The study investigated the specific mechanism by which oxocrebanine, the anti-hepatic cancer active ingredient in Stephania hainanensis, inhibits the proliferation of hepatic cancer cells. Firstly, methyl thiazolyl tetrazolium(MTT) assay, 5-bromodeoxyuridine(BrdU) labeling, and colony formation assay were employed to investigate whether oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells. Propidium iodide(PI) staining was used to observe the oxocrebanine-induced apoptosis of HepG2 and Hep3B2.1-7 cells. Western blot was employed to verify whether apoptotic effector proteins, such as cleaved cysteinyl aspartate-specific protease 3(c-caspase-3), poly(ADP-ribose) polymerase 1(PARP1), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), Bcl-2 homologous killer(Bak), and myeloid cell leukemia-1(Mcl-1) were involved in apoptosis. Secondly, HepG2 cells were simultaneously treated with oxocrebanine and the autophagy inhibitor 3-methyladenine(3-MA), and the changes in the autophagy marker LC3 and autophagy-related proteins [eukaryotic translation initiation factor 4E-binding protein 1(4EBP1), phosphorylated 4EBP1(p-4EBP1), 70-kDa ribosomal protein S6 kinase(P70S6K), and phosphorylated P70S6K(p-P70S6K)] were determined. The results of MTT assay, BrdU labeling, and colony formation assay showed that oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells in a dose-dependent manner. The results of flow cytometry suggested that the apoptosis rate of HepG2 and Hep3B2.1-7 cells increased after treatment with oxocrebanine. Western blot results showed that the protein levels of c-caspase-3, Bax, and Bak were up-regulated and those of PARP1, Bcl-2, and Mcl-1 were down-regulated in the HepG2 cells treated with oxocrebanine. The results indicated that oxocrebanine induced apoptosis, thereby inhibiting the proliferation of hepatic cancer cells. The inhibition of HepG2 cell proliferation by oxocrebanine may be related to the induction of protective autophagy in hepatocellular carcinoma cells. Oxocrebanine still promoted the conversion of LC3-Ⅰ to LC3-Ⅱ, reduced the phosphorylation levels of 4EBP1 and P70S6K, which can be reversed by the autophagy inhibitor 3-MA. It is prompted that oxocrebanine can inhibit the proliferation of hepatic cancer cells by inducing autophagy. In conclusion, oxocrebanine inhibits the proliferation of hepatic cancer cells by inducing apoptosis and autophagy.
Humans ; Apoptosis/drug effects* ; Autophagy/drug effects* ; Cell Proliferation/drug effects* ; Hep G2 Cells ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Caspase 3/genetics*

Compounds isolated from Epimedium include the total flavonoids of Epimedium , icariin, and its metabolites (icaritin, icariside I, and icariside II), which have similar molecular structures. Modern pharmacological research and clinical practice have proved that Epimedium and its active components have a wide range of pharmacological effects, especially in improving sexual function, hormone regulation, anti-osteoporosis, immune function regulation, anti-oxidation, and anti-tumor activity. To date, we still need a comprehensive source of knowledge about the pharmacological effects of Epimedium and its bioactive compounds on the male reproductive system. However, their actions in other tissues have been reviewed in recent years. This review critically focuses on the Epimedium , its bioactive compounds, and the biochemical and molecular mechanisms that modulate vital pathways associated with the male reproductive system. Such intrinsic knowledge will significantly further studies on the Epimedium and its bioactive compounds that protect the male reproductive system and provide some guidances for clinical treatment of related male reproductive disorders.
Male ; Epimedium/chemistry* ; Humans ; Genitalia, Male/drug effects* ; Flavonoids/therapeutic use* ; Animals

