Protective effects of the salt-induced kinase inhibitor HG-9-91-01 on sepsis-associated cognitive dysfunction in mice and the underlying mechanisms / 中南大学学报(医学版)

Objective:Sepsis-associated cognitive dysfunction is a common complication in patients with sepsis and lack of effective treatment.Its pathological mechanisms remain unclear.Salt-induced kinase(SIK)is an important molecule in the regulation of metabolism,immunity,and inflammatory response.It is associated with the development of many neurological diseases.This study aims to investigate the expression of SIK in the hippocampus of septic mice,and to evaluate the role and mechanism of the SIK inhibitor HG-9-91-01 in sepsis-associated cognitive dysfunction. Methods:Firstly,C57BL/6 mice were randomly divided into a control group(Con group)and a sepsis model group[lipopolysaccharide(LPS)group].The model group was injected intraperitoneally with LPS at a dose of 8 mg/kg and the Con group was injected with an equal volume of normal saline.Hippocampal tissues were harvested at 1,3,and 6 days after injection and the expressions of SIK1,SIK2,and SIK3 were detected by real-time fluorescence quantitative PCR(qPCR)and Western blotting.Secondly,C57BL/6 mice were randomly divided into a Con group,a LPS group,and a SIK inhibitor group(HG group).The LPS and HG groups were injected with LPS to establish a sepsis model;in the HG group,HG-9-91-01(10 mg/kg)was injected intraperitoneally at 3-6 days after LPS injection,and the LPS group was injected with the same volume of vehicle.Cognitive function was assessed at 7-11 days after LPS injection using the Morris water maze(MWM).Hippocampal tissues were harvested after the behavioral tests,and the mRNA levels of inflammatory factors and microglial markers were assessed by qPCR.The protein levels of inducible nitric oxide synthase(iNOS),CD68,ionized calcium binding adaptor molecule 1(Iba-1),N-methyl-D-aspartate(NMDA)receptor(NR)subunit,cAMP response element-binding protein(CREB)-regulated transcription coactivator 1(CRTC1),and insulin-like growth factor 1(IGF-1)were detected by Western blotting.Immunohistochemistry(IHC)was used to detect the expression of Iba-1 positive cells in the CA1,CA3 and dentate gyrus(DG)of the hippocampus,followed by Sholl analysis. Results:Compared with the Con group,the mRNA and protein levels of SIK1,SIK2,and SIK3 in the hippocampus were increased in the LPS group(all P<0.05).Compared with the Con group,mice in the LPS group had a significantly longer escape latency,a lower percentage of target quadrant dwell time and a reduced locomotor speed(all P<0.05);the HG group had a decreased escape latency and an increased percentage of time spent in the target quadrant in comparison with the LPS group(both P<0.05).The mRNA levels of inflammatory factors[tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)],and the M1-type microglial markers iNOS and CD68 in the hippocampus of the LPS group were increased in comparison with the Con group,while the M2-type microglial markers CD206 and arginase-1(Arg-1)were decreased.Compared with the LPS group,the mRNA levels of TNF-α,IL-1β,IL-6,and iNOS were downregulated,while the levels of CD206 and Arg-1 were upregulated in the HG group(all P<0.05).The protein levels of iNOS,CD68,and Iba-1 in the hippocampus of the LPS group were increased in comparison with the Con group,but they were downregulated in the HG group in comparison with the LPS group(all P<0.05).The number of Iba-1 positive cells in CA1,CA3,and DG of the hippocampus was increased in the LPS group in comparison with the Con group,but they were decreased in the HG group in comparison with the LPS group(all P<0.05).Sholl analysis showed that the number of intersections at all radii between 8-38 μm from the microglial soma was decreased in the LPS group in comparison with the Con group(all P<0.05).Compared with the LPS group,the number of intersections at all radii between 14-20 μm was significantly increased in the HG group(all P<0.05).The protein levels of NR subunit NR1,NR2A,NR2B,and IGF-1 were downregulated in the hippocampus of the LPS group in comparison with the Con group,while the expression of phosphorylated CRTC1(p-CRTC1)was increased.Compared with the LPS group,the levels of NR1,NR2A,NR2B,and IGF-1 were upregulated,while p-CRTC1 was downregulated in the HG group(all P<0.05). Conclusion:SIK expression is upregulated in the hippocampus of septic mice.The SIK inhibitor HG-9-91-01 ameliorates sepsis-associated cognitive dysfunction in mice,and the mechanism may involve in the activation of the CRTC1/IGF-1 pathway,inhibition of neuroinflammation,and enhancement of synaptic plasticity.

Effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice / 新乡医学院学报

Objective To investigate the effect of microglia inhibitor Pexidartinib on reconsolidation of remote contextual fear memory in mice.Methods Twelve healthy C57BL/6 male mice were randomly divided into experimental group and control group,with 6 mice in each group.The mice in the two groups underwent contextual fear conditioning training in the contextual fear response box to establish the contextual fear models,and the freezing time of mice after each footshock was recorded.After 7 days,the mice in the experimental group were fed with food containing Pexidartinib formulation PLX3397,while the mice in the control group were fed with regular food until the end of behavioral experiment.On the 16th day after contextual fear conditioning training,the mice were put back into the contextual fear box for recalling the fear memory without any stimulation.The mice were taken out after 5 minutes of free exploration,and the freezing time of mice during this period was recorded.At 24 h after the fear memory was recalled,the mice were again placed in the contextual fear box,allowing them to explore freely for 3 minutes,and the freezing time of mice during this period was recorded;the fear response of mice was indicated by the percentage of freezing time.After the behavioral experiment,the mice were anesthetized by intraperitoneal injection of pentobarbital sodium,and three mice from the two groups were taken and rapidly opened the chest to expose their hearts,the perfusion needle was inserted into the left ventricle from the tip of heart and then was perfuse with 40 g·L-1 paraformaldehyde(pH=7.4)which precooled at 4 ℃ until the involuntary convulsion disappeared and the body limbs of mice were stiff.The brain tissue of mice was taken and fixed with paraformaldehyde solution,and then placed in sucrose solution for dehydration.The brain tissue of mice was coated with tissue embedding agent.The number of microglial cells in the hippocampus of mice in the two groups was detected by immunohistochemistry.The remaining mice in the two groups were taken and quickly decapitated to obtain brain tissues,and the hippocampus tissues of two sides were separated on ice.The expressions of phosphorylated bromodomain-containing protein 4(pBRD4),gasdermin D(GSDMD)and mixed lineage kinase domain-like protein(MLKL)in the hippocampus of mice were detected by Western blot.Results In the stage of contextual fear conditioning training,the percentage of freezing time of mice in two groups increased with the increase of the number of footshock(P＜0.05),but there was no significant difference in the percentage of freezing time of mice after the first,second,third,fourth and fifth footshock between the two groups(P＞0.05).There was no significant difference in the percentage of freezing time of mice between the two groups during recall period(P＞0.05).The percentage of freezing time of mice in the experimental group was significantly lower than that in the control group at the phase of memory test(P＜0.05).The number of microglia in CA1,CA3 and DG regions of hippocampus of mice in the experimental group was significantly lower than that in the control group(P＜0.05).The relative expressions of pBRD4,GSDMD and MLKL in hippocampus of mice in the experimental group were significantly lower than those in the control group(P＜0.05).Conclusion Microglia inhibitor Pexidartinib can injury the reconsolidation of remote contextual fear memory,which may be related to its inhibition of microglial cell activation and the down-regulation of the expressions of pBRD4,GSDMD and MLKL.

Environmental enrichment ameliorates anxiety and depression-like behaviors in paternally stressed offspring mice through upregulation of hippocampal LASP1 / 西安交通大学学报(医学版)

