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Objective:To study the crosstalk between the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) - X-box binding protein 1 (XBP1) pathway in oxygen-glucose deprivation/reoxygenation (OGD/R)-injured mouse hippocampal neuronal cell line HT22.Methods:The OGD/R-injured HT22 cell model was used to observe the changes of the indicators of endoplasmic reticulum stress (ERS), cell viability, and apoptosis at different OGD/R time points (0, 3, 6, 12, and 24 hours). HT22 cells in the logarithmic growth phase were randomized into blank control group, control+ATF6 activator (AA147) group, control+IRE1 inhibitor (4μ8c) group, OGD/R model group, OGD/R+AA147 group and OGD/R+4μ8c group (10 μmol/L AA147 or 16 μmol/L 4μ8c was given during the whole process in the AA147 group and 4μ8c group). Western blotting was used to detect the expression of ERS-related proteins [glucose-regulated protein 78 (GRP78), phosphorylated-inositol-requiring enzyme 1 (p-IRE1), and phosphorylated-eukaryotic translation initiation factor-2α (p-eIF2α)], and apoptosis-related proteins (Bcl-2, Bax, caspase-3, and cleaved caspase-3). The mRNA of ERS-related genes, and ATF6 [homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (Herpud1), protein disulfide isomerase associated 4 (Pdia4) and Sel-1 suppressor of lin-12-like (Sel1L)] and spliced XBP1 [XBP1s, include DnaJ heat shock protein family member B9 (Erdj4), Sec24 related gene family, member D (Sec24d) and signal sequence receptor, gamma (Ssr3)] induced transcriptional response-related genes were measured by real-time quantitative polymerase chain reaction (RT-qPCR). A cell counting kit-8 (CCK-8) assay was used to detect the viability of HT22 cells. Immunofluorescence was utilized to test the expression of cleaved caspase-3.Results:Compared with the blank control group, the expression of ERS-related proteins p-IRE1 and p-eIF2α were significantly increased at 12 hours and 3 hours following OGD/R, respectively (p-IRE1/β-actin: 2.09±0.10 vs. 1.00±0.00, p-eIF2α/β-actin: 1.39±0.11 vs. 1.00±0.00, both P < 0.01). The mRNA expressions of ERS-related genes [ATF6, XBP1s, unspliced XBP1 (XBP1u), activating transcription factor 4 (ATF4), CCAAT/EBP homologous protein (CHOP)] were also upregulated in different OGD/R timepoint in HT22 cells, which indicated ERS was activated in OGD/R-stimulated HT22 cells. Compared with the OGD/R model group, the expression of protein p-IRE1 was not changed, but the mRNA of XBP1s and XBP1u were obviously downregulated in the OGD/R+AA147 group [XBP1s (2 -ΔΔCt): 0.76 (0.71, 0.92) vs. 1.13 (1.03, 1.29), XBP1u (2 -ΔΔCt): 0.29±0.05 vs. 0.52±0.04, both P < 0.01], whereas the expressions of XBP1s-induced transcriptional response downstream genes did not change significantly. Compared with the OGD/R model group, the protein of short-form ATF6 (sATF6) and GRP78 were not changed after administration of 4μ8c, neither was the mRNA expression of ATF6-induced transcriptional response-related genes. These results showed that the mRNA expression of XBP1s and XBP1u were inhibited by AA147-induced activation of ATF6, but no crosstalk was observed between the transcriptional response induced by ATF6 and XBP1s. Compared with the blank control group, the cell viability decreased significantly at OGD/R 3 hours [(44.64±5.12) % vs. (99.13±5.76) %, P < 0.01], the ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly increased at OGD/R 3 hours and OGD 0 hour, respectively (Bax/Bcl-2: 6.15±1.65 vs. 1.00±0.00, cleaved caspase-3/caspase-3: 17.48±2.75 vs. 1.00±0.00, both P < 0.01), which indicated that apoptosis was activated in OGD/R-treated HT22 cells. Compared with the OGD/R model group, the cell viability decreased significantly [(36.52±17.78)% vs. (69.90±9.43)%, P < 0.01], and the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly upregulated in the OGD/R+AA147 group in HT22 cells (Bax/Bcl-2: 2.06±0.31 vs. 1.10±0.25, cleaved caspase-3/caspase-3: 3.35±0.59 vs. 0.55±0.09, both P < 0.01). Conclusion:Under our experimental conditions, no obvious crosstalk between the transcriptional response induced by ATF6 and XBP1s was observed, while ATF6 activation induced by AA147 suppressed mRNA expression of XBP1s and XBP1u and promoted cell death in OGD/R-treated HT22 cells.
