1.Risk factor analysis for postoperative pulmonary infections with multidrug-resistant bacteria in patients with oral squamous cell carcinoma undergoing flap repair surgery
WANG Qian ; PENG Hui ; ZHANG Liyu ; YANG Zongcheng ; WANG Yuqi ; PAN Yu ; ZHOU Yu
Journal of Prevention and Treatment for Stomatological Diseases 2025;33(7):554-562
Objective:
To investigate the distribution patterns and risk factors for multidrug-resistant bacterial pulmonary infections in patients with oral squamous cell carcinoma (OSCC) undergoing flap reconstruction surgery, and to provide evidence for infection prevention and treatment in this population.
Methods:
This study was approved by the institutional medical ethics committee. We retrospectively analyzed sputum culture results, antimicrobial susceptibility testing data, and clinical records of 109 OSCC patients undergoing flap reconstruction. Chi-square tests were employed to identify pathogens and risk factors for multidrug-resistant bacteria (MDR) in postoperative pulmonary infections. Multivariate logistic regression analysis was conducted to determine MDR risk factors and establish a nomogram prediction model. The model’s discriminatory power, accuracy, and clinical utility were evaluated using receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA).
Results:
Among the 109 patients, 52 had negative sputum cultures and 57 tested positive, of whom 14 developed multidrug-resistant (MDR) pulmonary infections. Chi-square analysis revealed that blood transfusion, pre-existing pulmonary diseases, operation time ≥ 490 min, intraoperative blood loss ≥ 400 mL, and abnormal BMI were significant risk factors for postoperative MDR infections (P < 0.05). Multivariate logistic regression identified pre-existing pulmonary diseases, intraoperative blood loss ≥ 400 mL, abnormal BMI, and operative duration ≥ 490 min as independent risk factors for MDR infections (P < 0.05). The nomogram prediction model for MDR infections demonstrated an area under the ROC curve (AUC) of 0.874 (95% CI: 0.775-0.973). The calibration plot showed good agreement between predicted and observed outcomes. DCA indicated a net clinical benefit when the threshold probability for high-risk MDR infections ranged from 0.000 to 0.810. Common MDR pathogens included MDR Pseudomonas aeruginosa, MDR Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii (CRAB), and methicillin-resistant Staphylococcus aureus (MRSA).
Conclusion
Among OSCC patients undergoing flap reconstruction, MDR pulmonary infections were predominantly caused by gram-negative bacteria (including CRAB, MDR Pseudomonas aeruginosa, and MDR Klebsiella pneumoniae along with the gram-positive pathogen MRSA. Pre-existing pulmonary comorbidities, prolonged surgery duration (≥ 490 min), significant intraoperative blood loss (≥ 400 mL), and abnormal BMI were confirmed as independent risk factors for these MDR infections. The nomogram predictive model incorporating these four variables demonstrated clinically reliable accuracy in risk stratification for postoperative MDR pulmonary infections in this patient population.
