OBJECTIVE To determine the contents of 15 components in Aurantii Fructus Immaturus from different origins （Citrus aurantium， C. junos， C. aurantium Linn.， C. sinensis Osb.， C. sinensis）， screen differential components， and provide references for the quality evaluation of Aurantii Fructus Immaturus. METHODS HPLC method was adopted to determine the contents of synephrine， N-methyltyramine， 5，7-dihydroxychromone-7-neohesperidoside， neoponcirin， narirutin， naringin， hesperidin， neohesperidin， naringenin， hesperetin， sinensetin， nobiletin， tangeretin， 5-demethylnobiletin， and auraptene in 46 batches of Aurantii Fructus Immaturus from different origins. The determination was performed on Waters Symmetry C18 column with mobile phase consisted of acetonitrile-0.1% formic acid （gradient elution） at the flow rate of 1.0 mL/min； column temperature was set at 40 ℃ ， detection wavelength was 284 nm， and sample injection volume was 5 μL. The differences between different origins of Aurantii Fructus Immaturus were analyzed by cluster analysis， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA）， and differential components were screened. RESULTS The linear relationships of the aforementioned 15 components were all good within the tested mass concentration ranges （all r＞0.999 0）. The RSDs for precision， stability （24 h）， and repeatability tests were all less than 2.00%. The average recovery rate ranged from 91.1% to 103.9% （all RSDs＜3.10%）. Cluster analysis， PCA， and OPLS-DA revealed that C. sinensis Osb. and C. sinensis were clustered into one category， while C. aurantium，C. junos and C. aurantium Linn. were clustered into another category. The variable importance projection values for neohesperidin， auraptene， naringin， neoponcirin， tangeretin， hesperidin， sinensetin， and 5，7-dihydroxychromone-7-neohesperidoside were all greater than 1. CONCLUSIONS In this study， the contents of 15 components in Aurantii Fructus Immaturus from different origins are determined， and 8 differential components， including neohesperidin， auraptene， naringin， and neoponcirin， are screened out.

G protein-coupled receptor kinase 2 (GRK2) participates in the phosphorylation and desensitization of G protein-coupled receptor (GPCR), impacting various biological processes such as inflammation and cell proliferation. Dysregulated expression and activity of GRK2 have been reported in multiple cells in rheumatoid arthritis (RA). However, whether and how GRK2 regulates synovial hyperplasia and fibroblast-like synoviocytes (FLSs) proliferation is poorly understood. In this study, we investigated the regulation of GRK2 and its biological function in RA. We found that GRK2 transmembrane activity was increased in FLSs of RA patients and collagen-induced arthritis (CIA) rats. Additionally, we noted a positive correlation between high GRK2 expression on the cell membrane and serological markers associated with RA and CIA. Immunoprecipitation-mass spectrometry and pull-down analyses revealed tumor necrosis factor receptor-associated factor 2 (TRAF2) as a novel substrate of GRK2. Furthermore, surface plasmon resonance (SPR) and molecular docking assays determined that the C-terminus of GRK2 binds to the C-terminus of TRAF2 at the Gln340 residue. GRK2 knockdown and the GRK2 inhibitor CP-25 attenuated synovial hyperplasia and FLS proliferation in CIA both in vitro and in vivo by decreasing GRK2 membrane expression and activity. Mechanistically, increased GRK2 transmembrane activity contributed to the recruitment of TRAF2 on the cell membrane, promoting GRK2-TRAF2 interactions that facilitate the recruitment of the E3 ubiquitin ligase TRIM47 to TRAF2. This enhanced TRAF2 Lys63 polyubiquitylation and induced nuclear factor (NF)-κB activation, leading to synovial hyperplasia and abnormal proliferation of FLSs. Our study provides a mechanistic and preclinical rationale for further evaluation of GRK2 as a therapeutic target for RA.