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Objective:To observe the effect of modified Shengxian Decoction on extravascular lung water index (EVLWI) and lung injury prediction score (LIPS) in patients with acute respiratory distress syndrome (ARDS) caused by sepsis.Methods:Prospective cohort study. A total of 200 patients with ARDS caused by sepsis who were hospitalized in Baoshan Branch, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from January 2019 to May 2021 were selected and divided into the observation group and control group by random number table method, with 100 in each group. The patients in the control group were given rountin western medicine treatment according to the guidelines, and the patients in the observation group were treated with modified Shengxian Decoction on the basis of the treatment in the control group. Both groups were treated for 7 days as a course of treatment. The PH value, oxygen volume index (FiO 2), oxygen partial pressure (PaO 2), arterial carbon dioxide partial pressure (PaCO 2) of the two groups before and after treatment, calculate the oxygenation index (PaO 2/FiO 2) were observed and compared. The C-reactive protein (hs-CRP), interleukin-6 (IL-6) levels were observed by ELISA, the procalcitonin (PCT) levels was detected by double antibody sandwich immunoluminescence method. The APACHE Ⅱ score and LIPS score, EVLWI and cardiac index (CI) of the two groups were observed and compared. The mechanical ventilation time and ICU hospitalization time of the two groups were compared. Results:After treatment, the PaCO 2 level [(37.15 ± 5.42) mmHg vs. (38.24 ± 3.24) mmHg, t=2.03] of the observation group was significantly lower than that of the control group, and the oxygenation index (292.34 ± 78.91 vs. 236.54 ± 70.58, t=5.27) was significantly higher than that of the control group ( P<0.05). After treatment, the levels of hs-CRP [(35.21 ± 6.73) mg/L vs. (48.97 ± 8.52) mg/L, t=12.67], IL-6 [(40.57 ± 8.51) ng/L vs. (47.61 ± 9.97) ng/L, t=5.37] and PCT [(0.75 ± 0.21) μg/L vs. (1.14 ± 0.38) μg/L, t=8.98] in the observation group were significantly lower than those in the control group ( P<0.01). After treatment, the APACHE Ⅱscore (11.14 ± 0.54 vs. 14.67 ± 0.89, t=33.91], LIPS score (2.21 ± 0.73 vs. 4.59 ± 0.88, t=20.82), and EVLWI [(6.19 ± 0.42) ml/kg vs. (8.24 ± 0.78) ml/kg, t=23.14) of the observation group were significantly lower than those in the control group, and the CI level [(4.49 ± 1.27) L/(min?m 2) vs. (3.61 ± 0.88) L/(min?m 2), t=5.70] was significantly higher than that of the control group ( P<0.01). The mechanical ventilation time and ICU stay in the observation group were shorter than those in the control group ( t=3.66, 5.74, P<0.01). Conclusion:The modified Shengxian Decoction can reduce the level of inflammation indexes in patients with ARDS caused by sepsis, reduce EVLWI and LIPS scores, improve blood gas analysis indexes, and shorten the time of mechanical ventilation and ICU hospitalization.
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Objective:This study aimed to investigate the present status of professional identity in internship nursing students and to find the moderate role of mindfulness in the relationship between neuroticism and professional identity.Methods:This was a cross sectional investigation. From October to December 2021, 819 internship nursing students from four hospitals of Shandong Province were invited to participate in the study and to finish a questionnaire survey including the general information questionnaire, Professional Identity Questionnaire for Nurse Students (PIQNS), the Big Five Inventory(BFI) and Mindful Attention Awareness Scale (MAAS).Results:The average score of PIQNS was (61.41 ± 11.59). Pearson correlation analysis showed that professional identity was negatively associated with neuroticism ( r=-0.405, P<0.01) and positively associated with mindfulness ( r=0.301, P<0.01). Neuroticism was negatively associated with mindfulness ( r=-0.439, P<0.01). The hierarchical regression showed that mindfulness level could moderate the correlation between neuroticism and professional identity. Furthermore, the Johnson-Neyman technique revealed that the moderating role was significant only when the positive level was greater than 47.085. Conclusions:The professional identity of internship nursing students deserves attention. Mindfulness level could moderate the correlation between neuroticism and professional identity. It is suggested that clinical managers to carry out targeted training, to enhance the professional identity of internship nursing students during internship.
