ObjectiveTraditional Chinese medicine (TCM) constitutes a valuable cultural heritage and an important source of antitumor compounds. Poria (Poria cocos (Schw.) Wolf), the dried sclerotium of a polyporaceae fungus, was first documented in Shennong’s Classic of Materia Medica and has been used therapeutically and dietarily in China for millennia. Traditionally recognized for its diuretic, spleen-tonifying, and sedative properties, modern pharmacological studies confirm that Poria exhibits antioxidant, anti-inflammatory, antibacterial, and antitumor activities. Pachymic acid (PA; a triterpenoid with the chemical structure 3β-acetyloxy-16α-hydroxy-lanosta-8,24(31)-dien-21-oic acid), isolated from Poria, is a principal bioactive constituent. Emerging evidence indicates PA exerts antitumor effects through multiple mechanisms, though these remain incompletely characterized. Neuroblastoma (NB), a highly malignant pediatric extracranial solid tumor accounting for 15% of childhood cancer deaths, urgently requires safer therapeutics due to the limitations of current treatments. Although PA shows multi-mechanistic antitumor potential, its efficacy against NB remains uncharacterized. This study systematically investigated the potential molecular targets and mechanisms underlying the anti-NB effects of PA by integrating network pharmacology-based target prediction with experimental validation of multi-target interactions through molecular docking, dynamic simulations, and in vitro assays, aimed to establish a novel perspective on PA’s antitumor activity and explore its potential clinical implications for NB treatment by integrating computational predictions with biological assays. MethodsThis study employed network pharmacology to identify potential targets of PA in NB, followed by validation using molecular docking, molecular dynamics (MD) simulations, MM/PBSA free energy analysis, RT-qPCR and Western blot experiments. Network pharmacology analysis included target screening via TCMSP, GeneCards, DisGeNET, SwissTargetPrediction, SuperPred, and PharmMapper. Subsequently, potential targets were predicted by intersecting the results from these databases via Venn analysis. Following target prediction, topological analysis was performed to identify key targets using Cytoscape software. Molecular docking was conducted using AutoDock Vina, with the binding pocket defined based on crystal structures. MD simulations were performed for 100 ns using GROMACS, and RMSD, RMSF, SASA, and hydrogen bonding dynamics were analyzed. MM/PBSA calculations were carried out to estimate the binding free energy of each protein-ligand complex. In vitro validation included RT-qPCR and Western blot, with GAPDH used as an internal control. ResultsThe CCK-8 assay demonstrated a concentration-dependent inhibitory effect of PA on NB cell viability. GO analysis suggested that the anti-NB activity of PA might involve cellular response to chemical stress, vesicle lumen, and protein tyrosine kinase activity. KEGG pathway enrichment analysis suggested that the anti-NB activity of PA might involve the PI3K/AKT, MAPK, and Ras signaling pathways. Molecular docking and MD simulations revealed stable binding interactions between PA and the core target proteins AKT1, EGFR, SRC, and HSP90AA1. RT-qPCR and Western blot analyses further confirmed that PA treatment significantly decreased the mRNA and protein expression of AKT1, EGFR, and SRC while increasing the HSP90AA1 mRNA and protein levels. ConclusionIt was suggested that PA may exert its anti-NB effects by inhibiting AKT1, EGFR, and SRC expression, potentially modulating the PI3K/AKT signaling pathway. These findings provide crucial evidence supporting PA’s development as a therapeutic candidate for NB.

Aim To study the effects of high-fat diet on testicular germ cell apoptosis in mice through endoplasmic reticulum stress. Methods C57BL/6J male mice were assigned into normal group and high-fat diet group randomly, with six mice in each group. The mice in normal group or high-fat diet group were fed with regular or high-fat diet continuously for five months. The mice were weighed, anesthetized, and euthanized to collect testicular and epididymal tissue for analysis. The testicular tissue was weighed and their indices were calculated. Epididymal tissue was collected for semen analysis. The morphological alterations of testicular tissue were observed using hematoxylin-eosin ( HE ) staining. The apoptosis of germ cells was detected by TUNEL staining and the apoptotic indices were calculated. The expression levels of apoptosis and endoplasmic reticulum stress-related proteins in testicular tissue were detected by Western blot. The protein expression and localization of GRP78 in testicular tissue were further detected by immunofluorescence. Results The results showed that compared to the normal group, the high-fat diet group had a significant increase in body weight, a significant decrease in testicular index, sperm concentration, and sperm vability, loose arrangement of germ cells, significant thinning of the seminiferous epithelium, no significant change in the diameter of seminiferous tubules, a significant increase in germ cell apoptosis , with an increased apoptosis index, and significant increase in expression of Bax and cleaved-caspase-12,and a significant decrease in Bcl-2 protein expression. The expression levels of GRP78 , p-IREl, XBP1, and ATF6a proteins were significantly up-regulated, while p-PERK, p-eIF2a, ATF4 protein expression showed no significant changes. Immunofluorescence results further showed a significant increase in the expression of GRP78 protein in the testicular tissue,with no significant changes in the expression location. Conclusions High-fat diet can induce the apoptosis of mouse testicular germ cells, and the mechanism may be related to the activation of endoplasmic reticulum stress IRE1 and ATF6 signaling pathway.

