Objective:To investigate the protective effect of racanisodamine (654-2) on lung epithelial cell injury induced by X-ray in mice and unravel the underlying mechanism.Methods:Mouse alveolar epithelial cells MLE-12 were used to establish radiation-induced lung injury (RILI) model in vitro and divided into 4 groups as follows: control (no irradiation), radiation (16 Gy radiation), treatment 1 (16 Gy radiation + 2 μmol/L 654-2), treatment 2 (16 Gy radiation + 10 μmol/L 654-2), and inhibitor (16 Gy radiation + 10 μmol/L 654-2 + 2 μmol/L ML385), respectively. Cells were sampled at different time points after radiation. Cell senescence was detected with senescence-associated β-galactosidase (SA-β-Gal) staining. Cell colony-forming ability was detected to observe the recovery capability of cells after treatment. The expression levels of p21, p16, phosphorylated histone H2AX(γH2AX), nuclear factor erythroid-2 related factor 2 (Nrf2), Nrf2 Ser40 site phosphorylation (p-Nrf2), p62, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) proteins were measured by Western blot. Cell apoptosis and the production of intracellular reactive oxygen species (ROS) were determined by flow cytometry. The expression levels of glutathione (GSH) and superoxide dismutase (SOD) were detected according to the manufectuer instructions. The expression levels of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) mRNA were determined by real time reverse transcription PCR. Measurement data were expressed as Mean ±SD. Comparison between two groups was conducted by independent sample t-test, and comparison among multiple groups was performed by one-way ANOVA. Results:Compared with the radiation group, the proportion of cells with positive staining of SA-β-Gal was significantly lower and cell senescence were alleviated in the treatment 1 and 2 groups (all P<0.001). Compared with the radiation group, the expression level of γH2AX protein was significantly down-regulated ( P=0.037), cell apoptosis rate was significantly decreased ( P=0.026), the proliferation capacity of MLE-12 was enhanced ( P=0.004), GSH ( P=0.002) and SOD ( P<0.001) activity was enhanced and ROS production was declined ( P=0.001) in the treatment 2 group. The expression levels of Nrf2 and p-Nrf2 in total protein were up-regulated over the time of 654-2 intervention. Meanwhile, the expression levels of antioxidant proteins of NQO1 and HO-1 were up-regulated and that of GCLC and GCLM mRNA was also up-regulated. There were no significant differences in the number of cells with positive staining of SA-β-Gal ( P=0.145) and ROS production ( P=0.317) between the inhibitor and radiation groups after supplement of ML385, small-molecule inhibitor of Nrf2. Conclusion:654-2 can activate the Nrf2 pathway, enhance cell antioxidant capacity and inhibit cell senescence, thereby playing a protective role on radiation- induced lung injury.

Objective:To investigate the protective effect of racanisodamine on lung injury in mice exposed to irradiation.Methods:C57BL/6 mice were randomly divided into control group, racanisodamine group, 18 Gy irradiation group (model group) and racanisodamine combined with 18 Gy irradiation group (treatment group), with 5 mice in each group. The mice in the treatment group received racanisodamine (5 mg/kg) intraperitoneally 3 d before irradiation and contained the whole experiments. Then, single chest irradiation of 18 Gy X-rays was performed both in the model and treatment groups. The racanisodamine group and treatment group received racanisodamine intraperitoneally once a day until 6 weeks after irradiation. The mice were killed at 6 weeks after irradiation. The lung histopathology was observed by HE staining. Serum and bronchial alveolar lavage fluid (BALF) inflammatory cytokines such as TNF-α, IL-1β and IL-6 were determined by ELISA method. Cell senescence was detected by SA-β-Gal staining. The expressions of Nrf2, p-Nrf2 and p62 in lung tissue were performed by immunehistochemistry and Western blot assays.Results:Compared with the model group, the scores of HE staining were decreased ( t=8.66, P<0.01), the number of infiltrated inflammatory cells in BALF were decreased ( t=10.70, P<0.01), and protein concentration in BALF had lower levels ( t=6.75, P<0.01), the serum TNF-α, IL-1β and IL-6 were decreased significantly ( t=8.17, 4.58, 6.54, P<0.01), the activity of SA-β-gal was decreased, and the expressions of Nrf2, p-Nrf2 were enhanced ( t=6.42, 7.30, P<0.01), while the expression of p62 was reduced ( t=4.62, P<0.01) in the treatment group. Conclusions:Racanisodamine plays the protective effect of radiation-induced lung injury by alleviating inflammation associating with the activating of Nrf2-related pathway, which reversed radiation-induced cell senescence.

