Pharmacokinetic study on the interaction between succinic acid and irbesartan in rats and its potential mechanism

ContextSuccinic acid and irbesartan are commonly used drugs in cardiovascular disease treatment. The interaction might occur during their co-administration, which was still unclear.ObjectiveTo reveal the effect of succinic acid on the metabolism of irbesartan and its potential mechanism.Materials and methodsThe Sprague-Dawley rats (n = 6) were treated with a single dose of 30 mg/kg irbesartan (control) or the co-administration with the pre-treatment of 200 mg/kg succinic acid for 7 d. The effect of succinic acid on the metabolic stability and the activity of CYP2C9 was evaluated in rat liver microsomes.ResultsSuccinic acid increased the AUC (5328.71 ± 959.31 μg/L × h vs. 3340.23 ± 737.75 μg/L × h) and prolonged the half-life of irbesartan (from 12.79 ± 0.73 h to 20.59 ± 6.35 h). The T_max (2.83 ± 0.75 h vs. 3.83 ± 1.10 h) and clearance rate (3.46 ± 1.13 L/h/kg vs. 6.91 ± 1.65 L/h/kg) of irbesartan was reduced by succinic acid. Consistently, succinic acid improved the metabolic stability (half-life from 23.32 ± 3.46 to 27.35 ± 2.15 min, intrinsic clearance rate from 59.43 ± 6.12 to 50.68 ± 5.64 μL/min/mg protein). Succinic acid was also found to inhibit the activity of CYP2C9 with the IC₅₀ value of 13.87 μM.Discussion and conclusionsSuccinic acid increased the system exposure of irbesartan via inhibiting CYP2C9. The experiment design of this study also provides a reference for the further validation of this interaction in humans.     

Salidroside orchestrates metabolic reprogramming by regulating the Hif-1α signalling pathway in acute mountain sickness

ContextRhodiola crenulata (Hook. f. et Thoms.) H. Ohba (Crassulaceae) is used to prevent and treat acute mountain sickness. However, the mechanisms underlying its effects on the central nervous system remain unclear.ObjectiveTo investigate the effect of Rhodiola crenulata on cellular metabolism in the central nervous system.Materials and methodsThe viability and Hif-1α levels of microglia and neurons at 5% O₂ for 1, 3, 5 and 24 h were examined. We performed the binding of salidroside (Sal), rhodiosin, tyrosol and p-hydroxybenzyl alcohol to Hif-1α, Hif-1α, lactate, oxidative phosphorylation and glycolysis assays. Forty male C57BL/6J mice were divided into control and Sal (25, 50 and 100 mg/kg) groups to measure the levels of Hif-1α and lactate.ResultsMicroglia sensed low oxygen levels earlier than neurons, accompanied by elevated expression of Hif-1α protein. Salidroside, rhodiosin, tyrosol, and p-hydroxybenzyl alcohol decreased BV-2 (IC₅₀=1.93 ± 0.34 mM, 959.74 ± 10.24 μM, 7.47 ± 1.03 and 8.42 ± 1.63 mM) and PC-12 (IC₅₀=6.89 ± 0.57 mM, 159.28 ± 8.89 μM, 8.65 ± 1.20 and 8.64 ± 1.42 mM) viability. They (10 μM) reduced Hif-1α degradation in BV-2 (3.7-, 2.5-, 2.9- and 2.5-fold) and PC-12 cells (2.8-, 2.8-, 2.3- and 2.0-fold) under normoxia. Salidroside increased glycolytic capacity but attenuated oxidative phosphorylation. Salidroside (50 and 100 mg/kg) treatment increased the protein expression of Hif-1α and the release of lactate in the brain tissue of mice.ConclusionsThese results suggest that Sal induces metabolic reprogramming by regulating the Hif-1α signalling pathway to activate compensatory responses, which may be the core mechanism underlying the effect of Rhodiola crenulata on the central nervous system.     

Combination of paeoniflorin and calycosin-7-glucoside alleviates ischaemic stroke injury via the PI3K/AKT signalling pathway