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Objective:To investigate the effects of of anti tumor necrosis factor-α (TNF-α) in adjuvant treatment of strangulated intestinal obstruction combined with ischemic intestinal necrosis.Methods:From February 2011 to August 2016 in Huadu District People′s Hospital Affiliated with Southern Medical University, 122 patients with strangulated intestinal obstruction combined with ischemic intestinal necrosis were selected and were equally divided into the experimental group and control group with 61 cases in each group according to the random draw envelope principle. Conventional surgical resection and anastomosis was used in control group, the postoperative anti TNF-α therapy was given for 2 weeks based on the treatment in control group.Results:All patients completed surgery and there were no serious complications during operation.The postoperative anal exhaust time and symptom remission time in experimental group were significantly lower than those in control group: (2.14 ± 0.41) d vs. (6.24 ± 1.28) d and (3.54 ± 0.77) d vs. (6.99 ± 0.91) d ( P<0.05). The incidence of postoperative 14 d complications such as anastomotic leakage, wound infection, anastomotic stenosis and pulmonary infection in the experimental group was 4.9%(3/61), and that of the control group was 18%(11/61), and the incidence of postoperative complications in the experimental group was significantly lower than that in the control group ( P<0.05). The postoperative 1d and 7 d serum TNF-α content in the experimental group was significantly lower than that in the control group ( P<0.05). The postoperative 14 d anal function in the experimental group was significantly better than that in the control group ( P<0.05). MRASP and MSP of postoperative 14 d in experimental group were all significantly higher than those in the control group: (80.24 ± 11.39) mmHg (1 mmHg=0.133 kPa) vs. (76.24 ± 12.11) mmHg, (231.98 ± 45.29) mmHg vs. (226.39 ± 41.87) mmHg ( P<0.05). Conclusions:The anti TNF-α in adjuvant treatment of strangulated intestinal obstruction combined with ischemic intestinal necrosis can promote the recovery of clinical symptoms and inhibit the release of TNF-α. It also can reduce the incidence of postoperative complications and improve gastrointestinal motility of patients.
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The objective was to investigate the effect of kinsenoside (Kin) treatments on macrophage polarity and evaluate the resulting protection of chondrocytes to attenuate osteoarthritis (OA) progression. RAW264.7 macrophages were polarized to M1/M2 subtypes then administered with different concentrations of Kin. The polarization transitions were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), confocal observation and flow cytometry analysis. The mechanism of Kin repolarizing M1 macrophages was evaluated by Western blot. Further, macrophage conditioned medium (CM) and IL-1 were administered to chondrocytes. Micro-CT scanning and histological observations were conducted on anterior cruciate ligament transection (ACLT) mice with or without Kin treatment. We found that Kin repolarized M1 macrophages to the M2 phenotype. Mechanistically, Kin inhibited the phosphorylation of IB, which further reduced the downstream phosphorylation of P65 in nuclear factor-B (NF-B) signaling. Moreover, Kin inhibited mitogen-activated protein kinases (MAPK) signaling molecules p-JNK, p-ERK and p-P38. Additionally, Kin attenuated macrophage CM and IL-1-induced chondrocyte damage. , Kin reduced the infiltration of M1 macrophages, promoted M2 macrophages in the synovium, inhibited subchondral bone destruction and reduced articular cartilage damage induced by ACLT. All the results indicated that Kin is an effective therapeutic candidate for OA treatment.
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Bone defects caused by trauma, tumour resection, infection and congenital deformities, together with articular cartilage defects and cartilage-subchondral bone complex defects caused by trauma and degenerative diseases, remain great challenges for clinicians. Novel strategies utilising cell sheet technology to enhance bone and cartilage regeneration are being developed. The cell sheet technology has shown great clinical potential in regenerative medicine due to its effective preservation of cell-cell connections and extracellular matrix and its scaffold-free nature. This review will first introduce several widely used cell sheet preparation systems, including traditional approaches and recent improvements, as well as their advantages and shortcomings. Recent advances in utilising cell sheet technology to regenerate bone or cartilage defects and bone-cartilage complex defects will be reviewed. The key challenges and future research directions for the application of cell sheet technology in bone and cartilage regeneration will also be discussed.
