ObjectiveTo establish a high-performance liquid chromatography（HPLC） for the quantitative analysis of multiple components in Gegen Qinlian tablets， and to comprehensively evaluate the quality of samples from different manufacturers by integrating chemical pattern recognition and technique for order preference by similarity to ideal solution（TOPSIS）， in order to provide a reference basis for quality evaluation and control of Gegen Qinlian tablets. MethodsHPLC was employed to determine the contents of 10 components in 28 batches of Gegen Qinlian tablets collected from 6 manufacturers， and taking the detection results as variables， SIMCA 14.1 and SPSS 26.0 were employed for cluster analysis（CA）， principal component analysis（PCA）， and orthogonal partial least squares-discriminant analysis（OPLS-DA） to identify key components affecting the quality. Then， TOPSIS analysis was employed to rank the quality of Gegen Qinlian tablets from the 6 manufacturers and establish a comprehensive quality evaluation method. ResultsA quantitative method for Gegen Qinlian tablets was established. After methodological validation， the method was found to be stable and reliable， and could be used for the quantitative analysis of this preparation. The contents of 3′-hydroxy puerarin， puerarin， 3′-methoxy puerarin， daidzein， coptisine hydrochloride， epiberberine， jatrorrhizine hydrochloride， berberine hydrochloride， palmatine hydrochloride and baicalin in 28 batches of samples were 3.58-7.35， 24.88-42.32， 4.20-9.36， 4.33-7.60， 2.52-6.44， 0.93-4.10， 0.58-3.05， 10.68-22.92， 0.82-4.82， 11.73-60.16 mg·g^-1， respectively. Among them， puerarin， berberine hydrochloride and baicalin all met the limit requirements for this preparation specified in the 2025 edition of the Pharmacopoeia of the People's Republic of China. CA and PCA clustered the 28 batches of samples into 5 categories， PCA extracted 2 principal components with a cumulative variance contribution rate of 90.588%， and OPLS-DA screened out 4 differential markers with variable importance in the projection（VIP） values>1.0， namely baicalin， 3′-hydroxy puerarin， coptisine hydrochloride and palmatine hydrochloride， which might be the main components affecting the quality of Gegen Qinlian tablets. TOPSIS analysis showed that the comprehensive score of each evaluation index（C_i） values of different manufacturers were different. Among them， the C_i of manufacturer B was ranked higher， indicating potentially superior quality， while the C_i of manufacturer A was ranked lower， suggesting potentially inferior quality. ConclusionThis study establishes a quantitative method for Gegen Qinlian tablets， and the content uniformity of the same manufacturer is good， while there are differences in the contents of active components among different manufacturers. Through the chemical pattern recognition analysis， it is found that the content differences of Gegen Qinlian tablets may be related to baicalin， 3′-hydroxy puerarin， coptisine hydrochloride and palmatine hydrochloride.

ObjectiveBased on the AMP-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway， this study aimed to observe the effect of the Huazhuo Jiedu prescription on renal fibrosis in 5/6 nephrectomy rats and explore its underlying mechanism. MethodsA total of 67 SPF-grade male SD rats were used， of which 11 were randomly selected as the normal group. A chronic renal failure （CRF） model was established using 5/6 nephrectomy. The successfully modeled rats were randomly assigned to the model group， losartan potassium group （4.5 mg·kg^-1）， and low- （1.175 g·kg^-1）， medium- （2.35 g·kg^-1） and high-dose （4.7 g·kg^-1） Huazhuo Jiedu prescription groups， with 9 rats per group. Each group received an equivalent volume of saline or the corresponding concentration of Huazhuo Jiedu prescription by gavage once daily for 8 weeks. Hematoxylin-eosin （HE） and Masson staining were used to observe renal tissue pathological changes. Transmission electron microscopy examined renal ultrastructure. Immunohistochemistry （IHC） detected expressions of α-smooth muscle actin （α-SMA） and transforming growth factor-β₁ （TGF-β₁）. Western blot analyzed expression levels of microtubule-associated protein Ⅰ light chain 3Ⅱ （LC3Ⅱ）， Beclin1， p62， AMPK， phosphorylated AMPK （p-AMPK）， mTOR， and phosphorylated mTOR （p-mTOR）. ResultsCompared with the normal group， the model group exhibited glomerular shrinkage， mesangial and interstitial thickening， and tubular vacuolar degeneration， with no evident autophagosomes or autophagolysosome structures. Expression levels of α-SMA and TGF-β₁ were significantly increased （P0.01）， while p-AMPK/AMPK， Beclin1， and LC3Ⅱ were significantly decreased （P0.01）， and p-mTOR/mTOR and p62 were significantly increased （P0.01）. Compared with the model group， the medium- and high-dose Huazhuo Jiedu prescription groups and the losartan potassium group showed varying degrees of pathological improvement. Autophagosomes with double- or multiple-layer membranes and autophagolysosomes with monolayer membranes containing undegraded organelles were observed. Renal α-SMA and TGF-β₁ protein expression levels were markedly reduced （P0.05， P0.01）， p-mTOR/mTOR and p62 were significantly decreased （P0.05， P0.01）， and p-AMPK/AMPK， Beclin1， and LC3Ⅱ expression levels were significantly increased （P0.05， P0.01）. ConclusionHuazhuo Jiedu prescription may improve renal fibrosis in 5/6 nephrectomy rats by regulating the AMPK/mTOR signaling pathway and enhancing autophagy.

