BACKGROUND:Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.Ferroptosis,a novel form of programmed cell death,is closely associated with the onset and progression of intervertebral disc degeneration;however,its precise mechanisms remain unclear.OBJECTIVE:To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells,thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.METHODS:Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide(0,25,50,100,and 200 μmol/L),and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay.Interventions with 100 μmol/L tert-butyl hydroperoxide,10 μmol/L RSL3,or dimethylsulfoxide were applied to nucleus pulposus cells,and cell proliferation was evaluated using the EdU assay.The expression levels of ferroptosis-related proteins(glutathione peroxidase 4,ferritin heavy chain 1,PTGS2,and ACSL4)and intervertebral disc degeneration marker proteins(matrix metalloproteinase 13 and Col2A)were analyzed via western blot and immunofluorescence.Additionally,reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe.Mitochondrial morphological changes were observed under transmission electron microscopy.RESULTS AND CONCLUSION:(1)tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells.(2)tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells.(3)tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1,while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2.(4)tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells.(5)Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide,including contraction,reduced cristae,and increased membrane density.(6)tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells.In conclusion,tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells,contributing to the development of intervertebral disc degeneration.This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

OBJECTIVE: To investigate the role and mechanism of the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon gene (cGAS/STING) pathway in oleic acid-induced acute lung injury (ALI) in mice. METHODS: Male wild-type C57BL/6J mice were randomly divided into five groups (each n = 10): normal control group, ALI model group, and 5, 50, 500 μg/kg inhibitor pretreatment groups. The ALI model was established by tail vein injection of oleic acid (7 mL/kg), while the normal control group received no intervention. The inhibitor pretreatment groups were intraperitoneally injected with the corresponding doses of cGAS inhibitor RU.521 respectively 1 hour before modeling. At 24 hours post-modeling, blood was collected, and mice were sacrificed. Lung tissue pathological changes were observed under light microscopy after hematoxylin-eosin (HE) staining, and pathological scores were assessed. Western blotting was used to detect the protein expressions of cGAS, STING, phosphorylated TANK-binding kinase 1 (p-TBK1), phosphorylated interferon regulatory factor 3 (p-IRF3), and phosphorylated nuclear factor-κB p65 (p-NF-κB p65) in lung tissue. Immunohistochemistry was performed to observe STING and p-NF-κB positive expressions in lung tissue. Serum interferon-β (IFN-β) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the normal control group, the ALI model group exhibited significant focal alveolar thickening, intra-alveolar hemorrhage, pulmonary capillary congestion, and neutrophil infiltration in the pulmonary interstitium and alveoli, along with markedly increased pathological scores (10.33±0.58 vs. 1.33±0.58, P < 0.05). Protein expressions of cGAS, STING, p-TBK1, p-IRF3, and p-NF-κB p65 in lung tissue significantly increased [cGAS protein (cGAS/β-actin): 1.24±0.02 vs. 0.56±0.02, STING protein (STING/β-actin): 1.27±0.01 vs. 0.55±0.01, p-TBK1 protin (p-TBK1/β-actin): 1.34±0.03 vs. 0.22±0.01, p-IRF3 protein (p-IRF3/β-actin): 1.23±0.02 vs. 0.36±0.01, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 1.30±0.02 vs. 0.53±0.02, all P < 0.05], positive expressions of STING and p-NF-κB in lung tissue were significantly elevated [STING (A value): 0.51±0.03 vs. 0.30±0.07, p-NF-κB (A value): 0.57±0.05 vs. 0.31±0.03, both P < 0.05], and serum IFN-β levels were also significantly higher (ng/L: 256.02±3.84 vs. 64.15±1.17, P < 0.05). The cGAS inhibitor pretreatment groups showed restored alveolar structural integrity, reduced inflammatory cell infiltration, and decreased hemorrhage area, along with dose-dependent lower pathological scores as well as the protein expressions of cGAS, STING, p-TBK1, p-IRF3 and p-NF-κB p65 in lung tissue, with significant differences between the 500 μg/kg inhibitor group and ALI model group [pathological score: 2.67±0.58 vs. 10.33±0.58, cGAS protein (cGAS/β-actin): 0.56±0.03 vs. 1.24±0.02, STING protein (STING/β-actin): 0.67±0.03 vs. 1.27±0.01, p-TBK1 protein (p-TBK1/β-actin): 0.28±0.01 vs. 1.34±0.03, p-IRF3 protein (p-IRF3/β-actin): 0.32±0.01 vs. 1.23±0.02, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 0.63±0.01 vs. 1.30±0.02, all P < 0.05]. Compared with the ALI model group, positive expressions of STING and p-NF-κB in lung tissue were significantly reduced in the 500 μg/kg inhibitor group [STING (A value): 0.40±0.01 vs. 0.51±0.03, p-NF-κB (A value): 0.43±0.02 vs. 0.57±0.05, both P < 0.05], and serum IFN-β levels were also markedly reduced (ng/L: 150.03±6.19 vs. 256.02±3.84, P < 0.05). CONCLUSIONS The cGAS/STING pathway is activated in oleic acid-induced ALI, leading to exacerbated inflammatory responses and increased lung damage. RU.521 can inhibit cGAS, thereby down-regulating the expression of pathway proteins and cytokines, and providing protection to lung tissue.
Animals ; Acute Lung Injury/chemically induced* ; Male ; Nucleotidyltransferases/metabolism* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Membrane Proteins/metabolism* ; Oleic Acid/adverse effects* ; Transcription Factor RelA/metabolism* ; Lung/pathology* ; Interferon Regulatory Factor-3/metabolism* ; Disease Models, Animal