ContextPaeoniflorin (PF) and calycosin-7-glucoside (CG, Paeonia lactiflora Pall. extract) have demonstrated protective effects in ischaemic stroke.ObjectiveTo investigate the synergistic effects of PF + CG on ischaemia/reperfusion injury in vivo and in vitro.Materials and methodsMale Sprague-Dawley rats were subjected to the middle cerebral artery occlusion/reperfusion (MCAO/R). After MCAO/R for 24 h, rats were randomly subdivided into 5 groups: sham, model (MCAO/R), study treatment (PF + CG, 40 + 20 mg/kg), LY294002 (20 mg/kg), and study treatment + LY294002. Males were given via intragastric administration; the duration of the in vivo experiment was 8 days. Neurologic deficits, cerebral infarction, brain edoema, and protein levels were assessed in vivo. Hippocampal neurons (HT22) were refreshed with glucose-free DMEM and placed in an anaerobic chamber for 8 h. Subsequently, HT22 cells were reoxygenated in a 37 °C incubator with 5% CO₂ for 6 h. SOD, MDA, ROS, LDH and protein levels were measured in vitro.ResultsPF + CG significantly reduced neurobehavioral outcomes (21%), cerebral infarct volume (44%), brain edoema (1.6%) compared with the MCAO/R group. Moreover, PF + CG increased p-PI3K/PI3K (4.69%, 7.4%), p-AKT/AKT (6.25%, 60.6%) and Bcl-2/BAX (33%, 49%) expression in vivo and in vitro, and reduced GSK-3β (10.5%, 9.6%) expression. In vitro, PF + CG suppressed apoptosis in HT22 cells and decreased ROS and MDA levels (20%, 50%, respectively).ConclusionsPF + CG showed a synergistic protective effect against ischaemic brain injury, potentially being a future treatment for ischaemic stroke.     

Cryptotanshinone possesses therapeutic effects on ischaemic stroke through regulating STAT5 in a rat model

ContextCryptotanshinone (CT), a lipophilic compound extracted from roots of Salvia miltiorrhiza Bunge (Lamiaceae) (Danshen), has multiple properties in diseases, such as pulmonary fibrosis, lung cancer, and osteoarthritis. Our previous findings suggest that CT plays a protective role in cerebral stroke. However, the molecular mechanisms underlying CT protection in ischaemic stroke remain unclear.ObjectiveThis study examines the effect of CT on ischaemic stroke.Materials and methodsWe used the middle cerebral artery occlusion (MCAO) rat (Sprague-Dawley rats, 200 ± 20 g, n = 5) model with a sham operation group was treated as negative control. MCAO rats were treated with 15 mg/kg CT using intragastric administration. Moreover, TGF-β (5 ng/mL) was used to treat MCAO rats as a positive control group.ResultsThe 50% inhibitory concentration (IC₅₀) of CT on CD4⁺ cell damage was 485.1 μg/mL, and median effective concentration (EC₅₀) was 485.1 μg/mL. CT attenuates the infarct region in the MCAO model. The percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells in the peripheral blood of the MCAO group was increased with CT treatment. The protein level of FOXP3 and the phosphorylation of STAT5 were recovered in the CD4⁺CD25⁺ Treg cells of model group after treated with CT. Importantly, the effects of CT treatment were blocked by treatment with the inhibitor STAT5-IN-1 in CD4⁺ T cells of the MCAO model.Discussion and conclusionOur findings not only enhance the understanding of the mechanisms underlying CT treatment, but also indicate its potential value as a promising agent in the treatment of ischaemic stroke. Further study will be valuable to examine the effects of CT on patients with ischaemic stroke.     

Icariin inhibits the expression of IL-1β, IL-6 and TNF-α induced by OGD/R through the IRE1/XBP1s pathway in microglia

ContextIcariin (ICA), a flavonol glycoside extracted from Epimedium brevicornum Maxim (Berberidaceae), has been proven to inhibit inflammatory response in ischaemic rats in our laboratory''s previous work. However, its underlying mechanism is still unclear.ObjectiveThis study investigates the effects of ICA on endoplasmic reticulum (ER) stress mediated inflammation induced by cerebral ischaemia–reperfusion (I/R) injury in vitro.Materials and methodsThe primary cultured microglia were treated with oxygen-glucose deprivation (OGD) for 2 h followed by a 24 h reoxygenation. ICA (0.37, 0.74 and 1.48 μmol/L) administration was performed 1 h prior OGD and acting through 2 h OGD. The control group was cultured in normal conditions. At 24 h after reoxygenation, the expression of IRE1α, XBP1u, XBP1s, NLRP3 and caspase-1 was detected by western blotting (WB) and quantitative real-time (qRT) PCR; the expression of p-IRE1α was examined by WB; the expression of IL-1β, IL-6 and TNF-α was measured by WB and enzyme-linked immunosorbent assay (ELISA).ResultsICA (0.37, 0.74 and 1.48 μmol/L) reduced the ratio of p-IRE1α/IRE1α, the mRNA level of IRE1α, the expression of XBP1u, XBP1s, NLRP3, caspase-1 at both the mRNA and protein level expression of IL-1β, IL-6 and TNF-α in OGD/R injured microglia. Overexpression of IRE1 significantly reversed the effects of ICA.Discussion and conclusionsThese results suggested that ICA might decrease the expression of IL-1β, IL-6 and TNF-α by inhibiting IRE1/XBP1s pathway. The anti-inflammatory effect of ICA may provide a potential therapeutic strategy for the treatment of brain injury after stroke.     

