Objective To explore the protective effect and related mechanism of Compound Baimai Powder(CBMP,a compound description of Mongolian medicine)on astrocytes after oxygen glu-cose deprivation and reoxygenation(OGD/R)injury.Methods Astrocyte model of OGD/R injury was subjected to simulate in vitro cerebral ischemia/reperfusion injury.Cultured astrocytes were randomly divided into normal group,OGD/R group,OGD/R+nimodipine group(10 μmol/L),OGD/R+low-and high-dose CBMP groups(25,50 μmol/L).Cell viability and apoptosis were de-tected with CCK-8 assay and flow cytometry,respectively.Western blotting was used to measure the expression levels of the proteins related to the nuclear factor erythroid-2-related factor-2(NRF2)/antioxidant response element and Janus kinase(J AK)/signal transducer and activator of transcription(STAT)signaling pathways.ELISA was employed to examine the levels of inflam-matory factors IL-1β,IL-6,and TNF-α,as well as oxidative stress molecules ROS,GSH,MDA and SOD.Results Compared to the normal group,the OGD/R group showed significant decreases in cell viability,NRF2 protein level,and SOD and GSH activities(P＜0.05,P＜0.01),and obvious increases in p-JAK and p-STAT proteins levels,contents of IL-1β,IL-6 and TNF-α,and ROS and MDA levels(P＜0.05,P＜0.01).High-dose CBMP treatment resulted in notably elevated cell via-bility and NRF2 protein level,while reduced levels of p-JAK[(1.20±0.20)vs(2.50±0.26)]and p-STAT[(1.15±0.25)vs(2.10±0.21)]proteins,IL-6[(30.33±5.20)vs(180.35±18.50)]and TNF-α[(50.12±8.24)vs(160.45±15.20)]when compared to the OGD/R group(P＜0.05,P＜0.01).Conclusion CBMP exerts protective effect on astrocytes against OGD/R injury.

PURPOSE: To investigate the regulatory role of nerve injury-induced protein 1 (NINJ1) in the anti-inflammatory function of human umbilical cord mesenchymal stem cells (hUC-MSCs) co-stimulated by interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). METHODS: hUC-MSCs were expanded in vitro using standard protocols, with stem cell characteristics confirmed by flow cytometry and multilineage differentiation assays. The immunomodulatory properties and cellular activity of cytokine-co-pretreated hUC-MSCs were systematically evaluated via quantitative reverse transcription RT-qPCR, lymphocyte proliferation suppression assays, and Cell Counting Kit-8 viability tests. Transcriptome sequencing, Western blotting and small interfering RNA interference were integrated to analyze the regulatory mechanisms of NINJ1 expression. Functional roles of NINJ1 in pretreated hUC-MSCs were elucidated through gene silencing combined with lactate dehydrogenase release assays, Annexin V/Propidium Iodide apoptosis analysis, macrophage co-culture models, and cytokine Enzyme-Linked Immunosorbent Assay. Therapeutic efficacy was validated in a cecal ligation and puncture-induced septic mouse model: 80 mice were randomly allocated into 4 experimental groups (n=20/group): sham group (laparotomy without cecal ligation); phosphate-buffered saline-treated group (cecal ligation and puncture (CLP) + 0.1 mL phosphate-buffered saline); hUC-MSCs (small interfering RNA (siRNA)-interferon-gamma and tumor necrosis factor-alpha co-stimulation (IT))-treated group (CLP + hUC-MSCs transfected with scrambled siRNA); and hUC-MSCs (siNINJ1-IT)-treated group (CLP + hUC-MSCs with NINJ1-targeting siRNA). RESULTS: hUC-MSCs demonstrated compliance with International Society for Cellular Therapy criteria, confirming their stem cell identity. IFN-γ/TNF-α co-pretreatment enhanced the immunosuppressive capacity of hUC-MSCs, accompanied by the reduction of cellular viability, while concurrently upregulating pro-inflammatory cytokines such as interleukin-6 and interleukin-1β. This co-stimulation significantly elevated NINJ1 expression in hUC-MSCs, whereas genetic silencing of NINJ1 effectively suppressed pro-inflammatory cytokine production and attenuated damage-associated molecular patterns release through inhibition of programmed plasma membrane rupture. Furthermore, the NINJ1 interference potentiated the ability of cytokine-pretreated hUC-MSCs to suppress LPS-induced pro-inflammatory responses in RAW264.7 macrophages. In cecal ligation and puncture-induced sepsis model, NINJ1-silenced hUC-MSCs exhibited enhanced therapeutic efficacy, manifested by reduced systemic inflammation and multi-organ damage. CONCLUSION Our findings shed new light on the immunomodulatory functions of cytokine-primed MSCs, offering groundbreaking insights for developing MSC-based therapies against inflammatory diseases via interfering the expression of NINJ1.
Mesenchymal Stem Cells/drug effects* ; Animals ; Interferon-gamma/pharmacology* ; Tumor Necrosis Factor-alpha/pharmacology* ; Humans ; Mice ; Umbilical Cord/cytology* ; Cells, Cultured ; Apoptosis ; Male

