ObjectiveTo observe the clinical efficacy of Sanren Runchang formula in treating constipation-predominant irritable bowel syndrome （IBS-C） by regulating the brain-gut axis and the effects of the formula on serum levels of 5-hydroxytryptamine （5-HT）， vasoactive intestinal peptide （VIP）， and substance P （SP）. MethodsA randomized controlled design was adopted， and 72 IBS-C patients meeting Rome Ⅳ criteria were randomized into observation and control groups （36 cases）.The observation group received Sanren Runchang formula granules twice daily， and the control group received lactulose oral solution daily for 4 weeks. IBS Symptom Severity Scale （IBS-SSS）， IBS Quality of Life Scale （IBS-QOL）， and Bristol Stool Form Scale （BSFS） were used to assess clinical symptoms， and bowel movement frequency was recorded. The Self-Rating Anxiety Scale （SAS） and Self-Rating Depression Scale （SDS） were employed to evaluate psychological status. ELISA was employed to measure the serum levels of 5-HT， VIP， and SP. ResultsThe total response rate in the observation group was 91.67% （33/36）， which was higher than that （77.78%， 28/36） in the control group （χ²=4.50， P<0.05）. After treatment， both groups showed increased defecation frequency and BSFS scores， decreased IBS-SSS total score， abdominal pain and bloating scores， IBS-QOL health anxiety， anxiety， food avoidance， and behavioral disorders scores， SAS and SDS scores， serum 5-HT and VIP levels， and increased SP levels （P<0.05， P<0.01）. Moreover， the observation group showed more significant changes in the indicators above than the control group （P<0.05， P<0.01）. The SP level showed no significant difference between the two groups. During the 4-week follow-up， the recurrence rate was 5.88% in the observation group and 31.25% in the control group. No adverse events occurred in observation group， and 2 cases of mild diarrhea occurred in the control group. ConclusionSanren Runchang formula demonstrated definitive efficacy in alleviating gastrointestinal symptoms and improving the psychological status and quality of life in IBS-C patients， with a low recurrence rate. The formula can regulate serum levels of neurotransmitters such as 5-HT and VIP， suggesting its potential regulatory effect on the brain-gut axis through modulating neurotransmitters and neuropeptides. However， its complete mechanism of action requires further investigation through detection of additional brain-gut axis-related biomarkers.

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 observe the clinical effectiveness and safety of Qingfei Shenshi Decoction （清肺渗湿汤） combined with western medicine for patients with bronchial asthma of heat wheezing syndrome， and to explore its potential mechanism of action. MethodsEighty-six participants with bronchial asthma of heat wheezing syndrome were randomly divided into treatment group and control group， each group with 43 participants. The control group received conventional western medicine， and the treatment group was additionally administered Qingfei Shenshi Decoction orally on the basis of the control group， 1 dose per day. Both groups were treated for 14 days. The primary outcome measure was clinical effectiveness； secondary outcome measures included traditional Chinese medicine （TCM） syndrome score， asthma control test （ACT） score， pulmonary function indices such as forced expiratory volume in 1 second （FEV₁）， forced vital capacity （FVC）， peak expiratory flow （PEF）， serum inflammatory factor levels including interleukin-4 （IL-4）， tumour necrosis factor-α （TNF-α）， and high-sensitivity C-reactive protein （hs-CRP）， and immune function indices including CD3⁺， CD4⁺， CD8⁺， CD4⁺/CD8⁺. All outcome measures were evaluated before and after treatment. Vital signs were monitored， and electrocardiography， blood routine， urine routine， liver function， and renal function tests were performed before and after treatment. Adverse events and reactions during the study were recorded. ResultsA total of 80 patients completed the trial with 40 in each group. The total clinical effective rate of the treatment group was 97.5% （39/40）， which was significantly higher than that of the control group （85.0%， 34/40， P＜0.05）. After treatment， both groups showed decreased TCM syndrome scores， IL-4， TNF-α， hs-CRP， and CD8⁺ levels， as well as increased ACT scores， CD3⁺， CD4⁺， CD4⁺/CD8⁺， FEV₁， FVC， and PEF levels （P＜0.05 or P＜0.01）. Moreover， the improvements in these indices were more significant in the treatment group than in the control group （P＜0.05 or P＜0.01）. No significant abnormalities in safety indicators were observed in either group， and no adverse events or reactions occurred. ConclusionQingfei Shenshi Decoction combined with conventional western medicine for patients with bronchial asthma of heat wheezing syndrome can effectively improve the clinical symptoms， pulmonary function， and clinical effectiveness， with good safety. Its mechanism may be related to reducing inflammatory factor levels and regulating T lymphocyte subsets to improve immune function.