【Objective】 To explore the role of environmental enrichment (EE) in paternal stress-induced anxiety and depression-like behaviors in offspring and its potential mechanisms. 【Methods】 Male C57BL/6 mice (F0) were treated with unpredictable chronic mild stress (UCMS) and subsequently mated with normal females to obtain F1 offspring mice. The standard environment (SE) and enriched environment (EE) were administered to F1-UCMS offspring mice during their early life (3-5 weeks of age). Anxiety-like behaviors were detected by open field test (OFT) and elevated plus-maze test (EPM); depression-like behaviors were detected via forced swimming test (FST) and sucrose preference test (SPT) at the age of 8 weeks. The expressions of LIM and SH3 domain protein 1 (LASP1) in the hippocampus of adult F1 offspring mice were detected by Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. 【Results】 Compared to F1 offspring of normal paternal (F1-Nor), F1 offspring mice of the stressed paternal (F1-UCMS) showed significantly anxiety-like behavior with reduced percentage of time spent in the central region of OFT and in the open arm of EPM (P<0.05); mice from the F1-UCMS group showed a significantly increased percentage of immobility in FST and a reduced percentage of sugar consumption in SPT (P<0.01), which demonstrated significant depression-like behaviors. Compared to the SE group, mice in the EE group had an increased percentage of time spent in the central region of the OFT [males: (7.44±0.75)% vs. (14.93±1.74)%, P<0.01; females: (8.89±1.06)% vs. (15.10±1.82)%, P<0.05] and an increased percentage of time in the open arm of EPM [males: (8.09±1.05)% vs. (14.15±1.88)%, P<0.05; females: (9.13±1.14)% vs. (14.04±1.37)%, P<0.05]. This indicated that EE ameliorated anxiety-like behavior in F1-UCMS mice with paternal stress. Compared to the SE group, mice in the EE group had an decreased percentage of immobility in FST [males: (58.63±4.51)% vs. (42.15±3.81)%, P<0.05; females: (57.96±4.19)% vs. (43.25±4.22)%, P<0.05] and an increased percentage of sugar consumption in SPT [males: (50.38±3.47)% vs. (70.39±3.12)%, P<0.01; females: (52.42±2.84)% vs. (69.99±3.55)%, P<0.01]. This indicated that EE ameliorated depression-like behavior in F1-UCMS mice with paternal stress. Hippocampal LASP1 expression was reduced in SE group compared to F1-Nor group (males: P<0.01; females: P<0.05), while LASP1 was increased in EE group compared to SE group (P<0.05) detected by RT-qPCR and Western blotting. 【Conclusion】 EE ameliorates paternal stress-induced anxiety and depression-like behaviors in F1-UCMS mice, and the mechanism may be associated with increased hippocampal LASP1 expression in F1-UCMS mice.

Cause of death of children with intraocular retinoblastoma in the Beijing Children's Hospital 2009-2017 / 中华眼底病杂志

Objective:To observe and analyze the clinical characteristics of children who died of intraocular retinoblastoma (RB).Methods:A retrospective clinical study. Fourteen children (23 eyes) with intraocular RB who died after receiving treatment in Beijing Children's Hospital from 2009 to 2017 were included in the study. Among the children, there were 7 males (10 eyes) and 7 females (13 eyes); 5 had unilateral and 9 had bilateral tumor. Age were 17.2±15.5 months. All children underwent RetCam examination. RB was staged according to the international intraocular RB classify. Among the 23 eyes, 1 eye was in stage B, 2 eyes were in stage C, 12 eyes in stage D, and 8 eyes in stage E. Treatment methods included a systemic (vincristine, etoposide and carboplatin) chemotherapy (VEC chemotherapy), enucleation surgery, and vitrectomy. The basic conditions including age, time of diagnosis, pathological diagnosis, treatment and main causes of death were retrospectively analyzed.Results:Among the 14 cases, the first symptom was leukemia in 12 cases, red eye in 1 case, and squintin in 1 case. Systemic VEC chemotherapy was used for 1-6 courses of treatment; 5 cases were enucleated, 3 cases underwent histopathological examination; 3 cases were treated with vitrectomy. Among the 3 cases who underwent histopathological examination, the sclera and optic nerve, optic nerve and optic disc were invasted respectively. Seven patients died of tumor metastasis and/or intracranial lesions (50.0%, 7/14); the median survival time was 19 months. Four patients died of treatment (28.6%, 4/14), including 3 patients died of chemotherapy-related side effects, and 1 died of organ failure after enucleation surgery (7.1%); the median survival time was 3.5 months. Early abandonment of treatment died in 3 cases (21.4%, 3/14); the median survival time was 15 months.Conclusion:Intracranial metastasis is the main cause of death in children with intraocular RB.

