Effects of CpG-oligodeoxynucleotides and dexamethasone on airway remodeling and expression of MMP-9 and TIMP-1 in murine model of chronic asthma

AIM: To observe the changes of airway inflammation and remodeling in a murine model of chronic asthma with CpG- oligodeoxynucleotides(CpG ODN) and dexamethasone (DXM) treatments.METHODS: BALB/c mice were sensitized and repeatedly challenged with ovalbumin.Pathological slides were prepared from left lung and stained with hematoxylin-eosin.WAmus (smooth muscle area),Wamuc (mucous area) and WAi (inner wall area) of the airway were measured and standardized by Pbm (basement membrane perimeter). The areas of collagen Ⅰand Ⅲ in the lung tissue were determined by using a Sirius red-polarizing microscopy morphometry method.Expressions of matrix metalloprotease-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were detected by immunohistochemistry.RESULTS: WAmus/Pbm,WAmuc/Pbm and WAi/Pbm decreased significantly in CpG ODN and DXM treated group when compared with asthma group (P<0.05).No statistical significance between CpG ODN and DXM treated group was observed (P>0.05).Collagen deposition in asthma group increased more than that in CpG ODN and DXM treated group (P<0.05).The expressions of MMP-9 and TIMP-1 were much higher in asthma group than those in CpG ODN and DXM treated group (P<0.05).It had no statistical significance between CpG ODN and DXM treated group (P>0.05).CONCLUSION: Airway remodeling occurrs in the chronic asthma.Early intervention with steroid or CpG might partially inhibit its process via lowering expressions of MMP-9 and TIMP-1 in chronic asthma.     

Effect of Kechuanning on airway remodeling and protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus

AIM:To investigate the effect of Kechuanning on airway remodeling and the protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus. METHODS:The asthmatic rat model induced by respiratory syncytial virus was established. The experimental rats were divided into normal group, asthma model group, low dose (0.33 mL/kg), middle dose (3.0 mL/kg) and high dose (10 mL/kg) of Kechuanning groups, and PD98059 (3 mg/kg) group. The airway responsiveness of the rats was measured by animal ventilator. The pathological changes of the lung tissues were observed by HE staining. PAS staining and Masson staining were used to observe goblet epithelial cells metaplasia and airway collagen deposition. The expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the lung tissues of the rats was detected by immunohistochemical staining. The protein levels of ERK1/2 and p-ERK1/2 were determined by Western blot. RESULTS:Compared with model group, the airway responsiveness of the rats in middle dose and high dose of Kechuanning groups was significantly decreased (P<0.01), the injury of lung tissues was significantly decreased, the goblet epithelial cells metaplasia and airway collagen deposition were significantly reduced (P<0.01), and the expression of MMP-9 and TIMP-1 in the lung tissues was also significantly decreased (P<0.01). In addition, the protein level of p-ERK1/2 in high dose of Kechuanning group was significantly decreased compared with model group (P<0.01). CONCLUSION:Kechuanning may treat asthma by regulating the expression of p-ERK1/2 in the lung tissues and improving the airway remodeling symptoms of asthmatic rats induced by virus.     

Expression of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and collagen type IV in lung of rats with multiple organ dysfunction syndrome

