AIM: To investigate the effects of PAR-2 agonist peptide on the proliferation and cytosolic calcium concentration ([Ca~(2+)]_c) in human hepatoma cells HepG2. METHODS: Human hepatoma cell line HepG2 was cultured. The cells were treated with PAR-2 agonist peptide SLIGKV-NH_2 and the reverse PAR-2 agonist peptide VKGILS-NH_2, respectively. The [Ca~(2+)]_c of hepatoma cells were measured by microfluorimetric techniques based on calcium indicator fura-2/AM. The influences on proliferation of hepatoma cells were determined by MTT method. The changes of cell cycle were evaluated by flow cytometry, and the changes of cyclin D1 mRNA expression were detected by RT-PCR. RESULTS: After treated with 50 μmol/L SLIGKV-NH_2, a rapid rise of [Ca~(2+)]_c in HepG2 cells was induced (P<0.01), percent S phase, G_2/M phase and proliferation index (PI) of HepG2 cells were elevated (P<0.01), and cyclin D1 mRNA expression was significantly upregulated (P<0.01). The proliferation rates of HepG2 cells treated with 1-50 μmol/L SLIGKV-NH_2 were significantly increased, and the effect was in a dose-dependent manner (P<0.01 or P<0.05). No statistical significance of the difference between VKGILS-NH_2 and control group was observed (P>0.05). CONCLUSION: PAR-2 agonist peptide induces the rise of [Ca~(2+)]_c in HepG2 cells, upregulates the expression of cyclin D1 mRNA, accelerates the progress of cell cycle, promotes the synthesis of DNA and the proliferation of hepatoma cells via activating PAR-2 in vitro.

Objective To establish the method and cardiovascular imaging in mice using ultrasound biomicroscopy(UBM) with anatomical confirmation by magnetic resonance (MR) imaging, and to evaluate the feasibility and value of UBM in mice cardiovascular imaging.Methods The mouse thoracic anatomy was elucidated using Signo 3.0T MR imaging on 2 healthy C57BL/6 adult male mice.The structures and flow of mice heart and great vessels in 16 healthy C57BL/6 adult male mice were showed and analyzed by Visualsonics Vevo 770 UBM through several acoustic windows, including left parasternal, right parasternal, apical regions, and suprasternalis.The systolic and diastolic function of mice heart were also evaluated.ResultsAnatomic characteristic of mice could be displayed by MR imaging.The heart was mostly located in left thoracic cavity.The angle between long axis of the heart and the midline of the chest was about 45 degrees.Left atrium,left ventricle, mitral valve and right ventricle were located on the left side of the midline of the chest.Aortic valve orifice, ascending aorta,right atrium and tricuspid valve were located on the right side of the midline of the chest.Left heart long axis view,left ventricular short axis view,ascending aorta long axis view,long axis view of aortic arch, aortic short axis view, right ventricular inflow tract long axis view, pulmonary artery long axis view and so on were obtained clearly using UBM.The structures of heart and great vessels were displayed and the cardiovascular parameters could be measured accurately,including left atrium,left ventricle,aorta and aortic arch,interventricular septum,right atrium, right ventricle, pulmonary artery, innominate artery, right internal carotid artery and the velocity in each valve orifices from Doppler flow spectrum.No significant difference was found in the measurements between different age groups.Conclusions The cardiovascular structures and hemodynamics could be displayed by UBM in mice when appropriate acoustic windows and sections were selected.This novel imaging protocol provides us a non-invasive way to follow atherosclerosis in genes knockout mice.

Objective To evaluate the relationship between muscle fibrosis and the displacement of the hyoid bone while swallowing among patients with nasopharyngeal carcinoma (NPC) after radiation therapy.Methods Twenty-six NPC patients with dysphagia were recruited.Shear wave elastography was performed to assess the fibrosis of the mylohyoid muscle.The horizontal and vertical displacement of the hyoid bone were measured based on the videofluoroscopic swallowing study,and the severity of dysphagia was determined using the penetration-aspiration scale (PAS).The correlation between the modulus value of the mylohyoid muscle and the horizontal and vertical displacement of the hyoid bone were analyzed,respectively.Receiver-operating characteristic (ROC) curve were constructed to assess the diagnostic ability of modulus value for the severity of dysphagia above grade 5 of PAS.Results For patients whose PAS was higher than grade 5,the modulus value of mylohyoid muscle was negatively correlated with the displacement of hyoid bone,while such correlation was absent for patients whose PAS grading were less than or equal to 5.Sensitivity and specificity by modulus value to detect dysphagia were 80％ and 81.8％,respectively,with the best cut-off value of 14.37 kPa.Conclusion The reduction of hyoid bone displacement can be partially attributed to muscle fibrosis for post-radiation NPC patients with severe dysphagia.The modulus value measured by Shear wave elastography could be used as a supplementary way to monitor the development of dysphagia.

