Resveratrol suppresses microglial activation and promotes functional recovery of traumatic spinal cord via improving intestinal microbiota.

Spinal cord injury (SCI) can change the intestinal microbiota pattern and corresponding metabolites, which in turn affect the prognosis of SCI. Among many metabolites, short-chain fatty acids (SCFAs) are critical for neurological recovery after SCI. Recent research has shown that resveratrol exerts anti-inflammatory properties. But it is unknown if the anti-inflammatory properties of resveratrol are associated with intestinal microbiota and metabolites. We thus investigate the alteration in gut microbiota and the consequent change of SCFAs following resveratrol treatment. The SCI mouse models with retention of gut microbiota (donor) and depletion of gut microbiota (recipient) were established. Fecal microbiota transplantation from donors to recipients was performed with intragastrical administration. Spinal cord tissues of mice were examined by H&E, Nissl, and immunofluorescence stainings. The expressions of the inflammatory profile were examined by qPCR and cytometric bead array. Fecal samples of mice were collected and analyzed with 16S rRNA sequencing. The results showed that resveratrol inhibited the microglial activation and promoted the functional recovery of SCI. The analysis of intestinal microbiota and metabolites indicated that SCI caused dysbiosis and the decrease in butyrate, while resveratrol restored microbiota pattern, reversed intestinal dysbiosis, and increased the concentration of butyrate. Both fecal supernatants from resveratrol-treated donors and butyrate suppressed the expression of pro-inflammatory genes in BV2 microglia. Our result demonstrated that fecal microbiota transplantation from resveratrol-treated donors had beneficial effects on the functional recovery of SCI. One mechanism of resveratrol effects was to restore the disrupted gut microbiota and butyrate.

Identification of mitochondrial function-associated lncRNAs in septic mice myocardium.

The present study aimed to analyze long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiles in septic mice heart and to identify potential lncRNAs and mRNAs that be responsible for cardiac mitochondrial dysfunction during sepsis. Mice were treated with 10 mg/kg of lipopolysaccharides to induce sepsis. LncRNAs and mRNAs expression were evaluated by using lncRNA and mRNA microarray or real-time polymerase chain reaction technique. LncRNA-mRNA coexpression network assay, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. The results showed that 1275 lncRNAs were differentially expressed in septic myocardium compared with those in the control group. A total of 2769 mRNAs were dysregulated in septic mice heart, most of which are mainly related to the process of inflammation, mitochondrial metabolism, oxidative stress, and apoptosis. Coexpression network analysis showed that 14 lncRNAs were highly correlated with 11 mitochondria-related differentially expressed mRNA. Among all lncRNAs and their cis-acting mRNAs, 41 lncRNAs-mRNA pairs (such as NONMMUG004378 and Apaf1 gene) were enriched in GO terms and KEGG pathways. In summary, we gained some specific lncRNAs and their potential target mRNAs that might be involved in mitochondrial dysfunction in septic myocardium. These findings provide a panoramic view of lncRNA and might allow developing new treatment strategies for sepsis.

Local Delivery of ß-Elemene Improves Locomotor Functional Recovery by Alleviating Endoplasmic Reticulum Stress and Reducing Neuronal Apoptosis in Rats with Spinal Cord Injury.

BACKGROUND/AIMS: Spinal cord injury (SCI) is a serious global problem that leads to permanent motor and sensory deficits. This study explores the anti-apoptotic and neuroprotective effects of the natural extract ß-elemene in vitro and in a rat model of SCI. METHODS: CCK-8 assay was used to evaluate cell viability and lactate dehydrogenase assay was used to evaluate cytotoxicity. A model of cell injury was established using cobalt chloride. Apoptosis was evaluated using a fluorescence-activated cell sorting assay of annexin V-FITC and propidium iodide staining. A rat SCI model was created via the modified Allen's method and Basso, Beattie, and Bresnahan (BBB) scores were used to assess locomotor function. Inflammatory responses were assessed via enzyme-linked immunosorbent assay (ELISA). Apoptotic and surviving neurons in the ventral horn were respectively observed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Nissl staining. Western blotting was used to measure protein expression. RESULTS: ß-elemene (20 µg/ml) promoted cell viability by activating phosphorylation of the PI3K-AKT-mTOR pathway. ß-elemene reduced CoCl2-induced cellular death and apoptosis by suppressing the expression levels of CHOP, cleaved-caspase 12, 78-kilodalton glucose-regulated protein, cleaved-caspase 3, and the Bax/Bcl-2 ratio. In the rat model of SCI, Nissl and TUNEL staining showed that ß-elemene promoted motor neuron survival and reduced neuronal apoptosis in the spinal cord ventral horn. BBB scores showed that ß-elemene significantly promoted locomotor behavioral recovery after SCI. In addition, ß-elemene reduced the ELISA-detected secretion of interleukin (IL)-6 and IL-1ß. CONCLUSION: ß-elemene reduces neuronal apoptosis by alleviating endoplasmic reticulum stress in vitro and in vivo. In addition, ß-elemene promotes locomotor function recovery and tissue repair in SCI rats. Thus, our study provides a novel encouraging strategy for the potential treatment of ß-elemene in SCI patients.

