ObjectiveTo investigate the material basis of the pathogenesis of cerebral ischemic injury with blood stasis and toxin syndrome and to explore the protective effects of Huayu Jiedu prescription （HYJDP） on neutrophil extracellular trap-related cell death （NETosis） in cerebral ischemic injury following acute cerebral infarction. MethodsSeventy-two Sprague-Dawley （SD） rats were randomly divided into six groups （n=12 per group）： sham operation （Sham） group， blood stasis and toxin model （Model） group， low-， medium-， and high-dose HYJDP groups （HYJDP-L， HYJDP-M， and HYJDP-H； 9， 18， and 36 g·kg^-1， respectively）， and butylphthalide （NBP） group （0.06 g·kg^-1）. Except for the Sham group， rats in all other groups were subjected to carrageenan/dry yeast combined with a modified intraluminal filament method to establish a focal cerebral ischemia model of the middle cerebral artery with blood stasis and toxin syndrome. Neurological function was evaluated at 24 h after modeling using the Zea-Longa neurological deficit score. Cerebral infarction rate was assessed by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Pathological morphology of brain tissue was observed using hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was used to determine serum levels of interleukin-8 （IL-8）， myeloperoxidase-DNA complexes （MPO-DNA）， and citrullinated histone H3 （CitH3）. Protein expression of phosphorylated phosphatidylinositol 3-kinase （p-PI3K）， protein kinase B （p-Akt）， mammalian target of rapamycin （p-mTOR）， sequestosome 1 （p62）， and CitH3 in brain tissue was detected by Western blot. Immunofluorescence （IF） was used to detect the expression of neutrophil-specific marker Ly6G， CitH3， and neuron-specific nuclear protein （NeuN） in brain tissue. ResultsCompared with the Sham group， neurological deficit scores and cerebral infarction rates in the model group were significantly increased （P<0.01 for both）. HE staining showed varying degrees of neuronal degeneration and necrosis， characterized by blurred neuronal structures， nuclear pyknosis and fragmentation， cytoplasmic dissolution into a vacuolated reticular pattern， and mild glial cell proliferation. ELISA results showed that serum levels of IL-8， MPO-DNA， and CitH3 were significantly increased （P<0.01）. Western blot analysis demonstrated decreased expression of p-PI3K， p-Akt， p-mTOR， and p62， while CitH3 expression was significantly increased （P<0.01）. IF results showed an increased number of NETs⁺ cells and a significant decrease in NeuN⁺ cells （P<0.01）. Compared with the Model group， neurological deficit scores in the HYJDP-H group were significantly decreased （P<0.05）， and cerebral infarction rates in the HYJDP-H and NBP groups were significantly reduced （P<0.01）. HE staining showed that brain tissue damage was markedly alleviated in the HYJDP-H group. ELISA results showed that levels of IL-8， MPO-DNA， and CitH3 were significantly decreased in the HYJDP-M， HYJDP-H， and NBP groups （P<0.01）. Western blot analysis showed that expression of p-PI3K， p-Akt， p-mTOR， and p62 was significantly increased in the HYJDP-H and NBP groups， while CitH3 expression was significantly reduced in all drug intervention groups （P<0.01）. IF results showed that the number of NETs⁺ cells was significantly decreased and the number of NeuN⁺ cells was significantly increased in all drug intervention groups （P<0.01）. ConclusionNETs may be the material basis of the pathogenesis of cerebral ischemic injury characterized by blood stasis and toxin. HYJDP can regulate the PI3K/Akt/mTOR signaling pathway， reduce the release of pro-inflammatory mediators and NETosis-related products， alleviate cerebral ischemic injury caused by autophagy-dependent NETosis， and thereby exert a neuroprotective effect.

