AIM:To investigate the modulatory role of E3 ubiquitin-protein ligase ITCH in Alzheimer disease(AD)-like pathology through the thioredoxin-interacting protein(TXNIP)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway using both in vivo and in vitro experimental models.METHODS:(1)Ten 5×FAD(AD model)mice and 10 wild-type(WT)mice at 2-,4-and 6-month-old were randomly allocated into AD and WT groups.Amyloid β-protein(Aβ)plaque burden in the brain was detected by thioflavin-S and immunofluorescence staining,with the latter method additionally applied to assess TXNIP protein expression.The protein levels of ITCH and TXNIP were determined by Western blot,while their interaction was verified by co-immunoprecipitation.(2)Mouse mi-croglia BV2 cells stimulated by lipopolysaccharide(LPS)were used to construct neuroinflammation model,and were di-vided into control(CON)group and LPS+ATP treatment group.The BV2 cells stimulated by Aβ were used to construct AD inflammation model.According to the different treatment time,they were divided into CON,and 12,24 and 48 h treatment groups.Western blot was used to evaluate the expression of ITCH,TXNIP,and NLRP3 inflammasome compo-nents(NLRP3 and caspase-1)as well as the downstream IL-1β.Adenovirus-mediated ITCH overexpression(OE-ITCH)in Aβ-stimulated BV2 cells comprised three experimental groups:negative control group,Aβ oligomer stimulation group,and OE-ITCH group,with subsequent immunoblotting of inflammatory mediators.RESULTS:The deposition of Aβ plaques in the cortex and hippocampus of 5×FAD transgenic mice exhibited an age-dependent progression(P<0.01).Compared with WT mice,the levels of TXNIP protein increased synchronously,and the levels of ubiquitin ligase ITCH was significantly down-regulated(P<0.05).Co-immunoprecipitation confirmed the interaction between ITCH and TXNIP proteins in the brain of 2-and 4-month-old 5×FAD mice,which exhibited marked attenuation by 4 months of age.In BV2 microglial models,Aβ/LPS stimulation provoked significant ITCH suppression,concurrently up-regulating TXNIP,core NLRP3 inflammasome components(NLRP3 and caspase-1),and downstream IL-1β(P<0.05).Overexpression of ITCH significantly inhibited Aβ-induced activation of TXNIP and NLRP3 and therelated inflammatory factors in BV2 cells.CONCLUSION:The results of in vitro and in vivo experiments showed that ITCH protein exerts effects against AD-like pathology by inhibiting the expression of TXNIP-NLRP3 signaling pathway.

AIM:To investigate the modulatory role of E3 ubiquitin-protein ligase ITCH in Alzheimer disease(AD)-like pathology through the thioredoxin-interacting protein(TXNIP)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway using both in vivo and in vitro experimental models.METHODS:(1)Ten 5×FAD(AD model)mice and 10 wild-type(WT)mice at 2-,4-and 6-month-old were randomly allocated into AD and WT groups.Amyloid β-protein(Aβ)plaque burden in the brain was detected by thioflavin-S and immunofluorescence staining,with the latter method additionally applied to assess TXNIP protein expression.The protein levels of ITCH and TXNIP were determined by Western blot,while their interaction was verified by co-immunoprecipitation.(2)Mouse mi-croglia BV2 cells stimulated by lipopolysaccharide(LPS)were used to construct neuroinflammation model,and were di-vided into control(CON)group and LPS+ATP treatment group.The BV2 cells stimulated by Aβ were used to construct AD inflammation model.According to the different treatment time,they were divided into CON,and 12,24 and 48 h treatment groups.Western blot was used to evaluate the expression of ITCH,TXNIP,and NLRP3 inflammasome compo-nents(NLRP3 and caspase-1)as well as the downstream IL-1β.Adenovirus-mediated ITCH overexpression(OE-ITCH)in Aβ-stimulated BV2 cells comprised three experimental groups:negative control group,Aβ oligomer stimulation group,and OE-ITCH group,with subsequent immunoblotting of inflammatory mediators.RESULTS:The deposition of Aβ plaques in the cortex and hippocampus of 5×FAD transgenic mice exhibited an age-dependent progression(P<0.01).Compared with WT mice,the levels of TXNIP protein increased synchronously,and the levels of ubiquitin ligase ITCH was significantly down-regulated(P<0.05).Co-immunoprecipitation confirmed the interaction between ITCH and TXNIP proteins in the brain of 2-and 4-month-old 5×FAD mice,which exhibited marked attenuation by 4 months of age.In BV2 microglial models,Aβ/LPS stimulation provoked significant ITCH suppression,concurrently up-regulating TXNIP,core NLRP3 inflammasome components(NLRP3 and caspase-1),and downstream IL-1β(P<0.05).Overexpression of ITCH significantly inhibited Aβ-induced activation of TXNIP and NLRP3 and therelated inflammatory factors in BV2 cells.CONCLUSION:The results of in vitro and in vivo experiments showed that ITCH protein exerts effects against AD-like pathology by inhibiting the expression of TXNIP-NLRP3 signaling pathway.