Altered Profiles of Gut Microbiota in Klebsiella pneumoniae-Induced Pyogenic Liver Abscess.

Intestinal microbiota plays a crucial role in preventing the colonization and invasion by pathogens, and disruption of microbiota may cause opportunistic infections and diseases. Pathogens often have strategies to escape from the colonization resistance mediated by microbiota, but whether they also modulate the microbiota composition is still a topic of investigation. In the present study, we addressed this question using an opportunistic pathogen, Klebsiella pneumoniae serotype K1, which is known to cause pyogenic liver abscess (KLA) in about 30% of mice. We examined the effect of K. pneumoniae infection on cecal microbiota composition by performing high-throughput 454 pyrosequencing of the hypervariable V3-V4 regions of bacterial 16S rRNA gene. Our data revealed that K. pneumoniae inoculation substantially changed the cecal microbiota composition when analyzed at the phylum, order, and family levels. Most strikingly, the KLA-infected mice had significantly increased abundance of Bacteroidales and Enterobacteriales and decreased abundance of Lactobacillales and Eggerthellales. Furthermore, by comparing the infected mice with or without KLA disease symptoms, we identified specific microbiota changes associated with the KLA disease induction. Especially, the KLA group had dramatically decreased sequence identical to Lactobacillus compared with non-KLA mice. These findings suggest that the pathogenic process of KLA infection may involve alteration of microbiota compositions, particularly reduction in Lactobacillus.

[Research advances in the relationship between iron deficiency and neurodevelopment in preterm infants].

Iron deficiency (ID) is the most common micronutrient deficiency in children. Due to insufficient iron storage at birth and rapid catch-up growth after birth, preterm infants tend to have a high incidence rate of ID. During the critical period of brain development, ID alters iron-dependent neurometabolism, neurochemistry, neuroanatomy, and gene/protein profiles. This affects the central nervous system and causes the change in neurocognitive and behavioral development. Iron supplementation in infancy cannot reverse neurodevelopmental impairment caused by perinatal ID. The influence of ID on neurodevelopment is time- and region-specific, and in the high-risk population, early diagnosis and optimal iron treatment may help with the recovery of brain function and improve quality of life and long-term prognosis in preterm infants.

[The Study of Spectral Characteristic of Coal Ash from Different Sources with Laser-Induced Breakdown Spectroscopy]

The samples with different carbon content are collected for quantitative analysis. One of the normal methods is the ignition of different coals according to the notice of fast ashing method instead of collecting coal ash in boiler. But there are some differences between fast ashing method in laboratory and actual boiler. It is necessary that the spectral deviation of coal ash from these two sources is studied as a guidance of quantitative analysis in carbon content. In present work, the intensity of the characteristic lines and plasma temperature were compared with different carbon content from these two processes. As a result, Fe, Mg, Al line strength of ash with fast ashing method is stronger and plasma temperature is lower than coal ash in boiler. Principal component analysis was processed, the results show that the difference of Fe, Mg, Al and Si content is the primary factor, and minerals in coal ash with fast ashing method may influence the spectral characteristic. The influence of mineral elements and mineral content on spectra for quantitative analysis with fast ashing method should be noticed.

[Influence of C-Fe Lines Interference Correction on Laser-Induced Breakdown Spectroscopy Measurement of Unburned Carbon in Fly Ash].

