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@#Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.
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【Objective】 To investigate the binding and carrying effects of human serum albumin (HSA) from various sources on sphingosine-1-phosphate (S1P). 【Methods】 Utilizing human plasma-derived HSA (pHSA) and recombinant HSA (rHSA) samples as the focal points of our investigation, LC-MS/MS technology was employed to meticulously compare and analyze the disparities in S1P content among the aforementioned samples. Subsequently, under physiological concentration conditions, S1P was directly introduced to HSA samples for loading processing, facilitating a comprehensive comparison of the binding efficacy of HSA from different sources to S1P. Within a serum-free culture setting, HSA samples from various sources were co-cultured with HUVEC cells. The alterations in S1P content within the cell culture supernatant across different treatment groups were meticulously analyzed, allowing for a nuanced comparison of the S1P carry effects exerted by HSA from different sources on cells.The interaction between HSA and S1P molecules from different sources was analyzed and their affinity was calculated using surface plasmon resonance (SPR) technology. Furthermore, leveraging AutoDock Vina software and the Molprophet platform, the molecular docking analysis of HSA and S1P was conducted, aiming to predict the key binding pocket domain of S1P within HSA. 【Results】 All pHSA samples exhibited detectable levels of S1P (ranging from 3.31±0.03 to 30.35±0.07 μg/L), with significant variations observed among pHSA samples from different manufacturers (P<0.001). Conversely, S1P was undetectable in all rHSA samples. Upon load treatment, the binding affinity of HSA from diverse sources to S1P demonstrated significant discrepancies (P<0.001), with rHSA exhibiting approximately double the average S1P loading compared to pHSA (ΔCrHSA=801.75±142.45 μg/L vs ΔCpHSA=461.94±85.73 μg/L; P<0.001, t=5.006). Co-culture treatment outcomes revealed a significant elevation in S1P concentration within the supernatant after 6 hours of co-culture across all HSA sample processing groups with HUVEC cells, while no changes were observed in the supernatant of the blank control group. Notably, significant differences in supernatant S1P concentration were observed among treatment groups at 6 h, 12 h, and 24 h (P<0.001). SPR analysis unveiled a stronger affinity of pHSA for S1P compared to rHSA (KDpHSA-S1P: 2.38E-06, KDrHSA-S1P: 3.72E-06). Molecular docking analysis and binding pocket prediction suggested that the key binding pocket of HSA and S1P may reside in the IB subdomain of the HSA molecule. 【Conclusion】 HSA from various sources exhibits distinct binding and carrying effects on S1P, which appear to be closely associated with the IB subdomain of the HSA molecule.
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@#Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.
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OBJECTIVE To explore the effects of alfentanil （ALF） on myocardial fibrosis in rats with acute myocardial infarction （AMI） by regulating sphingosine kinase 1 （SphK1）/sphingosine 1-phosphate （S1P） signaling pathway. METHODS Male SD rats were collected to construct AMI model by the ligation of anterior descending branch of left coronary artery. The successfully modeled rats were randomly divided into AMI model group （Model group）， ALF low-dose group （ALF-L group， 0.25 mg/kg ALF）， ALF high-dose group （ALF-H group， 0.5 mg/kg ALF）， high dose of ALF+SphK1 activator group （ALF-H+K6PC-5 group， 0.5 mg/kg ALF+1 μg/g K6PC-5）. At the same time， a sham operation group （Sham group） was set up to perform only chest opening/closing operations without ligating the anterior descending branch of left coronary artery， with 15 rats in each group. Rats in each drug group were intraperitoneally injected with the corresponding drug solution， once a day， for 4 consecutive weeks. Twelve hours after the last medication， cardiac function indicators [left ventricular systolic pressure （LVSP）， left ventricular ejection fraction （LVEF）， left ventricular systolic diameter （LVSD）， left ventricular fractional shortening （LVFS）] of rats were detected in each group； the condition of myocardial infarction， pathological changes in myocardial tissue， and degree of fibrosis were observed； serum levels of brain natriuretic peptide （BNP） and cardiac troponin Ⅰ （cTnⅠ） in rats were detected. The protein expressions of collagen Ⅰ ， collagen Ⅲ ， matrix metalloproteinase-2 （MMP-2）， SphK1 and S1P were alsodetected in the myocardial tissue of rats. RESULTS Compared with the Sham group， the arrangement of myocardial cells in the Model group was disordered， with a large number of inflammatory cells infiltrating. The levels of LVSP， LVFS and LVEF in the Model group were significantly reduced （P＜0.05）； LVSD level， myocardial infarction area， collagen volume fraction， serum levels of BNP and cTnⅠ， the protein expressions of collagen Ⅰ， collagen Ⅲ， MMP-2， SphK1 and S1P in myocardial tissue were significantly increased or enlarged （P＜0.05）. Compared with the Model group， the pathological changes and degree of fibrosis in the myocardial tissue of rats in each dose group of ALF were improved or relieved， while the quantitative indicators of rats in the ALF-H group were significantly improved and significantly better than those in ALF-L group （P＜0.05）. K6PC-5 could significantly reverse the improvement effect of high-dose ALF on the above quantitative indicators in rats （P＜0.05）. CONCLUSIONS ALF can reduce myocardial fibrosis and improve cardiac function in AMI rats， and the effect may be related to the inhibition of the SphK1/S1P signaling pathway.