Objective To understand the distribution and antimicrobial resistance changes of bacteria isolated from pleural and peritoneal effusion in Hunan Province,and provide reference for correct clinical diagnosis and rational antimicrobial use.Methods Data reported by member units of Hunan Provincial Antimicrobial Resistance Survei-llance System from 2012 to 2021 were collected.Bacteria antimicrobial resistance surveillance method was imple-mented according to technical scheme of China Antimicrobial Resistance Surveillance System(CARSS),and WHO-NET 5.6 software was used to analyze the data of bacteria isolated from pleural and peritoneal effusion as well as antimicrobial susceptibility testing results.Results From 2012 to 2021,a total of 28 934 bacterial strains were iso-lated from specimens of pleural and peritoneal effusions from member units of Hunan Provincial Antimicrobial Re-sistance Surveillance System,with 5 752 strains from pleural effusion and 23 182 from peritoneal effusion.The top five bacteria isolated from pleural effusion were Escherichia coli(n=907,15.8％),Staphylococcus aureus(n=535,9.3％),Klebsiella pneumoniae(n=369,6.4％),Staphylococcus epidermidis(n=452,7.9％),and Staphy-lococcus haemolyticus(n=285,5.0％).The detection rate of methicillin-resistant Staphylococcus aureus(MR-SA)from pleural effusion was 24.3％-39.2％,and that of methicillin-resistant coagulase negative Staphylococcus(MRCNS)was 58.8％-77.1％.The top five bacteria isolated from peritoneal effusion were Escherichia coli(n=8 264,35.6％),Klebsiella pneumoniae(n=2 074,9.0％),Enterococcus faecium(n=1 458,6.3％),Staphylo-coccus epidermidis(n=1 383,6.0％),and Pseudomonas aeruginosa(n=1 152,5.0％).The detection rate of MRSA from peritoneal effusion was 22.1％-52.4％,which presented a decreasing trend(P=0.004).The detec-tion rate of MRCNS was 60.4％-79.4％.The resistance rates of Enterobacterales from peritoneal effusion to ce-fazolin,cefuroxime,ceftriaxone and cefepime all showed decreasing trends(all P＜0.05).Vancomycin-,linezo-lid-,and teicoplanin-resistant Staphylococcus strains were not found in pleural and peritoneal effusions.The resis-tance rates of Enterococcus faecium to most tested antimicrobial agents were higher than those of Enterococcus fae-calis.The resistance rates of Enterobacterales to imipenem and meropenem were ≤8.5％.The resistance rates of non-fermentative Gram-negative bacilli to imipenem and meropenem were ≤43.3％.Conclusion The data structure of Hunan Antimicrobial Resistance Surveillance System for pleural and peritoneal effusions from 2012 to 2021 is relatively complete.The constituent and antimicrobial susceptibility of isolated pathogenic bacteria vary in different years.

Objective To understand the epidemiology of clinically isolated Acinetobacter baumannii(A.bauma-nnii)in Hunan Province.Methods Bacterial antimicrobial resistance surveillance was carried out according to the requirements of the technical program of National Antimicrobial Resistance Surveillance System.Clinical data of Acinetobacter spp.reported to Hunan Provincial Antimicrobial Resistance Surveillance System by multiple centers in Hunan Province from 2012 to 2021 were summarized and analyzed with reference to the standards of the American Clinical and Laboratory Standards Institute.Results A total of 169 438 strains of Acinetobacter spp.were detected during the 10-year period,with the detection rate of A.baumannii being the highest(82.74％).70 923 strains(53.63％)of carbapenem-resistant A.baumannii(CRAB)and 58 149 strains(43.97％)of carbapenem-sensitive A.baumannii(CSAB)were detected respectively.Both CRAB and CSAB were detected most frequently in the age group＞70 years,which were 34.44％and 32.02％,respectively.The percentage of CRAB and CSAB detected in the intensive care unit were 34.80％and 11.31％,respectively.CRAB and CSAB were mainly isolated from spu-tum/bronchoalveolar lavage fluid,followed by pus/secretion,urine,and blood.The resistance rates of CRAB to commonly used antimicrobial agents didn't change much during the 10-year period.Resistance rates of CRAB to ceftazidime and cefepime were both＞84％,to ampicillin/sulbactam and piperacillin/tazobactam were both＞82％,to aminoglycosides and quinolones were both＞59％,to minocycline and polymyxin B were 15.9％-25.0％and 1.3％-6.9％,respectively.CSAB were sensitive to commonly used antimicrobial agents.Conclusion The isolation rate of CRAB is high and there is no significant change in resistance to commonly used antimicrobial agents.

