Establishment of LC-MS/MS Methodology for the Determination of Rapamycin Concentration in Rat Whole Blood.

BACKGROUND: The establishment and validation of methods for testing biological samples are crucial steps in pharmacokinetic studies. Currently, several methodological reports have been published on the detection of rapamycin plasma concentrations. OBJECTIVE: The objective of this study was to explore an effective method for detecting rapamycin in rat whole blood biological samples. METHOD: In this study, we designed a rapid, sensitive, and specific liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) methodology for detecting rapamycin in rat whole blood biological samples. We comprehensively validated the specificity, linear range, lower limit of quantification (LLOQ), precision, accuracy, recovery, and stability of this method. RESULTS: The findings of this study confirmed the successful implementation of LC-MS/MS for the detection of rapamycin, demonstrating its sensitivity, specificity, and reliability in quantitative analysis. This method ensures the accuracy and reliability of subsequent study data through our validated LC-MS/MS approach. CONCLUSION: The results demonstrated the successful implementation of an LC-MS/MS method for sensitive, specific, and reliable quantitative analysis of rapamycin in rat whole blood samples. This method ensures the accuracy and reliability of subsequent study data. SIGNIFICANCE: The importance of this study lies in the successful establishment of a rapid, sensitive, and specific LC-MS/MS method for detecting rapamycin concentration in rat whole blood, ensuring the accuracy and reliability of subsequent research data. This provides a crucial tool and foundation for further understanding the metabolism and pharmacological effects of rapamycin in vivo, aiding in the advancement of drug research and clinical applications in related fields.

RIPK1 inhibition mitigates neuroinflammation and rescues depressive-like behaviors in a mouse model of LPS-induced depression.

BACKGROUND: Depression is often linked to inflammation in the brain. Researchers have been exploring ways to reduce this inflammation to improve depression symptoms. One potential target is a protein called RIPK1, which is known to contribute to brain inflammation. However, it's unclear how RIPK1 influences depression. Our study aims to determine whether RIPK1 inhibition could alleviate neuroinflammation-associated depression and elucidate its underlying mechanisms. METHODS: To investigate our research objectives, we established a neuroinflammation mouse model by administering LPS. Behavioral and biochemical assessments were conducted on these mice. The findings were subsequently validated through in vitro experiments. RESULTS: Using LPS-induced depression models, we investigated RIPK1's role, observing depressive-like behaviors accompanied by elevated cytokines, IBA-1, GFAP levels, and increased inflammatory signaling molecules and NO/H2O2. Remarkably, Necrostatin (Nec-1 S), a RIPK1 inhibitor, mitigated these changes. We further found altered expression and phosphorylation of eIF4E, PI3K/AKT/mTOR, and synaptic proteins in hippocampal tissues, BV2, and N2a cells post-LPS treatment, which Nec-1 S also ameliorated. Importantly, eIF4E inhibition reversed some of the beneficial effects of Nec-1 S, suggesting a complex interaction between RIPK1 and eIF4E in LPS-induced neuroinflammation. Moreover, citronellol, a RIPK1 agonist, significantly altered eIF4E phosphorylation, indicating RIPK1's potential upstream regulatory role in eIF4E and its contribution to neuroinflammation-associated depression. CONCLUSION: These findings propose RIPK1 as a pivotal mediator in regulating neuroinflammation and neural plasticity, highlighting its significance as a potential therapeutic target for depression.

Maternal prenatal systemic inflammation indexes predicts premature neonatal respiratory distress syndrome.

