ObjectiveBased on transcriptomics， to explore the mechanism of Hei Xiaoyaosan regulating the phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway to improve the learning and memory ability of insomnia rats with liver depression syndrome. MethodsSixty 8-week-old male SD rats were randomly divided into the blank group， model group， eszopiclone group （0.09 mg·kg^-1）， and low， medium， and high dose groups of Hei Xiaoyaosan （3.82， 7.65， 15.30 g·kg^-1）， with ten rats in each group. Except for the blank group， the other groups were induced insomnia rat model with liver depression by chronic restraint， tail clamping stimulation and intraperitoneal injection of p-chlorophenylalanine （PCPA）. Each treatment group received intragastric administration according to the specified dosage， once a day for 14 consecutive days. The pentobarbital sodium cooperative sleep test， open field test， and Morris water maze test were used to test the sleep quality， depressive-like behavior， and learning and memory abilities of rats. Additionally， enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of 5-hydroxytryptamine （5-HT）， γ-aminobutyric acid （GABA）， brain-derived neurotrophic factor （BDNF）， glial cell line-derived neurotrophic factor （GDNF） and nitric oxide （NO） in hippocampus. Hematoxylin-eosin （HE） staining was performed to observe pathological changes of the hippocampal tissue， while terminal deoxynucleotidyl transferase deoxyuridine triphosphate （dUTP） nick end labeling （TUNEL） was used to evaluate apoptosis of hippocampal neurons. Transcriptomic sequencing technology was employed to identify differentially expressed genes in hippocampus between the model group and the blank group， as well as between the medium-dose group of Hei Xiaoyaosan and the model group. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis were performed on the intersecting genes. Subsequently， the enriched key genes and signaling pathways were analyzed and verified. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was utilized to assess the mRNA expression levels of phosphatase and tensin homolog （PTEN）， B-cell lymphoma-2 （Bcl-2）-like protein 11 （BCL2L11）， and mitogen-activated protein kinase 1 （MAPK1） in hippocampus， and Western blot was employed to evaluate the protein expressions of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， Bcl-2， Bcl-2-associated X protein （Bax）， and cleaved Caspase-3 in the same tissue. ResultsCompared with the blank group， the model group exhibited a reduction in body weight， an increase in sleep latency， and a decrease in sleep duration （P<0.01）. Additionally， rats showed obvious depression-like behavior， and their learning and memory abilities decreased. Furthermore， the contents of 5-HT， GABA， NO， BDNF and GDNF in hippocampus decreased （P<0.01）. Histological examination revealed a disorganized cell arrangement in the CA1 region of the hippocampus， characterized by irregular cell shapes， a reduced cell count， deeply stained and pyknotic nuclei， increased vacuolar degeneration， and an elevated apoptosis rate （P<0.01）. Compared with the model group， the body weight of the high and medium dose groups of Hei Xiaoyaosan increased， the sleep latency shortened and the sleep time prolonged （P<0.05， P<0.01）. Additionally， depression-like behavior and learning and memory abilities of rats were significantly improved， the levels of 5-HT， GABA， NO， BDNF and GDNF in the hippocampus increased （P<0.05， P<0.01）. These interventions also ameliorated pathological damage in the hippocampal CA1 area and reduced the apoptosis of hippocampal neurons （P<0.01）. Transcriptomic sequencing results indicated that Hei Xiaoyaosan might exert a therapeutic effect by regulating PI3K/Akt pathway through key mRNAs such as PTEN， BCL2L11， and MAPK1. The roles of these key mRNAs and proteins within PI3K/Akt pathway were further validated. In comparison to the blank group， the expression levels of PTEN， BCL2L11 and MAPK1 mRNA in the hippocampus of rats in the model group were increased （P<0.01）， while the protein expression levels of p-PI3K， p-Akt and Bcl-2 were decreased （P<0.01）， and the protein expression levels of PTEN， Bax and cleaved Caspase-3 were increased （P<0.01）. Compared with the model group， the high-dose and medium-dose groups of Hei Xiaoyaosan could down-regulate the expressions of PTEN， BCL2L11 and MAPK1 mRNAs （P<0.01）， up-regulate the expressions of p-PI3K， p-Akt and Bcl-2 proteins （P<0.01）， and down-regulate the protein expressions of PTEN， Bax and cleaved Caspase-3 （P<0.05， P<0.01）. ConclusionHei Xiaoyaosan may regulate PI3K/Akt signaling pathway by down-regulating expressions of key genes such as PTEN， BCL2L11 and MAPK1， and thus improve the learning and memory abilities of insomnia rats with liver depression syndrome.