Gambogic amide inhibits angiogenesis by suppressing VEGF/VEGFR2 in endothelial cells in a TrkA-independent manner

ContextGambogic amide (GA-amide) is a non-peptide molecule that has high affinity for tropomyosin receptor kinase A (TrkA) and possesses robust neurotrophic activity, but its effect on angiogenesis is unclear.ObjectiveThe study investigates the antiangiogenic effect of GA-amide on endothelial cells (ECs).Materials and methodsThe viability of endothelial cells (ECs) treated with 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 μM GA-amide for 48 h was detected by MTS assay. Wound healing and angiogenesis assays were performed on cells treated with 0.2 μM GA-amide. Chicken eggs at day 7 post-fertilization were divided into the dimethyl sulfoxide (DMSO), bevacizumab (40 μg), and GA-amide (18.8 and 62.8 ng) groups to assess the antiangiogenic effect for 3 days. mRNA and protein expression in cells treated with 0.1, 0.2, 0.4, 0.8, and 1.2 μM GA-amide for 6 h was detected by qRT-PCR and Western blots, respectively.ResultsGA-amide inhibited HUVEC (IC₅₀ = 0.1269 μM) and NhEC (IC₅₀ = 0.1740 μM) proliferation, induced cell apoptosis, and inhibited the migration and angiogenesis at a relatively safe dose (0.2 μM) in vitro. GA-amide reduced the number of capillaries from 56 ± 14.67 (DMSO) to 20.3 ± 5.12 (62.8 ng) in chick chorioallantoic membrane (CAM) assay. However, inactivation of TrkA couldn’t reverse the antiangiogenic effect of GA-amide. Moreover, GA-amide suppressed the expression of VEGF and VEGFR2, and decreased activation of the AKT/mTOR and PLCγ/Erk1/2 pathways.ConclusionsConsidering the antiangiogenic effect of GA-amide, it might be developed as a useful agent for use in clinical combination therapies.     

Deciphering the molecular mechanism of tetrandrine in inhibiting hepatocellular carcinoma and increasing sorafenib sensitivity by combining network pharmacology and experimental evaluation

ContextThe mechanism of tetrandrine (TET) in hepatocellular carcinoma (HCC) progression and sorafenib (Sora) chemosensitivity deserves investigation.ObjectiveUsing network pharmacology approaches to elucidate the mechanisms of TET in HCC.Materials and methodsCCK-8, colony formation, and flow cytometry assays were used to measure cell phenotypes. BALB/c nude mice were divided into Control, Sora (10 mg/kg), TET (50 mg/kg), and TET + Sora (10 mg/kg Sora plus 50 mg/kg TET) groups to evaluate the antitumor effects of TET for 21 days. Sora and TET were given by intraperitoneal injection or oral gavage.ResultsFor SMMC7721 (IC₅₀ = 22.5 μM) and PLC8024 (IC₅₀ = 18.4 μM), TET (10, 20 μM) reduced colony number (0.68 ± 0.04- and 0.50 ± 0.04-fold, 0.56 ± 0.04- and 0.42 ± 0.02-fold), induced cell cycle arrest at G0/G1 stage (1.22 ± 0.03- and 1.39 ± 0.07-fold, 1.37 ± 0.06- and 1.55 ± 0.05-fold), promoted apoptosis (2.49 ± 0.26- and 3.63 ± 0.33-fold, 2.74 ± 0.42- and 3.73 ± 0.61-fold), and inactivated PI3K/AKT/mTOR signalling. Sora (10 μM) decreased cell proliferation, enhanced apoptosis, and inhibited PI3K/AKT/mTOR signalling, and these effects were further aggravated in the combination group. Activating PI3K/AKT/mTOR reversed the effects of TET on cell proliferation and Sora sensitivity. In the combination group, tumour volumes and weights were decreased to 202.3 ± 17.4 mm³ and 151.5 ± 25.8 mg compared with Sora (510.6 ± 48.2 mm³ and 396.7 ± 33.5 mg).Discussion and conclusionsTET enhances Sora sensitivity by inactivating PI3K/AKT/mTOR, suggesting the potential of TET as a chemosensitizer in HCC.     

Wasp venom from Vespa magnifica acts as a neuroprotective agent to alleviate neuronal damage after stroke in rats

