1.Quercetin ameliorates myocardial injury in diabetic rats by regulating L-type calcium channels.
Hongyan SUN ; Guoqing LU ; Chengwen FU ; Mengwen XU ; Xiaoyi ZHU ; Guoquan XING ; Leqiang LIU ; Yufei KE ; Lemei CUI ; Ruiyang CHEN ; Lei WANG ; Pinfang KANG ; Bi TANG
Journal of Southern Medical University 2025;45(3):531-541
OBJECTIVES:
To investigate the effects of quercetin on cuproptosis and L-type calcium currents in the myocardium of diabetic rats.
METHODS:
Forty SD rats were randomized into control group and diabetic model groups. The rat models of diabetes mellitus (DM) induced by high-fat and high-sugar diet combined with streptozotocin (STZ) injection were further divided into DM model group, quercetin treatment group, and empagliflozin treatment group (n=10). Blood glucose and body weight were measured every other week, and cardiac function of the rats was evaluated using echocardiography. HE staining, Sirius red staining, and wheat germ agglutinin (WGA) analysis were used to observe the changes in myocardial histomorphology, and serum copper levels and myocardial FDX1 expression were detected. In cultured rat cardiomyocyte H9c2 cells with high-glucose exposure, the effects of quercetin and elesclomol, alone or in combination, on intracellular CK-MB and LDH levels and FDX1 expression were assessed, and the changes in L-type calcium currents were analyzed using patch-clamp technique.
RESULTS:
The diabetic rats exhibited elevated blood glucose, reduced body weight, impaired left ventricular function, increased serum copper levels and myocardial FDX1 expression, decreased L-type calcium currents, and prolonged action potential duration. Quercetin and empagliflozin treatment significantly lowered blood glucose, improved body weight, and restored cardiac function of the diabetic rats, and compared with empagliflozin, quercetin more effectively reduced serum copper levels, downregulated FDX1 expression, and enhanced myocardial L-type calcium currents in diabetic rats. In H9c2 cells, high glucose exposure significantly increased myocardial expressions of FDX1, CK-MB and LDH, which were effectively lowered by quercetin treatment; Elesclomol further elevated FDX1, CK-MB and LDH levels in the exposed cells, and these changes were not significantly affected by the application of quercetin.
CONCLUSIONS
Quercetin ameliorates myocardial injury in diabetic rats possibly by suppressing myocardial cuproptosis signaling and restoring L-type calcium channel activity.
Animals
;
Quercetin/pharmacology*
;
Calcium Channels, L-Type/metabolism*
;
Diabetes Mellitus, Experimental/metabolism*
;
Rats, Sprague-Dawley
;
Rats
;
Myocytes, Cardiac/drug effects*
;
Myocardium/pathology*
;
Male
2.Tongue squamous cell carcinoma-targeting Au-HN-1 nanosystem for CT imaging and photothermal therapy.
Ming HAO ; Xingchen LI ; Xinxin ZHANG ; Boqiang TAO ; He SHI ; Jianing WU ; Yuyang LI ; Xiang LI ; Shuangji LI ; Han WU ; Jingcheng XIANG ; Dongxu WANG ; Weiwei LIU ; Guoqing WANG
International Journal of Oral Science 2025;17(1):9-9
Tongue squamous cell carcinoma (TSCC) is a prevalent malignancy that afflicts the head and neck area and presents a high incidence of metastasis and invasion. Accurate diagnosis and effective treatment are essential for enhancing the quality of life and the survival rates of TSCC patients. The current treatment modalities for TSCC frequently suffer from a lack of specificity and efficacy. Nanoparticles with diagnostic and photothermal therapeutic properties may offer a new approach for the targeted therapy of TSCC. However, inadequate accumulation of photosensitizers at the tumor site diminishes the efficacy of photothermal therapy (PTT). This study modified gold nanodots (AuNDs) with the TSCC-targeting peptide HN-1 to improve the selectivity and therapeutic effects of PTT. The Au-HN-1 nanosystem effectively targeted the TSCC cells and was rapidly delivered to the tumor tissues compared to the AuNDs. The enhanced accumulation of photosensitizing agents at tumor sites achieved significant PTT effects in a mouse model of TSCC. Moreover, owing to its stable long-term fluorescence and high X-ray attenuation coefficient, the Au-HN-1 nanosystem can be used for fluorescence and computed tomography imaging of TSCC, rendering it useful for early tumor detection and accurate delineation of surgical margins. In conclusion, Au-HN-1 represents a promising nanomedicine for imaging-based diagnosis and targeted PTT of TSCC.
