1.Effect of Danggui Buxuetang on PINK1/Parkin Signaling Pathway of Vascular Dementia Rats
Guifang QI ; Yue JIANG ; Yunxiang TAN ; Nanbu WANG ; Xinghua CHEN ; Ting WAN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):15-24
ObjectiveTo investigate the potential mechanism of Danggui Buxuetang (DBT) in the treatment of vascular dementia (VAD). MethodsSixty male SD rats were randomly assigned to the sham-operated group, model group, DBT low-, medium-, and high-dose groups, and the donepezil group. Except for the sham-operated group, rats in all other groups underwent bilateral common carotid artery ligation. After successful modeling, DBT was administered at doses of 9.2, 18.4, 36.8 g·kg-1 for the low-, medium-, and high-dose groups, respectively, while the donepezil group received 3 mg·kg-1 donepezil solution by gavage once daily. After 4 consecutive weeks of drug treatment, rats underwent the Morris water maze test, novel object recognition test, Nissl staining to observe hippocampal neurons, and immunofluorescence staining to detect the expression of neuronal nuclear protein (NeuN) in the hippocampus. Western blot was used to assess the expression of PTEN-induced kinase 1 (PINK1), Parkin, microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X protein (Bax). Transmission electron microscopy was used to observe hippocampal neuronal ultrastructure. Real-time PCR was used to detect the expression of NADPH oxidase subunits p22phox and p47phox in hippocampal tissues. The levels of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and total antioxidant capacity were measured to evaluate oxidative stress levels. ResultsIn the Morris water maze test, escape latency changed significantly over time in all groups except the model group. Compared with the sham-operated group, the model group showed significantly prolonged escape latency (P<0.01). Compared with the model group, rats in the DBT groups and the donepezil group exhibited significantly shorter escape latency (P<0.05, P<0.01). The number of crossings over the original platform was significantly reduced in the model group compared with the sham-operated group (P<0.01), whereas rats in the DBT and donepezil groups showed significantly increased platform crossings compared with the model group (P<0.05, P<0.01). Compared with the sham-operated group, exploration time of new objects was significantly reduced in the model group (P<0.01). Compared with the model group, exploration time of new objects increased significantly in the medium- and high-dose DBT groups and the donepezil group (P<0.05, P<0.01), while no significant change was observed in the low-dose DBT group. Compared with the high-dose DBT group, rats in the donepezil group had significantly prolonged escape latency and reduced platform crossings and new-object exploration time (P<0.05). Nissl staining showed decreased density of healthy neurons in the CA1 and CA3 regions of the hippocampus in the model group, with loss of Nissl bodies and nuclear atrophy or disappearance. In the high-dose DBT group, neuronal density in CA1 and CA3 increased, with neurons arranged closely and displaying normal morphology. Immunofluorescence showed that compared with the sham-operated group, the hippocampal NeuN⁺ cell count in the VAD model group was significantly decreased(P<0.01), compared with the VAD model group, the hippocampal NeuN⁺ cell count in the high-dose DBT group was significantly increased(P<0.01). Compared with the sham-operated group, the expression of PINK1, Parkin, LC3Ⅱ, and Bax proteins was significantly increased(P<0.01), while the expression of Bcl-2 was significantly decreased in the VAD model group(P<0.01). Compared with the VAD model group, the high-dose DBT group showed significantly decreased expression of PINK1, Parkin, LC3Ⅱ, and Bax proteins(P<0.01)and significantly upregulated Bcl-2 expression(P<0.01). The medium-dose DBT group exhibited significantly reduced expression of Parkin, LC3Ⅱ, and Bax proteins(P<0.05,P<0.01) and significantly increased Bcl-2 expression(P<0.01), while no statistically significant differences were observed in the low-dose DBT group. Transmission electron microscopy showed mitochondrial pyknosis, thickened cristae, increased electron density, and the presence of mitochondrial autophagy in the model group. In contrast, hippocampal neurons in the high-dose DBT group contained abundant mitochondria with intact morphology, clear cristae, and uniform matrix. Compared with the sham-operated group, total antioxidant capacity, SOD activity, and GSH levels were significantly decreased, while MDA levels were significantly increased in the model group (P<0.01). Compared with the model group, total antioxidant capacity and antioxidant levels (SOD, GSH) increased significantly, and MDA decreased significantly in the medium- and high-dose DBT groups (P<0.01), while no significant changes were observed in the low-dose DBT group. Compared with the sham-operated group, mRNA expression of p22phox and p47phox was significantly increased in the model group (P<0.01). Compared with the model group, expression of p22phox and p47phox was significantly decreased in the DBT groups (P<0.05, P<0.01). ConclusionDBT may exert neuroprotective effects by regulating PINK1/Parkin-mediated mitochondrial autophagy, thereby improving learning and memory abilities and treating VAD.