AIM:To investigate the impact of Bruton tyrosine kinase(BTK)on Alzheimer disease(AD)-like pathology through the NIMA(never in mitosis gene A)-related kinase 7(NEK7)-nucleotide-binding oligomerization do-main-like receptor protein 3(NLRP3)pathway.METHODS:5xFAD and wild-type(WT)mice aged 2,4 and 6 months were utilized to assess the expression of BTK,NEK7 and NLRP3 proteins in the hippocampus and cortex via Western blot and immunofluorescence.Co-immunofluorescence was conducted to identify the interaction between NEK7 and NLRP3 in the brains of 4-month-old mice.Three-month-old mice were divided into a control group and an ibrutinib treatment group,receiving intraperitoneal injections of ibrutinib(10 mg/kg)or solvent for 14 d,and were then subjected to behavioral as-sessments including learning and memory tests using the Morris water maze and Y-maze.Wild-type mice were induced with an AD model by intracerebroventricular injection of Aβ42.Morris water maze tests were performed after 14 d to eva-luate learning and memory,followed by measurement of BTK protein levels in the brain via Western blot.BV2 microglial cells were treated with ibrutinib,followed by LPS or Aβ42 stimulation.Western blot analysis was conducted to measure the protein levels of NEK7,NLRP3,BTK and p-BTK(Y223),while immunofluorescence was used to assess the protein expression of ASC,caspase-1,NEK7 and NLRP3.RESULTS:The levels of BTK,NEK7 and NLRP3 in the brains of 5×FAD mice were significantly elevated compared to WT mice,with observed interaction between NEK7 and NLRP3 in the 5xFAD mouse brains.Ibrutinib treatment significantly improved learning and memory functions in mice compared to the AD group.In BV2 cells,pre-treatment with ibrutinib effectively suppressed the NLRP3 inflammasome pathway and NEK7 proteins in response to Aβ42 stimulation.CONCLUSION:BTK plays a regulatory role in AD-like pathology through the NEK7-NLRP3 pathway both in vivo and in vitro.