Neonatal respiratory distress syndrome (NRDS) is one of the leading causes of neonatal mortality in low-income countries. It is caused by a lack of surface-active substances in the lungs, and the maternal inflammatory response plays an important role in the formation of surface-active substances in the fetal lungs. We aimed to investigate the correlation between maternal prenatal systemic inflammatory indices and respiratory distress syndrome in preterm neonates. This is a retrospective case-control study that collected data from all patients who delivered between 28 and 36 weeks of gestation at Longhua District People's Hospital in Shenzhen City and whose infants were admitted to the neonatal unit, newborns with respiratory distress syndrome were in the experimental group (NRDS group), and newborns without NRDS were in the control group (non-NRDS group). To minimize the effect of confounders on the results, propensity score matching was performed on baseline characteristics. Totally, 524 patients were included (93 in the NRDS group and 431 in the non-NRDS group), and 71 matched pairs (142 patients). The neutrophil-to-lymphocyte ratio (NLR), derived neutrophil-to-lymphocyte ratio (dNLR), systemic immune-inflammation index (SII), systemic inflammation response index (SIRI), aggregate index of systemic inflammation (AISI) and neutrophil lymphocyte to platelet ratio (NLPR) were higher in the NRDS group than in the non-NRDS group (p < 0.05). The ROC curves of NLR, dNLR, SII, SIRI, AISI and NLPR for the diagnosis of NRDS were plotted, and it was found that the combined diagnostic efficacy of these six systemic inflammatory markers was better (AUC: 0.643, P = 0.003). Patients were divided into two groups based on the cut-off values determined from the ROC curves, and analysis using binary regression models revealed that SII ≥ 1199.94 (OR, 2.554; 95% CI 1.245-5.239, P = 0.011) and NLPR ≥ 0.0239 (OR, 2.175; 95% CI 1.061-4.459, P = 0.034) were independent risk factors predicting NRDS. Maternal prenatal SII ≥ 1199.94 and NLPR ≥ 0.0239 are independent risk factors for NRDS, and clinicians may be used to prevent neonatal respiratory distress in advance to reduce the incidence of NRDS.

Ceftriaxone averts neuroinflammation and relieves depressive-like behaviors via GLT-1/TrkB signaling.

The beneficial effect of a beta-lactam antibiotic, Ceftriaxone (CEF), to improve depressive-like symptoms has been documented previously, attributed to its modulation of glutamate neurotransmission. Here, we aimed to determine whether CEF could improve LPS-altered glutamatergic signaling associated with neuroinflammation-allied depression. To assess our goals, we established a neuroinflammation-allied depression mice model by injecting lipopolysaccharides (LPS), followed by behavioral and biochemical analysis. LPS-treated mice displayed depressive symptoms, neuroinflammation, dysregulated glutamate and its transporter (GLT-1) expression, altered expression of astrocyte reactive markers (GFAP, cxcl10, steap4, GBP2, and SRGN), and dysregulated BDNF/TrkB signaling. However, these changes were rescued by CEF treatment, as we found decreased neuroinflammation, relief of depression symptoms, and improved GLT-1 and BDNF/TrkB signaling upon CEF treatment. Moreover, GLT-1 and BDNF/TrkB regulation role of CEF was validated by K252a and DHK treatment. In summary, the anti-depressive effects of glutamate modulators, like CEF, are closely related to their anti-inflammatory role.

EPO prevents neuroinflammation and relieves depression via JAK/STAT signaling.

AIMS: Erythropoietin (EPO) is a glycoprotein cytokine that exerts therapeutic potential on neurological disorders by promoting neurogenesis and angiogenesis. However, its role as an antidepressant via anti-inflammatory axes is poorly explored. Furthermore, chronic inflammation can induce neuroinflammation, concurrent with depressive-like behaviors that anti-inflammatory and antidepressant agents could avert. Here, we aimed to elucidate the antidepressant potential of Erythropoietin (EPO) in the LPS-induced depression model. MAIN METHODS: For in vivo analysis, mice were treated with LPS (2 mg/kg BW), Erythropoietin (EPO) (5000 U/kg/day), (Ruxolitinib,15 mg/kg), and K252a (25 µg/kg). Depressive-like behaviors were confirmed via behavior tests, including OFT, FST, SPT, and TST. Cytokines were measured via ELISA, while IBA-1/GFAP expression was determined by immunofluorescence. Further, the desired gene expression was measured by immunoblotting. For in vitro analysis, BV2 and N2a cell lines were cultured, treated with LPS, EPO, Ruxolitinib, and K252a, collected, and analyzed. KEY FINDINGS: LPS treatment significantly induced neuroinflammation accompanied by depression-like behaviors in mice. However, EPO treatment rescued LPS-induced changes by averting cytokine production, secretion, and glial cell activation and reducing depressive-like behaviors in mice. Surprisingly, EPO treatment ameliorated LPS-induced JAK2/STAT5 signaling impairment, as validated by JAK2-antagonism. Furthermore, synaptic and dendritic spine defects and BNDF/TrkB signaling upon LPS administration could be prevented by EPO treatment. SIGNIFICANCE: EPO could act as an antidepressant via its anti-inflammatory potential by regulating JAK2/STAT5 signaling.

Clinical Manifestations of Neonatal Hyperbilirubinemia Are Related to Alterations in the Gut Microbiota.

