MASH1 induces neuron transdifferentiation of adrenal medulla chromaffin cells / 中南大学学报(医学版)

OBJECTIVES@#Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms.@*METHODS@#Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA.@*RESULTS@#Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited.@*CONCLUSIONS@#MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.

Th17/Treg imbalance mediated by IL-8 in RSV-infected bronchial epithelial cells / 中南大学学报(医学版)

OBJECTIVE@#To explore the mechanisms for an increase in susceptibility of asthma induced by respiratory syncytial virus (RSV), to observe the expression of interleukin-8 (IL-8) in human bronchial epithelial cells (HBECs) after RSV infection and to invesigate the regulatory effect of IL-8 on Th17/Treg differentiation.
@*METHODS@#HBECs were divided into a control group and a RSV infected group. The RSVE-infected model of HBECs was established and examined. The expression of IL-8 mRNA was detected by real-time PCR, and the levels of IL-8 were measured by ELISA. Peripheral blood lymphocytes in healthy people were extracted and divided into a control group and an IL-8 treatment group. Based on concentration of IL-8 in RSV-infected HBECs, lymphocytes were treated by a matched concentration of human recombinant IL-8 for 24 h. The distribution of Th17 and Treg subsets in lymphocytes were examined by flow cytometry.
@*RESULTS@#The RSV-infected HBECs model was successfully established. The infected HBECs were still able to split and passage. The RSV could be detected in every passage in the infected cells. Virus particles indicated by bright yellow green fluorescence were seen under fluorescence microscope. Edema of mitochondrias, expansion of endoplasmic reticulum, fissure around nucleus and intracellular virus particles were all observed under electron microscope. The expression IL-8 mRNA were significantly enhanced in the RSV-infected group, and the level of IL-8 in the RSV-infected group was higher than that in the control group (P0.05).
@*CONCLUSION@#Over-secretion of IL-8 by the RSV-infected HBECs may promote the differentiation of Th17 subsets and maintain the Th17/Tred imbalance.

Low-intensity aerobic exercise training attenuates airway inflammation and remodeling in a rat model of steroid-resistant asthma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Aerobic exercise can improve symptoms, reduce airway inflammation, and even ameliorate airway remodeling in asthmatic animals and patients. However, previous studies have focused mainly on the effect of aerobic exercise on steroid-sensitive asthma (SSA). The goals of this study were to determine the effect of low-intensity aerobic exercise training on airway hyperresponsiveness, inflammation, and remodeling in a rat model of steroid-resistant asthma (SRA) and to identify the potential mechanisms underlying these effects.</p><p><b>METHODS</b>Endotoxin-free ovalbumin with or without lipopolysaccharide were applied to establish rat models of SRA and SSA, respectively. Airway hyperresponsiveness, inflammation, remodeling, expression of interleukin (IL)-25, IL-33, thymic stromal lymphopoietin (TSLP), high mobility group box-1 (HMGB1), and IL-17 in bronchoalveolar lavage fluid (BALF), and the role of dexamethasone (DXM) were compared between these two asthmatic rat models. The effect of low-intensity aerobic exercise training and anti-HMGB1 treatment on airway hyperresponsiveness, inflammation, and remodeling in SRA rats also was evaluated.</p><p><b>RESULTS</b>SRA rats developed neutrophil-dominated airway inflammation ((29.5±4.1)% of the total cell numbers in BALF), whereas SSA rats developed eosinophil-dominated airway inflammation ((24.0±6.1)% of the total cell numbers in BALF). Compared with SSA rats, SRA rats had more severe airway hyperresponsiveness, lower levels of IL-25 ((33.6±10.3) vs. (104.8±24.9) pg/ml), IL-33 ((87.5±25.0) vs. (226.6±40.7) pg/ml), and TSLP ((1 933.2±899.5) vs. (7 224.0±992.1) pg/ml), and higher levels of HMGB1 ((21.2±4.5) vs. (5.4±1.6) ng/ml) and IL-17 ((780.5±261.7) vs. (291.4±76.4) pg/ml) in BALF (all P < 0.05). However, there was no significant difference in goblet cell hyperplasia, subepithelial collagen thickness, and airway smooth muscle remodeling between the two groups. Compared with control SSA rats, airway hyperresponsiveness, inflammation, and remodeling in SRA rats were less sensitive to DXM treatment. Anti-HMGB1 treatment attenuated airway hyperresponsiveness, inflammation, and remodeling in SRA rats to a certain extent and was accompanied by lower levels of IL-17 ((369.2±126.7) vs. (780.5±261.7) pg/ml in control SRA rats) in BALF (P < 0.05). Low-intensity aerobic exercise training decreased the expression of both HMGB1 ((14.1±2.9) vs. (21.2±4.5) ng/ml in control SRA rats) and IL-17 ((545.3±148.6) vs. (780.5±261.7) pg/ml in control SRA rats) in BALF (all P < 0.05) and was accompanied by improved airway hyperresponsiveness, inflammation, and remodeling in SRA rats (all P < 0.05).</p><p><b>CONCLUSIONS</b>Low-intensity aerobic exercise training attenuated airway hyperresponsiveness, inflammation, and remodeling in a rat model of SRA. Decreased HMGB1 and IL-17 levels in BALF by aerobic exercise training at least partly contributed to the improvements of SRA.</p>

