UPLC-Q-TOF-MS/MS analysis of ophiobolins sesterterpenoids and bioactive analogs from Bipolaris eleusines.

In order to elucidate the mass fragmentation patterns and unveil more undescribed ophiobolin analogs, the mass fragmentation patterns of ophiobolins were analyzed based on UPLC-Q-TOF-MS/MS experiments. Different kinds of rearrangements (including McLafferty rearrangement) were the main cleavage patterns. Twenty-six (9-31) analogs were then tentatively characterized based on their mass analysis, and three undescribed ophiobolins (6-8) and a known analogue (5) were isolated in target. Compound 5 possesses a rare polycyclic carbon skeleton only recently reported, and compound 6 contains an undescribed lactone ring system fused with A/B ring at C-3/C-21, whereas compounds 7 and 8 have a peroxyl group in the side chain, which is the first reported in all ophiobolins. Compounds 5 and 7 displayed significant cytotoxicity against MCF-7 cancer cells.

UPLC-Q-TOF-MS/MS-Based Targeted Discovery of Chetomin Analogues from Chaetomium cochliodes.

Chetocochliodin M (5) containing a rare cage-ring and chetocochliodin N (6) featuring an unusual piperazine-2,3-dione ring system together with known analogues chetomin (1), chetoseminudin C (2), chetocochliodin I (3), and oidioperazine E (4) were targeted for purification from the fungus Chaetomium cochliodes using a UPLC-Q-TOF-MS/MS approach. The structures of the new compounds were elucidated using HR-ESI-MS, NMR, and ECD spectra. Compounds 1, 3, and 6 exhibited strong cytotoxic activities against A549 and HeLa cancer cell lines.

Isolation, Purification, and Structural Characterization of Polysaccharides from Codonopsis pilosula and Their Anti-Tumor Bioactivity by Immunomodulation.

The activity of polysaccharides is usually related to molecular weight. The molecular weight of polysaccharides is critical to their immunological effect in cancer therapy. Herein, the Codonopsis polysaccharides of different molecular weights were isolated using ultrafiltration membranes of 60- and 100-wDa molecular weight cut-off to determine the relationship between molecular weight and antitumor activities. First, three water-soluble polysaccharides CPPS-I (<60 wDa), CPPS-II (60-100 wDa), and CPPS-III (>100 wDa) from Codonopsis were isolated and purified using a combination of macroporous adsorption resin chromatography and ultrafiltration. Their structural characteristics were determined through chemical derivatization, GPC, HPLC, FT-IR, and NMR techniques. In vitro experiments indicated that all Codonopsis polysaccharides exhibited significant antitumor activities, with the tumor inhibition rate in the following order: CPPS-II > CPPS-I > CPPS-III. The treatment of CPPS-II exhibited the highest inhibition rate at a high concentration among all groups, which was almost as efficient as that of the DOX·HCL (10 µg/mL) group at 125 µg/mL concentration. Notably, CPPS-II demonstrated the ability to enhance NO secretion and the antitumor ability of macrophages relative to the other two groups of polysaccharides. Finally, in vivo experiments revealed that CPPS-II increased the M1/M2 ratio in immune system regulation and that the tumor inhibition effect of CPPS-II + DOX was superior to that of DOX monotherapy, implying that CPPS-II + DOX played a synergistic role in regulating the immune system function and the direct tumor-killing ability of DOX. Therefore, CPPS-II is expected to be applied as an effective cancer treatment or adjuvant therapy.

[Research progress on pharmacology of butylphthalide and its derivatives].

The butylphthalide(NBP), a colorless or light yellow viscous oily component isolated from celery seeds, has the effects of anti-inflammation, anti-oxidative stress, protecting blood-brain barrier, improving cerebral microcirculation, and promoting angiogenesis. It can protect the neurological function of patients with ischemic stroke through a variety of mechanisms, improve the symptoms of patients, and contribute to the long-term recovery of them. Therefore, independently developed in China, NBP was approved by State Food and Drug Administration for the clinical treatment of stroke patients in 2002. At the same time, owing to the complex multi-target pharmacological mechanism, NBP has been frequently used in clinical practice. As frequently verified, it has obvious effects in the treatment of other neurological diseases such as Alzheimer's disease, vascular dementia, Parkinson's disease, autoimmune diseases, depression, traumatic central nervous system injury. Moreover, it demonstrates significant pharmacological effects on non-neurological diseases such as diabetes mellitus and myocardial infarction. Therefore, this study summarizes the research progress on roles of NBP in nervous system diseases and non-nervous system diseases, and the pharmacological characteristics and mechanisms of NBP, which is expected to lay a basis for research on related targets.

