic administration of Suv and Lem further potentiated LTP in WT mice. In contrast, in APP-KI mice, Suv moderately and Lem markedly reduced LTP. These findings suggest that orexin receptor antagonists bidirectionally modulate LTP in WT and AD model mice.
Microglia exhibit phenotypic plasticity between anti-inflammatory M2 and pro-inflammatory M1 states, and the transition from M2 to M1 is implicated in the progression of acute brain injuries. However, the molecular mecha...Microglia exhibit phenotypic plasticity between anti-inflammatory M2 and pro-inflammatory M1 states, and the transition from M2 to M1 is implicated in the progression of acute brain injuries. However, the molecular mechanisms that regulate this phenotypic shift remain poorly understood. Zn, stored in presynaptic vesicles, is extracellularly released during pathological events, such as cerebral ischemia, and modulates microglial function. In this study, we aimed to investigate the role of extracellular Zn in the M2-to-M1 transition using BV2 microglial cells. Pretreatment with ZnCl during M2 polarization significantly suppressed lipopolysaccharide-induced production of interleukin (IL)-6 and tumor necrosis factor-α following the phenotypic shift. Among the zinc transporters, Zrt- and Irt-related protein 12 (ZIP12) expression was markedly upregulated by IL-4 stimulation, and siRNA-mediated knockdown of ZIP12 abolished the Zn-mediated suppression of pro-inflammatory cytokine production. Furthermore, ZIP12 knockdown reduced intracellular Zn accumulation in IL-4-treated microglia, as revealed by FluoZin-3 fluorescence. These findings indicate that extracellular Zn is taken up via ZIP12 during M2 polarization and subsequently acts to suppress pro-inflammatory cytokine production, thereby restraining the shift toward an M1 phenotype.
Social behaviors rely on the integration of multiple sensory cues. Among these, ultrasonic vocalizations (USVs) serve as primary auditory cues in rodents. Although the function of USVs has been extensively studied in pup...Social behaviors rely on the integration of multiple sensory cues. Among these, ultrasonic vocalizations (USVs) serve as primary auditory cues in rodents. Although the function of USVs has been extensively studied in pup-female and male-female interactions, their role in male-male interactions remain to be elucidated. We tested whether adult male mice recognize male-emitted USVs as social cues using a two-choice playback paradigm. We demonstrated that male mice preferred male-emitted USVs and that oxytocin signaling is essential for perceiving male-emitted USVs. This study provides new insights into the neural mechanisms of male-male social behavior.
AIMS: Blocking peripheral nerve growth factor (NGF) obviously alleviated neuropathic pain, however, the effect and mechanism of spinal NGF in neuropathic pain are remain controversial. This study further investigated the...AIMS: Blocking peripheral nerve growth factor (NGF) obviously alleviated neuropathic pain, however, the effect and mechanism of spinal NGF in neuropathic pain are remain controversial. This study further investigated the mechanism by which peripheral NGF is involved in neuropathic pain and found safe, natural compounds that target NGF to attenuate neuropathic pain. METHODS: Chronic constriction injury (CCI) of the sciatic nerve was used to instill neuropathic pain. Pain behaviors were assessed using Von Frey filaments and Hargreaves test. For in vitro studies, primary neurons and astrocytes were cultured. RT-PCR, immunofluorescence and Western blot were used to assess the cell signaling pathway. RESULTS: Blocking spinal NGF effectively reduced CCI-induced neuropathic pain and suppressed astrocyte activation both in vivo and in vitro. The trends of changes in spinal NGF and the astrocyte marker matched the trends of changes in pain thresholds in CCI. Moreover, NGF-induced hypersensitivity could be abolished by astrocyte inhibitor. NGF was found mainly expressed in spinal neurons, and that the NGF receptor tropomyosin receptor kinase A (TrkA) but not p75, was widely expressed in spinal astrocytes. Dopamine D1/D2 receptor complex could promote NGF expression in spinal neurons, which bind to TrkA to promote astrocytes activation via ASK1-JNK/NF-κB signaling. CONCLUSIONS: These findings imply that D1/D2 receptor complex promotes NGF secretion in spinal neurons, which bind to TrkA to promote astrocyte activation and neuropathic pain via ASK1/JNK/NF-κB pathway.
