ObjectiveTo investigate the migration and distribution characteristics of chemical constituents in blood and hippocampal tissues before and after vinegar processing of Citri Reticulatae Pericarpium Viride（CRPV）， and to explore the potential material basis and mechanisms underlying their regulatory effects on emotional disorders by comparing the effects of raw and vinegar-processed products of CRPV. MethodsUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） was employed to characterize and identify the chemical constituents of raw and vinegar-processed products of CRPV extracts， as well as their migrating components in blood and hippocampal tissues after oral administration. Reference standards， databases， and relevant literature were utilized for compound annotation， with data processing performed using PeakView 1.2 software. Seventy male C57BL/6 mice were randomly divided into seven groups， including the blank group， model group， diazepam group（2.5 mg·kg^-1）， raw CRPV low/high dose groups（0.6， 1.2 g·kg^-1）， and vinegar-processed CRPV low/high dose groups（0.6， 1.2 g·kg^-1）， with 10 mice per group. Except for the blank group， all other groups underwent chronic restraint stress（2 h·d^-1） for 20 d. Each drug-treated group received oral administration at the predetermined dose starting 10 d after modeling， with a total treatment duration of 10 d. Following model-based drug administration， mice underwent open-field， forced swimming， and elevated plus maze tests. After anesthesia with isoflurane， whole brains were collected from each group of mice， and hippocampi were dissected. Reactive oxygen species（ROS） level in hippocampal tissues was quantified by enzyme-linked immunosorbent assay（ELISA）. Hematoxylin-eosin（HE） staining was used to observe hippocampal tissue morphology. Immunofluorescence was performed to detect neuronal nuclei（NeuN） and peroxisome proliferator-activated receptor alpha（PPARα） expressions in hippocampal tissue. Then， pharmacodynamic evaluations were conducted to assess the effects of raw and vinegar-processed CRPV on mood disorders， exploring the potential mechanisms. ResultsVinegar processing caused significant changes in the chemical composition of CRPV， with 18 components showing increased relative content and 35 components showing decreased relative content. The primary changes occurred in flavonoid compounds， including 20 flavonoids， 20 flavonoid glycosides， 3 triterpenes， 3 phenolic acids， 1 alkaloid， and 6 other compounds. Twenty-one components were detected in blood（15 methoxyflavones， 4 flavonoid glycosides， and 2 phenolic acids）， with 17 shared between raw and vinegar-processed CRPV. Seven components reached hippocampal tissues（all common to both forms）. In regulating emotional disorders， Vinegar-processed CRPV exhibited superior antidepressant-like effects compared to raw products. HE staining revealed that both treatments improved hippocampal neuronal morphology， particularly in the damaged CA1 and CA3 regions. Immunofluorescence and ELISA analyses demonstrated that both raw and vinegar-processed CRPV significantly modulated NeuN and PPARα expressions in hippocampal tissue while alleviating oxidative stress induced by excessive ROS（P<0.05）. ConclusionThe chemical composition of CRPV undergoes changes after vinegar processing， but the migrating components in blood and hippocampus are primarily methoxyflavonoids. These components may serve as the potential material basis for activating the PPARα pathway， thereby negatively regulating ROS generation in the hippocampus， reducing oxidative stress， and promoting the development of NeuN-positive neurons. These findings provide experimental evidence for enhancing quality standards， pharmacodynamic material research， and active drug development of raw and vinegar-processed CRPV.