OBJECTIVE: To assess the effect of tumor cell lysate (TCL) with low high-mobility group B1 (HMGB1) content for enhancing immune responses of dendritic cells (DCs) against lung cancer. METHODS: TCLs with low HMGB1 content (LH-TCL) and normal HMGB1 content (NH-TCL) were prepared using Lewis lung cancer (LLC) cells in which HMGB1 was inhibited with 30 nmol/L glycyrrhizic acid (GA) and using LLC cells without GA treatment, respectively. Cultured mouse DCs were exposed to different doses of NH-TCL and LH-TCL, using PBS as the control. Flow cytometry was used to detect the expressions of CD11b, CD11c and CD86 and apoptosis of the stimulated DCs, and IL-12 levels in the cell cultures were detected by ELISA. Mouse spleen cells were co-cultured with the stimulated DCs, and the activation of the spleen cells was assessed by detecting CD69 expression using flow cytometry; TNF-β production in the spleen cells was detected with ELISA. The spleen cells were then co-cultured with LLC cells at the effector: target ratios of 5:1, 10:1 and 20:1 to observe the tumor cell killing. In the animal experiment, C57/BL6 mouse models bearing subcutaneous LLC xenograft received multiple injections with the stimulated DCs, and the tumor growth was observed. RESULTS: The content of HMGB1 in the TCL prepared using GA-treated LLC cells was significantly reduced (P < 0.01). Compared with NH-TCL, LH-TCL showed a stronger ability to reduce apoptosis (P < 0.001) and promote activation and IL- 12 production in the DCs. Compared with those with NH-TCL stimulation, the DCs stimulated with LH-TCL more effectively induced activation of splenic lymphocytes and enhanced their anti-tumor immunity (P < 0.05). In the cell co-cultures, the spleen lymphocytes activated by LH-TCL-stimulated DCs showed significantly enhanced LLC cell killing activity (P < 0.01). In the tumor-bearing mice, injections of LH-TCL-stimulated DCs effectively activated host anti-tumor immunity and inhibited the growth of the tumor xenografts (P < 0.05). CONCLUSION Stimulation of the DCs with LH-TCL enhances the anti-tumor immune activity of the DCs and improve the efficacy of DCbased immunotherapy for LLC in mice.
Animals ; Humans ; Mice ; Apoptosis ; Dendritic Cells/immunology* ; Glycyrrhizic Acid/pharmacology* ; HMGB1 Protein ; Lung Neoplasms/immunology*

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.
Animals ; Mice ; Humans ; Adenoviruses, Human/genetics* ; Escherichia coli/genetics* ; HEK293 Cells ; Isopropyl Thiogalactoside ; Blotting, Western ; Immunoglobulin G ; Antibodies, Monoclonal ; Antibody Specificity ; Mice, Inbred BALB C

Objective:To investigate genetic characteristics of human rhinovirus (HRV) in adult inpatients with pneumonia in autumn and winter in Bengbu, Anhui province, 2021.Methods:The pharyngeal swabs of inpatients with pneumonia in Bengbu were collected for the detection of 14 common respiratory pathogens by Real-time PCR during September to December 2021. VP4/VP2 coding regions of HRV positive samples were amplified by nested PCR and phylogenetic tree was constructed using MEGA7.0.Results:A total of 146 samples were collected from inpatients with pneumonia; 35.62% (52/146) samples were positive with at least one pathogen. The four viruses with high detection rate were HRV, adenovirus, human coronavirus OC43 and influenza B virus. HRV positive samples accounted for 44.23% (23/52) of the positive samples, among which 9 cases (39.13%, 9/23) co-infected with HRV. Phylogenetic analysis found that HRV infection were dominated by HRV-A and HRV-B groups. The analysis based on clinical syndrome found that the white blood cells count and the proportion neutrophils of patients with HRV co-infection were higher that of HRV single infection. The proportion of patients with hypertension, diabetes, mechanical ventilation and poor prognosis in the HRV co-infection group were higher than that of HRV single infection group ( P<0.05). Conclusions:HRV is the predominant pathogen among the adult inpatients with pneumonia in Bengbu. HRV-A and HRV-B groups are common. Patients accompanied by hypertension, diabetes were easily co-infected with HRV. Patients coinfeted with HRV are more likely to be mechanical ventilation and poor prognosis.