Objective:To study the effects of epidermal growth factor(EGF)on the coordinated motor behaviors and changes of nigrostriatal dopaminergic system under the impact of EGF in middle-aged rats.Methods:Healthy aged Sprague Dawley(SD)rats were subcutaneously administered EGF(0.1 mg/ml)in the dorsal region for 7 days.Adhe-sive removal test,tapered beam walking test and descending a wire mesh pole test were performed to detect the changes in the coordinated motor behaviors of rats as well as changes in the expression of tyrosine hydroxylase(TH)and dopa-mine transporter(DAT)of nigrostriatal dopaminergic system in rats.Results:Middle-aged rats showed reduced behav-ioral parameters of adhesive removal test,tapered beam walking test and descending a wire mesh pole test compared with those of adult rats(P<0.05),which were improved after treatment with EGF(P<0.05).The expression of TH and DAT in nigrostriatal dopaminergic system was down-regulated in middle-aged rats compared with that in adult rats(P<0.01),however,which were up-regulated after treatment with EGF(P<0.01).Conclusion:EGF can amelio-rate the decline in coordinated motor behaviors and increases the activity of nigrostriatal dopaminergic system in middle-aged rats.

Objective:To study the effects of epidermal growth factor(EGF)on the coordinated motor behaviors and changes of nigrostriatal dopaminergic system under the impact of EGF in middle-aged rats.Methods:Healthy aged Sprague Dawley(SD)rats were subcutaneously administered EGF(0.1 mg/ml)in the dorsal region for 7 days.Adhe-sive removal test,tapered beam walking test and descending a wire mesh pole test were performed to detect the changes in the coordinated motor behaviors of rats as well as changes in the expression of tyrosine hydroxylase(TH)and dopa-mine transporter(DAT)of nigrostriatal dopaminergic system in rats.Results:Middle-aged rats showed reduced behav-ioral parameters of adhesive removal test,tapered beam walking test and descending a wire mesh pole test compared with those of adult rats(P<0.05),which were improved after treatment with EGF(P<0.05).The expression of TH and DAT in nigrostriatal dopaminergic system was down-regulated in middle-aged rats compared with that in adult rats(P<0.01),however,which were up-regulated after treatment with EGF(P<0.01).Conclusion:EGF can amelio-rate the decline in coordinated motor behaviors and increases the activity of nigrostriatal dopaminergic system in middle-aged rats.