BACKGROUND AND PURPOSE: Neonatal hyperbilirubinemia, also known as neonatal jaundice, is a common and frequent clinical condition with a complex etiology that can lead to brain damage in severe cases. Early recognition of hyperbilirubinemia and timely intervention and treatment can help reduce the occurrence of sequelae. This study was conducted to identify whether the gut microbiota composition can distinguish neonates with hyperbilirubinemia. METHODS: Meconium samples were collected from 69 neonates with neonatal jaundice (NJ) and 69 age- and sex-matched neonates without clinically significant jaundice (healthy controls; HCs) for 16S rRNA gene sequencing and microbiome analysis. RESULTS: Compared with HCs, the Chao 1 richness index of the gut microbiota was significantly decreased in the NJ group. The relative abundance of the probiotic gut bacterium, Lactobacillus, was significantly lower in the NJ group than in the HC group, whereas the abundances of potentially harmful gut bacteria, such as Escherichia coli and Staphylococcus, were significantly higher in the NJ group than in HCs. Correlation of the gut microbiota and clinical indicators revealed a positive correlation between Escherichia coli/Staphylococcus and serum total bilirubin levels. Finally, the results of a random forest machine-learning method to evaluate the possibility of using NJ-associated gut microbiota compositions as potential NJ biomarkers revealed an area under the curve of 96.88%. CONCLUSIONS: The abundances of Escherichia coli and Staphylococcus were positively correlated with serum total bilirubin levels. Hence, the gut microbiota composition is a potential biomarker of NJ.

miR-214-3p Deficiency Enhances Caspase-1-Dependent Pyroptosis of Microglia in White Matter Injury.

White matter injury (WMI) is the most frequent impairment of neurodevelopment in preterm infants. Here, we report that the caspase-1 inflammasome is abundantly activated in the microglia of WMI mice and results in increased pyroptosis of microglia. Pharmacology inhibition of caspase-1 cleavage alleviated the pathogenesis of WMI mice. The expression of microRNA miR-214-3p was largely reduced in the microglia of WMI mice compared to controls. Compromised expression of miR-214-3p on microglia gives rise to the inflammasome activation and microglial pyroptosis. Treatment with miR-214-3p agomir is sufficient to relieve the white matter lesion and demyelination in WMI mice. miR-214-3p is able to bind to the 3' region of the NLRP-3 inflammasome compartment NEK7, preventing the transcription of NEK7 mRNA. As a result, in WMI mice, the lack of miR-214-3p leads to the accumulation of NEK7 which supports NLRP 3 inflammasome activation, microglial pyroptosis, and white matter pathogenesis.

Knockdown of G protein-coupled receptor-17 (GPR17) facilitates the regeneration and repair of myelin sheath post-periventricular leukomalacia (PVL).

The G protein-coupled receptor-17 (GPR17) plays an important role in regulating the differentiation of oligodendrocytes and remyelination, which is a key negative regulator of oligodendrocyte differentiation. The present study aimed to investigate the function of GPR17 in the white matter of periventricular leukomalacia (PVL) neonatal rats. The PVL model was established in 2-day old neonatal rats by intracerebral injection of LPS (1 mg/kg). Compared to sham, GPR17 was significantly upregulated, while Olig1 was significantly downregulated in the PVL group at 1 d, 3 days, and 7 days post-modeling. Compared to the negative control (NC) group, the expression of GPR17 was suppressed, while that of Olig1 was elevated in the siRNA-GPR17 group as time progressed; the opposite results were observed in the GPR17-overexpressed group. Decreased formation of myelin sheaths as well as poor structure and loose arrangement were observed in the PVL group. Similar observations were found in the PVL + siRNA-GPR17 group at 1 d and 3 days post-modeling. However, on day 7 post-modeling, a dramatic increase in the formation of myelin sheath as well as thicker myelin sheaths were observed in the PVL + siRNA-GPR17 group. The migration ability of oligodendrocyte progenitor cells (OPCs) isolated from animals was found to be significantly suppressed in the GPR17-overexpressed group, accompanied by the downregulation of Olig1. Taken together, the regeneration and repair of myelin sheaths post-PVL white matter injury were induced by downregulating the GPR17 gene, which elevated the expression of Olig1.

[Correlation between end-tidal carbon dioxide and partial pressure of arterial carbon dioxide in ventilated newborns].

