P38-DAPK1 axis regulated LC3-associated phagocytosis (LAP) of microglia in an in vitro subarachnoid hemorrhage model.

BACKGROUND: The phagocytosis and homeostasis of microglia play an important role in promoting blood clearance and improving prognosis after subarachnoid hemorrhage (SAH). LC3-assocaited phagocytosis (LAP) contributes to the microglial phagocytosis and homeostasis via autophagy-related components. With RNA-seq sequencing, we found potential signal pathways and genes which were important for the LAP of microglia. METHODS: We used an in vitro model of oxyhemoglobin exposure as SAH model in the study. RNA-seq sequencing was performed to seek critical signal pathways and genes in regulating LAP. Bioparticles were used to access the phagocytic ability of microglia. Western blot (WB), immunoprecipitation, quantitative polymerase chain reaction (qPCR) and immunofluorescence were performed to detect the expression change of LAP-related components and investigate the potential mechanisms. RESULTS: In vitro SAH model, there were increased inflammation and decreased phagocytosis in microglia. At the same time, we found that the LAP of microglia was inhibited in all stages. RNA-seq sequencing revealed the importance of P38 MAPK signal pathway and DAPK1 in regulating microglial LAP. P38 was found to regulate the expression of DAPK1, and P38-DAPK1 axis was identified to regulate the LAP and homeostasis of microglia after SAH. Finally, we found that P38-DAPK1 axis regulated expression of BECN1, which indicated the potential mechanism of P38-DAPK1 axis regulating microglial LAP. CONCLUSION: P38-DAPK1 axis regulated the LAP of microglia via BECN1, affecting the phagocytosis and homeostasis of microglia in vitro SAH model. Video Abstract.

Peroxiredoxin 2 Is a Potential Objective Indicator for Severity and the Clinical Status of Subarachnoid Hemorrhage Patients.

Purpose: We have demonstrated that peroxiredoxin 2 (Prx2) released from lytic erythrocytes and damaged neurons into the subarachnoid space could activate microglia and then result in neuronal apoptosis. In this study, we tested the possibility of using Prx2 as an objective indicator for severity of the subarachnoid hemorrhage (SAH) and the clinical status of the patient. Materials and Methods: SAH patients were prospectively enrolled and followed up for 3 months. Cerebrospinal fluid (CSF) and blood samples were collected 0-3 and 5-7 days after SAH onset. The levels of Prx2 in the CSF and the blood were measured by an enzyme-linked immunosorbent assay (ELISA). We used Spearman's rank coefficient to assess the correlation between Prx2 and the clinical scores. Receiver operating characteristic (ROC) curves were used for Prx2 levels to predict the outcome of SAH by calculating the area under the curve (AUC). Unpaired Student's t-test was used to analyze the differences in continuous variables across cohorts. Results: Prx2 levels in the CSF increased after onset while those in the blood decreased. Existing data showed that Prx2 levels within 3 days in the CSF after SAH were positively correlated with the Hunt-Hess score (R = 0.761, P < 0.001). Patients with CVS had higher levels of Prx2 in their CSF within 5-7 days after onset. Prx2 levels in the CSF within 5-7 days can be used as a predictor of prognosis. The ratio of Prx2 in the CSF and the blood within 3 days of onset was positively correlated with the Hunt-Hess score and negatively correlated with Glasgow Outcome Scale (GOS; R = -0.605, P < 0.05). Conclusion: We found that the levels of Prx2 in the CSF and the ratio of Prx2 in the CSF and the blood within 3 days of onset can be used as a biomarker to detect the severity of the disease and the clinical status of the patient.

Alpha-Ketoglutarate Alleviates Neuronal Apoptosis Induced by Central Insulin Resistance through Inhibiting S6K1 Phosphorylation after Subarachnoid Hemorrhage.