BACKGROUND:The specific mechanism of Gushukang,as a Chinese traditional patent medicine for the treatment of postmenopausal osteoporosis of kidney deficiency and blood stasis,needs further studies. OBJECTIVE:To investigate the effect of Gushukang on serum sex hormones,bone microstructure and estrogen receptor in postmenopausal osteoporosis. METHODS:Firstly,network pharmacological analysis was performed.The active ingredients and action targets of Gushukang and the targets of postmenopausal osteoporosis were obtained respectively.Cytoscape was used to construct the active ingredient-target network.STRING database and Cytoscape were used for protein-protein interaction analysis and screening of core targets.DAVID database was used for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of intersection targets.Then the ovariectomized Sprague-Dawley rats were used in the animal experiment.Gushukang was administered by gavage for 3 months.The serum estrogen level was detected by ELISA,the bone microstructure was detected by microCT,and the protein expression of estrogen receptor α and estrogen receptor β in bone tiusse was detected by western blot. RESULTS AND CONCLUSION:The network pharmacological research results identified 132 active ingredients and 150 targets of Gushukang and 1155 targets of postmenopausal osteoporosis.After intersections with 1155 postmenopausal osteoporosis targets,87 targets of active ingredients of Gushukang against postmenopausal osteoporosis were obtained.By constructing the active ingredient-target network,it was found that the active ingredients at the core were quercetin,kaempferol,luteolin,naringin and isorhamnetin,and the targets at the core were NCOA2,ESR2,AR,F2,ESR1 and PTGS1.The final targets obtained after the protein-protein interaction analysis and screening included MAPK8,ESR1,JUN,R3C1,RELA and FOS,of which ESR1 was the common core target obtained from the two analyses.KEGG enrichment analysis showed estrogen,tumor necrosis factor,apoptosis and other signaling pathways.Therefore,animal experiments focused on the effect of Gushukang on different subtypes of estrogen receptors in the estrogen signaling pathway.The results showed that in the Gushukang group,bone microstructure was significantly improved,serum estrogen level had no significant change,but the protein expression of estrogen receptor α and β in bone tissue was significantly increased.All the findings indicate that the mechanism of Gushukang in the treatment of postmenopausal osteoporosis may be related to its hormone-like effect and the enhancement of estrogen receptor expression.

Objective To evaluate the role of elastic fiber changes in predicting survival outcomes in intermediate-grade lung adenocarcinoma.Methods All pulmonary adenocarcinoma resections conducted between January 2009 and December 2009 were reviewed.Pathologically confirmed adenocarcinomas smaller than 3 cm were included in the present study.All cases were categorized into three elastic fiber patterns (EFP):complete loss as pattern Ⅰ (EFP Ⅰ),partial loss as pattern Ⅱ (EFP Ⅱ),normal and diffusely increase as pattern Ⅲ (EFP Ⅲ).Patients with different EFP were compared.Results One hundred and ninety four patients were included in this study,with 67 (34.5％),70 (36.1％) and 57 (29.4％) cases presenting as EFP Ⅰ,EFP Ⅱ,and EFP Ⅲ,respectively.Lymph nodal metastases occurred in 35.8％ (24/67),40.0％ (28/70),and 10.5％ (6/57) of EFP Ⅰ,EFP Ⅱ and EFP Ⅲ patterns,respectively.The percentage of EFP Ⅰ and Ⅱ increased with increasing tumor size,these patterns occurring in 55.1％ (38/69) of tumors ≤2.0 cm,and 79.2％ (99/125) of tumors 2.1-3.0 cm in sizes,respectively.The overall 5-year overall survival rate was 75.8％,and 67.2％ for EFP Ⅰ,68.6％ for EFP Ⅱ,and 94.7％ for EFP Ⅲ.Conclusion In patients with intermediate-grade lung adenocarcinoma,EFP should be formally recognized as a feature of tumor invasion,and its evaluation can help to recognize tumor invasive and access clinical prognosis.