Ischemic stroke is a prominent cause of disability and mortality worldwide, currently no drug therapy is helpful for post-stroke symptoms; thus, there is a need to develop effective treatment strategies. Peptide medicati...Ischemic stroke is a prominent cause of disability and mortality worldwide, currently no drug therapy is helpful for post-stroke symptoms; thus, there is a need to develop effective treatment strategies. Peptide medication development has advanced significantly in the recent years and due to its potential to modulate key molecular pathways involved in stroke pathophysiology. This review provides an overview of recent advances in peptide therapy for stroke. These peptides can exert neuroprotective effects by inhibiting excitotoxicity, oxidative stress, and apoptosis, while also promoting neuronal survival and synaptic plasticity. Furthermore, artificial intelligence (AI) with deep learning holds a promising technique in peptide generation by enabling the design of novel peptides with specific binding site of a protein, this may accelerate drug discovery processes through predictive modeling and high-throughput analysis. Overall, peptide therapy holds great potential for improving stroke outcomes and represents a promising avenue for the development of novel stroke treatments.
OBJECTIVE: A rapid and quantitative analytical method for the detection of isepamicin (ISP) in human plasma was developed utilizing ultra-high performance liquid chromatography coupled with tandem triple quadrupole mass...OBJECTIVE: A rapid and quantitative analytical method for the detection of isepamicin (ISP) in human plasma was developed utilizing ultra-high performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry (UHPLC-MS/MS). This method facilitates the monitoring of ISP plasma concentrations to examine distribution trends and the incidence of adverse reactions post-administration. Furthermore, the safety profile of ISP in clinical applications was evaluated from a therapeutic perspective by correlating ISP levels with patients' clinical recovery, thereby providing a reference framework for assessing ISP's safety. METHODS: The UHPLC-MS/MS analysis was conducted using a Waters XBridge BEH Amide column with a specifically formulated mobile phase. The gradient elution procedure, flow rate, and column temperature were optimized to achieve efficient sample separation and detection within a reduced time frame. Kanamycin was employed as an internal standard to ensure accurate quantification of ISP in plasma. Prior to analysis, plasma samples underwent a straightforward protein precipitation processing using acetonitrile. The method was validated in compliance with pertinent regulations to ensure its reliability. Specifically, the established method was validated in accordance with the relevant regulations outlined in the 2020 edition of the Chinese Pharmacopoeia and FDA bioassay methods. Patient samples receiving ISP were collected and subjected to comprehensive analysis. RESULTS: The UHPLC-MS/MS method exhibited exceptional performance in quantifying ISP, demonstrating acceptable level of precision and accuracy. The relative standard deviation (RSD%) for inter-day precision ranged from 4.58 % to 6.40 %, while the RSD% for intra-day precision ranged from 1.19 % to 5.84 %, the relative error (RE%) values for both precision measures were within the range of 0.64 %-10.45 %. Additionally, the matrix effect observed for ISP was approximately 114 %. The RSD% for stability was less than 10.28 %. The experimental results conformed to the acceptance criteria for bioanalytical methods as recommended by the Chinese Pharmacopoeia and the FDA. CONCLUSIONS: The UHPLC-MS/MS method is a robust tool for the determination of ISP, and the validated method has been successfully applied to monitor plasma drug concentrations in patients using ISP.
Impaired neutrophil function is thought to increase infection risk associated with smoking. We examined the effects of nicotine- and tar-free cigarette smoke extracts (CSEs) from combustible cigarettes (CCs) and heated t...Impaired neutrophil function is thought to increase infection risk associated with smoking. We examined the effects of nicotine- and tar-free cigarette smoke extracts (CSEs) from combustible cigarettes (CCs) and heated tobacco products (HTPs) on neutrophil-like HL-60 cells. HTP-derived CSEs exhibited lower cytotoxicity and milder impairment of neutrophil functions, including chemotaxis, reactive oxygen species production, phagocytosis, and neutrophil extracellular trap formation, than those of CC-derived CSEs. However, at higher concentrations, HTP-derived CSEs markedly impaired the cell viability and function. These results indicate that HTP emissions impair neutrophil functions at high concentrations, highlighting the need for cautious health risk evaluation.
Intracerebral hemorrhage is the second most common subtype of stroke, characterized by high mortality and disability rates. To date, the mechanism of brain injury caused by intracerebral hemorrhage remains unclear, and t...Intracerebral hemorrhage is the second most common subtype of stroke, characterized by high mortality and disability rates. To date, the mechanism of brain injury caused by intracerebral hemorrhage remains unclear, and there are no effective treatments to delay the progression of brain injury after intracerebral hemorrhage. Increasing evidence suggests that oxidative stress plays a crucial role in secondary injury induced by intracerebral hemorrhage, and ferroptosis plays a dominant role in the pathogenesis of brain injury after intracerebral hemorrhage. In this study, we demonstrated in an in vitro hemin-induced PC12 cell model that astaxanthin improved cell viability, inhibited oxidative stress after intracerebral hemorrhage, and suppressed ferroptosis by upregulating the expression of glutathione peroxidase 4 and solute carrier family 7a member 11. In an in vivo autologous blood injection intracerebral hemorrhage rat model, we confirmed that astaxanthin could resist oxidative stress, ferroptosis, and further inflammatory responses by upregulating the expression of glutathione peroxidase 4 and solute carrier family 7a member 11 through the Akt1-FoxO3a pathway to protect against brain injury after intracerebral hemorrhage.