BACKGROUND: The clinical impact of post-percutaneous coronary intervention (PCI) quantitative flow ratio (QFR) in patients treated with PCI for chronic total occlusion (CTO) was still undetermined. METHODS: All CTO vessels treated with successful anatomical PCI in patients from PANDA III trial were retrospectively measured for post-PCI QFR. The primary outcome was 2-year vessel-oriented composite endpoints (VOCEs, composite of target vessel-related cardiac death, target vessel-related myocardial infarction, and ischemia-driven target vessel revascularization). Receiver operator characteristic curve analysis was conducted to identify optimal cutoff value of post-PCI QFR for predicting the 2-year VOCEs, and all vessels were stratified by this optimal cutoff value. Cox proportional hazards models were employed to calculate the hazard ratio (HR) with 95% CI. RESULTS: Among 428 CTO vessels treated with PCI, 353 vessels (82.5%) were analyzable for post-PCI QFR. 31 VOCEs (8.7%) occurred at 2 years. Mean value of post-PCI QFR was 0.92 ± 0.13. Receiver operator characteristic curve analysis shown the optimal cutoff value of post-PCI QFR for predicting 2-year VOCEs was 0.91. The incidence of 2-year VOCEs in the vessel with post-PCI QFR < 0.91 (n = 91) was significantly higher compared with the vessels with post-PCI QFR ≥ 0.91 (n = 262) (22.0% vs. 4.2%, HR = 4.98, 95% CI: 2.32-10.70). CONCLUSIONS Higher post-PCI QFR values were associated with improved prognosis in the PCI practice for coronary CTO. Achieving functionally optimal PCI results (post-PCI QFR value ≥ 0.91) tends to get better prognosis for patients with CTO lesions.