Objective To explore the effect and mechanism of microRNA(miR)-155-5p on myocardial pyroptosis in rats with myocardial ischemia-reperfusion injury(IRI).Methods Sixty SD rats were randomly divided into sham group,IRI group,agomir-NC group,miR-155-5p agomir group,antagomir-NC group,and miR-155-5p antagomir group,with 10 rats in each group.Echocardiography was used to measure the left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular ejection fraction(LVEF),and left ventricular fractional shortening(LVFS)of rats.Enzyme-linked immune absorbent assay(ELISA)was used to detect the levels of creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH),and cardiac troponin T(cTnT)in serum,as well as the levels of interleukin(IL)-1β,IL-6,IL-18,and tumor necrosis factor(TNF)-α in myocardial tissue of rats.Hematoxylin-eosin staining was used to observe pathological changes in rat myocardial tissue.Real-time fluorescent quantitative polymerase chain reaction was used to detect the expression levels of miR-155-5p and silent information regulator 1(SIRT1)messenger RNA(mRNA)in myocardial tissue of rats.Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-155-5p and SIRT1.Western blot was used to detect the expression levels of SIRT1,NOD-like receptor protein 3(NLRP3),cleaved cysteine aspartate specific proteinase-1(Cleaved Caspase-1),and gasdermin D(GSDMD)proteins in myocardial tissue of rats.Results Compared with the sham group,the LVEDD and LVESD of rats in the IRI group were increased,LVEF and LVFS were decreased,serum levels of CK-MB,LDH,and cTnT were increased,IL-1β,IL-6,IL-18 and TNF-α levels in myocardial tissue were increased,myocardial tissue structure was severely damaged,myocardial fibers were disordered,relative expression of NLRP3,Cleaved Caspase-1,and GSDMD proteins were increased,and the relative expression of SIRT1 protein was decreased(all P<0.05/5).Compared with the IRI group,the rats in the miR-155-5p agomir group had increased LVEDD and LVESD,decreased LVEF and LVFS,increased serum levels of CK-MB,LDH,and cTnT,increased myocardial tissue levels of IL-1β,IL-6,IL-18,TNF-α,aggravated myocardial tissue lesions,increased relative expression of NLRP3,Cleaved Caspase-1,and GSDMD proteins,and decreased relative expression of SIRT1 protein,and the rats in the miR-155-5p antagomir group had decreased LVEDD and LVESD,increased LVEF and LVFS,decreased serum levels of CK-MB,LDH,and cTnT,decreased myocardial tissue levels of IL-1β,IL-6,IL-18,TNF-α,reduced myocardial tissue lesions,decreased relative expression of NLRP3,Cleaved Caspase-1,and GSDMD proteins,and increased relative expression of SIRT1 protein(all P<0.05/5).miR-155-5p was negatively correlated with the expression levels of SIRT1 in rat myocardial tissue,and SIRT1 was a target gene of miR-155-5p.Conclusions miR-155-5p may participate in the regulation of myocardial IRI in rats by targeting the downregulation of SIRT1 and promoting NLRP3-mediated myocardial pyroptosis.

Objective:To explore the clinical value of coronary artery calcification (CAC) detected by chest CT in early screening of coronary atherosclerotic heart disease (CAHD) in civil pilots.Methods:The physical examination data of 2 899 civil pilots were retrospectively analyzed. Pilots were divided into CAHD group and control group based on the results of coronary angiography (CAG). The health data were compared between 2 groups and the clinical value of CAC in the diagnosis of CAHD was analyzed by using binary Logistic regression model and receiver operating characteristic (ROC) curve.Results:Thirty-eight CAHD cases were diagnosed, and the remaining 2 861 were in the control group. Comparing to that of control group, the average age of the pilots in CAHD group was greater ( t=12.09, P<0.001), and the average total flying hours were longer ( Z=-7.68, P<0.001). The proportions of smoking, hyperlipidemia, diabetes, hypertension, fatty liver, obesity, carotid plaques, positive or suspiciously positive in submaximal treadmill exercise test, CAC, as well as the proportions of taking further requested coronary CT angiography and CAG were significantly higher in the CAHD group ( χ2=5.42-1 430.25, P<0.01 or <0.05). Logistic regression model showed that smoking ( OR=2.800, 95% CI: 1.074-7.301, P=0.035), obesity ( OR=3.336,95% CI:1.243-8.956, P=0.017), positive or suspiciously positive in submaximal treadmill exercise test ( OR=17.669, 95% CI: 2.923-106.756, P=0.002) and CAC ( OR=96.039, 95% CI: 11.439-806.396, P<0.001) were the independent risk factors for diagnosing CAHD. The ROC curve results suggested that the sensitivity and specificity of CAC for predicting CAHD was 97.4% and 93.1%, respectively, and the area under the ROC curve was 0.952 ( P<0.001). Conclusions:CAC detected by chest CT in physical examination is helpful for early screening of asymptomatic or atypical CAHD in civil pilots.