AIM: To determine the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of me-talloproteinase-1 (TIMP-1) and collagen type IV (IV-C) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. METHODS: Adult male Sprague-Dawley (SD) rats (n=40) were randomly divided into sham control group and cecal ligation and puncture (CLP) model group. The rats in CLP group were divided into 4 subgroups as different intervals (6 h, 12 h, 24 h and 48 h), and there were 8 rats in each group. The rat model of MODS was established by CLP. All rats were sacrificed at various intervals. The functions of the liver, kidney and lung were determined by blood biochemical and blood gas analysis. The morphological changes of the lung tissues were observed with HE staining. The serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, MMP-9 and TIMP-1 were measured by ELISA. The expression of MMP-9 and TIMP-1 in the lung tissues was detected by RT-PCR and immunohistochemistry, and the expression of IV-C in the lung tissues was detected by immunofluorescence and Western blot. RESULTS: Compared with sham control group, the functions of the liver, kidney and lung were damaged at different degrees in model groups. No histopathological change in the lung tissues of sham control group was found, and the lung injury was serious in model groups. Compared with sham control group, the serum levels of TNF-α, IL-1β, MMP-9 and TIMP-1 in model groups increased significantly (P<0.05) and peaked at the interval of 12~24 h after modeling (P<0.01). The expression of MMP-9 and TIMP-1 in the lung tissues of model groups increased, and peaked at 12 and 24 h, respectively (P<0.01). The protein level of IV-C in MODS 6 h group was not changed as compared with control group, while that at the interval of 12~48 h after modeling was significantly decreased and dropped to the lowest at 24 h (P<0.01). CONCLUSION: MMP-9 and TIMP-1 play important roles in lung injury of MODS rats by regulating the synthesis and decomposition of IV-C which is the main component of extracellular matrix.     

Cucurbitin E relieves airway inflammation by inhibiting MAPKs/NF-κB signaling pathways in asthmatic mice

AIMTo investigate the effects of cucurbitacin E on airway inflammation and the signaling pathways of MAPKs and NF-κB in asthmatic mice. METHODSHealthy mice (n=40) were randomly divided into control group, model group, low-dose cucurbitacin E group, high-dose cucurbitacin E group and dexamethasone group. Ovalbumin sensitization was used to induce asthma in the mice. The protein levels of p-JNK, p-ERK1/2, p-p38 MAPK and p-p65 in the lung tissues were determined by Western blot. RESULTSCompared with control group, the numbers of inflammatory cells, such as eosinophils, lymphocytes and neutrophils, were significantly increased in model group, and the activity of MAPKs and NF-κB signaling pathway-related proteins was significantly enhanced. Cucurbitin E at high dose attenuated airway inflammation in asthmatic mice, and significantly inhibited the activity of MAPKs and NF-κB signaling pathway-related proteins. Histopathological results showed proliferation of goblet cells and bronchial mucosal epithelial cells, infiltration of inflammatory cells in the alveoli, and narrow alveolar cavity in model group, while the pathological changes were significantly alleviated in cucurbitin E treatment groups. CONCLUSION Cucurbitin E improves airway inflammation in asthmatic mice, and its mechanism may be related to the inhibition of MAPKs and NF-κB signaling pathways.     

Relationship between matrix metalloproteinase-9 and tissue metalloproteinase inhibitor-1 and carotid atheromatous plaque stability

AIM: To investigate the correlation between matrix metalloproteinase-9 (MMP-9),tissue metalloproteinase inhibitor-1 (TIMP-1),MMP-9/TIMP-1 and carotid atheromatous plaque stability in cerebral infarction patients.METHODS: 80 patients with cerebral infarction were categorized as microemboli-negative group (n=70) and microemboli-positive group (n=10),20 normal human were served as control group.The MMP-9 and TIMP-1 levels in plasma were determined by mean of ELISA in 3 groups.RESULTS: The levels of MMP-9 and TIMP-1 in plasma were significantly higher in cerebral infarction patients than those in control group (P<0.01).The plasma MMP-9 content was positively correlated with TIMP-1 content (r=0.76，P<0.01).The ratio of MMP-9/TIMP-1 increased only in microemboli-positive patients (P<0.01).CONCLUSION: The results indicate that the plasma MMP-9 participates in pathophysiological process of cerebral infarction.The ratio of MMP-9/TIMP-1 shows a close relationship with carotid atheromatous plaque instability.     