Objective: Exploring the effect and mechanism of Xinyang Tablet on reduction of ferroptosis in myocardial cells from mice with chronic heart failure. Methods: Sixty C57BL/6J mice were randomly assigned to the sham, model, Xinyang Tablet low-dose (0.34 g/kg), Xinyang Tablet medium-dose (0.68 g/kg), Xinyang Tablet high-dose (1.36 g/kg), and perindopril (0.607 mg/kg) groups using a random number table method (10 mice in each group). Except for the sham group, all other groups underwent aortic arch constriction surgery to construct a chronic heart failure model. On the third day after completion of the modeling, each treatment group was administered the corresponding medication by gavage, while the sham and model groups were administered equal volumes of water by gavage once a day for eight consecutive weeks. After treatment, cardiac ultrasound was used to detect the structure and function of the mouse heart. Hematoxylin and eosin staining was used to detect pathological changes in mouse heart tissue. Masson staining was used to detect the proportion of fibrotic area of mouse heart tissue. Realtime fluorescence PCR was used to detect the mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), collagen 3α (Col3α), and myosin heavy chain 7 (MYH7) in mouse myocardial tissue. Transmission electron microscope was used to detect the ultrastructure of myocardial cell mitochondria. Reactive oxygen species (ROS) staining was used to detect the mean fluorescence intensity of ROS in myocardial tissue. Micro-determination was used to detect superoxide dismutase (SOD) activity in myocardial tissue. An immunofluorescence assay was used to detect the mean fluorescence intensity of phosphorylated histone deacetylase 2 (p-HDAC2) in myocardial cell. Western blotting was used to detect the protein expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), p-HDAC2, nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1), glutathione peroxidase 4 (GPX4), and cystine glutamate reverse transporter (xCT) in mouse myocardial tissue. Results: Compared to the sham group, the model group showed a decrease in left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), an increase in left ventricular end-systolic diameter(LVESD) and left ventricular end-diastolic diameter (LVEDD), an increase in the proportion of cardiac fibrosis area, an increase in relative expression levels of ANP, BNP, Col3α, and MYH7 mRNA, an increase in ROS mean fluorescence intensity, a decrease in SOD activity, an increase in mean fluorescence intensity of p-HDAC2, an increase in relative expression levels of p-HDAC2 and NOX1 proteins, and a decrease in relative expression levels of Nrf2, GPX4, and xCT proteins (P<0.05). Myocardial fibrosis lesions are obvious, with disordered mitochondrial arrangement, decreased volume and shrinkage, increased membrane density, and reduced mitochondrial cristae. Compared to the model group, the LVEF and LVFS of mice in each dose group of Xinyang Tablet and the perindopril group increased, LVESD and LVEDD decreased, the proportion of fibrotic area of heart tissue decreased, the relative expression levels of ANP, BNP, Col3α, MYH7 mRNA decreased, ROS mean fluorescence intensity decreased, SOD activity increased, mean fluorescence intensity of p-HDAC2 decreased, relative expression levels of p-HDAC2 and NOX1 proteins decreased, and relative expression levels of Nrf2 and xCT proteins increased (P<0.05). Myocardial fibrosis was reduced, the mitochondrial arrangement was more regular, the mitochondria enlarged, the membrane density was reduced, and mitochondrial cristae increased. Compared to the model group, the relative expression level of the GPX4 protein in myocardial tissue increased in the Xinyang Tablet medium-, high-dose, and the perindopril groups (P<0.05). Conclusion Xinyang Tablet can improve ferroptosis and ventricular remodeling in mice with chronic heart failure by regulating the HDAC2-mediated Nrf2 antioxidant pathway.