Objective:To explore the clinical value of coronary artery calcification (CAC) detected by chest CT in early screening of coronary atherosclerotic heart disease (CAHD) in civil pilots.Methods:The physical examination data of 2 899 civil pilots were retrospectively analyzed. Pilots were divided into CAHD group and control group based on the results of coronary angiography (CAG). The health data were compared between 2 groups and the clinical value of CAC in the diagnosis of CAHD was analyzed by using binary Logistic regression model and receiver operating characteristic (ROC) curve.Results:Thirty-eight CAHD cases were diagnosed, and the remaining 2 861 were in the control group. Comparing to that of control group, the average age of the pilots in CAHD group was greater ( t=12.09, P<0.001), and the average total flying hours were longer ( Z=-7.68, P<0.001). The proportions of smoking, hyperlipidemia, diabetes, hypertension, fatty liver, obesity, carotid plaques, positive or suspiciously positive in submaximal treadmill exercise test, CAC, as well as the proportions of taking further requested coronary CT angiography and CAG were significantly higher in the CAHD group ( χ2=5.42-1 430.25, P<0.01 or <0.05). Logistic regression model showed that smoking ( OR=2.800, 95% CI: 1.074-7.301, P=0.035), obesity ( OR=3.336,95% CI:1.243-8.956, P=0.017), positive or suspiciously positive in submaximal treadmill exercise test ( OR=17.669, 95% CI: 2.923-106.756, P=0.002) and CAC ( OR=96.039, 95% CI: 11.439-806.396, P<0.001) were the independent risk factors for diagnosing CAHD. The ROC curve results suggested that the sensitivity and specificity of CAC for predicting CAHD was 97.4% and 93.1%, respectively, and the area under the ROC curve was 0.952 ( P<0.001). Conclusions:CAC detected by chest CT in physical examination is helpful for early screening of asymptomatic or atypical CAHD in civil pilots.

Objective:To observe the effect of noninvasive positive pressure ventilation (NIPPV) and high-flow nasal cannula oxygen therapy (HFNC) on the prognosis of patients with coronavirus disease 2019 (COVID-19) accompanied with acute respiratory distress syndrome (ARDS).Methods:A retrospective study was conducted in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology when authors worked as medical team members for treating COVID-19. COVID-19 patients with pulse oxygen saturation/fraction of inspiration oxygen (SpO 2/FiO 2, S/F) ratio < 235, managed by medical teams [using S/F ratio instead of oxygenation index (PaO 2/FiO 2) to diagnose ARDS] from February to April 2020 were included. The patients were divided into NIPPV group and HFNC group according to their oxygen therapy modes. Clinical data of patients were collected, including general characteristics, respiratory rate (RR), fraction of FiO 2, SpO 2, heart rate (HR), mean arterial pressure (MAP), S/F ratio in the first 72 hours, lymphocyte count (LYM), percentage of lymphocyte (LYM%) and white blood cell count (WBC) at admission and discharge or death, the duration of dyspnea before NIPPV and HFNC, and the length from onset to admission. The differences of intubation rate, all-cause mortality, S/F ratio and RR were analyzed, and single factor analysis and generalized estimation equation (GEE) were used to analyze the risk factors affecting S/F ratio. Results:Among the 41 patients, the proportion of males was high (68.3%, 28 cases), the median age was 68 (58-74) years old, 28 cases had complications (68.3%), and 34 cases had multiple organ dysfunction syndrome (MODS, 82.9%). Compared with HFNC group, the proportion of complications in NIPPV group was higher [87.5% (21/24) vs. 41.2% (7/17), P < 0.05], and the value of LYM% was lower [5.3% (3.4%-7.8%) vs. 10.0% (3.9%-19.7%), P < 0.05], the need of blood purification was also significantly lower [0% (0/24) vs. 29.4% (5/17), P < 0.05]. The S/F ratio of NIPPV group gradually increased after 2 hours treatment and RR gradually decreased with over time, S/F ratio decreased and RR increased in HFNC group compared with baseline, but there was no significant difference in S/F ratio between the two groups at each time point. RR in NIPPV group was significantly higher than that in HFNC group after 2 hours treatment [time/min: 30 (27-33) vs. 24 (21-27), P < 0.05]. There was no significant difference in rate need intubation and hospital mortality between NIPPV group and HFNC group [66.7% (16/24) vs. 70.6% (12/17), 58.3% (14/24) vs. 52.9% (9/17), both P > 0.05]. Analysis of the factors affecting the S/Fratio in the course of oxygen therapy showed that the oxygen therapy mode and the course of illness at admission were the factors affecting the S/F ratio of patients [ β values were -15.827, 1.202, 95% confidence interval (95% CI) were -29.102 to -2.552 and 0.247-2.156, P values were 0.019 and 0.014, respectively]. Conclusion:Compared with HFNC, NIPPV doesn't significantly reduce the intubation rate and mortality of patients with COVID-19 accompanied with ARDS, but it significantly increases the S/F ratio of those patients.