ObjectiveFor prepubertal and urgently treated malignant tumor patients, ovarian tissue cryopreservation and transplantation represent more appropriate fertility preservation methods. Current clinical practices often involve freezing ovarian tissue with high concentrations of cryoprotectants (CPAs) and thawing with water baths. These processes lead to varying degrees of toxicity and devitrification damage to ovarian tissue. Therefore, this paper proposes optimized methods for vitrification of ovarian tissues based on sodium alginate hydrogel encapsulation and magnetic induction nanowarming technology. MethodsFirstly, the study investigated the effects of sodium alginate concentration, the sequence of hydrogel encapsulation and CPAs loading on vitrification efficiency of encapsulated ovarian tissue. Additionally, the capability of sodium alginate hydrogel encapsulation to reduce the required concentration of CPAs was validated. Secondly, a platform combining water bath and magnetic induction nanowarming was established to rewarm ovarian tissue under various concentrations of magnetic nanoparticles and magnetic field strengths. The post-warming follicle survival rate, antioxidant capacity, and ovarian tissue integrity were evaluated to assess the efficacy of the method. ResultsThe study found that ovarian tissue encapsulated with 2% sodium alginate hydrogel exhibited the highest follicle survival rate after vitrification. The method of loading CPAs prior to encapsulation proved more suitable for ovarian tissue cryopreservation, effectively reducing the required concentration of CPAs by 50%. A combination of 8 g/L Fe₃O₄ nanoparticles and an alternating magnetic field of 300 Gs showed optimal warming effectiveness for ovarian tissue. Combining water bath rewarming with magnetic induction nanowarming yielded the highest follicle survival rate, enhanced antioxidant capacity, and preserved tissue morphology. ConclusionSodium alginate hydrogel encapsulation of ovarian tissue reduces the concentration of CPAs required during the freezing process. The combination of magnetic induction nanowarming with water bath provides an efficient method ovarian tissue rewarming. This study offers novel approaches to optimize ovarian tissues vitrification.

ObjectiveTo optimize Tusizi prescription for ovarian reserve function based on the uniform design method combined with in vitro experiments and explore the underlying mechanisms of this prescription. MethodsThe uniform design method was adopted to design a 5-factor 11-level experiment on the water extract of Tusizi prescription. The cell-counting kit-8 （CCK-8） assay was employed to measure the viability of human ovarian granulosa cells （KGN cells） treated with Tusizi prescription extracts 1-11， and multivariate regression analysis was performed to determine the optimal herb ratio in this prescription. The potential targets of active ingredients in the prescription were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform （TCMSP） and encyclopedia of traditional Chinese medicine （ETCM）. The common targets shared by Tusizi prescription and diminished ovarian reserve （DOR） were selected and imported into search tool for the retrieval of interacting genes/proteins （STRING） to construct a protein-protein interaction （PPI） network and into gene function annotation database （DAVID） for gene ontology （GO） analysis. The CCK-8 assay was used to measure the viability of ovarian germline stem cells treated with hyperoside. The CCK-8 assay， 5-ethynyl-2'-deoxyuridine （EdU） staining， terminal-deoxynucleoitidyl transferase mediated nick-end labeling （TUNEL）， and enzyme-linked immunosorbent assay （ELISA） were employed to examine the proliferation， apoptosis， and estradiol （E₂） secretion of KGN cells treated with the water extract 11 of Tusizi prescription （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1） and the optimal prescription screened by uniform design. On this basis， the optimal prescription composition for maximizing the effect on ovarian reserve function was determined and preliminary insights into the underlying mechanisms of this prescription were gained. ResultsA total of 147 common targets were obtained from 278 targets of Tusizi prescription and 1 721 targets of DOR. GO analysis revealed 194 biological processes， primarily involving cellular responses to exogenous compound stimuli， negative regulation of apoptotic process， and positive regulation of cell proliferation. It identified 84 cellular components， including cell membrane， mitochondria， and neuronal cell body， as well as 144 molecular functions such as enzyme binding， estrogen response element binding， and nuclear estrogen receptor binding. The multivariate