Retinopathy of prematurity(ROP)is a leading cause of childhood blindness, with extremely preterm and very-low-birth-weight infants now constituting the main high-risk group. ROP progresses in two stages: early retinal microvascular degeneration and progressive vascular arrest, followed by abnormal neovascularization in the avascular area. Early oxidative and nitrosative stress—amplified by oxygen fluctuations and immature antioxidant defenses—drives the two-phase pathogenesis via hypoxia-inducible factor/vascular endothelial growth factor(HIF/VEGF), NOX/STAT3, and nuclear factor erythroid 2 related factor 2(Nrf2)-antioxidant response element(ARE)pathways, mediating apoptosis of endothelial cells, damage to barrier and pathological angiogenesis. This review systematically analyzes different oxygen-induced retinopathy(OIR)models, elucidates key signaling pathways including Notch, Wnt in physiological and pathological vascularization, with particular emphasis on the biphasic effects of Nrf2 and the differential roles of NOX signaling between phases. We also discuss the limitations of anti-VEGF therapy and oxygen management principles. Reactive oxygen species(ROS)play context-dependent roles across vaso-obliteration and neovascularization phases. Based on mechanistic insights, we propose future directions including combined/sequential interventions, ferroptosis and lipid peroxidation targeting, nano-delivery systems for enhanced bioavailability, and perinatal safety assessment strategies, aiming to provide translatable mechanistic basis for reducing pathological neovascularization while promoting physiological vascular development.

ObjectiveTo screen and evaluate the antidepressant compounds of Curcumae Rhizoma， and explore its mechanism of regulating the nuclear factor erythroid 2-related factor 2（Nrf2）/glutathione（GSH） peroxidase 4（GPX4）/GSH pathway from an antioxidant perspective. MethodsThe antioxidant activities in vitro of 11 characteristic components from Curcumae Rhizoma， including curcumol， curgerenone， curdione， curzerene， curcumenol， curcumenone， dehydrocurdione， isocurcumenol， furanodienone， furanodiene and zederone， were detected using 1，1-diphenyl-2-picrylhydrazyl（DPPH） and 2，2'-azinobis-（3-ethylbenzothiazoline-6-sulphonic acid） diammonium salt（ABTS） radical scavenging assays. The depression in Drosophila melanogaster was induced by chronic unpredictable mild stress（CUMS）， and W1118 wild-type male D. melanogaster were randomly divided into blank group， model group， curcumol group， curgerenone group， curdione group， curzerene group， curcumenol group，curcumenone group， dehydrocurdione group， isocurcumenol group， furanodienone group， furanodiene group， zederone group and fluoxetine group（10 μmol·L^-1）. The treatment groups received a dose of 0.1 g·L^-1 of 11 characteristic components from Curcumae Rhizoma， while the blank and model groups were administered equivalent volumes of solvent. The sucrose preference test， climbing test and forced swimming test were used to evaluate the behavioral indicators of depression in D. melanogaster. Liquid chromatography-mass spectrometry（LC-MS） was used to detect the levels of 5-hydroxytryptamine（5-HT） and dopamine（DA） in the brain of D. melanogaster， and the entropy weight method was used to comprehensively evaluate neurobehavioral and neurotransmitter indicators， resulting in the identification of the antidepressant active components of Curcumae Rhizoma. In addition， a mouse depression model was established by CUMS， and C57BL/6J mice were randomly divided into blank group， model group， low and high dose groups of curzerene（0.5， 1 mg·kg^-1）， and fluoxetine group（10 mg·kg^-1） to confirm the antidepressant effect of the optimal active ingredient by behavioral analysis. Flow cytometry was used to detect the content of reactive oxygen species（ROS） in the hippocampus of mice from each group. Enzyme-linked immunosorbent assay was used to detect the contents of adenosine triphosphate（ATP）， superoxide dismutase（SOD）， catalase（CAT） and GSH. Transmission electron microscope（TEM） was used to observe the effect of curzerene on the ultrastructure of mitochondria in hippocampal tissue. Western blot was performed to determine the level of Nrf2 protein， and Nrf2 inhibitor（ML385） was used to verify the relationship between the antidepressant effect of curzerene and regulation of Nrf2. Real time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to detect the effect of curzerene on the mRNA expression level of GPX. ResultsIn vitro antioxidant experiments showed that curzerene and curgerenone exhibited the most significant ability to scavenge free radicals， and comprehensive evaluation results of entropy weight method indicated that curzerene stood out as the most promising active component. Compared with the blank group， the model group exhibited a significant decrease in sucrose preference coefficient and the number of times entering the open field center（P<0.01）， as well as a significant increase in immobility time in the forced swimming and tail suspension tests（P<0.01）， and the ROS content in hippocampus significantly elevated（P<0.01）， while the ATP content significantly reduced（P<0.01）. In the hippocampal neurons of the model group， mitochondrial cristae were disordered， with vacuolation of the inner membrane and severe damage. Nrf2 protein expression level in the model group was significantly decreased（P<0.05）， and the antioxidant