In coal-fired plants, Unburned carbon (UC) in fly ash is the major determinant of combustion efficiency in coal-fired boiler. The balance between unburned carbon and NO(x) emissions stresses the need for rapid and accurate methods for the measurement of unburned carbon. Laser-induced breakdown spectroscopy (LIBS) is employed to measure the unburned carbon content in fly ash. In this case, it is found that the C line interference with Fe line at about 248 nm. The interference leads to C could not be quantified independently from Fe. A correction approach for extracting C integrated intensity from the overlapping peak is proposed. The Fe 248.33 nm, Fe 254.60 nm and Fe 272.36 nm lines are used to correct the Fe 247.98 nm line which interference with C 247.86 nm, respectively. Then, the corrected C integrated intensity is compared with the uncorrected C integrated intensity for constructing calibration curves of unburned carbon, and also for the precision and accuracy of repeat measurements. The analysis results show that the regression coefficients of the calibration curves and the precision and accuracy of repeat measurements are improved by correcting C-Fe interference, especially for the fly ash samples with low level unburned carbon content. However, the choice of the Fe line need to avoid a over-correction for C line. Obviously, Fe 254.60 nm is the best

Different metabolic profiles of K1 serotype and non-serotype K1 and K2 Klebsiella pneumoniae isolates in oral infection mice model.

K1 or K2 serotype Klebsiella pneumoniae isolate caused clinical pyogenic liver abscess (KLA) infection is prevalent in many areas. It has been identified that K1 or K2 serotype K. pneumoniae isolates caused KLA infection in mice by oral inoculation. In our study, K1 serotype K. pneumoniae isolate Kp1002 with hypermucoviscosity (HV)-positive phenotype caused KLA infection in C57BL/6 mice by oral inoculation. Simultaneously, non-serotype K1 and K2 isolate Kp1014 with HV-negative phenotype failed to cause KLA infection in the same manner. It seems that gastrointestinal tract translocation is the pathway by which K1 or K2 serotype K. pneumoniae caused KLA infection. Liquid chromatography-tandem mass spectrometry was used to further analyze metabolic profile changes in mice with KLA infection. Data showed that after Kp1002 or Kp1014 oral inoculation, serum Phosphatidylcholine (PC) and Lysophosphatidylcholine (LPC) levels significantly changed in mice. Some PC and LPC molecules showed changes both in the Kp1002 KLA group and the Kp1014 no-KLA group compared with the control group. The level of 18:1/18:2-PC significantly changed in the Kp1002 KLA group compared with the control group, but showed no change between the Kp1014 no-KLA group and the control group. The level of 18:1/18:2-PC might have been particularly affected by KLA infection caused by K1 serotype K. pneumoniae Kp1002. It may be a potential biomarker for KLA infection.

Improved myocardial function with supplement of levosimendan to Celsior solution.

Levosimendan is a calcium-sensitizing agent shown to prevent myocardical contractile depression in various heart diseases. In this study, we investigated the effect of levosimendan on cardiac dysfunction and apoptosis in hypothermic preservation rat hearts. Isolated rat hearts were preserved in Celsior solution with or without levosimendan. The left ventricular developed pressure (LVDP) recovery rate of isolated rat heart significantly decreased, and the apoptosis index increased after 9 hours of hypothermic preservation. Supplement Celsior solution with levosimendan (10 and 10 mole/L) enhanced the LVDP recovery rate and reduced apoptosis. Levosimendan inhibited the hypothermic preservation-induced calpain activation and cleavage of Bid. Levosimendam induced increased myocardial inducible nitric oxide synthase but not endothelial nitric oxide synthase expression. A selective inducible nitric oxide synthase inhibitor 1400W, and a mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-hydroxydecanoate but not a sarcolemmal KATP channel blocker HMR-1098 prevented improvement effect of levosimendam on LVDP recovery rate, abolished the inhibitory effect of levosimendan on hypothermic preservation-induced activation of calpain, cleavage of Bid, and apoptosis. These data suggested that Celsior solution supplement with levosimendan improved cardiac function recovery and reduced myocyte apoptosis in hypothermic preservation rat hearts.

[Influence of laser energy on measurement of unburned carbon in fly Ash particle flow].

The fly ash particle flow was produced by a screw feeder and then ablated by a pulse laser to create plasma. The emission spectra of fly ash were detected by laser-induced breakdown spectroscopy. The present paper focused on the influence of laser energy on the measurement of unburned carbon. Seven groups of pulse laser in the range of 40 to 130 mJ were used to ablate the fly ash particle flow. The results show that the carbon line intensity is increased linearly with the increases in laser energy, but the SNR of carbon line increases in the range of 40 to 90 mJ and then trends to saturation, while the elimination rate of false data decreases. In this experiment, laser energy ranging from 90 to 100 mJ can enhance the plasma emission signal and improve the true spectral data of fly ash particle flow. So laser energy has close correlations with the ablation of the particle flow and the carbon line intensity. Reasonable laser energy is good for the effective ablation of the fly ash particle flow to get plasma spectra signals with good SNR.