regression analysis revealed that when Tusizi prescription was composed of Cuscutae Semen， Lycii Fructus， Dioscoreae Rhizoma， Poria， and Nelumbinis Semen in a ratio of 27∶30∶17∶12∶14， the water extract of Tusizi prescription had the best effect of enhancing the viability of KGN cells. CCK-8 results showed that compared with the normal group， the hyperoside group demonstrated increased viability of ovarian germline stem cells （P<0.01）. The CCK-8， EdU， and ELISA results showed that compared with the normal group， the optimal prescription screened by uniform design and the water extract 11 of Tusizi prescription increased the proliferation and reduced the apoptosis of KGN cells （P<0.05， P<0.01）. ELISA results showed that compared with the normal group， the water extract 11 of Tusizi prescription promoted the E₂ secretion of KGN cells （P<0.05）， while the optimal prescription screened by uniform design had no significant effect on the E₂ secretion. ConclusionBoth the water extract 11 of Tusizi prescription （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1） and the optimal prescription screened by uniform design （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 27∶30∶17∶12∶14） can improve the ovarian reserve function， and the former has better effect. Tusizi prescription can modulate biological processes （such as cell proliferation and apoptosis） and molecular functions （such as enzyme binding and estrogen response element binding） through active components like hyperoside to promote the proliferation and E₂ secretion and inhibit the apoptosis of KGN cells， thereby protecting the ovarian reserve function.

Da Qinjiaotang is the 54^th formula of the 100 formulas in the Catalogue of Ancient Classical Formulas （the first batch） ，and it originated from the Collection of Writings on the Mechanism of Disease， Suitability of Qi， and Safeguarding of Life Discussed in Plain Questions. Da Qinjiaotang is composed of Gentiana macrophylla， Glycyrrhizae Radix et Rhizoma， Ligusticum chuanxiong， Angelica sinensis， Paeonia lactiflora， Asari Radix et Rhizoma， Notopterygium incisum， Saposhnikoviae Radix， Scutellariae Radix， Gypsum， Angelica dahurica， Atractylodis Macrocephalae Rhizoma， Rehmanniae Radix， Rehmanniae Radix Praeparata， Poria， and Angelicae Pubescentis Radix. It is a classical formula for treating strokes. Da Qinjiaotang is widely used in modern clinical practices for treating ischemic stroke， peripheral facial paralysis， cervical spondylosis， rheumatic arthritis， neurodermatitis， and other multisystem diseases. Therefore， following the Principles of Textual Research on the Key Information of Ancient Classical Famous Formulas， the authors collected the ancient Chinese medical literature of Da Qinjiaotang by the method of bibliometrics and screened out 177 valid data， involving 100 ancient books of traditional Chinese medicine. Based on the historical evolution， composition， dosage， method of preparation， and preparation of the original medicinal materials of Da Qinjiaotang， a systematic study was carried out. It was found that among the 175 records of the main diseases and syndromes， stroke （144） was the most， accounting for 82.29% of the total diseases and syndromes. Later generations mostly followed the practice of LIU Wansu in using Da Qinjiaotang to treat stroke caused by "weak blood and inability to nourish tendon"， featuring "hands and feet cannot move， stiff tongue hinders speaking"， as well as other symptoms， such as slant of the mouth， hemiplegia， numbness of the limbs， paroxysmal pain， and acerbic syncope. The treatment scope was expanded， covering tendon dryness， clonic convulsion， spasm syndrome， and arthralgia syndrome. At the same time， it was found that there was a controversy between "internal wind" and "external wind" in the treatment of stroke by Da Qinjiaotang. LIU Wansu thought that stroke was caused by internal factors， created the theory of "hot stroke"， and used Da Qinjiaotang to treat "internal wind". Many doctors in later generations focused on treating the "external wind" of "internal deficiency and evil". There were 76 valid data on the composition of drugs， 59 of which had doses for each drug. It was suggested to use the modern conversion dosage of the original formula， with 41.30 g per dose. The drug should be boiled in 600 mL water until 300 mL， decocted once， and taken in a warm state after removing the dregs anytime. Through the analysis and study of the ancient books about Da Qinjiaotang， the paper clarified its historical evolution and confirmed its key information， so as to provide the ancient literature evidence for the research and development of the classical famous formula Daqinjiaotan and its better clinical application.