Objective : To investigate the effect of laminarin(LAM) on nonproliferative diabetes retinopathy by high throughput sequencing(RNA-seq). Methods : The diabetes model was established by intraperitoneal injection of streptozotocin(STZ), and the effect of LAM on diabetic mice was observed.C57BL/6 mice were randomly divided into three groups: Control group, Model group, and LAM group, with 8 mice in each group. After 8 weeks of modeling, the LAM group received a 4-week intraperitoneal injection of LAM treatment. Changes in blood glucose and body weight of the three groups of mice were recorded, HE staining was performed to examine retinal lesions, and RNA-seq was used to identify differentially expressed genes(DEGs) in diabetic retinopathy(DR) under the action of STZ and LAM. Results : STZ successfully established the model of DR, and LAM reduced the blood sugar in diabetic mice to a certain extent and improved the pathological morphology of retinal structural looseness in diabetic mice. After RNA-seq analysis of DEGs, it was found that there were a total of 214 DEGs in the retina of the Model group mice compared to the Control group. Enrichment analysis revealed that DR could exacerbate the lesions through the PI3K Akt signaling pathway. There were a total of 42 DEGs in the retina of the Model group and LAM group mice, and enrichment showed that LAM improved the lesions through the neutrophil extracellular trap pathway. Early growth response factor 1(Egr1), FBJ osteosarcoma oncogene(Fos), nuclear receptor subfamily 4A member 1(Nr4a1), and salt-induced kinase 1(Sik1) were regulated by STZ, and LAM significantly regulated their expression, which might be closely related to LAM′s treatment of diabetic retinopathy. Conclusion DEGs can exacerbate the severity of diabetic retinopathyviathe PI3K-Akt signaling pathway. LAM can mitigate diabetic retinopathyviathe neutrophil extracellular trap pathway. Egr1, Fos, Nr4a1, and Sik1 are key genes involved in LAM treatment of STZ-induced DR.

Establishment of rat models of liver transplantation provides an ideal animal model for resolving the problems of postoperative complications and perioperative treatment of liver transplantation. With in-depth study of the establishment of rat models of liver transplantation, classic "two-cuff" technique has been gradually employed. However, poor surgical field, vascular torsion, biliary tract injury and long anhepatic phase remain unresolved in the process of liver transplantation using traditional techniques. At present, the rat models of liver transplantation at home and abroad are modified mainly from the reconstruction of four vital anatomic structures including the suprahepatic inferior vena cava, portal vein, infrahepatic inferior vena cava and bile duct. Therefore, the latest progress in the reconstruction of the suprahepatic inferior vena cava, portal vein, infrahepatic inferior vena cava and bile duct was reviewed, aiming to provide reference for the establishment of rat models of liver transplantation and promote further development of liver transplantation techniques.

Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

Background and Aims:In recent years,function-preserving proximal gastrectomy with reconstruction has become an important approach for the treatment of early gastric cancer.However,there is no standardized surgical technique,and the short-and long-term outcomes of various new procedures remain unclear.This study was performed to evaluate the safety and short-term efficacy of laparoscopic proximal gastrectomy plus esophagogastrostomy with single-flap technique for early gastric cancer. Methods:The clinical data and follow-up records of 7 patients who underwent laparoscopic proximal gastrectomy with single-flap esophagogastrostomy in the First Affiliated Hospital of Soochow University between December 2021 and December 2022 were retrospectively analyzed.Perioperative safety,postoperative reflux,anastomotic stricture at 6 months,and related nutritional parameters were assessed.The nutrition-related indicators of this group of patients were compared with those of 11 patients who underwent total gastrectomy with Roux-en-Y anastomosis for early gastric cancer during the same period. Results:All 7 patients successfully underwent laparoscopic proximal gastrectomy with single-flap esophagogastrostomy.The average operative time was(212.9±20.6)min,with anastomosis taking(54.7±10.5)min;the mean intraoperative blood loss was(28.6±9.0)mL.No Clavien-Dindo grade Ⅲ or higher complications were observed during hospitalization.None of the patients experienced significant reflux symptoms,although 1 patient developed anastomotic stricture 3 months after operation.There were no statistically significant differences in hemoglobin concentration,albumin level,prealbumin level,total protein concentration,and lymphocyte count between preoperative and 6-month postoperative measurements(all P＞0.05).Compared to patients who underwent total gastrectomy with Roux-en-Y anastomosis,those who had the proximal gastrectomy with single-flap esophagogastrostomy showed a lower percentage decrease in body weight,skeletal muscle area at the third lumbar vertebra(L3),visceral fat area at L3,and hemoglobin concentration at 1 year after operation(all P＜0.05). Conclusion:Laparoscopic proximal gastrectomy with single-flap esophagogastrostomy is a safe and feasible surgical option for early gastric cancer,offering effective anti-reflux outcomes while minimizing the risk of anastomotic stricture.This procedure has a lower impact on postoperative nutritional status compared to total gastrectomy.