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Objective:To investigate the diagnosis value of serum levels of soluble B7-H3(sB7-H3),trefoil factor 1(TFF1)and lipocalin-2(LCN-2)in breast cancer(BC)patients.Methods:From March 2020 to March 2023,196 newly diagnosed BC patients accepted by Huanghe San-menxia hospital were regarded as the study subjects and assigned to the BC group;84 patients with benign breast lesions and 76 physically healthy individuals served as the benign lesion group and the control group,respectively;clinical data were collected.ELISA method was applied to de-tect the serum levels of sB7-H3,TFF1,and LCN-2 in each group,and the relationship between the serum levels of the three and the clinical and pathological characteristics of BC patients was analyzed;Spearman method was applied to analyze the correlation between serum sB7-H3,TFF1,LCN-2 levels and clinical pathological characteristics in BC patients;receiver operating characteristic(ROC)curve was plotted to evaluate the diagnostic value of serum sB7-H3,TFF1,and LCN-2 levels for BC.Results:Compared with the reference group,the serum levels of sB7-H3,TFF1,and LCN-2 in the benign lesion group and BC group were obviously increased(P＜0.05),the serum levels of sB7-H3,TFF1,and LCN-2 in the BC group were obviously higher than those in the benign lesion group(P＜0.05);the serum levels of sB7-H3,TFF1,and LCN-2 were obviously elevated in BC patients with tumour diameter＞2 cm,TNM stage Ⅲ+Ⅳ,lymph node metastasis,and negative ER expression(P＜0.05),the serum levels of sB7-H3,TFF1 and LCN-2 were positively correlated with tumor diameter(r=0.463,0.442,0.481,P＜0.001),positive correla-tion with TNM stage(r=0.474,0.394,0.562,P＜0.001),positive correlation with lymph node me-tastasis(r=0.567,0.488,0.409,P＜0.001),and negative correlation with ER expression(r=-0.575,-0.534,-0.538,P＜0.001);the AUC for serum sB7-H3,TFF1,and LCN-2 alone for BC diagnosis were 0.817,0.814,and 0.830,respectively,and the combined diagnosis was superior to the separate diagnosis(AUC=0.911,95％CI:0.871-0.942).Conclusion:The serum levels of sB7-H3,TFF1 and LCN-2 in BC patients were significantly increased,which is of high clinical value for the diagnosis of BC.

Objective:To explore the genetic etiology and clinical phenotype of a child with Triadin knockout syndrome (TKOS), and to review the relevant literature of TKOS patients due to variants of TRDN gene. Methods:A child who was admitted to the Children′s Hospital of Xi′an Jiaotong University on March 19, 2023 due to sudden cardiac arrest 3 days earlier was selected as the study subject. Peripheral blood samples (2 to 3 mL) were collected from the child and her parents for the extraction of genomic DNA and whole exome sequencing (WES). Pathogenic variants were searched from databases such as the Genome Aggregation Database (gnomAD) and Online Mendelian Inheritance in Man (OMIM), and were assessed based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Sanger sequencing was carried out for family validation of the pathogenic variants. Using keywords such as " arrhythmias" " TRDN" and " Triadin" both in Chinese and English, relevant literature on TKOS patients due to variants of the TRDN gene was retrieved from the CNKI, Wanfang Data Knowledge Service Platform, and PubMed databases, and the time of literature retrieval was set from January 1, 2012 to December 1, 2023. This study has been approved by the Ethics Committee of the Affiliated Children′s Hospital of Xi′an Jiaotong University (No. 20230097), and informed consent was obtained from the parents of the child. Results:The child had experienced syncope and cardiac arrest after exercise. Electrocardiographic examination revealed QTc interval prolongation, T-wave inversion in precordial leads V1-V3, polymorphic ventricular premature beat (VPB), and ventricular tachycardia (VT) along with increased heart rate. WES and Sanger sequencing revealed that the child has harbored a homozygous c.463del(p.E155Kfs*20) variant of the TRDN gene, for which both of the parents were heterozygous. Based on the guidelines from the ACMG, the variant was classified as pathogenic (PVS1+ PM2+ PM3). The child was ultimately diagnosed with TKOS. In total 12 publications on TOKS cases caused by TRDN gene variants were retrieved, which involved 30 patients and 28 carriers of single heterozygous variant of the TRDN gene. Among the 30 TKOS patients, 20 had carried homozygous variants of the TRDN gene, and 10 had carried compound heterozygous variants, and all had exhibited significant clinical phenotype of arrhythmia, with most cases had experienced malignant arrhythmia induced by exercise and/or excitement during infancy or early childhood, leading to recurrent syncope and cardiac arrest. Of note, none of the 28 carriers of single heterozygous variant had abnormal clinical phenotype. Conclusion:The homozygous c.463del(p.E155Kfs20) variant of the TRDN gene probably underlay the pathogenesis of cardiac arrest in this child. Above discovery has enriched the mutational spectrum of the TRDN gene.This mutation may represent a genetic cause for cardiac arrest in children with TKOS.