2.Effects of stress-induced protein Sestrin2 on necroptosis of dendritic cells induced by lipopolysaccharide
Mengyao WU ; Renqi YAO ; Yu DUAN ; Lu WANG ; Liyu ZHENG ; Pengyi HE ; Ning DONG ; Yao WU ; Yongming YAO
Chinese Critical Care Medicine 2024;36(3):237-243
Objective:To investigate the effect of stress-induced protein Sestrin2 (SESN2) on necroptosis of mouse dendritic cell (DC) induced by lipopolysaccharide (LPS) combined with zVAD, a panaspartate-specific cysteine protease (caspase) inhibitor.Methods:The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured. The cells were stimulated with LPS for 0 hour, 6 hours, 12 hours, and 24 hours, and grouped according to the stimulation time points. Western blotting was performed to determine the protein expression of SESN2 in each group. Overexpression empty lentivirus (NC), SESN2 gene overexpression RNA sequence lentivirus (SESN2 LV-RNA), small interfering empty lentivirus (NS), and SESN2 gene small interfering RNA sequence lentivirus (SESN2 siRNA) were transfected into DC2.4 cells. After 72 hours of transfection, cell fluorescence expression was observed under the inverted fluorescence microscope. Cells in each transfection group were stimulated with LPS for 24 hours. The blank control groups were set up and cultured with phosphate buffered saline (PBS) for 24 hours. Western blotting was performed to measure SESN2 protein expression. In the same groups as above, cells were stimulated with LPS+zVAD for 24 hours. The blank control groups were set up and cultured with PBS for 24 hours. Western blotting was used to determine the expression of mixed lineage kinase domain-like protein (MLKL) and phosphorylated-MLKL (p-MLKL). The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy. The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining.Results:Compared to the LPS 0 hour group, the expression of SESN2 in the LPS 24 hours group showed a significant increase. Therefore, 24 hours was chosen as the subsequent stimulation time point. After successful lentivirus transduction and 24 hours of cultivation, the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group. The MLKL phosphorylation in the SESN2 LV-RNA+LPS+zVAD group was significantly lower than that in the NC+LPS+zVAD group. The MLKL phosphorylation levels in both the NS+LPS+zVAD group and the NC+LPS+zVAD group were obviously higher than those in the NS+PBS group and the NC+PBS group, respectively. Laser scanning confocal microscopy showed that the trends in quantity and fluorescence intensity of p-MLKL protein expressions were consistent with the above results. The results from flow cytometry analysis and Hoechst staining showed that the rates of cell necrotic apoptosis in SESN2 siRNA+LPS+zVAD group were significantly higher than those in NS+LPS+zVAD group [flow cytometry analysis: (30.800±1.153)% vs. (20.800±1.114)%, Hoechst staining: (75.267±0.451)% vs. (46.267±3.371)%, both P < 0.05], indicating that knocking down SESN2 further exacerbated the occurrence of necroptosis. The necrotic apoptosis rates in SESN2 LV-RNA+LPS+zVAD group were significantly lower than those in NC+LPS+zVAD group [flow cytometry analysis: (7.160±0.669)% vs. (19.240±2.322)%, Hoechst staining: (32.433±3.113)% vs. (48.567±4.128)%, both P < 0.05], indicating that overexpressing SESN2 reversed such response and markedly reduced the proportion of necroptotic cells compared to the corresponding empty vector group. Conclusion:SESN2 exhibits an inhibitory effect on necroptosis of DC in sepsis. Targeted SESN2 expression may regulate the process of DC-mediated immune response in sepsis.
3.Clinical and Animal Experimental Research on Kidney Deficiency Syndrome: A Review
Guimin LIU ; Jianhui SUN ; Jianliang LI ; Zeyue YU ; Liyu HAO ; Hanhui HUANG ; Zongyuan LI ; Aoao WANG ; Yang YANG ; Hongmei LI
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(23):269-280
Kidney deficiency syndrome is a common clinical syndrome in traditional Chinese medicine (TCM). With the progress of science and technology, clinical and animal experiments on kidney deficiency syndrome have made remarkable progress. Research on kidney deficiency and the nature of "kidney" involves a large number of physiological and pathological bases, which are closely related to physiological and pathological links in the human body, among which the neuroendocrine-immune network shares the closest relationship. However, there are still many challenges in modern research on kidney deficiency syndrome, such as expert consensus on clinical diagnostic criteria and evaluation indexes and optimization of animal experimental models. In the past decade, a large number of clinical and animal experiments have been reported in the literature on kidney deficiency syndrome, among which the literature focusing on the combination of disease and syndrome is predominant, and most of them focus on kidney Yang deficiency and kidney Yin deficiency, involving the exploration of many pathological mechanisms. Research on the mechanisms related to kidney deficiency syndrome encompasses multiple signaling pathways and various biochemical indicators, including the phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-erythroid 2-relatedfactor-2(PI3K/Akt/Nrf2) signaling pathway, the Toll-like receptor 4/myeloid differentiation factor88/nuclear factor-κB(TLR4/MyD88/NF-κB) signaling pathway, the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, Osteoprotectin/nuclear factor-κB receptor activator ligand/receptor activator of nuclear factor-κB (OPG/RANKL/RANK) signaling pathway. The biochemical indicators cover the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP) ratio, Na+-K+-ATPase activity, Ca2+-Mg2+-ATPase activity, adrenocorticotropic hormone (ACTH), polycorticosterone (CORT), 17-OHCS, and other sex hormone indicators, providing crucial reference values for diagnosing kidney Yang deficiency or kidney Yin deficiency. The literature related to kidney deficiency syndrome over the past decade was collated and excavated, with a view to providing a reference for research on kidney deficiency syndrome.