OBJECTIVE: To study the correlation between end-tidal carbon dioxide (PetCO2) and partial pressure of arterial carbon dioxide (PaCO2) in ventilated newborns. METHODS: Thirty-one ventilated newborn underwent mainstream PetCO2 monitoring; meanwhile, arterial blood gas analysis was performed. The correlation and consistency between PetCO2 and PaCO2 were assessed. RESULTS: A total of 85 end-tidal and arterial CO2 pairs were obtained from 31 ventilated newborns. The mean PetCO2 (41±10 mm Hg) was significantly lower than the corresponding mean PaCO2 (46±11 mm Hg) (P<0.01). There was a significant positive correlation between PetCO2 and PaCO2 (r=0.92, P<0.01). The overall PetCO2 bias was 5.1±4.3 mm Hg (95% limits of consistency, -3.3 to 13.6 mmHg), and 5% (4/85) of the points were beyond the 95%CI. When the oxygenation index (OI) was less than 300 mm Hg (n=48), there was a significant positive correlation between PetCO2 and PaCO2 (r=0.85, P<0.01); the PetCO2 bias was 5.9±4.3 mm Hg (95% limits of consistency, -2.6 to 14.5 mm Hg), and 4.2% (2/48) of the points were beyond the 95%CI. When the OI was more than 300 mm Hg (n=37), there was also a significant positive correlation between PetCO2 and PaCO2 (r=0.91, P<0.01); the PetCO2 bias was 4.1±4.1 mm Hg (95% limits of consistency, -3.9 to 12.1 mm Hg), and 5% (2/37) of the points were beyond the 95%CI. CONCLUSIONS: There is a good correlation and consistency between PetCO2 and PaCO2 in ventilated newborns.

Diphenyleneiodonium protects preoligodendrocytes against endotoxin-activated microglial NADPH oxidase-generated peroxynitrite in a neonatal rat model of periventricular leukomalacia.

The contribution of microglial activation to preoligodendroglial (preOL) damage in the central nervous system (CNS) is considered to be one of the principal causes of periventricular leukomalacia (PVL) pathogenesis. The present study explores the effect of diphenyleneiodonium (DPI), a NADPH oxidase (NOX) inhibitor, on protection of preOLs from bacterial lipopolysaccharide (LPS)-induced microglial toxicity in vivo and in vitro. In vitro, preOLs co-cultured with microglia exhibited increased preOL apoptosis, accompanied by overproduction of superoxide anion (O(2)(-)) and the formation of peroxynitrite (ONOO(-)) after LPS exposure. LPS also significantly up-regulated accumulation of activated microglial NOX subunits p67-phox and gp91-phox in the plasma membrane. Diphenyleneiodonium (DPI) (10µm) was found to significantly attenuate up-regulation of this NOX activity. In vivo, DPI was administered (1mg/kg/day) by subcutaneous injection for 3 days to two-day-old neonatal Sprague-Dawley rats subjected to intracerebral injection of LPS. Treatment with DPI within 24h of LPS injection significantly ameliorated white matter injury, decreasing preOL loss, O(2)(-) generation, and ONOO(-) formation, and inhibiting p67-phox, gp91-phox synthesis and p67phox membrane translocation in microglia. These results indicated that LPS-induced preOL apoptosis may have been mediated by microglia-derived ONOO(-). DPI prevented this LPS-induced brain injury, most likely by inhibiting ONOO(-) formation via NOX, thereby preventing preOL loss and immature white matter injury.

Curcumin protects pre-oligodendrocytes from activated microglia in vitro and in vivo.

Infection and inflammation leading to injury or death of pre-oligodendrocytes (preOLs) is one of the principal initiating mechanisms in the pathogenesis of preterm periventricular leukomalacia (PVL). The present study explores the possible protective effect of curcumin against the toxicity of lipopolysaccharide (LPS)-activated microglia on preOLs in vitro and in vivo. In vitro, preOLs in coculture with microglia exhibited increased apoptosis after exposure to LPS. LPS also induced significantly increased expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase (NOX) subunits, p67-phox and gp91-phox in microglia. Our results suggest that iNOS and NOX contribute to the apoptosis of preOLs by activated microglia. The potential anti-inflammatory effects of curcumin were tested to determine if they could help to minimize microglia-mediated damage. Curcumin (10 microg/ml) was found to significantly inhibit the apoptosis of preOL and expression of either iNOS or NOX in the LPS-activated microglia. In vivo, curcumin was administered (50 mg/kg/day, i.p.) to two-day-old neonatal Sprague-Dawley rats subjected to intracerebral injection of LPS. Treatment with curcumin either 1h before or immediately after LPS injection significantly ameliorated white matter injury and loss of preOLs, decreased activated microglia, and inhibited microglial expression of iNOS and translocation of p67phox and gp91phox to the microglial cell membranes in neonatal rat brains following LPS injection. These results suggest that curcumin has a protective effect on infection-driven white matter injury, which is associated with suppression of iNOS and NOX activation. Consequently, curcumin may have potential as a protective agent against immature white matter injury.