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Objective:To compare the efficacy of single-agent versus multi-agent adjuvant chemotherapy after radical gastrectomy for elderly patients with stage Ⅲ gastric cancer.Methods:The propensity score matching and retrospective cohort study were conducted. The clinicopatholo-gical data of 456 elderly patients with stage Ⅲ gastric cancer who underwent D 2 radical resection in the Renji Hospital affiliated to Shanghai Jiaotong University School of Medicine from January 2016 to December 2020 were collected. There were 343 males and 113 females, aged 71(range, 65?89)years. Of the 456 patients, 274 cases undergoing single-agent adjuvant chemotherapy after surgery were divided into single-agent chemotherapy group, 182 cases undergoing double-agent or triple-agent adjuvant chemotherapy after surgery were divided into multi-agent chemotherapy group. Observa-tion indicators: (1) propensity score matching and comparison of general data of patients between the two groups after matching; (2) adverse events during chemotherapy; (3) follow-up. Propensity score matching was done by the 1∶1 ratio, with the caliper value of 0.05. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. Measurement data with skewed distribution were represented as M(range). Count data were described as absolute numbers, and comparison between groups was conducted using the chi-square test or Fisher exact probability. Comparison of ordinal data between groups was conducted using the non-parameter rank sum test. The Kaplan-Meier method was used to draw survival curves and calculate survival rates, and the Log-Rank test was used for survival analysis. Results:(1) Propensity score matching and comparison of general data of patients between the two groups after matching. Of 456 patients, 306 cases were successfully matched, including 153 cases in the single-agent chemotherapy group and 153 cases in the multi-agent chemotherapy group. The elimination of age, age-adjusted Charlson comorbidity index, pathological TNM staging confounding bias ensured comparability between the two groups after propensity score matching. (2) Adverse events during chemotherapy. In terms of hematological adverse events, 6 cases in the single-agent chemotherapy group and 16 cases in the multi-agent chemotherapy group had neutropenia, showing a significant difference in the neutropenia ( χ2=4.90, P<0.05). In terms of non-hematological adverse events, cases with anorexia and nausea were 77 and 50 for the single-agent chemotherapy group, versus 96 and 69 for the multi-agent chemotherapy group, showing significant differences between the two groups ( χ2=4.80, 4.96, P<0.05). (3)Follow-up. All the 306 patients were followed up for 48(range, 8?61)months. The 5-year overall survival rates of the single-agent chemotherapy group and the multi-agent chemotherapy group were 36.08% and 38.31%, respectively, showing no significant difference between the two groups ( hazard ratio=0.93, 95% confidence interval as 0.70?1.20, P>0.05). Results of further analysis showed that the 5-year overall survival rates were 32.41% and 39.40% for 97 patients of the single-agent chemotherapy group and 97 patients with double-agent regimen of the multi-agent chemotherapy group, respectively, showing no significant difference between them ( hazard ratio=1.20, 95% confidence interval as 0.82?1.70, P>0.05). The 5-year overall survival rates were 43.15% and 37.11% for 56 patients of the single-agent chemotherapy group and 56 patients with triple-agent regimen of the multi-agent chemotherapy group, respectively, showing no significant difference between them ( hazard ratio=0.81, 95% confidence interval as 0.65?1.00, P>0.05). Conclusions:For adjuvant chemotherapy in elderly patients with stage Ⅲ gastric cancer, there is no significant survival advantage of double-agent or triple-agent chemotherapy over single-agent oral chemotherapy. However, there is a higher incidence of neutropenia, anorexia, ausea.