enzymes SOD， CAT and GSH contents were also significantly reduced（P<0.05， P<0.01）， and the gene expression levels of GPX1， GPX4 and GPX7 were significantly decreased（P<0.01）. Compared with the model group， the high-dose group of curzerene showed a significant increase in the sucrose preference coefficient and the number of times entering the open field center（P<0.05）， as well as a significant decrease in immobility time in the forced swimming and tail suspension tests（P<0.05， P<0.01）. The ROS content in the hippocampus of the high-dose group of curzerene was significantly reduced（P<0.01）， while the ATP content was significantly increased（P<0.05）. The neuronal mitochondrial damage in the hippocampus of the high-dose group of curzerene was alleviated， and the expression level of Nrf2 protein was significantly increased（P<0.05）. The Nrf2 inhibitor ML385 reversed the improvement of curzerene on depressive behaviors in CUMS mice. The GSH content in the hippocampal neurons of the high-dose group of curzerene was significantly increased（P<0.01）， while there were no significant differences in SOD and CAT contents. The expression level of GPX4 gene in the hippocampal neurons of the high-dose group of curzerene was significantly increased（P<0.05）， while there were no significant differences in other GPX genes. ConclusionCurzerene is the best component with antidepressant activity in Curcumae Rhizoma. It may improve mitochondrial dysfunction to exert its antidepressant effect by regulating Nrf2 and its downstream GPX4/GSH pathway rather than CAT or SOD pathways.

BACKGROUND:Preliminary clinical observations found that Jiawei Chunze Decoction is an effective formula for clinical treatment of urinary retention after spinal cord injury.Animal experiments have found that the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway is closely related to the degree of bladder dysfunction. OBJECTIVE:To further investigate the effects of Jiawei Chunze Decoction on bladder function and PI3K/Akt signaling pathway in rats with urinary retention. METHODS:Sixty female Sprague-Dawley rats were randomly divided into sham operation group,model group,Jiawei Chunze Decoction low-dose group,Jiawei Chunze Decoction high-dose group and agonist group.In the sham operation group,the spinal cord was exposed but not transected.In the other groups,the modified Hassan Shaker spinal cord transection method was used to prepare the model of sacral medullary injury.At 24 hours after modeling,the sham operation group and model group were intragastrically given equal volume of normal saline,Jiawei Chunze Decoction low-dose and high-dose groups were given Jiawei Chunze Decoction granules containing 14.4 and 28.8 g/kg,respectively,via intragastric administration for 4 weeks,and the agonist group was treated with an intraperitoneal injection of PI3K/Akt signaling pathway agonist 740Y-P at a dose of 0.02 mg/kg.After 4 weeks of treatment,the maximum bladder capacity,leakage point pressure and bladder compliance of rats in each group were detected by urine flow dynamics.The minimum bladder contraction tension and frequency of rats in each group were detected by detrusor pull test.The pathological changes of the rat bladder in each group were observed by hematoxylin-eosin staining.The concentrations of GRP78,CHOP and Caspase-12 in serum were detected by ELISA,and the mRNA and protein expressions of PI3K,Akt,GRP78,CHOP and Caspase-12 in bladder tissues were detected by RT-PCR and western blot,respectively. RESULTS AND CONCLUSION:Compared with the sham operation group,the maximum bladder volume,bladder compliance and minimum systolic tension of rats in the model group were increased(P<0.05),and the leakage point pressure and bladder contraction frequency were decreased(P<0.05);serum GRP78,CHOP,and Caspase-12 levels were also increased(P<0.05).The arrangement of bladder epithelial cells in the model group was disordered,and there was monocyte infiltration between cells,tissue edema,and detrusor tract atrophy.The mRNA and protein expressions of PI3K and Akt in bladder tissues were significantly decreased in the model group compared with the sham operation group,while those of GRP78,CHOP and Caspase-12 were increased(P<0.05).Compared with the model group,the maximum bladder volume,bladder compliance and minimum systolic tension of rats were decreased in the Jiawei Chunze Decoction low-dose,high-dose and agonist groups after 4 weeks of intervention(P<0.05),while the leakage point pressure and bladder contraction frequency were increased(P<0.05);serum GRP78,CHOP,Caspase-12 levels were decreased(P<0.05).The bladder epithelial cells in the three intervention groups were distributed evenly,arranged neatly,with less inflammatory cell infiltration and fuller detrusor muscle bundle.Compared with the model group,the mRNA and protein expressions of PI3K and Akt were increased in the three intervention groups,while those of GRP78,CHOP and Caspase-12 were decreased(P<0.05).The Jiawei Chunze Decoction high-dose group was better than the Jiawei Chunze Decoction low-dose group and shared the similar results with the agonist group.To conclude,Jiawei Chunze Decoction can improve the bladder function of rats with urinary retention after spinal cord injury,and the mechanism may be related to reducing the occurrence of endoplasmic reticulum stress in bladder tissue through the PI3K/Akt signaling pathway,and then alleviating apoptosis.