[The construction and identification of siRNA expression vector against hypoxia inducible factor -1 alpha in vitro].

OBJECTIVE: Previous studies have suggested that under hypoxic conditions hypoxia inducible factor-1 alpha (HIF-1 alpha) contributes to the progression of neonatal pulmonary hemorrhage (NPH) by increasing the expression of endothelin-1 (ET-1) gene. RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene-silencing mediated by double-stranded RNA. This new gene-silencing technique has recently been shown to be a powerful approach for studying gene function. The aim of this study was to construct the small interfering RNA (siRNA) eukaryotic expression vectors specific to human HIF-1 alpha gene (pSIREN-Shuttle HIF-1 alpha siRNAin order to observe its silencing effect on HIF-1 alpha under hypoxic conditions. METHODS: Six potential siRNA target sites (a-fspecific to human HIF-1 alpha gene were designed and synthesized to two complementary oligonucleotides (A-F) for each siRNA target site. Using a gene recombination technique, the recombinant expression vectors (A-F') were constructed by cloning the double strands oligonucleotide into RNAi-Ready pSIREN vector. The recombinant vectors were then transfected into the cultured human umbilical vein endothelial cells (HUVECs). After 48 hrs of culture, the cells were treated with CoCl2 (100 mu M) for 3 hrs. Expression of HIF-1 alpha mRNA and protein was detected using RT-PCR and Western blot. ET-1 level in cell culture supernates was detected using ELISA. RESULTS: The recombinant HIF-1 alpha siRNA eukaryotic expression vectors A'-F'respectively aiming at sites (a-f) were constructed successfully. Compared to the non-transfection group, liposome-mediated gene transfection of pSIREN-Shuttle HIF-1 alpha siRNA expression vectors into HUVECs obviously down-regulated the mRNA and protein levels of HIF-1 alpha, and partly decreased the ET-1 level in the B' and D' transfection groups. CONCLUSIONS: The specific pSIREN-Shuttle HIF-1 alpha siRNA expression vectors B' and D' aiming at b and d sites can inhibit the expression of HIF-1 alpha, thus decreasing the level of its target gene ET-1. This may be helpful to study the relationship between HIF-1 alpha and neonatal pulmonary hemorrhage in vivo in future.

[Effect of NADPH oxidase on hypoxia-inducible factor-1alpha and endothelin-1 expression in human umbilical endothelial cells and its possible mechanism].

OBJECTIVE: To study the effect of NADPH oxidase on hypoxia-inducible factor (HIF)-1alpha and endothelin (ET)-1 expression in human umbilical endothelia cells (HUVECs) and its possible mechanism. METHODS: Twenty-five bottles of HUVECs culture fluid were randomly assigned into five groups: group A (normoxic control), group B (hypoxic), group C (NADPH oxidase inhibitor apocynin + normoxic), group D (H2O2 which can degrade HIF-1alpha rapidly+hypoxic) and group E (H2O2+apocynin+normoxic), with five bottles in each group. The culture supernates were collected and the total protein was extracted 3 hrs after treatment. Western Blot and ELISA were used to detect the HIF-1alpha protein expression in HUVECs and the ET-1 level in the culture supernates respectively. RESULTS: There was a lower expression of HIF-1alpha protein (0.336 +/- 0.012) and lower ET-1 levels (5.87 +/- 2.22 pg/mL) in group A. The HIF-1alpha protein expression in groups B and C (0.773 +/- 0.018 and 0.888 +/- 0.022) and ET-1 levels (95.38 +/- 8.06 and 33.67 +/- 4.21 pg/mL) were noticeably higher than in group A (P < 0.05). The groups D and E had the HIF-1alpha protein expression levels similar to group A, but the ET-1 levels in group D (108.43 +/- 8.38 pg/mL) and group E (109.66 +/- 5.80 pg/mL) were significantly higher than in group A (P < 0.05). CONCLUSIONS: Hypoxia or apocynin can increase the HIF-1alpha and ET-1 expression in HUVECs. H2O2 can inhibit the HIF-1alpha expression but increase the ET-1levels. It is speculated that NADPH oxidase as an oxygen sensor regulates the HIF-1alpha expression by changing the intracellular redox reaction and that except HIF-1, H2O2 might contribute to ET-1 synthesis and release.