4.Analysis of the current status and associated factors of nutritional literacy among primary and secondary school students in Beijing
Chinese Journal of School Health 2024;45(11):1551-1554
Objective:
To understand the nutritional literacy level and associated factors of primary and secondary school students in Beijing, so as to provide a scientific basis for improving student nutrition.
Methods:
From October 2022 to May 2023, a multi stage cluster random sampling method was employed to select a total of 14 568 primary, junior and senior high school students from 16 districts (ecluding the Economic Technological Development area) in Beijing. Through a survey questionnaire on nutritional literacy and dietary hehavior of school age children, basic information as well as data on nutritional literacy levels across four dimensions:nutrition related knowledge concepts, food selection, food preparation, and food intake dimensions were obtained. The Wilcoxon rank sum test, Kruskal-Wallis test, Spearman correlation analysis, Chi square test and binary Logistic regression were used for the analysis.
Results:
The median total score of nutritional literacy among primary and secondary school students in Beijing was 68.8. Approximately 26.0% of primary and secondary school students achieved nutritional literacy standards. The median scores and rates of meeting the standards for nutrition related knowledge concepts, food selection, food preparation and food intake dimensions were 23.0, 42.1%; 17.0, 27.4%; 6.5, 33.5%; 23.0, 33.3%, respectively. There were positive correlations between all pairs of the four dimensions ( r=0.33-0.49, P <0.05). The results of multiple Logistic regression analysis showed that primary school students, junior high school students, female students, suburban students, caregivers with a college education level and a bachelor s degree or above were the positive arrelation factors that promoted the achievement of nutritional literacy standards ( OR =2.21, 1.39, 1.18, 1.27, 1.42, 1.66, P <0.05).
Conclusion
The literacy level of primary and secondary school students in Beijing needs to be significantly improved. School stage, gender, region and caregiver s education level are associated factors.
5.Effects of stimulator of interferon gene on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 in mouse dendritic cells under sepsis
Mengyao WU ; Pengyi HE ; Yu DUAN ; Liyu ZHENG ; Renqi YAO ; Qiyuan ZHOU ; Yu CHEN ; Ning DONG ; Yao WU ; Yongming YAO
Chinese Journal of Burns 2024;40(10):920-929
Objective:To investigate the effects of stimulator of interferon gene (STING) on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 (ACSL4) in mouse dendritic cells (DCs) under sepsis, providing a basis for improving the dysregulation of immune response in sepsis caused by factors such as wound infection.Methods:This study was an experimental research. The mouse DC line DC2.4 in the logarithmic growth phase (with passages 3-10) were divided into lipopolysaccharide (LPS) stimulation 0 h (unstimulated) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 18 h group, and LPS stimulation 24 h group according to the random number table (the same grouping method below), which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of phosphorylated STING (p-STING), STING, and ACSL4 in cells were determined by Western blotting. DC2.4 successfully transfected with lentivirus containing STING gene small interfering RNA (hereinafter referred to as siSTING) were divided into siSTING+phosphate buffer solution (PBS) group and siSTING+LPS group. DC2.4 successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. After being stimulated with PBS or LPS and cultured for 24 hours, the protein expressions of p-STING, STING, and ACSL4 in cells were determined as above. Cell lipid peroxidation degrees were observed using the lipid peroxidation assay kit, and cell apoptosis rates were detected using flow cytometry. The sample numbers in the above cell experiments were all 3. Eighty male C57BL/6J mice aged 6 to 8 weeks were divided into sham surgery+normal saline (NS) group, cecal ligation and puncture (CLP)+NS group, sham surgery+C-176 group, and CLP+C-176 group, with 20 mice in each group. Mice in the two C-176 groups were intraperitoneally injected with C-176, while mice in the two NS groups were intraperitoneally injected with an equivalent volume of NS. One hour later, sham surgery was performed on the mice in the two sham surgery groups, and