Neuroprotection of Oral Edaravone on Middle Cerebral Artery Occlusion in Rats.

Edaravone has been widely used in the treatment of acute ischemic stroke. However, there has been no oral preparation of edaravone in the clinic. In this study, we assessed the effect and possible mechanisms of oral edaravone on the middle cerebral artery occlusion (MCAO) model in rats. Highly bioavailable form of novel edaravone formulation developed using self-nanomicellizing solid dispersion strategy which showed up to 16.1-fold improved oral bioavailability was considered oral edaravone. The male rats (n = 84) were randomly divided into sham; model; oral edaravone in low dose (10 mg/kg), medium dose (20 mg/kg), and high dose (30 mg/kg); and edaravone by intraperitoneal administration group (IP group, 10 mg/kg). Rats were treated with different drugs 5 h after the operation, twice a day for 7 days. The behavioral data were dose-dependently improved by oral edaravone and sensorimotor functions of the high dose group were similar to those of the edaravone by IP route group. Furthermore, oral edaravone significantly reduced cerebral infarction area and downregulated the levels of caspase-3, GFAP, Iba1, 3-NT, and 4-HNE, whereas upregulated those of Vamp-2 and Map-2 in a dose-dependent manner. Especially effect of the high dose on these molecules was equal to that of edaravone by IP administration. Taken together, our data suggest that the improvement of sensorimotor deficits by oral edaravone in high doses after ischemia is similar to that in edaravone by IP administration. Neuroprotection of oral edaravone is at least partial by minimizing oxidative stress, the overactivation of glial cells, and the levels of the apoptosis-associated proteins, and alleviating synaptic damage in a dose-dependent manner.

Panax notoginsenoside Rb1 Restores the Neurotrophic Imbalance Following Photothrombotic Stroke in Rats.

Mature brain-derived neurotrophic factor (mBDNF) has neuroprotection in cerebral ischemia. Conversely, the precursor of brain-derived neurotrophic factor (proBDNF) has the opposite function to its mature form, inducing apoptosis. However, whether the neuroprotection of Panax notoginsenoside Rb1 (PNS-Rb1) on ischemic stroke is due to, at least partially, its modulation of suppressing proBDNF/P75NTR/sortilin or upregulation of mBDNF is not clear. To test this hypothesis, rats induced by photothrombotic stroke were treated with PNS-Rb1 100 mg/kg or nimodipine 1 mg/kg twice a day until 3, 7, and 14 days. Our data indicate that PNS-Rb1 significantly reduced cerebral infarction rate, proBDNF/P75NTR/sortilin, and plasminogen activator inhibitor-1 (PAI-1) protein levels, and improved sensorimotor dysfunctions induced by ischemic stroke, upregulation of BDNF/TrkB levels, and its processing enzymes (tissue plasminogen activator, tPA) in a time-dependent manner. Taken together, our findings indicate that the improvement of sensorimotor dysfunctions by PNS-Rb1 following ischemic stroke is made, at least partially, by activating the BDNF/TrkB and inhibiting proBDNF/sortilin/P75NTR.

Effects of Ginsenoside Rb1 on Expressions of Phosphorylation Akt/Phosphorylation mTOR/Phosphorylation PTEN in Artificial Abnormal Hippocampal Microenvironment in Rats.

Artificial abnormal microenvironment caused by microperfusion of L-glutamate (Glu) and Ca2+ in the hippocampus results in neuron damage, which is closely related to cerebral ischemia. Ginsenoside Rb1, a compound from Panax notoginseng, was previously used to counter the artificial abnormal hippocampal environment in a microperfusion model. In addition, while the Akt/mTOR/PTEN signaling pathway has been shown to mediate neuronprotection in cerebral ischemia, whether this pathway is involved in the neuroprotection of ginsenoside Rb1 is unknown. Here SH-SY5Y cells exposed to OGD/R injury in treated with LY294002, ginsenoside Rb1, ginsenoside Rb1+ LY294002. Expressions of phosphorylation (P-)Akt/P-mTOR/P-PTEN (24 h after OGD/R) were detected by Western blotting. Effects were examined via the memory function of rats (by Morris water maze test), morphological changes in pyramidal cell (by histology), and mRNA expression (by qRT-PCR) and phosphorylation (P-) (by Western blotting and immunohistochemical staining) of Akt, P-mTOR, and P-PTEN in the hippocampus. The memory deficit of rats and pyramidal cellular necrosis and apoptosis in the CA1 region of hippocampus after microperfusion of Glu and Ca2+ were dose dependently alleviated by ginsenoside Rb1.Moreover,Western blot showed that ginsenoside Rb1 increased the expressions of P-Akt, P-mTOR and reduced P-PTEN in vivo and vitro. Thus, the potent neuroprotection of ginsenoside Rb1 in artificial abnormal microenvironment is, at least partially, related to the activation of P-AKT/P-mTOR signaling pathway and inhibition of P-PTEN protein.