Objective To identify disulfidptosis-related gene(DRG)in acute myocardial infarction(AMI)by bioinformatics,analyze the molecular pattern of DRGs in AMI,and construct a DRGs-related prediction model.Methods AMI-related datasets were downloaded from the Gene Expression Omnibus database,and DRGs with differential expression were screened in AMI.CIBERSORT method was used to analyze the immune infiltration.Based on the differentially expressed DRGs,the AMI patients were classified into distinct subtypes via consensus clustering,followed by immune infiltration analysis,differential expression analysis,gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis,and gene set variation analysis.Weighted gene co-expression network analysis(WGCNA)was then performed to construct subtype-associated modules and identify hub genes.Finally,least absolute shrinkage and selection operator,random forest,and support vector machine-recursive feature elimination were used to screen feature genes to construct a DRGs-related prediction model.The model's diagnostic efficacy was evaluated by nomogram and receiver operating characteristic(ROC)curve analysis,followed by external validation.Results Nine differentially expressed DRGs were identified between AMI patients and controls.Based on the expression levels of these nine DRGs,AMI patients were divided into two DRGs subtypes,C1 and C2.Increased infiltration of monocytes,M0 macrophages,and neutrophils was observed in AMI patients and C1 subtype(all P<0.05),indicating a close correlation between DRGs and immune cells.There were 257 differentially expressed genes between the C1 and C2 subtypes,which were related to biological processes such as myeloid leukocyte activation and positive regulation of cytokines.Fcγ receptor-mediated phagocytosis and NOD-like receptor signaling pathway activity were enhanced in C1 subtype.WGCNA analysis suggested that the brown module exhibited the strongest correlation with DRG subtypes(r=0.67),from which 23 differentially expressed genes were identified.The feature genes screened by three machine learning methods were interpolated to obtain a DRGs-related prediction model consisting of three genes(AQP9,F5 and PYGL).Nomogram and ROC curves(AUC_train=0.891,AUC_test=0.840)showed good diagnostic efficacy.Conclusions DRGs were closely related to the occurrence and progression of AMI.The DRGs-related prediction model consisting of AQP9,F5 and PYGL may provide targets for the diagnosis and personalized treatment of AMI.
Humans ; Myocardial Infarction/diagnosis* ; Transcriptome ; Computational Biology ; Gene Expression Profiling ; ROC Curve ; Gene Regulatory Networks ; Nomograms ; Disulfidptosis

Objective To analyze the correlations of the expressions of miR-4500 and nerve growth factor(NGF)in breast cancer patients with preoperative magnetic resonance(MR)signs.Methods A total of 105 patients with breast cancer admitted to our hospital were selected,the mRNA expression levels of miR-4500 and NGF in breast cancer tissues and adjacent tissues of patients were detected by qRT-PCR,and the positive expression rates of NGF in breast cancer tissues and adjacent tissues were determined by immunohistochemistry method.The correlations of the expressions of miR-4500 and NGF in breast cancer tissues with clinicopathological features and MR signs of patients were analyzed.The targeting relationship between miR-4500 and NGF was predicted by the bioinformatics website,and the correla-tion between the expressions of the two was analyzed.Results Compared with the adjacent tissues,the expression level of miR-4500 in breast cancer tissue decreased(P<0.05),while the level of NGF mRNA and the positive expression rate of NGF increased(P<0.05).There was no significant difference in age,pathological type,tumor classification,parenchymal background,apparent diffusion coefficient or enhancement curve among different expressions of miR-4500 and NGF(P>0.05).The histological grade,lymph node metastasis,tumor diameter,tumor morphology,ring-like enhancement,and peritu-moral brain edema were related to the expressions of miR-4500 and NGF(P<0.05).The bioinformatics website prediction showed that miR-4500 and NGF had binding sites,and the expressions of miR-4500 and NGF mRNA in breast cancer tissues were negatively correlated(r=-0.576,P<0.05).Conclusion The expression of miR-4500 in breast cancer tissue is low,and the expression of NGF is high,which are correlated with the preoperative MR signs in patients,providing a molecu-lar basis for MR signs.

Objective To analyze the changes of the cost and structure of coronary intervention patients before and after the centralized procurement of coronary stents in Zhejiang Province.Methods It was conducted on the medical expenses of patients in the FM1 group(percutaneous coronary stent implantation)of a Class A tertiary hospital in Zhejiang Province from February to December in each 2020 and 2021.The number of cases was 1 403 and 1 698,re-spectively.A nonparametric test was conducted on the patient expenses before and after the reform.Results There are significant differences in total medical expenses,material expenses,treatment expenses,operation expenses and drug expenses before and after centralized purchase(P＜0.01);After the implementation of centralized procure-ment,material cost of patients decreased significantly,while the treatment cost increased;At the same time,the more coronary stents were placed,the greater the decrease in the total medical expenses and material expenses.Conclusion The centralized procurement of coronary stents can reduce the medical burden of patients,improve the structure of medical expenses,enhance the value of technical labor services of medical personnel,and promote DRG cost control.

