[Effect of proprotein convertase subtilisin/kexin type 9 on platelet activation associated with sepsis].

OBJECTIVE: To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis. METHODS: (1) Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. (2) Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting. RESULTS: (1) Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (µg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×109/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients (n = 20) was significantly higher than that in the non-thrombocytopenia patients (n = 37; µg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients (r = -0.340, P = 0.010). (2) Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×109/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/ß-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. CONCLUSIONS: PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.

Swimming exercise activates aortic autophagy and limits atherosclerosis in ApoE-/- mice.

BACKGROUND: The aim of this study was to investigate the beneficial effect of swimming exercise on autophagy and atherosclerosis in mice aorta, so as to clarify the possible causal relationship between autophagy activation and atherosclerosis. METHODS: The body weight was monitored regularly. Hematoxylin-eosin staining and Oil Red O staining was conducted to observe vascular morphology and plaque burden respectively. The levels of serum total cholesterol (TC), triglyceride (TG), soluble intercellular adhesion molecule-1 (sICAM-1), matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) was examined via Enzyme-linked immu-nosorbent assays (ELISA). The mRNA expression level of autophagy markers, including LC3 and Beclin-1, was examined by real-time quantitative polymerase chain reaction (RT-PCR). The expressions of LC3-II/LC3-I and Beclin-1 are detected by Western blotting and immunohistochemistry. RESULTS: Compared with the model group, long-term swimming exercise decreased the weight gain of ApoE-/- mice, improved the structural disorder of artery, reduced the load of atherosclerotic lesion, and attenuated the concentrations of serum TC, TG, sICAM-1, MMP-9, and IL-6. In addition, the expression of autophagy markers LC3 and Beclin-1 increased significantly at the mRNA and protein levels. CONCLUSION: Long-term swimming exercise could activate the autophagy and reduce atherosclerotic lesion in the aorta of ApoE-/- mice. Autophagy activation may be one of the mechanisms by which atherosclerosis is improved through exercise.

TRIM34 localizes to the mitochondria and mediates apoptosis through the mitochondrial pathway in HEK293T cells.

Tripartite motif 34 (TRIM34) is a member of TRIM family that can be highly induced by type I Interferon. Currently little is known about the subcellular localization and biological function of TRIM34. In the present study, confocal microscope assay showed that TRIM34 proteins were mainly distributed in the cytoplasm and part of TRIM34 proteins were localized to the mitochondria in human embryonic kidney 293T (HEK293T) cells. Western blot results demonstrated FLAG-TRIM34 could also be identified in the mitochondrial fractions of HEK293T cells transfected with the 5'FLAG-pcDNA3.1-TRIM34 vector. The CCK-8 assay further demonstrated that TRIM34 significantly decreased the viability of HEK293T cells. Nevertheless, TRIM34 had no apparent effect on the cell cycle distribution. Interestingly, flow cytometry showed that TRIM34 could obviously induce apoptosis in HEK293T cells. Moreover, we discovered that TRIM34 promoted apoptosis by inducing the loss of mitochondrial membrane potential (MMP) in HEK293T cells, leading to the release of cytochrome c from mitochondia. In short, these results demonstrate that TRIM34 proteins can localize to the mitochondria and induce apoptosis via the depolarization of MMP in HEK293T cells.

TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells.

Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.

[Tripartite motif-containing protein 34 (TRIM34) colocalized with micronuclei chromosome and hampers its movement to equatorial plate during the metaphase stage of mitosis].

