[Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells].

OBJECTIVE: To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs. RESULTS: The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs. CONCLUSION: lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

[Effects of over-expressing heat shock protein 60 on marrow mesenchymal stem cells in the treatment of phosgene-induced acute lung injury].

Objective: To investigate the effect of heat shock protein 60 (HSP60) overexpression on the ability of bone marrow mesenchymal stem cells (MSCs) and its therapeutic effect on rats with phosgene induced acute lung injury. Methods: HSP60 was transfected into MSCs by adenovirus. Western blot was used to measure the expressions of HSP60 before and after transfection. CCK-8 assay was used to detect the activity of MSCs, flow cytometry was used to detect the apoptotic ability of MSCs, and Transwell assay was used to observe the migration ability of MSCs. Sixty SPF grade male SD rats were randomly divided into control group, phosgene exposure group (inhalation of phosgene for 5 min) , MSCs group (phosgene exposure, MSCs treatment group) and transfected MSCs group (phosgene exposure, overexpression of HSP60 MSCs treatment group) . The pathological changes of lung were observed by lung pathological section, lung wet dry ratio, the degree of pulmonary edema, the total cell count and total protein content of alveolar lavage fluid, the inflammatory changes of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in BALF and serum were observed. The data were analyzed by Graphpad Prism 8.0 software. Paired comparisons were performed by non paired t-test. One way ANOVA was used for comparison between groups. Results: The proliferation ability of MSCs transfected with HSP60[A= (0.69±0.05) ] was significantly higher than that of MSCs not transfected with HSP60[A= (0.27±0.02) ] (P<0.05) . Compared with the phosgene exposure group, the pulmonary edema and inflammatory factor infiltration of MSCs group and MSCs transfected group were reduced. However, compared with MSCs group, the degree of pulmonary edema in MSCs transfected group was significantly improved, the levels of inflammatory factors IL-6 and TNF-α were significantly decreased, and the total protein content and total cell count in bronchoalveolar lavage fluid were less (P<0.05) . Conclusion: MSCs transfected with HSP60 can enhance the ability of proliferation, anti apoptosis, migration and the curative effect in rats with phosgene induced acute lung injury.

JMJD3 enhances invasiveness and migratory capacity of non-small cell lung cancer cell via activating EMT signaling pathway.

OBJECTIVE: This study aimed to explore the expression of JMJD3 in non-small cell lung cancer (NSCLC) and to further study its association with clinical features and prognosis of patients. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the level of JMJD3 in 46 pairs of NSCLC tissues and para-cancerous specimens. The relationship between JMJD3 level and clinical features of NSCLC and patients' prognosis was analyzed. And JMJD3 expression in NSCLC cells was further verified by qRT-PCR. In addition, JMJD3 knockdown model was constructed using siRNA in cell lines including A549 and SPC-A1, and the effect of JMJD3 on the biological function of NSCLC cells was analyzed by cell counting kit-8 (CCK-8) and transwell migration and invasive-ness assays. Lastly, Western blot was performed to explore the potential mechanism. RESULTS: In this investigation, qRT-PCR results indicated that JMJD3 expression in above-mentioned tumor tissues was conspicuously higher than that in normal tissues. In addition, compared with patients with low level of JMJD3, patients with high level of JMJD3 had a higher incidence of lymphatic metastasis and distant metastasis and a lower overall survival rate. Meanwhile, the proliferation, invasiveness and migratory capacity of cells in the sh-JMJD3 group was conspicuously decreased when compared with the cells in negative control group. Western Blot results indicated that the levels of key proteins in EMT signaling pathway such as E-cadherin, N-cadherin, Vimentin, TGF-ß, and MMP-9 were notably decreased in sh-JMJD3 group. Besides, the addition of TGF-ß cytokines synergistically promoted the malignant progression of NSCLC induced by JMJD3. CONCLUSIONS: JMJD3 expression was found conspicuously increased in NSCLC, which might be close relevant to NSCLC lymphatic or distant metastasis as well as patients' poor prognosis. Therefore, we speculated that JMJD3 could promote invasiveness and migratory capacity of non-small cell lung cancer cells by activating EMT process.

Clinical observation of Danhong Injection (herbal TCM product from Radix Salviae miltiorrhizae and Flos Carthami tinctorii) in the treatment of traumatic intracranial hematoma.