Effect of Dahuang Zhechong pills on arterial thrombosis in rabbits / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of Dahuang Zhechong pills (DZ) on arterial thrombotic model in vivo.@*METHODS@#Sixty-five rabbits were randomly divided into 7 groups: normal, model (collagen encapsulated thread-drawing),model+aspirin (ASA), model+clopidogrel (CP),model+ASA+CP, model+ low dosage DZ (DZL), and model+high dosage DZ (DZH). All rabbits except the normal group were fed with the drugs repectively for 8 days,and sacrificed at 2 hours after the last feeding, obtained aortae. The pathological changes in the aortae were observed under microscope,and the level of FDP, D-dimer and tissue factor (TF) were measured by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#The vascular vessels were filled with thrombi in the model group and the elastic membranes of the vessel wall were seriously injured. The arterial thrombi were observed around the vascular wall in the DZL group, but some of the thrombi were dissolved. The number of thrombi was remarkably decreased in the DZH group, and most thrombi were dissolved and the vascular intimal membranes were intact. Compared with the model group, the dry and wet weight of the thrombi and the level of D-dimer, FDP, and TF in the plasma were significantly attenuated (P0.05). The pathological changes in the vascular vessel and the elevation of plasma parameters in the DZL group were similar to those in the ASA and CP groups (P>0.05). The dry and wet weight, D-dimer, FDP, and TF in the plasma in the DZH group were significantly lower than those in the DZL group (P<0.01 or P<0.05, separatively), and closed to those in the ASA+CP group.@*CONCLUSION@#Dahuang Zhechong pills are potential novel anti-thromobotic agent for arterial thrombosis.

Experimental study on lung neoplasm model induced by 3, 4-benzopyrene pulmonary injection in rats / 中国肺癌杂志