This article introduces Professor LIU Xing's clinical experience in treatment of peripheral facial paralysis at the recovery and sequelae stages with the combination of acupotomy, cupping and herbal medication. Based on the analysis of etiology and pathogenesis of peripheral facial paralysis, Professor LIU believes that "invasion of pathogenic wind to collaterals and obstruction of qi and blood" is crucial. Therefore, the treatment focuses on "dispelling wind and harmonizing blood". The compound therapeutic mode is proposed, with acupotomy, cupping and herbal decoction involved, in which, "three-step sequential method of acupotomy" is predominated. Firstly, in the prone position, five "feng" (wind) points are stimulated in patient, Fengfu (GV16), Fengchi (GB20), Yifeng (TE17), Bingfeng (SI12) and Fengmen (BL12). Secondly, in the lateral position, three-facial points are stimulated (FaceⅠneedle: Yangbai [GB14]-Yuyao [EX-HN4]; Face Ⅱ needle: Sibai [ST2]-Quanliao [SI18]; Face Ⅲ needle: Jiache [ST6]-Dicang [ST4]) to restore the deviated facial muscles. Finally, in the supine, two Dantian points are stimulated on the forehead and chest, respectively (upper Dantian: Yintang [GV24⁺], middle Dantian: Danzhong [CV17]), to regulate qi and blood. As the adjunctive therapies, cupping is used to remove stasis, and herbal decoction is to harmonize the body interior. In view of holistic regulation, the treatment is administered in accordance with the affected meridians, so as to expel wind, remove obstruction in collaterals and regulate qi and blood.
Humans ; Facial Paralysis/drug therapy* ; Drugs, Chinese Herbal/administration & dosage* ; Acupuncture Therapy ; Male ; Female ; Middle Aged ; Adult ; Combined Modality Therapy ; Acupuncture Points ; Cupping Therapy ; Aged ; Young Adult

OBJECTIVE: To investigate the short-term effectiveness of transtibial pull-out technique combined with side-to-side suture technique in treatment of complete radial tear of lateral meniscus body. METHODS: Between May 2020 and August 2023, 15 patients with complete radial tear of lateral meniscus body were repaired by arthroscopic transtibial pull-out technique combined with side-to-side suture technique. There were 11 males and 4 females, with an average age of 25.2 years (range, 15-43 years). Twelve cases were acute injuries and 3 were chronic injuries. All patients had tenderness in the lateral compartment of the knee. No abnormal alignment was observed on the X-ray films of the knee. MRI showed the complete radial tear of lateral meniscus body without associated injuries such as anterior cruciate ligament or cartilage. Preoperative Lysholm score was 44.5±6.4, International Knee Documentation Committee (IKDC) subjective score was 40.2±8.4, Tegner score was 1.3±1.1, and visual analogue scale (VAS) score for pain was 5.1±1.1. The operation time, incision healing, and complications such as vascular/nerve injury were recorded. During follow-up, the range of motion of the knee and tenderness in the lateral compartment of the knee were observed. The knee function and pain were evaluated using Lysholm score, Tegner score, IKDC subjective score, and VAS score. X-ray films and MRI of the knee were reexamined to assess knee degeneration. RESULTS: The operation time was 60-145 minutes (mean, 89.6 minutes). All incisions healed by first intention, and no complication such as vascular/nerve injury occurred. All patients were followed up 17-56 months (mean, 38.4 months). All patients had no knee extension limitation and 3 cases had tenderness in the lateral compartment of the knee. At last follow-up, the Lysholm score, IKDC subjective score, Tegner score, and VAS score for pain were 85.3±7.8, 82.1±15.7, 4.7±1.2, and 1.5±1.0, respectively, which were superior to those before operation ( P<0.05). Imaging reexamination showed that the meniscus was reset at 1 day after operation, and there was no sign of knee degeneration at last follow-up. CONCLUSION Transtibial pull-out technique combined with side-to-side suture technique can effectively treat the complete radial tear of lateral meniscus body and obtain good short-term effectiveness.
Humans ; Male ; Female ; Adult ; Tibial Meniscus Injuries/surgery* ; Adolescent ; Young Adult ; Arthroscopy/methods* ; Treatment Outcome ; Suture Techniques ; Tibia/surgery* ; Menisci, Tibial/surgery* ; Magnetic Resonance Imaging

OBJECTIVE: To investigate the surgical method and short-term effectiveness of arthroscopy edge-to-edge #-shaped suture in the treatment of the complete radial tear of the lateral meniscus body. METHODS: The clinical data of 13 patients with complete radial tear of lateral meniscus body between May 2020 and August 2023 were retrospectively analyzed. There were 10 males and 3 females, aged 15-38 years (mean, 24.2 years). There were 11 cases of acute injury and 2 cases of chronic injury, with time from injury to admission ranging from 2 days to 5 months. All patients had tenderness in the lateral joint space, and 2 patients with chronic injury had positive McMurray's sign. All patients were treated with arthroscopic edge-to-edge #-shaped suture technique. The knee joint activity and tenderness in the lateral joint space were detected, and the healing of the incision and the occurrence of complications were observed. X-ray films and MRI of the knee joint were performed to evaluate joint degeneration and meniscus healing. Lysholm score, International Knee Documentation Committee (IKDC) subjective score, Tegner score, and visual analogue scale (VAS) score were used to evaluate the functional recovery before and after operation. RESULTS: The operation time ranged from 46 to 100 minutes (mean, 80.08 minutes). All the incisions healed by first intention, and no complication such as intraoperative vascular and nerve injury or postoperative infection occurred. All 13 patients were followed up 20-59 months (mean, 29.3 months). All patients had no limitation of knee extension and flexion. One patient with chronic injury continued to have tenderness in the lateral space of the knee joint, while the remaining patients had no tenderness, swelling, and locking in the lateral space. Immediate postoperative MRI of knee joint showed continuous recovery of the lateral meniscus. At last follow-up, no degenerative changes were observed in X-ray films of knee joint. Except for 1 patient with chronic injury, the MRI of the other patients showed the healing performance after lateral meniscus suture. Lysholm score, IKDC subjective score, Tegner score, and VAS score all significantly improved when compared with those before operation (P<0.05). CONCLUSION The edge-to-edge #-shaped suture technique can effectively repair the complete radial tear of the lateral meniscus body, and the short-term effectiveness is satisfactory.