Chutan-Jiedu decoction reverses resistance of lung cancer to gefitinib by processing EMT

AIM: To investigate the mechanism of Chutan-Jiedu decoction (CJD) reversing the resistance of lung cancer to gefitinib via epithelial-mesenchymal transition (EMT) pathway.METHODS: BALB/c nude mice (n=60) were selected to establish lung cancer xenograft model with human lung adenocarcinoma drug-resistant cell line H1975, which were randomly divided into 6 groups (10 mice per group):model group, gefitinib (0.04 g/kg) group, low-dose (13.52 g/kg) CJD group, middle-dose (27.04 g/kg) CJD group, high-dose (54.08 g/kg) CJD group, and combined medication group (27.04 g/kg CJD+0.04 g/kg gefitinib). The mice in each group were treated for 2 weeks before the tumor size and tumor weight were detected for the calculation of the tumor inhibitory rate. The mRNA and protein expression levels of E-cadherin, Snail and vimentin were determined by immunohistochemistry, Western blot and real-time PCR.RESULTS: Compared with model group and gefitinib group, the tumor size and the tumor weight in middle-dose CJD group, high-dose CJD group and combined medication group were decreased significantly (P<0.05). The results of immunohistochemistry, Western blot and real-time PCR showed that the expression of E-cadherin at mRNA and protein levels was increased significantly, while the expression of Snail and vimentin at mRNA and protein levels was decreased significantly (P<0.05).CONCLUSION: The growth of lung adenocarcinoma H1975 xenografts in nude mice is inhibited by CJD. In addition, the resistance of lung cancer to gefitinib is reversed. The mechanism may be related to the regulation of EMT-related protein expression.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Effects of ginsenoside Rh1 on expression of inflammatory factors in mouse asthma model

AIM: To observe the effects of ginsenoside Rh1 on the levels of inflammatory factors in serum and bronchoalveolar lavage fluid (BALF), and the pathological changes of the lung tissues in an experimentally induced mouse asthma model. METHODS: Male BALB/c mice (n=40) were divided into 4 groups:normal control group, asthma mo-del group, and low-dose (40 mg·kg^-1·d^-1) and high-dose (80 mg·kg^-1·d^-1) ginsenoside Rh1 groups. The bronchial asthma mouse model was established by the method of ovalbumin induction and excitation, and during the excitation period, the mice were daily treated with ginsenoside Rh1 for 2 weeks. At 24 h after the final dose of ginsenoside Rh1, the mice were sacrificed. The number of eosinophils (EOS) and the concentrations of interleukin (IL)-4, IL-5 and interferon (IFN)-γ in BALF were determined. The levels of IgG and IgE in serum were measured, and the expression of transforming growth factor (TGF)-β1 and the pathological changes in lung tissues were evaluated. RESULTS: Ginsenoside Rh1 inhibited the increases in the number of EOS and the concentrations of IL-4, IL-5, IFN-γ and IgE, reversed the increased expression of TGF-β1, and improved the pathological changes of the lung tissues in asthmatic mice. CONCLUSION: Ginsenoside Rh1 improves the immuno-inflammatory profile and pathological changes in the experimentally induced mouse asthma model, implying its potential therapeutic effect on asthma.     

Effects of Danggui Buxue decoction on endothelial progenitor cells,serum VEGF and SDF-1 in atherosclerotic rabbits

AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.     

Baicalin attenuates pancreatic fibrosis by inhibiting TGF-β1/TAK-NF-κB signaling pathway in chronic pancreatitis mice

AIM: To explore the role of transforming growth factor-β1 (TGF-β1)/TGF-β-activated kinase (TAK)-nuclear factor-κB (NF-κB) signaling pathway in chronic pancreatitis (CP) mice and the effect of baicalin on pancreatic fibrosis in the mice. METHODS: Kunming mice (n=58) were randomly divided into 3 groups, including control group, CP group and baicalin group. The mice in CP group and baicalin group were intraperitoneally injected with 20% L-arginine. After 2 weeks of CP, the mice in baicalin group were intraperitoneally injected with baicalin (100 mg/kg, once a day). At 2 weeks, 4 weeks and 6 weeks after modeling, the mice were anesthetized and sacrificed. The morphological changes of the pancreas were observed by HE and Masson staining. The serum level of TGF-β1 was analyzed by ELISA. The expression of fibronectin (FN) and NF-κB in the pancreas was observed by immunohistochemistry staining. The protein levels of transforming growth factor-β receptor type Ⅰ (TGF-βRⅠ), phosphorylated TAK1 (p-TAK1) and NF-κB in the pancreas were determined by Western blot. The mRNA expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloprotease-1 (TIMP-1) was detected by real-time PCR. RESULTS: At 2 weeks, 4 weeks and 6 weeks after intraperitoneal injection of L-arginine, the pancreatic tissues were obviously injured and exhibited different degrees of fibrosis, and FN expression was significantly increased. After treatment with baicalin, the degrees of pancreatic injury and fibrosis were significantly attenuated and the expression of FN was reduced (P<0.01). Compared with control group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 in the pancreas of CP group were significantly increased, and the expression of MMP-1 was decreased at each time point. In baicalin group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 were significantly decreased, and the expression of MMP-1 was markedly increased at the corresponding time points compared with CP group (P<0.01). CONCLUSION: Baicalin effectively atte-nuates pancreatic fibrosis by inhibiting the activation of TGF-β1/TAK-NF-κB signaling pathway and regulating the balance of MMP-1/TIMP-1.     