ObjectiveTo investigate the protective effect of Huayu Jiedu prescription against PC12 cell damage induced by a combination of neutrophil extracellular traps （NETs） and oxygen-glucose deprivation （OGD）. MethodsFirst， dimethyl sulfoxide （DMSO） was used to induce the differentiation of the human promyelocytic leukemia cell line （HL-60） into neutrophil-like cells （dHL-60）， and cell differentiation was assessed through Giemsa staining and flow cytometry. Next， phorbol myristate （PMA） was used to induce the formation of NETs in dHL-60 cells. The formation of NETs was assessed via SYTOX Green staining， and NETs were extracted by low-speed centrifugation. Finally， the NETs prepared through the conditioned medium were combined with OGD to induce a high-differentiation PC12 neuronal cell damage model through the co-culture method. This model was treated with the Huayu Jiedu prescription-containing serum. Cells were randomly allocated into a normal control group， a model （NETs + OGD） group， a traditional Chinese medicine （NETs + OGD + 5% Huayu Jiedu prescription-containing serum） group， and a NETosis inhibitor （100 mg·L^-1 DNase I + NETs + OGD） group. The cell counting kit-8 （CCK-8） assay was used to evaluate cell viability and screen the optimal intervention concentrations of NETs and Huayu Jiedu Formula-containing serum， as well as the optimal intervention time for OGD. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of myeloperoxidase-DNA complexes （MPO-DNA）， citrullinated histone H3 （CitH3）， and neutrophil elastase （NE）. Western blot was employed to quantify the protein levels of CitH3 and cleaved Caspase-3. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） staining and flow cytometry were employed to assess neuronal apoptosis. ResultsCompared with those in the normal control group， cells in the model group successfully differentiated into dHL60 cells after DMSO induction. Wright-Giemsa staining revealed that the cells in the model group had disappeared nucleoli， distinct reniform and lobulated changes of nuclei， highly condensed chromatin， and the cytoplasm containing pale purple fine granules. Flow cytometry showed that the mean fluorescence intensity （MFI） of CD11b in the model group was enhanced （P<0.01）. Compared with those in the normal control group， dHL60 cells in the model group successfully generated NETs after PMA induction. SYTOX Green staining revealed loss of cell membrane integrity， with chromatin released from the cytoplasm to form abundant extracellular， cloud-like fibrillar structures， and a marked increase in fluorescence intensity （P<0.01）. Through the CCK-8 assay for assessing the viability of highly differentiated PC12 cells， the optimal concentration for NETs was determined as 1 000 ng·L^-1， the optimal concentration of Huayu Jiedu formula-containing serum as 5%， and the optimal duration for OGD as 2 h. Compared with the normal control group， the model group showed elevated levels of MPO-DNA， CitH3， and NE （P<0.01） and upregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. Furthermore， TUNEL staining revealed an increased proportion of apoptotic cells （P<0.01） and flow cytometry showed an increased apoptosis rate （P<0.01） in the model group. Compared with the model group， the Huayu Jiedu formula-containing serum group and the NETosis inhibitor group exhibited declined levels of MPO-DNA， CitH3， and NE （P<0.05，P<0.01） and downregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. In addition， TUNEL staining showed a decrease in the proportion of apoptotic cells （P<0.01）， and flow cytometry revealed a decrease in the apoptosis rate （P<0.01）. ConclusionHuayu Jiedu prescription may exert a protective effect against ischemic neuronal damage by inhibiting NET formation or alleviating NET-induced neurotoxicity

ObjectiveTo investigate the protective effect of Huayu Jiedu prescription against PC12 cell damage induced by a combination of neutrophil extracellular traps （NETs） and oxygen-glucose deprivation （OGD）. MethodsFirst， dimethyl sulfoxide （DMSO） was used to induce the differentiation of the human promyelocytic leukemia cell line （HL-60） into neutrophil-like cells （dHL-60）， and cell differentiation was assessed through Giemsa staining and flow cytometry. Next， phorbol myristate （PMA） was used to induce the formation of NETs in dHL-60 cells. The formation of NETs was assessed via SYTOX Green staining， and NETs were extracted by low-speed centrifugation. Finally， the NETs prepared through the conditioned medium were combined with OGD to induce a high-differentiation PC12 neuronal cell damage model through the co-culture method. This model was treated with the Huayu Jiedu prescription-containing serum. Cells were randomly allocated into a normal control group， a model （NETs + OGD） group， a traditional Chinese medicine （NETs + OGD + 5% Huayu Jiedu prescription-containing serum） group， and a NETosis inhibitor （100 mg·L^-1 DNase I + NETs + OGD） group. The cell counting kit-8 （CCK-8） assay was used to evaluate cell viability and screen the optimal intervention concentrations of NETs and Huayu Jiedu Formula-containing serum， as well as the optimal intervention time for OGD. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of myeloperoxidase-DNA complexes （MPO-DNA）， citrullinated histone H3 （CitH3）， and neutrophil elastase （NE）. Western blot was employed to quantify the protein levels of CitH3 and cleaved Caspase-3. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） staining and flow cytometry were employed to assess neuronal apoptosis. ResultsCompared with those in the normal control group， cells in the model group successfully differentiated into dHL60 cells after DMSO induction. Wright-Giemsa staining revealed that the cells in the model group had disappeared nucleoli， distinct reniform and lobulated changes of nuclei， highly condensed chromatin， and the cytoplasm containing pale purple fine granules. Flow cytometry showed that the mean fluorescence intensity （MFI） of CD11b in the model group was enhanced （P<0.01）. Compared with those in the normal control group， dHL60 cells in the model group successfully generated NETs after PMA induction. SYTOX Green staining revealed loss of cell membrane integrity， with chromatin released from the cytoplasm to form abundant extracellular， cloud-like fibrillar structures， and a marked increase in fluorescence intensity （P<0.01）. Through the CCK-8 assay for assessing the viability of highly differentiated PC12 cells， the optimal concentration for NETs was determined as 1 000 ng·L^-1， the optimal concentration of Huayu Jiedu formula-containing serum as 5%， and the optimal duration for OGD as 2 h. Compared with the normal control group， the model group showed elevated levels of MPO-DNA， CitH3， and NE （P<0.01） and upregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. Furthermore， TUNEL staining revealed an increased proportion of apoptotic cells （P<0.01） and flow cytometry showed an increased apoptosis rate （P<0.01） in the model group. Compared with the model group， the Huayu Jiedu formula-containing serum group and the NETosis inhibitor group exhibited declined levels of MPO-DNA， CitH3， and NE （P<0.05，P<0.01） and downregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. In addition， TUNEL staining showed a decrease in the proportion of apoptotic cells （P<0.01）， and flow cytometry revealed a decrease in the apoptosis rate （P<0.01）. ConclusionHuayu Jiedu prescription may exert a protective effect against ischemic neuronal damage by inhibiting NET formation or alleviating NET-induced neurotoxicity

Objective:This study investigated the dynamic expression of lipocalin-2(LCN2)and its receptor,brain-type organic cation transporter protein(BOCT),in spinal cords of hSOD1G93A transgenic mice during disease pro-gression,providing potential targets for early anti-inflammatory therapy for ALS.Methods:Utilizing hSOD1G93A trans-genic mice and their wild-type littermates(WT)as animal models,this investigation examined the expression of LCN2 and BOCT at four distinct disease stages:pre-symptomatic stage(60 d),early-symptomatic stage(95 d),symptomatic stage(108 d),and late-symptomatic stage(122 d).Spinal cords were harvested,then RT-qPCR,Western blot,and immunofluorescence double-labeling techniques were employed to assess alteration expressions of LCN2 and BOCT.Ad-ditionally,BV2 cells transfected with the pcDNA3.1-G93A-SOD1 overexpression plasmid served as an in vitro hSOD1G93A BV2 microglial model.After stimulated with LPS for 24 hours,LCN2 mRNA and protein expression in hSOD1G93A BV2 microglial cells and its culture medium were measured by RT-qPCR and ELISA respectively,while BOCT expression was measured by Western blot.Results:Compared with WT mice littermates,increased expression of LCN2 mRNA was detected in the spinal cords of hSOD1G93A transgenic mice at 108 and 122 d.No significant differences were observed in LCN2 or BOCT protein expression in the spinal cords of hSOD1G93A transgenic mice from 60 to 122 d.Double-immunofluorescence labeling revealed co-localization of LCN2 and BOCT with the microglial marker Iba-1 in the ventral horn of lumbar spinal cord of hSOD1G93A transgenic mice from 95 to 122 d.In hSOD1G93A BV2 microglial model,LPS stimulation led to a significantly increased LCN2 mRNA expression and protein secretion.Conversely,there was no significant change in BOCT protein expression after LPS stimulation.Conclusion:Our findings suggest that during ALS progression,there is an enhanced expression and release of LCN2 from activated microglia,potentially exacerbating neuroinflammation and neuronal degeneration.