Botulinum toxin(BoNT)is currently the first-line method for treating focal dystonia,which causes muscle paralysis by chemical denervation.Recent neuroimaging studies have found that BoNT treatment could alter neuroplasticity in the brain of patients with focal dystonia.However,the specific central nervous system mechanisms have not been fully elucidated.To this end,here we review the neuroimaging studies on BoNT treatment for dystonia from three aspects:functional magnetic resonance imaging,structural magnetic resonance imaging,and positron emission tomography imaging.It suggests that BoNT may improve the symptoms of dystonia patients by affecting functional connectivity,microstructure,and metabolic levels of the cortex,basal ganglia,thalamus,and cerebellum,etc.Therefore,this review will provide a theoretical reference for further exploring the mechanism and developing potential therapeutic targets of dystonia.

Myocardial infarction can lead to permanent loss of cardiomyocytes and depressed pumping efficiency of the heart.Due to the extremely limited self-renewal potential of cardiomyocytes,the capability of cardiac repair after ischemic injury is extremely low.Mesenchymal stem cells,one of the widely sourced,easily isolated,extracted and cultured stem cells,are regarded as an ideal source of stem cells for the treatment of myocardial infarction owing to their beneficial paracrine effects.However,the cardioprotective efficacy of mesenchymal stem cells and their derivatives observed in animal models has not been fully translated into visible clinical benefits for patients with myocardial infarction.Recently,mesenchymal stem cell-based cardiac tissue engineering has been widely used in cardiac regenerative medicine and demonstrated excellent cardiac repair potential in infarcted hearts.This article reviews recent research progress on the application of tissue engineering strategies to improve the cardiac repair efficacy of mesenchymal stem cells and their derivatives,aiming to provide evidences for exploring potential strategies that can realize the efficient clinical benefits of mesenchymal stem cell-treatment.

Objective:To investigate the effect of dexamethasone mixed with ropivacaine for cervical nerve pathway blockade on the postoperative swallowing function in patients undergoing thyroid surgery.Methods:This was a prospective randomized controlled trial. One hundred twenty-four patients, regardless of gender, aged 18-60 yr, of American Society of Anesthesiologists Physical Status classification Ⅰor Ⅱ, who underwent elective thyroidectomy at Xuzhou Cancer Hospital from March to December 2023, were selected and divided into test group (DR group, n=62) and control group (R group, n=62)using a random number table method. After induction of total intravenous anesthesia, bilateral cervical nerve pathway blockade was performed, dexamethasone 5 mg and 0.25% ropivacaine 10 ml were injected in DR group, and 0.25% ropivacaine 10 ml was injected in group R. Main outcome measures: The swallowing function was assessed using Standard Swallowing Assessment scale at 1 and 6 days postoperatively, and the occurrence of swallowing dysfunction (abnormal in any indicator) was recorded. Secondary outcome measures: numerical rating scale scores were recorded immediately after tracheal extubation and at 4, 8, 12, 24 and 48 h after tracheal extubation in both static and swallowing states. Voice function was evaluated using the Voice Handicap Index-10 at 1 and 6 days postoperatively. The use of ketorolac tromethamine and occurrence of adverse reactions after surgery were recorded. Results:Sixty-one patients were finally included in DR group and 60 patients in R group. Compared with R group, the Standard Swallowing Assessment scale scores at 1 and 6 days after operation and incidence of swallowing disorders at 1 day after operation were significantly decreased, the numerical rating scale scores at 8 and 12 h after tracheal extubation in the static state and at 4-24 h after tracheal extubation in the swallowing state were decreased, the incidence of postoperative nausea and vomiting and Voice Handicap Index-10 scores at 1 day after operation were decreased, and the number of patients who used ketorolac tromethamine was reduced in DR group ( P<0.05). Conclusions:Dexamethasone mixed with ropivacaine for cervical nerve pathway blockade can improve the postoperative swallowing function in patients undergoing thyroid surgery.

