1.Yijingtang Reduces Ovarian Inflammatory Responses in Rat Model of Diminished Ovarian Reserve via TLR4/MyD88/NF-κB Signaling Pathway
Heng HU ; Jijun CHU ; Zhe LI ; Haijing CHU ; Jing YU ; Chengcheng LIANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):20-30
ObjectiveTo investigate the effect and mechanism of Yijingtang (YJT) in treating diminished ovarian reserve (DOR) in rats by regulating the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. MethodsFifty female SD rats with normal estrous cycles were randomly allocated into blank, model, low- and high-dose (12.579 and 25.158 g·kg-1, respectively) YJT, and dehydroepiandrosterone (7.487 5 mg·kg-1) groups, with 10 rats in each group. The rats in other groups except the blank group were administrated with the tripterygium glycosides tablet suspension (5 mg·kg-1) by gavage for 14 days for the modeling of DOR. The rats in the drug treatment groups were administrated with corresponding drugs by gavage from day 15 for 30 consecutive days, and those in the blank and model groups received equal volumes of distilled water. The vaginal exfoliated cell smears were observed to assess the changes in the estrous cycle. The wet weight of bilateral ovaries was weighed for calculation of the ovarian index. Hematoxylin-eosin staining was performed to observe the histopathological changes in the ovaries and the proportions of follicles at various levels were calculated. The serum levels of sex hormones [follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH)] and inflammatory factors [tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-10 (IL-10)] were determined by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction(Real-time PCR) was conducted to determine the mRNA levels of TLR4, MyD88, NF-κB profilin α (IκBα), NF-κB and inflammatory factors in the ovarian tissue. Western blot was employed to measure the protein levels of factors related to the TLR4/MyD88/NF-κB signaling pathway in the ovarian tissue. Immunofluorescence (IF) was used to detect the nuclear translocation of NF-κB p65 in the ovarian tissue. ResultsCompared with the blank group, the model group showed disturbed estrous cycles, increased inflammatory infiltration in the ovarian tissue, decreases in ovarian index and proportion of presinusoidal follicles, and an increase in the proportion of atretic follicles (P<0.05, P<0.01). In addition, the model group showed elevated serum levels of FSH, LH, TNF-α, and IL-1β, up-regulated mRNA levels of TLR4, MyD88, IκBα, NF-κB, TNF-α, and IL-1β and protein levels of TLR4, MyD88, p-IκBα, and p-NF-κB p65 (P<0.01), lowered serum levels of AMH, E2, and IL-10, down-regulated mRNA level of IL-10 (P<0.01), and massive nuclear translocation of NF-κB p65 in the ovarian tissue. Compared with the model group, dehydroepiandrosterone and low and high doses of YJT restored the disturbed estrous cycle, reduced inflammatory infiltration in the ovarian tissue, increased the ovarian index (P<0.01), and changed the follicular composition ratio (P<0.01). Furthermore, the drugs lowered the serum levels of FSH, LH, TNF-α, and IL-1β, down-regulated the mRNA levels of TLR4, MyD88, IκBα, NF-κB, TNF-α, and IL-1β and the protein levels of TLR4, MyD88, p-IκBα, and p-NF-κB p65 (P<0.05, P<0.01), raised the serum levels of AMH, E2, and IL-10, up-regulated the mRNA level of IL-10 (P<0.05, P<0.01), and reduced the nuclear translocation of NF-κB p65 in the ovarian tissue. ConclusionYJT may inhibit the release and expression of inflammatory factors by regulating the TLR4/MyD88/NF-κB signaling pathway to attenuate the inflammatory responses in the ovarian tissue, thereby improving the ovarian function in DOR rats.
2.Intraspecific variation of Forsythia suspensa chloroplast genome.