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ObjectiveTo investigate the effects of hydroxycamptothecin liposomes （LHCPT） on myocardial fibrosis in rats with heart failure by regulating the sphingosine kinase 1 （SphK1）/sphingosine-1-phosphate （S1P） signaling pathway. MethodsSD rats were divided into control group， model group， hydroxycamptothecin （HCPT） group， LHCPT group， captopril group， and LHCPT+K6PC-5 （SphK1 activator） group， with 12 rats in each group. The heart failure rat models in all groups except the control group were established by intraperitoneal injection of doxorubicin and then the corresponding drugs were given once a day. After four weeks， we applied color Doppler ultrasound to detect left ventricular end systolic diameter （LVESD）， left ventricular end diastolic diameter （LVEDD）， and left ventricular ejection fraction （LVEF） in rats； HE and Masson staining for myocardial pathological damage and myocardial fibrosis in rats， respectively； ELISA method for the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rat myocardial tissues； qRT-PCR for the expression of transforming growth factor-β1 （TGF-β1）， type I collagen （Collagen I）， and type Ⅲ collagen （Collagen Ⅲ） in rat myocardial tissues； Western blot for the expression of SphK1 and S1P proteins in rat myocardial tissues. ResultsCompared with the control group， the model group showed severe myocardial pathological damage and myocardial fibrosis， increased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and decreased LVEF （P<0.05）. Compared with the model group， the HCPT group， LHCPT group and captopril group showed alleviated myocardial pathological damage and myocardial fibrosis， decreased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and increased LVEF （P<0.05）. Compared with the LHCPT group， the LHCPT+K6PC-5 group showed aggravated myocardial pathological damage and myocardial fibrosis， increased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and decreased LVEF （P<0.05）. ConclusionLHCPT may inhibit myocardial fibrosis in heart failure rats by inhibiting the SphK1/S1P signaling pathway.
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Sphingolipid metabolism is widely involved in the functional regulation of different cells, and also plays an important role in ocular tissues.Sphingosine 1-phosphate (S1P) is the end product of sphingolipid metabolism and has been shown to play an important role in the onset and development of eye diseases.S1P signaling pathway is widely expressed in various ocular cells and is involved in the regulation of cell proliferation, differentiation, migration and apoptosis.S1P activates a variety of signaling pathways by binding to corresponding receptors and thus plays a wide range of physiological and pathological effects in the eye.Recent studies have found that the S1P signaling pathway can not only mediate the normal development of blood vessels and nerves in the eye, maintain the normal structure of the ocular tissues, and participate in the metabolism of lipids in the eye, but also has a close relationship with immune-related inflammatory response, pathological fibrosis, destruction of cell functional barrier and other related pathological changes.This paper mainly reviewed the basic overview of the S1P signaling pathway, its physiological role in the eye, and its role in the pathological changes of anterior and posterior segment diseases, so as to provide new directions and targets for the treatment of eye diseases.
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【Objective】 To investigate the effects and mechanisms of different doses of fingolimod (FTY720) on non-antibody-mediated transfusion-related acute lung injury (TRALI). 【Methods】 A TRALI mouse model was constructed using lipopolysaccharide (LPS) pre-stimulation and platelets (Plt) of different storage days for second strike. The success of the modeling was determined by protein concentration in lung tissue homogenates, myeloperoxidase (MPo) activity, lung wet/dry weight ratio (W/D ratio), lung tissue damage score and pathological sections. Ceramide and sphingosine-1-phosphate (S1P) contents in platelets of different storage days were detected. FTY720 was administered 1 h after LPS injection to investigate the role of FTY720 in TRALI. The expression levels of vascular endothelial cadherin (VE-cadherin) and zonula occludens-1 (ZO-1) were analyzed by WB. 