[A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment].

Objective: To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro, providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis. Methods: According to the classification of periodontal diseases of 2018, gingival tissues were collected from periodontally healthy subjects and patients with stage Ⅲ-Ⅳ, grade C periodontitis, who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology, School of Stomatology, The Fourth Military Medical University from April to May 2022. Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D (GSDMD), a hallmark protein of cell pyroptosis, in gingival tissues. Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks (ligation group). The alveolar bone resorption was determined by micro-CT (mice without ligation treatment were used as the control group), and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg) combined with adenosine triphosphate (ATP) at various concentrations of 0.5, 1.0, 2.5, 5.0, and 10.0 mg/L, respectively, and the 0 mg/L group was set as the control group at the same time. Scanning electron microscopy was used to observe the morphology of HUVECs. Western blotting was used to detect the expression of gasdermin D-N terminal domains (GSDMD-N) protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD. Cell counting kit-8 (CCK-8) was used to detect the proliferative ability of HUVECs, and propidium iodide (PI) staining was used to detect the integrity of cell membrane of HUVECs. Results: Immunohistochemistry showed that GSDMD in gingival tissues of periodontitis was mainly distributed around blood vessels and its expression level was higher than that in healthy tissues. Micro-CT showed that alveolar bone resorption around the maxillary second molar significantly increased in ligation group mice compared with control subjects (t=8.88, P<0.001). Immunofluorescence staining showed significant colocalization of GSDMD with CD31 in the gingival vascular endothelial cells in mice of ligation group. The results of scanning electron microscopy showed that there were pores of different sizes, the typical morphology of pyroptosis, on HUVECs cell membranes in the inflammatory environment simulated by ATP combined with different concentrations of LPS, and 2.5 mg/L group showed the most dilated and fused pores on cell membranes, with the cells tended to lyse and die. Western blotting showed that the expression of GSDMD-N, the hallmark protein of cell pyroptosis, was significantly higher in 2.5 and 5.0 mg/L groups than that in the control group (F=3.86, P<0.01). Immunofluorescence cell staining showed that the average fluorescence intensity of GSDMD in 2.5 mg/L group elevated the most significantly in comparison with that in the control group (F=35.25, P<0.001). The CCK-8 proliferation assay showed that compared to the control group (1.00±0.02), 0.5 mg/L (0.52±0.07), 1.0 mg/L (0.57±0.10), 2.5 mg/L (0.58±0.04), 5.0 mg/L (0.55±0.04), 10.0 mg/L (0.61±0.03) groups inhibited cell proliferation (F=39.95, P<0.001). PI staining showed that the proportion of positive stained cells was highest [(56.07±3.22)%] in 2.5 mg/L group (F=88.24, P<0.001). Conclusions: Endothelial cells undergo significant pyroptosis in both in vivo and in vitro periodontal inflammatory environments, suggesting that endothelial cell pyroptosis may be an important pathogenic factor contributing to the pathogenesis of periodontitis.

[Effects of caspase recruitment domain protein 9 gene knockout on airway inflammation in C57BL/6 mice with steroid resistant asthma].

Objective: To investigate the role of caspase recruitment domain protein 9 (CARD9) in airway injury and inflammation of steroid resistant asthma in C57BL/6 mice. Methods: C57BL/6 mice were divided into A group (control group), B group (model group) and C group (dexamethasone treatment group), with 6 mouse in each group using random number table. The mouse asthma model was established in B and C group by subcutaneous injection of ovalbumin (OVA)/complete Freund adjuvant (CFA) in the abdomen and OVA aerosol challenge, the pathological change and cell count in broncho alveolar lavage fluid (BALF) were detected in order to confirm the model as steroid resistant asthma, and the lung tissue inflammatory infiltration was scored. Western blot was used to detect the changes of CARD9 protein between the group A and B; then wild-type and CARD9 knockout mice were divided into D group (wild-type control group), E group (wild-type model group), F group (CARD9 knockout control group) and G group (CARD9 knockout model group), the following indicators were observed and compared after establishing steroid resistant asthma model separately: HE staining was used to observe the pathological changes of lung tissue, ELISA was used to detect the protein levels of interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-17(IL-17) in BALF, and RT-PCR was used to detect the mRNA levels of CXC motif chemokine ligand-10 (CXCL-10) and IL-17 in lung. Results: The inflammatory score (3.33±0.82 vs 0.67±0.52) and BALF total cell count [(10.13±4.83) ×105/ml vs (3.76±0.84) ×105/ml] in B group were higher than those in the A group with statistical significance (P<0.05). There was no significant difference between group C and group B in inflammatory infiltration score (2.83±0.75 vs 3.33±0.82) and BALF total cell count [(9.80±3.19) ×105/ml vs (10.13±4.83) ×105/ml] (P>0.05). Moreover the protein level of CARD9 was increased in the B group than A group (0.245±0.090 vs 0.047±0.014, P=0.004). Compared to E group and F group, more obviously inflammatory cells, neutrophils, eosinophils infiltration and tissue injury were observed in G group (P<0.05), so did the expression of IL-4 (P<0.05), IL-5 and IL-17. Meanwhile the mRNA expression levels of IL-17 and CXCL-10 also increased in lung tissue (P<0.05) of G group. Conclusion: CARD9 gene deletion may aggravate the steroid resistant of asthma by increasing neutrophil chemokines, such as IL-17 and CXCL-10, therefore increasing infiltration of neutrophils in C57BL/6 mice asthma model.

[The role of S100A8/RAGE and Caveolin-1 and the effect of roxithromycin on their expression in a rat model of neutrophilic asthma].