Neuronal apoptosis after subarachnoid hemorrhage (SAH) is believed to play an important role in early brain injury after SAH. The energy metabolism of neuron is closely related to its survival. The transient hyperglycemia caused by insulin resistance (IR) after SAH seriously affects the prognosis of patients. However, the specific mechanisms of IR after SAH are still not clear. Studies have shown that α-KG takes part in the regulation of IR and cell apoptosis. In this study, we aim to investigate whether α-KG can reduce IR after SAH, improve the disorder of neuronal glucose metabolism, alleviate neuronal apoptosis, and ultimately play a neuroprotective role in SAH-induced EBI. We first measured α-KG levels in the cerebrospinal fluid (CSF) of patients with SAH. Then, we established a SAH model through hemoglobin (Hb) stimulation with HT22 cells for further mechanism research. Furthermore, an in vivo SAH model in mice was established by endovascular perforation. Our results showed that α-KG levels in CSF significantly increased in SAH patients and could be used as a potential prognostic biomarker. In in vitro model of SAH, we found that α-KG not only inhibited IR-induced reduction of glucose uptake in neurons after SAH but also alleviated SAH-induced neuronal apoptosis. Mechanistically, we found that α-KG inhibits neuronal IR by inhibiting S6K1 activation after SAH. Moreover, neuronal apoptosis significantly increased when glucose uptake was reduced. Furthermore, our results demonstrated that α-KG could also alleviate neuronal apoptosis in vivo SAH model. In conclusion, our study suggests that α-KG alleviates apoptosis by inhibiting IR induced by S6K1 activation after SAH.

N-(2,4-Dinitro-phen-yl)-1,3-dimeth-oxy-isoindolin-2-amine.

In the title compound, C(16)H(16)N(4)O(6), the planes of the isoindole and dinitro-benzene groups make a dihedral angle between of 84.15 (8)°. The N atom of the isoindole group is displaced by 0.2937 (3) Šfrom the plane through the remaining atoms. An intra-molecular N-H⋯O inter-action occurs. In the crystal, inversion dimers linked by pairs of N-H⋯O hydrogen bonds occur.

2-[(E)-(2-Hy-droxy-naphthalen-1-yl)methyl-idene-amino]-isoindoline-1,3-dione.

The title compound, C(19)H(12)N(2)O(3), has two independent mol-ecules (A and B) in the asymmetric unit. There is an intra-molecular O-H⋯N hydrogen bond in each mol-ecule. The mean planes of the naphthalene [maximum deviations = 0.024 (3) and 0.030 (3) Šin A and B, respectively] and the isoindoline units [maximum deviations 0.009 (3) and 0.008 (3) Šin A and B, respectively] are almostly coplanar, with dihedral angles of 4.25 (9) ad 3.84 (9)° in mol-ecules A and B, respectively. The two independent mol-ecules are connected by π-π inter-actions [centroid-centroid distances 3.5527 (19) and 3.5627 (19) Å]. In the crystal, the A+B pairs are further connected via π-π inter-actions [centroid-centroid distances = 3.693 (2)-3.831 (2) Å], leading to the formation of columns propagating along the a-axis direction. The columns are linked via C-H⋯O inter-actions, leading to the formation of a three-dimensional network.

(E)-3-[(2-Hy-droxy-naphthalen-1-yl)methyl-idene-amino]-5-(morpholin-4-yl-meth-yl)-1,3-oxazolidin-2-one.

The title compound, C(19)H(21)N(3)O(4), crystallizes with two independent mol-ecules in the asymmetric unit. In both mol-ecules, there is an intra-molecular O-H⋯N hydrogen bond, which correlates with the fact that each mol-ecule adopts an E configuration with respect to the C=N bond. In the crystal, there are C-H⋯O and C-H⋯π inter-actions present.

(E)-2-[(2-Chloro-benzyl-idene)amino]-isoindoline-1,3-dione.

The title compound, C(15)H(9)ClN(2)O(2), adopts an E configuration about the C=N double bond. The mean plane of the isoindoline ring system [maximum deviation = 0.011 (2) Å] is inclined to the chloro-benzene ring by 22.62 (8)°. In the crystal, mol-ecules are connected by C-H⋯O hydrogen bonds, forming chains that propagate along [010].

(E)-1-[(2-Hy-droxy-1-naphth-yl)methyl-idene-amino]-imidazolidine-2,4-dione.