Effect of oridonin on invasion and migration of human lung cancer NCI-H460 cells

AIM：To investigate the effect of oridonin on the invasion and migration of human lung cancer NCI-H460 cells. METHODS：NCI-H460 cells were divided into high-dose (HD), middle-dose (MD) and low-dose (LD) oridonin groups (cultured with 40, 20 and 10 μmol/L of oridonin, respectively, as experimental groups), and normal (N) group (treated without oridonin as control). The cell growth was observed. The cell proliferation was detected by MTT assay. Boyden chamber was used to determine the cell invasive capacity. The cell migration was also measured. The levels of MMP-2 and MMP-9 were assayed by Western blotting. RESULTS：The cell counts in the experimental groups were lower than that in N group. The cell proliferation was inhibited as the inhibitory rates were 48.94%, 36.17% and 19.15% for HD group, MD group and LD group, respectively. The numbers of the invasive cells were 26.67±5.16 for HD group, 36.17±5.08 for MD group, and 44.33±5.50 for LD group. The migration rates in the experimental groups were lower than that in N group. The expression of MMP-2 and MMP-9 decreased dependent on the oridonin dose as follows: HD group < MD group < LD group < N group. CONCLUSION：Oridonin inhibits the invasion and migration of NCI-H460 lung cancer cells, and reduces the expression of MMP-2 and MMP-9.     

Effect of Qushi-Dizhuo decoction on urate transporters in hyperuricemia rats

AIM:To investigate the effects of Qushi-Dizhuo decoction (QSDZ) on the expression of uric acid transporters in rats with hyperuricemia. METHODS:Male Sprague-Dawley rats (n=60) were randomly divided into blank control group, model group, allopurinol group, and low-, middle- and high-dose QSDZ groups, with 10 rats in each. Except the blank control group, rats in the other groups were treated with adenine and potassium oxonate to prepare hyperuricemia rat model by intragastric administration once a day. Meanwhile, the rats in treatment groups were intragastric admi-nistrated with the corresponding preparation, after 28 days of continuous administration, the rats were sacrificed. Serum uric acid (UA) and creatinine (Cr) levels and xanthine oxidase (XOD) activity were measured. The morphological changes of renal tissues were observed by HE staining. The protein and mRNA expression of urate transporter1 (URAT1), glucose transporter 9 (GLUT9) and organic anion transporter 1 (OAT1) were detected by Western blot and real-time PCR. ATP-binding cassette subfamily Gmember 2 (ABCG2) and organic cation transporter 1 (OCT1) were also determined. RESULTS:Compared with the blank control group, the content of UA and Cr and the activity of XOD were significantly increased (P<0.05), the URAT1 and GLUT9 protein levels were significantly up-regulated, and the OAT1, ABCG2 and OCT1 protein levels were significantly down-regulated in model group (P<0.05). Compared with model group, the levels of serum UA and Cr and actioity of DXD were significantly decreased in QSDZ middle,high-dose group (P<0.05). The protein levels of URAT1 and GLUT9 were decreased, while the protein levels of OAT1, ABCG2 and OCT1 were increased in middle- and high-dose groups compared with model group (P<0.05). The mRNA expression of URAT1, GLUT9, OAT1, ABCG2, OCT1 was consistent with its protein expression trend. CONCLUSION:QSDZ decoction may through inhibiting the activity of XOD and regulating the expression of transporter to show its anti-hyperuricemia effect.     