Danhong Injection (DHI), a Chinese Materia Medica standardized product extracted from Radix Salviae Miltiorrhizae and Flos Carthami tinctorii, has the actions of promoting blood circulation and resolving stasis to promote regeneration. The clinical therapeutic effects of DHI on traumatic intracranial hematoma (TICH) were observed. Eighty patients with TICH were randomly assigned to trial group and a control group (40 patients per group), and all were administered with routine medication. Additionally, DIH was administered intravenously to patients in the trial group. Pre and post-treatment GCS was observed in the two groups, along with GOS after therapy. The intracranial hematoma absorption, hemorheological changes, and changes in coagulation indexes pre- and post-treatment were evaluated. The results indicated that GCS and GOS after therapy for the trial group were superior to those for the control group (p<0.05). There was a significant post-treatment difference in the intracranial hematoma absorption between the two groups (p<0.01). Each hemorheological index in the trial group improved significantly as compared with that of the control group (p<0.05 or p<0.01). The plasma levels of fibrinogen and D-dimer in the trial group were significantly decreased after therapy (p<0.01). These results suggest that DHI is conducive to the recovery of patients with TICH.

Altered histamine H3 receptor radioligand binding in post-mortem brain samples from subjects with psychiatric diseases.

BACKGROUND AND PURPOSE: Histamine is a modulatory neurotransmitter in the brain. Auto- and hetero-histamine H3 receptors are present in human brain and are potential targets of antipsychotics. These receptors may also display disease-related abnormalities in psychiatric disorders. Here we have assessed how histamine H3 receptors in human brain may be affected in schizophrenia, bipolar disorder, major depression. EXPERIMENTAL APPROACH: Histamine H3 receptor radioligand binding assays were applied to frozen post-mortem prefrontal and temporal cortical sections and anterior hippocampal sections from subjects with schizophrenia, bipolar disorder, major depression and matched controls. KEY RESULTS: Compared with the controls, increased H3 receptor radioligand binding was found in dorsolateral prefrontal cortex of schizophrenic subjects (especially the ones who were treated with atypical antipsychotics), and bipolar subjects with psychotic symptoms. No differences in H3 receptor radioligand binding were found in the temporal cortex. In hippocampal formation of control subjects, H3 receptor radioligand binding was prominent in dentate gyrus, subiculum, entorhinal cortex and parasubiculum. Decreased H3 binding was found in the CA4 area of bipolar subjects. Decreased H3 binding in CA2 and presubiculum of medication-free bipolar subjects was also seen. CONCLUSIONS AND IMPLICATIONS: The results suggest that histamine H3 receptors in the prefrontal cortex take part in the modulation of cognition, which is impaired in schizophrenic subjects and bipolar subjects with psychotic symptoms. Histamine H3 receptors probably regulate connections between hippocampus and various cortical and subcortical regions and could also be involved in the neuropathology of schizophrenia and bipolar disorder.

Nanoscale surface topography enhances cell adhesion and gene expression of madine darby canine kidney cells.

Substrate topography is one of the key factors that influence cell behavior, such as cell attachment, adhesion, proliferation and differentiation. In the present work, nanostructures were produced on polystyrene Petri dish by polarized laser irradiation with the wavelength of 266 nm and the energy of 3.0 mJ/cm2. Cell adhesion, growth and gene expression of Madine darby canine kidney (MDCK) cells cultured on smooth and nanogrooved substrates were investigated. The results indicated that cells preferred to adhere and grow on nanogrooved substrate. The distribution of cell cycle for cells on smooth substrates was different from that on nanogrooved substrate. The percentage of G1 phase cells on nanogrooved substrate (48.6 +/- 1.4%) was lower than that on smooth substrate (57.6 +/- 4.4%), while the percentage of cells on nanogrooved substrate in S (30.2 +/- 0.5%) and G2/M (21.2 +/- 1.1%) phase was higher than those on smooth substrate (25.1 +/- 1.5% and 17.3 +/- 3.3%, respectively). Moreover, the gene expression of cyclin D1 and keratin 18, which was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), was significantly enhanced by nanogrooves, with an increase of cyclin D1 mRNA by 98% and an increase of keratin 18 mRNA by 75%. In conclusion, the nanogrooved surface features on polystyrene could alter cell cycle and enhance gene expression of cyclin D1 and keratin 18 in MDCK cells, which partly explained the increased cell adhesion and growth on nanogrooved substrate.

The laminar histamine receptor system in human prefrontal cortex suggests multiple levels of histaminergic regulation.