Humans ; Male ; Female ; Adult ; Tibial Meniscus Injuries/surgery* ; Arthroscopy/methods* ; Retrospective Studies ; Adolescent ; Young Adult ; Suture Techniques ; Treatment Outcome ; Menisci, Tibial/surgery* ; Sutures ; Knee Joint/surgery* ; Magnetic Resonance Imaging

Objective To investigate how culture duration affects the expression of immune membrane proteins in human monocyte-derived dendritic cells (DCs) and their exosomes (DEXs). Methods Human monocytes were induced with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to differentiate into DCs and were subsequently matured with tumor necrosis factor α(TNF-α). Exosomes were isolated by ultracentrifugation, and DEXs were identified by transmission electron microscopy and Amnis imaging flow cytometry, which were also used to quantify the expression of immune membrane proteins on DCs and DEXs. Results On the 10th day of culture, DCs displayed high surface expression of CD11c, CD80, CD86, major histocompatibility complex class I (MHC-I), and MHC-II. Expression peaked at day 18(CD11c: 78.66%±20.33%, CD80: 76.41%±10.02%, CD86: 96.43%±0.43%, MHC-I: 84.71%±2.96%, MHC-II: 80.01%±7.03%). After day 24, the overall expression showed a declining trend, with statistically significant differences observed for all markers except CD80 and MHC-II. By day 30, 80% of the DCs still expressed CD80, CD86, and MHC-II. The expression of immune membrane proteins on DEX surfaces also reached its peak on day 18, followed by an overall decline with prolonged culture time, with statistically significant differences observed for all markers except CD80. Correlation analysis revealed a significant positive relationship between the expression levels of immune membrane proteins on DC and DEX surfaces (CD11c: r=0.98; CD80: r=0.65; CD86: r=0.82; MHC-I: r=0.86; MHC-II: r=0.93). Conclusion Human monocyte-derived DCs in vitro express high expression of immune membrane proteins and maintain stable expression over a specific period. The exosomes secreted by these cells similarly demonstrate high surface expression of immune membrane proteins, with temporal trends aligned with those of the parent DCs.
