The Crosstalk Between Immune Cells After Intracerebral Hemorrhage.

The inflammatory mechanism of intracerebral hemorrhage (ICH) has been widely studied, and it is believed that the regulation of this mechanism is of great significance to the prognosis. In the early stage of the acute phase of ICH, the release of a large number of inflammatory factors around the hematoma can recruit more inflammatory cells to infiltrate the area, further release inflammatory factors, cause an inflammatory cascade reaction, aggravate the volume of cerebral hematoma and edema and further destroy the blood-brain barrier (BBB), according to this, the crosstalk between cells may be of great significance in secondary brain injury (SBI). Because most of the cells recruited are inflammatory immune cells, this paper mainly discusses the cells based on the inflammatory mechanism to discuss their functions after ICH, we found that among the main cells inherent in the brain, glial cells account for the majority, of which microglia are the most widely studied and it can interact with a variety of cells, which is reflected in the literature researches on its pathogenesis and treatment. We believe that exploring multi-mechanism and multi-cell regulated drugs may be the future development trend, and the existing research, the comparison and unification of modeling methods, and the observation of long-term efficacy may be the first problem that researchers need to solve.

[Identification of chemical constituents in Simiao Yong'an Decoction based on UPLC-LTQ-Orbitrap-MS].

This study aims to identify the chemical constituents of Simiao Yong'an Decoction based on ultra-performance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS). The elution was performed through a UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 µm) with the mobile phase of water(containing 0.1% formic acid)-acetonitrile at a flow rate of 0.4 mL·min~(-1). LTQ-Orbitrap-MS with heat electrospray ion(HESI) source was employed to collect MS fragment information in the negative ion mode. A total of 72 compounds were identified based on reference substance comparison, fragmentation rules, accurate molecular weight, related reports and databases(MassBank and HMDB), including 30 iridoid glycosides, 9 organic acids, 15 flavonoids, 10 phenylpropanoids, 7 triterpenoids, and 1 saccharide. The method established in this study is comprehensive, rapid, and accurate, which can help summarize the fragmentation rules of constituents and provide reference for revealing the active constituents and pharmacodynamic mechanism of Simiao Yong'an Decoction.

Comparative Pharmacokinetics of Seven Major Compounds in Normal and Atherosclerosis Mice after Oral Administration of Simiao Yong'an Decoction.

Simiao Yong'an decoction (SMYAD), a classic traditional Chinese medicine formula, has been used to treat atherosclerosis (AS) in clinical in China, but its therapeutic mechanism and pharmacodynamic material basis are not clear. In this study, the AS model was caused by a high-fat diet and perivascular carotid collar placement (PCCP), and SMYAD was orally administered to the model and normal mice. A rapid, sensitive, selective, and reliable method using ultrahigh-performance liquid chromatography (UHPLC) system combined with a Q Exactive HF-X mass spectrometer (UHPLC-Q Exactive HF-X MS) was established and validated for the simultaneous determination of seven compounds, including harpagide, chlorogenic acid, swertiamarin, sweroside, angoroside C, liquiritin, and isoliquiritigenin in the plasma of normal and AS mice. The specificity, linearity, precision, accuracy, recovery, and stability of the method were all within the acceptable criteria. The results showed that some pharmacokinetic behaviors of harpagide, chlorogenic acid, and isoliquiritigenin were significantly different among the two groups of mice. The specific parameter changes were harpagide (AUC0-t and AUC0-∞ were 11075.09 ± 2132.38 and 16221.95 ± 5622.42 ng·mL-1·h, respectively; CLz/F was 2.45 ± 0.87 L/h/mg), chlorogenic acid (t 1/2 was 21.59 ± 9.16 h; AUC0-∞ was 2637.51 ± 322.54 ng·mL-1·h; CLz/F was 13.49 ± 1.81 L/h/mg) and isoliquiritigenin (AUC0-t and AUC0-∞ were 502.25 ± 165.65 and 653.68 ± 251.34 ng·mL-1·h, respectively; CLz/F was 62.16 ± 23.35 L/h/mg) were altered under the pathological status of AS. These differences might be partly ascribed to the changes in gastrointestinal microbiota, nonspecific drug transporters, and cytochrome P450 activity under the AS state, providing research ideas and experimental basis for pharmacological effects and pharmacodynamic material basis.

Mechanistic Insight into PPARγ and Tregs in Atherosclerotic Immune Inflammation.

