A quantitative method for the analysis of the multi-component contents in Marsdenia tenacissimae was established, and the quality differences were evaluated by principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), factor analysis (FA) and weighted technique for order preference by similarity to ideal solution (TOPSIS) method. The contents of chlorogenic acid, cryptochlorogenic acid, sinapic acid, tenacigenoside A, tenacissoside G, tenacissoside I, tenacissoside H, drevogenin A, betulinic acid and lupeol were determined by HPLC wavelength switching method. At the same time, the contents of alcohol-soluble extract and total ash were detected. PCA, OPLS-DA and FA methods were used to identify the origin of M. tenacissimae from different producing areas. According to the OPLS-DA model, the index weight was determined to construct the weighted TOPSIS evaluation model. The qualities of M. tenacissimae from different producing areas were analyzed by model scoring results. The contents of 12 indexes in 18 batches of M. tenacissimae varied to different degrees, and the repeatability and accuracy of the test method were satisfactory. PCA analysis divided 18 batches of M. tenacissimae into three categories. OPLS-DA identified five main potential quality markers, including tenacissoside A, tenacissoside I, lupeol, tenacissoside H and chlorogenic acid. The evaluation results of FA and weighted TOPSIS method were consistent, which showed that the quality of M. tenacissimae from Yunnan and Guizhou was better. The established multi-component quantitative analysis method is accurate and reliable, the chemometrics model has strong predictive ability, and the evaluation results of FA and weighted TOPSIS method are scientific and objective. The combination of the four methods can clearly determine the qualities of M. tenacissimae from different producing areas.

BACKGROUND:Due to the complex movement of the scapula,which is a six-degree-of-freedom activity in three-dimensional space,it is difficult to measure it accurately using traditional methods.The image and model matching technology based on dual-plane X-ray is a three-dimensional measurement method that has gradually developed and matured in recent years.Two high-speed cameras are used to project and shoot from orthogonal directions.Compared with a single perspective,this method has advantages in observation range and reduction of out-of-plane errors,and is suitable for the study of scapula kinematics.OBJECTIVE:X-ray biplane and image-model registration technology were used to explore the differences in scapular kinematics between normal individuals and patients with rotator cuff tears,providing a basis for the treatment and rehabilitation of rotator cuff tear patients.METHODS:From April 2023 to January 2024,10 patients with normal shoulders and 10 patients with rotator cuff tears who met the inclusion criteria were enrolled from Guanghua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine.The subjects underwent a shoulder CT scan to create a 3D model and a local scapular coordinate system.X-ray biplane images were taken during shoulder abduction with two C-arm machines.The 2D and 3D images were registered to compare scapular kinematic differences between the two groups at 0°,15°,30°,45°,60°,75°,and 90° of abduction,including scapular rotation angle and displacement distance.RESULTS AND CONCLUSION:(1)During shoulder abduction,the scapula of both groups showed upward rotation,but the upward rotation of the rotator cuff tear group was greater than that of the control group,and the difference was significant when the abduction was 30°-90°(P＜0.01).At the same time,the scapula internal rotation of both groups gradually increased,but the rotator cuff tear group was significantly greater than the control group when the abduction was 45°-90°(P＜0.01).In addition,the anteroposterior tilt of the scapula of the two groups was significantly different when the abduction was 15°-90°(P＜0.01).The scapula posterior tilt of the control group gradually increased during abduction,while the scapula of the rotator cuff tear group tilted forward except for a slight posterior tilt at 15°-30° abduction.(2)In terms of displacement,the upward displacement of the rotator cuff tear group was less than that of the control group during abduction,and the difference was significant at 15°-90°(P＜0.05),but there was no significant difference in lateral and anterior-posterior displacement between the two groups(P＞0.05).(3)Rotator cuff tear can cause scapular dyskinesis,characterized by increased upward rotation,internal rotation,and abnormal forward tilt during shoulder abduction.Identifying and addressing scapular dyskinesis is crucial for treating rotator cuff tear.

