ObjectiveThis study aimed to investigate the intervention effect of Renqing Mangjue on the malignant transformation of gastric mucosal epithelial cells induced by N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） and to explore its molecular mechanism in preventing precancerous lesions of gastric cancer based on the cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）/mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodsHuman gastric mucosal epithelial cells （GES-1） were initially induced by MNNG to establish a precancerous cell model （MC cells）. The effective concentration of MNNG for inducing malignant transformation in GES-1 cells was screened using the cell proliferation activity decection （CCK-8） assay， and the effective concentration of Renqing Mangjue for inhibiting the proliferation of transformed GES-1 cells was also determined. GES-1 cells were divided into a blank control group， a model group， and treatment groups with Renqing Mangjue at concentrations of 1， 3， 10， and 30 mg·L^-1. Furthermore， the effects of Renqing Mangjue on the migratory ability and epithelial-mesenchymal transition （EMT） characteristics of GES-1 malignant transformed cells were evaluated using Transwell migration assays， wound healing assays， and real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR）. Additionally， candidate chemical components and target sites of Renqing Mangjue were obtained from the TCMIP v2.0 database， and disease targets at various stages of gastric cancer precursors were sourced from the Gene Expression Omnibus （GEO） database. Pathway enrichment analysis was performed using the Metascape database to predict the potential mechanisms of action of Renqing Mangjue. Finally， the protective mechanism of Renqing Mangjue against gastric cancer precursors was validated through Western blot analysis. ResultsAt a concentration of 20 μmol·L^-1， MNNG exhibited an inhibition rate of approximately 50% on GES-1 cells （P<0.01）， and at this concentration， the GES-1 cells displayed biological characteristics indicative of malignant transformation. In contrast， Renqing Mangjue had no significant effect on the proliferation of normal GES-1 cells， but significantly inhibited the proliferation of MC cells （P<0.01） and markedly reduced their migratory capacity （P<0.01）. Moreover， it also increased the mRNA expression level of E-cadherin during the EMT process （P<0.05）， while inhibiting the expression of both N-cadherin and the transcription factor Snail mRNA （P<0.05， P<0.01）. Network predictions suggested that Renqing Mangjue may prevent gastric cancer precursors through modulating the cGMP/PKG and MAPK/ERK signaling pathways. Furthermore， Western blot results indicated that Renqing Mangjue upregulated the expression of PKG and NPRB （B-type natriuretic peptide receptor） proteins in the cGMP/PKG pathway （P<0.01）， while downregulating the expression of the downstream proteins MEK and ERK （P<0.05， P<0.01）. ConclusionIn summary， Renqing Mangjue can prevent gastric cancer precursors by inhibiting the proliferation and migration of malignant transformed GES-1 cells， thereby delaying the EMT process. The underlying mechanisms may be related to the activation of the cGMP/PKG pathway and the inhibition of the MEK/ERK signaling pathway.

ObjectiveTo explore the mechanism of Jiawei Wendantang in preventing and treating diabetic atherosclerosis by observing the effect of this prescription on the nuclear factor-κB / NOD-like receptor protein 3（NF-κB/NLRP3） pathway and related inflammatory cytokines in rat model of diabetic atherosclerosis. MethodFifty-four SPF-grade rats were randomized into blank， model， atorvastatin （0.9 mg·kg^-1·d^-1）， and high-， medium-， low-dose （18.2， 9.1， 4.55 g·kg^-1·d^-1， respectively） Jiawei Wendantang groups. The rats in other groups except the blank group were modeled for diabetic atherosclerosis by intraperitoneal injection of streptozotocin and feeding with a high-sugar high-fat diet， and those in the blank group were injected with an equal dose of citric acid buffer and fed with a regular diet. The drug administration lasted for 4 weeks， and the blood glucose level in the tail vein was measured every 6 days. After the last administration， the rats were anesthetized for sample collection. Enzyme-linked immunosorbent assay was employed to measure the serum levels of interleukin-18 （IL-18）， C-reactive protein （CRP）， tumor necrosis factor-α （TNF-α）， and intercellular adhesion molecule-1 （ICAM-1）. Western blot was employed to determine the relative protein levels of NF-κB p65， NLRP3， and ICAM-1 in the abdominal aorta. Real-time quantitative polymerase chain reaction was employed to determine the mRNA levels of NLRP3 and interleukin-1β （IL-1β） in the abdominal aorta. The pathological changes in the thoracic aorta were observed by hematoxylin-eosin staining. ResultCompared with the blank group， the model group showed elevated levels of IL-18， CRP， TNF-α， and ICAM-1 in the serum and blood glucose （P<0.05， P<0.01）， up-regulated protein levels of NF-κB p65， NLRP3， and ICAM-1 （P<0.01）， and up-regulated mRNA levels of NLRP3 and IL-1β （P<0.05）. Compared with model group， Jiawei Wendantang lowered the levels of IL-18， CRP， TNF-α， ICAM-1 and blood glucose （P<0.05， P<0.01）， down-regulated the protein levels of NF-κB p65， NLRP3， and ICAM-1 （P<0.01）， and down-regulated the mRNA levels of NLRP3 and IL-1β （P<0.05， P<0.01）. Moreover， Jiawei Wendantang alleviated the pathological injuries in the thoracic aorta. ConclusionJiawei Wendantang may modulate the NF-κB/NLRP3 signaling pathway to reduce the release and adhesion of inflammatory cytokines and regulate the blood glucose level to treat diabetic atherosclerosis.