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Régénération osseuse , Os et tissu osseux , Cartilage articulaire , Régénération , Ingénierie tissulaire , Structures d'échafaudage tissulairesRÉSUMÉ
Objective To observe the effects of diacetyldianhydrogalactito (DADAG) on the proliferation and apopotosis in lung cancer cell NCI-H460.Methods MTT assay was performed to determine the half inhibitory concentration of DADAG(2.88,5.75,11.50,23.00 and 46.00 μg/mL)for the NCI-H460 cells and colony formation assay was used to detect the ability of cell proliferation; through AO/EB staining method, morphological changes of apoptotic cell under fluorescent microscope were observed;cell apoptosis was detected by flow cytometry to test the effects of DADAG on NCI-H460;RT-PCR was used to detect the effect of DADAG on Bcl-2 and Bax mRNA expres-sion levels within the cell. Results Compared with that in blank control group, cell proliferation was inhibited in the group treated with DADAG; the number of colony formation decreased and AO/EB staining results showed that cell apoptosis was characterized by typical morphological changes such as swelling, shrinking and fragmentation. Flow cytometry detection results showed that apoptosis rate was increased in the group treated with DADAG(all P<0.05).RT-PCR results indicated that the expression of pro-apoptosis gene Bax was up-regulated,while expression of anti-apoptosis gene Bcl-2 down-regulated. Conclusions DADAG exhibits the inhibitory activity in lung cancer cell NCI-H460 and can induce the apoptosis.The potential mechanism maybe associated with up-regulating the ex-pression of Bax but down-regulating the expression of Bcl-2.
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The aim of this study was to investigate the effect and mechanisms of miR-29a in migration and inva-sion of human breast cancer MCF-7 cells in vitro. MCF-7 cells were treated with miR-29a mimic or miR-29a inhibitor to up-regulate/down-regulate the expression level of miR-29a. Wound-healing assay and transwell chamber were employed to determine cell migration and invasion in vitro. The target gene of miR-29a was predic-ted with the Targetscan7. 1 database and verified through luciferase reporter method. The effects of miR-29a on the expression of the potential target were detected by Western blot and real-time PCR. Results showed that in vitro migration and invasion ability of MCF-7 cells was increased significantly by miR-29a,which could target HBP1 in the 3′-UTR region. The protein expression of HBP1 was decreased by miR-29a overexpression. However, the alteration of miR-29a had no significant effect on the expression of HBP1 mRNA. The results validated that miR-29a,highly expressed in breast cancer,could down-regulate HBP1 ,which in turn promotes migration and invasion ability of breast cancer cells,thus promoting breast cancer metastasis.
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Objective To investigate the degree of psychological distress in patients with lower extremity arteriosclerosis obliterans, and to understand the causes and to analyze the influencing factors. Methods The general information questionnaire, psychological distress thermometer, family APGAR index and family burden scale questionnaire were used to conduct a cross-sectional survey in 145 patients with obliterans in hospital during the period from July 2015 to July 2017. Results The average score of psychological pain of all patients was (4.7±2.2) points, the patients with significant psychological distress was 102 cases, accounting for 70.3% (DT score≥4 points). The main reason caused by lower extremity arterial occlusive disease in patients with psychological distress among the top 10 were: pain, anxiety, sleep problems, physical activity, work/ school, restricted travel, numbness, fatigue, constipation, economic problems. Multiple linear regression analysis showed that the hospitalization, duration of illness, family care and family burden of the disease affected the scores of psychological pain in different seasons (P＜0.05). Conclusions The patients with lower extremity arteriosclerosis obliterans have severe psychological pain, so the targeted measures should be taken to prevent and treat the risk factors.