【Results】 Mice infused with stored 5-day Plt (d5Plt group) exhibited typical signs of TRALI, and the differences in lung tissue homogenate protein concentration (6 546.38±409.50) μg/mL, MPO activity (49.38±4.43) U/L, W/D ratio 4.79±0.21, and lung tissue damage score 7.24±0.38 from the rest of the groups were statistically significant (P<0.05). With the increase of platelet storage time, the ceramide content gradually increased and S1P content gradually decreased, and the ratio of the two was imbalanced. d5Plt showed statistically significant differences (P<0.01) in ceramide content (58.37±5.69) μmol/L and S1P content (149.81±4.86) nmol/L from the rest of the groups. After preventive administration of FTY720, 1 mg/kg FTY720 had no significant effect on TRALI mice, whose lung tissue homogenate protein concentration (6 170.26±545.50) μg/mL, MPO activity (45.97±4.79) U/L, W/D ratio 4.88±0.25, and lung tissue damage score 7.92±0.65 were significantly higher than those of the normal and LPS control groups (P<0.01). The low-dose (0.5, 0.2, and 0.1 mg/kg) FTY720 group alleviated lung injury, and its protein concentration, MPO activity, W/D ratio, and lung tissue injury score were significantly lower than those of the d5Plt group (P<0.05). Pathological sections also showed similar results. In terms of endothelial intercellular junction protein expression, the VE-cadherin expression levels in the 1 mg/kg FTY720 group were significantly lower than those in the normal and LPS control groups (P<0.05), and the VE-cadherin and ZO-1 expression levels in the low-dose (0.5, 0.2, and 0.1 mg/kg) FTY720 group were significantly higher than those in the d5Plt group (P<0.05), which tended to be normalized. 【Conclusion】 In this study, a TRALI mouse model was successfully established by one strike of LPS and two strikes of d5Plt. Low doses of FTY720 (0.5, 0.2, 0.1 mg/kg) were protective against TRALI, while high doses of FTY720 (1 mg/kg) may aggravate the symptoms of TRALI. This protective effect may be somewhat dependent on the expression of VE-cadherin and ZO-1.
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Aim To explore the effect of S1P/S1PR1 signaling pathway on high glucose(HG)-induced epithelial-mesenchymal transition of rat renal tubular epithelial cells and its possible mechanism. Methods Cells were treated with different concentrations of glucose, and intracellular S1P expression was detected by ELISA and S1PR1 protein expression was detected by Western blot. The cells were divided into normal control group, HG group and HG + siS1PR1 group. The expression of E-cadherin, Vimentin, Fibronectin and Twist mRNA were detected by RT-qPCR and E-cadherin, α-SMA, Vimentin, NLRP3, ASC and NF-κB protein expression were detected by Western blot, and the levels of reactive oxygen species(ROS) were detected by flow cytometry. The cells were divided into normal control group, S1P group and S1P + siS1PR1 group. Vimentin, Snail, α-SMA, NLRP3, ASC and NF-κB protein expressions were detected by Western blot, and ROS levels were measured by fluorescence microscopy. Results ELISA results showed that the content of S1P in cells increased significantly under high glucose stimulation. Western blot results showed that S1PR1 protein expression was significantly higher at 30 mmol · L
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The development of targeted oral drugs that can stably treat inflammatory bowel disease (IBD) is still a clinical problem to be solved. In recent years, studies have confirmed that sphingosine⁃1⁃phosphate (S1P)/S1P receptor pathway can regulate lymphocyte homing and immune regulation, inhibit intestinal inflammation, protect intestinal endothelial barrier, and affect intestinal microbial metabolism, which may play a key role in the treatment of IBD. This article reviewed the effect of S1P/S1P receptor pathway on IBD and its potential mechanism.