ObjectiveTo optimize Tusizi prescription for ovarian reserve function based on the uniform design method combined with in vitro experiments and explore the underlying mechanisms of this prescription. MethodsThe uniform design method was adopted to design a 5-factor 11-level experiment on the water extract of Tusizi prescription. The cell-counting kit-8 （CCK-8） assay was employed to measure the viability of human ovarian granulosa cells （KGN cells） treated with Tusizi prescription extracts 1-11， and multivariate regression analysis was performed to determine the optimal herb ratio in this prescription. The potential targets of active ingredients in the prescription were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform （TCMSP） and encyclopedia of traditional Chinese medicine （ETCM）. The common targets shared by Tusizi prescription and diminished ovarian reserve （DOR） were selected and imported into search tool for the retrieval of interacting genes/proteins （STRING） to construct a protein-protein interaction （PPI） network and into gene function annotation database （DAVID） for gene ontology （GO） analysis. The CCK-8 assay was used to measure the viability of ovarian germline stem cells treated with hyperoside. The CCK-8 assay， 5-ethynyl-2'-deoxyuridine （EdU） staining， terminal-deoxynucleoitidyl transferase mediated nick-end labeling （TUNEL）， and enzyme-linked immunosorbent assay （ELISA） were employed to examine the proliferation， apoptosis， and estradiol （E₂） secretion of KGN cells treated with the water extract 11 of Tusizi prescription （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1） and the optimal prescription screened by uniform design. On this basis， the optimal prescription composition for maximizing the effect on ovarian reserve function was determined and preliminary insights into the underlying mechanisms of this prescription were gained. ResultsA total of 147 common targets were obtained from 278 targets of Tusizi prescription and 1 721 targets of DOR. GO analysis revealed 194 biological processes， primarily involving cellular responses to exogenous compound stimuli， negative regulation of apoptotic process， and positive regulation of cell proliferation. It identified 84 cellular components， including cell membrane， mitochondria， and neuronal cell body， as well as 144 molecular functions such as enzyme binding， estrogen response element binding， and nuclear estrogen receptor binding. The multivariate regression analysis revealed that when Tusizi prescription was composed of Cuscutae Semen， Lycii Fructus， Dioscoreae Rhizoma， Poria， and Nelumbinis Semen in a ratio of 27∶30∶17∶12∶14， the water extract of Tusizi prescription had the best effect of enhancing the viability of KGN cells. CCK-8 results showed that compared with the normal group， the hyperoside group demonstrated increased viability of ovarian germline stem cells （P<0.01）. The CCK-8， EdU， and ELISA results showed that compared with the normal group， the optimal prescription screened by uniform design and the water extract 11 of Tusizi prescription increased the proliferation and reduced the apoptosis of KGN cells （P<0.05， P<0.01）. ELISA results showed that compared with the normal group， the water extract 11 of Tusizi prescription promoted the E₂ secretion of KGN cells （P<0.05）， while the optimal prescription screened by uniform design had no significant effect on the E₂ secretion. ConclusionBoth the water extract 11 of Tusizi prescription （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1） and the optimal prescription screened by uniform design （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 27∶30∶17∶12∶14） can improve the ovarian reserve function， and the former has better effect. Tusizi prescription can modulate biological processes （such as cell proliferation and apoptosis） and molecular functions （such as enzyme binding and estrogen response element binding） through active components like hyperoside to promote the proliferation and E₂ secretion and inhibit the apoptosis of KGN cells， thereby protecting the ovarian reserve function.