Objective:A mouse model of pancreatitis induced by coxsackievirus B3 (CVB3) was established. The pathological change of pancreas and the infiltration of Th17/Treg cells were observed.Methods:The BALB/c mice were inoculated intraperitoneally with CVB3 to induce acute viral pancreatitis model. Then the pathological changes of pancreas were observed by HE staining; the viral RNA load and relative expression of cytokines (IFN-γ, IL-6 and IL-17) mRNA were detected by q-PCR; the proportion of infiltrated CD45 + CD3 + T cells, CD4 + and CD8 + T cells, Th17 and Treg cells in the pancreas was determined by flow cytometry. Results:Three days after CVB3 infection, the viral RNA load in pancreas was the highest (0.96±0.18) and gradually decreased with prolongation of infection. Compared with the 3 dpi group, the viral RNA load in pancreas was decreased (0.96±0.18 vs. 0.62±0.14) at 7 dpi, but there was no statistically significant difference. In addition, the infiltration of immune cell in pancreas increased significantly after 7dpi and the pathological score >2. The percent of infiltrated Th17 cells (1.05±0.21 vs. 22.13±5.79) and Treg cells (3.11±0.78 vs. 8.25±1.30) among CD4 + T cells significantly increased after infection (P<0.05), and the Th17/Treg also increased (P<0.01). Compared with the control group, the relative mRNA expression of IFN-γ (1.05±0.23 vs. 672.6±47.67), IL-6 (1.00±0.38 vs. 68.28±4.57), and IL-17 (1.01±0.11 vs. 54.15±7.94) in pancreas increased at 7 days after CVB3 infection ( P<0.01). Conclusions:The infiltration of Th17/Treg cells and the expression of related cytokines related cytokines IL-6 and IL-17 mRNA were upregulated in pancreas, which promoted the process of CVB3-induced pancreatitis.

Objective:To analyze the association of respiratory syncytial virus infection with meteorological factors and to predict and explain the trends.Methods:Data of cases with severe acute respiratory infections in hospitalized children in Xuzhou City were collected from 2015-2021. Respiratory syncytial virus (RSV) was detected by real-time fluorescence polymerase chain reaction. The result were statistically analyzed using SPSS 26.0 software, including constructing a negative binomial regression model to explore meteorological factors that impact RSV detection and a multivariate time series model to predict its epidemiological trend from 2020 to 2021.Results:A total of 1 663 samples of children with severe acute respiratory infections were collected from 2015to 2021, of which 218 (13.1%) were positive for RSV. Seasonal effects on RSV detection were evident: there was a 1-year cycle with a peak in winter (December-February) and a trough in summer (June-August). The negative binomial regression analysis showed that monthly mean temperature, monthly mean relative humidity, and monthly total sunshine hours may influenced RSV detection. The prediction result of the time series model with sunshine hours as the covariate showed that the prediction was better for 2020, and the actual values were close to the predicted values. The expected trends in 2021 were consistent, but the actual values were higher than predicted.Conclusions:Monthly mean temperature, monthly mean relative humidity, and monthly total sunshine hours may influence RSV detection in the Xuzhou region.A prediction model can be built using data from 2015-2019, where deviations in the predicted values for 2021, reflecting that disease prevalence is multifactorial correlated, suggest a possible rise in RSV prevalence in the future.

Objective:To explore the effect of myricitrin on the injury of human retinal microvascular endothelial cells (HRMECs) induced by high glucose and its regulation mechanism.Methods:HRMECs were divided into normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml myricitrin groups.HRMECs transfected with pcDNA and pcDNA-circZNF292, respectively and then cultured in high-glucose medium containing 25 mmol/L D-glucose for 24 hours were assigned as pcDNA group and pcDNA-circZNF292 group.HRMECs transfected with siR-NC and siR-circZNF292, respectively and then cultured in medium containing 50.0 μg/ml myricitrin and 25 mmol/L D-glucose for 24 hours were assigned as myricitrin+ siR-NC group and myricitrin+ siR-circZNF292 group.The cell apoptosis rate was detected by flow cytometry.The concentration of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in cells were detected by enzyme-linked immunosorbent assay (ELISA) kits.The expression levels of circZNF292 and miR-23b-3p were detected by real-time fluorescence quantitative PCR.The targeting relationship between circZNF292 and miR-23b-3p was detected by dual-luciferase reporter assay.The relative expression levels of B-cell lymphoma-2 (bcl-2) and bcl-2-related X protein (bax) were assayed by Western blot.Results:Significant differences were found in the relative expressions of bax and bcl-2 proteins, cell apoptosis rate, MDA concentration, SOD activity, circZNF292 and miR-23b-3p among normal control group, high glucose group and 12.5 μg/ml, 25.0 μg/ml, 50.0 μg/ml myricitrin groups ( F=105.707, 111.835, 74.515, 109.651, 135.020, 219.919, 116.304; all at P<0.001).With the increase of myricitrin concentration, the relative expression levels of bax protein, cell apoptosis rate, MDA concentration and miR-23b-3p in cells gradually decreased, while the relative expression levels of bcl-2 protein, SOD activity and circZNF292 increased, with statistically significant differences among groups with different concentrations of myricitrin (all at P<0.05).In the co-transfected wild-type (WT)-circZNF292 cells, the relative luciferase activity in miR-23b-3p group was 0.35±0.03, which was lower than 0.96±0.09 in microRNA-negative control group, and the difference was statistically significant ( t=11.137, P<0.001).Compared with pcDNA group, the relative expression levels of bcl-2 protein, circZNF292 and MDA concentration in cells of pcDNA-circZNF292 group were significantly increased, and the relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and SOD activity were significantly decreased (all at P<0.05).The relative expression levels of bax protein, miR-23b-3p, cell apoptosis rate and MDA concentration were reduced and relative expression levels of bcl-2 protein, circZNF292 and SOD activity were enhanced in myricitrin group and myricitrin+ siR-NC group in comparison with high glucose group and myricitrin+ siR-circZNF292 group, showing statistically significant differences (all at P<0.05). Conclusions:Myricitrin can inhibit cell apoptosis and oxidative stress by regulating the expression of circZNF292/miR-23b-3p, thereby reducing the damage of HRMECs induced by high glucose.