Objective:To analyze the clinical characteristics and prognosis of dengue fever inpatients in Guangzhou area, so that to provide references for clinical diagnosis and treatment.Methods:The demographic data, laboratory findings, clinical manifestations, and outcomes of 116 dengue fever cases admitted to Guangzhou Red Cross Hospital, Jinan University and the First Affiliated Hospital, Jinan University from August 1st to December 21st, 2024 were retrospectively analyzed.Results:Among the 116 patients, 99 cases (85.34%) were classified as non-severe dengue, while 17(14.66%) progressed to severe dengue (SD). The cohort was comprised of 62(53.45%) males and 54(46.55%) females, with an age of 63.5(22.0) years, and the hospitalization duration was 6(4) days, with the fever duration of 6(2) days. Sixty-two patients (53.45%) were complicated with comorbidities, among which hypertension (66.13%(41/62)), diabetes mellitus (25.81%(16/62)) and coronary atherosclerotic heart disease (22.58%(14/62)) accounted for the highest proportion. Primary clinical manifestations included fever (113 cases, 97.41%), with 86.21%(100/116) experiencing fever ≥five days, followed by fatigue (92 cases, 79.31%), myalgia (65 cases, 56.03%), anorexia (56 cases, 48.28%), cough/sputum (55 cases, 47.41%), and headache (54 cases, 46.55%). Early laboratory findings revealed leukopenia (2.22(1.75)×10 9/L], lymphopenia (0.63(0.50)×10 9/L), and thrombocytopenia (77.00(57.00)×10 9/L). Among 17 SD cases, 17 cases experienced severe organ damage, two cases had severe gastrointestinal bleeding, and two cases had septic shock. Among the patients with severe organ damage, there were eight cases of severe cardiac injury, followed by six cases of severe respiratory system injury, four cases of multiple organ dysfunction syndrome, three cases of acute kidney injury, three cases of neurological injury, and three cases of disseminated intravascular coagulation. Severe cardiac injury was manifested with acute heart failure in five cases, malignant arrhythmia in three cases, stress cardiomyopathy in one case, and acute myocardial infarction in one case. Severe respiratory system injuries were manifested with acute respiratory distress syndrome in five cases, severe pneumonia in three cases, and acute pulmonary embolism in one case. Neurological damage was manifested with viral meningitis in one case, acute cerebral infarction in one case, and sensorineural hearing loss in one case. All patients were cured or discharged after symptomatic supportive treatment and active prevention and treatment of complications. Conclusions:In 2024, dengue fever inpatients in Guangzhou area predominantly consist of elderly patients, who are complicated with comorbidities and present a high proportion of severe cases. Severe patients are primarily characterized by organ damage, with cardiovascular system injury being the most common severe manifestation. Dengue patients have a good prognosis.