Humans ; Endovascular Aneurysm Repair ; Endovascular Procedures ; Blood Vessel Prosthesis Implantation ; Aortic Aneurysm, Abdominal/surgery* ; Treatment Outcome ; Postoperative Complications ; Risk Factors ; Aortic Rupture/surgery* ; Retrospective Studies

This article analyzed the mechanism of Danggui Sini Decoction(DSD) in improving kidney injury caused by blood stasis syndrome(BSS) in rats. Firstly, 32 female SD rats were randomly divided into the following four groups: a normal group and a BSS group, both receiving an equal amount of distilled water by gavage; a normal+DSD group and a BSS+DSD group, both receiving 5.103 g·kg~(-1) DSD orally for a total of 14 days. Daily cold water bath was given to establish the BSS model, and on the 14th day, BSS rats were subcutaneously injected with 0.8 mg·kg~(-1) adrenaline. Normal rats were subjected to the water bath at 37 ℃ and injected with an equal volume of distilled water. After the experiment, 24-hour urine, serum, and kidney samples were collected for metabolomic analysis, biochemical measurements, and hematoxylin-eosin(HE) staining. The study then employed ~1H-NMR metabolomic technology to reveal the metabolic network regulated by DSD in improving BSS-induced kidney injury and used network pharmacology to preliminarily elucidate the key targets of the effectiveness of DSD. Pathological and biochemical analysis showed that DSD intervention significantly reduced inflammation and abnormal levels of blood creatinine, blood urea nitrogen, and urine protein in the kidneys. Metabolomic analysis indicated that DSD attenuated BSS-induced kidney injury primarily by regulating 10 differential metabolites and three major metabolic pathways(taurine and hypotaurine metabolism, citrate cycle, and acetaldehyde and dicarboxylic acid metabolism). Network pharmacology analysis suggested that the protective effect of DSD against BSS-induced kidney injury might be related to two key genes, ATP citrate lyase(ACLY) and nitric oxide synthase 2(NOS2), and two main metabolic pathways, i.e., arginine biosynthesis, and arginine and proline metabolism. This study, from the perspective of network regulation, provides initial insights and evidence into the mechanism of DSD in improving kidney injury induced by BSS, offering a basis for further investigation into the molecular mechanisms underlying its efficacy.
Rats ; Female ; Animals ; Rats, Sprague-Dawley ; Network Pharmacology ; Drugs, Chinese Herbal/chemistry* ; Metabolomics ; Kidney ; Arginine ; Water

OBJECTIVE: To investigate protective effect of Cordyceps sinensis (CS) through autophagy-associated adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in acute kidney injury (AKI)-induced acute lung injury (ALI). METHODS: Forty-eight male Sprague-Dawley rats were divided into 4 groups according to a random number table, including the normal saline (NS)-treated sham group (sham group), NS-treated ischemia reperfusion injury (IRI) group (IRI group), and low- (5 g/kg·d) and high-dose (10 g/kg·d) CS-treated IRI groups (CS1 and CS2 groups), 12 rats in each group. Nephrectomy of the right kidney was performed on the IRI rat model that was subjected to 60 min of left renal pedicle occlusion followed by 12, 24, 48, and 72 h of reperfusion. The wet-to-dry (W/D) ratio of lung, levels of serum creatinine (Scr), blood urea nitrogen (BUN), inflammatory cytokines such as interleukin- β and tumor necrosis factor- α, and biomarkers of oxidative stress such as superoxide dismutase, malonaldehyde (MDA) and myeloperoxidase (MPO), were assayed. Histological examinations were conducted to determine damage of tissues in the kidney and lung. The protein expressions of light chain 3 II/light chain 3 I (LC3-II/LC3-I), uncoordinated-51-like kinase 1 (ULK1), P62, AMPK and mTOR were measured by Western blot and immunohistochemistry, respectively. RESULTS: The renal IRI induced pulmonary injury following AKI, resulting in significant increases in W/D ratio of lung, and the levels of Scr, BUN, inflammatory cytokines, MDA and MPO (P<0.01); all of these were reduced in the CS groups (P<0.05 or P<0.01). Compared with the IRI groups, the expression levels of P62 and mTOR were significantly lower (P<0.05 or P<0.01), while those of LC3-II/LC3-I, ULK1, and AMPK were significantly higher in the CS2 group (P<0.05 or P<0.01). CONCLUSION CS had a potential in treating lung injury following renal IRI through activation of the autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI.
Rats ; Male ; Animals ; AMP-Activated Protein Kinases/metabolism* ; Cordyceps/metabolism* ; Rats, Sprague-Dawley ; Kidney/pathology* ; Acute Kidney Injury/metabolism* ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Cytokines/metabolism* ; Acute Lung Injury/drug therapy* ; Mammals/metabolism*

