ObjectiveTo optimize and validate the optimal sequential alcohol-water extraction process of modified Banxia Xiexintang（MBXT） based on pharmacodynamic evaluation， combined with the G1-entropy weight method and Box-Behnken response surface methodology， and to systematically and comprehensively analyze the material basis of this formula， providing a scientific basis for its quality control and industrial production. MethodsRats were randomly divided into the normal group， model group， metformin group， and MBXT water extraction， water extraction and ethanol precipitation， sequential ethanol-water extraction groups. Except for the normal group， a polycystic ovary syndrome with insulin resistance（PCOS-IR） model was established in all rats via a high-fat diet combined with letrozole induction. Enzyme-linked immunosorbent assay（ELISA） biochemical assay kits and hematoxylin-eosin（HE） staining were used to compare sex hormone levels in serum and ovarian histopathology， thereby screening extraction process routes. Based on this， a comprehensive score was constructed using the G1-entropy weight method based on the transfer rates of index components（berberine hydrochloride and baicalin） and the dry extract rate. Box-Behnken response surface methodology was then utilized to optimize the extraction process parameters. Finally， the chemical constituents of the sample from the optimal process were qualitatively analyzed by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap mass spectrometry（UPLC-Q-Orbitrap-MS）. ResultsPharmacodynamic findings revealed that compared with the normal group， serum testosterone（T） and luteinizing hormone（LH） levels were significantly elevated in the model group， while estradiol（E₂） and follicle-stimulating hormone（FSH） levels were significantly decreased（P<0.01）， with polycystic changes observed in ovarian tissues. Compared with the model group， all treatment groups significantly reversed the changes in sex hormone levels， with the sequential ethanol-water extraction group showing the optimal effect in improving the aforementioned indicators and pathological morphology， followed by subsequent process optimization. The optimized process involved adding 12 times the amount of 70% ethanol for extracting twice， each lasting 120 min， and adding 12 times the amount of water for extracting thrice， each lasting 90 min. Validation test results showed that under optimal process conditions， the average transfer rates of berberine hydrochloride and baicalin were 76.05% and 93.38%， respectively. MS analysis identified a total of 377 compounds， including 112 flavonoids， 41 terpenoids， 28 organic acids， 22 coumarins， and 8 alkaloids， while elucidating the cleavage patterns of key components. ConclusionThe optimized sequential ethanol-water extraction process is stable and feasible， effectively preserving the material basis of MBXT for treating PCOS-IR. It further clarifies the main chemical composition of this formula， providing a scientific basis for the development and quality control of its preparations.

ObjectiveTo evaluate the therapeutic effects of modified Banxia Xiexintang（MBXT） on polycystic ovary syndrome with insulin resistance（PCOS-IR） rats and reveal its potential mechanisms based on the integrated analysis of transcriptomics and metabolomics. MethodsFemale SD rats were selected， and a PCOS-IR model was established by intragastric administration of letrozole combined with a high-fat diet for 21 days. The modeled rats were randomly divided into the model group， MBXT low-， medium-， and high-dose groups（6.62， 13.23， 26.46 g·kg^-1）， and metformin group（0.158 g·kg^-1）， with a normal group set up separately. After 14 days of administration， the estrous cycle was observed， ovarian morphology was examined by hematoxylin-eosin（HE） staining， and the levels of testosterone（T）， estradiol（E₂）， follicle-stimulating hormone（FSH）， and luteinizing hormone（LH） in serum were detected by enzyme-linked immunosorbent assay（ELISA）. Serum metabolites and ovarian tissue gene expression were detected using ultra-performance liquid chromatography-quadrupole-electrostatic orbitrap mass spectrometry（UPLC-Q-Orbitrap-MS） and RNA-Seq technology， respectively， followed by multi-omics integrated analysis. ResultsPharmacodynamic findings revealed that all MBXT dose groups could reversed abnormal estrous cycles in PCOS-IR rats， improve polycystic ovarian lesions， and normalize dysregulated serum hormone levels（T， LH， E₂， FS， P<0.05， P<0.01）. Metabolomic analysis revealed that compared with the model group， MBXT reversed 278 differential metabolites such as estrone and S-formylglutathione， mainly involving pathways such as steroid hormone biosynthesis， glutathione metabolism， and lipid peroxidation regulation. Transcriptomic analysis identified 434 differentially expressed genes， and enrichment analysis revealed that MBXT significantly regulated lipid peroxidation defense systems， including glutathione metabolism， peroxisome function， and fatty acid metabolism， thereby intervening in ferroptosis processes. It also engaged in inflammation-related pathways such as the chemokine signaling pathway. Integrated analysis revealed that both metabolomics and transcriptomics co-enriched metabolic pathways associated with ferroptosis and fatty acid metabolism. And key Hub genes［such as Ras-related C3 botulinum toxin substrate 2 gene（Rac2） and Fas ligand gene（Faslg）］ showed significant correlations with differential metabolites. ConclusionMBXT can effectively ameliorate reproductive dysfunction and metabolic disorders in PCOS-IR rats. Its mechanism may be related to remodeling the immune-metabolism network， particularly by regulating MHC-mediated immune responses， inhibiting local ovarian ferroptosis， and enhancing steroid hormone synthesis pathways.