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Objective: To observe the clinical effect of auricle reconstruction in adult patients with microtia and summarize the experience. Methods: Clinical data of adult patients with microtia who underwent total auricle reconstruction using the modified Nagata's two stage for microtia reconstruction from June 2016 to June 2021 were analyzed. A total of 41 adult patients (42 ears) with microtia were enrolled, including 30 males and 11 females, with the median age at the time of surgery of 37 years. Autogenous costal cartilage was used as the auricular framework for all patients in this group. The first stage surgery was performed according to the modified Nagata's two stage for microtia reconstruction procedure,cartilage auricular framework carving was performed by different methods according to the ossification state of adult costal cartilage. Six months following the primary operation, ear elevation and cranioauricular angle formation, retroauricular facial flap transfer and medium-thick skin grafting were performed in the second stage. Results: All patients successfully completed two stage operation. During the follow-up of 3 months and 24 months, all the 41 patients were satisfied with the morphology of reconstructed auricle. Conclusion: According to the costal cartilage status of adult patients, different costal cartilage carving techniques can be used for total auricle reconstruction to obtain ideal surgical results.
Male ; Female ; Humans ; Adult ; Surgical Flaps ; Congenital Microtia/surgery* ; Plastic Surgery Procedures ; Ear, External/surgery* ; Ear Auricle/surgery*

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.
Chromones ; Sodium Chloride ; Chromatography, Liquid ; Flavonoids ; Tandem Mass Spectrometry ; Thymelaeaceae

Lysine decarboxylase is a key enzyme involved in the upstream biosynthesis of lycopodium alkaloids (LAs) such as huperzine A, contributing to the decarboxylation of lysine to 1,5-pentanediamine (cadaverine). Three lysine decarboxylase genes (HsLDC-L1, HsLDC-L2, HsLDC-L3) were successfully cloned from Huperzia serrata using transcriptomic sequence data mining strategy combined with reverse transcription PCR. The physicochemical properties, secondary and tertiary structures, amino acid identities, and evolutionary relationship of the three LDCs were analyzed by online bioinformatics analysis platforms and DNAMAN, MEGA 7.0 software, revealing that all of these proteins had the conserved PLP binding domain and active site residues were completely conserved in LDCs. Phylogenetic analysis showed that these LDCs were located in the same branch as other known LDCs from LA-producing plants. Accordingly, the ORFs of these three HsLDCs were inserted into different expression plasmids for further expression in E. coli. However, only HsLDC-L1 was successfully expressed in E. coli BL21 (DE3) by inserting into a pCold TF vector. The recombinant protein was purified by Ni²⁺ affinity chromatography purification. HsLDC-L1 contains 469 amino acid residues, with a calculated molecular weight of 50.50 kDa. HsLDC-L1 expectedly catalyzed the decarboxylation of lysine to produce cadaverine. In addition, HsLDC-L1 can also catalyze the generation of putrescine from ornithine. However, it cannot catalyze the decarboxylation of tyrosine, phenylalanine, tryptophan and histidine. The results not only provide insight into the biosynthesis of LAs including huperzine A, but also provide a critical genetic element for the overproduction of Δ¹-piperideine and pelletierine, the essential biosynthetic precursors of LAs, using synthetic biology strategies.

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.