Da Qinjiaotang is the 54^th formula of the 100 formulas in the Catalogue of Ancient Classical Formulas （the first batch） ，and it originated from the Collection of Writings on the Mechanism of Disease， Suitability of Qi， and Safeguarding of Life Discussed in Plain Questions. Da Qinjiaotang is composed of Gentiana macrophylla， Glycyrrhizae Radix et Rhizoma， Ligusticum chuanxiong， Angelica sinensis， Paeonia lactiflora， Asari Radix et Rhizoma， Notopterygium incisum， Saposhnikoviae Radix， Scutellariae Radix， Gypsum， Angelica dahurica， Atractylodis Macrocephalae Rhizoma， Rehmanniae Radix， Rehmanniae Radix Praeparata， Poria， and Angelicae Pubescentis Radix. It is a classical formula for treating strokes. Da Qinjiaotang is widely used in modern clinical practices for treating ischemic stroke， peripheral facial paralysis， cervical spondylosis， rheumatic arthritis， neurodermatitis， and other multisystem diseases. Therefore， following the Principles of Textual Research on the Key Information of Ancient Classical Famous Formulas， the authors collected the ancient Chinese medical literature of Da Qinjiaotang by the method of bibliometrics and screened out 177 valid data， involving 100 ancient books of traditional Chinese medicine. Based on the historical evolution， composition， dosage， method of preparation， and preparation of the original medicinal materials of Da Qinjiaotang， a systematic study was carried out. It was found that among the 175 records of the main diseases and syndromes， stroke （144） was the most， accounting for 82.29% of the total diseases and syndromes. Later generations mostly followed the practice of LIU Wansu in using Da Qinjiaotang to treat stroke caused by "weak blood and inability to nourish tendon"， featuring "hands and feet cannot move， stiff tongue hinders speaking"， as well as other symptoms， such as slant of the mouth， hemiplegia， numbness of the limbs， paroxysmal pain， and acerbic syncope. The treatment scope was expanded， covering tendon dryness， clonic convulsion， spasm syndrome， and arthralgia syndrome. At the same time， it was found that there was a controversy between "internal wind" and "external wind" in the treatment of stroke by Da Qinjiaotang. LIU Wansu thought that stroke was caused by internal factors， created the theory of "hot stroke"， and used Da Qinjiaotang to treat "internal wind". Many doctors in later generations focused on treating the "external wind" of "internal deficiency and evil". There were 76 valid data on the composition of drugs， 59 of which had doses for each drug. It was suggested to use the modern conversion dosage of the original formula， with 41.30 g per dose. The drug should be boiled in 600 mL water until 300 mL， decocted once， and taken in a warm state after removing the dregs anytime. Through the analysis and study of the ancient books about Da Qinjiaotang， the paper clarified its historical evolution and confirmed its key information， so as to provide the ancient literature evidence for the research and development of the classical famous formula Daqinjiaotan and its better clinical application.

Breast cancer is a kind of malignant tumor with a complex mechanism， and its morbidity and mortality are increasing year by year， which seriously threatens women's health. At present， the main clinical treatments are surgical resection， radiotherapy， chemotherapy， and drug therapy， but they are often accompanied by side effects and adverse reactions， which affect the therapeutic effect. Traditional Chinese medicine （TCM） has the advantages of multi-component and multi-target treatment in the fight against breast cancer. The wnt/β-catenin signaling pathway is one of the classic pathways in cancer research. Abnormally activated Wnt/β-catenin signaling pathway inhibits β-catenin degradation by blocking the formation of Axin/glycogen synthase kinase 3β/adenomatous polyposis coli complex， thus promoting β-catenin nuclear metastasis， and it binds to T cell transcription factor/lymphoenhancer factor-1 to initiate downstream target genes and further interfere with the proliferation， migration， and invasion of tumor cells to affect the tumor process. Previous studies have shown that TCM monomers and compounds can mediate the Wnt/β-catenin signaling pathway to inhibit the malignant phenotype of breast cancer cells， thus playing an anti-breast cancer role， and the biochemical process involved in the regulation of therapeutic drugs has not been systematically combed. By analyzing and collating Chinese and foreign literature at the present stage， this paper discussed the association mechanism between Wnt/β-catenin signaling pathway and breast cancer and analyzed the internal mechanism of TCM monomers and compounds in mediating Wnt/β-catenin signaling pathway to exert anti-breast cancer effect. The statistical results showed that the flavonoids， alkaloids， and terpenoids in TCM monomers could target the Wnt/β-catenin signaling pathway and block the further development of malignant phenotype of breast cancer cells. TCM compounds with functions of clearing heat and detoxifying， promoting blood circulation and removing blood stasis， and tonifying kidney and liver were commonly used to intervene in the Wnt/β-catenin signaling pathway to prevent breast cancer. Compared with the current inhibitors of Wnt/β-catenin signaling pathway， the application of TCM monomers and compounds is expected to bring low-toxicity and high-efficiency breast cancer treatment drugs to the clinical practice， and the existing results provide a reference for the subsequent screening， research， and development of TCM small-molecule compounds and TCM compounds against breast cancer.