CLP surgery was performed on the mice in the two CLP groups to establish a sepsis model. At 24 h post-surgery, 10 mice from each group were sacrificed to extract spleen DCs, and protein expression, lipid peroxidation, and apoptosis rates were detected as above ( n=3). Hematoxylin-eosin staining was performed to observe pathological damage in the heart, liver, lung, and kidney tissue. The remaining 10 mice in each group were observed for survival within 7 days after surgery. Results:The protein expressions of p-STING, STING, and ACSL4, as well as the p-STING/STING ratio in DC2.4 in LPS stimulation 24 h group were significantly higher than those in LPS stimulation 0 h group ( P<0.05). After 24 h of culture, the protein expressions of p-STING, STING, and ACSL4 in DC2.4 in siSTING+LPS group and empty vector+PBS group were significantly lower than those in empty vector+LPS group ( P<0.05); the lipid peroxidation degrees of DC2.4 in siSTING+LPS group and empty vector+PBS group were weaker than those in empty vector+LPS group. The apoptosis rates of DC2.4 in empty vector+PBS group, empty vector+LPS group, siSTING+PBS group, and siSTING+LPS group were (15.7±3.0)%, (37.8±2.9)%, (13.1±2.1)%, and (20.6±1.8)%, respectively. The apoptosis rates of DC2.4 in empty vector+PBS group and siSTING+LPS group were significantly lower than that in empty vector+LPS group ( P<0.05). At 24 h post-surgery, the protein expressions of p-STING and ACSL4, and the p-STING/STING ratio in spleen DCs of mice in CLP+NS group were significantly higher than those in sham surgery+NS group and CLP+C-176 group ( P<0.05); the protein expression of STING in spleen DCs of mice in CLP+NS group was significantly higher than that in sham surgery+NS group ( P<0.05); the lipid peroxidation degrees of spleen DCs of mice in CLP+C-176 group and sham surgery+NS group were weaker than that in CLP+NS group. The apoptosis rates of spleen DCs of mice in sham surgery+NS group and CLP+C-176 group were significantly lower than that in CLP+NS group ( P<0.05), and the apoptosis rate of spleen DCs of mice in CLP+C-176 group was significantly higher than that in sham surgery+C-176 group ( P<0.05). Pathological tissue damage in the heart, liver, lung, and kidney of mice in CLP+NS group was significantly worse than that in sham surgery+NS group, while such damage in the above organs of mice in CLP+C-176 group was significantly alleviated compared with that in CLP+NS group. The survival ratio of mice in CLP+NS group within 7 days after surgery was significantly lower than that in sham surgery+NS group ( χ2=8.30, P<0.05). Conclusions:Under sepsis, STING activation in mouse DCs is significant, which enhances ACSL4-mediated ferroptosis. Inhibiting STING activation can significantly reduce ACSL4-mediated ferroptosis level in mouse DCs under sepsis, thereby improving the survival rate of septic mice.
6.Development and validation of an Assessment Scale of Proactive Health Behavior Ability for the Disabled Elderly in Nursing Homes
Yangli OU ; Xiaoyan LIAO ; Ying PENG ; Hong ZHANG ; Shaohua YIN ; Liyu CHEN ; Xue XIONG ; Xiuli YU ; Lifang TONG ; Yan XIE ; Dan HUO ; Jun SHEN
Chinese Journal of Nursing 2024;59(21):2579-2586
Objective To develop the assessment scale of proactive health behavior ability for the disabled elderly in nursing homes and to test its reliability and validity.Methods The first draft of the scale was formed by literature review,qualitative interviews and Delphi method.From December 2023 to March 2024,525 disabled elderly people from 9 nursing homes in Sichuan Province and Chongqing City were selected as the survey subjects,and item analysis and reliability and validity test were carried out on the scale.30 disabled elderly people were re-investigated after 2 weeks to calculate the retest reliability of the scale.Results The scale consisted of 4 dimensions and 27 items.Exploratory factor analysis extracted 4 common factors,with the cumulative vanance contribution rate of 65.992%,and confirmatory factor analysis showed that the modified model fitting index was within acceptable range.The content validity index at item level was 0.917-1.000,and that at scale level was 0.997.The Cronbach's α coefficient,test-retest reliability and split-half reliability of the total scale were 0.944,0.997 and 0.882,respectively.Conclusion The scale has good reliability and validity,and it can be used to evaluate the proactive health behavior ability of the disabled elderly in nursing homes.
