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.

Objective : To investigate the role of endoplasmic reticulum stress in the occurrence and development of fatty liver induced by high fat. Methods : In the high-fat Drosophila model, the high-fat group was fed with high-fat medium, while the control group was fed with normal medium; in the mouse fatty liver model, the high-fat group was fed with high-fat diet, and the control group was fed with normal diet; in the HepG2 cell steatosis model, the high-fat group was induced by palmitic acid(PA), and the control group was cultured with DMEM. The fat body size of the third instar larvae of Drosophila melanogaster was photographed. Steatosis in mice liver and HepG2 cells was observed by H&E and Oil Red staining. The expression levels of ATF6, Bip and CHOP in the third instar larvae, liver tissues of mice and HepG2 cells were analyzed by quantitative real-time polymerase chain reaction(qPCR) and Western blot. Results : In Drosophila model, fat body and fat storage were obviously increased in high fat fed flies when compared with control group. The formation of liver fat droplets and cells vacuolation were confirmed by H&E and Oil Red staining in mice livers fed with high fat and HepG2 cells with palmitic acid treatment. The expression levels of ATF6, Bip and CHOP were significantly increased in third instar larvae and mice livers fed with high fat and palmitic acid treated HepG2 cells with palmitic acid treatment. Conclusion High fat may induce the occurrence and development of hepatic steatosis by activating endoplasmic reticulum stress.

Objective : To generate heterozygous TRAF2 knockout mice, the CRISPR/Cas9 technology was successfully employed. These mice were served as a valuable model to explore the pathological mechanisms underlying inflammatory and immune disorders mediated by abnormal TNF-α-TRAF2 signaling and to develop new therapeutic targets. Methods : A vector targeting the knockout of the TRAF2 gene was constructed. Lead RNA and Cas9 Mrna were introduced into the fertilized eggs of C57BL/6JGpt mice through microinjection to mediate the TRAF2 gene mutation in mice. The mouse tail protein was extracted and the genotype of the F0 generation was determined by PCR and Western blot. TRAF2+/- mice were successfully obtained. F0 generation mice were backcrossed with C57BL/6JGpt wild-type mice to obtain stable TRAF2+/- mice for propagation and subsequent experiments. The body weight of TRAF2+/- mice was detected; Western blot was used to detect the expression of TRAF2 in the spleen, liver and kidney tissues of TRAF2+/- mice. The development of spleen, liver and kidney tissues in TRAF2+/- mice was detected by HE staining. Results : PCR identification using specific primers demonstrated that TRAF2+/- mice exhibited a target band at 679 bp. Western blot analysis results indicated that, compared with the WT group, the expression of TRAF2 in the tail protein of TRAF2+/- mice was significantly reduced(P+/- mice had a lower body weight compared to their littermate WT mice(P+/- mice was decreased(P+/- mice and WT mice. Conclusion The successful construction of TRAF2+/- mice has provided an important animal model for exploring the role of TRAF2 in developmental regulation, revealing the mechanism of inflammatory immune diseases mediated by abnormal TNF-α-TRAF2 signaling, and screening related drug targets.

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.