Objective:To investigate the application value of nectin-4 targeting radiotracer 68Ga-N188 in the diagnosis of pancreatic cancer. Methods:The prospective study was conducted. The clinicopathologic data of 16 patients diagnosed as pancreatic cancer on enhanced computed tomography (CT) who were admitted to the Peking University First Hospital from August to December 2022 were collected. There were 9 males and 7 females, aged (62±8)years. All patients underwent 18F-flurodeoxyglucose ( 18F-FDG) and 68Ga-N188 positron emission tomography (PET)/CT examination. Observation indicators: (1) distribution of 68Ga-N188 in different tissues and tumor primary lesion of patients; (2) expression of Nectin-4 and uptake of 68Ga-N188 in pancreatic cancer; (3) comparison of examination results between 68Ga-N188 and 18F-FDG PET/CT. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the independent sample t test. Count data were described as absolute numbers or percentages. Results:(1) Distribution of 68Ga-N188 in different tissues and tumor primary lesion of patients. Results of PET/CT examination showed that in 1 hour after injection, the maximum standard uptake value (SUVmax) and mean standard uptake value (SUVmean) of 68Ga-N188 in fat, muscle, skin, and brain tissues of 16 patients were 0.40±0.16 and 0.25±0.09, 0.68±0.20 and 0.44±0.12, 0.39±0.14 and 0.28±0.11, 0.09±0.04 and 0.05±0.02, respectively. In the tissues of the esophagus, liver, spleen, and pancreas, the above indicators were 1.53±0.48 and 1.16±0.31, 1.49±0.45 and 0.91±0.30, 1.40±0.30 and 1.02±0.24, 1.24±0.31 and 0.96±0.25, respectively. In tumor primary lesion, the above indicators were 3.28±1.02 and 2.14±0.62, respectively, showing significant differences in SUVmax and SUVmean compared with pancreatic tissue ( t=8.03, 6.75, P<0.05). The tumor background ratio in tumor primary lesion based on SUVmax was 1.82±0.58. (2) Expression of Nectin-4 and uptake of 68Ga-N188 in pancreatic cancer. Results of immunohistochemical staining in 16 patients showed that there were 7 patients with high Nectin-4 expression and 9 patients with low Nectin-4 expression. Results of PET/CT examination showed that the SUVmax of 68Ga-N188 in tumor primary lesion of the 7 patients with high Nectin-4 expression and 9 patients with low Nectin-4 expression were 3.77±1.10 and 2.64±0.68, showing a significant difference between them ( t=2.64, P<0.05). The SUVmax of 18F-FDG in tumor primary lesion of the 7 patients with high Nectin-4 expression and 9 patients with low Nectin-4 expression were 6.73±3.24 and 6.43±3.45, showing no significant difference between them ( t=0.17, P>0.05). (3) Comparison of examination results between 68Ga-N188 and 18F-FDG PET/CT. Of the 16 patients, cases with positive results of tumor primary lesion on 68Ga-N188 and 18F-FDG PET/CT were 14 and 11, respectively, for the 14 pancreatic cancer patients diagnosed by postoperative histopathology. Among them, cases with positive results of tumor primary lesion on 68Ga-N188 and 18F-FDG PET/CT were 3 and 1 for the 3 pancreatic cancer patients receiving evaluation for chemotherapy. The SUVmax of 18F-FDG in tumor primary lesion of the 3 patients with chemotherapy and the 11 patients without chemotherapy were 2.80±0.69 and 6.97±2.11, showing a significant difference between them ( t=3.29, P<0.05). The SUVmax of 68Ga-N188 in tumor primary lesion of the 3 patients with chemotherapy and the 11 patients without chemotherapy were 3.38±1.12 and 2.93±0.50, showing no significant difference between them ( t=0.66, P>0.05). Cases with positive results of lymph node metastases in 68Ga-N188 and 18F-FDG PET/CT were 6 and 4, respectively, for the 6 pancreatic cancer patients diagnosed with lymph node metastases by postoperative histopathology, and the SUVmax of 68Ga-N188 and 18F-FDG in lymph node metastases were 2.25±1.12 and 4.02±1.27. Conclusion:68Ga-N188 PET/CT can be used for imaging diagnosis of tumor primary lesion and lymph node metastases of pancreatic cancer.