OBJECTIVETo observe the regulatory effects of acupoint electric stimulation on the analgesic substances and the relevant indices of nerve-immunity-endocrine system in the patients undergoing general anesthesia anorectal operation.

METHODSOne hundred and fifty-six patients undergoing hemorrhoids and anal fistula operation were randomized into three groups, 146 cases were included in the analysis. In the No.1 group (48 cases), the conventional intravenous general anesthesia was applied. In the No.2 group (50 cases), besides the conventional intravenous general anesthesia, the acupoint transcutaneous electric stimulation was combined at Neiguan (PC 6), Shenmen (HT 7), Shangliao (BL 31) and Ciliao (BL 32). operation in the No.2 and No.3 groups were lower apparently than that in the No.1 group (<0.05,<0.01).

CONCLUSIONSDuring the general anesthesia anorectal operation, the acupoint transcutaneous electric stimulation achieves analgesic anesthesia through effectively promoting the release of body analgesic substance and reducing the stress level in the operation. With the comprehensive acupoint selection as Neiguan (PC 6) and Shenmen (HT 7) and the local acupoints, the therapeutic effects are better in comparison with the simple selection of local acupoints. In the No.3 group (48 cases), besides the conventional intravenous general anesthesia, the acupoint transcutaneous electric stimulation was combined at Shangliao (BL 31) and Ciliao (BL 32). The electric stimulation was maintained till the end of operation. The patients' saliva was collected 0.5 h before operation and 1 h after operation separately. The indices that reflect the body pain regulation and nerve-immune-endocrine secretion were detected, such as opiophin protein (OPI), secretory immunoglobulin A (SIgA), saliva amylase (sAA), cortisol (Cor) and tumor necrosis factor α (TNF-α). The pain degree was observed 1 h after operation.

RESULTSIn the No.2 group, OPI after operation was higher than that before operation (<0.05). The difference value of OPI in the No.2 group was higher apparently than that in the No.1 group and the No.3 group (both<0.05). SIgA after operation was higher than that before operation in the No.1 group (<0.05). The difference values of SIgA, sAA, Cor in the No.2 group were lower apparently than those in the No.1 group (<0.05,<0.01). TNF-αbefore and after operation and its difference value among the groups were not significant statistically (all>0.05). The pain degrees in 1 h after.

This article was to elucidate that the needling depth is closely related to the meridian qi, disease location, disease nature and needled area based on the records of needling depth in Nei Jing (Canon of Internal Medicine). Moreover, different depths will produce different therapeutic efficacies;meanwhile, improper depth may lead to grave consequences.

Objective To explore the mechanism of down-regulation of regulatory T cells (Treg) by immunization with attenuated activated autologous T cells. Methods Aulologous T cells were activated with ConA in vitro. Mice were immunized subcutaneously and inlraperitoneally every 5 days for 3 times (5 ×10~6 per time for each mouse), and the number and function of Treg were examined. PBS was subcutaneously injected for control group. Serum level of anti-mouse CD25 antibody was measured by ELISA. The number and function of Treg was detected by serum adoptive transfer and proliferation and inhibition assays. Results Compared with control group, there were less CD4~+ CD25~+ Foxp3~+ Treg in the mice after immunization (P < 0. 01), the immunosuppression ability decreased (P<0. 01), and the level of anti-CD25 antibody increased (P <0.01). Adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice (P<0. 01). Conclusion Immunization with attenuated activated autologous T cells induces more anti-CD25 antibody, which may further down-regulate CD4~+CD25~+Foxp3~+ Treg expansion and function in vivo.