Exosomes Derived from Endothelial Cells Inhibit Neointimal Hyperplasia Induced by Carotid Artery Injury in Rats via ROS-NLRP3 Inflammasome Pathway.

This study attempted to investigate whether exosomes derived from rat endothelial cells (EC-Exo) attenuate intimal hyperplasia after balloon injury using hematoxylin and eosin staining, immunohistochemistry, immunofluorescence staining, Evans blue staining, and Western blotting. The results indicated that EC-Exo inhibited intimal hyperplasia in the carotid artery after balloon injury, promoted re-endothelialization, and reduced vascular inflammation and ROS-NLRP3-mediated cell pyroptosis. Thus, EC-Exo can inhibit neointimal hyperplasia after carotid artery injury in rats presumably by inhibiting the ROS-NLRP3 inflammasome and phenotypic transformation of vascular smooth muscle cells.

[Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse].

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.

[To evaluate the disk position of the temporomandibular joint using calcium suppressed spectral-based CT images in the patients with temporomandibular disorder].

Objective: To evaluate the application of calcium suppressed (CaSupp) spectral CT technique in evaluating disk position and measuring the thickness of the posterior band of temporomandibular joint (TMJ). Methods: The twenty-three temporomandibular disorder patients [mean age 23(12~62) years, male/female=14/9] were performed with oblique sagittal and coronal proton density weighted imaging (PDWI) and spectral CT scans from February to July, 2019 in Department of Radiology, Hainan Hospital of General Hospital of Chinese PLA, and 45 TMJ joints were evaluated. The subjects were classified into two groups according to the scanning modalities: MRI measurement group and CaSupp spectral-based CT group. The CaSupp technique were applied with the spectral-based CT images and CaSupp images were generated. The oblique sagittal and coronal CaSupp imaged were reformatted by perpendicular to the long axis of the condyle. The TMJ disk positions were evaluated on oblique sagittal and coronal images, and the maximal disk thickness were measured on the oblique sagittal images. Results: The joint position was basically consistent on MRI and CaSupp images for the 45 TMJ joints. The intra-class coefficient value was 0.843 (0.712, 0.914) for the measurement of the posterior band of the TMJ disk between MRI and CaSupp images. Bland-Altman presented that the [95.6% (43/45)] points with the difference located in the 95% agreement interval. Wilcoxon paired text demonstrated that there was no significant different for the thickness of the posterior band between MRI [2.57 (1.76, 3.65) mm] and CaSupp images [2.67 (1.74, 4.56) mm] (P=0.07). Conclusions: The CaSupp spectral-based CT could be used to evaluated the TMJ disk position and the thickness of the posterior band.

Co-inhibitory immune checkpoints in head and neck squamous cell carcinoma.

The upregulation of co-inhibitory immune checkpoints hampers the immune response toward tumor cells and facilitates the tumor cells ability to evade immunosurveillance. Specific inhibitory immune checkpoint delivers inhibitory signals to T cells using multiple mechanisms. More in-depth understanding of the co-inhibitory immune checkpoints could be exploited for head and neck squamous cell carcinoma (HNSCC) treatment. In this review, we summarize the expression and the mechanism of partial co-inhibitory immune checkpoint signals and discuss targeting co-inhibitory immune checkpoints as an immunotherapeutic target for cancer therapy. This review may provide a better understanding of the co-inhibitory immune checkpoints and could promote applications of immunotherapy.

[Effect of picroside â ¡ on the expression of mitochondrial VDAC1 after cerebral ischemia/reperfusion in rats].

Objective: To explore the effect of picroside Ⅱ on the expression of mitochondrial voltage-dependent anion channel 1 (VDAC1) in rats after cerebral ischemiareperfusion. Methods: A total of 70 Wistar rats models with middle cerebral artery occlusionreperfusion (MCAO/R) were randomly divided into the sham group, model group, picroside (Picr) group, ruthenium red (RuR) group, RuR+ Picr group, Spermine (Sper) group, Sper+ Picr group (n=10 per group). Modified neurological severity scale (mNSS) was used to evaluated the neurobehavioral function, the expression of reactive oxygen species (ROS) in brain tissues were measured by enzyme-linked immunosorbent assay (ELISA), the morphology of brain tissues was observed by hematoxylin-eosin (HE) staining, the apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL), and the expressions of VDAC1 and endonuclease G (EndoG) were determined by immunohistochemical assay and Western blot. Results: Compared with the shame group, the mNSS scores (9.6±1.9), the expression of ROS[(47.6±2.7)U/ml], the apoptosis of neuron(23.8±2.8), and the expressions of VDAC1(0.94±0.06) and EndoG in cytoplasm (0.76±0.06) and nuclei(0.75±0.06)were enhanced in the model group (all P<0.05). The Picr group had obviously decreased mNSS scores (5.7±0.9), ROS expression[(35.6±2.2)U/ml], number of apoptotic cells (14.5±2.1), VDAC1 (0.63±0.06) and EndoG in cytoplasm (0.34±0.05) and nuclei (0.31±0.06)expressions compared to the model group (P<0.05). Conclusion: Picroside Ⅱ could attenuate cerebral I/R injury by down-regulating the expression of VDAC1 and inhibiting the EndoG release from mitochondria into cytoplasm.

[Effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit(+) cardiac stem cells apoptosis].

Objective: To explored the effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit(+) cardiac stem cells apoptosis. Methods: C-kit(+) cardiac stem cells were isolated from SD rats by the methods of enzyme digestion and magnetic bead. Cells were divided into the following experimental groups: (1) negative control mimics (NCM)group: cells were transfected with negative control miRNA-21 mimics for 48 hours; (2)mimics group: cells were transfected with miRNA-21 mimics for 48 hours; (3) NCM+ H(2)O(2) group: negative control miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 µmol H(2)O(2) for 2 hours; (4)mimics+ H(2)O(2) group: miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 µmol H(2)O(2) for 2 hours. mRNA of miRNA-21 was detected by RT-PCR. The apoptosis rate of C-kit(+) cardiac stem cells was determined using the annexin V-FITC/PI staining assay. Western blot was employed to measure the expression of apoptosis related proteins(Caspase-3, Bax, and Bcl-2). Results: (1) Compared with the NCM group, the mRNA expression level of miRNA-21 was significantly up-regulated in mimics group, while obviously down-regulated in NCM+ H(2)O(2) group(all P<0.05). Compared with the mimics group, the mRNA expression levels of miRNA-21 in mimics+ H(2)O(2) group was significantly downregulated (P<0.05), but remarkably upregulated compared with the NCM+ H(2)O(2) group(P<0.05). (2) Flow cytometry results indicated that the early apoptosis rates were similar between the NCM group and mimics group ((4.57±3.45)% vs. (5.13±3.21)%, P>0.05). Compared with the NCM group, the early apoptosis rates were remarkably increased ((79.07±5.75)% vs.(4.57±3.45)%, P<0.05) in NCM+ H(2)O(2) group. Compared with the mimics group, the early apoptosis was significantly up-regulated in the mimics+ H(2)O(2) group ((30.27±1.36)% vs.(5.13±3.21)%, P<0.05), which were further down-regulated in mimics+ H(2)O(2) group compared with the NCM+ H(2)O(2) group ((30.27±1.36)% vs.(79.07±5.75)%, P<0.05). (3) Western blot results showed similar protein expression of Caspase-3, Bax and Bcl-2 between NCM group and mimics group(all P>0.05). Compared with the NCM group, the Caspase-3 and Bax protein expression was significantly increased in NCM+ H(2)O(2) group (all P<0.05), but the protein expression level of Bcl-2 was similar between the 2 groups(P>0.05). The Caspase-3 and Bax protein expression was markedly decreased, while Bcl-2 apparently increased in the mimics+ H(2)O(2) group compared with the NCM+ H(2)O(2) group(all P<0.05). Conclusion: Overexpression of miRNA-21 protects the C-kit(+) cardiac stem cells from apoptosis caused by oxidative stress through downregulating proapoptotic and upregulating the antiapoptotic proteins.

STAT3 Induces Immunosuppression by Upregulating PD-1/PD-L1 in HNSCC.

Head and neck cancer is one of the most prevalent cancers around the world. Head and neck squamous cell carcinoma (HNSCC) accounts for nearly 90% of head and neck cancer. In recent years, significant advances have been made in immunotherapy for HNSCC. Although some clinical trials targeting immune checkpoints have shown success, the molecular mechanism for regulation of programmed death 1 (PD-1) and its ligand (PD-L1) is partially understood. In an effort to explore the effect of activation of signal transducers and activators of transcriptions (STAT3) on PD-1/PD-L1, the expression and correlation between phosphorylation of STAT3 and PD-1/PD-L1 were determined with immunostaining of human and mouse HNSCC tissue sections. PD-1/PD-L1 overexpression was found to be significantly associated with p-STAT3 in human and mouse HNSCC. Targeting STAT3 by a small molecule effectively inhibited the expression of PD-L1 in the CAL27 cell line. Furthermore, we found that blockade of STAT3 signaling downregulated PD-1/PD-L1 in a Tgfbr1/Pten 2cKO HNSCC mouse model. These findings suggest that STAT3 signaling plays an important role in PD-1/PD-L1 regulation and the antitumor immune response of HNSCC.

SATB1 promotes tumor metastasis and invasiveness in oral squamous cell carcinoma.

OBJECTIVE: Our aim is to evaluate the expression of SATB1 in human oral squamous cell carcinomas (OSCC) and its role in the invasiveness and metastasis of OSCC. SUBJECTS AND METHODS: A human OSCC tissue microarray was used to evaluate the expression pattern of SATB1. SATB1 mRNA knockdown was performed in human OSCC cell lines SCC25 and Cal27 to assess the function of SATB1 in the invasiveness and metastasis of OSCC. RESULTS: SATB1 is highly expressed in human OSCC determined by immunohistochemistry, and its nuclear/cytoplasmic ratio of histoscore is significantly correlated with patients' prognosis. Reduced cell motility, invasiveness, expression of epithelial to mesenchymal transition (EMT) markers (N-cadherin and ß-catenin), and elevated expression of epithelial markers were observed in SATB1-knockdown cells in in vitro studies. Depletion of SATB1 also restored a cobblestone-like morphology in TGF-ß1-treated cells. CONCLUSIONS: These findings suggest SATB1 may play an important role in OSCC invasiveness and metastasis.