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Objective To investigate the effects of the frozen embryo transplantation for patients with poor outcome of endometrial growth by using growth hormone (GH) intrauterine perfusion combined with replacement cycle in the treatment of thin endometrium.Methods This was a prospective study and study participants were consecutively recruited between Jun 2014 and September 2015.A total of 88 frozen thawed embryo transfer cycles was divided into two groups from the Reproductive Center of Hunan Provincial Maternal and Child Health Hospital.Group A were 63 hormone replacement therapy (HRT) cycles and Group B were 25 GH intrauterine perfusion combined HRT cycles.Results The endometrial thickness of 22 thin endometrium patients from Group B were increased above 7 mm on progesterone day.The endometrial thickness on transplant day of Group A was (9.28 ± 1.64) mm,which was significantly higher than Group B (7.9 ± 0.86) mm (P ＜ 0.05).The clinical pregnancy rate (50.79％ vs 52.0％),implantation rate 31.1％ vs 47.17％),miscarriage rate (9.38％ vs 15.38％) had no significant difference between Groups A and B.The endometrial thickness from 7 mm to 7.9 mm on transplant day,the clinical pregnancy rate (30.76％ vs 54.54％) had no significant difference in two groups (P ＞0.05),but the implantation rate of group A was significantly lower than that of group B (20％ vs 52.17％) (P ＜0.05).When the endometrial thickness was above 8 mm on transplant day,the clinical pregnancy rate (58.33％ vs 63.63％),implantation rate (36.36％ vs 50％) had no significant difference between groups A and B (P ＞ 0.05).Conclusions GH uterine cavity perfusion was a useful method for treatment of thin endometrium,and was helpful for improvement of endometrial thickness and receptivity,improved embryo implantation environment by assistance for HRT under the high estrogen levels.
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Objective To investigate the level of knowledge,attitude and practice(KAP)of pelvic floor dysfunction(PFD)and its influencing factors in pregnant women and provide reference for further health education for pregnant women. Methods Totally 637 pregnant women were investigated by Knowledge-Attitude-Practice (KAP)scale for PFD. Results The average scores of KAP in pregnant women were(31.68 ± 7.73),(16.41 ± 4.01) and (10.57 ± 3.00) respectively. About 85.6% of the pregnant women had interest in knowledge of PFD. Educational background,willing to understand knowledge of PFD,and gestational age were dominant factors affecting KAP of women with PFD. Conclusions The awareness rate of PFD and compliance rate of pelvic floor muscle function exercise in pregnant women are low. Effective measures should be taken to prevent PFD for pregnant women.
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Objective To investigate the enterovirus ( EV)-carrying status and the circulating se-rotypes in healthy children from inner and border areas of Yunnan Province in 2014 and 2015 and to analyze the genetic characteristics of echovirus 6 (ECHO6), ECHO25 and ECHO11 strains. Methods Stool sam-ples were collected from children less than 15 years old living in 6 to 7 counties of 3 inner prefectures/cities and 8 to 9 counties of border prefectures/cities. Altogether 921 samples were collected including 453 sam-ples in 2014 (213 samples in inner counties and 240 samples in border counties) and 468 samples in 2015 (195 samples in inner counties and 273 samples in border counties). Viruses were isolated from the stool samples and their serotypes were identified by gene sequencing. Results The numbers of EV strains isola-ted from the samples collected in inner counties and border counties in 2014 were 20 ( isolating rate:9.39%, 20/213) and 16 (isolating rate: 6. 67%, 16/240), respectively. The overall isolating rate for 2014 was 7. 95% (36/453). The predominant species was enterovirus B, accounting for 88. 89% of all iso-lated strains (32/36), followed by enterovirus A species (11. 11%, 4/36). No strains of enterovirus spe-cies C (including poliovirus) and D was detected in 2014. In total, 46 EV strains were isolated in 2015 with an overall isolating rate of 9. 83% (46/468), including 13 strains in inner counties (isolating rate:6. 67%, 13/195) and 33 strains in border counties (isolating rate:12. 09%, 33/273). Most of the strains were enterovirus B species, accounting for 78. 26% (36/46), followed by enterovirus C species (19. 57%, 9/46) and enterovirus A species (2. 17%, 1/46). Altogether 82 EV strains were isolated in 2014 and 2015 with an isolating rate of 8. 90% (82/921), of which 33 strains were isolated in inner counties (8. 09%, 33/408) and 49 strains were isolated in border counties (9. 55%, 49/513). Among the 82 EV strains, 9 strains were polioviruses (0. 98%, 9/921) and all of them were Sabin-like polioviruses. The rest of the strains were non-polio enterovirus (7. 93%, 73/921). Conclusion In 2014, the EV isolating rate in inner counties (9. 39%) was higher than that in border counties (6. 67%). However, the EV isolating rate in border counties (12. 09%) was higher than that in inner counties (6. 67%) in 2015. Enterovirus B was the predominant species in both 2014 and 2015. No wild type polioviruses and enterovirus D species were detec-ted. Polio-free status was maintained well in Yunnan Province.