BACKGROUND:Due to the complex movement of the scapula,which is a six-degree-of-freedom activity in three-dimensional space,it is difficult to measure it accurately using traditional methods.The image and model matching technology based on dual-plane X-ray is a three-dimensional measurement method that has gradually developed and matured in recent years.Two high-speed cameras are used to project and shoot from orthogonal directions.Compared with a single perspective,this method has advantages in observation range and reduction of out-of-plane errors,and is suitable for the study of scapula kinematics.OBJECTIVE:X-ray biplane and image-model registration technology were used to explore the differences in scapular kinematics between normal individuals and patients with rotator cuff tears,providing a basis for the treatment and rehabilitation of rotator cuff tear patients.METHODS:From April 2023 to January 2024,10 patients with normal shoulders and 10 patients with rotator cuff tears who met the inclusion criteria were enrolled from Guanghua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine.The subjects underwent a shoulder CT scan to create a 3D model and a local scapular coordinate system.X-ray biplane images were taken during shoulder abduction with two C-arm machines.The 2D and 3D images were registered to compare scapular kinematic differences between the two groups at 0°,15°,30°,45°,60°,75°,and 90° of abduction,including scapular rotation angle and displacement distance.RESULTS AND CONCLUSION:(1)During shoulder abduction,the scapula of both groups showed upward rotation,but the upward rotation of the rotator cuff tear group was greater than that of the control group,and the difference was significant when the abduction was 30°-90°(P＜0.01).At the same time,the scapula internal rotation of both groups gradually increased,but the rotator cuff tear group was significantly greater than the control group when the abduction was 45°-90°(P＜0.01).In addition,the anteroposterior tilt of the scapula of the two groups was significantly different when the abduction was 15°-90°(P＜0.01).The scapula posterior tilt of the control group gradually increased during abduction,while the scapula of the rotator cuff tear group tilted forward except for a slight posterior tilt at 15°-30° abduction.(2)In terms of displacement,the upward displacement of the rotator cuff tear group was less than that of the control group during abduction,and the difference was significant at 15°-90°(P＜0.05),but there was no significant difference in lateral and anterior-posterior displacement between the two groups(P＞0.05).(3)Rotator cuff tear can cause scapular dyskinesis,characterized by increased upward rotation,internal rotation,and abnormal forward tilt during shoulder abduction.Identifying and addressing scapular dyskinesis is crucial for treating rotator cuff tear.

Objective:To evaluate the relationship between the mechanism of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) in preventing brain neurotoxicity caused by multiple sevoflurane anesthesia and peroxisome proliferator-activated receptor gamma (PPARγ)/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) signaling pathway in neonatal mice.Methods:This study was performed in 2 parts. Part Ⅰ Using a random number table method, 6 C57BL/6 pregnant mice were assigned to receive a standard diet, 3 pregnant mice were assigned to receive a diet supplemented with fish oil from day 2 of gestation to day 14 after parturition (ω-3 PUFAs 300 mg were added to every 20 g of conventional diet). Healthy C57BL/6 mice of both sexes, aged 6 days, weighing 3-5 g, were selected after parturition. Seventeen neonatal pups from 3 pregnant mice that were fed a conventional diet were assigned to control group (C group), 17 neonatal pups from 3 pregnant mice that were fed a conventional diet were assigned to sevoflurane group (S group), and 17 neonatal pups from pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus sevoflurane group (PS1 group) using the random number table method. Part Ⅱ Four C57BL/6 pregnant mice were assigned to receive a diet supplemented with fish oil from day 2 of gestation to day 14 after parturition. After parturition, 12 neonatal pups from 2 pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus sevoflurane group (PS2 group), and 12 neonatal pups from 2 pregnant mice that were fed a diet supplemented with fish oil were assigned to ω-3 PUFAs plus PPARγ inhibitor GW9662 plus sevoflurane group (PGS group) using a random number table method. GW9662 (2 mg/kg) was intraperitoneally injected at 30 min before exposure to sevoflurane in PGS group. All offspring mice were breastfed until 21 days of age, after which they were housed separately from the mother and allowed ad libitum access to a conventional diet. S, PS1, PS2 and PGS groups inhaled 