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

BACKGROUND:Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.Ferroptosis,a novel form of programmed cell death,is closely associated with the onset and progression of intervertebral disc degeneration;however,its precise mechanisms remain unclear.OBJECTIVE:To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells,thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.METHODS:Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide(0,25,50,100,and 200 μmol/L),and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay.Interventions with 100 μmol/L tert-butyl hydroperoxide,10 μmol/L RSL3,or dimethylsulfoxide were applied to nucleus pulposus cells,and cell proliferation was evaluated using the EdU assay.The expression levels of ferroptosis-related proteins(glutathione peroxidase 4,ferritin heavy chain 1,PTGS2,and ACSL4)and intervertebral disc degeneration marker proteins(matrix metalloproteinase 13 and Col2A)were analyzed via western blot and immunofluorescence.Additionally,reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe.Mitochondrial morphological changes were observed under transmission electron microscopy.RESULTS AND CONCLUSION:(1)tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells.(2)tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells.(3)tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1,while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2.(4)tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells.(5)Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide,including contraction,reduced cristae,and increased membrane density.(6)tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells.In conclusion,tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells,contributing to the development of intervertebral disc degeneration.This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

Literature analysis,expert consultation,case analysis and other methods were used to establish a public hospital specialty operation evaluation system suitable for high-quality development,including 2 first-level indicators of medical ability and economic operation and 13 second-level indicators.Urology surgery in the pilot hospital was taken as an example to carry out surgical operation effect evaluation.To strengthen the evaluation of public hospital specialized operation,it should pay attention to the evaluation of medical capacity,strengthen the cooperation of functional departments,and improve the supporting policies of operation,for better promoting the development of high-quality operation of hospitals.

Objective:To investigate the correlations of melanin concentration hormone (MCH) in cerebrospinal fluid (CSF) and serum with sleep disorder, memory dysfunction and prognoses in patients with cerebral ischemic stroke (CIS).Methods:One hundred elderly CIS patients, admitted to Department of Neurology, He'nan Provincial People's Hospital from June 2021 to January 2022 were enrolled as CIS group, and 50 subjects collected from Physical Examination of the same hospital during the same period were enrolled as control group. MCH levels in the CSF and serum were detected by ELISA. Sleep quality was assessed by polysomnography and Pittsburgh Sleep Quality Index (PSQI). Memory function was assessed by Rivermead Behavioral Memory Test 2 nd Edition (BMT-II). Prognoses were assessed by modified Rankin Scale (mRS) 3 months after discharge. The clinical data and MCH levels of the two groups were compared; the differences in MCH levels among CIS patients with different degrees of sleep disorder, and different memory functions and prognoses were compared. Correlations of MCH level and sleep parameters with RBMT-II scores in these CIS patients were analyzed. Results:Compared with that in the control group, the proportion of patients with hypertension in CIS group was significantly higher ( P<0.05). Compared with the control group ([42.39±16.11] pg/mL), the serum MCH level in CIS group ([36.89±15.19] pg/mL) was statistically lower ( P<0.05). In CIS patients, patients with mild or severe sleep disorder had significantly decreased CSF MCH level compared with patients without sleep disorder ( P<0.05), patients with severe sleep disorder had significantly decreased CSF MCH level compared with patients with mild sleep disorder ( P<0.05); patients with severe sleep disorder had significantly decreased serum MCH level compared with patients without sleep disorder ( P<0.05); CSF MCH level was negatively correlated with PSQI scores, sleep latency and wake frequency ( P<0.05), and positively correlated with percentage of rapid eye movement ( P<0.05); serum MCH level in CIS patients was negatively correlated with PSQI scores and wake frequency ( P<0.05). In CIS patients, the CSF and serum MCH levels in patients with memory dysfunction was significantly lower compared with those with normal memory function ( P<0.05); a positive correlation was noted between RBMT-II scores and CSF MCH level ( P<0.05). In CIS patients, patients with poor prognosis had statistically lower CSF and serum MCH levels compared with those with good prognosis ( P<0.05). Conclusion:The serum MCH level in CIS patients is significantly decreased, which is closely related to the occurrence of sleep disorder and memory dysfunction after stroke; and they further affects the prognoses.