Metabonomics Application on Screening Serum Biomarkers of Golden Hamsters with Nonalcoholic Steatohepatitis Induced by High-Fat Diet.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a common liver injury which will develop into advanced fibrosis and cirrhosis. This study was designed to identify the different serum metabolites of NASH hamsters and predict the diagnosis biomarkers for NASH. METHODS: Golden hamsters were randomly divided into a control group that received a normal diet and a NASH group that received a high-fat diet (HFD). After 12 weeks of feeding, the body and liver weight of the hamsters were monitored. Serum biochemical parameters and liver histopathological changes were analyzed. Moreover, an untargeted metabolomics analysis based on a GCTOF/ MS system was performed to identify the serum differential metabolites between the NASH and control groups. RESULTS: The liver weight was increased in the NASH group, accompanied by significantly higher levels of serum TC, TG, ALT, AST, LDL-C, and lower HDL-C. HE, Masson, and oil red O staining showed the hepatocyte structure destroyed, lipid droplets accumulated, and fibers proliferated in the NASH group. Furthermore, 63 differential metabolites were identified by metabolomic analysis. Lipids and fatty acids were significantly up-regulated in the NASH group. The top 9 differential metabolites included cholesterol, methyl phosphate, taurine, alpha-tocopherol, aspartic acid, etc. Metabolites were mainly involved in amino acid metabolism (glycine, cysteine, taurine), spermine, fatty acid biosynthesis, urea cycle, bile acid metabolism pathways, etc. Conclusion: Metabonomics analysis identified 63 differential metabolites in the serum of NASH hamsters; among them, lipids and fatty acids had a key role and may be used as biomarkers for the early diagnosis of NASH.

Geniposide alleviates pressure overload in cardiac fibrosis with suppressed TGF-ß1 pathway.

BACKGROUND: Cardiac fibrosis is one of the main contributors to the pathogenesis of heart failure. Geniposide (GE), a major iridoid in gardenia fruit extract, has recently been reported to improve skeletal muscle fibrosis through the modulation of inflammation response. This investigation aimed to illuminate the cardio-protective effect and the potential mechanism of GE in cardiac fibrosis. MATERIAL AND METHODS: A transverse aortic contraction (TAC) induction mice model was established and GE (0 mg/kg; 10 mg/kg; 20 mg/kg; 40 mg/kg) was administered by oral gavage daily for 4 weeks. Hemodynamic parameters, Masson's trichrome stain, and hematoxylin-eosin (HE) staining were estimated and cardiomyocyte fibrosis, interstitial collagen levels, and hypertrophic markers were analyzed using qPCR and western blot. In vitro, H9C2 cells were exposed to the Ang II (1 µM) pretreated with GE (0.1 µM, 1 µM, and 10 µM). Cardiomyocyte apoptosis was detected. Moreover, the transforming growth factor ß1 (TGF-ß1)/Smad2 pathway was assessed in vivo and in vitro. RESULTS: GE significantly ameliorated TAC-induced cardiac hypertrophy, ventricular remodeling, myocardial fibrosis, and improved cardiac function in vivo, and it inhibited Ang II-induced cardiomyocyte apoptosis in vitro. We further observed that the inflammatory channel TGF-ß1/Smad2 pathway was suppressed by GE both in vivo and in vitro. CONCLUSION: These results indicate that GE inhibited myocardial fibrosis and improved hypertrophic cardiomyocytes with attenuated the TGF-ß1/Smad2 pathway and proposed to be an important therapeutic of cardiac fibrosis reduced by TAC.

Pretreatment with geniposide mitigates myocardial ischemia/reperfusion injury by modulating inflammatory response through TLR4/NF-κB pathway.