Correlation analysis of eye and neurological manifestations in 56 children with infantile gangliosideosis in China / 中华眼底病杂志

Objective:To observe and analyze the clinical characteristics and correlation between the eye and nervous system in children with infantile gangliosideosis.Methods:From November 2018 to January 2021, 3 children with infantile ganglion lipidosis diagnosed by genetic examination in the Department of Ophthalmology and Neurology, Beijing Children's Hospital of Capital Medical University, and through China National Knowledge Infrastructure and Wanfang database and The National Library of Medicine of the United States (PubMed) were searched, and 53 cases of Chinese infantile gangliosideosis diagnosed by gene, enzyme activity or pathological examination were selected and a total of 56 cases were included in the study. The searching time was from the establishment of the database to February 2021, and the search keywords are"gangliosideosis", "cherry-spot" macula and "Chinese". The demographic characteristics of 56 cases of children and other system manifestations were analyzed such as eyes, nervous system, skin, bones. According to the presence or absence of cherry-spot (CS) on the fundus examination, the children were divided into a fundus CS group (group A) and a fundus without CS group (group B), with 20 and 27 cases, respectively. The age of onset, gender, different types and neurological manifestations of the two groups of children were compared and analyzed. The non-parametric rank sum test was used for age comparison between groups; the χ2 test or Fisher's exact test were used for the comparison of gender, disease type and incidence between groups. Results:Among the 56 children, 27 were males and 29 were females; the median age of onset was 7.0 months. There were 33 and 23 cases of GM1 and GM2, respectively. Among 44 children with visual function examination records, 41 cases (93.2%, 41/44) were unable to follow the visual object. Of 47 children who underwent ocular fundus examination, 20 cases (42.6%, 20/47) had CS on the fundus. The main manifestations of the nervous system are neuromotor development regression or retardation (100%, 56/56), convulsions (58.1%, 25/43), and "startle" phenomena (89.7%, 26/29). Among 42 patients with brain magnetic resonance imaging examination records, 39 cases (92.9%) were abnormal. The incidence of "startle" and seizures in group A was higher than that in group B, and the difference was statistically significant ( χ2=5.815, 6.182, P=0.021, 0.013). Conclusios:Chinese infantile gangliosideosis is more common in GM1 type. Ocular visual impairment is the visual object as the main manifestation, the incidence of fundus CS is 42.6%, and the symptoms of neurological damage in children with CS are more severe.

Matrix metalloproteinase regulates natural killer cells function in methicilin-resistant staphylococcus aureus sepsis / 中华急诊医学杂志

Objective:To investigate the characteristics of natural killer (NK) cell subsets and function in methicillin-resistant staphylococcus aureus (MRSA) sepsis, and to assess the influence of matrix metalloproteinase (MMP) to NK cell function in MRSA sepsis patients.Methods:Twenty-one MRSA sepsis patients who were hospitalized in our department between January 2017 and June 2018 were enrolled. Eleven healthy individuals were served as healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated. NK cell subsets were investigated by flow cytometry. NK cell function was assessed by measuring CD107a, CD69, and CD16 expression in co-culture system between PBMCs and different target cells. MMP mRNA was semi-quantified by real-time PCR in purified NK cells. The influence of NK cell function was assessed by measuring CD107a expression in co-culture system between NK cells with MMP inhibitor stimulation and target cells.Results:There was no significant difference of total NK cell percentage between healthy controls and MRSA sepsis patients ( P>0.05). CD56brightCD16 -NK [(5.36±1.02)% vs (4.30±0.89)%] and CD56 -CD16 +NK [(24.04±2.92)% vs (9.70±1.54)%] percentage was elevated ( P<0.05), while CD56dimCD16 +NK percentage [(71.22±13.03)% vs (87.64±7.05)%, P<0.01] was reduced in MRSA sepsis. NK cells recognized and killed target cells via different receptors upon activation. CD107a [(33.55±3.84)% vs (25.34±6.20)%] and CD69 percentage [(14.96±1.47)% vs(18.80±1.49)%] was decreased ( P<0.0001), while CD16 MFI was increased [(247.1±50.31) vs (189.4±57.54), P<0.01] in MRSA sepsis patients in comparison with healthy controls. MMP-1/2/3/9 mRNA relative levels were elevated in purified NK cells from MRSA sepsis patients ( P<0.01). Inhibition of MMP in NK cells from MRSA sepsis patients promoted CD107a percentage [(33.67±8.03)% vs (25.87±6.23)%, P=0.018]. Conclusions:NK cell subsets imbalance and exhaustion is existed in MRSA sepsis, which might be due to the MMP-induced down-regulation of antibody-dependent cell-mediated cytotoxicity.

