Safety Assessment of L-ß-Aminoisobutyric Acid (L-BAIBA): Subchronic Toxicity Study in Sprague Dawley Rats.

L-3-Aminoisobutyric acid (L-BAIBA) is an endogenous compound in human metabolism when thymine and valine undergo catabolism. L-BAIBA represents one of the two isomers of BAIBA in biological systems. BAIBA has been shown to reduce body fat percentage via an increase in fatty acid oxidation and a decrease in hepatic lipogenesis. However, no toxicological effects of L-BAIBA in animals or humans have been established. The present study was designed to evaluate the safety and toxic potentials of this compound, where L-BAIBA was administered orally to Sprague Dawley rats at 100, 300, and 900 mg/kg/day for 90 days. No treatment-related adverse effects were observed in any of the treatment groups. Based on the results, the No-Observed-Adverse-Effect Level (NOAEL) of L-BAIBA was 900 mg/kg/day.

Neuroprotective effects of Ginkgolide B in focal cerebral ischemia through selective activation of prostaglandin E2 receptor EP4 and the downstream transactivation of epidermal growth factor receptor.

The present study was undertaken to identify whether prostaglandin E2 receptor is the potential receptor/binding site for Ginkgolide A, Ginkgolide B, Ginkgolide K, and Bilobalide, the four main ingredients of the Ginkgo biloba L., leaves. Using functional assays, we identified EP4, coupled with Gs protein, as a target of Ginkgolide B. In human neuroblastoma SH-SY5Y cells suffered from oxygen-glucose deprivation/reperfusion, Ginkgolide B-activated PKA, Akt, and ERK1/2 as well as Src-mediated transactivation of epidermal growth factor receptor. These resulted in downstream signaling pathways, which enhanced cell survival and inhibited apoptosis. Knockdown of EP4 prevented Ginkgolide B-mediated Src, epidermal growth factor receptor (EGFR), Akt, and ERK1/2 phosphorylation and neuroprotective effects. Moreover, Src inhibitor prevented Ginkgolide B-mediated EGFR transactivation and the downstream Akt and ERK1/2 activation, while the phosphorylation of PKA induced by Ginkgolide B was not affected, indicating Ginkgolide B might transactivate EGFR in a ligand-independent manner. EP4 knockdown in a rat middle cerebral artery occlusion (MCAO) model prevented Ginkgolide B-mediated infarct size reduction and neurological assessment improvement. At the same time, the increased expressions of p-Akt, p-ERK1/2, p-PKA, p-Src, and p-EGFR and the deceased expression of cleaved capases-3 induced by Ginkgolide B in cerebral cortex were blocked due to EP4 knockdown. In conclusion, Ginkgolide B exerts neuroprotective effects in rat MCAO model through the activation of EP4 and the downstream transactivation of EGFR.

Identification of TaBADH-A1 allele for improving drought resistance and salt tolerance in wheat (Triticum aestivum L.).

Drought and salt stress can strongly affect the growth and development of wheat. Wheat adapts to drought and salt stress through osmotic regulation. Betaine aldehyde dehydrogenase (BADH) is a key enzyme in the synthesis of betaine, an osmotic regulator. We cloned a region of the TaBADH-A1 promoter and genomic DNA that included the introns and exons, from four Chinese wheat cultivars. Following the analysis of TaBADH-A1 genomic DNA and promoter sequence polymorphisms of 4 cloned and 15 cultivars from the database, 7 haplotypes of TaBADH-A1 gene were identified. We divided the 7 haplotypes with a 254 bp insertion or deletion (indel) into two main alleles, BADH-A1a and BADH-A1b. Meanwhile, a molecular marker was developed based on the 254 bp indel of the third intron of TaBADH-A1 gene. Expression levels of BADH-A1b were found to be significantly higher than those of BADH-A1a under drought and salt stress conditions. Betaine accumulation was significantly higher in wheat containing BADH-A1b compared to BADH-A1a under drought and salt stress. We also identified that the average relative germination and survival rates of wheat with the BADH-A1b allele were significantly higher than wheat with the BADH-A1a allele. The results reveal that wheat containing BADH-A1b has stronger drought and salt tolerance than wheat with BADH-A1a. Meanwhile, the geographic distribution and frequency of the TaBADH-A1 locus alleles indicate that BADH-A1a has been preferred in Chinese wheat breeding programs, while BADH-A1b, associated with favorable stress tolerance, has been neglected. The results of this study provide evidence for an excellent candidate allele for marker-assisted selection of new wheat cultivars with increased salt tolerance and drought resistance.

Nucleoredoxin Gene TaNRX1 Positively Regulates Drought Tolerance in Transgenic Wheat (Triticum aestivum L.).