OBJECTIVES: Laryngeal cancer (LC) is a globally prevalent and highly lethal tumor. Despite extensive efforts, the underlying mechanisms of LC remain inadequately understood. This study aims to conduct an innovative bioinformatic analysis to identify hub genes that could potentially serve as biomarkers or therapeutic targets in LC. METHODS: We acquired a dataset consisting of 117 LC patient samples, 16 746 LC gene RNA sequencing data points, and 9 clinical features from the Cancer Genome Atlas (TCGA) database in the United States. We employed weighted gene co-expression network analysis (WGCNA) to construct multiple co-expression gene modules. Subsequently, we assessed the correlations between these co-expression modules and clinical features to validate their associations. We also explored the interplay between modules to identify pivotal genes within disease pathways. Finally, we used the Kaplan-Meier plotter to validate the correlation between enriched genes and LC prognosis. RESULTS: WGCNA analysis led to the creation of a total of 16 co-expression gene modules related to LC. Four of these modules (designated as the yellow, magenta, black, and brown modules) exhibited significant correlations with 3 clinical features: The age of initial pathological diagnosis, cancer status, and pathological N stage. Specifically, the yellow and magenta gene modules displayed negative correlations with the age of pathological diagnosis (r=-0.23, P<0.05; r=-0.33, P<0.05), while the black and brown gene modules demonstrated negative associations with cancer status (r=-0.39, P<0.05; r=-0.50, P<0.05). The brown gene module displayed a positive correlation with pathological N stage. Gene Ontology (GO) enrichment analysis identified 77 items, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 30 related signaling pathways, including the calcium signaling pathway, cytokine-cytokine receptor interaction, neuro active ligand-receptor interaction, and regulation of lipolysis in adipocytes, etc. Consequently, central genes within these modules that were significantly linked to the overall survival rate of LC patients were identified. Central genes included CHRNB4, FOXL2, KCNG1, LOC440173, ADAMTS15, BMP2, FAP, and KIAA1644. CONCLUSIONS This study, utilizing WGCNA and subsequent validation, pinpointed 8 genes with potential as gene biomarkers for LC. These findings offer valuable references for the clinical diagnosis, prognosis, and treatment of LC.
Humans ; Laryngeal Neoplasms/genetics* ; Rosaniline Dyes ; Biomarkers ; Adipocytes ; Gene Regulatory Networks ; Gene Expression Profiling