ContextAcute ischaemic stroke (AIS) is a major cause of disability and death, which is a serious threat to human health and life. Wasp venom extracted from Vespa magnifica Smith (Vespidae) could treat major neurological disorders.ObjectiveThis study investigated the effects of wasp venom on AIS in rats.Material and methodsWe used a transient middle cerebral artery occlusion (MCAO) model in Sprague-Dawley rats (260–280 g, n = 8–15) with a sham operation group being treated as negative control. MCAO rats were treated with wasp venom (0.05, 0.2 and 0.6 mg/kg, i.p.) using intraperitoneal injection. After treatment 48 h, behavioural tests, cortical blood flow (CBF), TTC staining, H&E staining, Nissl staining, TUNEL assay, immunohistochemistry (IHC) and ELISA were employed to investigate neuroprotective effects of wasp venom.ResultsCompared with the MCAO group, wasp venom (0.6 mg/kg) improved neurological impairment, accelerated CBF recovery (205.6 ± 52.92 versus 216.7 ± 34.56), reduced infarct volume (337.1 ± 113.2 versus 140.7 ± 98.03) as well as BBB permeability as evidenced by changes in claudin-5 and AQP4. In addition, function recovery of stroke by wasp venom treatment was associated with a decrease in TNF-α, IL-1β, IL-6 and inhibition activated microglia as well as apoptosis. Simultaneously, the wasp venom regulated the angiogenesis factors VEGF and b-FGF in the brain.ConclusionsWasp venom exhibited a potential neuroprotective effect for AIS. In the future, we will focus on determining whether the observed actions were due to a single compound or the interaction of multiple components of the venom.     

Effect of dihydromyricetin on hepatic encephalopathy associated with acute hepatic failure in mice

ContextHepatic encephalopathy (HE) is a complex neuropsychiatric disease caused by liver failure. Dihydromyricetin (DMY) is a traditional medicine used to treat liver injury.ObjectiveTo investigate the effects of dihydromyricetin (DMY) on hepatic encephalopathy associated with acute hepatic failure mice models established by thioacetamide (TAA) exposure.Materials and methodsFemale BALB/c mouse were randomly divided into control, DMY, TAA, and TAA + DMY groups (n = 8). The first two groups were intraperitoneally injected with saline or 5 mg/kg DMY, respectively. The last two groups were injected with 600 mg/kg TAA to establish HE models, and then the mice in the last group were treated with 5 mg/kg DMY. Neurological and cognition functions were evaluated 24 and 48 h after injection. Mice were sacrificed after which livers and brains were obtained for immunoblot and histopathological analysis, while blood was collected for the analysis of liver enzymes.ResultsIn the TAA + DMY group, ALT and AST decreased to 145.31 ± 12.88 U/L and 309.51 ± 25.92 U/L, respectively, whereas ammonia and TBIL decreased to 415.67 ± 41.91 μmol/L and 3.31 ± 0.35 μmol/L, respectively. Moreover, MDA decreased to 10.74 ± 3.97 nmol/g, while SOD and GST increased to 398.69 ± 231.30 U/g and 41.37 ± 21.84 U/g, respectively. The neurological score decreased to 2.87 ± 0.63, and the number of GFAP-positive cells decreased to 41.10 ± 1.66. Furthermore, the protein levels of TNF-α, IL-6, and GABA_A in the cortex decreased.ConclusionsWe speculate that DMY can serve as a novel treatment for HE.     

Taxifolin,a novel food,attenuates acute alcohol-induced liver injury in mice through regulating the NF-κB-mediated inflammation and PI3K/Akt signalling pathways

ContextTaxifolin (TAX) has effective anti-inflammatory, antioxidant and hepatoprotective activities, but its potential mechanism has not been revealed.ObjectiveTo evaluate the potential protective effect of TAX on acute alcohol-induced liver injury in mice.Materials and methodsAlcoholic liver injury model was established by oral alcohol in mice, and randomly distributed in five groups (n = 10): Normal group (oral saline only); Alcohol group (concentration of fermented alcohol: 56%, 6 mL/kg); TAX groups, mice were orally administered with alcohol, and then TAX with doses of 20, 40, 80 mg/kg, respectively. Oral administration was conducted for 6 weeks.ResultsTAX treatment illustrated that the level of alanine aminotransferase (ALT) was reduced to 65.90 ± 2.26 U/L and aspartate aminotransferase (AST) to 33.28 ± 5.62 U/L compared with alcohol group (ALT 124.51 ± 4.40 U/L, AST 61.70 ± 4.09 U/L), while superoxide dismutase (SOD) was increased to 49.81 ± 2.39 U/mg and glutathione (GSH) to 8.16 ± 0.44 μmol/g, but MDA was reversed to 2.53 ± 0.24 nmol/mg. Histopathological examination showed TAX treatment alleviated alcohol-induced hepatocyte necrosis and inflammatory infiltration. Meanwhile, Western blot and rt-PCR indicated TAX reduced IL-6 to 2.49 ± 0.25 pg/mL and TNF-α to 1.79 ± 0.20 pg/mL, and inhibiting NF-κB activation in liver. Moreover, TAX reversed alcohol-induced apoptosis by regulating the expression of PI3K/Akt and its downstream apoptotic factors.ConclusionsThe research provides novel evidence of the hepatoprotective effect of TAX on alcohol-induced liver injury, while also providing the possibility for future treatment of alcoholic liver disease.     