BACKGROUND:Targeted therapy based on nuclear transcription factor kappa B signaling pathway to explore neuroinflammation is increasingly worth exploring,and the advantages of Chinese medicine such as many targets,wide range,rich mechanisms,and few side effects have great potential in the treatment of various diseases. OBJECTIVE:Based on the nuclear transcription factor kappa B signaling pathway,this paper systematically expounded and summarized the research progress of kaempferol,safflower yellow,baicalin,and triptolide in the treatment of neuroinflammation after spinal cord injury. METHODS:Search terms"spinal cord injury,inflammation,anti-inflammatory,traditional Chinese medicine monomer,monomeric compound,NF-κB signaling pathway,flavonoids,glycosides,phenols,esters,alkaloids"were searched in CNKI and PubMed databases.Totally 67 articles were finally included. RESULTS AND CONCLUSION:(1)The role of nuclear transcription factor kappa B signaling pathway in the nervous system is complex and diverse,which can regulate neutrophils,microglia,astrocytes,and macrophages,and mediate the occurrence and development of inflammation after injury.(2)The effects of traditional Chinese medicine monomers such as baicalin on the degradation of nuclear transcription factor kappa B inhibitory protein,the inhibition of phosphorylation process by safflowerin on nuclear transcription factor kappa B signaling pathway,and the inhibition of kaempferol on nuclear transcription factor kappa B signaling pathway p65 nuclear translocation can reduce the impact of inflammatory response on the body,thereby promoting the recovery of neurological function.(3)The nuclear transcription factor kappa B signaling pathway can promote inflammation and immune cell migration and activation in the early stage of injury,and can promote the repair of injury site and the occurrence of fibrosis in the middle and late stages of injury.Appropriate activation of the nuclear transcription factor kappa B signaling pathway can promote the release of inflammatory factors,improve the antioxidant capacity of cells,and promote the activation of immune cells,but the over-activated nuclear transcription factor kappa B signaling pathway can easily lead to the occurrence and continuation of chronic inflammation and the inhibition of apoptosis.(4)Future research can further explore how to accurately regulate the activation level of nuclear transcription factor kappa B signaling pathway,how to achieve precise intervention for nervous system inflammation and injury,and can also focus on the preparation of traditional Chinese medicine monomers and the mechanism of action of traditional Chinese medicine monomers on signaling pathways,in order to provide more effective treatment strategies for the rehabilitation and functional recovery of neurological diseases.