Objective To explore the protective effect and related mechanism of Compound Baimai Powder(CBMP,a compound description of Mongolian medicine)on astrocytes after oxygen glu-cose deprivation and reoxygenation(OGD/R)injury.Methods Astrocyte model of OGD/R injury was subjected to simulate in vitro cerebral ischemia/reperfusion injury.Cultured astrocytes were randomly divided into normal group,OGD/R group,OGD/R+nimodipine group(10 μmol/L),OGD/R+low-and high-dose CBMP groups(25,50 μmol/L).Cell viability and apoptosis were de-tected with CCK-8 assay and flow cytometry,respectively.Western blotting was used to measure the expression levels of the proteins related to the nuclear factor erythroid-2-related factor-2(NRF2)/antioxidant response element and Janus kinase(J AK)/signal transducer and activator of transcription(STAT)signaling pathways.ELISA was employed to examine the levels of inflam-matory factors IL-1β,IL-6,and TNF-α,as well as oxidative stress molecules ROS,GSH,MDA and SOD.Results Compared to the normal group,the OGD/R group showed significant decreases in cell viability,NRF2 protein level,and SOD and GSH activities(P＜0.05,P＜0.01),and obvious increases in p-JAK and p-STAT proteins levels,contents of IL-1β,IL-6 and TNF-α,and ROS and MDA levels(P＜0.05,P＜0.01).High-dose CBMP treatment resulted in notably elevated cell via-bility and NRF2 protein level,while reduced levels of p-JAK[(1.20±0.20)vs(2.50±0.26)]and p-STAT[(1.15±0.25)vs(2.10±0.21)]proteins,IL-6[(30.33±5.20)vs(180.35±18.50)]and TNF-α[(50.12±8.24)vs(160.45±15.20)]when compared to the OGD/R group(P＜0.05,P＜0.01).Conclusion CBMP exerts protective effect on astrocytes against OGD/R injury.

Objective: To detect pathogenic gene variants in two Chinese families with cone-rod dystrophy(CORD). Methods: After the informed consent and comprehensive ophthalmic examinations for the patients, 3 mL peripheral blood was taken from the patients’ blood vessel and DNA was extracted. The DNA was sequenced by whole-exome sequencing technology and variants were analyzed. Results: Two novel compound heterozygous AIPL1 variants were detected in two patients, which were c. 923T>C(p.L308P) and c. 421C>T(p.Q141X) variants in Family 1, c. 572T>C(p.L191P) and c. 421C>T(p.Q141X) in Family 2 . Conclusion The results supported that AIPL1 gene variants are the main cause of the two CORD families. Whole-exome sequencing technology is a useful tool in the clinical differentiated diagnosis and genetic counseling for CORD patients.

Objective To investigate the effect of triptolide (TP) on the expression of hypoxia inducible factor 1 alpha (HIF1α) and vascular endothelial growth factor (VEGF) in the human umbilical vein endothelial cells (HUVECs) under hypoxia. Methods (1) HUVECs were treated with 0, 40, 80, 160 and 320 nmol/L TP (named with hypoxia group, TP40 group, TP80 group, TP160 group and TP320 group, respectively) under the hypoxic condition (37℃, 5%CO2, 1%O2, 94%N2) for culturing 12 hours. Meanwhile, cells cultured under normoxia condition (without TP added) were set as the normoxia group. Western blot assay was used to detect the expression of HIF1αin each group. (2) The cells were divided into normal control group, hypoxia group and TP80 group. The immunofluorescence method was performed to detect the localization of HIF1α in cells. (3) Expressions of VEGF were detected by Western blot assay in TP80 group and hypoxia group. (4) The cells were divided into hypoxia group, TP80 group, TP80+KF20 group (80 nmol/L TP and 20μmol/L KC7F2), and TP80+KF30 group (80 nmol/L TP and 30 μmol/L KC7F2). After 12-hour culturing, Western blot assay was used to detect the expressions of HIF1α and VEGF in each group. Results (1) Under the normoxia condition, no HIF1α was detected in HUVECs. The expression level of HIF1αwas significantly increased in TP80 group than that in hypoxia group (P<0.05), while there was no significant change in expression of hypoxia HIF1αin TP160 group and TP320 group compared with that of hypoxic group. (2) The immunofluorescence result showed that HIF1α was mainly expressed in the nucleus. (3) The expression of VEGF was significantly increased in TP80 group than that in hypoxia group (P < 0.05). (4) After the intervention of KC7F2, HIF1αand VEGF expression levels were significantly decreased in the TP80+KF20 group and the TP80+KF30 group than those in the TP80 group (P<0.05). Conclusion TP can improve the expression of HIF1αand VEGF to accelerate the proliferation of endothelial cells under hypoxia condition.