Objective·To analyze the expression of mitochondrial related gene SFXN3(sideroflexin 3)in head and neck squamous cell carcinoma(HNSCC)and its effect on cell proliferation.Methods·Mitochondrial related genes and TCGA-HNSCC dataset were obtained from public databases to identify the target gene SFXN3 by using bioinformatic methods.The expression of SFXN3 in HNSCC patient samples was analysed by using the UALCAN database,and survival analyses of patients with different SFXN3 expression levels were performed according to TCGA-HNSCC cohort and GEO cohorts(GSE65858,GSE41613 and GSE27020).By using TCGA-HNSCC cohort and GEO cohorts(GSE40020,GSE210287),the differences in SFXN3 expression levels between therapeutic responders and non-responders were compared.Quantitative real-time PCR(qRT-PCR)was used to verify the expression of SFXN3 in the collected HNSCC tumor and para-tumor tissues,and the expression level of SFXN3 in human normal oral epithelial cells and HNSCC tumor cell lines was detected.With the construction of stable-SFXN3-knockdown HNSCC cell lines,the effect of SFXN3 on the proliferation ability of HNSCC cells was observed by using the Incucyte live-cell imaging analysis system and plate colony formation assay.Transcriptome sequencing was performed on the total RNA of stably-SFXN3-knockdown cells and control cells,and pathway enrichment analysis was performed on the genes with differentially down-regulated expression in knockdown group compared with control group.Results·SFXN3 was highly expressed in tumor tissues of HNSCC patients(P=0.000),and the prognosis of patients in the SFXN3-high-expression group was poor(all P＜0.05).The expression level of SFXN3 in the non-responders was higher than that in the responders(P=0.008),indicating an unfavorable prognosis.High expression of SFXN3 was verified in the collected HNSCC tumor tissues and HNSCC cell lines(all P＜0.05).After the knockdown of SFXN3 expression,the proliferation ability of HNSCC cells decreased,and the number of plate clones decreased(all P＜0.05).RNA sequencing showed that the differentially expressed down-regulated genes in HNSCC cells in the SFXN3-knockdown group were enriched in DNA replication,cell cycle,mitochondrial translation,mitochondrial RNA metabolic process and mitochondrial gene expression pathways.Conclusion·SFXN3 is highly expressed in HNSCC and it has negative correlation with the prognosis of patients.The proliferation ability and plate colony formation ability are inhibited in SFXN3-knockdown HNSCC cells,and these may work by affecting mitochondria function.

ObJective·To evaluate the dentofacial phenotype in non-syndromic tooth agenesis(NSTA)patients with paired box gene 9(PAX9)mutation.Methods·Patients with NSTA who visited the Department of Second Dental Center of Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,between January 2016 and December 2023 received whole-exome sequencing to screen PAX9 mutation.The location and number of missing teeth were evaluated by oral pantomography,and dentofacial deformities were evaluated by X-ray cephalometrics.Results·Seven patients with PAX9 mutation were included in the study,including 3 males(42.9％)and 4 females(57.1％).The patients were 7-31 years old at first visit,with a mean age of(19.7±8.0)years old.All the 7 patients were PAX9 heterozygotes,of which 4 were missense and 3 were frameshift.The average number of missing teeth was 15.9±2.9.The number of missing teeth in maxilla(9.6±2.6)was slightly higher than that in mandible(6.3±2.4)(P=0.030).Maxillary second molar(100.0％),maxillary canine(85.7％)and mandibular second premolar(85.7％)were the three most common missing teeth,while mandibular lateral incisor(14.3％)and mandibular canine(14.3％)were the two least missing teeth.Patients with frameshift mutation had more missing teeth(18.3±2.1)than those with missense mutation(14.0±1.8)(P=0.032).X-ray cephalometrics analysis results showed that the angle sella-nasion-subspinale(SNA),angle nasion-subspinale-subspinale-porion(NA-Apo)and sella-nasion(S-N)in adult patients with PAX9 mutation were significantly lower than the normal reference values,suggesting a shorter anterior cranial base and maxillary length.The frankfort horizontal plane-nasion-porion(FH-NPo)was higher than the reference value,and the Y-axis was lower than the reference value,indicating a more prognathic mandible.The angle subspinale-nasion-supramental(ANB)was lower than the reference value,indicating a skeletal angle Ⅲ malocclusion.The angle upper central incisor-nasion-subspinale(angle U1-NA)was higher than the reference value,indicating a lip inclination of maxillary central incisor.The angle lower central incisor-mandibular plane(IMPA)and lower central incisor-nasion-supramental(L1-NB)were lower than the reference values,indicating a retroclination of the mandibular central incisor,and crossbite in the maxillary and mandibular anterior teeth.Conclusion·The dentofacial phenotype of PAX9-mutated patients with NSTA is reported comprehensively.It is helpful to improve the understanding of the role of PAX9 in human maxillofacial development.