Tongue Neoplasms/diagnostic imaging*
;
Carcinoma, Squamous Cell/diagnostic imaging*
;
Animals
;
Gold/chemistry*
;
Mice
;
Photothermal Therapy/methods*
;
Tomography, X-Ray Computed
;
Photosensitizing Agents
;
Metal Nanoparticles
;
Humans
;
Cell Line, Tumor
3.Expert consensus on reprocessing of medical ultrasound probes
Xi YAO ; Luzeng CHEN ; Anhua WU ; Liubo ZHANG ; Chunyan MA ; Li WANG ; Huixue JIA ; Xun HUANG ; Meng CAI ; Qing ZHANG ; Tao CHEN ; Hongwen FEI ; Yunxi LIU ; Guiqiu CHEN ; Xiaodong GAO ; Xin LI ; Baohua LI ; Guoqing HU ; Ping LIANG ; Liuyi LI
Chinese Journal of Infection Control 2025;24(3):301-307
Medical ultrasound technology is widely used for diagnosis and therapy in clinical practice.Ultrasound probes,which are directly contact with patients,pose a potential risk of pathogen transmission.This expert consen-sus was developed by a multidisciplinary team based on international guidelines,standards in China,and the results of a national survey,aiming to reduce the risk of healthcare-associated infection through standardizing reprocessing of medical ultrasound probes,and formulating consensus recommendations with the Delphi method.The consensus clarifies the reprocessing principles for three types of ultrasound probes of different infection risks:external-use ul-trasound probes,interventional percutaneous ultrasound probes,and internal-use ultrasound probes,puts forward systematic suggestions on the reprocessing standards and disinfection levels of ultrasound probe isolation covers and coupling agents,the reprocessing procedures and methods of ultrasound probes,as well as architectural layout and management of reprocessing,so as to provide a scientific prevention and control framework for ensuring ultrasound diagnosis and therapy safety.
4.Catheter-directed thrombolysis for acute entire lower extremity deep venous thrombosis: a comparative study of calf deep vein and contralateral femoral venous access
Jian WANG ; Cheng QIAN ; Guoqing NI ; Maofeng GONG ; Liang LIU ; Peng PENG ; Libing GAO ; Jianping GU ; Guoping CHEN
Chinese Journal of Radiology 2025;59(5):577-585
Objective:To compare the clinical efficacy of catheter-directed thrombolysis (CDT) via the contralateral femoral vein approach (CFVA-CDT) and the calf deep vein approach (CVA-CDT) in the treatment of acute mixed-type lower extremity deep vein thrombosis (DVT).Methods:Patients treated with CFVA-CDT and CVA-CDT for acute mixed-type DVT were retrospectively collected from January 2018 to December 2021, totaling 49 and 32 patients, respectively. The relevant technical indicators, thrombolysis rates in the iliac-femoral vein segment and femoral-popliteal vein segment, clinical efficacy, and the incidence of lower extremity deep vein patency, venous valve insufficiency, and post-thrombotic syndrome (PTS), as well as the severity of chronic venous disease in the affected limb (VCSS score) during a 2-year follow-up period were retrospectively compared between the two venous access CDT groups. The t-test was used for comparing quantitative data, while the chi-square test or Fisher′s exact test was used for categorical data.Results:During CFVA-CDT procedure, 6-8 F vascular sheaths were used, and balloon dilation of 2~6 mm was more frequently employed (65.31%, 32/49) to expand venous stenosis/occlusion segments before successful sheath placement compared to the CVA-CDT group (37.50%, 12/32), and the difference was statistically significant ( P=0.014). In the CVA-CDT group, 31.25% (10/32) of patients had a maximum sheath size of 6 F, while the remainder used 4 or 5 F sheaths. Among them, 34.38% (11/32) of patients required re-puncture of the popliteal or femoral vein for larger sheaths (≥8 F) for thrombus aspiration and subsequent endovascular treatment during or after thrombolysis. The effective thrombolysis rates (≥50%) in the iliac-femoral vein segment were not significantly different between the two groups ( P=0.778). The effective thrombolysis rate of the femoral-popliteal venous segment is related to the presence or absence of popliteal vein opacification on lower extremity venous antegrade venography. There was no significant difference between the groups when the popliteal vein was visualized ( P=1.000). While the popliteal