2.Research progress and clinical challenges in immunosuppressive regimens for xenotransplantation
Yu ZHANG ; Kun WANG ; Xuyuan ZHU ; Yuxiang CHEN ; Tao LI ; Xiaojie MA ; Hongtao JIANG
Organ Transplantation 2026;17(1):28-35
As a pivotal strategy to alleviate the shortage of organ donors, xenotransplantation has achieved remarkable advances in both pre-clinical and clinical studies in recent years, driven by continuous optimization of gene modification techniques and immunosuppressive regimens. Nevertheless, clinical translation still confronts formidable challenges, including rejection and heightened infection risks, which severely compromise long-term graft survival. Consequently, the role of immunosuppressive regimens in xenotransplantation has become increasingly prominent. This article summarizes the mechanisms underlying xenogeneic immune rejection, the latest developments in immunosuppressive regimens, cutting-edge strategies for inducing immune tolerance and the major hurdles facing clinical xenotransplantation. It delves into potential optimization strategies and directions for future clinical research, aiming to offer theoretical insights and practical guidance for the safe and effective application of clinical xenotransplantation.
3.Changes and Trends in the microbiological-related standards in the Chinese Pharmacopoeia 2025 Edition
FAN Yiling ; ZHU Ran ; YANG Yan ; JIANG Bo ; SONG Minghui ; WANG Jing ; LI Qiongqiong ; LI Gaomin ; WANG Shujuan ; SHAO Hong ; MA Shihong ; CAO Xiaoyun ; HU Changqin ; MA Shuangcheng, ; YANG Meicheng
Drug Standards of China 2025;26(1):093-098
Objective: To systematically analyze the revisions content and technological development trends of microbiological standards in the Chinese Pharmacopoeia (ChP) 2025 Edition, and explore its novel requirements in risk-based pharmaceutical product lifecycle management.
Methods: A comprehensive review was conducted on 26 microbiological-related standards to summarize the revision directions and scientific implications from perspectives including the revision overview, international harmonization of microbiological standards, risk-based quality management system, and novel tools and methods with Chinese characteristics.
Results: The ChP 2025 edition demonstrates three prominent features in microbiological-related standards: enhanced international harmonization, introduced emerging molecular biological technologies, and established a risk-based microbiological quality control system.
Conclusion: The new edition of the Pharmacopoeia has systematically constructed a microbiological standard system, which significantly improves the scientificity, standardization and applicability of the standards, providing a crucial support for advancing the microbiological quality control in pharmaceutical industries of China.
4.Research on interview outline design and quality control methods based on grounded theory for physician prescribing behavior
Liyuan TAO ; Li WANG ; Xinli XIANG ; Lu YANG ; Songleng DUAN ; Dechun JIANG
China Pharmacy 2025;36(5):524-528
OBJECTIVE To establish a interview outline design process and quality control evaluation method based on grounded theory, providing ideas for qualitative research interview outline design in medical fields. METHODS A literature review was conducted to understand the current research status; a preliminary interview outline was developed around the research content. The triangulation method, group evaluation, expert review and pre-interview were adopted to execute the interview outline and conduct quality control. The evaluation indicators and target values were formulated (an average score for the overall quality evaluation of all indicators ≥4.5, and an average score for individual indicators ≥4.00) to evaluate the effect of the interview outline. Taking the research on the mechanism of physicians’ prescribing behavior under the background of Diagnosis Related Groups (DRGs) payment as an example, the methodological contents of above interview outline were applied in practical research. RESULTS The interview outline included basic information and interview questions. The interview questions were divided into three parts:influencing factors survey, promoting and hindering factors of standardizing physician prescription behavior, and communication, with a total of 12 questions. After being reviewed by members of the research group, experts review and pre- interview, a total of 9 people participated in the quality control evaluation of the interview outline. The overall evaluation score was 4.94 (>4.50), and the average score of each indicator was greater than 4.00, indicating that the quality of the outline met the requirements for the interview and could be used for the formal interview. CONCLUSIONS The established interview outline design and quality control method based on grounded theory provides ideas for the qualitative research interview outline design in the medical field, and lays the foundation for further using grounded theory to study the influencing factors and mechanisms of physician prescription behavior under the DRG background.