3% sevoflurane in 40% oxygen for 2 h daily on postnatal days 6, 7 and 8. C group inhaled only 40% oxygen instead. Y maze test was performed on days 33 after birth. The rotarod test was performed on day 35 after birth. After the behavioral testing, the expression of PPARγ, PGC1α, mitofusin-1 (MFN1), MFN2, dynamin-related protein 1 (DRP1), interleukin-6 (IL-6), interleukin-1 β (IL-1 β), and tumor necrosis factor-α (TNF-α) was detected by Western blot, the ultrastructure of mitochondria in hippocampal neurons was observed with a transmission electron microscope, and the mitochondrial membrane potential (MMP), level of reactive oxygen species (ROS) and content of ATP were determined.Results:Part Ⅰ Compared with C group, the time of stay at the new-arm and time spent on the rotarod were significantly shortened, the percentage of movement distance in the new-arm was decreased, the expression of PPARγ, PGC1α, MFN1 and MFN2 in the hippocampus was down-regulated, the expression of DRP1, IL-6, IL-1β and TNF-α in the hippocampus was up-regulated, the MMP and content of ATP were decreased, and the level of ROS was increased in S group ( P<0.05). Compared with S group, the time of stay at the new-arm and time spent on the rotarod were significantly prolonged, the percentage of movement distance in the new-arm was increased, the expression of PPARγ, PGC1α, MFN1 and MFN2 in the hippocampus was up-regulated, the expression of DRP1, IL-6, IL-1β and TNF-α in the hippocampus was down-regulated, the MMP and content of ATP were increased, and the level of ROS was decreased in PS1 group ( P<0.05). Part Ⅱ Compared with PS2 group, the time of stay at the new-arm and time spent on the rotarod were significantly shortened, the percentage of movement distance in the new-arm was decreased, the MMP and content of ATP were decreased, the level of ROS was increased, and the expression of IL-6, IL-1β and TNF-α was up-regulated ( P<0.05). Conclusions:The mechanism by which ω-3 PUFAs prevent brain neurotoxicity caused by multiple sevoflurane anesthesia is related to the activation of the PPARγ/PGC1α signaling pathway and alleviation of mitochondrial dysfunction and neuroinflammation in neonatal mice.

Objective:To evaluate the role of docosahexaenoic acid (DHA) in long-term cognitive impairment after multiple sevoflurane anesthesia in newborn mice.Methods:Clean-grade healthy male C57BL/6 mice, aged 6 days, were used in this study. Ten mice were divided into 2 groups ( n=5 each) by the random number table method: control group (group C) and sevoflurane group (group S). The animals inhaled 3% sevoflurane for 2 h at 6, 7 and 8 days after birth. The DHA content was detected by ultra-high performance liquid chromatography-mass spectrometry at 9 days of age. Fifty-two mice were selected and divided into 4 groups ( n=13 each) by a random number table method: control+ normal saline group (group C+ S), sevoflurane anesthesia + normal saline group (group S+ S), control+ DHA group (group C+ D), and sevoflurane anesthesia+ DHA group (group S+ D). The sevoflurane anesthesia method was the same as the one mentioned above. DHA 50 mg/kg was administered by intragastric gavage from postnatal days 6-19 (at 6, 7 and 8 days after birth, 2 h before anesthesia) in C+ D and S+ D groups. The equal volume of normal saline was given instead in C+ S group and S+ S group. The novel object recognition test was conducted at 37 days of age, and the Morris water maze test was performed at 42 days of age. The corpus callosum and hippocampal tissues were isolated at 47 days of age for examination of the ultrastructure of myelin (with a transmission electron microscope) and for determination of the expression of myelin basic protein (MBP) in hippocampal tissues (by Western blot). The G-ratio was calculated. Results:Compared with group C, the content of DHA in hippocampal tissues was significantly decreased in group S ( P<0.05). Compared with group C+ S, the discrimination index was significantly decreased, the percentage of duration of staying at the target platform quadrant and the number of crossing the original platform were decreased, the expression of MBP was down-regulated, and the G-ratio in the original platform and hippocampus was increased in S+ S group ( P<0.05). Compared with group S+ S, the discrimination index was significantly increased, the percentage of duration of staying at the target platform quadrant and the number of crossing the original platform were increased, the expression of MBP was up-regulated, and the G-ratio in the original platform and hippocampus was decreased in S+ D group ( P<0.05). Conclusions:The mechanism of long-term cognitive impairment following multiple sevoflurane anesthesia may be related to a decrease in the content of DHA, which subsequently leads to myelin structural damage in neonatal mice.