vein was not visualized, the CVA-CDT group (75.0%, 15/20) was significantly better than the CFVA-CDT group (34.38%, 11/32), and the difference was statistically significant ( P=0.004). There was no significant difference in clinical efficacy between the two groups ( P=0.819). During follow-up, the femoral-popliteal vein patency rate in the CVA-CDT group (87.50%, 28/32) was significantly higher than in the CFVA-CDT group (44.90%, 22/49), the difference was statistically significant ( P<0.001). Conclusions:Successful CFVA-CDT requires the assistance of more ancillary devices, while the use of larger sheaths is more limited in CVA-CDT due to the smaller caliber of the calf deep veins. The presence or absence of popliteal vein opacification on lower extremity venous antegrade venography may influence the effective thrombolysis of the femoral-popliteal venous segment thrombus in patients with acute mixed deep vein thrombosis (DVT) treated with CFVA-CDT and CVA-CDT. Compared to CFVA-CDT, CVA-CDT can improve the patency rate of the femoral-popliteal venous segment.
5.Multi dimensional assessment study on the accuracy and clinical application of mainstream and sidestream monitoring technologies for partial pressure of end tidal carbon dioxide in monitoring devices
Sheng CHENG ; Fei WANG ; Guoqing WANG ; Nanhai WANG ; Dehao ZHU ; Zhijie LIU
China Medical Equipment 2025;22(8):1-6,16
Objective:To investigate the measurement differences between mainstream and sidestream partial pressure of end tidal carbon dioxide(PetCO2)monitoring techniques in mechanically ventilated patients,as well as the correlation with partial pressure of arterial blood carbon dioxide(PaCO2)and clinical applicability,and to provide a basis for the rational selection of monitoring methods in the clinic.Methods:A total of 169 patients who underwent invasive mechanical ventilation in the operating room of the First Affiliated Hospital of Bengbu Medical University from August to November 2024 were selected to be monitored by three monitoring techniques of mainstream PetCO2,sidestream PetCO2 and PaCO2,respectively,and the 243 sets of valid pairwise data monitored by the three monitoring techniques were recorded,analyzed for correlation and deviation,and performed PaCO2 and PetCO2 were stratified and analyzed.Likert scales were used to analyze the subjective ratings of the 3 monitoring techniques of mainstream PetCO2,sidestream PetCO2,and PaCO2 by the 31 health care professionals who used the study.Results:Both mainstream and sidestream PetCO2 correlated with PaCO2(r=0.554,0.515,P<0.05),and mainstream PetCO2 correlated better than sidestream;mainstream PetCO2 was lower than the concurrent PaCO2 by 6.00 mmHg,and the 95%limit of agreement(95%LoA)was±6.28 mmHg,both lower than that of sidestream by 8.24 mmHg and±7.41 mmHg.8.24 mmHg and±7.41 mmHg.The dispersion of sidestream PetCO2 monitoring values was greater,especially at high PaCO2 levels.Mainstream method PetCO2 had the highest correlation with PaCO2 in gynecologic patients(r=0.820,P<0.05),and sidestream method PetCO2 underestimated PaCO2 more markedly in gastrointestinal and hepato-biliary-pancreatic surgery patients.The deviation of oxygenation index≤300 mmHg was significantly greater than that of patients with oxygenation index>300 mmHg,and the tendency of underestimation was more significant at high PaCO2.31 healthcare experts evaluated that the mainstream PetCO2 monitoring method was outstanding in terms of accuracy and real-time,and that the sidestream method of PetCO2 monitoring was advantageous in terms of ease of operation and management of consumables.Conclusion:In patients who receive mechanical ventilation,the precision,accuracy and real-time of mainstream PetCO2 monitoring are better,which is suitable for patients with rapid changes in disease condition or patients at risk of hypercapnia.The sidestream method is suitable for non-intubated or transported patients,but it is easy to be underestimated at high PaCO2,and it needs to be corrected regularly in combination with arterial blood gas.The clinical application should rationally choose monitoring method after a comprehensive assessment based on the patient's disease condition,oxygenation status,and the scenario of use.