ObjectiveTo investigate the mechanism of modified Banxia Xiexintang（MBXT） in improving ovarian dysfunction in polycystic ovary syndrome with insulin resistance（PCOS-IR） rats by inhibiting ferroptosis through the adenosine monophosphate（AMP）-activated protein kinase（AMPK）/fatty acid synthase（FASN）/glutathione peroxidase 4（GPX4） signaling pathway. MethodsSeventy-six female SD rats were randomly divided into a normal group（n=13） and a modeling group（n=63）. The modeling group established a PCOS-IR model by intragastric administration of letrozole combined with a high-fat diet for 21 days. After successful modeling， these rats were randomly divided into the model group， MBXT low-， medium-， and high-dose groups（6.62， 13.23， 26.46 g·kg^-1）， metformin group（0.158 g·kg^-1）， and high-dose of MBXT combined with ferroptosis inducer Erastin group（15 mg·kg^-1）， with 10 rats in each group. After 14 days of intervention， ovarian pathological morphology was observed by hematoxylin-eosin（HE） staining， the mitochondrial ultrastructure of granulosa cells was observed by transmission electron microscopy（TEM）， ovarian reactive oxygen species（ROS） levels were detected by dihydroethidium（DHE） probe， biochemical methods were used to detect Fe²⁺， malondialdehyde（MDA）， glutathione（GSH） and other indicators in ovarian tissues， serum sex hormone and insulin levels were measured by enzyme-linked immunosorbent assay（ELISA）， and the protein expressions of AMPK， FASN， acyl-CoA synthetase long-chain family member 4（ACSL4）， GPX4， and solute carrier family 7 member 11（SLC7A11） in ovarian tissues were detected by Western blot. ResultsCompared with the normal group， the model group showed polycystic changes in the ovaries， with atrophy of mitochondria in granulosa cells and increased membrane density. Serum levels of testosterone（T）， luteinizing hormone（LH）， and insulin were significantly increased（P<0.01）. The levels of ROS， MDA， 4-hydroxynonenal（4-HNE）， and Fe²⁺ in ovarian tissues were significantly elevated（P<0.01）， while adenosine triphosphate（ATP）， GSH， and reduced nicotinamide adenine dinucleotide phosphate （NADPH） levels were significantly decreased（P<0.01）. The phosphorylation levels of AMPK and acetyl-CoA carboxylase （ACC）， as well as the protein expressions of SLC7A11， GPX4， and ferroptosis suppressor protein 1（FSP1） were significantly downregulated（P<0.01）， whereas the expressions of FASN， ACSL4， and nuclear receptor coactivator 4（NCOA4） were significantly upregulated（P<0.01）. Compared with the model group， MBXT intervention at various doses improved the above pathological changes and biochemical indicators in a dose-dependent manner， with the high-dose group showing the most significant effect（P<0.01）. Compared with the MBXT high-dose group， the high-dose of MBXT combined with ferroptosis inducer Erastin group restored ovarian ferroptosis characteristics in rats， with increased ROS and lipid peroxidation products， and altered expressions of key proteins（P<0.05， P<0.01）. ConclusionMBXT can effectively improve ovarian function and metabolic disorders in PCOS-IR rats. Its mechanism may be related to activating the AMPK/ACC signaling pathway， downregulating FASN and ACSL4 to reduce lipid peroxidation substrates， and restoring glucose-6-phosphate dehydrogenase/phosphoglycerate dehydrogenase（G6PD/PHGDH） metabolic flux to enhance the GPX4/FSP1 antioxidant defense system， thereby inhibiting ferroptosis in ovarian granulosa cells.