Effects of damage on endothelin production in epithelial cells and sub-epithelial fibroblasts

AIM: To investigate the level of ET-1 produced by cultured human bronchial epithelial cells (HBECs) under injury and the effects of injured HBECs on ET-1 production in sub-epithelial fibroblasts. The interaction between ET-1 and matrix metalloproteinase-9(MMP-9) was detected in HBECs under damage. The purpose of the study is to evaluate the effect of injured HBECs related to ET-1 release on airway remodeling in asthma. METHODS: ET-1 level was detected in supernatants from cultured HBECs 12 h after being treated with either mechanical scraping or LPS stimulation or mechanical scraping plus LPS, as well as from subepithelial fibroblasts cocultured with mechanical damaged HBECs. It was also measured in the supernatant from HBECs transfected with MMP-9 expression plasmid. MMP-9 activity was assessed in supernatants from HBECs stably transfected with pEGFPc1 -antisense-ET-1 converting enzyme(ECE) RNA. RESULTS: It was found that there was an increase in ET-1 level in supernatants from HBECs either treated with mechanical scraping plus LPS or transiently transfected with MMP-9 plasmid, as well as from sub-epithelial fibroblasts cocultured with mechanical scraping HBECs compared with that in controls. Gelatin zymography showed a obviously attenuated gelatinolytic activity of MMP-9 in conditioned media of HBECs expressing antisense ECE RNA after mechanical damage. CONCLUSIONS: Airway epithelial cells under injury are able to overproduce ET-1 as well as initiate ET-1 release from sub-epithelial fibroblasts, MMP-9 produced by injured bronchial epithelial cells may also increase ET-1 processing leading to ET-1 production further. The interaction between ET-1 and MMP-9, both of which enhanced in damaged HBECs, may play an important role in airway inflammation related to airway remodeling in asthma.     

Kinetics of pathologic changes in bleomycin-induced murine pulmonary fibrosis model

AIM: To Investigate the kinetics of pathologic changes in bleomycin-induced pulmonary fibrosis in rats. METHODS: Sixty male SD rats were randomized as a negative control group and pulmonary fibrosis model groups (B3, B7, B14, B28, B56 sub-groups). Except for control group, rats in the other groups were intratracheally administered with bleomycin. Animals in pulmonary fibrosis model groups were sacrificed on day 3, 7, 14, 28 and 56. The sections of the right lung were stained by HE, Masson and sirius red. The left lung was weighed and its hydroxyproline content was assayed. The mRNAs of TGF-β1, MMP-9 and TIMP-1 in the lung homogenate were measured by semi-quantitative RT-PCR. The expressions of TGF-β1, MMP-9 and TIMP-1 in lungs were observed by immunohistochemistry. RESULTS: (1) The content of lung hydroxyproline in pulmonary fibrosis model groups was significantly increased than that in control group (P<0.05). The pulmonary inflammation in pulmonary fibrosis model groups was significantly serious than that in control group, pulmonary fibrosis in B14, B28 and B56 groups was also significantly serious than that in control group. (2) A small quantity of TGF-β1, MMP-9 and TIMP-1mRNA were measured in normal lung, and the expression increased significantly after administration of bleomycin. Different expressions of TGF-β1, MMP-9 and TIMP-1 in different days after bleomycin administration were observed. CONCLUSION: The pathological changes in different days after bleomycin administration are different. TGF-β1, MMP-9 and TIMP-1 may play important roles in the pathogenesis of pulmonary fibrotic process.     