Drought is the main abiotic stress factor limiting the growth and yield of wheat (Triticum aestivum L.). Therefore, improving wheat tolerance to drought stress is essential for maintaining yield. Previous studies have reported on the important role of TaNRX1 in conferring drought stress tolerance. Therefore, to elucidate the regulation mechanism by which TaNRX1 confers drought resistance in wheat, we generated TaNRX1 overexpression (OE) and RNA interference (RNAi) wheat lines. The results showed that the tolerance of the OE lines to drought stress were significantly enhanced. The survival rate, leaf chlorophyll, proline, soluble sugar content, and activities of the antioxidant enzymes (catalase, superoxide dismutase, and peroxidase) of the OE lines were higher than those of the wild type (WT); however, the relative electrical conductivity and malondialdehyde, hydrogen peroxide, and superoxide anion levels of the OE lines were lower than those of the WT; the RNAi lines showed the opposite results. RNA-seq results showed that the common differentially expressed genes of TaNRX1 OE and RNAi lines, before and after drought stress, were mainly distributed in the plant-pathogen interaction, plant hormone signal transduction, phenylpropane biosynthesis, starch and sucrose metabolism, and carbon metabolism pathways and were related to the transcription factors, including WRKY, MYB, and bHLH families. This study suggests that TaNRX1 positively regulates drought stress tolerance in wheat.

Ultrafiltration strategy combined with nanoLC-MS/MS based proteomics for monitoring potential residual proteins in TCMIs.

Traditional Chinese medicine injections (TCMIs) containing complex constituents frequently cause unpredictable adverse reactions. The residual heterologous proteins in TCMIs may be one kind of the sensitized constituents. However, few methods were developed to identify and monitor the residual proteins of TCMIs in industry. Here, we described a method combining the advantages of ultrafiltration and mass spectrometry-based proteomics for monitoring the potential residual proteins in Re Du Ning injection (RDNI) intermediates and preparations. We identified and quantified both de novo peptides and the proteins matched against databases of three raw plants by using PEAKS software. Interesting, we found there was a significant decrease of peptides and proteins in No. 3-5 of RDNI intermediates and some even disappeared. Besides, we found this method could greatly reduce the interference of contaminants in proteomics experiments. The rapid and accurate method proposed in this paper could be used for monitoring potential residual proteins in TCMIs to guarantee their quality and safety.

Novel Therapeutic Targets for Hypoxia-Related Cardiovascular Diseases: The Role of HIF-1.

Insufficient oxygen availability (hypoxia) is a precursor to numerous cardiovascular diseases, including atherosclerosis, pulmonary hypertension, and heart failure. The main site of hypoxic injury in the human body is the mitochondria, where oxygen acts as the final electron acceptor in the process of oxidative phosphorylation. Hypoxia-inducible factor (HIF) is activated in hypoxic conditions and acts as an important modulator of diverse target genes in the human body. The downstream genes of HIF include vital modulators of cardiovascular-related signaling pathways. Therefore, it is hypothesized that HIF represents a potential therapeutic target for the treatment and prevention of cardiovascular diseases. In this short review, we introduce the pathophysiology of hypoxic injury in cardiovascular disease, and we conclude from convincing evidence that HIF can modulate relevant cardioprotective signaling pathways.

Chip-based serum proteomics approach to reveal the potential protein markers in the sub-acute stroke patients receiving the treatment of Ginkgo Diterpene Lactone Meglumine Injection.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. is a kind of traditional Chinese medicinal material with a long history. Its main active ingredients, ginkgolides, can be used for the treatment of stroke and other cardio-cerebrovascular diseases. Ginkgo Diterpene Lactone Meglumine Injection (GDLI), a modernized TCM, has attracted much attention because of its neuroprotective and anti-inflammatory properties. AIM OF THE STUDY: To uncover the effects of GDLI on ischemic stroke patients, as well as the underlying biomarkers involved in sub-acute stroke. MATERIALS AND METHODS: We used a state-of-the-art targeted proteomics chip to investigate the association between numerous serum proteins (1101 proteins) and the sub-acute phase post-ischemic stroke. Then, the relative proteins of anti-apoptosis, anticoagulant, and neuroprotection of GDLI were verified in animal models. RESULTS: Compared with the serum from healthy volunteers, we identified 15 up-regulated proteins and 26 down-regulated proteins (FC ≥ 1.5) involved in inflammatory response, immune response, and nervous system development in the sub-acute ischemic stroke. The pro-inflammatory proteins, such as IL17, MSP-R, G-CSF-R, TLR3, MIP-3ß, TNFRSF19, and TNFRSF12, were significantly increased in serum, illustrating that the chronic inflammatory state was evident in the sub-acute stage of ischemic stroke. However, the common pro-inflammatory proteins, such as IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, and IL-10, known to be up-regulated in acute stroke, had close or lightly lower levels than healthy humans (FC ≥ 1.5, P > 0.05). And some cytokines (IL3, CCL13, TNFRSF3, IL10 R beta, HLA-A, IL-1 F8/FIL1 eta, TNFRSF8, CCL18) were also markedly down-regulated in the sub-acute phase of stroke. These proteins are highly associated with the onset of stroke-induced immunosuppression and post-stroke infection. Moreover, we noticed that Ginkgo Diterpene Lactone Meglumine Injection (GDLI) treatment for 14 days was helpful to the recovery of patients in the subacute period. After the treatment of GDLI, it was observed that several inflammatory cytokines (i.e. IL-17 and IL-28A), chemokine (i.e. CCL14), and Coagulation Factor III were reduced. Meanwhile, the anti-inflammatory cytokines (IL-10 R alpha, GREMLIN, and Activin C) and neurotrophic factors (Neurturin and IGFBP2) were found to be up-regulated in stroke patients through self-control observation. Finally, we identified the IGFBP2 as a novel marker in the animal models. CONCLUSIONS: In summary, the potential markers in sub-acute stroke patients were highly different from known protein markers in the acute phase of ischemic stroke. The serum protein IGFBP2 could be novel biomarkers for the treatment of GDLI in sub-acute stroke patients. Our present findings provide an innovative insight into the novel treatment of GDLI in ischemic stroke therapy.