5.Reference threshold and offspring short-term security of in vitro fertilization-embryo transfer sperm DNA fragmentation index based on live birth
Chao ZHOU ; Shuxian WANG ; Chunmei YU ; Guangyu YU ; Yueyuan JIANG
Chinese Journal of Tissue Engineering Research 2025;29(1):111-119
BACKGROUND:There is a significant correlation between sperm DNA fragmentation index and fertilization,embryonic development potential,embryo implantation,miscarriage,and offspring safety.However,its clinical reference value is affected by many factors,resulting in extremely limited clinical significance.This study took live birth as the outcome,corrected other confounding factors through propensity score matching,constructed the best clinical cutoff value of sperm DNA fragmentation index and live birth,and conducted internal and external tests on it,which has good predictive value and clinical application efficiency. OBJECTIVE:To investigate the reference threshold and offspring short-term security of in vitro fertilization-embryo transfer sperm DNA fragmentation index based on live birth. METHODS:A total of 1 921 patients who received in vitro fertilization and embryo transfer in Changzhou Maternal and Child Health Area Hospital from May 2019 to May 2021 were selected.On the basis of tendency matching tolerance of 0.02 and propensity score matching of 1:1,540 cases were successfully matched in each live birth group and non-live birth group,and the model group was established.135 patients who received in vitro fertilization and embryo transfer in Nanxishan Hospital of Guangxi Zhuang Autonomous Region were selected as the external validation group.The optimal clinical cutoff value of sperm DNA fragmentation index for live birth was investigated by the receiver operating characteristic curve.The accuracy and clinical application efficacy of the cutoff value were evaluated by restricted cubic spline curve,standard curve,clinical decision curve,clinical impact curve and internal and external validation tests. RESULTS AND CONCLUSION:(1)The DNA fragmentation index of sperm in the non-live birth group was significantly higher than that in the live birth group and had a significant negative correlation with live birth(r=-0.444,P<0.001).(2)Receiver operating characteristic curve results showed that the optimal cut-off value of DNA fragmentation index for live birth was 24.33%;the area under the curve was 0.775(0.746,0.804);the specificity was 72.60%;the sensitivity was 78.90%,and the accuracy was 75.70%.(3)Restricted cubic spline curve fitting the results of Logistic regression showed that when the sperm DNA fragmentation index was greater than 24.57%,the risk of clinical non-live birth increased.(4)The probability of Logistic regression analysis results showed that sperm DNA fragmentation index was a risk factor for live birth[OR(95%CI)=0.916(0.904,0.928),P<0.001],and when sperm DNA fragmentation index was greater than 27.78%,the probability of clinical live birth would be less than 50%.With the increase of sperm DNA fragmentation index by 1 unit,the probability of a live birth fell by 8.4%.(5)Internal and external to the validation of the clinical cutoff value showed that the cutoff point had certain clinical predictive value and accuracy.(6)Clinical decision curve and clinical impact curve results exhibited that the prediction model based on the clinical cut-off value had the maximum clinical net benefit value when the threshold probability was 0.22-0.73,and the ratio of loss to gain within the threshold probability range was always less than 1,which confirmed that the prediction model had good clinical application effectiveness.(7)The results of sperm DNA fragmentation index and offspring short-term security analysis showed that sperm DNA fragmentation index had no significant differences with preterm birth,body weight,deformity and sex.(8)These findings suggest that the optimal clinical cut-off value of sperm DNA fragmentation index for in vitro fertilization-embryo transfer live birth was 24.33%.The established clinical prediction model has good differentiation,accuracy and clinical application effectiveness.Sperm DNA fragmentation index has no significant impact on offspring short-term security,but large samples and long-term follow-up evaluation are still needed.