Isorhamnetin inhibits ovalbumin-induced pulmonary inflammation in asthmatic mice

AIM To investigate the effect of isorhamnetin on pulmonary inflammation in asthmatic mice and to analyze its primary mechanism. METHODS BALB/c mice were randomly divided into normal control group, asthma model group, isorhamnetin low-dose （50 mg/kg） treatment group and isorhamnetin high-dose （150 mg/kg） treatment group. Ovalbumin （OVA） was used to establish a mouse asthma model. HE staining and PAS staining were used to observe the pathological changes of lung tissue. The contents of cysteinyl leukotriene1 （CysLT1）, cystelinyl leukotriene receptor 1 （CysLTR1）, interleukin-4 （IL-4）, IL-5, IL-13 and tumor necrosis factor-α （TNF-α） in bronchoalveolar lavage fluid （BALF） were measured by ELISA. The expression of nuclear factor of activated T cells 4（NFATc4） in lung tissue was detected by immunohistochemical staining. The protein expression of NFATc4, intercellular cell adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule-1 （VCAM-1） was determined by Western blot. RESULTS Isorhamnetin improved histopathological changes in OVA-induced asthma model mice, reduced the contents of CysLT1, CysLTR1, IL-4, IL-5, IL-13 and TNF-α in BALF, and reduced NFATc4, ICAM-1 and VCAM-1 expression in lung tissue （P<0.05）. CONCLUSION Isorhamnetin inhibits the inflammatory response of lung tissue in asthmatic model mice, and the mechanism may be related to the inhibition of the calcineurin/NFATc4 signaling pathway.     

Dynamic observation of asthma model induced by allergen in mice from acute inflammation to chronic remodeling

AIM: To dynamically observe and compare the relative changes of the indexes from the process of acute inflammation to chronic remodeling in asthmatic mice induced by ovalbumin (OVA).METHODS: Female BALB/c mice (n=60) were randomly divided into normal control group and asthma group. The mice in asthma group were sensitized and challenged by OVA, while the mice in normal group received equal volume of normal saline (NS). The challenge was performed for 3 consecutive days from the 21th day to observe the response of acute inflammation, and then the mice in different groups were challenged once per week for 5 weeks. Detailed comparisons of the dynamic changes of cell infiltration, cytokine expression and airway remodeling were conducted.RESULTS: Compared with NS group, the mice in OVA group showed a predominantly eosinophilic infiltration into the airway lumen, increased production of Th2-type cytokines, secretion of epithelial mucus and deposition of subepithelial collagen. In OVA challenge groups, the levels of inflammatory cells and inflammatory factors were remarkably higher in 24 d group, whereas the most obvious changes of goblet cell hyperplasia and airway remodeling were observed in 52 d group.CONCLUSION: Acute asthma model is sufficiently induced by 3 consecutive days of OVA challenge protocol, which is accompanied with high levels of inflammatory cells and inflammatory factors. The OVA challenge protocol once per week for 5 weeks could induce a chronic asthma model with obvious airway remodeling.     

Effects of PFOA exposure on inflammatory mediators IL-4, IFN-γ and glucocorticoid receptor in asthmatic mice

AIM: To investigate the effects of perfluorooctanoic acid (PFOA) exposure on the changes of asthmatic mouse airway inflammation, inflammatory mediators interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum, and glucocorticoid receptor (GR) expression in the lung tissue.METHODS: BALB/c mice (n=30) were randomly divided into 5 groups:normal control (C) group, asthma (A) group, asthma+low-dose PFOA (AP10) group, asthma+ mode-rate-dose PFOA (AP50) group and asthma+high-dose PFOA (AP100) group. Asthma model and PFOA exposure model of mice were established according to the grouping. The animals were sacrificed and their lungs were collected for HE staining, transmission electron microscopy, Western blot and immunohistochemical staining. ELISA was applied to detect the levels of IL-4 and IFN-γ in the serum.RESULTS: HE staining of the lungs showed that the asthmatic mice, compared with the normal control mice, had obvious mucus secretion around the airways and infiltration of inflammatory cells around airways and blood vessels, and the effects were much more marked in AP groups. Ultrastructural alteration of the lung tissues in the asthmatic mice were indicated by transmission electron microscopy. Compared with C group, the results of ELISA in A group and AP groups proved that IL-4 in the serum was increased and IFN-γ was decreased significantly (P<0.05). Compare with A group, IL-4 was significantly increased and IFN-γ was decreased in AP100 group (P<0.05), and no difference of those between AP10 group and AP50 group was found. The results of Western blot indicated that GR protein expression in the asthmatic mice were decreased compare with the normal mice (P<0.05), and no difference of that among A group and AP groups was observed. Immunohistochemical staining manifested that GR protein was mainly located in the cytoplasm of bronchial columnar epithelial cells, airway smooth muscle cells and vascular smooth muscle cells.CONCLUSION: Acute airway PFOA exposure in asthmatic mice dose-dependently exacebates lung inflammation by inducing Th2 type immune responses, promotes infiltration of inflammatory cells and mucus secretion around the airways and blood vessels, and destroys the ultrastructure of the lung tissues.     