Objective:To evaluate the relationship between preoperative gut microbiota and post-operative ventilator-associated pneumonia (VAP) in patients undergoing coronary artery bypass grafting.Methods:This was a secondary analysis of a previous research project study. Patients who received invasive mechanical ventilation treatment after elective off-pump coronary artery bypass grafting at the Second Hospital of Hebei Medical University from April to September 2023 were selected and divided into VAP group and non-VAP group based on whether VAP occurred after surgery. Fecal samples were collected from patients before surgery, and 16S rRNA gene sequencing was used to analyze the characteristics of preoperative gut microbiota in the two groups. The differences in the diversity of gut microbiota between the two groups were compared. The linear discriminant analysis was used to identify the gut microbiota with significant differences between groups (differential bacteria), and logistic regression analysis was conducted to assess the association between differential bacteria and VAP. Receiver operating characteristic curves were drawn to analyze the predictive value of the differential bacteria for VAP.Results:A total of 79 patients were finally included, with 25 in VAP group and 54 in non-VAP group. The Beta diversity analysis showed statistically significant differences between VAP group and non-VAP group (pseudo- F=2.00, P=0.002). The linear discriminant analysis indicated that Bifidobacterium, Blautia and Megamonas were enriched in non-VAP group, while Klebsiella was enriched in VAP group. Multivariate logistic regression analysis showed that the relative abundance of Bifidobacterium was a protective factor for postoperative VAP ( OR=0.32, 95% confidence interval [ CI] 0.15-0.71, P=0.005), and the relative abundance of Klebsiella was a risk factor for postoperative VAP ( OR=2.49, 95% CI 1.143-5.43, P=0.022). The area under the receiver operating characteristic curve of the relative abundance of Bifidobacterium for predicting VAP was 0.80 (95% CI 0.69-0.90, P<0.001) and of the relative abundance of Klebsiella was 0.70 (95% CI 0.57-0.83, P=0.005). Conclusions:Bifidobacterium is a protective factor, while Klebsiella is a risk factor for postoperative VAP in patients undergoing coronary artery bypass grafting, and the relative abundance of both bacteria has a certain predictive value for VAP.