6.Biomechanical characteristics of walking in patients with idiopathic scoliosis:cross-sectional analysis of three-dimensional motion capture
Yanyun GOU ; Meijin HOU ; Zheng JIANG ; Shaoqing CHEN ; Xiang CHEN ; Yuzhan GAO ; Xiangbin WANG
Chinese Journal of Tissue Engineering Research 2025;29(3):471-477
BACKGROUND:The biomechanical analysis of scoliosis cases is limited,with only independent analysis focusing on the spine or lower limbs,thus lacking a comprehensive evaluation of the multidimensional body.As a result,it becomes challenging to reflect the movement relationship between the trunk and lower limbs during daily activities,which hinders comprehensive clinical treatment guidance. OBJECTIVE:To explore the relationship between different segments of the spine and the kinematics/kinetics of the lower limbs during gait activities by measuring spinal kinematics in scoliosis patients,to provide a comprehensive and multi-level assessment of the biomechanical differences between scoliosis patients and the normal population,consequently offering evidence-based guidance for the prevention and treatment of scoliosis. METHODS:A cross-sectional study was conducted from July 2020 to June 2021 at the Rehabilitation Hospital Affiliated to Fujian University of Traditional Chinese Medicine in Fuzhou University City.A total of 28 scoliosis patients and 28 normal individuals in the same age group were included.Three-dimensional motion capture system was used to capture gait kinematic data at a sampling frequency of 100 Hz.Two force plates(AMTI 400600,sampling frequency 1 500 Hz)were embedded in a 10-meter-long 2.4-meter-wide level ground walkway(with an effective data collection length of 4 m)to collect kinetic data.The differences in spatial-temporal parameters,kinematics,and kinetics of gait between the two groups were compared.Immediately after inclusion,all subjects underwent full spinal X-ray measurements to compare the differences between the scoliosis and normal groups. RESULTS AND CONCLUSION:(1)Patients with scoliosis exhibited reduced relative rotational range of motion between the shoulder and trunk,as well as between the thorax and pelvis,compared to the normal group(P<0.05).However,the rotational range of motion in the pelvis was larger in patients with scoliosis compared to the normal group(P<0.05).(2)Patients with scoliosis showed decreased range of motion in the hip and knee joints,as well as reduced peak torque in hip joint flexion and extension,and lower peak values of ground reaction forces in the concave and convex directions,in comparison to the normal group(P<0.05).(3)Patients with scoliosis demonstrated greater asymmetry indices in knee joint range of motion,relative rotational range of motion between the shoulder and trunk,and between the thorax and pelvis,when compared to the normal group(P<0.05).(4)These findings illustrate a rigid movement pattern among the shoulder,thorax,and pelvis in patients with scoliosis during level walking.There is a reduction in range of motion in the hip and knee joints,as well as decreased peak torque values in hip joint flexion and extension,and ground reaction forces in the concave and convex directions.These characteristics can serve as foundational elements for assessing rehabilitation and developing treatment plans.
