Integrated multi-omics analysis reveals molecular changes associated with chronic lipid accumulation following contusive spinal cord injury.

Functional and pathological recovery after spinal cord injury (SCI) is often incomplete due to the limited regenerative capacity of the central nervous system (CNS), which is further impaired by several mechanisms that sustain tissue damage. Among these, the chronic activation of immune cells can cause a persistent state of local CNS inflammation and damage. However, the mechanisms that sustain this persistent maladaptive immune response in SCI have not been fully clarified yet. In this study, we integrated histological analyses with proteomic, lipidomic, transcriptomic, and epitranscriptomic approaches to study the pathological and molecular alterations that develop in a mouse model of cervical spinal cord hemicontusion. We found significant pathological alterations of the lesion rim with myelin damage and axonal loss that persisted throughout the late chronic phase of SCI. This was coupled by a progressive lipid accumulation in myeloid cells, including resident microglia and infiltrating monocyte-derived macrophages. At tissue level, we found significant changes of proteins indicative of glycolytic, tricarboxylic acid cycle (TCA), and fatty acid metabolic pathways with an accumulation of triacylglycerides with C16:0 fatty acyl chains in chronic SCI. Following transcriptomic, proteomic, and epitranscriptomic studies identified an increase of cholesterol and m6A methylation in lipid-droplet-accumulating myeloid cells as a core feature of chronic SCI. By characterizing the multiple metabolic pathways altered in SCI, our work highlights a key role of lipid metabolism in the chronic response of the immune and central nervous system to damage.

Bioinformatics analysis identified apolipoprotein E as a hub gene regulating neuroinflammation in macrophages and microglia following spinal cord injury.

Macrophages and microglia play important roles in chronic neuroinflammation following spinal cord injury (SCI). Although macrophages and microglia have similar functions, their phagocytic and homeostatic abilities differ. It is difficult to distinguish between these two populations in vivo, but single-cell analysis can improve our understanding of their identity and heterogeneity. We conducted bioinformatics analysis of the single-cell RNA sequencing dataset GSE159638, identifying apolipoprotein E (APOE) as a hub gene in both macrophages and microglia in the subacute and chronic phases of SCI. We then validated these transcriptomic changes in a mouse model of cervical spinal cord hemi-contusion and observed myelin uptake, lipid droplets, and lysosome accumulation in macrophages and microglia following SCI. Finally, we observed that knocking out APOE aggravated neurological dysfunction, increased neuroinflammation, and exacerbated the loss of white matter. Targeting APOE and the related cholesterol efflux represents a promising strategy for reducing neuroinflammation and promoting recovery following SCI.

New strain of Pediococcus pentosaceus alleviates ethanol-induced liver injury by modulating the gut microbiota and short-chain fatty acid metabolism.

BACKGROUND: Intestinal dysbiosis has been shown to be associated with the pathogenesis of alcoholic liver disease (ALD), which includes changes in the microbiota composition and bacterial overgrowth, but an effective microbe-based therapy is lacking. Pediococcus pentosaceus (P. pentosaceus) CGMCC 7049 is a newly isolated strain of probiotic that has been shown to be resistant to ethanol and bile salts. However, further studies are needed to determine whether P. pentosaceus exerts a protective effect on ALD and to elucidate the potential mechanism. AIM: To evaluate the protective effect of the probiotic P. pentosaceus on ethanol-induced liver injury in mice. METHODS: A new ethanol-resistant strain of P. pentosaceus CGMCC 7049 was isolated from healthy adults in our laboratory. The chronic plus binge model of experimental ALD was established to evaluate the protective effects. Twenty-eight C57BL/6 mice were randomly divided into three groups: The control group received a pair-fed control diet and oral gavage with sterile phosphate buffered saline, the EtOH group received a ten-day Lieber-DeCarli diet containing 5% ethanol and oral gavage with phosphate buffered saline, and the P. pentosaceus group received a 5% ethanol Lieber-DeCarli diet but was treated with P. pentosaceus. One dose of isocaloric maltose dextrin or ethanol was administered by oral gavage on day 11, and the mice were sacrificed nine hours later. Blood and tissue samples (liver and gut) were harvested to evaluate gut barrier function and liver injury-related parameters. Fresh cecal contents were collected, gas chromatography-mass spectrometry was used to measure short-chain fatty acid (SCFA) concentrations, and the microbiota composition was analyzed using 16S rRNA gene sequencing. RESULTS: The P. pentosaceus treatment improved ethanol-induced liver injury, with lower alanine aminotransferase, aspartate transaminase and triglyceride levels and decreased neutrophil infiltration. These changes were accompanied by decreased levels of endotoxin and inflammatory cytokines, including interleukin-5, tumor necrosis factor-α, granulocyte colony-stimulating factor, keratinocyte-derived protein chemokine, macrophage inflammatory protein-1α and monocyte chemoattractant protein-1. Ethanol feeding resulted in intestinal dysbiosis and gut barrier disruption, increased relative abundance of potentially pathogenic Escherichia and Staphylococcus, and the depletion of SCFA-producing bacteria, such as Prevotella, Faecalibacterium, and Clostridium. In contrast, P. pentosaceus administration increased the microbial diversity, restored the relative abundance of Lactobacillus, Pediococcus, Prevotella, Clostridium and Akkermansia and increased propionic acid and butyric acid production by modifying SCFA-producing bacteria. Furthermore, the levels of the tight junction protein ZO-1, mucin proteins (mucin [MUC]-1, MUC-2 and MUC-4) and the antimicrobial peptide Reg3ß were increased after probiotic supplementation. CONCLUSION: Based on these results, the new strain of P. pentosaceus alleviated ethanol-induced liver injury by reversing gut microbiota dysbiosis, regulating intestinal SCFA metabolism, improving intestinal barrier function, and reducing circulating levels of endotoxin and proinflammatory cytokines and chemokines. Thus, this strain is a potential probiotic treatment for ALD.