This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Glucosidases are an indispensable class of enzymes in the sugar metabolism of organisms. To investigate the biological functions and expression patterns of α-glucosidases (AGLUs) and β-glucosidases (BGLUs), we identified the two family members in the genome of melon (Cucumis melo). The number, location on chromosomes, gene structure, subcellular localization, conserved motifs, and phylogenetic relationship of the two family members were analyzed. Based on the cis-acting elements in the promoter region and protein interaction models, their functions were preliminarily predicted. Furthermore, the gene expression of the two family members was determined by qRT-PCR. The results showed that the melon genome contained five AGLU family members on five chromosomes, and all of the five members were located in the extracellular matrix, with the amino acid sequence lengths ranging from 899 aa to 1 060 aa. The melon genome carried 18 BGLU family members on 8 chromosomes, and all the members were located in the cell membrane or cytoplasm, with the amino acid lengths ranging from 151 aa to 576 aa. The qRT-PCR results showed that the expression of about 50% of the genes was down-regulated upon cold stress. CmAGLU5 and CmBGLU7 may be key members of the two families, respectively, in response to cold stress. The expression of all members of the two families was up-regulated under abscisic acid (ABA), high salt, and drought stress. In the AGLU family, CmAGLU3 was the key gene in response to ABA and high salt stress, while CmAGLU4 was the key gene in response to drought stress. In the BGLU family, CmBGLU18 was the key gene in response to ABA, while CmBGLU6 was the key gene in response to high salt and drought stress.
beta-Glucosidase/metabolism* ; Phylogeny ; alpha-Glucosidases/metabolism* ; Gene Expression Regulation, Plant ; Cucurbitaceae/enzymology* ; Multigene Family ; Cucumis melo/enzymology* ; Stress, Physiological

Objective:To evaluate the role of fibrinogen in perioperative neurocognitive disorder (PND) in aged mice.Methods:Sixty SPF healthy male C57BL/6J mice, aged 16-18 months, weighing 25-30 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), PND group (group P), urokinase group (group U) and PND+ urokinase group (group PU). Abdominal surgery was performed under 3% sevoflurane anesthesia to establish the mouse model of PND. In PU group, urokinase 20 000 U/kg was intraperitoneally administered at 1 h after surgery, once a day, for 5 consecutive days. In group U, urokinase was intraperitoneally injected once a day for 5 consecutive days without anesthesia and surgery. The cognitive function was assessed after operation using the novel object recognition test (discrimination index) and the Morris water maze test (frequency of crossing the original platform and percentage of the time spent in the target quadrant). The expression of occludin, claudin-5, fibrinogen and ionized calcium-binding adapter molecule 1 (Iba-1) and CD11b in hippocampal tissues was detected using Western blot, the area of fibrinogen extravascular deposits was measured and the morphology of microglia was observed using the immunofluorescence staining, and the mRNA expression of pro-inflammatory factors (interleukin-1beta, tumor necrosis factor-alpha, and inducible nitric oxide synthase), anti-inflammatory factors (interleukin-4 and arginase-1), and chemokines (chemokine 2 and chemokine ligand 10) in hippocampal tissues was detected by quantitative real-time polymerase chain reaction after surgery. Results:Compared with group C, the parameters of cognitive function were significantly decreased, the expression of occludin and claudin-5 was down-regulated, the expression of fibrinogen was up-regulated, the area of fibrinogen extravascular deposits was increased, the number of branches was decreased and the average process length was shortened in the microglia around fibrinogen deposits, the expression of Iba-1 and CD11b was up-regulated, the mRNA expression of proinflammatory cytokines and chemokines was up-regulated, and the mRNA expression of the anti-inflammatory factors was down-regulated in group PND ( P<0.05). Compared with group PND, the parameters of cognitive function were significantly increased, the expression of occludin and claudin-5 was up-regulated, the expression of fibrinogen was down-regulated, the area of fibrinogen extravascular deposits was decreased, the number of branches was increased and the average process length was prolonged in the microglia around fibrinogen deposits, the expression of Iba-1 and CD11b was down-regulated, the mRNA expression of proinflammatory cytokines and chemokines was down-regulated, and the mRNA expression of the anti-inflammatory factors was up-regulated in group PU ( P<0.05). Conclusions:Fibrinogen deposits in the brain parenchyma through the damaged blood-brain barrier after anesthesia and surgery and participates in the development of PND, and the underlying mechanism may be related to the promotion of microglial activation and the induction of neuroinflammation in aged mice.