7.Ferroptosis and osteoporosis
Cheng YANG ; Weimin LI ; Dongcheng RAN ; Jiamu XU ; Wangxiang WU ; Jiafu XU ; Jingjing CHEN ; Guangfu JIANG ; Chunqing WANG
Chinese Journal of Tissue Engineering Research 2025;29(3):554-562
BACKGROUND:It has also been confirmed that ferroptosis is closely related to a variety of musculoskeletal diseases,such as rheumatoid arthritis,osteosarcoma,and osteoporosis.The pathophysiological mechanisms of ferroptosis and osteoporosis need to be further studied and elucidated to broaden our understanding of iron metabolism and osteoporosis.It will provide research ideas for the future elucidation of new mechanisms of osteoporosis and the development of new technologies and drugs for the treatment of osteoporosis. OBJECTIVE:To provide an overview of the current status of research on ferroptosis in osteoporosis,to provide a new direction for future research on the specific molecular mechanisms of osteoporosis,and to provide more effective and better options for osteoporosis treatment strategies. METHODS:The first author used the computer to search the literature published from 2000 to 2024 in CNKI,WanFang,VIP,and PubMed databases with search terms"ferroptosis,iron metabolism,osteoporosis,osteoblast,osteoclast,bone metabolism,signal pathway,musculoskeletal,review"in Chinese and English.A total of 68 articles were finally included according to the selection criteria. RESULTS AND CONCLUSION:(1)Ferroptosis is a new type of cell death discovered in recent years,which is usually accompanied by a large amount of iron accumulation and lipid peroxidation during cell death,and its occurrence is iron-dependent.This is distinctly different from several types of cell death that are currently being hotly studied(e.g.,cellular pyroptosis,necrotic apoptosis,cuproptosis,and autophagy).(2)Intracellular iron homeostasis is manifested as a balance between iron uptake,export,utilization,and storage.The body's iron regulatory system includes systemic and intracellular regulation.The main factor of systemic regulation is hepcidin produced by hepatic secretion,and cellular regulation depends on the iron regulatory protein/iron response element system.Of course,intracellular iron homeostasis can be controlled by other factors,such as hypoxia,cytokines,and hormones.(3)Lipid peroxidation causes oxidative damage to biological membranes(plasma membrane and internal organelle membranes),lipoproteins,and other lipid-containing molecules.Polyunsaturated fatty acid-containing phospholipids are important targets of lipid peroxidation.Free polyunsaturated fatty acid is an important substrate for lipid oxidation and can bind to the phospholipid bilayer,leading to over-oxidation and thus triggering lipid apoptosis.(4)Several studies have shown that osteoblasts are overloaded with iron in different ways,resulting in the accumulation of unstable ferrous iron and the generation of reactive oxygen species and lipid peroxides,causing ferroptosis of osteoblasts and ultimately a decrease in bone formation,affecting bone homeostasis and the development of osteoporosis.(5)Osteoclasts are large multinucleated cells formed by the fusion of mononuclear macrophage cell lines or bone marrow mesenchymal stem cells induced by nuclear factor-κB ligand receptor activator,and they have the function of bone resorption.Iron ions can promote osteoclast differentiation and bone resorption through the production of intracellular lipid reactive oxygen species,while iron chelators can inhibit osteoclast formation in vitro and thus affect the occurrence and development of osteoporosis.
8.SR9009 combined with indolepropionic acid alleviates inflammation in C2C12 myoblasts through the nuclear factor-kappa B signaling pathway
Huihui JI ; Xu JIANG ; Zhimin ZHANG ; Yunhong XING ; Liangliang WANG ; Na LI ; Yuting SONG ; Xuguang LUO ; Huilin CUI ; Ximei CAO
Chinese Journal of Tissue Engineering Research 2025;29(6):1220-1229
BACKGROUND:Rev-erbα is involved in the regulation of inflammation,but pharmacological activation of Rev-erbα increases the risk for cardiovascular diseases.To reduce the relevant risk,an exploration on SR9009,a Rev-erbα agonist,combined with other drugs to relieve inflammation in skeletal myoblasts was conducted,laying the theoretical foundation for the treatment of inflammation-associated skeletal muscle atrophy. OBJECTIVE:To investigate the relationship of SR9009,indolepropionic acid and nuclear factor-κB signaling pathways in lipopolysaccharide-induced C2C12 myoblasts. METHODS:(1)C2C12 myoblasts were induced to differentiate in the presence of lipopolysaccharide(1 μg/mL).RNA-seq and KEGG pathway analysis were used to study signaling pathways.(2)C2C12 myoblast viability was assessed using the cell counting kit-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,cells were categorized into control group,lipopolysaccharide(1 μg/mL)group,SR9009(10 μmol/L)+lipopolysaccharide group,indolepropionic acid(80μmol/L)+lipopolysaccharide group,and SR9009+indolepropionic acid+lipopolysaccharide group.ELISA was employed to measure protein expression levels of interleukin-6 in the cultured supernatant.Real-time quantitative PCR were employed to measure mRNA expression levels of interleukin-6,tumor necrosis factor α,TLR4 and CD14.Western blot assay were employed to measure protein expression levels of NF-κB p65 and p-NF-κB p65.