Humans ; Dendritic Cells/immunology* ; Exosomes/immunology* ; Monocytes/metabolism* ; Cells, Cultured ; Time Factors ; B7-1 Antigen/metabolism* ; Membrane Proteins/immunology* ; Cell Culture Techniques/methods* ; B7-2 Antigen/metabolism* ; Cell Differentiation ; CD11c Antigen/metabolism* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology*

OBJECTIVES: Hypoxia/reoxygenation (H/R) injury is a critical pathological process during liver transplantation. Kazinol B has known anti-inflammatory, anti-apoptotic, and metabolic regulatory properties, but its protective mechanism in H/R-induced liver injury remains unclear. This study aims to investigate the protective effects and underlying mechanisms of Kazinol B in H/R-induced hepatocyte injury. METHODS: An ischemia-reperfusion model was established in healthy adult male Sprague-Dawley rats, and an in vitro H/R model was created using cultured hepatocytes. Hepatocytes were treated with Kazinol B (0-100 μmol/L) to assess cytotoxicity and protective effects. Cell viability was evaluated using the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays. Expression of apoptosis-related proteins, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad), and cleaved caspase-3, was detected by Western blotting. Reactive oxygen species (ROS) levels were assessed via fluorescence probes, and inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured using enzyme-linked immunosorbent assay (ELISA). TdT-mediated nick end labeling (TUNEL) staining was performed to assess DNA damage and apoptosis. RESULTS: Kazinol B had no significant effect on hepatocyte viability at 0-50 μmol/L, but showed cytotoxicity at 100 μmol/L (P<0.05). At 0.1-20 μmol/L, Kazinol B significantly improved cell survival, reduced LDH release, decreased apoptosis, and attenuated DNA damage (all P<0.001). At 10 μmol/L, Kazinol B markedly down-regulated Bad and cleaved caspase-3 (both P<0.05), and up-regulated Bcl-2 (P<0.01). It also dose-dependently reduced ROS levels and inflammatory cytokines TNF-α and IL-1β (all P<0.01). Both in vitro and in vivo, Kazinol B inhibited activation of the c-Jun N-terminal kinase (JNK) pathway without affecting extracellular regulated protein kinase (ERK) signaling (P>0.05). TUNEL staining showed that the protective effect of Kazinol B against apoptosis was partially reversed by the JNK agonist anisomycin (P<0.01). CONCLUSIONS Kazinol B mitigates hepatocyte injury induced by H/R by inhibiting the JNK signaling pathway. Its protective effect is associated with suppression of oxidative stress and inflammation, indicating its potential as a hepatoprotective agent.
Animals ; Hepatocytes/pathology* ; Rats, Sprague-Dawley ; Male ; Rats ; Reperfusion Injury/prevention & control* ; Apoptosis/drug effects* ; Reactive Oxygen Species/metabolism* ; MAP Kinase Signaling System/drug effects* ; Cell Survival/drug effects* ; Cell Hypoxia ; Cells, Cultured

Objective·To analyze the clinicopathologic characteristics of patients with kidney-involved diffuse large B-cell lymphoma(DLBCL),including clinical characteristics,pathological characteristics,gene mutation profiles,and prognostic factors.Methods·One hundred and forty-nine patients with kidney-involved DLBCL,admitted to Ruijin Hospital,Shanghai Jiao Tong University School of Medicine from July 2005 to November 2021,were retrospectively analyzed for their clinicopathological data,survival and prognostic factors,which included therapeutic methods,clinical outcomes,staging,etc.Gene mutation profiles were evaluated by targeted sequencing of 54 lymphoma-related genes.Prognostic factors were also analyzed based on the information mentioned above.Results·A total of 149 kidney-involved DLBCL cases were included,of which 89 patients(58.4%)were aged over sixty,121 patients(81.2%)were staged Ann Arbor Ⅲ?Ⅳ,27 patients(18.1%)had an Eastern Cooperative Oncology Group(ECOG)performance status of two or more,121 patients(81.2%)had elevated serum lactate dehydrogenase(LDH)level,111 patients(74.5%)had extranodal invasion in at least two organs and 131 patients(87.9%)scored over 2 points on the international prognosis index(IPI).The estimated 5-year overall survival(OS)rate and progression-free survival(PFS)rate of kidney-involved DLBCL patients were 52.2%and 50.4%respectively.Univariate analysis revealed that elevated serum LDH levels were an adverse prognostic factor for both OS(P=0.048)and PFS(P=0.033).In pathological characteristics,145 patients(97.3%)belonged to DLBCL,not otherwise specified(NOS)and 39 patients(26.3%)belonged to germinal center B-cell(GCB)according to Hans classification.Among 144 patients who could be evaluated for clinical outcomes,87 patients(60.4%)got complete response(CR).Targeted sequencing data from 75 kidney-involved DLBCL patients showed high mutation frequency in PIM1(n=23,31%),MYD88(n=22,29%),CD79B(n=21,28%)and KMT2D(n=18,24%),with CD79B mutation indentified as an adverse prognostic factor for OS in patients with kidney-involved DLBCL(P=0.034).Conclusion·Elevated serum LDH level is an adverse prognostic factor in patients with kidney-involved DLBCL.The prognosis of patients with CD79B mutations is poor.