BACKGROUND:Leonurine has many biological activities such as improving microcirculation,anti-oxidation,anti-apoptosis,scavenging free radicals,anti-inflammation,and anti-fibrosis,and can promote osteogenic differentiation of bone marrow mesenchymal stem cells,which has the potential to be applied in the treatment of periodontitis. OBJECTIVE:To explore the anti-inflammatory and osteogenic effects of leonurine loading into chitosan/sodium glycerophosphate/sodium alginate hydrogel. METHODS:(1)Chitosan/sodium glycerophosphate/sodium alginate hydrogel(blank hydrogel)and chitosan/sodium glycerophosphate/sodium alginate/leonurus alkali hydrogel were prepared respectively.RAW 264.7 and MC3T3-E1 cells were inoculated with the two kinds of hydrogel.The cytotoxicity of hydrogels was detected by CCK-8 assay and live/dead cell staining.(2)RAW 264.7 cells were cultured in five groups.The blank group was cultured for 24 hours routinely.The lipopolysaccharide group was treated with lipopolysaccharide.The simple hydrogel group was treated with lipopolysaccharide and blank hydrogel.The drug-loaded hydrogel group was treated with lipopolysaccharide and drug-loaded hydrogel.The inhibitor group was treated with lippolysaccharide,drug-loaded hydrogel,and PI3K inhibitor LY294002.24 hours later,mRNA expression of inflammation-related factors was detected by qRT-PCR.Western blot assay was utilized to detect the protein expression of inflammation-related factors and PI3K/AKT signaling pathway.(3)MC3T3-E1 cells were inoculated in four groups.The blank group was cultured without any material.The simple hydrogel group was treated with blank hydrogel.The drug-loaded hydrogel group was treated with drug-loaded hydrogel.The inhibitor group was treated with drug-loaded hydrogel and PI3K inhibitor LY294002 for 7 days.Alkaline phosphatase staining was performed.mRNA expression levels of osteogenic factors were detected by qRT-PCR.The protein expression levels of the PI3K/AKT signaling pathway were detected by western blot assay. RESULTS AND CONCLUSION:(1)The results of CCK-8 assay and live/dead cell staining showed that the two kinds of hydrogels had no cytotoxic effect and had good cytocompatibility.(2)Compared with the blank group,the mRNA and protein expression levels of interleukin 6,tumor necrosis factor α,and interleukin 1β were significantly increased(P<0.05),and the protein expression levels of p-AKT,p-PI3K,p-p65,and p-IκBα were significantly increased in the lipopolysaccharide group(P<0.05).Compared with lipopolysaccharide group,mRNA and protein expression levels of the above indexes were decreased in drug-loaded hydrogel group(P<0.05).Compared with the drug-loaded hydrogel group,the mRNA and protein expression levels of the above indexes were decreased in the inhibitor group(P<0.05).(3)The activity of alkaline phosphatase in drug-loaded hydrogel group was higher than that in the blank group,simple hydrogel group,and inhibitor group(P<0.05).Compared with blank group,the mRNA expression levels of alkaline phosphatase,Runx2,osteocalcin,and type I collagen were increased(P<0.05),and the protein expression levels of p-AKT and p-PI3K were increased in the simple hydrogel group(P<0.05).Compared with the simple hydrogel group,the mRNA and protein expression levels of the above indexes were increased in the drug-loaded hydrogel group(P<0.05).Compared with the drug-loaded hydrogel group,the mRNA and protein expression levels of the above indexes were decreased in the inhibitor group(P<0.05).(4)These findings conclude that chitosan/sodium glycerophosphate/sodium alginate/leonurine hydrogel has anti-inflammatory and osteogenic effects,which may be related to the regulation of PI3K/AKT signaling pathway.