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.

CONTEXT: Folium 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. OBJECTIVE: This study explores the potential mechanisms of Xingxiong injection in vivo or in vitro. MATERIALS AND METHODS: Sprague-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. RESULTS: Xingxiong 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-1ß (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. CONCLUSIONS: Xingxiong 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.

Guan Xin Dan Shen formulation protects db/db mice against diabetic cardiomyopathy via activation of Nrf2 signaling.

Guan Xin Dan Shen formulation (GXDSF) is a widely used treatment for the management of coronary heart disease in China and is composed of three primary components: Dalbergiae odoriferae Lignum, Salviae miltiorrhizae Radix et Rhizoma and Panax notoginseng Radix et Rhizoma. However, the potential use of GXDSF for the management of diabetic cardiomyopathy (DCM) has not been previously assessed. The present study aimed to assess the effects of GXDSF on DCM, as well as the underlying mechanism. In the present study, db/db mice were used. Following treatment with GXDSF for 10 weeks, fasting blood glucose, insulin sensitivity, serum lipid levels and cardiac enzyme levels were detected. Cardiac pathological alterations and cardiac function were assessed by performing hematoxylin and eosin staining and echocardiograms, respectively. TUNEL assays were conducted to assess cardiomyocyte apoptosis. Additionally, reverse transcription­quantitative PCR and western blotting were performed to evaluate the expression of apoptosis­associated genes and proteins, respectively. In the model group, the db/db mice displayed obesity, hyperlipidemia and hyperglycemia, accompanied by noticeable myocardial hypertrophy and diastolic dysfunction. Following treatment with GXDSF for 10 weeks, serum triglyceride levels were lower and insulin sensitivity was enhanced in db/db mice compared with the model group, which indicated improvement in condition. Cardiac hypertrophy and dysfunction were also improved in db/db mice following treatment with GXDSF, resulting in significantly increased left ventricular ejection fraction and fractional shortening compared with the model group. Following treatment with metformin or GXDSF, model­induced increases in levels of myocardial enzymes were decreased in the moderate and high dose groups. Moreover, the results indicated that, compared with the model group, GXDSF significantly inhibited cardiomyocyte apoptosis in diabetic heart tissues by increasing Bcl­2 expression and decreasing the expression levels of Bax, cleaved caspase­3 and cleaved caspase­9. Mechanistically, GXDSF enhanced Akt phosphorylation, which upregulated antioxidant enzymes mediated by nuclear factor erythroid 2­related factor 2 (Nrf2) signaling. Collectively, the results of the present study indicated that GXDSF attenuated cardiac dysfunction and inhibited cardiomyocyte apoptosis in diabetic mice via activation of Akt/Nrf2 signaling. Therefore, GXDSF may serve as a potential therapeutic agent for the management of DCM.

Notoginsenoside R1 activates the NAMPT-NAD+-SIRT1 cascade to promote postischemic angiogenesis by modulating Notch signaling.

Nicotinamide phosphoribosyltransferase (NAMPT) maintains mitochondrial function and protects against cerebral ischemic injury by improving energy metabolism. Notoginsenoside R1 (R1), a unique constituent of Panax notoginseng, has been shown to promote the proliferation and tube formation of human umbilical vein endothelial cells. Whether R1 has proangiogenesis on the activation of NAMPT in ischemic stroke remains unclear. The purpose of this study was to investigate the pharmacodynamic effect and mechanism of R1 on angiogenesis after ischemic stroke. We used male Sprague-Dawley (SD) rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered via intraperitoneal (i.p.) injection immediately after ischemia induction. The promotion of R1 on angiogenesis were detected by immunofluorescence staining, 3D stereoscopic imaging and transmission electron microscopy detection. HBMEC cells were pretreated with different concentrations of R1 for 12 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, scratch assay, EdU staining and tube formation were determined. Western blot analyses of proteins, including those involved in angiogenesis, NAMPT-SIRT1 cascade, VEGFR-2, and Notch signaling, were conducted. We showed that R1 significantly restored cerebral blood flow, improved mitochondrial energy metabolism and promoted angiogenesis. More importantly, incubation with 12.5-50 µM R1 significantly increased the migration, proliferation and tube formation of HBMECs in vitro. The promotion of R1 on angiogenesis were associated with the NAMPT-NAD+-SIRT1 cascade and Notch/VEGFR-2 signaling pathway, which was partially eliminated by inhibitors of NAMPT and SIRT1. We demonstrated that R1 promotes post-stroke angiogenesis via activating NAMPT-NAD+-SIRT1 cascade. The modulation of Notch signaling and VEGFR-2 contribute to the post-stroke angiogenesis. These findings offer insight for exploring new therapeutic strategies for neurorestoration via R1 treatment after ischemic stroke.