(3)After Rev-erbα was knocked down by siRNA,knockdown efficiency was assessed by RT-qPCR.And mRNA levels of interleukin-6 and tumor necrosis factor α were also measured. RESULTS AND CONCLUSION:Compared with the blank control group,lipopolysaccharide time-dependently inhibited myofibroblast fusion to form myotubes,the mRNA expression levels of interleukin-6 and tumor necrosis factor α were elevated,and the level of interleukin-6 in the cell supernatant was significantly increased.The results of KEGG pathway showed that the nuclear factor-κB signaling pathway was activated by lipopolysaccharide.Indolepropionic acid exhibited significant suppression of C2C12 myoblasts viability when its concentration exceeded 80 μmol/L.Indolepropionic acid and SR9009 inhibited the activation of NF-κB signaling pathway,thereby played an anti-inflammatory role,and suppressed the mRNA expression levels of interleukin-6,tumor necrosis factor α,TLR4 and CD14.Compared with the lipopolysaccharide group,the ratio of p-NF-κB p65/NF-κB p65 protein expression were downregulated.SR9009 combined with indolepropionic acid notably reduced lipopolysaccharide-induced inflammation,further downregulated the mRNA expression levels of interleukin-6,tumor necrosis factor α,TLR4 and CD14.The ratio of p-NF-κB p65/NF-κB p65 protein expression was significantly lower than that in the SR9009+lipopolysaccharide group or indolepropionic acid+lipopolysaccharide group.Rev-erbα increases time-dependently with lipopolysaccharide induction.The knockdown efficiency of Rev-erbα by siRNA reached over 58%,and lipopolysaccharide was added after Rev-erbα was successfully knocked down.Compared with the lipopolysaccharide group,the mRNA expression levels of interleukin-6 and tumor necrosis factor α were significantly up-regulated.These results conclude that Rev-erbα may act as a promising pharmacological target to reduce inflammation.SR9009 targeted activation of Rev-erbα combined with indolepropionic acid significantly inhibits the nuclear factor-κB signaling pathway and attenuates the inflammatory response of C2C12 myofibroblasts.Moreover,the combined anti-inflammatory effect is superior to that of the intervention alone.
9.An experimental method for simultaneous extraction and culture of primary cortical neurons and microglial cells from SD rats
Longcai HE ; Wenxue SONG ; Jiang MING ; Guangtang CHEN ; Junhao WANG ; Yidong LIAO ; Junshuan CUI ; Kaya XU
Chinese Journal of Tissue Engineering Research 2025;29(7):1395-1400
BACKGROUND:Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders,and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction.It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously. OBJECTIVE:To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. METHODS:Newborn suckling SD rats were taken within 24 hours.The brain was removed and placed in a dish with DMEM,and the pia mater was removed for later use.Primary neurons were extracted from the same brain tissue,and then the remaining brain tissue was used to extract microglial cells.The whole process was performed on ice.Extraction and culture steps of primary cortical neurons:The cerebral cortex was taken 2.0-3.0 mm with forceps,and the tissue was digested with papain for 20 minutes.After aborting digestion,the blown tissue presented an adherent tissue suspension.The supernatant cell suspension was obtained,filtered,and dispensed into 15 mL centrifuge tubes.After centrifugation and re-suspension,the cells were inoculated onto 6-well plate crawls coated with L-polylysine.Neuronal morphology was observed at 1-day intervals,and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7.Microglia extraction and culture steps:The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction,digested by trypsin for 20 minutes.After digestion was stopped,the tissue was blown to a homogenate,and then the homogenate was transferred to the culture bottle for culture.On day 14,the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours.Microglial cells were shed in the supernatant.Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining. RESULTS AND CONCLUSION:(1)After 24 hours of culture,the neurons were adherent to the wall,the cytosol was enlarged,and some neurons developed synapses.After 3 and 5 days of culture,the cytosol was further enlarged,and most of the neurons were in the form of synapses,and some neurons were growing in clusters.On day 7,neuronal synapses were prolonged and thickened,and they were connected with each other to form a network.The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining.(2)The cells grew close to the wall on day 1 of culture.On days 3,5,and 7,the density of microglial cells was small,and the cell morphology was bright oval or round,but the cells basically grew in clumps on the upper layer of other cells.On day 10,the density of microglial cells increased significantly.On day 14,microglial cells grew in dense clumps on the upper layer of other cells,and then they could be isolated and purified.The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence;their purity was greater than 95%.(3)The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity,good morphology,and high viability.