Imeglimin, a novel oral antidiabetic drug, has been suggested to affect mitochondrial functions in some types of cells and tissues, however, it has never been investigated whether aging has an impact on its pharmacologic...Imeglimin, a novel oral antidiabetic drug, has been suggested to affect mitochondrial functions in some types of cells and tissues, however, it has never been investigated whether aging has an impact on its pharmacological effects in the brain. In this study, we investigated whether imeglimin directly affects functions of brain mitochondria and whether its intraperitoneal injection protects against ischemic brain injury in young, middle-aged and aged Wistar rats. We found that direct addition of imeglimin to mitochondria isolated from young and middle-aged rat brains suppressed oxidative phosphorylation and activities of mitochondrial Complexes I and IV. The opposite, stimulating effect on Complex II activity was observed within the same groups. Injection of imeglimin 24 h before simulated brain ischemia in vitro reduced infarct size only in young and middle-aged rat groups. In the aged rat group, imeglimin did not reduce cerebral infarct size nor directly modulate mitochondrial respiration and activities of the complexes. In conclusion, we provided novel evidence on potential effects of imeglimin in the brain by demonstrating a direct stimulating effect on mitochondrial Complex II activity and age-dependent protective effects against brain injury under in vitro simulated ischemia.
Cisplatin is a commonly used chemotherapy drug that can effectively treat a variety of cancers, but it often causes severe side effects, including nephrotoxicity, ototoxicity, and gastrointestinal toxicity, which signifi...Cisplatin is a commonly used chemotherapy drug that can effectively treat a variety of cancers, but it often causes severe side effects, including nephrotoxicity, ototoxicity, and gastrointestinal toxicity, which significantly affects patients' quality of life. Lutein is a natural carotenoid known for its potent antioxidant properties. Recent literature supports the beneficial effects of lutein supplements in conditions such as retinal degeneration, cardiovascular disease, and liver damage, emphasizing its broad anti-inflammatory capabilities. However, the mechanism by which cisplatin causes intestinal inflammation and the protective effect of lutein against this remain unknown. Here, we investigated the potential protective effect of lutein against cisplatin-induced intestinal epithelial injury. Our results proved that cisplatin significantly decreased cell viability, enhanced ROS generation, and activated inflammatory signaling pathways involving p38, ERK, and NF-κB in IEC-6 cells. Pretreatment with lutein markedly suppressed ROS production, reduced p38 and ERK phosphorylation, prevented NF-κB activation, and consequently attenuated inflammatory cytokine expression. These findings establish lutein as a promising dietary strategy to reduce cisplatin-induced intestinal inflammation, supporting its therapeutic potential for improving chemotherapy tolerance.
Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by mutations in the dystrophin gene. Although the C57BL/10 background mdx mouse (B10-mdx) model is widely used for DMD research, it presents...Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by mutations in the dystrophin gene. Although the C57BL/10 background mdx mouse (B10-mdx) model is widely used for DMD research, it presents milder symptoms than observed in human patients. In contrast, the DBA/2N-mdx model exhibits more severe pathology, making it a promising model for evaluating disease mechanisms and therapies. In this study, we employed a 24-h behavioral monitoring system to investigate spontaneous locomotor activity and gait characteristics in DBA/2N-mdx mice. We observed significantly reduced movement and shorter active periods during the dark (active) phase at 4 and 8 weeks of age in DBA/2N-mdx mice compared to controls. Subsequent gait analysis revealed shorter walking distances, slower speeds, and reduced body extension during straight walking. These findings suggest that the DBA/2N-mdx mouse model exhibits distinct behavioral abnormalities that parallel DMD symptoms in humans. Our noninvasive, continuous monitoring approach provides an innovative method for assessing motor impairments and may facilitate more accurate preclinical assessments of potential therapies for DMD.