6.Guideline for the prevention of intraoperative acquired pressure injury in paraplegic patients with spinal cord injury (version 2025)
Aijun XU ; Shuixia LI ; Bo CHEN ; Mengyuan YE ; Lejiao LANG ; Ning NING ; Lin ZHANG ; Changqing LIU ; Zhonglan CHEN ; Weihu MA ; Weishi LI ; Xiaoning WANG ; Dongmei BIAN ; Jiancheng ZENG ; Xin WANG ; Yuan GAO ; Yaping CHEN ; Jiali CHEN ; Yun HAN ; Xiuting LI ; Yang ZHOU ; Xiaojing SU ; Qiong ZHANG ; Tianwen HUANG ; Ping ZHANG ; Hua LIN ; Xingling XIAO ; Ruifeng XU ; Fanghui DONG ; Bing HAN ; Luo FAN ; Yanling PEI ; Suyun LI ; Xiaoju TAN ; Rongchen GUO ; Yefang ZOU ; Xiaoyun HAN ; Junqin DING ; Yi WANG ; Shuhua DENG ; Jinli GUO ; Yinhua LIANG ; Yuan CEN ; Xiaoqin LIU ; Junru CHEN ; Haiyang YU ; Lunlan LI ; Ying REN ; Yunxia LI ; Jianli LU ; Ying YING ; Lan WEI ; Yin WANG ; Qinhong XU ; Yanqin ZHANG ; Yang LYU ; Shijun ZHANG ; Sui WENJIE ; Sanlian HU ; Shuhong YANG ; Guoqing LI ; Jingjing AN ; Baorong HE ; Leling FENG
Chinese Journal of Trauma 2025;41(6):530-541
Paraplegia caused by spinal cord injury is a serious neurological complication, for which surgery is currently the main treatment method. Due to different surgical approaches, patients are usually expected to maintain a passive prone position for a long time or switch between the supine and prone positions. Affected by multiple factors such as neurogenic sensory disorders, pathological changes in muscle tone and operative duration, the risk of intraoperative acquired pressure injury (IAPI) is significantly increased. Current clinical prevention strategies for IAPI in these patients predominantly focus on localized pressure relief during positioning, lacking systematic, standardized comprehensive prevention protocols or evidence-based guidelines. To address it, Department of Nursing, Orthopedics Branch, China International Exchange and Promotive Association for Medical and Health Care, Spinal Trauma Professional Committee, Orthopedics Branch, Chinese Medical Doctor Association, Nursing Group of Spine and Spinal Cord Professional Committee of Chinese Association of Rehabilitation Medicine organized experts in relevant fields to formulate Guideline for the prevention of intraoperative acquired pressure injury in paraplegic patients with spinal cord injury ( version 2025), based on evidence-based medical evidence and latest research results and clinical practice at home and abroad. Eleven recommendations were put forward from the aspects of preoperative risk assessment, intraoperative prevention strategies, postoperative handover and monitoring, and supportive mechanisms for IAPI prevention, aiming to standardize the prevention measures and management strategies of IAPI in paraplegic patients with spinal cord injury and accelerate the recovery of patients and improve the therapeutic effect.