Mechanism of MALAT1 induced osimertinib resistance in HCC827 lung cancer cells / 中华肿瘤杂志

Objective@#To test the effect of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) and/or osimertinib on the proliferation and apoptosis of HCC827 cells, and explore the potential mechanism of MALAT1 induced resistance to osimertinib.@*Methods@#We transfected HCC827 cells with LV-vector or LV-over/MALAT1. Stable transfected cells (HCC827/Vector, HCC827/MALAT1) were selected by adding puromycin. HCC827/MALAT1 cells were further transfected with the shRNA-negative control (NC) or shRNA-human epidermal growth factor receptor 3 (ERBB3) plasmid. The effects of overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib on the proliferation of HCC827 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. Cell apoptosis induced by MALAT1 overexpression, knockdown of ERBB3 and/or osimertinib treatment were analyzed by flow cytometry analysis. The expressions of EGFR and ERBB3 signal pathway related proteins in HCC827 cells treated with overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib treatment were detected by western blot.@*Results@#The MTT assay showed that sensitivity to osimertinib of HCC827/MALAT1 cells were significantly repressed. The 50% inhibitive concentration (IC50) of osimertinib >4 000 nmol/L in HCC827/MALAT1 cells. However, knockdown of ERBB3 facilitated the anti-proliferation effect of osimertinib, and the IC50 of osimertinib in shRNA-ERBB3 cells was (17.27±3.21) nmol/L. The results of flow cytometry analysis showed that the apoptotic rate of HCC827/MALAT1 cells induced by 10 nmol/L osimertinib was (8.38±0.92)%, significantly lower than (27.17±5.83)% of knockdown of ERBB3 (P<0.01). Western blotting showed that the expression of p-ERBB3, p-AKT and p-extracellular regulated protein kinases (ERK) in HCC827/MALAT1 cells was markedly up-regulated, while the expression of p-epithelial growth factor receptor (EGFR) was inhibited. The expressions of p-ERBB3, p-AKT and p-ERK were marginally affected by osimertinb. However, osimertinib downregulated the expressions of p-EGFR, p-ERBB3, p-AKT and p-ERK in ERBB3 deleted cells.@*Conclusions@#MALAT1 confers resistance to osimertinb in HCC827 cells by activating of the ERBB3/PI3K/AKT and ERBB3/MAPK/ERK signaling pathways.

Upregulation of PLOD2 promotes invasion and metastasis of osteosarcoma cells / 中华肿瘤杂志

Objective@#To investigate the relationship of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression and the clinical characteristics of osteosarcoma, and explore the potential mechanism of tumour metastasis promoted by PLOD2.@*Methods@#The expression of PLOD2 in osteosarcoma tissues and paired adjacent tissues were detected by immunohistochemistry and qRT-PCR. Correlation of PLOD2 expression in osteosarcoma with the clinical pathologic features was analyzed by Chi square test and Kaplan-Meier analysis.Fibrillar collagen formation and collagen deposition in the tumor tissues were detected by picrosirius red staining. We transfected U-2OS cells with LV-vector, LV-over/PLOD2, sh-NC and sh-PLOD2. The expression of PLOD2 was detected by qRT-PCR. The impact of POLD2 on U-2OS cell invasion was determined by wound-healing assay and Transwell migration assay. The expressions of PLOD2/FAK/JAK2-STAT3 signal pathway related proteins were detected by western blotting.@*Results@#The high expression level of PLOD2 in osteosarcoma tissues was 72.5%, significantly higher than 0% in paired adjacent noncancerous tissues (P<0.01), the expression of PLOD2 was positively correlated with lymph node metastasis, pulmonary metastasis and poor outcome (P<0.01). The same results were also observed in qRT-PCR assay. The median survival time of patients with high expression of PLOD2 protein was 13 months, significantly shorter than 32 months of patients with low expression of PLOD2 (P<0.05). The result of picrosirius red staining showed that the percentage of collagen fiber deposition in the osteosarcoma tissue with high level of PLOD2 was (74.43+ 9.63)%, significantly higher than (9.67±1.28)% in tissue with low expression of PLOD2 (P<0.001). The result of wound-healing and Transwell migration assay showed that over-expression of PLOD2 markedly promoted the invasion, however, knockdown of PLOD2 suppressed the invasion of U-2OS cells (both P<0.01). The result of western blotting showed that over-expression of PLOD2 significantly increased the expression levels of p-FAK, p-JAK2, p-STAT3, but knockdown PLOD2 decreased the levels of p-FAK, p-JAK2, p-STAT3 in U-2OS cells.@*Conclusions@#Up-regulation of PLOD2 in osteosarcoma is correlated with lymphatic and distant metastasis. PLOD2 promotes invasion and metastasis of osteosarcoma might through FAK/JAK2-STAT3 signal pathway.