Panax notoginsenoside saponins Rb1 regulates the expressions of Akt/ mTOR/PTEN signals in the hippocampus after focal cerebral ischemia in rats.

Panax notoginsenoside saponins Rb1 (PNS-Rb1) is an important active ingredient of panax notoginseng for effective treatment of cerebrovascular diseases. However, the mechanism underlying its actions in the state of cerebral ischemia is still unclear. We asked whether the potential neuroprotection of PNS-Rb1 on the brain is due to, at least partially, its modulation of AkT/mTOR/PTEN signalling pathway along with down-regulation of caspase-3 in rats subjected to phototrombic stroke. To test this hypothesis, rats with induced photothrombotic stroke were treated with PNS-Rb1 (applied in three different doses, 25 mg/kg, 50 mg/kg,100 mg/kg, respectively) or saline, while sham operated rats injected with saline were used as the control. Our results indicate that PNS-Rb1 significantly alleviated the morphological lesion concomitant with improvement of cognitive and sensorimotor deficits induced by ischemic stroke. Moreover, immunohistochemistry and Western blot analyses showed that PNS Rb1 in a dose dependent manner increased the expressions of P-Akt, P-mTOR and reduced P-PTEN and caspase-3. The present study suggests that the improvement of cognitive and sensorimotor deficits by PNS-Rb1 is made, at least partially, by the modulation of the Akt/mTOR/PTEN signalling pathway.

CIDE gene expression in adipose tissue, liver, and skeletal muscle from obese and lean pigs.

The expression of the cell death-inducing DNA fragmentation factor α-like effector (CIDE) family including Cidea, Cideb, and Cidec was significantly increased in mouse and human models of obesity. However, there was less information on these genes' expression in pigs. Here, we hypothesized that different fat accumulation between lean (Duroc×Landrace×Yorkshire gilts, DLY) and obese (Lantang) pigs was attributed to porcine CIDE-modulating lipid metabolism. Our data showed that Cidea and Cidec were expressed at a high level in adipose tissue, and at a relatively high level in skeletal muscle, whereas Cideb was mainly expressed in the liver in both breeds of pig. Lantang pigs had higher white adipose and skeletal muscle Cidea and Cidec mRNA abundance, and hepatic and muscle Cideb mRNA than DLY pigs. Lipid metabolism-related genes including sterol regulatory element binding protein 1c (SREBP-1c), hepatocyte nuclear factor-4α (HNF-4α), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), fatty acid synthase (FASN), diacylglycerol O-acyltransferase 1 (DGAT1), and DGAT2 showed a higher expression level in adipose tissue from obese pigs than in that from lean pigs. Lantang pigs exhibited higher mRNA abundance for liver SREBP-1c, HNF-4α, and PGC-1α, and higher skeletal muscle SREBP-1c, HNF-4α, PGC-1α, and DGAT2 expression, as compared with DLY pigs. However, the perlipin2 mRNA levels in adipose tissues, liver, and skeletal muscle were significantly lower in obese pigs than in their lean counterparts. Furthermore, plasma non-esterified fatty acid (NEFA), glucose, and triacylglycerol (TAG) levels were greater in obese pigs than in lean pigs. Finally, data from correlation analysis further found that CIDE mRNA expression was positively correlated with back fat thickness (BFT), abdominal fat mass (AFM), and the levels of NEFA, TAG, and glucose in the two breeds. Collectively, these data revealed that the porcine CIDEs possibly modulated lipid metabolism and contributed to the development of fat deposition and obesity in Lantang pigs.

Influence of low protein diets on gene expression of digestive enzymes and hormone secretion in the gastrointestinal tract of young weaned piglets.