Yu-Han LI ; Lin-Lin CAO ; Chang GUO ; Yi-Heng WANG ; Dan LIU ; Jia-Hui SUN ; Sheng WANG ; Gang-Min ZHANG ; Wen-Pan DONG
China Journal of Chinese Materia Medica 2025;50(8):2108-2115
Forsythia suspensa is a traditional Chinese medicine and a commonly used landscaping plant. Its dried fruit is used in medicine for its functions of clearing heat, removing toxins, reducing swelling, dissipating masses, and dispersing wind and heat. It possesses extremely high medicinal and economic value. However, the genetic differentiation and diversity of its wild populations remain unclear. In this study, chloroplast genome sequences were obtained from 15 wild individuals of F. suspensa using high-throughput sequencing technology. The sequence characteristics and intraspecific variations were analyzed. The results were as follows:(1) The full length of the F. suspensa chloroplast genome ranged from 156 184 to 156 479 bp, comprising a large single-copy region, a small single-copy region, and two inverted repeat regions. The chloroplast genome encoded a total of 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes.(2) A total of 166-174 SSR loci, 792 SNV loci, and 63 InDel loci were identified in the F. suspensa chloroplast genome, indicating considerable genetic variation among individuals.(3) Population structure analysis revealed that F. suspensa could be divided into five or six groups. Both the population structure analysis and phylogenetic reconstruction results indicated significant genetic variation within the wild populations of F. suspensa, with no obvious correlation between intraspecific genetic differentiation and geographical distribution. This study provides new insights into the genetic diversity and differentiation within F. suspensa species and offers additional references for the conservation of species diversity and the utilization of germplasm resources in wild F. suspensa.
Genome, Chloroplast
;
Forsythia/classification*
;
Phylogeny
;
Genetic Variation
;
Chloroplasts/genetics*
;
Microsatellite Repeats
3.Application progress on functional insoles in the prevention and treatment of diabetic foot.
Heng-Yu LIU ; Zhen-de JIANG ; Yao-Kuan RUAN ; Qiu-Ju LI ; Si-Yuan CHEN ; Shun-Yu WEI ; Nan MEI ; Chou WU ; Fei CHANG
China Journal of Orthopaedics and Traumatology 2025;38(9):969-975
Diabetic foot (DF) is one of the most serious chronic complications of diabetes. The incidence rate among global diabetes patients is as high as 15% to 25%, and about 50% of patients will develop contralateral foot ulcers within 5 years after the first unilateral ulcer. As a non-invasive prevention and control solution, the application progress of functional insoles is mainly reflected in the following aspects:(1) Material innovation. The application of new composite materials and smart materials has significantly enhanced the pressure reduction effect and comfort. (2) Structural optimization. The development of multi-layer design and local pressure reduction structure has achieved more precise pressure distribution regulation. (3) Manufacturing process. 3D printing and parametric design have enabled the personalized customization of functional insoles. (4) Intelligent monitoring. It integrates functions such as pressure sensing and temperature monitoring, achieving real-time monitoring and early warning of foot conditions. Clinical research has confirmed that personalized functional insoles could reduce the incidence of foot ulcers and shorten the healing time of ulcers. At present, the research hotspots mainly focus on the development of smart materials, the construction of multi-functional integration and remote monitoring systems. However, in-depth research is still needed in the aspects of biomechanical mechanisms, standardized evaluation systems and long-term efficacy assessment. The development of future functional insoles should focus on the coordinated advancement of "personalization-intelligence-standardization", with the aim of providing more effective solutions for the prevention and treatment of DF.
Humans
;
Diabetic Foot/therapy*
;
Foot Orthoses
4.Clinical study of the lower limb constitutional alignment restoration in patients undergoing robotic-assisted functionally aligned total knee arthroplasty.
Heng ZHANG ; Yu CHEN ; Bo-Wen LI ; Feng QIAN ; Jian-Sheng ZHOU
China Journal of Orthopaedics and Traumatology 2025;38(10):994-1000
OBJECTIVE:
To compare and analyze the early clinical efficacy of robotic-assisted functionally aligned total knee arthroplasty (RFA-TKA) and manual mechanically aligned total knee arthroplasty (MMA-TKA).
METHODS:
A retrospective analysis was conducted on 58 patients with end-stage knee osteoarthritis who underwent total knee arthroplasty (TKA) between February 2024 and January 2025. According to the different surgical methods, the patients were divided into the RFA-TKA group and the MMA-TKA group.There were 26 patients in the RFA-TKA group, including 7 males and 19 females, aged from 58 to 80 years old with an average of (69.08±5.93) years old;robotic-assisted functional alignment was adopted in this group. The MMA-TKA group consisted of 32 patients, including 9 males and 23 females, aged from 53 to 78 years old with an average of (66.59±7.76) years old;manual mechanical alignment was used in this group. Surgical indicators of the two groups were compared, including operation time, hemoglobin loss, and soft tissue release. Postoperative clinical outcomes were evaluated using Knee Society score-the knee dcore (KSS-K) and the visual analogue scale (VAS) for knee pain. Postoperative radiological measurements micluded, the hip-knee-ankle (HKA) angle, mechanical lateral distal femoral angle (mLDFA), and medial proximal tibial angle (MPTA) were measured.