Objective To investigate the relationship between sleep fragmentation(SF)parameters and left ventricular structure in a community-based elderly population using data collected from the Multi-Ethnic Study of Atherosclerosis(MESA)cohort.Methods A total of 1 404 participants from the MESA cohort who underwent polysomnography(PSG)and cardiac magnetic resonance(CMR)were included.PSG was used to assess SF parameters,including wake after sleep onset(WASO),arousal index-total(ArI-total),arousal index-rapid eye movement(ArI-REM),and arousal index-non-rapid eye movement(ArI-NREM).Left ventricular end-diastolic mass(LVEDM)was measured via CMR.Linear regression was employed to analyze the relationship between SF parameters and left ventricular mass to height ratio(LVHi).Results Univariate linear regression analysis showed that WASO[β 0.134;95％confi-dence interval(CI)0.023-0.052;P＜0.001],ArI-total(β 0.184;95％CI 0.203-0.362;P＜0.001),ArI-REM(β 0.116;95％CI 0.100-0.260;P＜0.001)and ArI-NREM(β 0.175;9 5％CI 0.176-0.323;P＜0.001)were positively correlated with LVHi.After adjust-ment for confounding factors in multivariate linear regression analysis,WASO(β 0.045;95％CI 0.001-0.024;P=0.033)was positively correlated with LVHi.WASO(β 0.089;95％CI 0.006-0.034;P=0.006)was significantly associated with increased LVHi in the female group but not in the male group.Conclusion In an ethnically diverse cohort,WASO is significantly associated with increased LVHi after adjustment for potential confounders,especially in the female elderly population.

Objective:Hypoxia is a common pathological phenomenon,usually caused by insufficient oxygen supply or inability to use oxygen effectively.Hydroxylated and methoxylated flavonoids have significant anti-hypoxia activity.This study aims to explore the synthesis,antioxidant and anti-hypoxia activities of 6-hydroxygenistein(6-OHG)and its methoxylated derivatives. Methods:The 6-OHG and its methoxylated derivatives,including 4',6,7-trimethoxy-5-hydroxyisoflavone(compound 3),4',5,6,7-tetramethoxyisoflavone(compound 4),4',6-imethoxy-5,7-dihydroxyisoflavone(compound 6),and 4'-methoxy-5,6,7-trihydroxyisoflavone(compound 7),were synthesized by methylation,bromination,methoxylation,and demethylation using biochanin A as raw material.The structure of these products were characterized by 1hydrogen-nuclear magnetic resonance spectroscopy(1H-NMR)and mass spectrometry(MS).The purity of these compounds was detected by high pressure chromatography(HPLC).The antioxidant activity in vitro was investigated by 1,1-diphenyl-2-picrylhydrazyl radical(DPPH)free radical scavenging assay.PC12 cells were divided into a normal group,a hypoxia model group,rutin(1×10-9-1×10-5 mol/L)groups,and target compounds(1×10-9-1×10-5 mol/L)groups under normal and hypoxic conditions.Cell viability was detected by cell counting kit-8(CCK-8)assay,the target compounds with excellent anti-hypoxia activity and the drug concentration at the maximum anti-hypoxia activity were screened.PC12 cells were treated with the optimal concentration of the target compound or rutin with excellent anti-hypoxia activity,and the cell morphology was observed under light microscope.The apoptotic rate was determined by flow cytometry,and the expressions of hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)were detected by Western blotting. Results:The structure of 6-OHG and its 4 methylated derivatives were correct,and the purity was all more than 97%.When the concentration was 4 mmol/L,the DPPH free radical removal rates of chemical compounds 7 and 6-OHG were 81.16%and 86.94%,respectively,which were higher than those of rutin,the positive control.The removal rates of chemical compounds 3,4,and 6 were all lower than 20%.Compared with the normal group,the cell viability of the hypoxia model group was significantly decreased(P<0.01).Compared with the hypoxia model group,compounds 3,4,and 6 had no significant effect on cell viability under hypoxic conditions.At all experimental concentrations,the cell viability of the 6-OHG group was significantly higher than that of the hypoxia model group(all P<0.05).The cell viability of compound 7 group at 1×10-7 and 1×10-6 mol/L was significantly higher than that of the hypoxia model group(both P<0.05).The anti-hypoxia activity of 6-OHG and compound 7 was excellent,and the optimal drug concentration was 1×10-6 and 1×10-7 mol/L.After PC12 cells was treated with 6-OHG(1×10-6 mol/L)and compound 7(1×10-7 mol/L),the cell damage was reduced,the apoptotic rate was significantly decreased(P<0.01),and the protein expression levels of HIF-1α and VEGF were significantly decreased in comparison with the hypoxia model group(both P<0.01). Conclusion:The optimized synthesis route can increase the yield of 6-OHG and obtain 4 derivatives by methylation and selective demethylation.6-OHG and compound 7 have excellent antioxidant and anti-hypoxia activities,which are related to the structure of the A-ring ortho-triphenol hydroxyl group in the molecule.