Geniposide (GEN), a medical herb, is known for its therapeutic applications in cardiovascular diseases, though its efficacy in treating myocardial ischemia/reperfusion injury (MI/RI) is yet to be fully elucidated. This study is an endeavor to explore the potential protective mechanism of GEN against MI/RI. To simulate the MI/RI condition, the left anterior descending artery was occluded for 30 min, followed by a reperfusion period of 120 min in a rat model. Three dosages (50, 100, or 150 mg/kg) of GEN were intraperitoneally injected to the Sprague-Dawley rats once a day, for seven days before the ligation of the artery. The rats were categorized into sham group, MI/RI group, and three different dosages GEN-treated groups. As the results showed, the pretreatment with GEN mitigated myocardial injury, reduced infarct volume, inhibited apoptosis, enhanced superoxide dismutase activity, and decreased malondialdehyde and myeloperoxidase activity, as well as serum creatine kinase-MB and lactate dehydrogenase levels. Moreover, GEN ameliorated MI/RI by downregulating protein expression of toll-like receptor 4, myeloid differentiation primary response 88, and p-nuclear factor-κB. In conclusion, the pretreatment of GEN may be considered as a potential therapeutic option for MI/RI.

Liver Metabolomics Analysis Revealing Key Metabolites Associated with Different Stages of Nonalcoholic Fatty Liver Disease in Hamsters.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is not only the top cause of liver diseases but also a hepatic-correlated metabolic syndrome. This study performed untargeted metabolomics analysis of NAFLD hamsters to identify the key metabolites to discriminate different stages of NAFLD. METHODS: Hamsters were fed a high-fat diet (HFD) to establish the NAFLD model with different stages (six weeks named as the NAFLD1 group and twelve weeks as the NAFLD2 group, respectively). Those liver samples were analyzed by untargeted metabolomics (UM) analysis to investigate metabolic changes and metabolites to discriminate different stages of NAFLD. RESULTS: The significant liver weight gain in NAFLD hamsters was observed, accompanied by significantly increased levels of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Moreover, the levels of TG, LDL-C, ALT, and AST were significantly higher in the NAFLD2 group than in the NAFLD1 group. The UM analysis also revealed the metabolic changes; 27 differently expressed metabolites were detected between the NAFLD2 and NAFLD1 groups. More importantly, the levels of N-methylalanine, allantoin, glucose, and glutamylvaline were found to be significantly different between any two groups (control, NAFLD2 and NAFLD1). Receiver operating characteristic curve (ROC) curve results also showed that these four metabolites are able to distinguish control, NAFLD1 and NAFLD2 groups. CONCLUSION: This study indicated that the process of NAFLD in hamsters is accompanied by different metabolite changes, and these key differently expressed metabolites may be valuable diagnostic biomarkers and responses to therapeutic interventions.

IFN-γ-STAT1-mediated CD8+ T-cell-neural stem cell cross talk controls astrogliogenesis after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) causes nearly all patients to suffer from protracted disabilities. An emerging therapeutic strategy involving the recruitment of endogenous neural stem cells (NSCs) has been developed. However, endogenous NSCs in the adult spinal cord differentiate into mostly astrocytes after traumatic injury, forming glial scars, which is a major cause of regeneration failure in SCI. Thus, understanding which factors drive the activation and differentiation of endogenous NSCs after SCI is critical for developing therapeutic drugs. METHODS: The infiltration, state, and location of CD8+ T cells in spinal cord after traumatic injury were analyzed by flow cytometry and immunofluorescence (IF) staining. The Basso Mouse Scale (BMS) scores and rotarod testing were used for motor behavioral analysis. NSCs were co-cultured with CD8+ T cells. EdU assay was used to detect proliferative cells. Western blotting was used to analyze the expression levels of STAT1, p-STAT1, and p27. ChIP-seq and ChIP-qRT-PCR analyses were used to detect the downstream of STAT1. Nestin-CreERT2::Ai9 transgenic mice were used to genetic lineage tracing of Nestin+ NSCs after SCI in vivo. RESULTS: A prolonged increase of activated CD8+ T cells occurs in the injured spinal cords. The behavioral analysis demonstrated that the administration of an anti-CD8 antibody promotes the recovery of locomotor function. Then, we discovered that CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1 pathway in vitro. ChIP-seq and ChIP-qRT-PCR analysis revealed that STAT1 could directly bind to the promoters of astrocyte marker genes GFAP and Aldh1l1. Genetic lineage tracing of Nestin+ NSCs demonstrated that most NSCs differentiated into astrocytes following SCI. Depleting CD8+ T cells reduced the differentiation of NSCs into astrocytes and instead promoted the differentiation of NSCs into oligodendrocytes. CONCLUSION: In conclusion, CD8+ T cells suppressed the proliferation of NSCs and promoted the differentiation of NSCs into astrocytes by the IFN-γ-STAT1-GFAP/Aldhl1l axis. Our study identifies INF-γ as a critical mediator of CD8+ T-cell-NSC cross talk and a potential node for therapeutic intervention in SCI.