7.Predictive value of pre-treatment circulating tumor DNA genomic landscape in patients with relapsed/refractory multiple myeloma undergoing anti-BCMA CAR-T therapy: Insights from tumor cells and T cells
Rongrong CHEN ; Chunxiang JIN ; Kai LIU ; Mengyu ZHAO ; Tingting YANG ; Mingming ZHANG ; Pingnan XIAO ; Jingjing FENG ; Ruimin HONG ; Shan FU ; Jiazhen CUI ; Simao HUANG ; Guoqing WEI ; He HUANG ; Yongxian HU
Chinese Medical Journal 2025;138(19):2481-2490
Background::B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T (CAR-T) therapy yield remarkable responses in patients with relapsed/refractory multiple myeloma (R/RMM). Circulating tumor DNA (ctDNA) reportedly exhibits distinct advantages in addressing the challenges posed by tumor heterogeneity in the distribution and genetic variations in R/RMM.Methods::Herein, the ctDNA of 108 peripheral blood plasma samples from patients with R/RMM at the First Affiliated Hospital, School of Medicine, Zhejiang University was thoroughly investigated before administration of anti-BCMA CAR-T therapy to establish its predictive potential. Flow cytometry is used primarily to detect subgroups of T cells or CAR-T cells.Results::In this study, several tumor and T cell effector-mediated factors were considered to be related to treatment failure by an integrat analysis, including higher percentages of multiple myeloma (MM) cells in the bone marrow ( P = 0.0125), lower percentages of CAR-T cells in the peripheral blood at peak ( P = 0.0375), and higher percentages of CD8 + T cells ( P = 0.0340). Furthermore, there is a substantial correlation between high ctDNA level (>143 ng/mL) and shorter progression-free survival (PFS) ( P = 0.007). Multivariate Cox regression analysis showed that high levels of ctDNA (>143 ng/mL), MM-driven high-risk mutations (including IGLL5 [ P = 0.004], IRF4 [ P = 0.024], and CREBBP [ P = 0.041]), number of multisite mutations, and resistance-related mutation ( ERBB4, P = 0.040) were independent risk factors for PFS. Conclusion::Finally, a ctDNA-based risk model was built based on the above independent risk factors, which serves as an adjunct non-invasive measure of substantial tumor burden and a prognostic genetic feature that can assist in predicting the response to anti-BCMA CAR-T therapy.
8.Analysis of the Effects of Tongfu Xiefei Formula on Serum Inflammatory Cytokines in Patients with Acute Exacerbation of Chronic Obstructive Pulmonary Disease Based on the Theory of Simultaneous Treatment of Lung and Intestine
Guoqing ZHU ; Yan ZHOU ; Xianzhen LI ; Lili TANG ; He CHEN ; Zhiliang LIU
Journal of Guangzhou University of Traditional Chinese Medicine 2025;42(6):1340-1348
Objective To investigate the clinical efficacy of Tongfu Xiefei Formula in treating patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD)based on the theory of simultaneous treatment of lung and intestine,and to observe its effects on serum inflammatory cytokines.Methods A retrospective study was conducted on 134 AECOPD patients treated at the Department of Pulmonary Diseases of Traditional Chinese Medicine,Tangshan Hospital of Traditional Chinese Medicine from December 2020 to December 2022.The patients were divided into an observation group and a western medicine group based on the treatment plans,with 67 cases in each group.The western medicine group received conventional western medical treatment,while the observation group was given Tongfu Xiefei Formula orally in addition to the western medical treatment.The course of treatment covered 2 weeks.Before and after treatment,the two groups were observed in the changes of the modified Medical Research Council(mMRC)dyspnea scale scores,COPD Assessment Test(CAT)scores,lung function indicators,arterial blood gas analysis indicators,and serum inflammatory cytokine levels.The clinical efficacy,total incidence of adverse reactions,and hospitalization time were compared between the two groups.Results(1)After 2 weeks of treatment,the total effective rate in the observation group was 95.52%(64/67),compared to 79.10%(53/67)in the western medicine group.The intergroup comparison(tested by the chi-square test)showed that the efficacy of the observation group was significantly superior to that of the western medicine group(P<0.01).(2)After treatment,the mMRC scores and CAT scores in both groups were significantly decreased(P<0.05),and the decrease in the observation group was significantly superior to that in the western medicine group(P<0.01).(3)After treatment,lung function indicators of the forced expiratory volume in one second(FEV1),forced vital capacity(FVC),and their ratio(FEV1/FVC)in both groups were significantly increased(P<0.05),and the increase in the observation group was significantly superior to that in the western medicine group(P<0.01).