Atherosclerosis (AS) is the main pathological cause of acute cardiovascular and cerebrovascular diseases, such as acute myocardial infarction and cerebral apoplexy. As an immune-mediated inflammatory disease, the pathogenesis of AS involves endothelial cell dysfunction, lipid accumulation, foam cell formation, vascular smooth muscle cell (VSMC) migration, and inflammatory factor infiltration. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in lipid metabolism, inflammation, and apoptosis by antagonizing the Wnt/ß-catenin pathway and regulating cholesterol efflux and inflammatory factors. Importantly, PPARγ-dependant fatty acid uptake is critical for metabolic programming. Activated PPARγ can exert an anti-atherosclerotic effect by inhibiting the expression of various inflammatory factors, improving endothelial cell function, and restraining the proliferation and migration of VSMCs. Regulatory T cells (Tregs) are the only subset of T lymphocytes that have a completely negative regulatory effect on the autoimmune response. They play a critical role in suppressing excessive immune responses and inflammatory reactions and widely affect AS-associated foam cell formation, plaque rupture, and other processes. Recent studies have shown that PPARγ activation promotes the recruitment of Tregs to reduce inflammation, thereby exerting its anti-atherosclerotic effect. In this review, we provide an overview of the anti-AS roles of PPARγ and Tregs by discussing their pathological mechanisms from the perspective of AS and immune-mediated inflammation, with a focus on basic research and clinical trials of their efficacies alone or in combination in inhibiting atherosclerotic inflammation. Additionally, we explore new ideas for AS treatment and plaque stabilization and establish a foundation for the development of natural PPARγ agonists with Treg recruitment capability.

Folate polyglutamylation eliminates dependence of activity on enzyme concentration in mitochondrial serine hydroxymethyltransferases from Arabidopsis thaliana.

The reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT) is the major one-carbon unit source for essential metabolic processes. The Arabidopsis thaliana genome encodes seven SHMT isozymes localized in mitochondria, plastids, nuclei, and the cytosol. Knowledge of the biochemical properties of each isozyme is central to understanding and manipulating one-carbon metabolism in plants. We heterologously expressed and purified three recombinant SHMTs from A. thaliana (AtSHMTs) putatively localized in mitochondria (two) and the cytosol (one). Their biochemical properties were characterized with respect to the impact of folate polyglutamylation on substrate saturation kinetics. The two mitochondrial AtSHMTs, but not the cytosolic one, had increased turnover rates at higher (>0.4ng/µL) enzyme concentrations in the presence of monoglutamylated folate substrates, but not in the presence of pentaglutamylated folate substrates. We found no experimental support for a change in oligomerization state over the range of enzyme concentration studied. Modeling of the enzyme structures presented features that may explain the activity differences between the mitochondrial and cytosolic isozymes.

Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions.

One-carbon metabolism in plants: characterization of a plastid serine hydroxymethyltransferase.

SHMT (serine hydroxymethyltransferase; EC 2.1.2.1) catalyses reversible hydroxymethyl group transfer from serine to H4PteGlun (tetrahydrofolate), yielding glycine and 5,10-methylenetetrahydrofolate. In plastids, SHMTs are thought to catalytically direct the hydroxymethyl moiety of serine into the metabolic network of H4PteGlun-bound one-carbon units. Genes encoding putative plastid SHMTs were found in the genomes of various plant species. SHMT activity was detected in chloroplasts in pea (Pisum sativum) and barley (Hordeum vulgare), suggesting that plastid SHMTs exist in all flowering plants. The Arabidopsis thaliana genome encodes one putative plastid SHMT (AtSHMT3). Its cDNA was cloned by reverse transcription-PCR and the encoded recombinant protein was produced in Escherichia coli. Evidence that AtSHMT3 is targeted to plastids was found by confocal microscopy of A. thaliana protoplasts transformed with proteins fused to enhanced green fluorescent protein. Characterization of recombinant AtSHMT3 revealed that substrate affinity for and the catalytic efficiency of H4PteGlu1-8 increase with n, and that H4PteGlu1-8 inhibit AtSHMT3. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate with one and five glutamate residues inhibited AtSHMT3-catalysed hydroxymethyl group transfer from serine to H4PteGlu6, with the pentaglutamylated inhibitors being more effective. Calculations revealed inhibition with 5-methyltetrahydrofolate or 5-formyltetrahydrofolate resulting in little reduction in AtSHMT3 activity under folate concentrations estimated for plastids.

An HPLC-based fluorometric assay for serine hydroxymethyltransferase.

We have developed a novel HPLC-based fluorometric assay for serine hydroxymethyltransferase activity. In this assay, the 5,10-CH(2)-H(4)PteGlu formed by serine hydroxymethyltransferase activity is reduced to 5-CH(3)-H(4)PteGlu using NaBH(4). Then the fluorescent assay components are separated by reversed-phase chromatography under isocratic conditions and 5-CH(3)-H(4)PteGlu is quantified by comparison with standards. We show that this assay can be used to measure serine hydroxymethyltransferase activity at 10(-8) to 10(-3)M (6R,S)-H(4)PteGlu.