[Experimental study of M2 microglia transplantation promoting spinal cord injury repair in mice].

Objective: To investigate the effect of M2 microglia (M2-MG) transplantation on spinal cord injury (SCI) repair in mice. Methods: Primary MG were obtained from the cerebral cortex of 15 C57BL/6 mice born 2-3 days old by pancreatic enzyme digestion and identified by immunofluorescence staining of Iba1. Then the primary MG were co-cultured with interleukin 4 for 48 hours (experimental group) to induce into M2 phenotype and identified by immunofluorescence staining of Arginase 1 (Arg-1) and Iba1. The normal MG were harvested as control (control group). The dorsal root ganglion (DRG) of 5 C57BL/6 mice born 1 week old were co-cultured with M2-MG for 5 days to observe the axon length, the DRG alone was used as control. Forty-two 6-week-old female C57BL/6 mice were randomly divided into sham group ( n=6), SCI group ( n=18), and SCI+M2-MG group ( n=18). In sham group, only the laminae of T 10 level were removed; SCI group and SCI+M2-MG group underwent SCI modeling, and SCI+M2-MG group was simultaneously injected with M2-MG. The survival of mice in each group was observed after operation. At immediate (0), 3, 7, 14, 21, and 28 days after operation, the motor function of mice was evaluated by Basso Mouse Scale (BMS) score, and the gait was evaluated by footprint experiment at 28 days. The spinal cord tissue was taken after operation for immunofluorescence staining, in which glial fibrillary acidic protein (GFAP) staining at 7, 14, and 28 days was used to observe the injured area of the spinal cord, neuronal nuclei antigen staining at 28 days was used to observe the survival of neurons, and GFAP/C3 double staining at 7 and 14 days was used to observe the changes in the number of A1 astrocytes. Results: The purity of MG in vitro reached 90%, and the most of the cells were polarized into M2 phenotype identified by Arg-1 immunofluorescence staining. M2-MG promoted the axon growth when co-cultured with DRGs in vitro ( P<0.05). All groups of mice survived until the experiment was completed. The hind limb motor function of SCI group and SCI+M2-MG group gradually recovered over time. Among them, the SCI+M2-MG group had significantly higher BMS scores than the SCI group at 21 and 28 days ( P<0.05), and the dragging gait significantly improved at 28 days, but it did not reach the level of the sham group. Immunofluorescence staining showed that compared with the SCI group, the SCI+M2-MG group had a smaller injury area at 7, 14, and 28 days, an increase in neuronal survival at 28 days, and a decrease in the number of A1 astrocytes at 7 and 14 days, with significant differences ( P<0.05). Conclusion: M2-MG transplantation improves the motor function of the hind limbs of SCI mice by promoting neuron survival and axon regeneration. This neuroprotective effect is related to the inhibition of A1 astrocytes polarization.

Esculetin inhibits the pyroptosis of microvascular endothelial cells through NF-κB /NLRP3 signaling pathway.

The effect of Esculetin on pyroptosis and its possible mechanism in endothelium were explored. 10 µg/mL LPS and 0.5 mM ATP were used to stimulate the rat intestinal microvascular endothelial cells. Then add different concentrations of Esculetin (20µM, 40 µM) to the culture medium containing LPS and ATP culturing for 24 h. The expression of p-NF-κB p65, NF-κB p65, I-κB, p-I-κB, NLRP3, ASC, caspase-1, and gasdermin-D were detected by Western blot, and the release level of IL-18 and IL-1ß were measured by ELISA. The NLRP3 inhibitor MCC950 was used at the concentration of 10 µM for 4 h to disentangle the potential mechanism of the influence of Esculetin on pyroptosis. In our experiments, the expression of gasdermin-d and important proteins of NF-κB and NLRP3 signaling pathways were inhibited by Esculetin. Besides, Esculetin also attenuated the morphological changes like swelling rupture and pores on the membrane caused by pyroptosis thereby protecting cells from being damaged by pyroptosis. Combining with the effect of Esculetin on proteins above and its protective effect on cell morphology, we believe that Esculetin has an anti-pyroptosis effect. The inhibiting pyroptosis effects mentioned above are similar to MCC950, which means the anti-pyroptosis effects of Esculetin are associated with the NLRP3 signaling pathway. In conclusion, Esculetin inhibits the pyroptosis of microvascular endothelial cells through the NF-κB/NLFP3 signaling pathway and is expected to be conducive in treating pyroptosis-related diseases.

[Significance of monitoring pulmonary arterial pressure and cardiac function in neonates with pulmonary or extra-pulmonary acute respiratory distress syndrome].