RESULTS:
All patients were followed up for more than 3 months. There was no statistically significant difference in the operation time between two groups (P>0.05). The hemoglobin loss in the RFA-TKA group on the first day after surgery was (5.08±4.07) g·L-1, which was less than that of the MMA-TKA group (14.03±12.49) g·L-1, and the difference was statistically significant (P<0.05). The number of patients who underwent soft tissue release in the RFA-TKA group was 5 cases, which was less than 32 cases in the MMA-TKA group, and the difference was statistically significant (P<0.05). At 3 months after surgery, the HKA angle (177.71±1.05)°, mLDFA (88.30±0.64)° and MPTA (87.53±1.47)° of the RFA-TKA group were all smaller than those of the MMA-TKA group (179.19±0.57)°, (89.14±0.59)° and(89.27±0.62)° respectively, with statistically significant differences (all P<0.05). The KSS-K of the RFA-TKA group was (92.50±3.64) points, which was higher than that of the MMA-TKA group(86.22±3.38) points, and the difference was statistically significant(P<0.05). For the VAS score of knee pain during walking, the RFA-TKA group (0.31±0.62) points was lower than the MMA-TKA group (1.03±1.12) points, and the difference was statistically significant (P<0.05).
CONCLUSION
Domestic robot-assisted functionally aligned TKA effectively restores the constitutional alignment of the lower limb, reduces the need for soft tissue release, minimizes intraoperative blood loss, alleviates postoperative pain, and achieve superior early clinical outcomes.
Humans
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Male
;
Female
;
Arthroplasty, Replacement, Knee/methods*
;
Aged
;
Middle Aged
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Aged, 80 and over
;
Retrospective Studies
;
Robotic Surgical Procedures/methods*
;
Osteoarthritis, Knee/physiopathology*
;
Lower Extremity/physiopathology*
5.Comparison of application and efficacy of domestic HURWA and imported Smith & Nephew Cori robots in total knee arthroplasty.
Ming-You WANG ; Zhuo-Dong TANG ; Yu-Ping LAN ; Heng XIAO ; Ming-Li WANG ; Xun-Zhou SONG ; Hong-Ping WANG
China Journal of Orthopaedics and Traumatology 2025;38(10):1027-1036
OBJECTIVE:
Investigation on the clinical application of HURWA robot and Smith & Nephew Cori robot in total knee arthroplasty(TKA).
METHODS:
A retrospective analysis was performed on 84 patients with knee osteoarthritis who underwent robotic-assisted TKA (RATKA) between June 2023 and March 2025. According to the different robotic systems used, the patients were divided into the domestic HUARUN robotic-assisted total knee arthroplasty group (HRATKA group) and the Smith & Nephew Cori robotic-assisted total knee arthroplasty group (CRATKA group). There were 42 patients in the HRATKA group, including 16 males and 26 females; the age ranged from 56 to 73 years old, with an average of (64.70±8.30) years old;the body mass index (BMI) was (25.10±2.30) kg·m-2;21 cases were on the right side and 21 cases on the left side;in terms of Kellgren-Lawrence(K-L) classification, there were 15 cases of Grade Ⅲ and 27 cases of Grade Ⅳ;the disease duration ranged from 3 to 25 years, with an average of (15.5±7.5) years. The CRATKA group also included 42 patients, with 14 males and 28 females;the age ranged from 58 to 74 years old, with an average of (65.60±7.50) years old;the BMI was (24.50±2.70) kg·m-2; 20 cases were on the right side and 22 cases on the left side;regarding K-L classification, there were 11 cases of Grade Ⅲ and 31 cases of Grade Ⅳ;the disease duration ranged from 2 to 26 years, with an average of (16.5±8.8) years. Collect general data of all patients, including age, gender, height, weight, surgical site, K-L classification, incision length, and operation time. To evaluate prosthesis position, compare the frontal tibia component (FTC) angle, lateral femoral component (LFC) angle, lateral tibia component (LTC) angle, and frontal femoral component angle between the two groups of patients after surgery. Measure the deviation of the hip-knee-ankle (HKA) angle to assess lower limb alignment. Additionally, compare the following indicators between the two groups:Knee Society Score (KSS), Visual Analogue Scale (VAS) for pain, knee range of motion (ROM), hemoglobin (HB) level, hematocrit (HCT) level, complication rate, and in-hospital satisfaction.