Oxaliplatin-induced peripheral neuropathy (OIPN) is a dose-limiting toxicity with limited countermeasures. Basic research has revealed that omeprazole, a proton pump inhibitor (PPI), exerts preventive effects against OIP...Oxaliplatin-induced peripheral neuropathy (OIPN) is a dose-limiting toxicity with limited countermeasures. Basic research has revealed that omeprazole, a proton pump inhibitor (PPI), exerts preventive effects against OIPN. In this study, we evaluated whether other PPIs exert similar effects via in vitro and in vivo experiments. Notably, esomeprazole, lansoprazole, and rabeprazole, classified as PPIs, prevented oxaliplatin-induced cultured F11 neuronal cell damage, and repeated PPI administration prevented mechanical allodynia in rats. However, vonoprazan, a potassium ion-competitive acid blocker, did not exert such effects. Overall, our results highlight the class effects of PPIs against OIPN.
Inhibitors of histone deacetylases (HDACs) suppress retinal angiogenesis by interrupting the vascular endothelial growth factor (VEGF)-mammalian target of rapamycin complex 1 (mTORC1) pathway in proliferating endothelial...Inhibitors of histone deacetylases (HDACs) suppress retinal angiogenesis by interrupting the vascular endothelial growth factor (VEGF)-mammalian target of rapamycin complex 1 (mTORC1) pathway in proliferating endothelial cells. To investigate the underlying mechanisms, we examined the effects of valproic acid (VPA) and vorinostat on the distribution of VEGF protein and phosphorylated S6 protein, an indicator of mTORC1 activity, in the neonatal mouse retina, an experimental model of retinal angiogenesis. Newborn mice were subcutaneously injected with VPA, vorinostat, or vehicle once daily from postnatal day (P) 0 to P3. Their eyes were collected at P4. Compared to vehicle-treated mice, retinal vascularization was delayed, and the number of proliferating vascular cells was reduced in front of the retinal vasculature in VPA- and vorinostat-treated mice. In P4 mice, a single injection of VPA or vorinostat reduced VEGF expression on the retinal surface at 2 and 6 h after injection. Both drugs reduced mTORC1 activity in proliferating endothelial cells. The proteasome inhibitor, MG132, suppressed the VPA- and vorinostat-induced reduction in VEGF expression on the retinal surface. These results suggest that HDAC inhibitors suppress retinal angiogenesis by preventing endothelial cell proliferation and accelerating VEGF protein degradation in a proteasome-dependent manner.
The NLRP3 inflammasome is primarily expressed and activated in microglial and endothelial cells. Extensive research has been conducted on the activation of NLRP3 inflammasomes by microglial cells leading to pyroptosis. H...The NLRP3 inflammasome is primarily expressed and activated in microglial and endothelial cells. Extensive research has been conducted on the activation of NLRP3 inflammasomes by microglial cells leading to pyroptosis. However, there have been no reports on the activation of NLRP3 inflammasomes in brain vascular endothelial cells in patients with Huntington's disease (HD) or HD animal models, leading to blood-brain barrier (BBB) disruption. We herein found that BBB leakage increased and the expression of tight junction proteins significantly decreased after transfecting the mutant Huntingtin protein (mHtt) Q74 plasmid into the mouse brain microvascular endothelial cell line bEnd.3. mHtt promoted the activation of NLRP3 by brain vascular endothelial cells, and increased the expression of the pyroptosis-related proteins. This resulted in a decrease in the expression of the NeuN in the brain of hHTT130 transgenic mice. Furthermore, by downregulating NLRP3 in Q74-transfected bEnd.3 cells or in hHTT130 mouse brain vascular endothelial cells, BBB disruption and endothelial cell pyroptosis were alleviated, the number of surviving neurons was significantly increased. In conclusion, mHtt can activate the NLRP3 inflammasome in brain microvascular endothelial cells to induce endothelial cell pyroptosis, thereby disrupting the function of the BBB, leading to neuronal damage.
Sodium-glucose co-transporter 2 inhibitors (SGLT2i) and angiotensin receptor blockers (ARBs) each have renoprotective effects in chronic kidney diseases, including Alport syndrome. Here, we investigated the combination o...Sodium-glucose co-transporter 2 inhibitors (SGLT2i) and angiotensin receptor blockers (ARBs) each have renoprotective effects in chronic kidney diseases, including Alport syndrome. Here, we investigated the combination of SGLT2i dapagliflozin and ARBs with different antiproteinuric strength - losartan (weak) and olmesartan (strong) - in Col4a5 G5X Alport mice. Dapagliflozin enhanced the renoprotective effect of losartan but not of olmesartan. Olmesartan alone suppressed the decline in renal function and prolonged survival similarly to losartan plus dapagliflozin. These findings suggest that the add-on effectiveness of dapagliflozin varies depending on the ARB, and that their combination needs careful evaluation for maximum benefit.