Effects of Xiaoyaosan on imiquimod induced psoriatic lesions and depression neurotransmitters in mouse model

AIM: To observe the effect of Xiaoyaosan decoction on the psoriatic lesions and depression neurotransmitters induced by imiquimod in mice. METHODS: BALB/c male mice were randomly divided into control group, model group, methotrexate group and Xiaoyaosan high, medium and low dose groups, 6 mice in each group. Imiquimod (IMQ, 5%) was used on the back of the animals to induce psoriasis-like lesions in the mice. The psoriasis area and seve-rity index (PASI) were evaluated for daily scoring. The sugar water preference experiment was conducted to explore the behavioral differences in the mice. The morphological changes and epidermal thickness of the lesions were observed under light microscopy. Immunohistochemical method was used to detect the expression of CD3 on T lymphocyte surface. The expression of Ki67 in the skin lesions was detected by immunofluorescence. The contents of monoamine neurotransmitters such as adrenaline (AD), gamma-aminobutylic acid (GABA), glutamate (Glu), dopamine (DA) and their metabolites in the hippocampus and hypothalamus of mice were detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). RESULTS: Compared with model group, the back skin lesions of Xiaoyaosan each dose group and methotrexate group were significantly improved, and the PASI score and epidermal thickness were both lower than those in model group (P<0.05). The expression levels of Ki67 and CD3⁺ T cells in Xiaoyaosan group and methotrexate group were lower than those in model group (P<0.05). Compared with model group, the body mass change range of Xiaoyaosan high-dose group and blank control group was significantly smaller than that in model group (P<0.05). The sugar water preference rate in blank control group was significantly higher than that in model group (P<0.01). Compared with model group, the sugar water preference rate in methotrexate and Xiaoyaosan groups showed a certain increase trend, but no statistical diffe-rence was observed. Compared model group, the levels of 3, 4-Dihydroxypheny-lacetic acid (DOPAC), AD, GLU and GABA levels in the mouse hippocampus in blank control group were decreased significantly (P<0.01), while the levels of DA and homovanillic acid (HVA) had no significant difference (P>0.05). No significant difference of DA, DOPAC, HVA and GLU levels in the mouse hypothalamus was observed between blank control group and model group (P>0.05), while the content of AD and GABA in the mouse hypothalamus in blank control group was lower than that in model group. The AD content of the hypothalamus in high-dose Xiaoyaosan group was significantly higher than that in model group (P<0.01), and the HVA content of the hypothalamus in low-dose Xiaoyaosan group was significantly higher than that in model group (P<0.01). PASI score was negatively correlated with the content of DOPAC, AD, GLU and GABA in the hippocampus and the content of AD, GLU and GABA in the hypothalamus, those were, the more severe the back skin lesion was, the lower the expression of depression-related neurotransmitters were, indicating the aggravation of depression in the mice. CONCLUSION: Xiaoyaosan improves the skin lesions induced by imiquimod in the mice with psoriasis, improves the behavior of depression in the mice with psoriasis, and up-regulates the expression of depression-related monoamine neurotransmitters. The expression of depression-related neurotransmitters is negatively correlated with the skin lesions induced by imiqumod in the mice with psoriasis. The degree of depression is increased with the aggravation of psoriatic lesions.     

Expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in lung tissue of hypercapnia rat model

AIM: To study the expression of matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2) and the tissue inhibitor of metalloproteinase(TIMP-1) in the lung tissue of the hypercapnia rat.METHODS: Forty Wistar rats were randomly divided into a control group (group A, n=20) and hypercapnia group (group B, n=20). Group B received mix gas exposure (6% CO2, 21% O2, 72% N2) 7 h daily for 4 weeks. The parameters we would examine were as follow: arterial blood gas; the mean pulmonary artery pressure;MMP-2,MMP-9, TIMP-1, and NE activity in lung tissue. Masson pigmentation of elasticity fibre was analyzed by computer image analyzer. Histopathological changes of lung tissue were observed under light microscope. The protein expression of MMP (MMP-2, MMP-9) and TIMP (TIMP-1) in lung tissue were determined by immunocytochemistry.RESULTS: Decompensate respiratory acidosis (pH=7.20±0.04, PaCO2=7.84±0.15) developed in group B. The mean pulmonary artery pressure were similar between groups B and A (P>0.05). Tissue edema in the lung, endothelial cell damage of the small blood vessels, pulmonary micro thrombus formations and increased pulmonary capillary permeability were observed in group B. NE activity increased significantly (P<0.01). However, no significant change of MMP-2, MMP-9, TIMP-1 activity was found in group B and group A (P>0.05). There was significant decrease in the relative content of elasticity fibre in lung tissue in group B compared to group A (P<0.01). The expression of MMP-2 protein in the lung tissue of group B was lower than that in group A (P<0.01), but the expression of both MMP-9 and TIMP-1 proteins in the lung tissue in group B were higher than those in group A (P<0.01).CONCLUSION: Hypercapnia rat model is successfully reproduced by exposure of animals to the mix gas exposure (6% CO2, 21% O2, and 72% N2). The pulmonary artery pressure is not affected by hypercapnia. High concentration of CO2 causes increase of NE activity and decrease in the relative content of elasticity fibre. High concentration of CO2 causes the increase of MMP-2 protein expression and decrease in the MMP-9 and TIMP-1 protein expression.     

Effects of pirenzepine on expression of MMP-2 and TIMP-2 in stroma of sclera in chick myopic eyes

AIM:To observe the effect of intravitreal injection of pirenzepine on form-deprivation myopia in chicks. METHODS:Forty-one day old chicks were randomly divided into 4 groups:normal control group, form deprivation group, vehicle control group and pirenzepine injection group. The right eyes of all chicks were used as experimental eyes. The deprived eyes in vehicle control group and pirenzepine injection group received daily intravitreal injection of vehicle control solution(0.01 mol/L PBS) and pirenzepine(1%in PBS), respectively. Optical examinations such as refraction, axial length and equatorial diameter were made at the end of the 5th day. The mRNA and protein levels of matrix metalloproteinase 2(MMP-2) and tissue inhibitor of metalloproteinase-2(TIMP-2), and the activity of MMP-2 were detected by RT-PCR, Western blotting and zymography analysis,respectively. RESULTS:Refraction, axial length and equatorial diameter of the eyes in pirenzepine injection group were significantly lower than those in form deprivation group and vehicle control group(P<0.05), but those were higher(P<0.05) and the eyes were relatively myopic as compared with normal control group. The mRNA expression, protein le vels and activity of MMP-2 in pirenzepine group were significantly higher than those in normal control group(P<0.01), and were significantly lower than those in form deprivation group and vehicle control group(P<0.01, P<0.01). The mRNA and protein levels of TIMP-2 in pirenzepine group were significantly lower than those in normal control group(P<0.01), and was significantly higher than those in form deprivation group and vehicle control group(P<0.01, P<0.01). CONCLUSION:Intravitreal injection of pirenzepine may partly prevent form-deprivation myopia by modulating the expression of MMP-2 and TIMP-2 in the fibrous layer of sclera.