Objective: To explore the role of S100A8, the receptor for advanced glycation endproducts (RAGE) and Caveolin-1 in neutrophilic asthmatic rats, and to further study the intervention of roxithromycin and the possible mechanisms. Methods: Male Brown Norway rats were randomly assigned to a control group, an asthma group and a Roxithromycin group. The asthmatic rat model was established by intraperitoneal injection of ovalbumin (OVA) and Freund's complete adjuvant (FCA) mixture, and aerosol inhalation of OVA. Rats in the Roxithromycin group were given roxithromycin injection 30 mg/kg 30 minutes before each challenge. Rats in the control and the asthma groups were replaced with equal volumes of saline, respectively. Bronchoalveolar lavage fluid (BALF) neutrophil percentage (Neu%) and pathological changes of pulmonary tissue (hematoxylin-eosin, HE staining) were measured to confirm the establishment of asthmatic models. The concentration of inflammatory cytokines and S100A8 were quantified by enzyme-linked immunosorbent assay (ELISA), and the expression of Caveolin-1 and RAGE at protein levels were detected by immunohistochemistry and Western blot. Results: Neu% in BALF of the asthma group was significantly higher than those of the control group, and Neu% in the Roxithromycin group was lower than the asthma group (all P<0.01). Pulmonary histology revealed that there were a large number of inflammatory cells infiltrated in the bronchial and perivascular, pulmonary interstitial and alveolar spaces, and the bronchial wall and smooth muscles were thickened obviously in the asthma group. Rats in the Roxithromycin group showed milder inflammation and airway remodeling change than the asthma group. There was no obvious pathological damage in the control group. The concentration of IL-6 and IL-17 in BALF and serum of rats in the asthma group were significantly higher than those in the control group (P<0.01), and Roxithromycin inhibited the high expression of these cytokines (P<0.05). The expression of S100A8 and RAGE in the asthma group were significantly higher than those in the control group [(20.6±4.4) vs (7.1±2.0) ng/L; (885±118) vs (462±102) ng/L; (14.2±1.7) vs (7.6±1.8) ng/L; (774±166) vs (406±69) ng/L, all P<0.05], and Roxithromycin inhibited the high expression of these proteins [(14.3±3.7) vs (20.6±4.4) ng/L; (650±53) vs (885±118) ng/L; (10.4±1.2) vs (14.2±1.7) ng/L; (560±64) vs (728±72) ng/L] (all P<0.05). Meanwhile, the expression of Caveolin-1 in the asthma group was significantly lower than that in the control group (P<0.01), and Roxithromycin up-regulated its expression (P<0.01). Correlation analysis showed that there was a significantly positive correlation between the expression of S100A8 and RAGE (r=0.706, P<0.01), while there was a significantly negative correlation between the expression of S100A8 and Caveolin-1 (r=-0.775, P<0.01), and between the expression of Caveolin-1 and RAGE (r=-0.919, P<0.01). Conclusion: S100A8 and Caveolin-1 may play an important role in neutrophilic asthma via RAGE, and Roxithromycin may exerts anti-inflammatory effects and inhibition of airway remodeling partly through this signaling pathway.

InAs/GaAs quantum dot semiconductor saturable absorber for controllable dual-wavelength passively Q-switched fiber laser.

We experimentally demonstrate the first use of 1550-nm InAs/GaAs quantum dot semiconductor saturable absorber mirror (QD-SESAM) in the dual-wavelength passively Q-switched (QS) erbium doped fiber (EDF) laser. The dual-wavelength QS lasing was obtained at a pump threshold of 180 mW with the average output power of 2.2 mW and the spacing between the two lasing wavelengths is 14 nm. A large absorption ranging from 1520 to 1590 nm has been realized when no substrate rotation was employed during the molecular beam epitaxy growth of the QD-SESAM indicating the potential to generate a 60 nm spacing of the dual-wavelength QS lasing peaks by changing the positions in the QD-SESAM and replacing EDF by co-doped fiber as gain medium. These results have provided a new opportunity towards achieving the stable and wide wavelength-tunable dual-modes fiber lasers.

Effect of intravesical botulinum neurotoxin-A injection on detrusor hyperreflexia in spinal cord injured patients.

PURPOSE: To evaluate the effects of Botulinum Toxin A injection into the detrusor muscle on various voiding parameters in spinal cord injured patients with neurogenic detrusor hyperreflexia Materials and methods: 24 patients with spinal cord injuries who had detrusor overactivity and urinary incontinence and were refractory to oral medications, were injected 300 IU of BTX-A into the detrusor muscle. The pre-and post-treatment evaluations included determination of bladder urinary continence status, frequency/volume chart of CIC, Incontinence Quality of Life questionnaire (I-QOL) and patient satisfaction. The urodynamic parameters measured included maximum cystometric capacity (MCC), reflex detrusor volume (RDV) and maximum detrusor pressure during bladder contraction (MDP) were analyzed at the outset and during the follow-up (2, 6, and 24 weeks) examinations. RESULTS: The evaluation of urodynamic parameters during follow-up examinations (2, 6 and 24 weeks) revealed significant increase in mean reflex volume (p<0.05) and cystometric capacity (p<0.05), on the other detrusor pressure decreased significantly (p<0.05). In majority of patients there was considerable reduction in incontinence episodes and no complications or side effects were reported. Most of the patents were satisfied with the treatment. CONCLUSION: The use of Botulinum toxin type A for treatment of neurogenic detrusor overactivity in spinal cord injured patients is safe and efficacious. In our 24-week study period, there was significant improvement in most urodynamic parameters with consistence and subjective satisfaction indicated by the treated patients.