BACKGROUND:Knee osteoarthritis(KOA)is a common chronic inflammatory disease that causes damage to joint cartilage and surrounding tissues.Immune cells play an important role in the immune-inflammatory response in knee osteoarthritis,but the specific mechanisms involved are still not fully understood. OBJECTIVE:To evaluate the potential causal relationship between 731 immune cell phenotypes and the risk of knee osteoarthritis using Mendelian randomization. METHODS:Summary statistics of genome-wide association studies(GWAS)for 731 immune cell phenotypes(from GCST0001391 to GCST0002121)obtained from the GWAS catalog and GWAS data for knee osteoarthritis from the IEUGWAS database(ebi-a-GCST007090)were used.Inverse variance-weighted method,MR-Egger regression,weighted median method,weighted mode method,and simple mode method were employed to investigate the causal relationship between immune cells and knee osteoarthritis.Sensitivity analyses were conducted to assess the reliability of the Mendelian randomization results.Reverse Mendelian randomization analysis was also performed using the same methods. RESULTS AND CONCLUSION:The forward MR analysis indicated significant causal relationships(FDR<0.20)between knee osteoarthritis and four immune cell phenotypes,namely CD27 on CD24+CD27+in B cells(OR=1.026,P=0.000 26,Pfdr=0.18),CD33 on CD33dim HLA DR-in myeloid cells(OR=1.014,P=0.000 50,Pfdr=0.18),and CD45RA+CD28-CD8br%CD8br in Treg cells(OR=1.001,P=0.000 78,Pfdr=0.18),and PDL-1 on monocytes in mononuclear cells(OR=0.952,P=0.000 98,Pfdr=0.18).These immune cell phenotypes showed direct positive or negative causal associations with the risk of knee osteoarthritis.Reverse Mendelian randomization analysis revealed no significant causal relationships(FDR<0.20)between knee osteoarthritis as exposure and any of the 731 immune cell phenotypes.The results of sensitivity analysis show that the P-values of the Cochran's Q test and the MR-Egger regression method for bidirectional Mendelian randomization were both greater than 0.05,indicating that there is no significant heterogeneity and pleiotropy in the causal effect analysis between immune cell phenotypes and knee osteoarthritis.To conclude,there may be four potential causal relationships between immune cell phenotypes,such as CD27 on CD24+CD27+cells,CD33 on CD33dim HLA DR-cells,CD45RA+CD28-CD8br%CD8br cells,and PDL-1 on monocytes,and knee osteoarthritis.These findings provide valuable clues for studying the biological mechanisms of knee osteoarthritis and exploring early prevention and treatment strategies.They also offer new directions for the development of intervention drugs.

BACKGROUND:The repair process of myocardial injury involves complex cellular and molecular mechanisms,especially mitochondrial calcium homeostasis,macrophage autophagy and pyroptosis pathways.Traditional Chinese medicine(TCM)has shown significant clinical efficacy in improving myocardial injury,but its mechanism of action needs to be thoroughly investigated. OBJECTIVE:To investigate the role of mitochondrial calcium homeostasis-mediated macrophage autophagy and pyroptosis pathways in myocardial injury,and to summarize the progress of TCM in this field. METHODS:A computerized search was performed for relevant literature from the database inception to March 2024 in the Web of Science,PubMed and CNKI.The search terms were"mitochondrial calcium homeostasis,macrophage autophagy,macrophage pyroptosis,traditional Chinese medicine,myocardial injury,myocardial injury reperfusion"in Chinese and English.Through literature review,we analyzed the relationship between mitochondrial calcium homeostasis and macrophage autophagy and pyroptosis,explored the mechanism of their roles in myocardial injury,and summarized the pathways of multi-targeted,multi-pathway effects of TCM. RESULTS AND CONCLUSION:The maintenance of mitochondrial calcium homeostasis has been found to be closely related to the normal function of cardiomyocytes.Macrophages can participate in the repair process of myocardial injury through autophagy and pyroptosis pathways.Autophagy contributes to cell clearance and regulation of inflammatory response,while pyroptosis affects myocardial repair by releasing inflammatory factors.TCM regulates mitochondrial calcium homeostasis and macrophage function through multiple mechanisms.For example,astragalosid regulates calcium homeostasis by lowering mitochondrial membrane potential and inhibiting cytochrome C,and epimedium glycoside plays a role in reducing β-amyloid deposition.In addition,herbal compounds and single drugs promote myocardial repair by activating or inhibiting specific signaling pathways,such as PI3K/AKT and nuclear factor-κB signaling pathways.Future studies should focus on the interactions between mitochondrial calcium homeostasis,autophagy and pyroptosis pathways,as well as how TCM can exert therapeutic effects through these pathways to provide new strategies and drugs for the treatment of myocardial injury.