OBJECTIVE: To evaluate the dynamic changes of pulmonary arterial pressure (PAP) and cardiac function in neonates with pulmonary or extra-pulmonary acute respiratory distress syndrome (ARDSp/ARDSexp). METHODS: An observational study was conducted. A total of 128 neonates with ARDS admitted to neonatology department of the Affiliated Yancheng Hospital of Southeast University Medical College from January 2016 to December 2020 were enrolled, with 67 neonates in ARDSp group and 61 neonates in ARDSexp group. After starting mechanical ventilation, oxygenation index [OI, OI = mean airway pressure (Pmean)×fraction of inspired oxygen (FiO2)/arterial partial pressure of oxygen (PaO2)×100], PAP, cardiac function parameters [cardiac index (CI), left ventricular ejection fraction (LVEF), right ventricular Tei (RV-Tei)], and plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) were compared between the two groups; the incidence of pulmonary arterial hypertension [PAH, pulmonary artery systolic pressure (PASP) was more than 35 mmHg (1 mmHg = 0.133 kPa) or more than 2/3 of the systolic blood pressure of the body circulation] of neonates was recorded. The correlation between PAP and NT-proBNP was analyzed by Pearson correlation method. The dynamically changes in PAP and RV-Tei before and after using Milrinone in neonates with ARDSp and ARDSexp combined with moderate-severe PAH (PASP 50-69 mmHg was moderate, and PASP ≥ 70 mmHg was severe) were observed. The duration of mechanical ventilation, total length of hospital stay and prognosis were recorded; Kaplan-Meier survival curve was drawn to analyze the 28-day survival of the two groups. RESULTS: The occurrence rate of PAH in ARDSp group was significantly higher than that in ARDSexp group (97.01% vs. 70.49%, P < 0.01). OI, PAP, NT-proBNP and RV-Tei were also higher [OI: 17.61±6.12 vs. 11.04±5.35, PAP (mmHg): 64.27±9.54 vs. 53.61±6.47, NT-proBNP (ng/L): 23 126.32±1 485.14 vs. 18 624.24±1 647.15, RV-Tei: 0.61±0.22 vs. 0.52±0.19, all P < 0.05], but there was no significant difference in CI or LVEF between the two groups. Pearson correlation analysis showed that PAP was significantly positively correlated with NT-proBNP (r = 0.918, P < 0.01). There were 97 ARDS neonates with moderate-severe PAH with 63 in ARDSp group and 34 in ARDSexp group. Both PAP and RV-Tei in the two group showed a decreasing trend with the prolongation of Milrinone treatment, the decrease was more significant in the ARDSexp group compared with ARDSp group, the difference was statistically significant at 72 hours of treatment [PAP (mmHg): 38.42±8.95 vs. 45.67±13.32, RV-Tei: 0.58±0.19 vs. 0.61±0.13, both P < 0.05]; there was no significant difference in PAP or RV-Tei before extubation between the two groups. The duration of mechanical ventilation and the total length of hospital stay in ARDSp group were significantly longer than those in ARDSexp group [duration of mechanical ventilation (days): 10.12±1.36 vs. 6.31±1.31, total length of hospital stay (days): 16.52±3.25 vs. 13.12±3.57, both P < 0.01]. Kaplan-Meier survival curve showed that neonate in ARDSp group had a significantly lower 28-day cumulative survival rate as compared with ARDSexp group (82.09% vs. 95.01%; Log-Rank test: χ2 = 5.062, P = 0.025). CONCLUSIONS: Both PAP and RV-Tei were significantly increased in neonates with ARDS, PAP in neonates with ARDSp were significantly higher than that in neonates with ARDSexp. Dynamic monitoring of PAP and RV-Tei can reflect the severity of ARDS in neonates, and targeted intervention of pulmonary surfactant combined with Milinone for improving oxygenation and reducing PAP is one of the effective methods for the treatment of PAH.

Cu-Doped-ZnO Nanocrystals Induce Hepatocyte Autophagy by Oxidative Stress Pathway.

As a novel nanomaterial for cancer therapy and antibacterial agent, Cu-doped-ZnO nanocrystals (CZON) has aroused concern recently, but the toxicity of CZON has received little attention. Results of hematology analysis and blood biochemical assay showed that a 50 mg/kg dosage induced the increase in white blood cells count and that the concentration of alanine aminotransferase (ALT), superoxide dismutase (SOD), catalase (CAT), and Malonaldehyde (MDA) in the serum, liver, and lungs of the CZON group varied significantly from the control mice. Histopathological examinations results showed inflammation and congestion in the liver and lung after a single injection of CZON at 50 mg/kg. A transmission electron microscope (TEM) result manifested the autolysosome of hepatocyte of mice which received CZON at 50 mg/kg. The significant increase in LC3-II and decrease in p62 of hepatocyte in vivo could be seen in Western blot. These results indicated that CZON had the ability to induce autophagy of hepatocyte. The further researches of mechanism of autophagy revealed that CZON could produce hydroxyl radicals measured by erythrocyte sedimentation rate (ESR). The result of bio-distribution of CZON in vivo, investigated by ICP-OES, indicated that CZON mainly accumulated in the liver and two spleen organs. These results suggested that CZON can induce dose-dependent toxicity and autophagy by inducing oxidative stress in major organs. In summary, we investigated the acute toxicity and biological distribution after the intravenous administration of CZON. The results of body weight, histomorphology, hematology, and blood biochemical tests showed that CZON had a dose-dependent effect on the health of mice after a single injection. These results indicated that CZON could induce oxidative damage of the liver and lung by producing hydroxyl radicals at the higher dose.