Effects of notoginseng leaf triterpenes on small molecule metabolism after cerebral ischemia/reperfusion injury assessed using MALDI-MS imaging.

BACKGROUND: Notoginseng leaf triterpenes (PNGL) is believed to have neuroprotective effects via the inhibition of inflammatory response and neuronal apoptosis. However, its mechanisms underlying the anti-ischemia/reperfusion (I/R) injury effects on the regulation of small molecule metabolism in rat brain remains unclear. The purpose of this study was thus to explore the mechanisms of PNGL on the regulation of small molecule metabolism in rat brain after I/R injury using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). METHODS: As a model of in vivo cerebral I/R injury, male Sprague-Dawley (SD) rats were established with a middle cerebral artery occlusion/reperfusion (MCAO/R) model after PNGL administration with 40 mg·kg-1 through intraperitoneal injection (i.p.) for 7 days. We assessed the neurological behavior, regional cerebral blood flow (r CBF), neuron injury, and spatial distribution of metabolic small molecules. RESULTS: Our in vivo results suggested that PNGL increased cerebral blood flow and relieved neurological dysfunction. Furthermore, using MALDI-MSI, we demonstrated that PNGL regulated 16 endogenous small molecules implicated in metabolic networks including tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) metabolism, malate-aspartate shuttle, metal ions, and antioxidants underwent noticeable changes after reperfusion for 24 h. CONCLUSIONS: PNGL is a novel cerebrovascular agent that can improve cerebral blood flow and attenuate adverse neurological disorders. The mechanisms are closely correlated with relative metabolic pathways, which offers insight into exploring new mechanisms in PNGL for the treatment of cerebral I/R injury.

Xuesaitong injection (lyophilized) combined with aspirin and clopidogrel protect against focal cerebral ischemic/reperfusion injury in rats by suppressing oxidative stress and inflammation and regulating the NOX2/IL-6/STAT3 pathway.

BACKGROUND: Combination of aspirin (ASA) and clopidogrel (CLP) [dual antiplatelet therapy (DAPT)] has been limited in reducing early recurrent stroke events. Xuesaitong injection (lyophilized) (XST) made of total saponins from P. notoginseng, which significantly improves cerebral circulation and has been widely used in clinical applications for decades to treat and prevent ischemic stroke. Here, we confirmed the protective effect and mechanism of XST combined with DAPT (XST+ASA+CLP) on cerebral ischemia/reperfusion injury, exploring their better pharmacological action for clinical patients. METHODS: Sprague-Dawley rats (SD rats) (n=9 in each group) were randomly assigned to three groups and pretreated with XST, ASA+CLP, or XST+ASA+CLP for 7 days. Then rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. Therapeutic effect of XST+ASA+CLP was measured by infarct volume, neurological behavior and regional cerebral blood flow (rCBF). Inhibition of neuronal apoptosis and glial cells was determined by immunofluorescent staining. We studied the protein levels of neurotrophic factors, neuroplasticity-related factors, oxidative stress indicators and inflammatory factors by ELISA assay. RESULTS: XST+ASA+CLP group showed significant reduction in infarct volumes and neurological deficit scores. XST+ASA+CLP group also had higher levels in rCBF and synaptic growth, and showed remarkable inhibition of microglia and astrocytes activation and the neuronal apoptosis. In addition, XST+ASA+CLP group had lower levels of NADPH, protein carbonyl, 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHdG) and several inflammatory cytokines. Moreover, XST+ASA+CLP group also had lower levels of NOX2, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and p-STAT3/STAT3. CONCLUSIONS: These results demonstrate that a combination of XST, ASA, and CLP effectively protected rats against middle cerebral artery occlusion/reperfusion (MCAO/R) injury by suppressing the NOX2/IL-6/ STAT3 pathway. These novel findings provide theoretical basis and experimental evidence for the rationality of clinical combined use of drugs in the treatment of ischemic stroke.