Idiopathic pulmonary fibrosis is a progressive, highly lethal disease with limited treatment options. It is characterized by fibroblast-to-myofibroblast transformation, excessive ECM proliferation and collagen deposition...Idiopathic pulmonary fibrosis is a progressive, highly lethal disease with limited treatment options. It is characterized by fibroblast-to-myofibroblast transformation, excessive ECM proliferation and collagen deposition, leading to the destruction of normal lung architecture and function. As a constituent of Rhodiola rosea L., rosiridin is a monomer with significant structural compatibility, conferring strong therapeutic potential. This bioactive compound mitigates oxidative stress-driven pathology and reverses its resultant damage in various diseases. However, its potential protective effects against bleomycin-induced IPF and the underlying mechanisms remain unclear. This study aimed to investigate the role and mechanism of rosiridin in IPF. Rosiridin attenuated TGF-β1-induced oxidative stress and inflammatory responses in lung epithelial cells and suppressed apoptosis associated with pulmonary fibrosis. Hematoxylin and eosin (HE) staining and Masson's trichrome staining showed that rosiridin improved pathological lung changes, reduced oxidative stress, and alleviated pulmonary fibrosis in a dose-dependent manner. Transcriptomic analysis revealed that rosiridin inhibited JAK protein activation, reduced the transformation of fibroblasts into myofibroblasts, and suppressed the secretion of proinflammatory and profibrotic cytokines. These findings suggest that rosiridin mitigates pulmonary fibrosis through modulation of the STAT3/NF-κB/SMAD3 signaling pathways. Rosiridin may represent a promising therapeutic candidate for the treatment of IPF.
Prostate cancer is the second most common cancer in men. Although androgen deprivation therapy is initially effective, resistance inevitably develops. Most patients eventually progress to castration-resistant prostate ca...Prostate cancer is the second most common cancer in men. Although androgen deprivation therapy is initially effective, resistance inevitably develops. Most patients eventually progress to castration-resistant prostate cancer, a stage with limited treatment options and poor prognosis. Rho kinases (ROCK1 and ROCK2) have been implicated in cancer progression, but their therapeutic targeting remains limited. This study examined the pathological roles of ROCK1 and ROCK2 in epithelial-mesenchymal transition (EMT) and proliferation of prostate cancer cells. ROCK1 expression was comparable between human prostate epithelial cells (PrECs) and androgen-independent prostate cancer cells, PC-3 and DU145. In contrast, ROCK2 expression was higher in PC-3 cells than in PrECs and DU145 cells. EMT marker analysis revealed that PC-3 cells exhibited decreased E-cadherin and increased N-cadherin and Snail expression. ROCK2 knockdown reversed this EMT phenotype, reducing cell proliferation, migration, 3D tumor spheroid formation, and spheroid cell viability. Similar inhibitory effects were observed by the ROCK2-selective blocker KD025 (IC = 422 nM). Furthermore, ROCK2 deficiency attenuated the tumor growth of PC-3 cells in a xenograft mouse model. These findings indicate that ROCK2 promotes EMT process and tumor progression in PC-3 cells. Targeting ROCK2 may represent a promising therapeutic strategy for androgen-independent prostate cancer.
Nephrin is crucial for the formation of the glomerular slit diaphragm, which is the final filtration barrier in the kidney. A mutation in the NPHS1 gene that codes for nephrin causes congenital nephrotic syndrome of the...Nephrin is crucial for the formation of the glomerular slit diaphragm, which is the final filtration barrier in the kidney. A mutation in the NPHS1 gene that codes for nephrin causes congenital nephrotic syndrome of the Finnish type (CNF). Most missense mutations render nephrin non-functional due to the defective nephrin trafficking to the cell membrane. Pharmacological approaches that induce the expression of nephrin on the cell membrane are feasible, but therapeutic development is hampered by the lack of a high-throughput screening (HTS) system. Here, we developed a nanoluciferase HiBiT-based HTS platform to quantify the cell membrane expression of a nephrin mutant. This evaluation system reflected the previously reported results of various nephrin mutant localization. Using this system, we screened and identified 10 compounds that promoted the expression of the nephrin E725D mutant on the cell membrane. Moreover, the phosphorylation and N-glycosylation of nephrin, which are modifications that indicate its cell surface localization, correlated with the luminescence values of HiBiT-Nephrin in the compound screening. Consequently, this HiBiT-Nephrin evaluation system could be a new platform for predicting the pathogenicity of variants and searching for therapeutic agents for CNF.