Objective To examine whether tripartite motif-containing protein 34 (TRIM34) is colocalized with micronuclei and investigate the influence on the movement of micronuclei chromosome in mitosis. Methods The eukaryotic expression vector TRIM34-pEGFP-N3 was constructed, identified and then transfected into HEK293T cells. With 4', 6-diamidino-2-phenylindole 2HCI (DAPI) staining, the colocalization between TRIM34 and micronuclei was observed under a fluorescence microscope. Moreover, MitoTracker(R)Deep Red was used to identify the colocalization between the complex of TRIM34-micronulei and mitochondria under a confocal microscope. Finally, the effect of TRIM34 on the movement of micronuclei chromosome in mitosis was examined. Results DNA sequencing confirmed that the vector TRIM34-pEGFP-N3 was constructed successfully. A fluorescence microscope revealed that TRIM34 could be colocalized with micronuclei in HEK293T cells transfected with TRIM34-pEGFP-N3. In the same manner, a confocal microscope distinctly showed that TRIM34 was colocalized with micronuclei similarly in appearance. However, there was no distinguished colocalization relationship between the complex of TRIM34-micronulei and mitochondria. Interestingly, the micronuclei chromosome conjugated with TRIM34 was hardly transferred to equatorial plate during the metaphase stage of mitosis. Conclusion TRIM34 is colocalized with micronuclei chromosome and hampers its movement to equatorial plate in mitosis.

Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A.

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22's targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.

[Expressions and co-localization of HIV capsid protein p24 and TRIM22 in HEK293T cells].

OBJECTIVE: To observe the co-localization of human immunodeficiency virus (HIV) capsid protein p24 and tripartite motif containing 22 (TRIM22) in HEK293T cells. METHODS: The retroviral packaging vector pLP1 was used as the template of p24. After the amplification by PCR, the sequence of p24 was cloned into the eukaryotic expression vector pDsRed-Monomer-N1. The recombinant vector was confirmed by colony PCR, double restriction enzyme digestion and DNA sequencing. HEK293T cells were co-transfected with the vector pDsRed-Monomer-N1-p24 together with pEGFP-N3-TRIM22. After 24 hours, the co-localization of p24-DsRed-Monomer and TRIM22-EGFP was detected under a fluorescence microscope. RESULTS: Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the eukaryotic expression vector pDsRed-Monomer-N1-p24 was constructed successfully. Fluorescence microscope showed that p24-DsRed-Monomer was co-localized with TRIM22-EGFP in HEK293T cells. CONCLUSION: HIV capsid protein p24 is co-localized with TRIM22 in HEK293T cells.

[Changes and the clinical significance of CD4⁺ CD25⁺ regulatory T cells and Th17 cells in peripheral blood of infants with respiratory syncytial virus bronchiolitis].

AIM: To observe the percentages of CD4(+);CD25(+); regulatory T cells (Tregs) and Th17 cells and the levels of IL-10, TGF-ß and IL-17 in peripheral blood of infants with respiratory syncytial virus (RSV) bronchiolitis. The relationship between above cells, cytokines and RSV bronchiolitis was determined. METHODS: Thirty-three infants with RSV bronchiolitis, twenty-eight infants with non-RSV pneumonia and twenty-six healthy infants were enrolled. The percentages of Tregs and Th17 cells in peripheral blood were detected by flow cytometer (FCM), and the levels of IL-10, TGF-ß and IL-17 in plasma were determined by ELISA. RESULTS: The percentage of Tregs and the levels of IL-10 and TGF-ß in infants with RSV bronchiolitis were significantly lower than those in infants with non-RSV pneumonia and healthy infants (P<0.05), while the percentage of Th17 cells and the level of IL-17 in infants with RSV bronchiolitis were significantly higher than those in infants with non-RSV pneumonia and healthy infants (P<0.05). CONCLUSION: The imbalance between Tregs and Th17 cells in peripheral blood of infants with RSV bronchiolitis may be one of the pathogenesis of RSV bronchiolitis.

[Construction of Trim6 eukaryotic expression vector and its expression in HEK293 cells].

AIM: To construct the recombinant eukaryotic expression vector pcDNA3.1 (+)-Trim6, and observe its expression in HEK293T cells in vitro. METHODS: The total RNA was isolated from HeLa cells. After amplification with reverse transcription polymerase chain reaction (RT-PCR), the target sequences were cloned into the pcDNA3.1(+). The recombinant vector was confirmed by restriction enzyme digestion, PCR and sequencing. Then it was transfected into HEK293T cells.After 24 hours, the Trim6 expression was detected by Western blot. RESULTS: The results of the restriction enzyme digestion, PCR and sequencing confirmed the vector was constructed successfully, and it can express Trim6 protein in HEK293T cells. CONCLUSION: The vector is constructed successfully, which establishes the foundation for future research on the effect of Trim6.