Neuroprotective Effect of Stroke Pretreated Mesenchymal Stem Cells Against Cerebral Ischemia/Reperfusion Injury in Rats.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to enhance neurological recovery after stroke. A rat middle cerebral artery occlusion model was designed to assess neuroprotective effects of stroke pretreated MSCs on cerebral ischemia/reperfusion injury. METHODS: MSCs were isolated and cultured in medium with 10% fetal bovine serum, normal control serum, or stroke serum (SS). MSCs were then injected into rats (n = 6 in each group) 1 day after middle cerebral artery occlusion, and feeding continued for 28 days. A battery of behavioral tests, 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, enzyme-linked immunosorbent assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were used to assess neural injury. To detect enhancement of neuronal regeneration and angiogenesis, immunofluorescence and Western blotting were performed to assess expression of trophic factors and growth factors. RESULTS: After treatment, behavior of rats improved significantly. Infarction area, brain lesion, and apoptosis cells were significantly decreased in the SS-MSCs group compared with the other groups. SS-MSCs also modulated inflammation by attenuating inflammatory cytokines. Furthermore, the number of neurogenesis-positive cells and expression of trophic factors and growth factors were significantly higher in the SS-MSCs group compared with the others. MSCs cultured with fetal bovine serum and normal control serum showed differences in expression of trophic factors and growth factors, but the results were not as good as with SS-MSCs. CONCLUSIONS: Administration of SS-MCSs after reperfusion led to neuroprotection by inducing the recovery process, including improving pathological changes, behavioral improvement, neurogenesis, suppression of apoptosis and inflammation, and angiogenesis.

Single-cell RNA sequencing analysis reveals the relationship of bone marrow and osteopenia in STZ-induced type 1 diabetic mice.

INTRODUCTION: Type 1 diabetes (T1D) is a multifactorial autoimmune disease. Broad knowledge about the genetics, epidemiology and clinical management of T1D has been achieved, but understandings about the cell varieties in the bone marrow during T1D remain limited. OBJECTIVES: We aimed to present a profile of the bone marrow cells and reveal the relationship of bone marrow and osteopenia in streptozotocin (STZ)-induced T1D mice. METHODS: The whole bone marrow cells from the femurs and tibias of healthy (group C) and STZ-induced T1D mice (group D) were collected for single-cell RNA sequencing analysis. Single-cell flow cytometry and immunohistochemistry were performed to confirm the proportional changes among bone marrow neutrophils (BM-neutrophils) (Cxcr2+, Ly6g+) and B lymphocytes (Cd19+). X-ray and micro-CT were performed to detect bone mineral density. The correlation between the ratio of BM-neutrophils/B lymphocytes and osteopenia in STZ-induced T1D mice was analyzed by nonparametric Spearman correlation analysis. RESULTS: The bone marrow cells in groups C and D were divided into 12 clusters, and 249 differentially expressed genes were found. The diversity of CD45+ immune cells between groups C and D were greatly affected: the proportion of BM-neutrophils showed a significant increase while the proportion of B lymphocytes in group D showed a significant decrease. X-ray and micro-CT analyses confirmed that osteopenia occurred in group D mice. In addition, the results of single-cell flow cytometry and correlation analysis showed that the ratio of BM-neutrophils/B lymphocytes negatively correlated with osteopenia in STZ-induced T1D mice. CONCLUSION: A single-cell RNA sequencing analysis revealed the profile and heterogeneity of bone marrow immune cells in STZ-induced T1D mice for the first time. The ratio of BM-neutrophils/B lymphocytes negatively correlated with osteopenia in STZ-induced T1D mice, which may enhance understanding for treating T1D and preventing T1D-induced osteopenia.

Macrophages induce cardiomyocyte ferroptosis via mitochondrial transfer.