Calenduloside E Ameliorates Myocardial Ischemia-Reperfusion Injury through Regulation of AMPK and Mitochondrial OPA1.

Calenduloside E (CE) is a natural triterpenoid saponin isolated from Aralia elata (Miq.) Seem., a well-known traditional Chinese medicine. Our previous studies have shown that CE exerts cardiovascular protective effects both in vivo and in vitro. However, its role in myocardial ischemia/reperfusion injury (MIRI) and the mechanism involved are currently unknown. Mitochondrial dynamics play a key role in MIRI. This study investigated the effects of CE on mitochondrial dynamics and the signaling pathways involved in myocardial ischemia/reperfusion (MI/R). The MI/R rat model and the hypoxia/reoxygenation (H/R) cardiomyocyte model were established in this study. CE exerted significant cardioprotective effects in vivo and in vitro by improving cardiac function, decreasing myocardial infarct size, increasing cardiomyocyte viability, and inhibiting cardiomyocyte apoptosis associated with MI/R. Mechanistically, CE restored mitochondrial homeostasis against MI/R injury through improved mitochondrial ultrastructure, enhanced ATP content and mitochondrial membrane potential, and reduced mitochondrial permeability transition pore (MPTP) opening, while promoting mitochondrial fusion and preventing mitochondrial fission. However, genetic silencing of OPA1 by siRNA abolished the beneficial effects of CE on cardiomyocyte survival and mitochondrial dynamics. Moreover, we demonstrated that CE activated AMP-activated protein kinase (AMPK) and treatment with the AMPK inhibitor, compound C, abolished the protective effects of CE on OPA1 expression and mitochondrial function. Overall, this study demonstrates that CE is effective in mitigating MIRI by modulating AMPK activation-mediated OPA1-related mitochondrial fusion.

Anti-ischemia/reperfusion injury effects of notoginsenoside R1 on small molecule metabolism in rat brain after ischemic stroke as visualized by MALDI-MS imaging.

Ischemic stroke is a syndrome of severe neurological responses that cause neuronal death, damage to the neurovascular unit and inflammation. Notoginsenoside R1 (NG-R1) is a neuroprotective drug that is commonly used to treat neurodegenerative and cerebrovascular diseases. However, its potential mechanisms on the regulation of small molecule metabolism in ischemic stroke are largely unknown. The aim of this study was to explore the potential mechanisms of NG-R1 on the regulation of small molecule metabolism after ischemic stroke. Here, we found that NG-R1 reduced infarct size and improved neurological deficits by ameliorating neuronal damage and inhibiting glial activation in MCAO/R rats. Furthermore, using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we clarified that NG-R1 regulated ATP metabolism, the tricarboxylic acid (TCA) cycle, the malate-aspartate shuttle, antioxidant activity, and the homeostasis of iron and phospholipids in the striatum and hippocampus of middle cerebral artery occlusion/reperfusion (MCAO/R) rats. In general, NG-R1 is a promising compound for brain protection from ischemic/reperfusion injury, possibly through the regulation of brain small molecule metabolism.

[Effect of Guanxin Danshen Formulation on diabetic kidney disease in db/db mice through regulation of Nrf2 pathway].

Diabetic kidney disease(DKD) has become a primary cause of end-stage kidney disease, without any effective treatment available. In this study, we assessed the protective effect of Guanxin Danshen Formulation(GXDSF) on diabetic nephropathy in db/db mice. The db/m and db/db mice were randomly divided into 4 groups: control group, model group, metformin group, and GXDSF group. After 8 weeks' treatment with GXDSF, metformin or normal saline, the mice were sacrificed, and the blood and kidney tissues were collected for the further analysis. Compared with the model group, TG, TCH and LDL levels significantly decreased in the GXDSF group. The results from HE and PAS staining showed that db/db mice exhibited abnormal kidney tissues with increased glomerular volume, basement-membrane thickening and mesangial cell proliferation, which could be significantly alleviated by GXDSF treatment. GXDSF treatment also reduced serum creatinine and BUN. Meanwhile, GXDSF treatment markedly elevated GSH-PX levels, while reduced LDH and MDA levels in the kidney tissues. Western blot assay showed that GXDSF evidently up-regulated protein levels of ERα and p-Akt, and subsequently promoted HO-1 expression mediated by Nrf2. These data collectively indicated that GXDSF protects db/db mice against DN by regulating ERα and Nrf2-mediated HO-1 expression.