Objective To compare rotational techniques used by Chinese and world elite men’s shot put athletes, so as to provide scientific references for Chinese male shot putters to improve their sports performance and results in international competitions. Methods Three-dimensional (3D) kinematics data from Chinese male shot putters in actual competitions were obtained, and kinematic parameters of Chinese and world elite men’s shot put athletes were calculated and compared. Namely, shot velocities and hip-shoulder separation angles at five critical instants of rotational techniques: right foot off, left foot off, right foot touchdown, left foot touchdown and release, as well as phase durations and shot travel distances during four critical phases: first single support, air-born flight, second single support and final delivery. ResultsCompared with world elite athletes, Chinese male shot putters using rotational techniques had significantly lower vertical release velocity, longer air-borne duration, smaller hip-shoulder separation angles at left foot off and touchdown instant, and longer shot travel distance during air-borne flight, but shorter shot travel distance during final shot delivery. Conclusions The difference in lower extremity strength is a primary casue leading to different sports performance between Chinese and world elite male shot putters. The technique differences in Chinese and world elite male shot putters mainly lie in different phase timing and shot travel distances during different techinique phases.

Objective To compare the surgical effects between minimally invasive anterior ondontoid screw fixation assisted by our self-designed odontoid guider and open anterior ondontoid screw fixation in the treatment of odontoid fractures of Anderson-D'Alonzo type Ⅱ.Methods From July 2011 to July 2016,28 adults with odontoid fracture of Anderson-D'Alonzo type Ⅱ were treated at Department Ⅱ of Spinal Surgery,Hospital of 89 Army Group of Chinese PLA.Of them,15 were treated by minimally invasive anterior ondontoid screw fixation assisted by our self-designed odontoid guider (guider group) while the other 13 by open anterior ondontoid screw fixation (open group).All the patients were male,aged from 31 to 59 years (average,42.7 years).The 2 groups were compared in terms of incision length,operation time,intraoperative bleeding,intraoperative fluoroscopic frequency and hospital stay.Results There were no significant differences between the 2 groups of patients in their preoperative general data,indicating they were compatible (P ＞ 0.05).All the patients were followed up for 12 to 45 months (average,22.1 months).The internal fixation was in good place and clinical union achieved in all the 28 patients.The incision length (2.2 ± 0.1 cm),operation time (45.0 ± 3.1 min),intraoperative bleeding (29.0 ± 2.3 mL) and intraoperative fluoroscopic frequency (15.5 ± 1.9 times) for the guider group were all significantly less than those (2.9 ±0.7 cm,61.6±3.8 min,51.6±3.9 mL and 21.7±3.2 times,respectively) for the open group (P ＜0.05),but there was no significant difference between the 2 groups in hospital stay (6.5 ± 0.5 d versus 6.5 ± 0.6 d) (P ＞ 0.05).Conclusion In the treatment of odontoid fractures of Anderson-D'Alonzo type Ⅱ,compared with open anterior ondontoid screw fixation,the minimally invasive anterior ondontoid screw fixation assisted by our self-designed odontoid guider may lead to a smaller incision,shorter operation time,less blood loss and a lower fluoroscopic frequency.