INTRODUCTION: Mitochondrial transfer is a new cell-to-cell communication manner. Whether the mitochondrial transfer is also involved in the macrophage infiltration-induced cardiac injury is unclear. OBJECTIVES: This study aimed to determine whether macrophage mitochondria can be transferred to cardiomyocytes, and to investigate its possible role and mechanism. METHODS: Mitochondrial transfer between macrophages and cardiomyocytes was detected using immunofluorescence staining and flow cytometry. Cellular metabolites were analyzed using LC-MS technique. Differentially expressed mRNAs were identified using RNA-seq technique. RESULTS: (1) After cardiomyocytes were cultured with macrophage-conditioned medium (COND + group), macrophage-derived mitochondria have been found in cardiomyocytes, which could be blocked by dynasore (an inhibitor of clathrin-mediated endocytosis). (2) Compared with control (CM) group, there were 545 altered metabolites found in COND + group, most of which were lipids and lipid-like molecules. The altered metabolites were mainly enriched in the ß-oxidation of fatty acids and glutathione metabolism. And there were 4824 differentially expressed mRNAs, which were highly enriched in processes like lipid metabolism-associated pathway. (3) Both RNA-seq and qRT-PCR results found that ferroptosis-related mRNAs such as Ptgs2 and Acsl4 increased, and Gpx4 mRNA decreased in COND + group (P < 0.05 vs CM group). (4) The levels of cellular free Fe2+ and mitochondrial lipid peroxidation were increased; while GSH/GSSG ratio, mitochondrial aspect ratio, mitochondrial membrane potential, and ATP production were decreased in cardiomyocytes of COND + group (P < 0.05 vs CM group). All the above phenomena could be blocked by a ferroptosis inhibitor ferrostatin-1 (P < 0.05). CONCLUSION: Macrophages could transfer mitochondria to cardiomyocytes. Macrophage-derived mitochondria were internalized into cardiomyocytes through clathrin- and/or lipid raft-mediated endocytosis. Uptake of exogenous macrophage mitochondria induced cardiomyocyte injury via triggering ferroptosis.

Single-cell transcriptome analysis reveals the immune heterogeneity and the repopulation of microglia by Hif1α in mice after spinal cord injury.

Neuroinflammation is regarded as a vital pathological process in spinal cord injury (SCI), which removes damaged tissue, secretes cytokines, and facilitates regeneration. Repopulation of microglia has been shown to favor recovery from SCI. However, the origin and regulatory factors of microglia repopulation after SCI remain unknown. Here, we used single-cell RNA sequencing to portray the dynamic transcriptional landscape of immune cells during the early and late phases of SCI in mice. B cells and migDCs, located in the meninges under physiological conditions, are involved in immune surveillance. Microglia quickly reduced, and peripheral myeloid cells infiltrated three days-post-injury (dpi). At 14 dpi, microglia repopulated, myeloid cells were reduced, and lymphocytes infiltrated. Importantly, genetic lineage tracing of nestin+ and Cx3cr1+ cells in vivo showed that the repopulation of microglia was derived from residual microglia after SCI. We found that residual microglia regress to a developmental growth state in the early stages after SCI. Hif1α promotes microglial proliferation. Conditional ablation of Hif1α in microglia causes larger lesion sizes, fewer axon fibers, and impaired functional recovery in the late stages after SCI. Our results mapped the immune heterogeneity in SCI and raised the possibility that targeting Hif1α may help in axon regeneration and functional recovery after SCI.

Protective effects of the aqueous extract of Scutellaria baicalensis against acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells.

CONTEXT: Scutellaria baicalensis Georgi (Labiatae) (SbG), one of the fifty fundamental herbs of Chinese herbology, has been reported to have anti-asthmatic, antifungal, antioxidative, and anti-inflammatory activities. OBJECTIVE: This study was designed to determine the protective effects of the extract of SbG against the acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The MTT reduction assay was employed to determine cell viability. The total cellular glutathione (GSH) level was detected using a colorimetric GSH assay kit. Cellular GSH production was conducted by detecting the mRNA expression levels of γ-glutamylcysteine ligase catalytic subunit and modifier subunit. RESULTS: Concentration-dependent cytotoxic effects of acrolein were observed while SbG could effectively protect the acrolein-induced oxidative damage. The protective mechanism was investigated, showing that the increased GSH content in the SbG-incubated HUVE cells was associated with the protective effects of SbG-treated cells. Further RT-PCR data confirmed the elevated mRNA expressions of GSH synthesis enzymes. DISCUSSION AND CONCLUSION: The current study strongly indicated that SbG could be a potential antioxidant against oxidative stress in treating cardiovascular diseases.