Ginsenoside Rc attenuates myocardial ischaemic injury through antioxidative and anti-inflammatory effects

ContextPanax ginseng C. A. Meyer (Araliaceae) is a famous Asian medicine. Ginsenoside Rc is a component isolated from Panax ginseng.ObjectiveThis study evaluates the effect of ginsenoside Rc on myocardial ischaemic injury.Materials and methodsMale Swiss mice were subcutaneously injected with 50 mg/kg isoproterenol once a day for three days. Ginsenoside Rc (10, 20, or 40 mg/kg) was intragastrically administered 1 h after isoproterenol injection. The mice in the control group were subcutaneously injected with normal saline and intragastrically given 0.5% CMC-Na. CK-MB and troponin T were assayed. Histopathological examination of myocardium was conducted. The expression of Nrf2, GCLC, GCLM and HO-1 in heart tissues was evaluated by Western blot.ResultsIn myocardial ischaemic mice, ginsenoside Rc reduced the levels of CK-MB (197.1 ± 15.7, 189.9 ± 19.0, 184.0 ± 14.4 vs. 221.6 ± 27.9) and troponin T (10.3 ± 1.7, 9.5 ± 1.3, 8.7 ± 1.7 vs. 13.4 ± 2.4). Ginsenoside Rc attenuated the necrosis and inflammatory cells infiltration in myocardium. Furthermore, ginsenoside Rc not only decreased the contents of MDA, TNF-α but also increased GSH level in the heart tissues. The expression of Nrf2, GCLC, GCLM and HO-1 was significantly increased in the animals treated with ginsenoside Rc. ML385, an Nrf2 inhibitor, blocked partially the ginsenoside Rc-mediated cardioprotective effect. Ginsenoside Rc attenuated myocardial ischaemic injury in mice, which may be, in part, through its antioxidative and anti-inflammatory effects.ConclusionsThis study indicated that ginsenoside Rc might be a novel candidate for treatment of myocardial ischaemia.     

Rhodiola rosea polysaccharides promote the proliferation of bone marrow haematopoietic progenitor cells and stromal cells in mice with aplastic anaemia

ContextThe effects of Rhodiola rosea L. (Crassulaceae) polysaccharides (RRPs) on haematopoiesis are poorly understood.ObjectiveTo determine the effects of RRPs on haematopoiesis in mice with aplastic anaemia.Materials and methodsAplastic anaemia was induced in Kunming mice by ⁶⁰Coγ (2.0 Gy) irradiation and cyclophosphamide administration (50 mg/kg/day for 3 consecutive days; intraperitoneal injection). The in vivo effects of RRPs (10, 20, and 40 mg/kg; intraperitoneal injection) on haematopoiesis were analyzed using peripheral blood tests, histopathological examination of haematopoietic tissues, culture of haematopoietic progenitors and bone marrow stromal cells (BMSCs), and Western blotting of Fas and Fas ligand (FasL). The in vitro effects of RRPs on bone-marrow haematopoietic progenitors and BMSCs were also evaluated.ResultsCompared to anaemic controls, high-dose RRPs (40 mg/kg) significantly increased red blood cells (8.21 ± 0.57835 versus 6.13 ± 1.34623 × 10¹²/L), white blood cells (5.11 ± 1.6141 versus l.54 ± 1.1539 × 10⁹/L), and BMSCs (10.33 ± 1.5542 versus 5.87 ± 3.1567 × 10¹²/L) in mice with aplastic anaemia (all p < 0.01). High-dose RRPs significantly increased the formation of colony-forming unit-granulocyte macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E; p < 0.01). Fas and FasL protein expression in BMSCs decreased after RRPs administration. Especially at the high dose, RRPs (150 μg/mL) significantly promoted in vitro CFUs-E, BFUs-E, and CFUs-GM formation. RRPs (150–300 μg/mL) also promoted BMSC proliferation.Discussion and conclusionsRRPs helped to promote haematopoietic recovery in mice with aplastic anaemia, facilitating haematopoietic tissue recovery. This study indicated some mechanisms of the haematopoietic regulatory effects of RRPs. Our findings provide a laboratory basis for clinical research on RRPs.     

Paeonol attenuates heart failure induced by transverse aortic constriction via ERK1/2 signalling