Study on the influence of challenging-barrier pressure on the professional benefit of emergency nurses / 中国实用护理杂志

Objective To investigate the status of emergency nurses'challenging-barrier pressure and professional benefits, and analyze the influence of challenging-barrier pressure on professional benefits. Methods A total of 262 emergency nurses in 13 general hospitals of Shenyang were investigated by the Challenging-Barrier Pressure Scale and the Nurses' Professional Benefit Scale. Results Emergency nurses challenging pressure score was (3.15±0.86) points, the barrier pressure score was (2.91±0.72) points , and professional benefit score was (48.52±8.55) points. The Pearson correlation analysis showed that the emergency nurses' professional benefits and barrier pressure was negatively correlated (r =-0.471, P < 0.01), the emergency nurses' job benefits and challenging pressure was positively correlated (r=0.550, P<0.01). Herarchical regression analysis showed that after the control of demographic data, challenging-barrier pressure emergency nurses could independently explain the professional benefit of 32.90% of the variance (△R2=0.329). Conclusions The challenging-barrier pressure is an important factor influencing the professional benefit of emergency nurses. The positive role of challenging pressure should be used to improve the professional benefit and enthusiasm of the nurses in the emergency department.

A systematic review of the mechanisms and influence factors of cancer-related fatigue / 中国医师杂志

Cancer-related fatigue (CRF) is one of the most common symptoms in clinical cancer patients.It is associated with cancer itself or with cancer-related therapies.It is a persistent and subjective tiredness experience.It not only affects the patient;s physical,psychological and physical condition,social function,quality of life,but also may cause interruption of cancer-related treatment and affect the patient's life.However,the etiology and mechanism of CRF are not fully understood.Many foreign scholars believe that the occurrence of CRF may be related to anemia,obesity,tumor type,insomnia,inflammatory cytokines,hypothalamic-pituitary-adrenal axis and so on.As cancer patients demand higher quality of life,CRF are increasingly receiving medical attention.In this paper,the related factors and mechanisms of CRF are stated.

Mechanism of long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1 induced invasion and metastasis of esophageal cancer cell EC-109 / 中华肿瘤杂志

Objective@#To investigate the effect and mechanism of long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1, (LncRNA-MALAT1) on invasion and metastasis of esophageal cancer cell EC-109.@*Methods@#EC-109 cells were transfected with lentiviral vector carrying short hairpin RNA of MALAT1( shRNA-MALAT1) or a nonspecific shRNA control (shRNA-control). The expressions of MALAT1, microRNA-200a, ZEB1 and ZEB2 were detected by qRT-PCR. The effect of shRNA-MALAT1 on invasion of EC-109 cells was determined by transwell assay. The expressions of components of epithelial-msenchymal transition pathway in EC-109 cells were determined by immunofluorescence array and western blotting. The expression relationship between MALAT1 and miR-200a in EC-109 cells was detected by dual-luciferase reporter assay.@*Results@#The result of qRT-PCR showed that the expressions levels of MALAT1, ZEB1 and ZEB2 in shRNA-MALAT1 group were 0.43±0.06, 0.64±0.04 and 0.51±0.04, respectively, significantly lower than 0.97±0.08, 1.06±0.07 and 0.98±0.05 in shRNA-control group and 1 in control group, respectively(all P<0.05). Transwell assay showed that the number of invaded cells in shRNA MALAT1 group was (96.81±10.43) per low-power field, markedly lower than that of (278.44±13.28) per low-power field in shRNA-control group (P<0.01). Immunofluorescence staining and Western blotting showed that MALAT1 downregulation significantly reduced the expressions of proteins related to EMT signal pathway in EC-109 cells.Dual luciferase reporter assay showed that compared to negative control, the activities of luciferase reporter in EC-109 cells co-transfected with pmirGLO-MALAT1-wt and miR-200a were significantly down-regulated. While co-transfected pmirGLO-MALAT1-mut with miR-200a mimics had no effect on the luciferase reporter activities of MALAT1.@*Conclusion@#LncRNA MALAT1 functions as a competing endogenous RNA to regulate the expressions of ZEB1 and ZEB2 by sponging miR-200a and promotes invasion and migration of esophageal cancer cells through inducing epithelial-mesenchymal transition.