Scutellarin protects against myocardial ischemia-reperfusion injury by suppressing NLRP3 inflammasome activation.

BACKGROUND: Activation of NLRP3 inflammasome plays a key role in cardiac dysfunction for acute myocardial ischemia-reperfusion injury. Scutellarin (Scu) is a flavonoid purified from Erigeron breviscapus. Whether Scu has any influence on the activation of NLRP3 inflammasome in cardiomyocytes remains unknown. PURPOSE: We aimed to examine the therapeutic effect of Scu on cardiomyocyte ischemia-reperfusion (I/R) injury and its effect on NLRP3 inflammasome in rats with acute myocardial I/R injury and anoxia/reoxygenation (A/R)-induced H9c2 injuries. METHODS: Heart injuries were induced through 30 min of ischemia followed by 24 h of reperfusion. Scu was intraperitoneally administered 15 min before vascular ligation. Effects of Scu on cardiac injury were detected by echocardiograms, TTC staining, and histological and immunohistochemical analyses. The effects of Scu on biochemical parameters were analyzed. H9c2 cells were pretreated with different concentrations of Scu for 6 h before A/R exposure. Afterward, cell viability, LDH release, and Hoechst 33342 and peromide iodine double staining were determined. Western blot analyses of proteins, including those involved in autophagy, NLRP3, mTOR complex 1 (mTORC1), and Akt signaling, were conducted. RESULTS: In vivo study revealed that Scu improved diastolic dysfunction, ameliorated myocardium structure abnormality, inhibited myocyte apoptosis and inflammatory response, and promoted autophagy. Scu reduced NLRP3 inflammasome activation, inhibited mTORC1 activity, and increased Akt phosphorylation. In vitro investigation showed the same results. The Scu-mediated NLRP3 inflammasome and mTORC1 inhibition and cardioprotection were abolished through the genetic silencing of Akt by siRNA. CONCLUSIONS: The cardioprotective effect of Scu was achieved through its anti-inflammatory effect. It suppressed the activation of NLRP3 inflammasome. In addition, inflammasome restriction by Scu was dependent on Akt activation and mTORC1 inhibition.

Shenkang Injection protects against diabetic nephropathy in streptozotocin (STZ)-induced mice through enhancement of anti-oxidant and anti-inflammatory activities.

Objective: To investigate the protective effects and possible mechanisms of Shenkang Injection (SKI) on the diabetic nephropathy in streptozotocin-induced mice. Methods: STZ with the feeding of high fat diet (HFD) was used to induce diabetic mice. The balb/c mice and diabetic mice were then randomly divided into five groups: (1) control group, (2) model group, (3) alprostadil (Alp, 1.5 µg/kg) group, (4) SKI (30 ml/kg) group, (5) Alp (1.5 µg/kg) + SKI (15 ml/kg) group. After six weeks' treatment, blood, urine and kidney tissues were collected for biochemical assay, ELISA assay, and pathological analysis. Results: Diabetic mice exhibited evident manifestations of diabetic nephropathy (DN), as indicated by increased 24-h urine volume, urinary albumin and kidney weight index (P < 0.01), which could be attenuated by SKI treatment (P < 0.01). SKI was further found to improve abnormal morphology in glomerulus with increased glomerular volume and to decrease urinary N-acetyl-b-D-glucpsaminidase (NAG), ß2-microglobulin (ß2-MG), and kidney injury molecules-1 (KIM-1) levels (P < 0.05, P < 0.01). Plasma levels of anti-oxidant enzymes significantly reduced in the diabetic mice, and those decreases could be reversed by SKI and Alp treatments. Additionally, SKI obviously suppressed the diabetes-induced increases of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α) (P < 0.01). Meanwhile, SKI was found to effectively attenuate the diabetes-induced coagulation dysfunction, as evidenced by lengthening prothrombin and thrombin time, and decreasing plasma levels of fibrinogen (FIB), 6-K-PGF1α and thromboxane B2 (TXB2) (P < 0.05, P < 0.01). With SKI and Alp combined treatment, the anti-oxidant activities and improvements of coagulation dysfunction were enhanced. Conclusion: SKI possesses a remarkable property to prevent diabetic nephropathy. The improvements of kidney function and hypercoagulability by SKI were enhanced with Alp combined treatment. The molecular mechanisms underlying the protection of SKI against DN may be related to enhancing the anti-oxidant and anti-inflammatory activities, and improving the coagulation dysfunction.