The title compound, C(14)H(11)N(3)O(3), adopts an E or trans configuration with respect to the C=N bond. In the mol-ecule there is an intra-molecular O-H⋯N hydrogen bond involving the hy-droxy substituent at the 2-positon of the naphthalene ring and the adjacent methyl-ene-amino N atom. The mol-ecule is roughly planar, the dihedral angle between the naphthalene and imidazolidine-2,4-dione mean planes being 8.4 (1)°. In the crystal, pairs of N-H⋯O hydrogen bonds link mol-ecules into inversion dimers. These dimers are futher linked via C-H⋯O inter-actions, forming a three-dimensional network.

Methyl 3-[(E)-(2-hy-droxy-1-naphth-yl)methyl-idene]carbazate.

The title compound, C(13)H(12)N(2)O(3), has an E configuration with respect to the C=N bond: the conformation is stabilized by an intramolecular O-H⋯N hydrogen bond. In the crystal, an N-H⋯O interaction links the molecules into a C(4) chain along [100].

1,2-Bis(4-nitro-benzo-yl)hydrazine.

The title mol-ecule, C(14)H(10)N(4)O(6), crystallizes with one half-mol-ecule in the asymmetric unit; the mid-point of the N-N bond lies on an inversion centre. The nitro and amide groups are twisted with respect to the benzene ring, making dihedral angles of 14.6 (5) and 31.1 (5)°, respectively. In the crystal structure, mol-ecules are linked through N-H⋯O hydrogen bonding between the imino and carbonyl groups.

(E)-N'-(3-Fluoro-benzyl-idene)-2-hydroxy-benzohydrazide.

The title compound, C(14)H(11)FN(2)O(2), adopts an E or trans configuration with respect to the C=N bond. An intra-molecular N-H⋯O hydrogen bond contributes to the relatively planarity of the mol-ecular conformation; the two benzene rings are inclined to one another by 12.5 (2)°. In the crystal structure, inter-molecular O-H⋯O hydrogen bonds link the mol-ecules into chains running parallel to the c axis.

(E)-2-Hydroxy-naphthalene-1-carb-al-de-hyde semicarbazone.

The title compound, C(12)H(11)N(3)O(2), adopts an E or trans configuration with respect to the C=N bond. There is an intra-molecular O-H⋯N hydrogen bond involving the hydroxyl H atom and an N atom of the hydrazine group. In the crystal structure, mol-ecules are connected via N-H⋯O hydrogen bonds, forming a three-dimensional network.

[Study on the interaction between BSA and nicotine].

The interaction of BSA and NIC was investigated by absorption spectra, fluorescence spectroscopy and synchronous fluorescence spectroscopy. In the system of sodium phosphate dibasic-citric acid of 0.1 mol(-1) x L(-1), fluorescence titration showed that the fluorescence intensity of BSA at 342 nm was quenched when NIC was added, NIC was capable of binding with BSA to form a 1:1 complex and the quenching mechanism of BSA affected by NIC was shown to be a static quenching procedure by calculating the binding number n and binding constant K. NIC decreased the intensity of the characteristic absorption peak of BSA, showing that the binding of NIC to BSA had strong impact on protein conformation with the decrease in a-helical content of the protein. Synchronous fluorescence indicated that the binding of NIC to BSA is near tryptophan subunit.

Determination of nitrofuran metabolites in shrimp by high performance liquid chromatography with fluorescence detection and liquid chromatography-tandem mass spectrometry using a new derivatization reagent.

A high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for the simultaneous determination of total nitrofuran metabolite residues (furazolidone, furaltadone, nitrofurantoin, and nitrofurazone) in shrimp was developed. The method involves the acid hydrolysis of protein-bound metabolites, followed by the derivatization of the freed metabolites with the new fluorescent derivatization reagent 2-hydroxy-1-naphthaldehyde (HN) and subsequent liquid-liquid extraction (LLE). Separation is achieved on a YMC-Pack Polymer C18 column under alkaline conditions, and the high fluorescence intensity of the derivatives at an emission wavelength Em=463nm (Ex=395nm) enables, for the first time, their simultaneous determination in shrimp at concentrations as low as 1µg/kg by HPLC-FLD. The method was validated using blank shrimp fortified with all four metabolites at 0.5, 1.0 and 2.0µg/kg. Recoveries were >87% with relative standard deviations of <8.1% for all four metabolites. Furthermore, the results obtained by HPLC-FLD were in very good agreement with those obtained by LC-MS/MS analysis.