BACKGROUND:Research has demonstrated a close association between ferroptosis and vascular dementia.Tongmai Kaiqiao Pill has a certain effect on improving the cognitive function of vascular dementia patients,but its mechanism is unclear. OBJECTIVE:To explore the interventional effects and molecular mechanisms of Tongmai Kaiqiao Pill for vascular dementia based on the regulation of ferroptosis by the nuclear factor erythroid-2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4)signaling pathway. METHODS:Among eighty-four SD male rats,12 rats were used as the sham-operated group,and the rest of them were prepared as a model of vascular dementia by the modified 2-VO method,and then randomly divided into the model group,the Tongmai Kaiqiao Pills high-,moderate-,and low-dosage(27.6,13.8,and 6.9 g/kg)groups,the combined group(Tongmai Kaiqiao Pill high-dosage+ML385,20 mg/kg),and the donepezil hydrochloride group(0.45 mg/kg).The drug was given once a day by intragastric administration.The combined group was also intraperitoneally injected Nrf2 inhibitor ML385,once a day,for 4 weeks.Morris water maze was used to detect the learning memory ability of rats.Hematoxylin-eosin staining was used to observe the histopathological changes in the hippocampus of rats in each group.Colorimetric assay was used to detect the content of reduced glutathione,ferrous ion(Fe2+),and malondialdehyde in the serum of rats.Prussian blue staining was used to detect the iron deposition in the hippocampal tissue of rats.Transmission electron microscopy was used to observe the ultrastructural changes of mitochondria in rat hippocampal tissues.Western blot assay was used to detect the protein expression levels of Nrf2,HO-1,GPX4,XCT,and ferritin heavy chain 1(FTH1)in rat hippocampal tissues. RESULTS AND CONCLUSION:(1)In comparison to the sham operation,rats in the model group exhibited a significantly prolonged latency period(P<0.05)and a reduced number of platform crossings(P<0.05).Additionally,the hippocampal tissues of these rats displayed loosely organized structure,deeply stained cell nuclei,and solidified or lysed chromatin.Ferri ions aggregated in CA1 region.There were atrophied mitochondria with dissolved cristae and thickened mitochondrial membranes.Fe2+,malondialdehyde,and reduced glutathione levels in rat serum were found to be elevated(P<0.05).A significant reduction in the expression of GPX4,HO-1,XCT,Nrf2,and FTH1 proteins was detected in the hippocampus(P<0.05).(2)Compared to the model group,the average escape latency of the rats was significantly reduced following intervention with Tongmai Kaiqiao Pills and donepezil hydrochloride(P<0.05),with an increased number of platform crossings(P<0.05).Hippocampal neurons showed significant recovery.Notably,iron aggregation in the CA1 region was significantly reduced,and mitochondrial structure and function were improved.There were significant reductions in Fe2+and malondialdehyde levels,while the levels of GPX4,HO-1,XCT,Nrf2,and FTH1 in rat hippocampal tissues,and reduced glutathione in serum were significantly increased(P<0.05).(3)The high-dose Tongmai Kaiqiao Pills exhibited a treatment effect comparable to that of donepezil hydrochloride(P>0.05),with a significant prolongation of water maze escape latency(P<0.05),a reduced number of platform crossings(P<0.05),and insignificant neuronal pathological changes in the CA1 area.However,the combined group showed increased iron deposition,elevated malondialdehyde and Fe2+levels in blood serum(P<0.05),reduced glutathione content(P<0.05),hippocampal tissue mitochondrial atrophy,and reduced expression of Nrf2,XCT,HO-1,GPX4,and FTH1 proteins(P<0.05).Within a certain range,higher doses of Tongmai Kaiqiao Pills demonstrated a more pronounced effect,comparable to the efficacy of high-dose donepezil hydrochloride.(4)It is concluded that Tongmai Kaiqiao Pills have been shown to mitigate histopathological changes in the rat hippocampus and enhance cognitive function in rats with vascular dementia.The mechanism of action is likely associated with the suppression of ferroptosis through the activation of the Nrf2/HO-1/GPX4 signaling pathway.