Xuezhitong capsule, an extract of Allium macrostemon Bunge, exhibits reverse cholesterol transport and accompanies high-density lipoprotein levels to protect against hyperlipidemia in ApoE-/- mice.

BACKGROUND: Xuezhitong capsules (XZT) are derived from Xie Bai and used for abnormal lipid homeostasis treatment through maintained metabolic balance. However, their mechanisms are largely unknown. Here, we mainly assessed the contribution of reverse cholesterol transport (RCT) and the accompanying increase in the high-density lipoprotein (HDL) effects of XZT to cholesterol dysfunction amelioration in mice. METHODS: We assessed serum lipids by using enzymatic kits. We observed atherosclerotic plaque formation by hematoxylin-eosin (HE) and Oil Red O staining. We studied the lipid metabolism, fatty acid synthase (FAS), HDL, low-density lipoprotein receptor (LDLR), triglyceride (TG) metabolic enzyme expression levels, and RCT function in various tissues upon stimulation with high-fat diet, XZT, and some positive drugs by ELISA. RESULTS: After 34 weeks of high-fat diet administration, blood lipids levels increased because attenuated by XZT treatment (800 and 1,600 mg/kg, i.g.). XZT improved the lipid metabolism instability, induced RCT activation, and subsequently increased the HDL levels in hyperlipidemic mice (P<0.05). FAS (P<0.05) and LDLR (P<0.01) levels also remarkably improved. The effects of XZT were closely associated with RCT activation and the accompanying increase in the HDL levels, as characterized by XZT-induced preservation in ATP-binding cassette transporter member 1 (ABCA1), scavenger receptor class B type 1 (SRB1), acyl coenzyme A: cholesterol acyltransferase (ACAT), lecithin cholesterol acyltransferase (LCAT), apolipoprotein A I (ApoA1) and apolipoprotein B (ApoB). However, XZT showed no effect on high fat diet-activated TG metabolic enzyme expression levels (P>0.05). CONCLUSIONS: XZT are promising drugs in balancing the cholesterol dysfunction from hyperlipidemia through RCT activation and accompanying increase in HDL levels.

[Preventive and therapeutic effects of Keluoxin Capsules on early diabetic retinopathy in db/db mice].

The aim of this paper was to investigate the preventive effects of Keluoxin Capsules(KLX) on diabetic retinopathy in db/db mice. One hundred male db/db diabetic mice(45-55 g, 8 weeks) were randomly divided into 5 groups(model, KLX low dose, KLX middle dose, KLX high dose, Dobesilate) and 20 male C57 BL/KsJdb~(+/+) were taken as control group. Body weight and fasting blood-glucose were detected every week. Mice were administrated with saline(control and model group), KLX(780, 1 560, 3 120 mg·kg~(-1)·d~(-1), ig), Dobesilate(195 mg·kg~(-1)·d~(-1), ig) for 20 weeks, respectively. At the end of the administration, optical coherence tomography, fundus fluorescein angiography and electroretinogram of the retina were measured. The eyeball was extirpated and retina was isolated to make paraffin section, followed by HE staining and glial fibrillary acidic protein(GFAP) immunohistochemistry. The results indicated that KLX has no obvious effect on body weight and fasting blood level in db/db mice. However, KLX could significantly regulate the thickness of retinal ganglion layer and inner plexiform layer. KLX was able to remarkably reduce the quantity of diabetic microvessel. Meanwhile, KLX could notably improve retinal function. Moreover, KLX could observably modulate the cell arrangement and edema in each layer. There was no markable difference in retina according to the immunochemistry assay. In the present study, KLX exert marked preventive effects on diabetic retinopathy in db/db mice, which provided an experimental evidence for clinical use.

The clickable activity-based probe of anti-apoptotic calenduloside E.