Vaccination is a main measure for protecting chickens against Marek's disease,while it is not able to suppress the infection,proliferation,transmission,and virulence enhancement on Marek's disease virus.Inhibiting the proliferation of Marek's disease virus in chicken is therefore an im-portant option for enhancing defense effectiveness.In this study,a compound,DUBs-IN-1,was found to inhibit the activity of MDV049,a protease encoded by Marek's disease virus,via screening a protease inhibitor library using MDV049 as target and ubiquitin probe.Molecular docking re-vealed that DUBs-IN-1 can interact with the residues which formed the catalytic pocket of MDV049,blocking the interaction between Ub substrate and the catalytic center of MDV049,then suppress the activity of MDV049 with competitive inhibition.Using the CPE model,it was found that DUBs-IN-1 at the concentration of 0.35 and 0.70 μmol/L significantly inhibited the CPE in-duced by Marek's disease virus in CEF cells.Quantitative analysis revealed that DUBs-IN-1 inhibi-ted the proliferation of Marek's disease virus in CEF cells(P＜0.01).Furthermore,it was found that the administration of 80 and 150 pg/(kg·d)of DUBs-IN-1 in chicken infected by Marek's disease virus significantly inhibited the proliferation of MDV in T cells(P＜0.01).In summary,this study demonstrated that the compound DUBs-IN-1 is able to inhibit the proliferation of Marek's disease virus in chicken cells,laying a theoretical and practical foundation for further de-velopment of the drugs against Marek's disease virus.

Objective:To explore the risk factors of recurrence after resection of thigh soft tissue malignant tumors and the evaluation of the postoperative lower limb function.Methods:A total of 211 patients who underwent thigh soft tissue malignant tumor resection in the Department of Orthopaedics of the second affiliated Hospital of Medical College of Zhejiang University from May 2011 to May 2021 were retrospectively analyzed, including 117 males and 94 females, aged 53 (43, 65) years. There were 59 cases of atypical/well-differentiated liposarcoma, 30 cases of other types of liposarcoma, 33 cases of malignant fibrous histiocytoma, 19 cases of fibrosarcoma, 12 cases of rhabdomyosarcoma, 12 cases of leiomyosarcoma, 9 cases of synovial sarcoma, and 37 cases of others. The tumor involvement of muscle, bone, blood vessel, nerve tissue and intermuscular compartment, postoperative Musculoskeletal Tumor Society (MSTS) functional score and recurrence were recorded. The relationship between anatomical structure and postoperative recurrence was analyzed by Cox logistic regression to determine risk factors; the cumulative recurrence rate of different compartment groups (medial compartment, anterior compartment, and posterior compartment) were compared; The Box plot was used to compare the postoperative function of patients with resection of different anatomical structures (e.g., vastus lateralis, biceps femoris, femur, etc.).Results:A total of 34 out of 211 patients relapsed, with a recurrence rate of 16.1%. The recurrence time ranged from 2.6 months to 91.6 months after operation, with a median recurrence time of 37.0 (18.2, 52.8) months. Three independent risk factors were found to be associated with recurrence, namely: pathological grade [ HR=3.86, 95% CI(1.75, 8.51)], involvement of vastus intermedius [ HR=3.05, 95% CI(1.53, 6.06)], and involvement of vastus medialis [ HR=3.17, 95% CI(1.56, 6.41)]. The recurrence rate of patients with anterior chamber tumor resection was 35.3%, which was higher than that of patients without tumor resection (16.2%), and the difference was statistically significant ( P=0.020). There was no significant difference in recurrence rate between patients with medial chamber tumor resection and patients without tumor resection ( P>0.05). The recurrence rate of patients with posterior compartment tumor resection was 12.3%, which was lower than that of unresectable patients (37.6%), and the difference was statistically significant ( P=0.002).The postoperative MSTS score of 167 patients averaged 26±3.2 points (9-30 points). After intraoperative resection of part of the femur, vastus intermedius, vastus medialis, and rectus femoris, the patient's function was relatively poor [corresponding median MSTS score: 25 (23, 28), 26 (24 28), 26 (24,28), 26(24, 27)]. Conclusion:The risk factors for local recurrence after resection of thigh soft tissue malignant tumors include: pathological grade of the tumor, and whether the vastus intermedius or vastus medialis is involved. Anterior compartment tumors have a higher recurrence rate after surgery. If the tumor involves the above-mentioned anatomical structures, more attention should be paid to the risk of local recurrence after surgery.