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With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
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Animaux , Mâle , Capra , Cellules souches , Spermatogonies/métabolisme , Prolifération cellulaire , Cytométrie en flux , Testicule/métabolismeRésumé
The present study aimed to investigate the protective effect and underlying mechanism of Platycladi Semen oil(SP) on Aβ_(25-35)-induced brain injury in mice to provide a theoretical basis for the clinical treatment of Alzheimer's disease(AD). Male Kunming(KM) mice were randomly divided into a control group, a model group(brain injection of Aβ_(25-35), 200 μmol·L~(-1), 0.15 μL·g~(-1)), a positive drug group(donepezil, 10 mg·kg~(-1)), and low-and high-dose SP groups(0.5 and 1 mL·kg~(-1)). Learning and memory ability, neuronal damage, levels of Aβ_(1-42)/Aβ_(1-40), p-Tau, related indicators of apoptosis and oxidative stress, and immune cells, and protein and mRNA expression related to the sphingosine kinase 1(SPHK1)/sphingosine-1-phosphate(S1P)/sphingosine-1-phosphate receptor 5(S1PR5) signaling pathway of mice in each group were determined. In addition, compounds in SP were analyzed by gas chromatography-mass spectrometry(GC-MS). The mechanism of SP against AD was investigated by network pharmacology, 16S rDNA gene sequencing for gut microbiota(GM), and molecular docking techniques. The results showed that SP could improve the learning and memory function of Aβ_(25-35)-induced mice, reduce hippocampal neuronal damage, decrease the levels of Aβ_(1-42)/Aβ_(1-40), p-Tau, and indicators related to apoptosis and oxidative stress in the brain, and maintain the homeostasis of immune cells and GM. Network pharmacology and sequencing analysis for GM showed that the therapeutic effect of SP on AD was associated with the sphingolipid signaling pathway. Meanwhile,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid, the components with the highest content in SP, showed good binding activity to SPHK1 and S1PR5. Therefore, it is inferred that SP exerts anti-apoptosis and antioxidant effects by regulating GM and inhibiting SPHK1/S1P/S1PR5 pathway, thereby improving brain injury induced by Aβ_(25-35) in mice. Moreover,(Z,Z,Z)-9,12,15-octadecatrienoic acid and(Z,Z)-9,12-octadecadienoic acid may be the material basis for the anti-AD effect of SP.
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Souris , Animaux , Mâle , Sperme/métabolisme , Microbiome gastro-intestinal , Pharmacologie des réseaux , Acide linoléique , Simulation de docking moléculaire , Maladie d'Alzheimer/génétique , Lésions encéphaliquesRésumé
BACKGROUND The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can infect common mice inducing significant pathological lung lesions and inflammatory responses. This substantially mimics coronavirus disease 19 (COVID-19) infection and pathogenesis in humans. OBJECTIVES To characterise the effects of recombinant SARS-CoV-2 S1 receptor-binding domain (RBD) peptide in murine macrophage and microglial cells' immune activation compared with classical PAMPs in vitro. METHODS Murine RAW 264.7 macrophages and BV2 microglial cells were exposed to increasing concentrations of the RBD peptide (0.01, 0.05, and 0.1 µg/mL), Lipopolysaccharide (LPS) and Poly(I:C) and evaluated after two and 24 h for significant markers of macrophage activation. We determined the effects of RBD peptide on cell viability, cleaved caspase 3 expressions, and nuclear morphometry analysis. FINDINGS In RAW cells, RBD peptide was cytotoxic, but not for BV2 cells. RAW cells presented increased arginase activity and IL-10 production; however, BV2 cells expressed iNOS and IL-6 after RBD peptide exposure. In addition, RAW cells increased cleaved-caspase-3, apoptosis, and mitotic catastrophe after RBD peptide stimulation but not BV2 cells. CONCLUSION RBD peptide exposure has different effects depending on the cell line, exposure time, and concentration. This study brings new evidence about the immunogenic profile of RBD in macrophage and microglial cells, advancing the understanding of SARS-Cov2 immuno- and neuropathology.
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Objective:To design a modified S1PR3 specific agonist, GPS-725.017, and investigate its protective effect on acute lung injury by promoting macrophage clearance of bacteria.Methods:A short peptide derived from the intracellular region of S1PR3 receptor was named GPS725.017, which was modified with norleucine (Nle) and myristicacid (myr) at its N terminus. Mice were divided into the sham operation group, solvent group and GPS-725.017 treatment group. The acute lung injury model was induced by endotracheal injection of E. coli (5×10 6 CFU), and the experimental group was treated with GPS-725.017 (10 mg/kg). The 48-h survival rate of mice was recorded. After 5 h of modeling, the bacterial load and inflammatory cytokines in peripheral blood and lung were detected, and Vps34 protein content in alveolar macrophages was determined by Western blot. After 12-h of modeling, lung tissues were collected for H&E staining and pathological scores. Results:Compared with the solvent group, the survival rate of mice in the GPS-725.017 treatment group was significantly improved ( P<0.01), the bacterial CFU in blood and alveolar lavage fluid was significantly lower than that in the solvent group ( P<0.001), and the levels of TNF-α and IL-1β in blood and alveolar lavage fluid were significantly lower than those in the solvent group ( P<0.001). Western blot showed that the expression level of Vps34 protein in alveolar macrophages was significantly higher than that in the solvent group ( P<0.01). Histopathology result showed that the pathological damage of lung in the treatment group was significantly less than that in the solvent group ( P<0.001). Conclusions:The modified synthetic S1PR3 specific agonist GPS-725.017 could specifically activate the S1PR3 receptor on the membrane of alveolar macrophages and up-regulate the expression level of intracellular Vps34 protein, which can promote the removal of bacteria in alveolar macrophages, significantly reduce the degree of lung injury and improve the survival rate in ALI mice.