CONTEXT: Calenduloside E (CE), one of the primary natural products found in Aralia elata (Miq.) Seem. (Araliaceae), possesses prominent anti-apoptotic potential. A previous study found that one of the anti-apoptotic CE targets is heat shock protein 90 AB1 (Hsp90AB1) by probe CE-P, while the other targets of CE still need to be identified with more efficient probes. OBJECTIVE: This study investigates CE analogue (CEA) as one clickable activity-based probe for use in exploring anti-apoptotic CE targets. MATERIALS AND METHODS: Pretreatment of HUVECs with CEA (1.25 µM) for 8 hr, followed by ox-LDL stimulation for 24 h. Flow cytometry analysis and JC-1 staining assays were performed The kinetic constant measurements were tested by the Biacore T200, CM5 Sensor Chip which was activated by using sulpho-NHS/EDC. Ligands were dissolved and injected with a concentration of 12.5, 6.25, 3.125, 1.56, 0.78 and 0 µM. RESULTS: CEA was confirmed to possess an anti-apoptotic effect. The probable targets of CE/CEA were calculated, and as one of the higher scores proteins (Fit values: 0.88/0.86), Hsp90 properly got our attention. Molecular modelling study showed that both CE and CEA could bind to Hsp90 with the similar interaction, and the docking scores (S value) were -7.61 and -7.33. SPR assay provided more evidence to prove that CEA can interact with Hsp90 with the KD value 11.7 µM. DISCUSSION AND CONCLUSIONS: Our results suggest that clickable probe CEA could alleviate ox-LDL induced apoptosis by a similar mechanism of anti-apoptotic CE, and afforded the possibility of identifying additional anti-apoptotic targets of CE.

Protective effects of Myrica rubra flavonoids against hypoxia/reoxygenation-induced cardiomyocyte injury via the regulation of the PI3K/Akt/GSK3ß pathway.

Myrica rubra is well known for its delicious taste and high nutritional value. The present study investigated the potential protective effects and mechanisms of M. rubra flavonoids (MRF) extract on isoproterenol (ISO)­induced myocardial injury in rats and hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes. An in vivo study revealed that MRF decreased serum cardiac enzyme levels, ameliorated pathological heart alterations and increased the antioxidant potential. The in vitro investigation demonstrated that MRF inhibited cell death, reactive oxygen species (ROS) accumulation, mitochondrial membrane depolarization, apoptosis rate and caspase­3 activation and enhanced the Bcl­2/Bax ratio during H/R injury. These effects were accompanied by the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase (GSK)­3ß. Further mechanism studies demonstrated that LY294002, a specific inhibitor of phosphoinositide 3­kinase (PI3K), abolished the MRF­mediated cardioprotection against H/R­induced apoptosis and ROS overproduction. Collectively, these results suggested that MRF exerts cardioprotective effects by attenuating oxidative damage and cardiomyocyte apoptosis most likely via a PI3K/Akt/GSK3ß­dependent mechanism.

Cytochrome P450 3A Enzymes Are Key Contributors for Hepatic Metabolism of Bufotalin, a Natural Constitute in Chinese Medicine Chansu.

Bufotalin (BFT), one of the naturally occurring bufodienolides, has multiple pharmacological and toxicological effects including antitumor activity and cardiotoxicity. This study aimed to character the metabolic pathway(s) of BFT and to identify the key drug metabolizing enzyme(s) responsible for hepatic metabolism of BFT in human, as well as to explore the related molecular mechanism of enzymatic selectivity. The major metabolite of BFT in human liver microsomes (HLMs) was fully identified as 5ß-hydroxylbufotalin by LC-MS/MS and NMR techniques. Reaction phenotyping and chemical inhibition assays showed that CYP3A4 and CYP3A5 were key enzymes responsible for BFT 5ß-hydroxylation. Kinetic analyses demonstrated that BFT 5ß-hydroxylation in both HLMs and human CYP3A4 followed the biphasic kinetics, while BFT 5ß-hydroxylation in CYP3A5 followed substrate inhibition kinetics. Furthermore, molecular docking simulations showed that BFT could bind on two different ligand-binding sites on both CYP3A4 and CYP3A5, which partially explained the different kinetic behaviors of BFT in CYP3A4 and CYP3A5. These findings are very helpful for elucidating the phase I metabolism of BFT in human and for deeper understanding the key interactions between CYP3A enzymes and bufadienolides, as well as for the development of bufadienolide-type drugs with improved pharmacokinetic and safety profiles.