ContextPaeonol (PAE) is the main phytochemical from Cortex Moutan. Its main pharmacological effects are anti-inflammatory and antioxidant, but its cardioprotective effect is unclear.ObjectiveThe study investigates the effects and underlying mechanisms of PAE on transverse aortic constriction (TAC)-induced heart failure (HF) in mice.Materials and methodsC57BL/6 mice were randomly divided into five groups: sham, TAC, PAE10 (TAC + PAE 10 mg/kg), PAE20 (TAC + PAE 20 mg/kg) and PAE 50 (TAC + PAE 50 mg/kg). Paeonol was intragastrically administered to mice for 4 weeks. Mice were anaesthetized with pentobarbital sodium and underwent cardiac echocardiography using echocardiography system. Serum levels of atrial natriuretic peptide (ANP), tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Myocardial apoptosis was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining. Haematoxylin–eosin (H&E) and Masson’s staining were used for histopathological evaluation. Western and quantitative real-time PCR (qRT-PCR) were performed to detect levels of apoptosis and fibrosis-related proteins.ResultsEchocardiography showed PAE improved cardiac function (LVEF: TAC, 52.3±6.8%; PAE20, 65.8±3.6%; PAE50, 71.4±2.5%) and H&E staining showed PAE alleviated myocardial injury (TAC: 1170.3 ± 134.6 μm²; PAE50: 576.0 ± 53.5 μm²). Western and qRT-PCR results showed that PAE down-regulated the levels of ANP, BNP and α-MHC. In addition, TUNEL and western results showed PAE significantly inhibited apoptosis. Masson and western results showed PAE inhibited cardiac hypertrophy. Western results showed the ERK1/2/JNK pathway could be inhibited by PAE.Discussion and conclusionsPaeonol regulates ERK1/2/JNK to improve cardiac function, which provides theoretical support for the extensive clinical treatment of HF.     

Metabolomics coupled with network pharmacology study on the protective effect of Keguan-1 granules in LPS-induced acute lung injury

ContextKeguan-1 (KG-1) plays a vital role in enhancing the curative effects, improving quality of life, and reducing the development of acute lung injury (ALI).ObjectiveTo unravel the protective effect and underlying mechanism of KG-1 against ALI.Materials and methodsC57BL/6J mice were intratracheally instilled with lipopolysaccharide to establish the ALI model. Then, mice in the KG-1 group received a dose of 5.04 g/kg for 12 h. The levels of proinflammatory cytokines, chemokines, and pathological characteristics were determined to explore the effects of KG-1. Next, untargeted metabolomics was used to identify the differential metabolites and involved pathways for KG-1 anti-ALI. Network pharmacology was carried out to predict the putative active components and drug targets of KG-1 anti-ALI.ResultsKG-1 significantly improved the levels of TNF-α (from 2295.92 ± 529.87 pg/mL to 1167.64 ± 318.91 pg/mL), IL-6 (from 4688.80 ± 481.68 pg/mL to 3604.43 ± 382.00 pg/mL), CXCL1 (from 4361.76 ± 505.73 pg/mL to 2981.04 ± 526.18 pg/mL), CXCL2 (from 5034.09 ± 809.28 pg/mL to 2980.30 ± 747.63 pg/mL), and impaired lung histological damage. Untargeted metabolomics revealed that KG-1 significantly regulated 12 different metabolites, which mainly related to lipid, amino acid, and vitamin metabolism. Network pharmacology showed that KG-1 exhibited anti-ALI effects through 17 potentially active components acting on seven putative drug targets to regulate four metabolites.Discussion and conclusionsThis work elucidated the therapeutic effect and underlying mechanism by which KG-1 protects against ALI from the view of the metabolome, thus providing a scientific basis for the usage of KG-1.     

Inhibitory effects of (±)-propranolol on excitation-contraction coupling in isolated soleus muscles of the rat

1. The effect of a β-adrenoceptor antagonist, propranolol, was investigated on excitation-contraction coupling in small, intact bundles of soleus muscle fibres from the rat.
2. (±)-Propranolol significantly inhibited twitch and tetanic tension with IC₅₀ values of 6.7 μM and 3.5 μM, respectively.
3. (+)-Propranolol (which has 100 times less β-blocking activity than the (±) form) was approximately one third as effective as the (±) form at inhibiting isometric tension.
4. (±)-Propranolol (20 μM) had no significant effect on the amplitude of caffeine contractures, suggesting that it did not directly inhibit Ca²⁺ release from the sarcoplasmic reticulum.
5. The resting membrane potential measured after 15 min perfusion with 20 μM (±)-propranolol was not significantly different from control. However, this concentration of (±)-propranolol significantly reduced both the peak amplitude and the maximum rate of rise of the action potential. Both effects were only partially reversible after extensive washing.
6. (±)-Propranolol perfusion caused a modest reduction in the amplitude of sub-maximal K⁺ contractures at concentrations (5  μM) that markedly depressed tetanic tension.
7. The results indicate that (±)-propranolol can decrease isometric tension independently of β-receptor occupation by (i) reducing the amplitude and rate of rise of the action potential and (ii) by directly inhibiting excitation-contraction coupling. The relatively low IC₅₀ for the ‘membrane-stabilizing'' action of propranolol on tetanic tension (3.5 μM), combined with the ability of the drug to accumulate gradually in biological membranes, may contribute to a peripheral component of the tremorolytic and fatigue-inducing actions of propranolol on skeletal muscle.