The goal of this study was to identify MSX1 gene variants in multiple Chinese families with nonsyndromic oligodontia and analyse the functional influence of these variants. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variants in five families with nonsyndromic oligodontia, and a series of bioinformatics databases were used for variant confirmation and functional prediction. Phenotypic characterization of the members of these families was described, and an in vitro analysis was performed for functional evaluation. Five novel MSX1 heterozygous variants were identified: three missense variants [c.662A>C (p.Q221P), c.670C>T (p.R224C), and c.809C>T (p.S270L)], one nonsense variant [c.364G>T (p.G122*)], and one frameshift variant [c.277delG (p.A93Rfs*67)]. Preliminary in vitro studies demonstrated that the subcellular localization of MSX1 was abnormal with the p.Q221P, p.R224C, p.G122*, and p.A93Rfs*67 variants compared to the wild type. Three variants (p.Q221P, p.G122*, and p.A93Rfs*67) were classified as pathogenic or likely pathogenic, while p.S270L and p.R224C were of uncertain significance in the current data. Moreover, we summarized and analysed the MSX1-related tooth agenesis positions and found that the type and variant locus were not related to the severity of tooth loss. Our results expand the variant spectrum of nonsyndromic oligodontia and provide valuable information for genetic counselling.
Anodontia/genetics* ; Humans ; MSX1 Transcription Factor/genetics* ; Pedigree ; Whole Exome Sequencing

Objective To investigate correlation between QT interval(QT),corrected QT interval(QTc) and metabolic syndrome(MS). Methods Residents who participated in our survey concerning atherosclerosis and related diseases conducted in Shenyang were included. They accomplished questionnaire,physical examination, laboratory tests and electrocardiography test. We divided them into MS group and non-metabolic syndrome (NMS)group according to International Diabetes Federation(IDF)diagnostic criteria for MS. QT interval was measured from the standard 12-lead electrocardiogram. QTc was calculated by using Bazett and Fridericia equations. We analyze correlation of QT ,QTc and MS. Results A total of 739 residents who were 35~64 years old were included. Individuals with MS had longer QTcB and QTcF than NMS group[(415.8 ± 31.9)ms vs.(410.1 ± 32.1)ms, (407.2± 29.1)ms vs.(402.6 ± 28.8)ms,P<0.05]. The more the number of abnormal MS parameters they had, the longer the QT,QTcB and QTcF they had. Regression analysis showed that QT was associated with serum potassium,smoking,blood glucose,and LDL,and QTcB and QTcF were associated with hypertension,waist circumference and blood potassium. Conclusions MS is associated with corrected QTc. Careful ECG monitoring among persons with MS for early detection of a long corrected QT interval may prevent severe and often fatal arrhythmias or sudden death.