<p><b>BACKGROUND</b>There is still disadvantage in animal model for lung cancer study, no matter what is xenograft nude mice or rats/mice lung neoplasm induced by carcinogenesis.The purpose of the current investigation is to explore a simple and convenient and reliable method for lung neoplasm animal model.</p><p><b>METHODS</b>The prepared 36 female Sprague Dawley (SD) rats, with the range from 180 to 220g of body weight, were randomly divided into two groups, each group included 18 rats. In the study group, the rats were given 2mg of 3, 4-benzopyrene soluting in 0.2mL corn oil every two-weekly pulmonary injection for 4 times through right middle-chest percutaneous puncture under control of anaesthesia by pentobarbital sodium i.p. The controls were only given 0.2mL corn oil injection simultaneously. The rats were sacrificed after suffering from dyspnea, and the survived rats were sacrificed at narcotism in 1 year after the first 3, 4-benzopyrene toxicosis. Lung, brain, liver, esophagus and stomach of all rates were anatomized in search of tumor.</p><p><b>RESULTS</b>No lung neoplasm was found in the control group within 1-year observation. The earliest dyspnea caused by lung malignant neoplasm was observed in 16 week after the first injection in a rat treated with 3, 4-benzopyrene oil, then 10 rats were sacrificed and found with malignant neoplasm in theirs right lung when the rats suffered from dyspnea, and 1 rat was found a huge tumour in its right lower limb in 26 weeks after treatment. The other 6 rats were sacrificed in 1 year after the first 3, 4-benzopyrene oil treatment, in which 1 rat was found with a malignant neoplasm in its right lung and 5 rats were normal. No tumor was found in brain, liver, esophagus and stomach. Out of these 18 rats, 12 (66.67%) lung malignant neoplasms and 1 limb malignant neoplasm were found within 1 year in the experimental group which yielded a total cancerogenic rate of 72.22% (13/18). The lung neoplasms included: 2 poor-differentitaed squamous cell carcinomas, 3 adenocarcinomas and 7 undifferentiated carcinomas.</p><p><b>CONCLUSIONS</b>The results suggest that 3,4-benzopyrene pulmonary injection by percutaneous puncture may provide an efficiency method for lung neoplasm model established in rats.</p>

Neuro-Immuno-Endocrine Modulation by Nerve Growth Factor in Asthma / 生物化学与生物物理进展

Nerve growth factor, a kind of neurotrophic factor, plays an important role in neuronal development, differentiation, survival and neurogenesis, and is considered as a link between neuroendocrine system and immune system in asthma attack. The possible mechanism of effects of nerve growth factor on asthma is as follows: (1) nerve growth factor changes airway innervation, and facilitates the synthesis and release of neurotransmitter in nerve terminal, which will contribute to airway remodeling; (2) nerve growth factor induces eosinophils aggregation, proliferation and releasing inflammatory factor, which will lead to the abnormality of immunologic response; (3) nerve growth factor triggers the redundancy of adrenal medullary cells, which results in adrenal medullary cell to nerve cell transition, and then the impairment of chromaffin cell endocrine secretion function. As a result, the concentrations of adrenaline in circulation are not competent to relieve the bronchoconstriction in asthmatic attack.

Synthesis and identification of antigenic conjugates of podophyllotoxin / 药学学报

Aim To synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody. Methods The hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits. Results The results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA1, 40.3 for Hapten-BSA2, 17.8 for Hapten-OVA1, and 54.2 for Hapten-OVA2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC50 value of 2.21 μg·mL -1 with a detection limit of 0.12 μg·mL -1. Conclusion Antigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.

Dysfunction of releasing adrenaline in primary cultured AMCC due to functional reduncdency primed by nerve growth factor / 中国实用内科杂志

Objective To investigate the possible mechanism of the biologic ethology effects of NGF and SP(substance P)on primary cultured adrenaline medullary chromaffin cells(AMCC).Methods To establish primary cultured AMCC by means of enzyme digestion and purify the cells by means of isopycnic gradient centrifugation and differential plating.To observe the morphological and ultrastructural changes after the addition of NGF and SP,and detect the concentration of adrenaline and noradrenaline in serum by ELISA.Results Confervaceous processes could be observed after 2 d of addition of NGF to the culture and the processes strenched longer as days went by the observation of electron microscopy.there are some drumstick-like and villiform processes in the cell membrane and some vesiculation be observed near the cell membrane of the primary cultured AMCC cells after the addition of NGF.The bioblast was abundant but the structure was not clear in the intracytoplasm and the concentration of adrenaline were decreased(P