ObjectiveTo investigate the intervention effect of Qufeng Gutong Babu ointment （QFGT） on rats with osteoarthritis （OA） with cold-dampness obstruction， and preliminarily clarify its mechanism. MethodSD male rats were divided into 6 groups， namely， the blank group， model group， positive control drug Huoxue Zhitong ointment （HXZTG） group （1.26 cm²·d^-1）， and low， medium， and high-dose QFGT group （75， 150， 300 mg·d^-1）. OA model was prepared by joint cavity injection of papain and L-cysteine. On the second day of modeling， climate factors were applied to establish an animal model of combination of disease and syndrome of OA rats with cold-dampness obstruction. Standard VonFrey fiber was used to evaluate the threshold of mechanical pain. Weight bearing difference score and joint function score of both hind limbs were recorded. Hematoxylin-eosin （HE） staining and safranine fixation green staining were used to observe the pathological changes and cartilage degeneration of rat knee joint. Immunohistochemistry （IHC） was used to detect the expression of interleukin-1β （IL-1β）， interleukin-8 （IL-8）， tumor necrosis factor-α （TNF-α）， matrix metalloproteinase-9 （MMP-9）， and cathepsin K （CTSK）. Western blot was used to detect the protein expression of kinase B （Akt）， phosphorylated protein kinase B （p-Akt）， phosphatidylinositol 3-kinase （PI3K）， nuclear factor 1 （NFATc1）， MMP-9， and CTSK in T cells. ResultCompared with the normal group， the model group showed significant mechanical pain sensitivity reaction after modeling （P<0.01）， and the weight bearing difference of both hind limbs and joint function score were significantly increased （P<0.05， P<0.01）. Compared with the model group， both the high-dose QFGT group and the HXZTG group significantly reduced the mechanical pain sensitivity， weight difference， and joint function score of rats （P<0.05， P<0.01）， and the medium-dose QFGT group also improved the joint function to a certain extent， and the degeneration of the knee joint cartilage of rats was significantly reduced （P<0.05， P<0.01）. QFGT and HXZTG both inhibited the protein expression of IL-1β， IL-8， TNF-α， MMP-9， CTAK， PI3K， p-Akt， Akt， and other related proteins in articular cartilage of rats with OA to a certain extent （P<0.05， P<0.01）. ConclusionQFGT can inhibit the release of inflammatory factors and matrix metalloproteinases by inhibiting the PI3K/Akt signal pathway in articular articular cartilage of rats with OA with cold-dampness obstruction， thus ultimately weakening local cartilage degeneration and improving joint function.

Direct acid hydrolysis of Dioscorea zingiberensis rhizomes for preparation of diosgenin is wildly used in the traditional industry， which uses a large amount of inorganic acid catalysts， with high wastewater discharge and serious environmental pollution. Therefore， exploring clean and efficient preparation methods and processes has become an inevitable choice to realize the sustainable development of industrial production of diosgenin. Herein， the author reviewed and analyzed the research progress and problems of enzymatic hydrolysis， microbial transformation and modified acid hydrolysis in the preparation of diosgenin from D. zingiberensis rhizomes during the last ten years， and their application prospects are analyzed. Enzymatic hydrolysis has mild reaction conditions， but the yield of diosgenin is low， the economic cost is high， and the purification process of active enzyme is complicated. Microorganism shows specific activity to the substrate and high efficiency for diosgenin production， and microbial transformation is clean and environmentally friendly， but microbial transformation is time-consuming and the metabolic intermediates are complicated. For the modified acid hydrolysis， two-phase acid hydrolysis can reduce the amount of acid catalyst， and sulfonic acid-functionalized ionic liquid displays good recyclable performance by replacing the traditional inorganic acid， however， the wastewater discharge should still be considered. Solid acid catalysts are non-corrosive and easy to be recycled， but the need to use ethanol as the reaction solvent has certain safety hazards， and the catalyst preparation process is cumbersome. In conclusion， exploring clean and efficient conversion methods is an important research trend for preparation of diosgenin from D. zingiberensis rhizomes. For the enzymatic hydrolysis， the key glycoside hydrolases in the bioconversion process should be explored in depth， the conversion pathway of enzymatic saponins and enzyme specificity should be fully elucidated， and efforts should be made to improve the efficiency of enzymatic hydrolysis. For the microbial transformation， we should accelerate its industrial application process based on selecting and breeding efficient transformation strains， and optimizing stable transformation systems and processes， and in-depth investigation of the mechanism of microbial transformation， fully elucidating the specific key hydrolases and its catalytic properties， and striving to improve the efficiency of microbial transformation. For the modified acid hydrolysis， novel acid catalytic system with simple structure， stable performance and good biodegradability should be explored and applied， which can effectively solve the problems of environmental pollution and production safety.