7.A qualitative study on dilemma of"Giving Care"for the disabled elderly in nursing homes from perspective of active health
Hong ZHANG ; Jun SHEN ; Qianying JIA ; Xiuli YU ; Lifang TONG ; Miao LI ; Xiaoyan LIAO ; Yangli OU ; Liyu CHEN ; Qiu CHEN ; Zhifan BAI
Chinese Journal of Nursing 2023;58(22):2761-2768
Objective This study aims to explore the formation of the dilemma of"giving care"for disabled elderly people in nursing homes from the perspective of active health.Methods Purposive sampling was used to select caregivers and disabled elderly people from a nursing home in Chongqing as research subjects from July 2022 to December 2022.One-on-one in-depth interviews were conducted and Colaizzi's 7-step thematic analysis method was employed to collect and analyze the interview data.Results 4 themes were identified:①the dominance of the"giving"care concept,including the cultural thoughts of filial piety,passive acceptance of care characteristics and consumer psychology regarding paid services;②insufficient"participation"care ability,including lack of knowledge regarding active health and a weakening of skills to promote participation;③the hindrance to"transformation"of care models,including objective limitations in terms of human resources and delays in adapting aging environments;and ④the decline in"utilization"of internal abilities,including excessive avoidance of potential risks,a heavy workload of care and poor quality of individual care.Conclusion The formation of the dilemma of"giving care"for disabled elderly people in nursing homes is affected by multiple factors such as social background,service system,supply resources,and management mode.Transforming disabled elderly individuals from a state of"passive giving care"to"active participation in their health"is an important measure to realize the concept of positive aging and healthy aging.
8.Value of Chemoradiotherapy based on the depth of invasion for the prognosis of oral squamous cell carcinoma
DUAN Xianjie ; WENG Haiyan ; SHEN Guodong ; SI Chengyun ; AN Xingfei ; ZHANG Liyu ; ZHOU Yu
Journal of Prevention and Treatment for Stomatological Diseases 2023;31(3):186-191
Objective :
To explore the prognostic value of chemoradiotherapy based on the depth of invasion (DOI) in patients with oral squamous cell carcinoma.
Methods :
Patients with oral squamous cell carcinoma who received surgical treatment in a hospital from 2008 to 2016 were enrolled. The chi-square test was used to compare the effects of DOI on postoperative cervical lymph node metastasis and local recurrence. The effects of chemoradiotherapy on postoperative cervical lymph node metastasis, local recurrence, and survival were analyzed based on the DOI.
Results:
A total of 111 patients with oral squamous cell carcinoma were included in this study. The postoperative local recurrence rate (P<0.05) and cervical lymph node metastasis rate (P<0.05) of patients with 5 mm < DOI ≤ 10 mm and DOI > 10 mm were significantly higher than those with DOI ≤ 5 mm. The time of postoperative recurrence was concentrated within two years after the operation. The greater the DOI was, the shorter the time to postoperative recurrence (P<0.05). The addition of postoperative chemoradiotherapy did not significantly improve the postoperative local recurrence rate, cervical lymph node metastasis or survival rate of patients with different DOIs (P > 0.05).
Conclusion
DOI has important predictive value for postoperative recurrence, cervical lymph node metastasis and survival rate. However, DOI cannot be used as an independent index to guide whether chemoradiotherapy is needed after oral cancer surgery.