With the development of posterior chamber phakic intraocular lenses implantation and the constant improvement of the implantable collamer lens(ICL), ICL V4c implantation has become one of the main methods for correcting moderate and high myopia. Vault is an important indicator to evaluate the security of posterior chamber intraocular lens implantation. In recent years, optimizing surgical procedures to obtain the ideal vault in ICL V4c implantation surgery has become a research hotspot. This paper aims to provide help for improving surgical safety by summarizing and analyzing the optimized programs of ICL V4c implantation surgery. The focus will be on preoperative examination, intraoperative surgical design, and postoperative follow-up.

Using beta-2 adrenergic receptor, 5-hydroxytryptamine and angiotensin II type 1 receptor as control, we here established a method for rapid prediction of the initial position amino acids of N-terminal, C-terminal, intracellular loops, extracellular loops and transmembrane (TM) regions in G protein-coupled receptors (GPCRs), and successfully predicted the structure of Mas-related G protein-coupled receptors X3 (MRGPRX3). To achieve this purpose, nanoluciferase (Nluc) was inserted into the different sites of these GPCRs′ sequence by sequence and ligation-independent cloning (SLIC) method, and the luminescence value were measured to distinguish the different parts of GPCRs. The results showed that luminescence values of NLuc luciferase at TM region were less than 100 000, and the values were higher than 1 000 000 at N terminal, C terminal, or extracellular loops and intracellular loops, and the values were between 100 000 and 500 000 at junction. The predicted MRGPRX3 structure was analyzed in detail and was compared with AlphaFold predicted structure. In conclusion, this method could provide useful information of GPCR structure model for the ligand virtual screening, and could provide certain experimental basis for structural pharmacology.

ObjectiveTo establish a mutation library of rifampicin resistance gene rpoB. MethodsThe ΔrpoB attB：：rpoB strain of Mycobacterium smegmatis （M. smegmatis） be constructed by homologous recombination and L5 attB phage integration site exchange. Based on the L5 attB plasmid exchange system and resistance selection medium， 48 clones are selected to verify plasmid replacement efficiency. Degenerate primers are designed every 3 bases in the rifampicin resistance determining region （RRDR）， and a full-coverage mutation library of 81 bases in RRDR region is obtained by PCR amplification. The library fragments are seamlessly cloned into the vector and transformed into Escherichia coli （E. coli）to form an E. coli mutation library. Based on the principle of plasmid exchange， the mutant plasmid library is transformed into the M. smegmatis strain ΔrpoB attB：：rpoB， and the original L5 attB site plasmid is replaced to form the M. smegmatis mutant library. The genotype of the library are determined by genome extraction， library construction and high-throughput sequencing. ResultsCompared with the wild-type rpoB gene （5 600 bp）， the amplified fragment of the rpoB knockout strain is 2 200 bp， which proved that the ΔrpoB attB：：rpoB conditional knockout strain of M. smegmatis is successfully constructed. The success rate of plasmid replacement is 100%. There were 540 kinds of single amino acid mutations in both E. coli library and M. smegmatis library， 5 301 kinds of multi-point mutations in E. coli library， and 853 kinds of multi-point mutations in M. smegmatis library. The correlation coefficient between E. coli library and M. smegmatis library is 0.84. ConclusionsWe have developed a strategy to construct a library of mutants targeting the essential mycobacterial gene rpoB， and successfully established a mutant library of rifampicin resistance gene rpoB.