     

Bradykinin-evoked sensitization of neuropeptide release from afferent neurons in the guinea-pig lung

1. It has been shown that bradykinin (BK) causes sensitization of airway sensory neurons and an enhancement of the cough reflex in guinea-pigs. In the present study, the guineapig isolated perfused lung was used to investigate the possible enhancement by BK of histamine-evoked neuropeptide release from peripheral terminals of primary afferent neurons, and to determine the contribution of cyclooxygenase products of arachidonate metabolism to this effect.
2. The lung was perfused with oxygenated physiological salt solution containing peptidase inhibitors (thiorphan, bestatin and captopril, 1 μM each). BK and histamine were added to the perfusate for 10 and 5 min, respectively.
3. BK alone (0.1 μM) evoked the release of 10.35±2.4 fmol immunoreactive calcitonin gene-related peptide (CGRP), histamine alone (100 μM) evoked the release of 12.7±1.6 fmol CGRP. Stimulation with 100 μM histamine in the presence of 0.1 μM BK (added 5 min before histamine and present during histamine) evoked the release of 67.1±5.3 fmol CGRP.
4. Prostaglandin (PG) release was stimulated by BK (418±71 pmol 15-keto-13,14-dihydro-PGF_2α and 345±59 pmol 6-keto-PGF_1α), and, to a lesser extent, by histamine (36.1±7.4 pmol 15-keto-13,14-dihydro-PGF_2α, and 24.6±3.9 pmol 6-keto-PGF_1α). Prostaglandin release induced by histamine in the presence of BK was not significantly higher than with BK alone.
5. Indomethacin (5 μM) as well as the bradykinin B₂ receptor antagonist HOE140 (icatibant, 1 μM) inhibited prostaglandin release following stimulation with histamine in combination with BK. CGRP release evoked by histamine in combination with BK was attenuated by indomethacin and HOE140 to 22.1±7.8 fmol and 16.4±3.8 fmol, respectively, significantly less than the value obtained in control experiments (67.1±5.3 fmol).
6. The results suggest that BK-induced stimulation of prostaglandin synthesis results in facilitation of histamine–evoked release of pro-inflammatory neuropeptides from afferent neurons, a mechanism that probably becomes relevant during inflammation, and that can be blocked by a bradykinin B₂ receptor antagonist.

     

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine,barium, ouabain and 6-hydroxydopamine in chromaffin cells

1. Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154, did not enhance LDH release. Both lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (0.3–10 μM) decreased the veratridine-induced LDH release.
2. Penfluridol did not increase LDH release at concentrations 0.003–1 μM; 3–10 μM increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μM) did not protect against the cytotoxic effects of veratridine; at 1 μM, 15% protection was produced. Higher concentrations (3–10 μM) enhanced the cytotoxic effects of veratridine.
3. Ba²⁺ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μM lubeluzole and fully counteracted by 1 μM penfluridol. {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba²⁺.
4. Ouabain (10 μM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μM) and the lower concentrations of penfluridol (0.003–0.3 μM) prevented the ouabain cytotoxic effects. At higher concentrations (3 μM), penfluridol increased drastically the ouabain cytotoxic effects.
5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μM) had no cytoprotective effects against 6-OHDA.
6. Lubeluzole (3 μM), {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (3 μM) and penfluridol (1 μM) blocked the current through Na⁺ channels in voltage-clamped chromaffin cells (I_Na) by around 20–30%. Ca²⁺ current through Ca²⁺ channels (I_Ca) was inhibited 57% by lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 were readily reversible.
7. Lubeluzole (3 μM) induced reversible blockade of the oscillations of the cytosolic Ca²⁺, [Ca²⁺]_i, in fura-2-loaded cells exposed to 30 or 100 μM veratridine. Penfluridol (1 μM) inhibited those oscillations in an irreversible manner.
8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na⁺ and Ca²⁺ entry, as well as the [Ca²⁺]_i oscillations. The Ba²⁺ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na⁺/Ca²⁺ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.

     

Isovitexin alleviates acute gouty arthritis in rats by inhibiting inflammation via the TLR4/MyD88/NF-κB pathway

ContextThe prevalence of gout has greatly increased, and it has become the most common inflammatory arthritis in men. Isovitexin possesses anti-inflammatory and antioxidant properties.ObjectiveWe explored the effects of isovitexin on rats with acute gouty arthritis (GA).Materials and methodsFifty-four Sprague-Dawley rats were assigned to five groups: sham, model, positive (colchicine, 0.3 mg/kg), isovitexin (100 mg/kg), TLR4 inhibitor (TAK-242, 3 mg/kg) and isovitexin + TAK-242. The gait of rats and the ankle joint swelling index were monitored. The levels of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, and pathological changes in the synovial tissues were determined.ResultsIsovitexin significantly reduced the ankle joint swelling index at day 7 compared to that in the model group (4.39 ± 1.01 vs. 6.09 ± 1.31). Moreover, isovitexin alleviated the infiltration of inflammatory cells and ameliorated the proliferation of synovial cells. The levels of TNF-α (93.42 ± 5.02 pg/mL), IL-1β (25.46 ± 1.91 pg/mL) and IL-6 (194.71 ± 7.92 pg/mL) in the isovitexin group were significantly lower than in the model group (129.39 ± 5.43, 39.60 ± 2.71 and 223.77 ± 5.35 pg/mL). The expression of TLR4, MyD88 and p-NF-κB-p65 was remarkably decreased after isovitexin and colchicine treatment. The effect of isovitexin was similar to that colchicine. Furthermore, the combination of isovitexin and TAK-242 had better effect, and there was no significantly difference with colchicine treatment.Discussion and conclusionsIsovitexin ameliorates joint inflammation in acute GA via the TLR4/MyD88/NF-κB pathway. Isovitexin may be a potential substitute medicine for GA.     