Pretreatment With Bacillus cereus Preserves Against D-Galactosamine-Induced Liver Injury in a Rat Model.

Bacillus cereus (B. cereus) functions as a probiotic in animals, but the underlying mechanisms remain unclear. We aim to evaluate the protective effects and definite mechanism by which orally administered B. cereus prevents D-galactosamine (D-GalN)-induced liver injury in rats. Twenty-one Sprague-Dawley rats were equally assigned into three groups (N = 7 animals per group). B. cereus ATCC11778 (2 × 109 colony-forming units/ml) was administered to the B. cereus group via gavage, and phosphate-buffered saline was administered to the positive control (PC) and negative control (NC) groups for 2 weeks. The PC and B. cereus groups received 1.1 g/kg D-GalN via an intraperitoneal injection to induce liver injury. The blood, terminal ileum, liver, kidney and mesenteric lymph nodes (MLNs) were collected for histological examinations and to evaluate bacterial translocation. Liver function was also determined. Fecal samples were collected for deep sequencing of the 16S rRNA on an Illumina MiSeq platform. B. cereus significantly attenuated D-GalN-induced liver injury and improved serum alanine aminotransferase (ALT) and serum cholinesterase levels (P < 0.05 and P < 0.01, respectively). B. cereus modulated cytokine secretion, as indicated by the elevated levels of the anti-inflammatory cytokine interleukin-10 (IL-10) in both the liver and plasma (P < 0.05 and P < 0.01, respectively) and the substantially decreased levels of the cytokine IL-13 in the liver (P < 0.05). Pretreatment with B. cereus attenuated anoxygenic bacterial translocation in the veins (P < 0.05) and liver (P < 0.05) and upregulated the expression of the tight junction protein 1. The gut microbiota from the B. cereus group clustered separately from that of the PC group, with an increase in species of the Ruminococcaceae and Peptococcaceae families and a decrease in those of the Parabacteroides, Paraprevotella, and Desulfovibrio families. The potential probiotic B. cereus attenuated liver injury by restoring the gut flora balance and enhancing the intestinal barrier function.

[Ultracytochemical observation of the intracellular localization of H+-adenosine triphosphatase].

OBJECTIVE: To observe the ultracytochemical localization of H(+)-adenosine triphosphatase (H(+)-ATPase) in the cell organelles. METHODS: The localization of H(+)-ATPase in the cell organelles was observed in the hepatocytes and renal cells of Wistar rats using routine ultracytochemical methods. RESULTS: H(+)-ATPase activities were observed on the lysosomal membrane and nuclear envelope of the hepatocytes and proximal tubule epithelial cells of the nephron in Wistar rats. CONCLUSION: This finding supports the hypothesis that H(+)-ATPase (V-ATPase) is present on the plasma membrane and in the endomembrane system.

[Nephrotoxicity of tacrolimus and preventive effect of diltiazem: experiment with rats].

OBJECTIVE: To study the nephrotoxicity tacrolimus (FK506) at the therapeutic dose the preventive effect of diltiazem (Dil), a calcium antagonist against the FK506-induced pathological changes. METHODS: 24 Sprague-Dawley male rats were randomly divided into 4 equal groups: cyclosporine A (CsA) group, undergoing treatment of CsA at the therapeutic dose after kidney transplantation (25 mg x kg(-1) x d(-1)) for 4 weeks, FK506 group treated with FK506 (0.8 mg x kg(-1) x d(-1)), FK506 + Dil group treated with FK506 (0.8 mg x kg(-1) x d(-1)) and Dil at the dose of 8 mg x kg(-1) x d(-1), and control group. Four weeks later body weight was measured and 24 h urine sample was collected. Then the rats were killed. Their kidneys underwent light and transmission electron microscopy. RESULTS: The body weight ad weight gain, and the weights of both kidney of the CsA group were all significantly lower than those of the other 3 groups (all P < 0.05), and there were not significant differences in there parameters among the other 3 groups. The serum creatinine levels of the FK506 and CsA groups were (36.0 +/- 2.6) and (34.2 +/- 4.5) micromol/L respectively, both significantly higher than those of the FK506 + Dil and control groups [(28.5 +/- 2.1) and (29.2 +/- 3.428) micromol/L respectively, all P < 0.05], however, there was no significant difference between the FK506 + Dil and control groups. The creatinine clearance rate of the FK506 and CsA groups were (0.63 +/- 0.45) and (0.58 +/- 0.39) ml x min(-1) x 100 g(-1) respectively, significantly lower than those of the FK506 + Dil and control groups [(1.55 +/- 0.91) and (1.02 +/- 0.62) mlxmin(-1) x 100 g(-1) respectively, all P < 0.05]. Pathological examination showed epithelial cell cloudy swelling and vacuolization and interstitial fibrosis in the renal tubules, mitochondria swelling and vacuolization in renal tubular epithelial cells, renal arteriole hyalinization, and foot cell conjugation glomerulus, mitochondria swelling and vacuolization in the FK506 and CsA groups, and such changes were relatively mild in the FK506 + Dil group. CONCLUSION: FK506 at renal transplantation therapeutic dose, as well as CsA, induces pathological changes in renal tissues and ultrastructural organization. Dil is able to prevent FK506-induced these pathological changes.