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Objective To explore the change of apoptosis factor Caspase-3 and Bcl-2 in the injured segment of rat with spinal cord injury after inhibiting lentivirus expression of inflammation factor TNF-α. To study the relationship between Caspase-3, Bcl-2, Bax and TNF-α in spinal cord injury. Mthods Spinal cord contusion model was prepared by Allen method. The relation between tumor necrosis factor alpha and Bcl-2, was predicted by the method of GeneMANIA bioinformatics. The RNA which was packaged by lentivirus constructed the RNA interference model of tumour necrosis factor alpha. After interference of tumor necrosis factor alpha, we used the method of QRT-PCR to assays the mRNA expression of Caspase-3 and Bcl-2 in spinal cord and detect of the localization of Caspase-3 and Bcl-2 by immunohistochemistry. Statistical analysis with SPSS17.0. Results SD rats had paraplegia and urinate retentaion because of spinal cord injury. The result of QRT-PCR showed that in the seventh day after SCC, the expression of Caspase-3 reduced significantly (P 0.05). Immunohistochemistry experiment results showed that Caspase-3 Bcl-2 and Bax immunoreactive cells were observed in the neurons and glial cells of both white matter and gray matter in the spinal cord. The results were the same with QRT-PCR.. Conclusion TNF-α in rats after SCC can effectively regulate the ratio of Bcl-2 and Bax , and then regulate the expression of Caspase-3 , which may affect the function of apoptosis and function recovery after spinal cord injury.
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Objective To investigate the effect of statin on trabecular bone microstructure by using trabecular bone score (TBS), a new type of bone microstructure evaluation index. Methods A total of 253 middle and aged patients hospitalized in the First Affiliated Hospital of Nanjing Medical University between January 2014 and March 2016 were retrospectively analyzed. According to whether statin was used or not, patients were divided into two groups: 90 patients in the statin use group (statin was use for more than 1 year) and 163 in the control group (not taken any statin). Serum biochemical indicators, such as triacylglycerol, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, alkaline phosphatase, fasting blood glucose and 25 hydroxy vitamin D, were compared between the two groups. Dual energy X-ray absorptiometry (DXA) was used to measure the bone mineral density (BMD) of lumbar spine and femoral neck. TBS was calculated with TBS iNsight? software, and the DXA image of lumbar spine were analyzed. Results Values of total cholesterol and low density lipoprotein cholesterol were significantly lower in statin group compared with those of control group (P0.05). There was higher lumbar spine BMD statin group compared to that of control group (g/cm2:1.04 ± 0.19 vs. 0.96 ± 0.14, P0.05). Conclusion Statin increases lumbar spine BMD and improves trabecular bone microstructure in middle and aged people.
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Objective To investigate fluoride-induced inflammation and nuclear factor-κB (NF-κB) signaling pathway in cultured human acute monocytic leukemia cells (THP-1).Methods In vitro cultured THP-1 cells were used as a model of microglia.After cultured with different concentrations of [0 (negative control group),10,50,100,500,1 000 and 5 000 μmol/L] sodium fluoride (NaF) for 48 h,the survival of cells was detected by CCK8.THP-1 cells were divided into 3 groups:control group,low dose and high dose fluoride groups according to the results of CCK8 assay,and then treated with different concentrations of sodium fluoride (0,500,5 000 μmol/L) for 48 h,concentrations of inflammatory cytokines,such as Interleukin-lβ (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) in THP-1 mononuclear cell culture medium.The protein levels of IκBα,phospho-NF-κB p65 and phospho-IκB-α were detected by Western blotting.Results THP-1 cells were treated with different concentrations of sodium fluoride (500,1 000,5 000 μ mol/L) for 48 h.Fluoride group THP-1 cell survival rate [(73.21 ± 3.67)％,(31.40 ± 4.56)％,(0.40 ± 0.24)％] was lower than that of the negative control group [(100.00 ± 0.00)％,all P ＜ 0.01].Compared to the control groups [(0.36 ± 0.07),(31.07 ± 0.81)ng/L],significant increases of the inflammatory cytokines IL-1β [(1.42 ± 0.79),(19.47 ± 2.90)ng/L] and TNF-α [(61.06 ± 2.20),(172.72 ± 2.29)ng/L] were detected in culture medium in low-fluoride and high fluoride groups,respectively.Interestingly,compared to the control groups [(100.00 ± 5.48)％,(100.00 ± 14.82)％],significant increases of phospho-NF-κB p65 [(113.71 ± 8.99)％,(134.74 ± 1.93)％] and phospho-IκB-α [(152.61 ± 14.16)％,(176.91 ± 7.95)％] were observed in both low-fluoride and high fluoride groups.Meanwhile,the protein level of IκBα in high fluoride group [(63.53 ± 9.67)％] was significantly lower than that of the control group [(100.00 ± 10.99)％,P ＜ 0.01].Furthermore,significant positive correlation was detected between increased IL-1β,TNF-α and phospho-NF-κB p65 (r =0.74,0.72,all P ＜ 0.05).Conclusions Excessive fluoride can induce microglial cells to release inflammatory cytokines and activate nuclear factor-κB signaling pathway.The release of inflammatory cytokines and activation of the signaling pathway may be one of the mechanisms of the damage of the central nervous system caused by sodium fluoride.