Aspirin relieves the calcification of aortic smooth muscle cells by enhancing the heat shock response

ContextVascular calcification is a major complication of chronic renal failure, which has been identified as an active process partly driven by osteogenic transition of vascular smooth muscle cells (VSMCs). Aspirin could prevent cardiomyocyte damage by inducing heat shock response.ObjectiveThis study investigates the effect of aspirin on alleviating VSMC calcification.Materials and methodsAn in vitro VSMC calcification model was established by 10-day calcification induction in osteogenic medium. VSMCs were grouped as following: control group (normal medium), calcified group (osteogenic medium) and treated group (osteogenic medium with 1 or 4 mmol/L aspirin). VSMC calcification was evaluated by calcified nodules formation, intracellular calcium concentration and osteoblastic marker (OPN and Runx2) expression.ResultsAfter 10-day culture, the intracellular calcium concentration in calcified group was significantly higher than that in control group (1.16 ± 0.04 vs. 0.14 ± 0.01 μg/mg, p < 0.01), but significantly reduced in 1 mmol/L aspirin treated group (0.74 ± 0.05 μg/mg, p < 0.01), and 4 mmol/L aspirin treated group (0.93 ± 0.03 μg/mg, p < 0.01). The elevated expression of OPN and Runx2 induced by osteogenic medium was significantly relieved after 1 or 4 mmol/L aspirin treatment. The expression of HSF1, HSP70 and HSP90 was decreased in calcification-induced VSMCs, but significantly increased after treatment of aspirin. Furthermore, inhibition of HSP70 (or HSP90) by small-molecule inhibitor or small interfering RNA could partially abolish the anti-calcification effect of aspirin, proved by the changes of intracellular calcium concentration and osteoblastic marker expression.Discussion and conclusionsAspirin could relieve the calcification of VSMCs partially through HSP70- or HSP90-mediated heat shock response. These findings expanded the understanding of aspirin pharmacology, and imply that local induction expression of HSPs might be a potential therapeutic strategy for the prevention and therapy of vascular calcification.     

Folium Ginkgo extract and tetramethylpyrazine sodium chloride injection (Xingxiong injection) protects against focal cerebral ischaemia/reperfusion injury via activating the Akt/Nrf2 pathway and inhibiting NLRP3 inflammasome activation

ContextFolium Ginkgo extract and tetramethylpyrazine sodium chloride injection (Xingxiong injection) is a compound preparation commonly used for treating cerebral ischaemia/reperfusion injury in ischaemic stroke in China. However, its potential mechanisms on ischaemic stroke remain unknown.ObjectiveThis study explores the potential mechanisms of Xingxiong injection in vivo or in vitro.Materials and methodsSprague-Dawley (SD) rats were randomly assigned to five groups: the sham (normal saline), the model (normal saline) and the Xingxiong injection groups (12.5, 25 or 50 mL/kg). The rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 14 d. Xingxiong injection was administered via intraperitoneal (i.p.) injection immediately after ischaemia induction for 14 d. Afterwards, rats were sacrificed at 14 d induced by administration of Xingxiong injection.ResultsXingxiong injection significantly reduces infarct volume (23%) and neurological deficit scores (93%) compared with the MCAO/R group. Additionally, Xingxiong injection inhibits the loss in mitochondrial membrane potential (43%) and reduces caspase-3 level (44%), decreases NOX (41%), protein carbonyl (29%), 4-HNE (40%) and 8-OhdG (41%) levels, inhibits the expression of inflammatory factors, such as TNF-α (26%), IL-1β (34%), IL-6 (39%), MCP-1 (36%), CD11a (41%) and ICAM-1 (43%). Moreover, Xingxiong injection can increase p-Akt/Akt (35%) and Nrf2 (47%) protein expression and inhibit NLRP3 (42%) protein expression.ConclusionsXingxiong injection prevents cerebral ischaemia/reperfusion injury via activating the Akt/Nrf2 pathway and inhibiting NLRP3 inflammasome. These findings provide experimental evidence for clinical use of drugs in the treatment of ischaemic stroke.