RESULTS:
All patients successfully completed the surgery as scheduled, and all were followed up after the operation. The follow-up period ranged from 5 to 17 months with an average of (11.2±6.1) months. There were 4 cases of venous thrombosis in the HRATKA group and 3 cases in the CRATKA group;each group had 2 cases of wound exudation. No mechanical-related complications, pulmonary embolism, or other severe complications occurred. Comparison of the incision length and hospital stay between the HRATKA group and the CRATKA group showed no statistically significant difference (P>0.05). The operation time in the HRATKA group was (96.80±7.10) minutes, which was longer than that in the CRATKA group (90.10±8.80) minutes, and the difference was statistically significant (P<0.05). In the HRATKA group, the HKA angle was (178.93±1.11) degree, the FFC angle was (89.00±0.91)°, and the LFC angle was (7.31±2.17) degree;the corresponding values in the CRATKA group were (178.05±1.34)°, (87.88±1.74)°, and (10.60±2.84) degree respectively. The differences in these three indicators between the two groups were all statistically significant (P<0.05). However, there were no statistically significant differences in the FTC angle or LTC angle between the two groups (P>0.05). There was also no statistically significant difference in the total perioperative blood loss between the two groups (P>0.05). At 3 days after surgery, the VAS score for movement in the HRATKA group (5.95±1.45) points was higher than that in the CRATKA group (4.50±0.97) points, with a statistically significant difference (P<0.05);at 90 days after surgery, there was no statistically significant difference in the movement VAS score between the two groups (P>0.05). Additionally, no statistically significant differences were observed between the two groups in the KSS, ROM at 3 and 90 days after surgery, or satisfaction degree during hospitalization (all P>0.05).
CONCLUSION
The domestic HURWA robot demonstrates excellent performance in osteotomy efficiency and lower limb alignment recovery. The Smith & Nephew Cori robot has a significant advantage in soft tissue assessment and joint stability optimization. Both robotic systems offer high-quality surgical treatments that significantly improve short-term knee function.
Humans
;
Male
;
Female
;
Arthroplasty, Replacement, Knee/instrumentation*
;
Aged
;
Middle Aged
;
Retrospective Studies
;
Robotic Surgical Procedures/methods*
;
Osteoarthritis, Knee/surgery*
6.Protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on a yorkshire model of brain injury after traumatic blood loss.
Xiang-Yu SONG ; Yang-Hui DONG ; Zhi-Bo JIA ; Lei-Jia CHEN ; Meng-Yi CUI ; Yan-Jun GUAN ; Bo-Yao YANG ; Si-Ce WANG ; Sheng-Feng CHEN ; Peng-Kai LI ; Heng CHEN ; Hao-Chen ZUO ; Zhan-Cheng YANG ; Wen-Jing XU ; Ya-Qun ZHAO ; Jiang PENG
Chinese Journal of Traumatology 2025;28(6):469-476
PURPOSE:
To investigate the protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on ischemic hypoxic injury of yorkshire brain tissue caused by traumatic blood loss.
METHODS:
This article performed a random controlled trial. Brain tissue of 7 yorkshire was selected and divided into the sub-low temperature anterograde machine perfusion group (n = 4) and the blank control group (n = 3) using the random number table method. A yorkshire model of brain tissue injury induced by traumatic blood loss was established. Firstly, the perfusion temperature and blood oxygen saturation were monitored in real-time during the perfusion process. The number of red blood cells, hemoglobin content, NA+, K+, and Ca2+ ions concentrations and pH of the perfusate were detected. Following perfusion, we specifically examined the parietal lobe to assess its water content. The prefrontal cortex and hippocampus were then dissected for histological evaluation, allowing us to investigate potential regional differences in tissue injury. The blank control group was sampled directly before perfusion. All statistical analyses and graphs were performed using GraphPad Prism 8.0 Student t-test. All tests were two-sided, and p value of less than 0.05 was considered to indicate statistical significance.