Effect of rat bone marrow mesenchymal stem cells on dynamic changes of inflammatory factors and apoptosis index during hepatocarcinogenesis / 中国组织工程研究

BACKGROUND:Bone marrow mesenchymal stem cel transplantation has not been thoroughly reported on its effects on apoptosis in hepatoma carcinoma cel s and inflammatory factor level. OBJECTIVE:To investigate the effect of rat bone marrow mesenchymal stem cel s on dynamic change of inflammatory factors and cel apoptosis during hepatocarcinogenesis. METHODS:Sixty healthy Sprague-Dawley rats were divided randomly into healthy group (n=30), control group (n=30) and transplantation group (n=30). Healthy group was given ordinary feed and normal water, while other groups were given diethylnitrosamine solution in drinking water to induce liver cancer models. Then, rats in the transplantation group were subjected to bone marrow mesenchymal stem cel transplantation via the tail vein. Two weeks after cel transplantation, CXCL5, interleukin-8 and interleukin-6 levels were tested by ELISA, mRNA level of hepatocyte nuclear factor 1αdetected by RT-PCR, expression of Bcl-2 and Bax in liver tissue measured by immunohistochemical method, and liver cancer cel apoptosis index detected by TUNEL technique. RESULTS AND CONCLUSION:After modeling, the expressions of CXCL5, interleukin-8 and interleukin-6 in the control group were significantly higher than those in the healthy group (P0.05). Bone marrow mesenchymal stem cel transplantation significantly up-regulated the mRNA level of hepatocyte nuclear factor 1αin the liver tissue that was decreased obviously after modeling (P<0.05). In addition, the expression of Bcl-2 was reduced, while the expression of Bax and the apoptosis index increased significantly in the transplantation group compared with the control group (P<0.05). These findings indicate that bone marrow mesenchymal stem cel transplantation contributes to hepatocyte differentiation and regeneration in liver cancer rats by reducing serum inflammatory factor levels and promoting apoptosis in hepatoma carcinoma cel s.

Features of full field electroretinogram responses in children with early type 1 diabetes / 中华眼底病杂志

Objective To observe the features of the full field electroretinogram (FF-ERG) in type 1 diabetes (T1D) children without diabetic retinopathy (DR).Methods Retrospective case study.Forty-one T1D children and 25 age-matched normal controls underwent a complete ophthalmic examination,including best-corrected visual acuity,refraction,intraocular pressure,slit lamp,fundus photography,indirect ophthalmoscopy,and spectral domain optical coherence tomography to exclude DR.All FF-ERG tests were performed by an experienced technician.The ERG series includes six protocols:dark-adapted 0.01 ERG (r-b 0.01);dark-adapted 3 ERG (mix-a 3.0,mix-b 3.0);dark-adapted 10 ERG (mix-a 10.0,mix-b 10.0);dark-adapted oscillatory potentials (OPS);light-adapted 3 ERG (c-a 3.0,c-b 3.0);light-adapted 30 Hz flicker (30 Hz FP) ERG.To compare the amplitudes and implicit times of the FF-ERG between the T1D and control group children.Results Compared with the control subjects,the FF-ERG amplitudes decreased and the implicit times increased in T1D.Except for r-b 0.01 (t =-0.228,P＞0.05),the amplitudes of other FF-ERGs were all significantly attenuated (t =-1.664,-3.645,-4.324,-6.123,-5.846,-12.9,-14.4,-5.23;P＜0.05) in T1D children.The implicit times of mix-b 3.0,mix-b 10.0,c-b 3.0 and OP2 significantly increased (t=5.242,2.879,5.378,3.506;P＜0.05).The implicit times of r-b 0.01,mix-a 3.0,mix-a 10.0,c-a 3.0 and 30Hz FP changes were not significantly (t=2.331,1.677,0.557,0.84,0.064;P ＞ 0.05).Conclusion The FF-ERG amplitudes decreased and implicit times increased in T1D children compared with the control normal subjects.