10.Effect of Complanatoside A on the apoptosis of articular chondrocytes
Lu YIN ; Chuanfeng JIANG ; Junjie CHEN ; Ming YI ; Zihe WANG ; Houyin SHI ; Guoyou WANG ; Huarui SHEN
Chinese Journal of Tissue Engineering Research 2025;29(8):1541-1547
BACKGROUND:Chondrocyte apoptosis is an important factor in the development of osteoarthritis,and Complanatoside A has a flavonoid effect,which can inhibit apoptosis of various cells,but its effect on chondrocyte apoptosis and the mechanism of action are not clear. OBJECTIVE:To investigate the intrinsic association and mechanism of Complanatoside A in chondrocyte apoptosis based on the Wnt/β-catenin signaling pathway. METHODS:(1)The cartilage tissues of the femur and tibia transected during knee arthroplasty were collected,and chondrocytes were isolated,cultured in vitro,and identified.(2)Cell counting kit-8 was used to detect the optimal intervention concentration of Complanatoside A in the concentration range of 0-160 μmol/L.(3)Chondrocytes were divided into blank group,sodium nitroprusside(1.5 mmol/L)-induced group,and sodium nitroprusside(1.5 mmol/L)+Complanatoside A(5 μmol/L)group.The viability and apoptosis rate of the cells in each group were detected by cell counting kit-8 and flow cytometry.The expression of type Ⅱ collagen and SOX9 was detected by immunofluorescence staining.The expression of apoptosis-related proteins and Wnt/β-catenin pathway proteins was detected by western blot assay. RESULTS AND CONCLUSION:The cells extracted in vitro were cultured and stained,and were clearly identified as chondrocytes.Complanatoside A had no obvious cytotoxicity to chondrocytes in the concentration range of 0-80 μmol/L,and significantly improved the chondrocyte viability in the concentration range of 2.5-10 μmol/L,especially when the concentration was 5 μmol/L.The apoptotic rate of chondrocytes was higher in the sodium nitroprusside-induced group than the blank control group,while the apoptotic rate was lower in the sodium nitroprusside+Complanatoside A group than the sodium nitroprusside-induced group.The fluorescence intensity of type Ⅱ collagen and SOX9 in chondrocytes was weaker in the sodium nitroprusside-induced group than the blank control group,while the fluorescence intensity of type Ⅱ collagen and SOX9 in the sodium nitroprusside+Complanatoside A group was higher than that of the sodium nitroprusside-induced group.In the sodium nitroprusside-induced group,the protein expression of Bax,Caspase-3,matrix metalloproteinase 13,Wnt3a,Wnt5a and β-catenin was higher than that of the blank control group,while the protein expression of Bcl-2 was lower than that of the blank control group.In the sodium nitroprusside+Complanatoside A group,except for the protein expression of Bcl-2 which was higher than that of the sodium nitroprusside-induced group,the expression of the other aforementioned proteins was lower than that of the sodium nitroprusside-induced group.To conclude,Complanatoside A has a certain inhibitory effect on chondrocyte apoptosis,which could regulate apoptosis-related proteins and promote the expression of chondrocyte regulatory factors,and presumably might play a role through inhibiting the Wnt/β-catenin signaling pathway.

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