Ginkgo diterpene lactones inhibit cerebral ischemia/reperfusion induced inflammatory response in astrocytes via TLR4/NF-κB pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. (Ginkgoaceae) is a traditional Chinese medicine known to treating stroke and other cardio-cerebrovascular diseases for thousands of years in China. Ginkgo diterpene lactones (GDL) attracted much attention because of their neuroprotective properties. AIM OF THE STUDY: To uncover the effects of GDL, which consist of ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), on ischemic stroke, as well as the underlying molecular mechanisms. MATERIALS AND METHODS: We used middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) models mimicking the process of ischemia/reperfusion in vivo and in vitro, respectively. Anticoagulant effects of GDL were investigated on platelet activating factor (PAF), arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation both in vivo and in vitro. We also evaluated the effects of GDL on lipopolysaccharide (LPS)-induced inflammatory response in primary cultured rats' astrocytes. Infarct size, neurological deficit score, and brain edema were measured at 72 h after MCAO. Immunohistochemistry was utilized to analyze neurons necrosis and astrocytes activation. Expression of pro-inflammatory cytokines, including tumor necrotic factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected using enzyme-linked immunosorbent assay (ELISA) and real time PCR. The levels of toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) were assessed by real time PCR or Western blot. RESULTS: Compared with MCAO/R rats, GDL significantly reduced infarct size and brain edema, improved neurological deficit score. Meanwhile, GDL suppressed platelet aggregation, astrocytes activation, pro-inflammatory cytokines releasing, TLR4 mRNA expression and transfer of NF-κB from cytoplasm to nucleus. Furthermore, GDL alleviated OGD/R injury and LPS-induced inflammatory response in primary astrocytes, characterized by promoting cell viability, decreasing lactate dehydrogenase (LDH) activity, and inhibiting IL-1ß and TNF-α releasing. CONCLUSIONS: In summary, GDL attenuate cerebral ischemic injury, inhibit platelet aggregation and astrocytes activation. The anti-inflammatory activity might be associated with the downregulation of TLR4/NF-κB signal pathway. Our present findings provide an innovative insight into the novel treatment of GDL in ischemic stroke therapy.

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway.

BACKGROUND AND PURPOSE: Endoplasmic reticulum (ER) stress is increasingly recognized as an important causal factor of many diseases. Targeting ER stress has now emerged as a new therapeutic strategy for treating cardiovascular diseases. Here, we investigated the effects and underlying mechanism of ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) on cardiac ER stress. EXPERIMENTAL APPROACH: Cell death, apoptosis and ER stress-related signalling pathways were measured in cultured neonatal rat cardiomyocytes, treated with the ER stress inducers tunicamycin, hydrogen peroxide and thapsigargin. Acute myocardial infarction was established using left coronary artery occlusion in mice, and infarct size was measured by triphenyltetrazolium chloride staining. Echocardiography was used to assess heart function and transmission electron microscopy for evaluating ER expansion. KEY RESULTS: Ginkgolide K (GK) significantly decreased ER stress-induced cell death in both in vitro and in vivo models. In ischaemic injured mice, GK treatment reduced infarct size, rescued heart dysfunction and ameliorated ER dilation. Mechanistic studies revealed that the beneficial effects of GK occurred through enhancement of inositol-requiring enzyme 1α (IRE1α)/X box-binding protein-1 (XBP1) activity, which in turn led to increased ER-associated degradation-mediated clearance of misfolded proteins and autophagy. In addition, GK was also able to partly repress the pro-apoptotic action of regulated IRE1-dependent decay and JNK pathway. CONCLUSIONS AND IMPLICATIONS: In conclusion, GK acts through selective activation of the IRE1α/XBP1 pathway to limit ER stress injury. GK is revealed as a promising therapeutic agent to ameliorate ER stress for treating cardiovascular diseases.