Objective:To explore the risk factors of intensive care unit-acquired weakness (ICU-AW) and the characteristics of Medical Research Council (MRC) score and electromyogram.Methods:A case control study was conducted. Patients with mechanical ventilation ≥ 7 days and MRC score admitted to department of respiratory and critical care medicine of China-Japan Friendship Hospital from September 2018 to January 2020 were enrolled, and they were divided into ICU-AW group (MRC score < 48) and non-ICU-AW group (MRC score ≥ 48) according to MRC score. The general situation, past medical history, related risk factors, MRC score, respiratory support mode, laboratory examination results, electromyogram examination results, ICU-AW related treatment, outcome and length of ICU stay were collected, and the differences between the two groups were compared. The risk factors of ICU-AW were analyzed by binary multivariate Logistic regression, and the characteristics of MRC score and electromyogram were analyzed.Results:A total of 60 patients were enrolled in the analysis, including 17 patients in ICU-AW group and 43 patients in non-ICU-AW group. Univariate analysis showed that there were significant differences in acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score, sequential organ failure assessment (SOFA) score, brain natriuretic peptide (BNP), blood urea nitrogen (BUN) on the first day of ICU admission and the ratio of invasive mechanical ventilation between ICU-AW group and non-ICU-AW group [APACHEⅡ score: 21 (18, 25) vs. 18 (15, 22), SOFA score: 7 (5, 12) vs. 5 (3, 8), BNP (ng/L): 364.3 (210.1, 551.2) vs. 160.1 (66.8, 357.8), BUN (mmol/L): 9.9 (6.2, 17.0) vs. 6.0 (4.8, 9.8), invasive mechanical ventilation ratio: 88.2% vs. 46.5%, all P < 0.05]. Binary multivariate Logistic regression analysis showed no independent risk factor for ICU-AW. The average MRC score of 17 ICU-AW patients was 33±11. The limb weakness was symmetrical, and the proximal limb weakness was the main manifestation. Electromyography examination showed that the results of nerve conduction examination in ICU-AW patients mainly revealed that the amplitude of compound muscle action potential (CMAP) and sensory nerve action potentials (SNAP) were decreased, and the conduction velocity was slowed down; needle electromyography showed increased area of motor unit potential (MUP), prolonged time limit and a large number of spontaneous potentials. Prognosis evaluation showed that compared with non-ICU-AW group, patients in ICU-AW group underwent more tracheotomy (70.6% vs. 11.6%), longer length of ICU stay (days: 57±52 vs. 16±8), and more rehabilitation treatment (58.8% vs. 14.0%), and the differences were statistically significant (all P < 0.01). Conclusions:The occurrence of ICU-AW may be related to high APACHEⅡ score and SOFA score, high levels of BNP and BUN on the first day of ICU admission and the proportion of invasive mechanical ventilation, but the above factors are not independent risk factors for ICU-AW. The MRC score of ICU-AW patients was characterized by symmetrical limb weakness, mainly proximal limb weakness; in electromyography examination, the nerve conduction examination results mainly showed that CMAP and SNAP amplitude were decreased, and conduction velocity was slowed down; needle electromyography examination showed increased MUP area, prolonged duration and a large number of spontaneous potentials.

Objective: To determine the effect of transplanting bone marrow mononuclear cells (BMMCs) on the expression of glial fibrillary acidic protein (GFAP) and Nogo-A around the ischemic foci after focal cerebral ischemia and reperfusion, and to study any role of BMMCs in nerve function recovery. Methods: BMMCs were isolated from the bone marrow of Sprague-Dawley rats. Cerebral ischemia and reperfusion was performed using a nylon thread to occlude the right middle cerebral artery for 2h followed by 24h of reperfusion. The qualified models were selected according to the Longa scale. The 48 models selected were randomly divided into a model group and an observation group, each of 24. Each group was further divided into 7d, 14d and 21d subgroups. 100μl of BMMCs (5×10⁶ /ml) were slowly injected into the ischemic lateral striata of the observation group. The rats in the model group were similarly injected, but with buffered saline solution. The rats were evaluated using the Longa scale after 7d, 14d and 21d. The rats were then sacrificed and the brain was resected. Immunohistochemical assays quantified the expression of GFAP and Nogo-A around the ischemic foci. Results: Compared with the model group, the rats in the observation group showed less neurological deficit on the 21st day, significantly greater expression of GFAP and significantly less Nogo-A expression on days 14 and 21. Nogo-A expression on the 21st day was also significantly lower than on the 14th day in the observation group. Conclusion BMMC transplantation can promote recovery from nerve damage after focal cerebral ischemia, which is probably related to enhanced expression of GFAP and restrained expression of Nogo-A in the brain tissues surrounding ischemic lesions.