(4)After treatment,the oxygen saturation(SaO2)and arterial oxygen partial pressure(PaO2)levels in both groups significantly increased(P<0.05),while the arterial carbon dioxide partial pressure(PaCO2)level was significantly decreased(P<0.05).The increase in SaO2 and PaO2 levels and the decrease in PaCO2 level in the observation group were significantly superior to those in the western medicine group(P<0.01).(5)After treatment,the levels of serum inflammatory cytokines of tumor necrosis factor α(TNF-α),C-reactive protein(CRP),and interleukin-6(IL-6)in both groups were significantly decreased(P<0.05),and the decrease in the observation group was significantly superior to that in the western medicine group(P<0.01).(6)The total incidence of adverse reactions in the observation group was 2.99%(2/67),compared to 5.97%(4/67)in the western medicine group,with no statistically significant difference between the two groups(P>0.05).(7)The observation group had a significantly shorter hospitalization time than the western medicine group,and the difference was statistically significant(P<0.05).Conclusion Tongfu Xiefei Formula,formulated based on theory of simultaneous treatment of lung and intestine,is effective and safe on relieving symptoms such as dyspnea in AECOPD patients,improving lung function,correcting arterial blood gas disorders,inhibiting the release of inflammatory factors,shortening treatment time,while causing no serious adverse reactions.
9.Effects of sacral neuromodulation on urodynamic parameters during the storage phase in patients with neurogenic bladder
Haichao LIU ; Guoqing CHEN ; Peng ZHANG ; Fan ZHANG ; Baihui WANG ; Fei ZHOU ; Yanhe JU
Journal of Modern Urology 2025;30(12):1075-1079
Objective To explore the effects of sacral neuromodulation (SNM) on urodynamic parameters during the storage phase in patients with neurogenic bladder (NB), so as to provide reference for evaluating the efficacy of SNM. Methods A total 49 NB patients undergoing SNM at our hospital during Oct.2012 and May 2025 were enrolled. Baseline data and video-urodynamic parameters were collected. Changes in maximum cystometric capacity, maximum detrusor pressure during storage phase, and bladder compliance before and after treatment were assessed. Improvements in detrusor overactivity (DO) and vesicoureteral reflux (VUR) were also analyzed. Results Among the 49 patients,27 were male and 22 were female, with a mean age of (37.41±15.15) years, a median disease duration of 5.0 (2.0,15.5) years, and a median follow-up of 11 (1,32) months. Up to 37 patients (75.5%) received permanent sacral nerve pulse generator implantation (permanent implant group), while the remaining 12 were classified as the non-permanent implant group. Before and after the test period, all patients showed a significant increase in maximum cystometric capacity [ (218.0 (93.0,358.5) mL vs.300.0 (238.5, 400.0) mL, P<0.001], a decrease in maximum detrusor pressure during the filling phase [32.0 (13.5,71.0) cmH_2 O vs. 20.0 (9.0,50.0) cmH_2 O, P<0.001], and an improvement in bladder compliance [11.8 (8.3,25.6) mL/cmH_2 O vs.26.7 (8.6,44.1) mL/cmH_2O, P<0.001]. In the permanent implant group, comparisons before and after the test period showed an increase in maximum bladder capacity [ (239.16±147.23) mL vs. (312.24±121.83) mL, P<0.001], a decrease in maximum detrusor pressure during filling[32.0 (15.0,58.0) cmH_2 O vs.15.0 (9.0,41.0) cmH_2 O, P<0.05], and improved bladder compliance [10.8 (8.3,23.6) mL/cmH_2 O vs.28.6 (8.6,41.4) mL/cmH_2 O, P<0.001]. No statistically significant differences in these parameters before and after the test period were observed in the non-permanent implant group (P>0.05). A total of 17 patients in the permanent implant group underwent follow-up video urodynamics. Compared to pre-test values, significant improvements were observed in maximum detrusor pressure during filling, and bladder compliance both at the end of the test period and at the last follow-up (P<0.05). However, no statistically significant differences were found in maximum cystometric capacity, maximum detrusor pressure during filling, and bladder compliance between the end of the test period and the last follow-up (P>0.05). Among the 49 patients,21 had DO and 20 had VUR. Both DO and VUR showed improvement after the test period and at the last follow-up. Conclusion SNM can effectively improve storage function in NB patients, ameliorate detrusor overactivity and bladder compliance, and relieve or eliminate VUR in some patients. Long-term follow-up confirms that SNM provides stable therapeutic effects, demonstrating significant clinical value.