OBJECTIVE: To explore the incidence, clinical features, genetic variant characteristics and prognosis of Glutaric acidemia type I (GA1) among neonates from Henan Province. METHODS: A total of 814 625 neonates undergoing screening for inherited metabolic diseases by tandem mass spectrometry (MS/MS) at the Third Affiliated Hospital of Zhengzhou University from January 2016 to December 2022 were selected as the study subjects. A retrospective method was adopted to collect the clinical data of the patients. Whole exome sequencing was carried out to detect GCDH gene variants in individuals with positive results by GA1 newborn screening, and Sanger sequencing was used to verify the candidate variants. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the pathogenicity of candidate variants was rated. This study was approved by the Medical Ethics Committee of the Hospital (Ethics Number: 2019 Medical Ethics Review No. 67). RESULTS: Eight cases of GA1 were diagnosed among the 814 625 neonates. Blood glutaryl carnitine (C5DC) and urine glutaric acid (GA) levels of the 8 children were higher than the normal reference values. In total 12 variants were detected, all of which were missense variants. c.1064G>A (p.Arg355His) was the most common one, accounting for 21.4% (3/14). Three GCDH gene variants, including 1297G>C (p.Ala433Pro), c.467G>A (p.Gly156Asp) and c.1125T>G (p.Cys375Trp), were previously unreported. REVEL software analysis predicted that all of the three variants were harmful. 3D protein structure modeling indicated that the three variants may cause amino acid residue alterations, and c.1297G>C (p.Ala433Pro) and c.1125T>G (p.Cys375Trp) may result in increase in hydrogen bonds and affect the function of GCDH protein. By December 2023, one of the eight children had deceased, and another child had severe clinical symptoms with poor prognosis. Six children had a good prognosis, of which two had mild motor development delay and four had normal development without clinical symptoms. CONCLUSION The incidence of GA1 in newborns screened by MS/MS in Henan Province is 1/101 828, and the carrier rate of pathogenic GCDH variants is 1/160. The c.1064G>A (p.Arg355His) may be the hotspot variant of the GCDH gene among children with GA1 in Henan. Discovery of the three novel variants has enriched the mutational spectrum of the GCDH gene and provide a basis for the early diagnosis, treatment, prognosis and genetic counseling of this disease.
Humans ; Amino Acid Metabolism, Inborn Errors/epidemiology* ; Glutaryl-CoA Dehydrogenase/chemistry* ; Infant, Newborn ; Female ; Neonatal Screening/methods* ; Male ; Brain Diseases, Metabolic/epidemiology* ; China/epidemiology* ; Retrospective Studies ; Mutation ; Genetic Variation ; Glutarates

Objective:To investigate the anatomical feasibility of the endoscopic transorbital approach (ETOA) to the orbital apex and lateral middle cranial fossa, to identify stable and recognizable surgical landmarks under endoscopic visualization, and to provide morphometric data for preoperative planning and intraoperative navigation.Methods:Stepwise anatomical dissection was performed on five formalin-fixed cadaveric heads and one fresh arterially injected cadaveric specimen to simulate the ETOA using a 0° endoscope. Key structures and their anatomical relationships were observed and recorded. Additionally, high-resolution CT scans of 50 adults were retrospectively analyzed. Three-dimensional reconstructions and measurements were performed using Mimics 17.0 software. Spatial validation was performed using 17 dry skulls to verify the consistency and reliability of osseous anatomical landmarks.Results:Cadaveric dissection identified the meningo-orbital band, superior orbital fissure, optic canal, foramen rotundum, and foramen ovale as reliable surgical landmarks for the ETOA. A topographic map of the surgical region was established based on the endoscopic view. CT measurements revealed the following distances (Mean±SD): the midpoint of the supraorbital rim to the foramen rotundum (57.31±3.59) mm and foramen ovale (71.46±3.42) mm; the lateral orbital rim to the lateral edge of the superior orbital fissure (37.38±2.52) mm; the distance from the superior orbital fissure to the optic canal (9.98±1.49) mm; and the distance from the anterior ethmoidal artery to the optic canal (19.98±2.05) mm. These measurements were consistent with dry skull data, indicating that these osseous landmarks had stable spatial relationships and were suitable for intraoperative localization.Conclusions:The ETOA provides favorable anatomical accessibility and clinical feasibility for lesions involving the orbital apex and lateral skull base. Key osseous structures demonstrate high identifiability and stable spatial relationships, serving as critical references for intraoperative navigation and preoperative pathway planning. The quantitative anatomical framework established in this study provides critical morphometric support for minimally invasive surgery targeting lesions in this region.