9.Mechanism of Huashi Baidu Prescription in Treating Influenza Viral Pneumonia Based on Transcriptome
Zhongchao SHAN ; Jianhui SUN ; Jianliang LI ; Zeyue YU ; Liyu HAO ; Yurong DENG ; Hairu HUO ; Hongmei LI ; Luqi HUANG
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(18):54-61
ObjectiveTo clarify the therapeutic effect of Huashi Baidu prescription on pneumonia in mice caused by influenza A (H1N1) virus and explore its mechanism based on the transcriptome. MethodA mouse influenza viral pneumonia model was built by intranasal infection with influenza A virus, and mice were continuously administered the drug for five days, so as to investigate the general condition, lung index, viral load, pathological morphology of lung tissue, survival time, and prolongation rate of survival time of mice and clarify the therapeutic effect of Huashi Baidu prescription on influenza viral pneumonia. Transcriptome technology was used to detect the differentially expressed genes in the lung tissue of mice in the model group and the normal group, as well as the Huashi Baidu prescription group and the model group, and the potential core target of the Huashi Baidu prescription for the treatment of influenza viral pneumonia was screened. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to verify the effect of Huashi Baidu prescription on the mRNA expression level of core target genes. ResultCompared with the normal group, the lung index and viral load in the lung tissue of the model group were significantly increased (P<0.05, P<0.01). Compared with the model group, the high-dose group of Huashi Baidu prescription significantly prolonged the survival time of mice infected with influenza A virus (P<0.05) and significantly reduced the lung index value of mice (P<0.05) and the viral load of lung tissue. The high-dose, medium-dose, and low-dose groups of Huashi Baidu prescription could significantly reduce lung tissue inflammation, blood stasis, swelling, and other pathological changes in mice (P<0.05, P<0.01). Transcriptome analysis of lung tissue showed that core genes were mainly enriched in the nuclear transcription factor-κB (NF-κB) signaling pathway, interleukin-17 (IL-17) signaling pathway, cytokine-cytokine receptor interaction, and other pathways after the intervention of Huashi Baidu prescription. TRAF6, NFKBIA, CCL2, CCL7, and CXCL2 were the top five node genes with combined score values. Real-time PCR validation showed that Huashi Baidu prescription significantly downregulated the mRNA expression of key genes TRAF6 and NFKBIA in the NF-κB signaling pathway, as well as chemokines CCL2, CCL7, and CXCL2 (P<0.05, P<0.01). ConclusionHuashi Baidu prescription has a therapeutic effect on influenza viral pneumonia, possibly by inhibiting the expression of key nodes TRAF6 and NFKBIA in the NF-κB signaling pathway and that of chemokines CCL2, CCL7, and CXCL2, reducing the recruitment of inflammatory cells and viral load, and exerting anti-influenza viral pneumonia effects.
10.Influence of family with sequence similarity 134, member B-mediated reticulophagy on lipopolysaccharide-induced apoptosis of mouse dendritic cells
Yu DUAN ; Renqi YAO ; Liyu ZHENG ; Ning DONG ; Yao WU ; Yongming YAO ; Xingui DAI
Chinese Journal of Burns 2023;39(9):857-866
Objective:To investigate the influence of family with sequence similarity 134, member B (FAM134B)-mediated reticulophagy on lipopolysaccharide (LPS)-induced apoptosis of mouse dendritic cells (DCs), so as to provide a basis for improving the immune suppression of sepsis caused by wound infection and other factors.Methods:The experimental research methods were used. The DC line DC2.4 of the 3 rd to 10 th passage in the logarithmic growth stage was collected for experiments. DCs were divided into LPS stimulation 0 h (no stimulation) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group, which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of FAM134B, microtubule-associated protein 1 light chain 3B (LC3B), and transporter protein SEC61B were determined by Western blotting, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated ( n=3). DCs were divided into phosphate buffer solution (PBS) group and LPS group for corresponding treatment. After 24 hours of culture, the expression of FAM134B and its co-localization with lysosomal probes and LC3B were detected using immunofluorescence method, while the number of autolysosomes in cells were observed through transmission electron microscope. DCs were divided into the FAM134B-knockdown