To investigate dietary protein level effects on digestive mechanisms, weaned piglets were fed for 45 d with diets containing 20%, 17%, or 14% crude protein (CP) supplemented to meet requirements for essential amino acids. This article describes the influence of dietary protein on gastrointestinal hormones and expression of an array of digestive enzymes in the gastrointestinal tract and pancreas. Results indicated that there were no significant differences in expression of enzymes involved in carbohydrate digestion, except for maltase in the duodenum. In the jejunum, amylase expression in pigs fed 20% CP was much higher than that in pigs fed other diets (P<0.05) and maltase expression in those fed 17% CP was higher than that in other treatments (P<0.05). Although there were no remarkable differences in expression of aminopeptidase in the small intestine or carboxypeptidase in the pancreas (P>0.05), there was a trend towards higher expression of various proteases in pigs fed 17% CP. The duodenal expression of enteropeptidase in diets with 14% and 17% CP was significantly higher than that with 20% CP (P<0.05), but treatment differences did not existed in jejunum (P>0.05). The expression of GPR93 as a nutrient-responsive G protein-coupled receptor in 14% and 17% CP diets was significantly higher than that in 20% CP diet in the small intestine (P<0.05). The expressions of genes for pancreatic enzymes, lipase and elastase, were significantly higher in pigs fed diets with low CP, while similar trends occurred for carboxypeptidase, chymotrypsin and amylase. Conversely, the gastric expressions of pepsinogen A and progastricsin were lower with the 17% CP diet. Differences between treatments were found in the gastric antral contents of cholecystokinin and somatostatin: both increased in pigs fed 17% CP, accompanied by decreased content of motilin, which was also seen in plasma concentrations. These patterns were not reflected in duodenal contents. In general, 17% dietary CP was beneficial to the digestion of nutrient substance in the gastrointestinal tract.

Conformation Selective Antibody Enables Genome Profiling and Leads to Discovery of Parallel G-Quadruplex in Human Telomeres.

G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (<3 nt). By using D1, telomeric parallel G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo.

Blocking the binding of WT1 to bcl-2 promoter by G-quadruplex ligand SYUIQ-FM05.

At present, wt1, a Wilms' tumor suppressor gene, is recognized as a critical regulator of tumorigenesis and a potential therapeutic target. WT1 shows the ability to regulate the transcription of bcl-2 by binding to a GC-rich region in the promoter, which can then fold into a special DNA secondary structure called the G-quadruplex. This function merits the exploration of the effect of a G-quadruplex ligand on the binding and subsequent regulation of WT1 on the bcl-2 promoter. In the present study, WT1 was found to bind to the double strand containing the G-quadruplex-forming sequence of the bcl-2 promoter. However, the G-quadruplex ligand SYUIQ-FM05 effectively blocked this binding by interacting with the GC-rich sequence. Our new findings are significant in the exploration of new strategies to block WT1's transcriptional regulation for cancer-cell treatment.

A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region.

The guanine-rich sequences are able to fold into G-quadruplexes in living cells, making these structures promising anti-cancer drug targets. In the current study, we identified a small molecule, Ber8, from a series of 9-substituted berberine derivatives and found that it could induce acute cell growth arrest and senescence in cancer cells, but not in normal fibroblasts. Further analysis revealed that the cell growth arrest was directly associated with apparent cell cycle arrest, cell senescence, and profound DNA damage at the telomere region. Significantly, our studies also provided evidence that Ber8 could stabilize endogenous telomeric G-quadruplexes structures in cells. Ber8 could then induce the delocalization of TRF1 and POT1 from the telomere accompanied by a rapid telomere uncapping. These results provide compelling insights into direct binding of telomeric G-quadruplexes and might contribute to the development of more selective, effective anticancer drugs.

Targeting G-quadruplex nucleic acids with heterocyclic alkaloids and their derivatives.

G-Quadruplex nucleic acids or G-quadruplexes (G4s) are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. They are widely distributed in functional regions of the human genome, such as telomeres, ribosomal DNA (rDNA), transcription start sites, promoter regions and untranslated regions of mRNA, suggesting that G-quadruplex structures may play a pivotal role in the control of a variety of cellular processes. G-Quadruplexes are viewed as valid therapeutic targets in human cancer diseases. Small molecules, from naturally occurring to synthetic, are exploited to specifically target G-quadruplexes and have proven to be a new class of anticancer agents. Notably, alkaloids are an important source of G-quadruplex ligands and have significant bioactivities in anticancer therapy. In this review, the authors provide a brief, up-to-date summary of heterocyclic alkaloids and their derivatives targeting G-quadruplexes.