Human prefrontal cortex is essential for high brain functions and its activity is modulated by multiple neurotransmitters, including histamine. However, the histamine receptors in this brain area have not been systematically studied so far. In situ hybridization and receptor binding autoradiography were employed to map and quantify the mRNA expression and receptor binding of three of the four histamine receptors (H(1), H(2), H(3)). mRNA expression and receptor binding of these three histamine receptors displayed characteristic laminar distribution patterns. Both H(1) and H(3) receptor mRNAs were mainly expressed in the deeper layers (H(1) in laminae V and VI; H(3) in lamina V), where most of the corticothalamic projections originate, whereas H(2) receptor mRNA was primarily expressed in the superficial layer II. Receptor ligand binding of these three histamine receptors displayed relatively even distribution patterns throughout the gray matter. However, higher densities of H(1) and H(3) receptor radioligand binding sites were seen in the middle layers III and IV that receive abundant thalamic inputs and where some of the apical dendrites of the deep-layer pyramidal neurons terminate, whereas higher density of H(2) receptor radioligand binding sites was seen in the superficial layers I-III. The results, together with data on histaminergic regulation of thalamic oscillations suggest that histamine regulates both cortico-cortical and thalamo-cortical circuits. As histamine receptors are also abundant in thalamus, histamine may be involved also in human diseases of the thalamocortical system.

The histaminergic system in human thalamus: correlation of innervation to receptor expression.

The mRNA expression of three histamine receptors (H1, H2 and H3) and H1 and H3 receptor binding were mapped and quantified in normal human thalamus by in situ hybridization and receptor binding autoradiography, respectively. Immunohistochemistry was applied to study the distribution of histaminergic fibres and terminals in the normal human thalamus. mRNAs for all three histamine receptors were detected mainly in the dorsal thalamus, but the expression intensities were different. Briefly, H1 and H3 receptor mRNAs were relatively enriched in the anterior, medial, and part of the lateral nuclei regions; whereas the expression level was much lower in the ventral and posterior parts of the thalamus, and the reticular nucleus. H2 receptor mRNA displayed in general very low expression intensity with slightly higher expression level in the anterior and lateropolar regions. H1 receptor binding was mainly detected in the mediodorsal, ventroposterolateral nuclei, and the pulvinar. H3 receptor binding was detected mainly in the dorsal thalamus, predominantly the periventricular, mediodorsal, and posterior regions. Very high or high histaminergic fibre densities were observed in the midline nuclear region and other nuclei next to the third ventricle, ventroposterior lateral nucleus and medial geniculate nucleus. In most of the core structures of the thalamus, the fibre density was very low or absent. The results suggest that histamine in human brain regulates tactile and proprioceptory thalamocortical functions through multiple receptors. Also, other, e.g. visual areas and those not making cortical connections expressed histamine receptors and contained histaminergic nerve fibres.

Conditioning with high-dose cyclophosphamide may not be sufficient to provide a long-term remission of paroxysmal nocturnal hemoglobinuria following syngeneic peripheral blood stem cell transplantation.

A patient with paroxysmal nocturnal hemoglobinuria (PNH) received a syngeneic peripheral blood stem cell transplant (PBSCT) with high-dose cyclophosphamide (CY) conditioning. He had a reasonable engraftment and complete hematologic recovery. However, at 12 months after PBSCT, he became symptomatic and peripheral blood cells were almost entirely composed of glycosylphosphatidylinositol-anchored proteins deficient cells. This case suggests that high-dose CY may not exert a significant effect on PNH clones in the long term, although it had been effective in allogeneic BMT. In view of the possible autoimmune basis, it seems to be necessary to include other immunosuppressive therapy including ALG in addition to CY.

National food contamination monitoring programmes-levels of mercury, lead and cadmium in Chinese foods.

Six hundred and thirty-four food samples of six kinds were collected from Beijing, Shanghai, Jiangsu, Sichuan, Jilin and Guangdong areas, China and Hg, Pb, Cd contents were determined. The results showed that their levels in the Chinese foods were low; their levels in meat, egg, milk and fish were generally below the national hygienic standard. The average daily dietary intake of Hg, Pb, Cd were 7.25 micrograms, 103.77 micrograms and 30.72 micrograms respectively and they were all less than the ADI established by WHO.

[A study on cadmium content in foods of six provinces and cities].

Contents of cadmium in six kinds of foods were monitored in 626 specimens from Beijing, Shanghai, Jiangsu, Sichuan, Jilin and Guangdong in 1992, with proportions of 95% for rice, 87.8% vegetables, and 100% wheat flour, meat, eggs fish and dairy products, respectively, up to the national hygienic standards. The average weekly intake of cadmium from foods of residents in six monitored areas was lower than that of the provisional allowable intake set by WHO.