Objective To fabricate a novel tissue-engineered cartilage with adipose-derived stem cells (ADSCs) seeded on the chitosan hydrogel scaffold to repair articular cartilage defect.Methods Adipose tissue and costal cartilage were harvested from New Zealand rabbits,and ADSCs in passage one and chondrocytes were obtained after the samples were digested and cultured in vitro.ADSCs were digested,suspended,seeded onto the sterile chitosan gel,and cultured in vitro for 1 week to fabricate the tissue-engineered cartilage.The defects were respectively filled with the tissue-engineered cartilage (composite group),chondrocyte suspension (cell group),chitosan gel (material group) and nothing at all (control group).At postoperative 12 weeks,cartilage repair was evaluated using the gross examination,histological staining,immunohistochemical staining and international cartilage repair society (ICRS) histological score.Results Effect of cartilage repair in composite group was significantly better compared to other groups.The regenerated tissue in composite group seemed tightly bound in normal tissue,with similar structure and extracellular matrix secretion.ICRS histological score in composite group was (13.89 ± 0.14) points,which differed significantly from (7.06 ± 0.19) points in control group,(7.14 ± 0.22) points in cell group and (7.46 ± 0.26) points in material group (P ＜0.01).Conclusion The tissue-engineered cartilage with ADSCs seeded onto the chitosan hydrogel is effective for repair of articular cartilage defect.

Objective The adverse reactions of levonorgestrel releasing intrauterine system ( LNG-IUS) has been emphasized with the increase of its indications.The study was to investigate the feasibility of interventional treatment in treating uterine adenomyosis with LNG-IUS by observing its adverse reactions, trying to reduce its adverse reaction, increase its utilization ratio and service life and improve its efficacy. Methods Retrospective analysis was made on 67 patients with uterine adenomyosis who were willing to accept LNG-IUS treatment from January 2012 to December 2013 in our hospital.The patients were randomly divided into observation group ( n=34) and control group( n=34) according to different therapies.The observation group were given interventional treatment immedi-ately after the placement of LNG-IUS, that is to take oral non-steroidal anti-inflammatory drug ( indomethacin, 25 mg, 1 pill each time, three times a day) for 7 consecutive days and desogestrel-ethinylestradiol ( marvelon, 1 pill a day) for 21 consecutive 21 days, 3 con-secutive cycles;while the control group were only given communications without any special treatment.Observation and record were made on related adverse reactions at 1 month, 3 months and 6 months after the treatment. Results At 1 month, 3 months and 6 months after the interventional treatment, the incidence rates of abnormal uterine bleeding between two groups were of significant difference ( 23.5%vs 60.6%, 11.7%vs 36.4%, 0%vs 12.1%, P<0.05) in the control group. At 6 month after the treatment, statistical difference was found in the total incidence rates of amenorrhea, ovarian cysts, dislocation and out of place of IUD between these two groups ( 23.5%vs 6.1%, 2.9%vs 21.2%, 2.9%vs 18.2%, P<0.05). Conclusion The research has indicated that the adoption of interventional treatment after placing LNG-IUS can obviously reduce the occurrence of the main adverse reactions, so it is feasible in clinical application.

Objective Construction of human IL‐4 recombinant expression vector and then conduct the expression ,purification and identification of human recombinant IL‐4 .Methods the open reading frame of IL‐4 was amplified by nest PCR with total RNA from PBMC of healthy volunteer .And then the amplified IL‐4 was inserted into pET101/D‐TOPO ,transformed into BL21 ,ex‐pressed ,purified and indentified .Results The size of amplified open reading frame of IL‐4 was about 460 bp and the sequence was correct .After transformed into BL21 ,the IL‐4 clone with higher expression level was selected by selection of different clones insert‐ed with IL‐4 and the size of expressed ,purified IL‐4 was about 28 × 103 .Western blot results showed that the size of single band was identical with the expected protein .Conclusion Human IL‐4 recombinant protein was got successfully .