Coixol Suppresses NF-κB, MAPK Pathways and NLRP3 Inflammasome Activation in Lipopolysaccharide-Induced RAW 264.7 Cells.

Coixol, a plant polyphenol extracted from coix (Coix lachryma-jobi L.var.ma-yuen Stapf), has not been investigated for its anti-inflammatory effect. In this study, using a lipopolysaccharide (LPS)-induced macrophage cell model, we observed that coixol can effectively reduce the expression of interleukin (IL)-1ß, IL-6, IL-18, tumor necrosis factor (TNF)-α, nitric oxide (NO), inducible nitric oxide synthases (iNOS), and cyclooxygenase (COX)-2, but had no effect on the expression of the anti-inflammatory mediator IL-10. Furthermore, we found that coixol inhibits mitogen-activated protein kinases (MAPKs), nuclear transcription factor κ B (NF-κB) pathways, and NOD-like receptor protein (NLRP) 3 inflammasome activation. In conclusion, the present study demonstrates that coixol exerts certain anti-inflammatory effects by inhibiting the expression of pro-inflammatory mediators in vitro. The mechanism of this effect was in part related to its ability to inhibit the activation of NF-κB, MAPKs pathways, and NLRP3 inflammasome.

Kaempferol inhibits multiple pathways involved in the secretion of inflammatory mediators from LPS­induced rat intestinal microvascular endothelial cells.

Inflammatory bowel disease (IBD) is a chronic, idiopathic inflammatory disease of the small and/or large intestine. Endothelial expression of inflammatory mediators, including cytokines and adhesion molecules, serves a critical role in the initiation and progression of IBD. The dietary flavonoid, kaempferol, has been reported to inhibit expression of inflammatory mediators; however, the underlying mechanisms require further investigation. In the present study, a novel molecular mechanism of kaempferol against IBD was identified. The potential anti­inflammatory effect of kaempferol in a cellular model of intestinal inflammation was assessed using lipopolysaccharide (LPS)­induced rat intestinal microvascular endothelial cells (RIMVECs), and an underlying key molecular mechanism was identified. RIMVECs were pretreated with kaempferol of various concentrations (12.5, 25 and 50 µM) followed by LPS (10 µg/ml) stimulation. ELISA was used to examine the protein levels of tumor necrosis factor­α (TNF­α), interleukin­1ß (IL­1ß), IL­6, intercellular adhesion molecule-1 (ICAM­1) and vascular cell adhesion molecule-1 (VCAM­1) in the supernatant. Protein expression levels of Toll­like receptor 4 (TLR4), nuclear factor­κB (NF­κB) p65, inhibitor of NF­κB, mitogen­activated protein kinase p38 and signal transducer and activator of transcription (STAT) in cells were measured by western blotting. Kaempferol significantly reduced the overproduction of TNF­α, IL­1ß, interleukin­6, ICAM­1 and VCAM­1 induced by LPS, indicating the negative regulation of kaempferol in TLR4, NF­κB and STAT signaling underlying intestinal inflammation. The present results provide support for the potential use of kaempferol as an effective therapeutic agent for IBD treatment.

[CT manifestations of the thymic carcinoid and literature review].

OBJECTIVE: To investigate the CT manifestations of thymic carcinoid and assess the diagnostic value of CT for this disease. METHODS: CT and clinical findings of 5 patients (4 males and 1 female, average age 41 years) with histologically confirmed thymic carcinoid were retrospectively analyzed. RESULTS: The clinical findings of the 5 patients showed no specificity, and none of the patients presented with carcinoid syndrome. The tumors were relatively large (mean size on the largest planar of 11.7 cm x 7.6 cm) with heterogeneous density, and showed necrosis or cystic degeneration in the tumor. The lesions showed uneven enhancement in contrast-enhanced imaging and displayed linear enhancement of the blood vessels in the tumors in 3 cases with unclear tumor margins. The adjacent major vessels were displayed in 4 cases (the superior vena cava in 2 and brachiocephalic vein in 4 cases), and 5 showed mediastinal and/or root of the neck lymphatic metastasis. None of the cases have lung or other site metastasis. CONCLUSION: The CT findings of the thymic carcinoid have some characteristics, and can be helpful in the diagnosis.

[Correlation between magnetic resonance imaging findings and proliferating cell nuclear antigen expression in peripheral lung cancer].

OBJECTIVE: To study the correlation between magnetic resonance imaging (MRI) findings and proliferating cell nuclear antigen (PCNA) expression in peripheral lung cancer. METHODS: The expression of PCNA was detected by means of SABC immunohistochemistry in 45 cases of surgically and pathologically confirmed peripheral lung cancer. The correlation between PCNA expression in the tumors and the MRI findings was analyzed. RESULTS: PCNA expression was correlated to the differentiation, tumor size, lobulation, and mediastinal lymph node metastasis of the tumors (P<0.05), but not to the histological type, clinical stage, pleural retraction, spiculation, or signal feature. CONCLUSION: Correlations are found between MRI findings of lung cancer and abnormal expression of PCNA.