Objective:To study the relationship between neutrophil to lymphocyte ratio (N/L) and traditional cardio‐vascular risk factors among community 35~64‐year‐old residents .Methods :A total of 1884 residents (548 males and 1336 females) from urban Shenyang city received baseline condition questionnaire on cardiovascular diseases and re‐lated diseases from Apr 2011 to Feb 2012. According to presence of cardiovascular risk factors or not ,subjects were divided into healthy control group (n=675) and risk factor group (n=1209);according to number of risk factors , risk factor group was further divided into one risk factor group (n=491) ,two risk factors group (n=263) and ≥3 risk factors group (n=455) .Morning blood sample and urine sample were retained to measure blood and urine rou‐tine ,blood glucose and blood lipid profile etc in all subjects .N/L was compared and analyzed among all groups .Re‐sults:Among patients with only one of following risk factors [hypertension ,diabetes mellitus (DM) ,dyslipidemia and obesity] ,N/L levels of patients with hypertension or DM were significantly higher than that of healthy control group [1.55(1.15 ,1.95) ,1.60(1.21 ,2.07) vs .1.45(1.09 ,1.91)] , P<0.05 both ,and there were no significant difference between any other one risk factor group and healthy control group , P>0.05 all .Among risk factor sub‐groups ,N/L level of ≥3 risk factors group was significantly higher than that of two risk factors group [1.57(1.16 , 2.04) vs .1.41(1.07 ,1.89) ,P<0.05] ,and there was no significant difference between any other two groups (P>0.05) .Conclusion:N/L significantly related to hypertension or DM ,and N/L level of ≥3 risk factors group was sig‐nificantly higher than that of two risk factors group ,N/L is helpful to assess risk of cardiovascular diseases .

BACKGROUND AND OBJECTIVEHuman achaete-scute homolog 1 (hASH1) gene plays a critical role in development of the central nervous system, automatic nervous system, adrenal medullary chromaffin cells, thyroid C cells and pulmonary neuroendocrine cells. The aim of this study is to determine hASH1 gene expression in the normal lung tissue and various types of lung tumors, to analyze whether its expression correlated with pulmonary neuroendocrine markers, and to explore the possibility of hASH1 as clinical pathological markers in the neuroendocrine tumors compared with previous neuroendocrine tumor markers.

METHODShASH1, Chromogranin A, Synaptophysin and CD56 expression were examined in lung tumor specimens (lung inflammatory pseudotumor, squamous cell carcinoma, adenocarcinomas, large cell carcinoma, typical carcinoids, atypical carcinoids, large cell neuroendocrine carcinomas and small cell lung carcinoma and corresponding normal lung specimens) using immunohistochemistry (S-P method). Western blot and reverse transcription polymerase chain reaction (RT-PCR) assay were applied to detect the expressions of hASH1 protein and mRNA in lung cancer tissues.

RESULTShASH1 expression was positive in 2/16 (12.5%) typical carcinoids, 15/20 (75%) atypical carcinoids, 6/10 (60%) large cell neuroendocrine carcinomas and 31/40 (77.5%) small cell lung carcinoma, respectively, but not in any normal lung tissue (0/10), lung inflammatory pseudotumor (0/49), squamous cell carcinoma (0/30), adenocarcinomas (0/30) or large cell carcinoma (0/20). There was a significant difference in hASH1 expression between typical carcinoids and atypical carcinoids (P < 0.01), but not in large cell neuroendocrine carcinomas and small cell lung carcinoma (P > 0.05). hASH1 expression highly closely correlated with Chromogranin A, Synaptophysin and CD56 expression (P < 0.05).

CONCLUSIONhASH1 is a new kind of highly specific markers of pulmonary neuroendocrine tumours, and may be applied to clinical pathology diagnosis of the pulmonary neuroendocrine tumors.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Carcinoma, Large Cell ; genetics ; metabolism ; pathology ; Carcinoma, Neuroendocrine ; genetics ; metabolism ; pathology ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Humans ; Immunohistochemistry ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Neuroendocrine Tumors ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; genetics ; metabolism ; pathology