BACKGROUND:Primary osteoporosis has a high incidence,but the pathogenesis is not fully understood.Currently,there is a lack of effective early screening indicators and treatment programs. OBJECTIVE:To further explore the mechanism of primary osteoporosis through comprehensive bioinformatics analysis. METHODS:The primary osteoporosis data were obtained from the gene expression omnibus(GEO)database,and the differentially expressed genes were screened for Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.In addition,the differentially expressed genes were subjected to protein-protein interaction network to determine the core genes related to primary osteoporosis,and the least absolute shrinkage and selection operator algorithm was used to identify and verify the primary osteoporosis-related biomarkers.Immune cell correlation analysis,gene enrichment analysis and drug target network analysis were performed.Finally,the biomarkers were validated using qPCR assay. RESULTS AND CONCLUSION:A total of 126 differentially expressed genes and 5 biomarkers including prostaglandins,epidermal growth factor receptor,mitogen-activated protein kinase 3,transforming growth factor B1,and retinoblastoma gene 1 were obtained in this study.GO analysis showed that differentially expressed genes were mainly concentrated in the cellular response to oxidative stress and the regulation of autophagy.KEGG analysis showed that autophagy and senescence pathways were mainly involved.Immunoassay of biomarkers showed that prostaglandins,retinoblastoma gene 1,and mitogen-activated protein kinase 3 were closely related to immune cells.Gene enrichment analysis showed that biomarkers were associated with immune-related pathways.Drug target network analysis showed that the five biomarkers were associated with primary osteoporosis drugs.The results of qPCR showed that the expression of prostaglandins,epidermal growth factor receptor,mitogen-activated protein kinase 3,and transforming growth factor B1 in the primary osteoporosis sample was significantly increased compared with the control sample(P<0.001),while the expression of retinoblastoma gene 1 in the primary osteoporosis sample was significantly decreased compared with the control sample(P<0.001).Overall,the study screened and validated five potential biomarkers of primary osteoporosis,providing a reference basis for further in-depth investigation of the pathogenesis,early screening and diagnosis,and targeted treatment of primary osteoporosis.

BACKGROUND:The granulosa cell state greatly affects the quality of oocytes.As the increased proportion of aged women in assisted reproduction,granulosa cells from patients of different ages are examined at transcript levels in order to better assess oocyte quality. OBJECTIVE:To investigate the expression changes of mRNA and long non-coding RNA in granulosa cells from the young and aged patients. METHODS:Waste cumulus granulosa cells obtained during the assisted reproductive cycle.Young group was in 25-35 years old,and the aged group was in 38-45 years old.Granulosa cells collected from the young and aged females were analyzed using RNA sequencing,three replications for each group. RESULTS AND CONCLUSION:(1)The RNA sequencing analysis results showed that the average sequencing volume of samples was more than 14 G;the data quality Q30 after quality control was more than 93%;the average mapping rate of data was 98.4%,which indicates that the data had good quality.(2)The results of principal component analysis and correlation analysis showed that the samples of the aged group and the young group could be clearly distinguished.(3)The difference analysis results showed that compared with the young group,a total of 410 differentially expressed mRNAs were detected in the aged group(167 up-regulated and 243 down-regulated).GO analysis results showed that the down-regulated genes were mainly enriched in regulatedexocytosis and the emetabolicprocess.GSEA analysis results showed that secretion-related pathways in the aged group were down-regulated.(4)Compared with the young group,662 differentially expressed long non-coding RNAs were detected in the aged group,and 1 772 protein-coding genes were directly regulated by these long non-coding RNAs,among which 59 genes overlaped with differentially expressed genes.The results showed that the expression of secretion-related genes and pathways in granulosa cells of the aged group was down-regulated,thus affecting oocyte quality.At the same time,these down-regulated genes were regulated by long non-coding RNA.Therefore,the expression of long non-coding RNA might be related to age.