RESULTS:
The contents of red blood cells and hemoglobin during perfusion were maintained at normal levels but more red blood cells were destroyed 3 h after the perfusion. The blood oxygen saturation of the perfusion group was maintained at 95% - 98%. NA+ and K+ concentrations were normal most of the time during perfusion but increased significantly at about 4 h. The Ca2+ concentration remained within the normal range at each period. Glucose levels were slightly higher than the baseline level. The pH of the perfusion solution was slightly lower at the beginning of perfusion, and then gradually increased to the normal level. The water content of brain tissue in the sub-low and docile perfusion group was 78.95% ± 0.39%, which was significantly higher than that in the control group (75.27% ± 0.55%, t = 10.49, p < 0.001), and the difference was statistically significant. Compared with the blank control group, the structure and morphology of pyramidal neurons in the prefrontal cortex and CA1 region of the hippocampal gyrus were similar, and their integrity was better. The structural integrity of granulosa neurons was destroyed and cell edema increased in the perfusion group compared with the blank control group. Immunofluorescence staining for glail fibrillary acidic protein and Iba1, markers of glial cells, revealed well-preserved cell structures in the perfusion group. While there were indications of abnormal cellular activity, the analysis showed no significant difference in axon thickness or integrity compared to the 1-h blank control group.
CONCLUSIONS
Mild hypothermic machine perfusion can improve ischemia and hypoxia injury of yorkshire brain tissue caused by traumatic blood loss and delay the necrosis and apoptosis of yorkshire brain tissue by continuous oxygen supply, maintaining ion homeostasis and reducing tissue metabolism level.
Animals
;
Perfusion/methods*
;
Disease Models, Animal
;
Brain Injuries/etiology*
;
Swine
;
Male
;
Hypothermia, Induced/methods*
7.The Enhancing Effects and Underlying Mechanism of Ionizing Radiation on Adipogenic Differentiation of Mesenchymal Stem Cells via Regulating Oxidative Stress Pathway.
Fu-Hao YU ; Bo-Feng YIN ; Pei-Lin LI ; Xiao-Tong LI ; Jia-Yi TIAN ; Run-Xiang XU ; Jie TANG ; Xiao-Yu ZHANG ; Wen-Jing ZHANG ; Heng ZHU ; Li DING
Journal of Experimental Hematology 2025;33(1):246-254
OBJECTIVE:
To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells (MSCs).
METHODS:
Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with 60Co, and the radiation dose rate was 0.98 Gy/min. Bulk RNA-seq was performed on control and irradiated MSCs. The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis. Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro, and real-time fluorescence quantitative PCR (qPCR) was used to detect the expression differences of key regulatory factors Cebpa, Lpl and Pparg after radiation treatment. At the same time, qPCR and Western blot were used to detect the effect of inhibition of Nrf2, a key factor of antioxidant stress pathway, on the expression of key regulatory factors of adipogenesis. Moreover, the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR.
RESULTS:
Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways. The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs, with high expression of Cebpa, Lpl and Pparg, as well as oxidative stress-related gene Nrf2. Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation. Notably, the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs. In addition, irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs.
CONCLUSION
Ionizing radiation promotes adipogenesis of MSCs in mice, and oxidative stress pathway participates in this effect, blocking Nrf2 further promotes the adipogenesis of MSCs. Additionally, irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.
Mesenchymal Stem Cells/cytology*
;
Oxidative Stress/radiation effects*
;
Animals
;
Adipogenesis/radiation effects*
;
Mice
;
Radiation, Ionizing
;
Cell Differentiation/radiation effects*
;
Humans
;
NF-E2-Related Factor 2/metabolism*
;
PPAR gamma
;
Cells, Cultured
8.Establishment and Application of an in Vitro Cellular Model of Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells with Serum Injury in aGVHD Mouse.
Run-Xiang XU ; Pei-Lin LI ; Jia-Yi TIAN ; Jie TANG ; Bo-Feng YIN ; Fu-Hao YU ; Fei-Yan WANG ; Xiao-Tong LI ; Xiao-Yu ZHANG ; Wen-Rong XIA ; Heng ZHU ; Li DING
Journal of Experimental Hematology 2025;33(1):255-261
OBJECTIVE:
To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs).
METHODS:
The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×107 per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×107 per mouse) and spleen lymphocytes (2×106 per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR).
RESULTS:
An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced.
CONCLUSION
The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals
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Mesenchymal Stem Cells/cytology*
;
Mice
;
Mice, Inbred BALB C
;
Mice, Inbred C57BL
;
Adipogenesis
;
Female
;
Cell Differentiation
;
Graft vs Host Disease/blood*
;
Bone Marrow Cells/cytology*
;
PPAR gamma/metabolism*
;
Disease Models, Animal
;
CCAAT-Enhancer-Binding Protein-alpha/metabolism*
9.Preparation and Evaluation of Clinical-Grade Human Umbilical Cord-Derived Mesenchymal Stem Cells with High Expression of Hematopoietic Supporting Factors.