OBJECTIVE To study the spectru m-toxicity relationship of in vitro hepatotoxicity of aqueous extract from Euodia rutaecarpa. METHODS The aqueous extract from 16 batches of E. rutaecarpa from different habitats were prepared. The fingerprints of aqueous extract from E. rutaecarpa were established by ultra high performance liquid chromatography （UPLC） method and Similarity Evaluation System of TCM Fingerprint （2012A edition ），and common peaks were identified and the similarity was evaluated. Using normal human hepatocytes L 02 as subject ，inhibitory effect of aqueous extract from 16 batches of E. rutaecarpa to them were investigated. The spectrum-toxicity relationship of UPLC fingerprint of aqueous extract from E. rutaecarpa with the hepatotoxicity of hepatocytes L 02 was analyzed by grey relational analysis （GRA）and partial least squares regression analysis （PLSR）. The corresponding compound of the chromatographic peak with the greatest correlation with the in vitro hepatotoxicity of E. rutaecarpa were isolated ，prepared and identified. RESULTS There were 27 common peaks in UPLC fingerprints of aqueous extract from 16 batches of E. rutaecarpa ，with similarity of 0.375-0.995. Totally 9 peaks were confirmed ，i.e. neochlorogenic acid （peak 5），chlorogenic acid （peak 9），cryptochlorogenic acid （peak 10），caffeic acid （peak 12），rutin （peak 16），hyperin（peak 17），dehydroevotarine（peak 19），evotarine（peak 24），rutecarpine（peak 25）. The aqueous extract from 16 batches of E. rutaecarpa showed significant inhibitory effect on the growth of L 02 cells（P＜0.05 or P＜0.01），and the inhibitory rate ranged from 6.68％ to 67.95％. GRA showed that there were 18 common peaks with correlation degree greater than 0.8，which were peak 8＞peak 3＞peak 23＞peak 7＞peak 4＞peak 9＞peak 12＞peak 2＞peak 19＞peak 6＞ 4928381。E-mail：799247687@qq.com peak 15＞peak 5＞peak 1＞peak 17＞peak 21＞peak 26＞ peak 20＞peak 14 in descending order of correlation degree. PLSR showed that there were 14 peaks with regression coefficient＞0 and variable importance projection value ＞1，and the order of regression coefficient was peak 8＞peak 3＞peak 23＞ peak 2＞peak 7＞peak 4＞peak 12＞peak 9＞peak 19＞peak 5＞peak 17＞peak 26＞peak 10＞peak 15. Peak 8 had the greatest correlation with in vitro hepatotoxicity，and the corresponding compound of this peak was identified as 6-O-trans caffeoyl gluconic acid. CONCLUSIONS The in vitro hepatotoxicity of aqueous extract from E. rutaecarpa is the result of multiple component interaction，among which 6-O-trans caffeoyl gluconic acid shows closest relation with in vitro hepatotoxicity.

Betacoronavirus ; Coronavirus Infections ; Humans ; Pandemics ; Pneumonia, Viral ; Single-Cell Analysis

OBJECTIVE: To explore the molecular basis for a pedigree affected with coagulation factor V (FV) deficiency. METHODS: Clinical data of the patient and his family members was analyzed. Targeted capture and next-generation sequencing (NGS) and Sanger sequencing were carried out to detect potential variant of the FV gene. RESULTS: The patient presented with jaundice and prolonged prothrombin time (PT) and activated partial thromboplastic time (APTT). V factor activity measured only 0.1% of the normal level, though the patient had no sign of bleeding. A paternal heterozygous variant c.653T>C (p.F218S) and a maternal heterozygous variant c.3642_3643del (p.P1215Rfs*175) were identified in the FV gene of the patient. His elder brother was a heterozygous carrier of the c.653T>C (p.F218S) variant. c.653T>C(p.F218S) was a known pathogenic variant, while the c.3642_3643del (p.P1215Rfs*175) variant was unreported previously. CONCLUSION Mutations of the FV gene probably underlie the hereditary coagulation factor V deficiency in this patient. NGS combined with Sanger sequencing has detected potential variant with efficiency and provided a reliable basis for clinical and prenatal diagnosis for this family.
Aged ; Factor V ; Factor V Deficiency ; genetics ; Genetic Variation ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree ; Phenotype