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Objective:To evaluate the efficacy of concurrent radiotherapy combined with S-1 (CCRT) versus radiotherapy (RT) alone in elderly patients with esophageal cancer by Meta-analysis.Methods:The Cochrane Library, PubMed, Web of science, EMbase, CBM, CNKI, VIP and Wanfang database were searched. The eligible studies were subject to evaluation of methodological quality. The Meta-analysis was performed by the Revman 5.3 software.Results:A total of 1693 patients were enrolled in 23 studies. The results showed that CCRT increased the incidence of CR [ OR=2.08,95% CI (1.66-2.61), P<0.001] and PR [ OR=1.31,95% CI (1.08-1.60), P=0.007] and total response rate [ OR=2.99,95% CI (2.37-3.77), P<0.001]. Furthermore, CCRT improved the 1-year survival rate [ OR=2.56, 95% CI (1.94-3.38), P<0.001] and 2-year survival rate [ OR=2.33, 95% CI (1.77-3.08), P<0.001]. Meanwhile, CCRT reduced the incidence of leucopenia, thrombocytopenia, radioactive esophagitis, nausea and vomiting (all P<0.05), but there was no significant difference in the incidence of anemia and radiation pneumonia between two groups (both P>0.05). Conclusions:Available evidence suggests that CCRT combined with S-1 can improve therapeutic efficacy and prolong survival time in elderly patients with esophageal cancer, but CCRT may increase the incidence of treatment-related side effects. Due to the limitations of the number and quality of the included studies, the above conclusions need to be verified by more high-quality studies.
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Objective:To compare the clinical efficacy and safety of continuous dosing or alternate-day dosing of apatinib combined with SOX regimen as first-line treatment for patients with advanced gastric cancer.Methods:A total of 52 patients with human epidermal growth factor receptor 2 (HER2) negative and inoperable locally advanced or advanced gastric cancer who were pathologically diagnosed from January 2018 to January 2021 in the Second Affiliated Hospital of Shandong First Medical University were collected. The patients were divided into continuous dosing group and alternate-day dosing group by random number table method. The continuous dosing group received apatinib (250 mg, once a day) combined with SOX regimen (S-1+oxaliplatin); the alternate-day dosing group received apatinib (250 mg, once every other day) combined with SOX regimen. Twenty-one days were a cycle, and the efficacy was evaluated after 2 cycles. After 4-6 cycles, patients with stable disease received apatinib and S-1 for maintenance therapy. The therapeutic effects and adverse reactions of the two groups were compared.Results:The curative effect could be evaluated in 51 patients, including 26 in the continuous dosing group and 25 in the alternate-day dosing group. The disease control rates in the continuous dosing group and the alternate-day dosing group were 84.6% (22/26) and 76.0% (19/25) ( χ2 = 0.60, P = 0.499), and the median progression-free survival time was 7.50 months (95% CI 6.17-8.83 months) and 8.30 months (95% CI 6.99-9.61 months) ( χ2 = 0.71, P = 0.401), and the median overall survival time was 15.50 months (95% CI 11.30-19.69 months) and 15.60 months (95% CI 13.63-17.57 months) ( χ2 = 1.82, P = 0.177). The main adverse reactions in the two groups were leukopenia, thrombocytopenia, hypertension, nausea, vomiting, fatigue, hand-foot syndrome, proteinuria, liver and kidney damage. The incidence rates of ≥grade 3 adverse reactions in the continuous dosing group and the alternate-day dosing group were 42.3% (11/26) and 12.0% (3/25), and the difference was statistically significant ( χ2 = 4.46, P = 0.035). Conclusions:The efficacy of continuous dosing or alternate-day dosing of apatinib combined with SOX regimen as first-line treatment for advanced gastric cancer is similar, but the incidence of ≥grade 3 adverse reactions in alternate-day dosing group is lower, which improves the compliance and tolerance of patients.