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<p><b>BACKGROUND</b>Recent studies on bone have shown an endocrine role of the skeleton, which could be impaired in various human diseases, including osteoporosis, obesity, and diabetes-associated bone diseases. As a sensor and regulator of energy metabolism, AMP-activated protein kinase (AMPK) may also play an important role in the regulation of bone metabolism. The current study aimed to establish the expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types.</p><p><b>METHODS</b>Reverse transcription-polymerase chain reaction (PCR) for relative quantification, real-time PCR for absolute quantification, and Western blotting were used to investigate the gene expression profiles and phosphorylation patterns of AMPK subunits in several mesenchymal cell types, including primary human mesenchymal stem cells (hMSCs) and hFOB, Saos-2, C3H/10T1/2, MC3T3-E1, 3T3-L1, and C2C12 cells.</p><p><b>RESULTS</b>AMPKα1 and AMPKβ1 mRNAs were abundantly expressed in all cell types. AMPKγ1 mRNA was abundantly expressed in C3H/10T1/2, MC3T3-E1, 3T3-L1, and C2C12 but not detected in human-derived cell types. AMPKγ2 mRNA was mildly expressed in all cell types. AMPKα1 protein was highly expressed in all cell types and AMPKα2 protein was highly expressed only in hFOB and Saos-2 cells. AMPKβ1 protein was abundantly expressed in all cell types except for Saos-2, in which AMPKβ2 protein overwhelmed AMPKβ1 expression. AMPKγ1 and AMPKγ2 proteins were expressed in C3H/10T1/2, MC3T3-E1, 3T3-L1, and C2C12 cells and only AMPKγ2 protein was expressed in hMSCs, hFOB and Saos-2 cells. AMPKα was phosphorylated at Thr172 and Ser485 and AMPKβ1 was phosphorylated at Ser108 and Ser182 in all cell types with a specific pattern in each cell type.</p><p><b>CONCLUSION</b>The combination of AMPK α, β, and γ subunits and phosphorylation of AMPKα (Thr172 and Ser485) and AMPKβ1 (Ser108 and Ser182) showed a specific pattern in each cell type.</p>
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Animaux , Humains , Souris , AMP-Activated Protein Kinases , Génétique , Métabolisme , Lignée cellulaire , Cellules souches mésenchymateuses , PhosphorylationRÉSUMÉ
Objective To explore the optimum flow shear stress and mass transport for the construction of tissue-engineered bone.Methods The β-tricalcium phosphate (β-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (HBMMSCs) were cultured in perfusion bioreactor.When the same flow rate was applied,the flow shear stress was separately 1×,2× and 3×.When the same flow shear stress was applied,the flow rates were separately 3 ml/min,6 ml/min and 9 ml/min.Cell proliferation was measured by MTT method.The construction of tissue-engineered bone was evaluated by measuring alkaline phosphatase (AKP) activity,secretion of osteopontin (OP) and osteocalcin (OC),and the mineralization of extracellular matrix (ECM).The flow shear stress and the mass transport were obtained using computational fluid dynamics.Results When the flow rate was same,the most cell proliferation was found in 2× group.The AKP activity and secretion of OC was higher in 2× and 3× groups than in those in 1× group.After 28days,the highest amount of mineralization of ECM was found in 3× group.When the flow shear stress was same,the AKP activity was highest in 6 ml/min group.After 28 days,secretion of OC and formation of mineralized ECM was highest in 3 ml/min group.When the flow rate was same,the flow shear stress was separately 0.004-0.007 Pa,0.009-0.013 Pa and 0.013-0.018 Pa.When the flow shear stress was same,the flow rate was separately 0.267-0.384 mm/s,0.521-0.765 mm/s and 0.765-1.177 mm/s.Conclusion When the tissue-engineered bone was constructed,0.013-0.018 Pa flow shear stress and 0.267-0.384 mm/s mass transport velocity could improve the construction of the tissue-engineered bone in vitro.