10.Biological properties of bacterial outer membrane vesicles surface-displaying PD-L1 nanobodies and their disrupting effects on PD-1/PD-L1 signaling pathway
Zhimin LI ; Mingge HUO ; Longxue GUAN ; Fanlin GU ; Dandan LIANG ; Zhuorui LIU ; Guoqing WANG ; Xingang GUAN
Journal of Jilin University(Medicine Edition) 2025;51(5):1407-1414
Objective:To prepare the bacterial outer membrane vesicles(OMV)that can express programmed death ligand 1(PD-L1)nanobody on surface,and to discuss its structural characteristics,cell compatibility,intracellular distribution,and its blocking effect on the programmed cell death protein-1(PD-1)/PD-L1 signaling axis.Methods:The pET28a-ClyA-PD-L1nb prokaryotic expression vector was constructed and transformed into Escherichia coli BL21(DE3)competent cells;the OMV was isolated from the BL21(DE3)monoclonal colonies transformed with the PD-L1nb expression vector by ultracentrifugation;the protein purification was performed using the histidine(His)tag;transmission electron microscope and nanoparticle size analyzer were used to analyze and identify the OMV;the OMV isolated from the BL21(DE3)monoclonal colonies transformed with the PD-L1nb expression vector was used as experimental group;the OMV isolated from the untransformed BL21(DE3)monoclonal colonies was used as control group;Western blotting method was used to detect the expression levels of ClyA-PD-L1nb fusion protein in the OMV in two groups;cell counting kit-8(CCK-8)assay was used to detect the activities of mouse macrophage RAW 264.7 cells,mouse triple-negative breast cancer 4T1 cells,and human embryonic kidney HEK293T cells after treated with OMV;fluorescence imaging technology was used to observe the tumor cell endocytosis of OMV;flow cytometry was used to detect the binding effect of OMV to the PD-L1 on surface of the tumor cells in PBS group,OMV-PD-L1nb group,and aPD-L1+OMV-PD-L1nb group.Results:The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)results showed that after induction of Escherichia coli,significantly thickened protein bands appeared near the predicted relative molecular mass(about 49 000),and after purification,no obvious impurity proteins existed in the lanes;the OMV-PD-L1nb with a size of about 120 nm was isolated by ultracentrifugation,and it presented a uniform spherical structure under transmission electron microscope;the Western blotting results showed that the specific band of ClyA-PD-L1nb was detected in the OMV in experimental group;the CCK-8 assay results showed that after treated with different concentrations of OMV,the viabilities of the RAW 264.7 cells,4T1 cells,and HEK293T cells were all close to 100%;the fluorescence imaging results showed that OMV-PD-L1nb was endocytosed by 4T1 cells and dispersed in the cytoplasm;compared with OMV-PD-L1nb group,the average fluorescence intensity in the cells in aPD-L1+OMV-PD-L1nb group was significantly decreased(P<0.001).Conclusion:The OMV surface-displaying PD-L1nb,OMV-PD-L1nb,is successfully prepared and isolated;OMV-PD-L1nb shows good compatibility on mouse macrophage cells,tumor cells,and human embryonic kidney cells,can be endocytosed by tumor cells,and successfully blocks the PD-1/PD-L1 signaling pathway.

Result Analysis
Print
Save
E-mail