Jie TANG ; Pei-Lin LI ; Xiao-Yu ZHANG ; Xiao-Tong LI ; Fu-Hao YU ; Jia-Yi TIAN ; Run-Xiang XU ; Bo-Feng YIN ; Li DING ; Heng ZHU
Journal of Experimental Hematology 2025;33(3):892-898
OBJECTIVE:
To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics.
METHODS:
Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR).
RESULTS:
The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method.
CONCLUSION
Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans
;
Mesenchymal Stem Cells/metabolism*
;
Umbilical Cord/cytology*
;
Cell Differentiation
;
Female
;
Cell Proliferation
;
Cells, Cultured
;
Chemokine CXCL12/metabolism*
;
Angiopoietin-1/metabolism*
;
Vascular Cell Adhesion Molecule-1/metabolism*
;
Stem Cell Factor/metabolism*
;
Flow Cytometry
;
Pregnancy
10.Yijingtang Reduces Ovarian Inflammatory Responses in Rat Model of Diminished Ovarian Reserve via TLR4/MyD88/NF-κB Signaling Pathway
Heng HU ; Jijun CHU ; Zhe LI ; Haijing CHU ; Jing YU ; Chengcheng LIANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):20-30
ObjectiveTo investigate the effect and mechanism of Yijingtang (YJT) in treating diminished ovarian reserve (DOR) in rats by regulating the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. MethodsFifty female SD rats with normal estrous cycles were randomly allocated into blank, model, low- and high-dose (12.579 and 25.158 g·kg-1, respectively) YJT, and dehydroepiandrosterone (7.487 5 mg·kg-1) groups, with 10 rats in each group. The rats in other groups except the blank group were administrated with the tripterygium glycosides tablet suspension (5 mg·kg-1) by gavage for 14 days for the modeling of DOR. The rats in the drug treatment groups were administrated with corresponding drugs by gavage from day 15 for 30 consecutive days, and those in the blank and model groups received equal volumes of distilled water. The vaginal exfoliated cell smears were observed to assess the changes in the estrous cycle. The wet weight of bilateral ovaries was weighed for calculation of the ovarian index. Hematoxylin-eosin staining was performed to observe the histopathological changes in the ovaries and the proportions of follicles at various levels were calculated. The serum levels of sex hormones [follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH)] and inflammatory factors [tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-10 (IL-10)] were determined by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction(Real-time PCR) was conducted to determine the mRNA levels of TLR4, MyD88, NF-κB profilin α (IκBα), NF-κB and inflammatory factors in the ovarian tissue. Western blot was employed to measure the protein levels of factors related to the TLR4/MyD88/NF-κB signaling pathway in the ovarian tissue. Immunofluorescence (IF) was used to detect the nuclear translocation of NF-κB p65 in the ovarian tissue. ResultsCompared with the blank group, the model group showed disturbed estrous cycles, increased inflammatory infiltration in the ovarian tissue, decreases in ovarian index and proportion of presinusoidal follicles, and an increase in the proportion of atretic follicles (P<0.05, P<0.01). In addition, the model group showed elevated serum levels of FSH, LH, TNF-α, and IL-1β, up-regulated mRNA levels of TLR4, MyD88, IκBα, NF-κB, TNF-α, and IL-1β and protein levels of TLR4, MyD88, p-IκBα, and p-NF-κB p65 (P<0.01), lowered serum levels of AMH, E2, and IL-10, down-regulated mRNA level of IL-10 (P<0.01), and massive nuclear translocation of NF-κB p65 in the ovarian tissue. Compared with the model group, dehydroepiandrosterone and low and high doses of YJT restored the disturbed estrous cycle, reduced inflammatory infiltration in the ovarian tissue, increased the ovarian index (P<0.01), and changed the follicular composition ratio (P<0.01). Furthermore, the drugs lowered the serum levels of FSH, LH, TNF-α, and IL-1β, down-regulated the mRNA levels of TLR4, MyD88, IκBα, NF-κB, TNF-α, and IL-1β and the protein levels of TLR4, MyD88, p-IκBα, and p-NF-κB p65 (P<0.05, P<0.01), raised the serum levels of AMH, E2, and IL-10, up-regulated the mRNA level of IL-10 (P<0.05, P<0.01), and reduced the nuclear translocation of NF-κB p65 in the ovarian tissue. ConclusionYJT may inhibit the release and expression of inflammatory factors by regulating the TLR4/MyD88/NF-κB signaling pathway to attenuate the inflammatory responses in the ovarian tissue, thereby improving the ovarian function in DOR rats.

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