Effects of mycophenolate mofetil on the expression of monocyte chemoattractant protein-1 and fibronectin in high glucose cultured human mesangial cells.

The effects of high glucose on the expression of monocyte chemoattractant protein-1 (MCP-1) and the main component of the extracellular matrix, fibronectin (FN), were explored in human mesangial cells (HMCs), along with the intervention effects of mycophenolate mofetil (MMF) on these indicators. Cultured HMCs were divided into five groups: 1) normal control group (5 mM glucose); 2) high glucose group (30 mM glucose); 3) mannitol osmotic pressure control group (5 mM glucose + 25 mM mannitol); 4) high glucose + MMF-10 group (30 mM glucose + 10 µg/mL MMF); 5) high glucose + MMF-100 group (30 mM glucose + 100 µg/mL MMF). At 24, 48, and 72 h, reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay methods were used to detect the effects of MMF on MCP-1 mRNA and protein and FN expression in HMCs under high glucose conditions. MCP-1 mRNA and protein expressions and FN secretion significantly increased in HMCs of the high glucose group compared with the normal control group (P < 0.01), with the highest expression observed at 48 h. MMF could reduce the MCP-1 mRNA and protein and FN expression levels (P < 0.01), and the inhibition occurred in a dose- and time-dependent manner (P < 0.05). In conclusion, MMF could inhibit MCP-1 expression and the secretion of FN, indicating that it may delay the progression of glomerulosclerosis and interstitial fibrosis in diabetic nephropathy to ultimately achieve protective effects on the kidney.

[The role of pentoxifylline in acute lung injury complicated by pancreatitis].

Caerulein-induced acute pancreatitis in rats commonly complicated ARDS-like acute lung injury. Acute pancreatitis induced by caerulein in the circulating neutrophil-depleted rat by hydroxyrea or with the administration of SOD, CAT or Pentoxifylline, the wet lung weight, lung capillary endothelial permeability decrease significantly compared to the caerulein group (P < 0.05). There are no lung morphologic evidences of neutrophil sequestration, interstial edema, intralveolar hemorrhage that seen in caerulein infusion animals. But it has no effect against the development of acute pancreatitis. It suggested that neutrophil and neutrophil-derived oxygen radical are the important mediators of acute lung injury complicated by pancreatitis.

[Lung biopsies with light microscopic and ultrastructural observations in 34 cases of diffuse pulmonary diseases].

Lung biopsies with light microscopic and ultrastructural observations were performed in 34 cases of diffuse pulmonary diseases. Open lung biopsy 2 cases, transfiberoptic bronchoscopy lung biopsy 32 cases. There were 19 (56%) idiopathic interstitial fibrosis, 3 (9%) pulmonary alveolar proteinosis, 3 (9%) pulmonary sarcoidosis, 1 (3%) idiopathic pulmonary hemosiderosis, 2 (6%) pulmonary mycosis, 1 (3%) alveolar cell carcinoma, 2 (6%) pulmonary tuberculosis and 3 (9%) pulmonary collagen diseases. Diagnosis was based on clinical data and pathologic manifestation. Lung biopsy with light and electronic microscopic examination is useful in etiologic diagnosis and differential diagnosis for diffuse pulmonary diseases.

[Analysis of multiple stepwise regression for prognosis in patients with bacterial pneumonias in elderly].

This paper was based on the material of 100 patients with elderly bacterial pneumonias, doing analysis of multiple stepwise regression for factors effecting prognosis with IBC-PC electronic computer. 100 patients (82 men, 18 women), average ages were 69.6 +/- 7.9 (60-90) years. According to the materials of clinical selected 39 items of possible affect prognosis factors. Item is independent variable (xi). The prognosis is emergency variable (y). Between both were doing analysis of regression, factors of significance were selected to multiple regression equation. In order to decide the closer relations degree between the xi and the y the F values were regulated. Using quantitative analysis and measure, the primary factors were founded. When F value is equal to 1, selected 22 factors related to prognosis. F value is equal to 2, selected factors decrease to 12, F value is equal to 3, selected factors were 6 items. Respectively, F value is 4, 5, selected factors were 5 items, they have very close regression coefficient. That showed the equation was tendency stable. According to the big or small standard regression coefficient, the sequence of affecting prognosis factors were course of disease, upper digestive tract haemorrhage, dielectric disorder, respiratory failure and gram-negative bacilli infection. The result of analysis showed that the key factors were: treatment and diagnosis without delay, pay great attention to examine of pathogen and management of complication.