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Objective:To evaluate the efficacy and safety of the combination of apatinib and S-1 for treatment of patients with advanced gastric cancer, in order to provide clinical therapy reference for advanced gastric cancer.Methods:Clinical trials were retrieved from China National Knowledge Infrastructure (CNKI) , Chinese Science and Technology Journal Database (CSTJ) , Wanfang Medical Network, VIP Journal Database (VIP) , China Biomedical Literature Database (CBMdisc) , Cochrane Library, PubMed, etc., searched from Jan. 2010 to Oct. 2019. The experimental group were given apatinib combined with S-1, and the control group received S-1 monotherapy. Two sets of RCT in patients with advanced gastric cancer were collected. Researchers first screened literature, data extraction and to assess the risk of bias, then made Meta analysis with RevMan5.3 software, the test level was α=0.05.Results:A total of 12 Meta analysis of randomized RCT were selected from the group, including 561 cases of patients. The results showed that objective response rate (ORR) and disease control rate (DCR) of the experimental group was higher than those of the control group [ (RD=0.16, 95% CI: 0.08-0.23, P<0.0001; RD=0.21,95% CI: 0.14-0.29, P<0.00001) ]; There was no significant difference in nausea and vomiting, hand-foot syndrome, fatigue, diarrhea, thrombocytopenia, neutropenia, leukopenia, neuro-toxicity and mucositis between the two groups. The rate of hypertension, proteinuria, hemoglobin of the experimental group decrease was higher than that of the control group [ (OR=6.21, 95% CI: 1.92-20.13, P=0.002; OR = 10.57,95% CI: 5.06-22.04, P<0.00001; OR=2.84, 95% CI:1.25-6.48, P=0.01) ]; and there was a significant heterogeneity in hypertension among them ( P=0.008, I 2=63) . Conclusion:Compared with S-1 alone, the treatment effect of S-1 combined with targeted drug apatinib can significantly improve ORR and DCR of patients with advanced gastric cancer.
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Objective:To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus (MV) vector on gene expression and virus replication.Methods:The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome (the upstream of the N gene, between P and M genes, between H and L genes), and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N, P, and L proteins, respectively. The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses. S1 proteins expressed by the recombinant viruses were identified by RT-PCR, indirect immunofluorescence assay, Western blot and ELISA. Growth kinetics of the recombinant viruses were analyzed.Results:Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene. Two recombinant viruses, MV-M-S1 and MV-L-S1, were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes, or H and L genes, and could express S1 protein. MV-M-S1 expressed more S1 protein than MV-L-S1, but the titer of MV-M-S1 was lower.Conclusions:Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication. This study provided reference for the subsequent construction of MV vector.
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【Objective】 To compare the clinical efficacy of percutaneous transforaminal endoscopic decompression （PEID） and percutaneous interlaminar endoscopic decompression （PETD） in the treatment of L5-S1 lateral recess stenosis. 【Methods】 We selected the patients in our center diagnosed with L5-S1 lateral recess stenosis from March 2018 to October 2019 and divided them into Group A and Group B according to the principle of prospective， single-blind， and randomized control （A： PETD； B： PEID）. The operation was performed by the same senior surgeon with mature spinal endoscopy technology. We recorded the basic information， operation duration， usage count of C-arm， hospital stay， VAS score and ODI index of lower back and lower limbs before operation and 3 days， 1 month， 1 year and the last follow-up after the operation， and the operative excellent and good rates （the last follow-up）. The angle of bony lateral recess was measured during pre- and postoperative CT. 【Results】 A total of 95 patients （A： n=48； B： n=47） successfully completed the operation and were followed up for at least 1 year. The two groups did not significantly differ in age， gender， hospital stay， or complication by lumbar intervertebral disc herniation， but PEID group had significantly shorter operation duration and fewer usage counts of C-arm （P<0.001）. VAS score of lower back and lower limbs， and ODI index were significantly reduced at 3 days，1 month， 1 year and the last follow-up after the operation， with no significant difference between the two groups at the same time； no statistical difference was found between the two groups in operative excellent and good rates at the last follow-up （P>0.05）. The postoperative bony side recess angle was significantly improved （P<0.05）， while there was no significant difference in either pre- or postoperative bony side recess angle between the two groups （P>0.05）. 【Conclusion】 Both PEID and PETD are effective strategies in the treatment of L5-S1 lateral recess stenosis and can achieve good clinical outcomes.
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The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus (PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3'-end. The PCR product was cloned into the expression vector pET-21b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3 (sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.