group that were transfected with lentivirus containing small interfering RNA (siRNA) sequence of FAM134B gene and the empty vector group with empty lentivirus transfected. At post transfection hour 72, the fluorescence expression of cells was observed under the inverted fluorescence phase contrast microscope, meanwhile, the normally cultured DCs were set as blank control group, and the same observation was performed at the corresponding time point. DCs were divided into PBS alone group and LPS alone group, DCs successfully transfected with lentivirus containing siRNA sequence of FAM134B gene were divided into FAM134B-knockdown+PBS group and FAM134B-knockdown+LPS group, and DCs successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. These cells were stimulated correspondingly and cultured for 24 hours. The protein expression of FAM134B was detected using Western blotting ( n=3); the apoptotic rate of cells was determined by flow cytometry ( n=3); the situation of apoptosis was observed by Hoechst staining, and the apoptotic rate was calculated ( n=5); the protein expressions of cleaved cysteine aspartic acid specific protease-3 (caspase-3), B cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were detected using Western blotting, and the ratio of Bax/Bcl-2 was calculated ( n=5). Data were statistically analyzed with one-way analysis of variance (ANOVA), least significant difference test, and ANOVA for factorial design. Results:Compared with those in LPS stimulation 0 h group, the protein expressions of FAM134B of cells in LPS stimulation 12 h group and LPS stimulation 24 h group were significantly increased ( P<0.05), the protein expressions of SEC61B of cells in LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 24 h group, and LPS stimulation 72 h group were significantly decreased ( P<0.05), and the ratios of LC3B-Ⅱ/LC3B-Ⅰ of cells in LPS stimulation 24 h group and LPS stimulation 72 h group were obviously increased ( P<0.05). As the most significant changes of three proteins were seen in the cells of LPS stimulation 24 h group, 24 h was used as the duration of subsequent LPS stimulation. After 24 hours of culture, the expression of FAM134B and its co-localization with LC3B and lysosomal probes in the cells of LPS group were all significantly enhanced, with a significant increase in the number of autolysosomes in comparison with those in PBS group. Both the empty vector group and the FAM134B-knockdown group showed high intensity fluorescence in the cells at post transfection hour 72, but the blank control group showed no fluorescence in the cells at the corresponding time point. After 24 hours of culture, the protein expression of FAM134B of cells in FAM134B-knockdown+PBS group was significantly lower than the expressions in PBS alone group and empty vector+PBS group (with P values all <0.05), the protein expression of FAM134B of cells in FAM134B-knockdown+LPS group was significantly lower than the expressions in LPS alone group and empty vector+LPS group (with P values all <0.05), the protein expression of FAM134B of cells in LPS alone group was significantly higher than that in PBS alone group ( P<0.05), while the protein expression of FAM134B of cells in empty vector+LPS group was significantly higher than that in empty vector+PBS group ( P<0.05). After 24 hours of culture, flow cytometry assay revealed that the apoptotic rate of cells in PBS alone group, LPS alone group, empty vector+PBS group, empty vector+LPS group, FAM134B-knockdown+PBS group, and FAM134B-knockdown+LPS group were (13.3±0.8)%, (32.6±4.3)%, (17.0±1.5)%, (51.7±3.3)%, (52.4±3.1)%, and (62.3±2.6)%, respectively. After 24 hours of culture, compared with those in LPS alone group and empty vector+LPS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in FAM134B-knockdown+LPS group ( P<0.05); compared with those in the corresponding PBS treatment group, namely, PBS alone group, empty vector+PBS group, and FAM134B-knockdown+PBS group, the protein expression of cleaved caspase-3, the ratio of Bax/Bcl-2, and the apoptotic rates of cells detected by flow cytometry and Hoechst staining were significantly increased in LPS alone group, empty vector+LPS group, and FAM134B-knockdown+LPS group ( P<0.05). Conclusions:The activation of reticulophagy mediated by FAM134B in mouse DCs is enhanced and peaked in 24 hours under LPS stimulation, and the activated reticulophagy has a significant inhibitory effect on cell apoptosis.


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