Objective:To explore the protective effect of nerve plane-oriented laparoscopic total mesorectal excision (NPO+ LTME) for postoperative urinary and sexual function in patients with rectal cancer.Methods:Retrospective analysis was performed on rectal cancer patients who received surgical treatment at Renmin Hospital of Wuhan University from Jan 2016 to Dec 2018, including 114 patients in the NPO+ LTME group and 92 patients in the laparoscopic TME combined with pelvic autonomic nerve preservation (LTME+ PANP) group. Surgical and tumor-related indicators were recorded and compared between the two groups, and postoperative urination and sexual function were followed up.Results:There was no significant difference in baseline indicators between the two groups ( P>0.05). The operative time of the two groups was (150±7) min and (154±7) min, respectively ( t=3.585, P<0.05). Intraoperative bleeding was (9±3) ml and (15±6) ml ( t=7.654, P<0.05), respectively.Three months after surgery, the rate of urinary dysfunction in the NPO+ LTME group was lower than that in the LTME+ PANP group ( Z=2.549, P<0.05), but there was no difference between the two groups 6 and 12 months after surgery ( Z=0.814, P>0.05 and Z=1.275, P>0.05). At 3, 6 and 12 months after surgery, the erectile function in NPO+ LTME group was better than that in LTME+ PANP group ( Z=4.917, P<0.05; Z=4.947, P<0.05 and Z=4.081, P<0.05); The rate of ejaculation dysfunction was also lower than that of the LTME+ PANP group ( Z=4.464, P<0.05; Z=4.948, P<0.05 and Z=4.434, P<0.05); In addition, postoperative female sexual function was superior to LTME+ PANP group ( Z=2.532, P<0.05; Z=2.364, P<0.05; Z=2.076, P<0.05). Conclusion:NPO+ LTME has good surgical safety and also has certain advantages for patient sexual function and early urinary function protection.

Objective To summarize the characteristics of deltoid ligament rupture and explore the feasibility and shortterm clinical outcomes of targeted suture anchor repair technique according to the rupture site.Methods From May 2011 to October 2014,19 cases of complete deltoid ligament rupture (17 males and 2 females) were recruited in this study,with an average age of 34.15± 1.23 years (ranged from 15 to 60 years).According to Lauge-Hansen classification,there were 7 cases of pronation external rotation grade ⅣV injury,including 3 cases of Maisonnuve fracture;1 case of pronation abduction type Ⅲ degree injury,1 case of pronation abduction grade ⅣV injury;and 10 cases of supination external rotation grade ⅣV injury.According to AO / OTA classification,there were 9 cases of 43B type injury and 10 cases of 43C type injury.According to the rupture site of deltoid ligament,the targeted suture anchor repair surgery was operated respectively.Early mobilization with the help of hinged ankle brace was encouraged.The evaluation at last follow-up was based on the American Orthopedic and Ankle Association (AOFAS) criteria of ankle and hindfoot,and the visual analogue scale (VAS) scoring system.Results Nineteen patients were all followed up for 24 to 48 months,with an average of 30.42±5.11 months.Fourteen cases (73.7％,14/19) with talus end avulsion were treated by double suture anchor in the talus,with continuous locking suture of the avulsed end.Four cases (21.1％,4/19) with middle part rupture were treated by double suture anchor in the talus,with the sutures crossing three bone tunnels at the medial malleolus.One case (5.3％,1/19) with medial malleolus end avulsion was treated by single suture anchor at the medial malleolus,with continuous locking suture of the avulsed end.At the last follow-up,the AOFAS score was ranged from 70 points to 96 points,with an average of 90.53 points,and excellent in 16 cases,good in 2 cases,fair in 1 case,excellent and good rate was 94.7％.The VAS score was ranged from 0 to 2 points,with an average of 0.42 point.No wide medical clear space was detected.But traumatic arthritis was happened in 2 patients.Conclusion The targeted suture anchor repair technique according to the rupture site was a save technique in treating deltoid ligament rupture,which is conducive to early postoperative functional exercise,with excellent short-term clinical outcomes and few complications.