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Objective To explore the effects of alendronate on adipogenic differentiation of bone marrow stremal cells (BMSCs) and the role of mitogen-activated protein kinases (MAPK) signal pathway in this process. Methods BMSCs were derived from 9-month-old ovariectomized SD rats and exposed to 0.01, 0.1, 1, 10 μmol/L of alendronate for 2 weeks. The number of BMSCs was counted under light microscope after oil red O staining. The expression of peroxisome proliferators activated receptor-γ, 2 (PPAR-γ2) was measured by RT-PCR. The effect of alendronate on MAPK signal pathway was detected by Western blot. Results After two weeks of induction of BMSCs by alendronate, BMSCs with positive oil red O staining significantly decreased as the increase of the concentration of alendronate (P <0.01), so did the expression of PPAR-γ2. The expression level of PPAR 2 increased when exposing BMSCs to ERK1/2 or JNK specific inhibitors, PD98059 and SP600125 for two weeks. However, the expression level of PPAR 2 decreased when exposing BMSCs to SB203580 (an inhibitor of p38) for two weeks. Condusion Alendronate can inhibit adipogenic differentiation of BMSCs derived from ovariectomized rats in a doso-dependent manner by activating ERK1/2 and JNK rather than p38.
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The purpose of the study was to observe the effect of rapamycin (RAPA) on the differentiation and maturation of rat bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs from Wistar rats were cultured with granulocyte-macrophage colony-stimulating factor plus interleukin-4 in the presence or absence of RAPA (20 ng/mL), and stimulated with lipopolysaccharide (LPS) for 24 h before cells and supernatants were collected. Surface phenotype of BMDCs was flow-cytometrically detected to determine the expression of maturation markers, MHC class II and CD86. Supernatants were analyzed for the production of IL-12 and IFN-gamma cytokines by using ELISA. BMDCs were co-cultured with T cells from Lewis rats and mixed lymphocyte reaction was assessed by MTT method. The morphology of BMDCs stimulated with LPS remained immature after RAPA pretreatment. RAPA significantly decreased the CD86 expression, impaired the IL-12 and IFN-gamma production of BMDCs stimulated with LPS, and inhibited the proliferation of allogeneic T cells. In conclusion, RAPA can inhibit the maturation of BMDCs stimulated with LPS in terms of the morphology, surface phenotype, cytokine production, and ability of BMDCs to stimulate the proliferation of allogeneic T cells in vitro.
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We conducted studies to confirm the hypothesis that the cellular damage occurring around implanted biphasic bioceramics could be related to a micro-particles release because of an insufficient sintering. An in vitro cytotoxicity study was performed on four biphasic ceramic (BCP) samples. Without the treatment of extraction medium, a cytotoxicity was observed, although after centrifugation this cytotoxicity disappeared in all samples. (2) Micro-particles of HA, beta-TCP and 40%beta-TCP/60%HA mixture were used for a cell inhibition study. A decrease of cell viability was observed with the increase in particles concentration. At 10000 particles/ cell, the viability and proliferation were completely inhibited. (3) HA, beta-TCP and BCP ceramic granules were implanted in rabbit femoral cavities for 12 weeks. No degradation of HA granules was observed. The degradation was higher for beta-TCP (40%) than for BCP (5%). On the other hand, new bone formation was significantly higher for beta-TCP (21%) and HA (18%) than for BCP (12%). Much more micro-particles were formed around BCP granules than around beta-TCP, and were phagocytosed by macrophages. The release of ceramic micro-particles could be related to the sintering process. BCP ceramics have to be sintered at only 1160 degrees C. Consequently, HA microparticles of BCP ceramic are incompletely sintered and easily released after immersion or implantation. The microparticles could be at the origin of local inflammation and cell damage and could perhaps modify osteogenesis. Particular attention must be paid to this problem with regard to BCP ceramics because of the sintering difficulties of this bioceramic.