Objective To investigate the effects of Zuogui Jiangtang Tongmai Prescription on short-chain fatty acids(SCFAs)and G protein-coupled receptor 109A(GPR109A)in diabetic atherosclerotic mice;To explore its mechanism of improving diabetic atherosclerosis.Methods ApoE-/-mice were fed with high sugar and high fat diet and intraperitoneal injection of streptozotocin to establish atherosclerosis model of diabetes.After modeling,the mice were randomly divided into the model group,Western medicine group(metformin hydrochloride+atorvastatin),and Zuogui Jiangtang Tongmai Prescription low-,medium-and high-dosage groups(19,38,76 g/kg);and the other C57BL/6J mice were set as the control group,with 6 mice in each group.Each group was given solution for gavage,once a day for 4 weeks.Blood glucose of the mice were detected,HE staining was used to observe the morphology of the aorta,TG,TC,LDL-C and HDL-C contents were detected by fully automated biochemistry analyzers,ELISA was used to detect the contents of serum HbA1c,fasting insulin(FINS),interleukin(IL)-1β,IL-6 and tumor necrosis factor(TNF)-α,gas chromatography was used to detect the content of intestinal SCFAs,and RT qPCR and Western blot were used to detect the mRNA and protein expressions of GPR109A in ileal tissue and nuclear factor(NF)-κB p65 in aortic tissue,respectively.Results Compared with the control group,obvious plaques and inflammatory cells infiltration were seen in the aorta of the model group,the blood glucose and serum HbA1c,TG,TC,LDL-C,IL-1β,IL-6 and TNF-α contents increased(P<0.01),FINS and HDL-C content decreased(P<0.01),intestinal acetate,propionate,butyric acid,isobutyric acid and isovaleric acid contents decreased(P<0.01),GPR109A mRNA and protein expression in ileal tissue decreased,NF-κB p65 mRNA and protein expression in aortic tissue increased(P<0.01).Compared with the model group,the aortic plaque area and inflammatory cells infiltration were significantly improved in each drug intervention group,the blood glucose and serum HbA1c,TG,TC,LDL-C,IL-1β,IL-6 and TNF-α contents decreased(P<0.05,P<0.01);the FINS and HDL-C content increased(P<0.05,P<0.01),intestinal acetic,propionic,butyric acid,isobutyric acid and isovaleric acid contents increased(P<0.05,P<0.01),GPR109A mRNA and protein expression in ileal tissue increased and NF-κB p65 mRNA and protein expression in ileal tissue decreased decreased(P<0.05,P<0.01).Conclusion Zuogui Jiangtang Tongmai Prescription can improve glucolipid metabolism and inflammatory response in diabetic atherosclerotic mice,which may be related to regulating SCFAs/GPR109A pathway.

ObjectiveTo explore the regulation of hypothalamic-pituitary-gonadal （HPG） axis by modified Erxian decoction in rats with perimenopausal syndrome （PMS） and to further analyze the expression of proteins related to the silent information regulator 1 （SIRT1）/hypothalamic kisspeptin （Kisspeptin）/gonadotropin-releasing hormone （GnRH） signaling pathway in the arcuate nucleus region （ARC） of the hypothalamus， so as to reveal the potential target of action and molecular biological mechanism of modified Erxian decoction for the treatment of perimenopausal syndrome. MethodsAn animal model was established via the incomplete castration method， with successful modeling confirmed by the exfoliated cervical cell smear method. The 48 rats were divided into six groups based on the randomization principle after successful modeling， including a sham operation group， a model group， an estradiol valerate group （0.09 mg∙kg^-1∙d^-1）， high-， medium-， and low-dose modified Erxian decoction groups （7.614， 3.807，1.903 5 g∙kg^-1∙d^-1）， with 8 rats in each group. The estradiol valerate group and the high-， medium- and low-dose modified Erxian decoction groups were continuously administered by gavage for 28 days， and the indicators were detected 24 hours after the last administration. Body weights and uterine indices were measured. The pathological changes of the uterus were observed by hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the levels of estradiol （E₂）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and gonadotropin-releasing hormone （GnRH）. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to determine the expression levels of SIRT1， Kisspeptin， kisspeptin receptor （GPR54）， and GnRH in the ARC region of the hypothalamus and gonadotropin-releasing hormone receptor （GnRH-R） in pituitary. ResultsCompared with the sham operation group， rats in the model group had a significantly increased body weight （P0.01）， reduced wet weight and index of uterus （P0.01）， endometrial thinning or atrophy， glandular atrophy， and a decreasing number of glands. Additionally， serum levels of E₂ and the expression of SIRT1 in the ARC region of the hypothalamus significantly decreased （P0.01）. Serum levels of FSH， LH， and GnRH， the expression of Kisspeptin， GPR54， and GnRH in the ARC region of the hypothalamus， and GnRH-R in pituitary significantly increased （P0.01）. Compared with the model group， the estradiol valerate group and the high-， medium-dose modified Erxian decoction groups had significantly reduced body weight， serum levels of FSH， LH， and GnRH， and expression of Kisspeptin， GPR54， and GnRH in the ARC region of the hypothalamus and GnRH-R in pituitary （P0.05， P0.01） and significantly increased wet weight and index of uterus， serum level of E₂， and expression of SIRT1 in the ARC region of the hypothalamus （P0.05， P0.01）. In addition， they showed thickened endometrium， increased number of endometrial glands， and improved glandular atrophy. ConclusionModified Erxian decoction regulates the function of the HPG axis through multi-targets， and its mechanism of action may be related to the up-regulation of the expression of SIRT1 in the ARC region of the hypothalamus， the inhibition of the over-activation of the Kisspeptin/GnRH signaling pathway， the regulation of the expression of GnRH-R in the pituitary， the restoration of secretion balance of gonadotropins， and the elevation of the estrogen level. This study provides an experimental basis for the interpretation of the scientific connotation of modified Erxian decoction in the treatment of perimenopausal syndrome and a theoretical reference for the development of a novel therapeutic strategy based on the SIRT1/Kisspeptin/GnRH pathway.

Objective:To explore the incidence, clinical features, genetic variant characteristics and prognosis of Glutaric acidemia type I (GA1) among neonates from Henan Province.Methods:A total of 814 625 neonates undergoing screening for inherited metabolic diseases by tandem mass spectrometry (MS/MS) at the Third Affiliated Hospital of Zhengzhou University from January 2016 to December 2022 were selected as the study subjects. A retrospective method was adopted to collect the clinical data of the patients. Whole exome sequencing was carried out to detect GCDH gene variants in individuals with positive results by GA1 newborn screening, and Sanger sequencing was used to verify the candidate variants. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the pathogenicity of candidate variants was rated. This study was approved by the Medical Ethics Committee of the Hospital (Approval Number: 2019 Medical Ethics Review No. 67). Results:Eight cases of GA1 were diagnosed among the 814 625 neonates. Blood glutaryl carnitine (C5DC) and urine glutaric acid (GA) levels of the 8 children were higher than the normal reference values. In total 12 variants were detected, all of which were missense variants. c. 1064G>A (p.Arg355His) was the most common one, accounting for 21.4% (3/14). Three GCDH gene variants, including 1297G>C (p.Ala433Pro), c. 467G>A (p.Gly156Asp) and c. 1125T>G (p.Cys375Trp), were previously unreported. REVEL software analysis predicted that all of the three variants were harmful. 3D protein structure modeling indicated that the three variants may cause amino acid residue alterations, and c. 1297G>C (p.Ala433Pro) and c. 1125T>G (p.Cys375Trp) may result in increase in hydrogen bonds and may affect the function of GCDH protein. By December 2023, one of the eight children had deceased, and another child had severe clinical symptoms with poor prognosis. Six children had a good prognosis, of which two had mild motor development delay and four had normal development without clinical symptoms. Conclusion:The incidence of GA1 in newborns screened by MS/MS in Henan Province is 1/101 828, and the carrier rate of pathogenic GCDH variants is 1/160. The c. 1064G>A (p.Arg355His) may be the hotspot variant of the GCDH gene among children with GA1 in Henan. Discovery of the three novel variants has enriched the mutational spectrum of the GCDH gene and provide a basis for the early diagnosis, treatment, prognosis and genetic counseling of this disease.