Objective·To explore EDAR(ectodysplasin A receptor)gene variants that lead to congenital tooth agenesis,and preliminarily analyze the reasons why variants in EDAR can cause both syndromic and non-syndromic tooth agenesis.Methods·Patients with congenital tooth agenesis admitted to the Department of 2nd Dental Center,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine and their family members were included,and genomic DNA from their peripheral blood was extracted for whole exome sequencing(WES).After preliminary screening,PolyPhen-2,Mutation Taster,and Provean were used to predict the harmfulness of potential variants.The screened variants in patients and their families were verified by Sanger sequencing.Conservation analysis of variants was performed,and Swiss-Model was used to analyze the changes in the three-dimensional structure of EDAR.The teeth and syndromic phenotype of the patients and their family members were investigated.Results·Among the included congenital tooth agenesis patients,five patients with EDAR mutations were found,one with EDAR frameshift mutation c.368_369insC(p.L123fs)and the other four with EDAR missense mutations.Two of these four patients were diagnosed as non-syndromic tooth agenesis(NSTA),resulted from c.77C＞A(p.A26E)homozygous mutation and c.380C＞T(p.P127L)heterozygous mutation,respectively.The other two patients with two variants were diagnosed as hypohidrotic ectodermal dysplasia(HED).One compound heterozygous missense mutation patient carried EDAR c.77C＞T(p.A26V)from her father andEDAR c.1281G＞C(p.L427F)from her mother;the other patient with both EDAR and EDA mutations carried EDAR c.1138A＞C(p.S380R)heterozygous mutation and EDA c.1013C＞T(p.T338M)hemizygous mutation.Both variants were from his mother and were reported to be related with NSTA.Two of these missense mutations,EDAR c.1281G＞C(p.L427F)and EDAR c.77C＞A(p.A26E),had not been reported before.The missense mutations affected the protein's spatial conformation by altering the polarity,charge,or volume of the amino acid residues.The frameshift mutation caused a non-triplet base addition,which probably led to protein truncation or degradation.Conclusion·Two new EDAR missense mutations are discovered.An NSTA patients with EDAR homozygous mutations and an HED patient with both EDA and EDAR mutations are reported.It expands the understanding of pathogenic mechanisms of EDAR mutations causing HED and NSTA.

Objective To investigate the impact of radiotherapy simulant Zeocin induced double-stranded RNA(dsRNA)on double-stranded RNA-dependent protein kinase(PKR)activation and explore its molecular mechanism in inhibiting the growth and migration of malignant gliomas.Methods We employed scratch assays,colony formation assays,and CCK8 assays to assess the impact of Zeocin on glioma growth and migration inhibition.Western blot and RT-qPCR were employed to measure the expression levels of methyl-modifying enzymes in glioma cells.The J2 antibody was used to detect endogenous dsRNA in malignant glioma cells treated with Zeocin.Western blot was used to assess the phosphorylation levels of PKR and eukaryotic initiating factor 2α(eIF2α).Results Zeocin significantly inhibited growth and migration of glioma cells;Zeocin treatment induced the production of endogenous dsRNA in malignant glioma cells;as the concentration of Zeocin increased,phosphorylated PKR and eIF2αprotein level also significantly increased;Zeocin downregulated the total m6A level of glioma cells in a dose-dependent manner;Zeocin selectively downregulated the protein level of methylase METTL14;Zeocin had no effect on mRNA levels of m6A modifying enzymes.Conclusion Zeocin probably triggered more dsRNA by downregulating the m6A level of RNA in malignant glioma cells,which in turn activated the PKR/eIF2α pathway,ultimately leading to tumor growth inhibition.