Bufalin reverses hepatocyte growth factor-induced resistance to afatinib in H1975 lung cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism.</p><p><b>METHODS</b>The afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot.</p><p><b>RESULTS</b>The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly.</p><p><b>CONCLUSIONS</b>Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.</p>

Clinical characteristics of infantile malignant osteopetrosis with eye disease / 中华实验眼科杂志

Background Malignant osteopetrosis is an extremely rare dense bone disease,and sometimes features ocular disease and cranial nerve palsy.This disease received high attention because of its poor prognosis.And whether the eye manifestation improved after treatment is a problem for concern.Objective This study was to clarify the clinical manifestation,treatment and prognosis of malignant osteopetrosis associated with ocular disease.Methods A retrospective study was adopted.Two children with malignant osteopetrosis associated with eye symptoms were collected from Beijing Children Hospital.The systemic and ocular medical examinations were performed on the patients,including physical examination,hematology laboratory examination,abdominal B ultrasound and bone X ray radiography,external ocular examination,flash visual evoked potential (F-VEP) and CT of orbit.Bone marrow hematopoietic stem cell transplantation was employed and 5-year following-up was cinducted on the chidren.Results The children showed increased bone density,systemic bone sclerosis,basilar thickening,abnormalities of hematology indexes,anemia,hepatosplenomegaly,optical canal stenosis and abnormality of F-VEP P2 wave.In addition,optical disc pale,facial paralysis and paralytic esotropia were seen in a female child and alternating strabismus was found in another boy.After successful treatment,the systemic symptoms remitted in both children,but the eye findings remained unchanged in the female child during the follow-up duration.However,the strabismus diminished in the male child.The optical bone canal widening to 1.9 mm 1 year and 3.2 mm 5 years after treatment in the female child.Conclusions Strabismus and eye disease are the signs of malignant infantile osteopetrosis and reflections of the impairment of the central nervous system.The pathogenetic mechanism of malignant osteopetrosisrelated eye disease is below understanding now.Early bone marrow hematopoietic stem cell transplantation for malignant osteopetrosis can offer the best chance of long-term survival and improve the prognosis of eye diseases.

Clinical study of recombinant human erythropoietin in treatment of lung cancer chemotherapy-related anemia / 中国医师进修杂志

ObjectiveTo evaluate the effect and safety of recombinant human erythropoietin (rhEPO) in treatment of lung cancer chemotherapy-related anemia.MethodsNinety-eight lung cancer chemotherapy-related anemia patients were divided into treatment group and control group with 49 cases each by random digits table method.The patients in treatment group were given rhEPO and chalybeate.The patients in control group were merely given chalybeate.The hemoglobin (Hb),hematocrit,allogeneic blood transfusion rate and quality of life between two groups were observed and compared.ResultsThree cases were rejected in treatment group,and 3 cases with anergy and dizzy and 2 cases with local injection site pain and sclerosis recovered spontaneously.Hb and hematocrit showed downward trend after treatment in control group,but there was no significant differences (P ＞ 0.05).Hb and hematocfit had upgrade trend after treatment in treatment group,and there were significant differences between after 4 - 8 months treatment and before treatment (P ＜ 0.05 ).The allogeneic blood transfusion rate was 24.5％ (12/49) in control group and 6.5％ (3/46) in treatment group,and there was significant difference between two groups (P ＜ 0.05 ).The quality of life in treatment group was increased compared with that in control group.There were significant differences in the effective rate after 4 or 8 weeks treatment between two groups [52.2％(24/46) vs.6.1％(3/49) and 95.7％ (44/46) vs.20.4％ ( 10/49 )].ConclusionsrhEPO is effective and safe in treatment of lung cancer chemotherapy-related anemia.rhEPO has little adverse reaction and can improve the quality of life.