[Cloning and sequence analysis of the preprotein cDNA of human bone morphogenetic protein 12].

OBJECTIVE: To clone the cDNA of the preprotein cDNA of human bone morphogenetic protein 12 (hBMP12). METHODS: Two primers were designed according to hBMP12 sequence reported in GenBank. The hBMP12 preprotein cDNA was obtained by reverse transcriptional (RT)-PCR from the mRNA extracted from human placenta, followed by cloning into pTARGETTM plasmid and sequence analysis of the plasmid pT(ARGE)T(TM)-BMP12. RESULTS: DNA agarose gel electrophoresis showed that the product of RT-PCR was about 920 bp, as was consistent with the result of PCR detection of the recombinant plasmid. The result of sequence analysis was in agreement with the reported hBMP12 sequence in GenBank. CONCLUSION: The preprotein of hBMP12 cDNA has been successfully cloned with correct sequence.

[Morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin artificial tendon].

OBJECTIVE: To observe the morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin (HHK) artificial tendon. METHODS: Rabbit models of injured tendon were established in which implantation of HHK artificial tendon was performed to observe the formation of autogenous tendon under light microscope and electron microscope at 3, 6, 9, 12 and 16 weeks after HHK implantation. RESULTS AND CONCLUSION: During autogenous tendon formation induced by HHK artificial tendon, the tendon cells of the impaired end of the tendon and beneath the tendon membrane dedifferentiate and are capable of collagen secretion, followed by the formation of typesI,II and III collagen fibrils, and eventually, the majority of the tendon cells disappear with the collagenization of the tendon.

Degradation of human hair keratin scaffold implanted for repairing injured skeletal muscles.

OBJECTIVE: To observe the in vivo degradation process of human hair keratin (HHK) scaffold after implantation in rabbits. METHODS: Seven New Zealand rabbits were divided into 4 groups including a control group and 3 operation groups. HHK scaffold was implanted, after partial resection of the skeletal muscles, in rabbits of the 3 operation groups, followed by observation 1, 3, and 6 weeks later respectively. Routine morphological observation, histochemistry with ubiquitin and electron microscopy were performed. HHK scaffold incorporated 3 types of HHK with different degradation speeds, respectively designated types F, B, and Z. RESULTS: Light microscopic observation revealed that human hair cuticles began to strip off at the first postoperative week, and the material was homogeneous on the surface of which macrophagocytes and multinuclear giant cells adhered. At the third week HHK scaffold was degraded into particles as seen under electron microscope and was phagocytosed by macrophagocytes and multinuclear giant cells. Ubiquitin enzymatic histochemistry demonstrated that macrophagocytes, multinuclear giant cells were positive at the first week. At sixth week, further degradation of HHK scaffold occurred when newly generated muscles were seen beside the HHK. CONCLUSIONS: HHK scaffold is initially degraded extracellularly by ubiquitin system into particles, which are phagocytosed by the cells and degraded by the cooperation of lysosome and ubiquitin; meanwhile the satellite cells are activated, beginning to proliferate and eventually fused into newly generated muscle fibers.

Differentiation and telomerase activity of human mesenchymal stem cells.

OBJECTIVE: To study the differentiation of human mesenchymal stem cells (MSCs) derived from human fetal bone marrow and observe its telomerase activity. METHODS: MSCs were isolated from fetal bone marrow of the femur followed by cell culture and amplification. In situ hybridization was employed to detect telomerase activity in the MSCs. Subcutaneous implantation of MSCs into nude mice was performed to observe their differentiation potentials 4 weeks after the implantation. RESULTS: Human MSCs were found positive for telomerase activity and they showed the potential to differentiate into such tissue cells as of the bone, cartilage, adipose, skeletal muscle, tendon-like tissue and unmyelinated nerve fiber-like bundles. CONCLUSION: Human MSCs are multipotent stem cells that may differentiate into many types of tissue cells with high levels of telomerase activity.