Effect of BCG on experimental asthma in a guinea pigmodel of allergen sensitization

AIM: To examine the effect of bacillus calmette-guerin (BCG) on experimental asthma in guinea pigs. METHODS: Guinea pigs were sensitized with BCG and then with ovalbumin (ip). Two weeks later, guinea pigs were challenged with ovalbumin (OVA) aerosol inhalation. Thirty one Guinea pigs were divided into three groups at random control group,OVA-treated group, BCG and OVA-treated group.RESULTS: Ovalbumin inhalation caused a marked airway infiltration of eosinophils and all the animals exhibit asthmatic symptoms. Pretreatment with BCG induced typical increase in lymphocytes and monocytes in peripheral blood and in bronchoalveolar lavage fluid (BALF). BCG markedly inhibited eosinophil infiltration and attenuated the asthmatic symptoms. CONCLUSION: These data suggest that BCG exerts an inhibitory effect on asthmatic inflammation.     

Changes of K+ channels of outer hair cells in guinea pig cochlea with streptomycin ototoxicity

AIM:To study the changes of K⁺ channels of outer hair cells in guinea pig cochlea with streptomycin ototoxicity. METHODS:Auditory brainstem responses (ABR) and whole-cell patch clamp techniques were used.RESULTS:(1) The body weight of guinea pigs with streptomycin ototoxicity decreased significantly; (2) The ABR threshold markedly increased in streptomycin group (Ⅱ,Ⅲ);(3)The number of dissociated outer hair cells of guinea pigs (Ⅱ,Ⅲ) was lower than that of control (Ⅰ); (4) Streptomycin decreased the Ca²⁺-sensitive K⁺ currents and delayed outward K⁺ currents distinctly; (5) There was no significant difference of K⁺ currents between Ⅰ and Ⅱ/Ⅲ. CONCLUSION:These results suggest that the inhibition of K⁺ channels is the basis of streptomycin ototoxicity, but not the direct reason for cell death.     

Histamine receptor antagonist prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig

AIM:To investigate the effect of histamine receptor antagonist on airway remodeling and acid-base imbalance in asthma of guinea pig. METHODS:Guinea pigs were divided into 5 groups: the normal control group, the asthma model group, the continued asthma model group, histamine group and histamine receptor antagonist group. For each group, the content of histamine, Na+, Cl-, PaO2, PaCO2, pH, AB, SB in serum, and thickness of airway mucosa and smooth muscle cell layer were measured and compared with each other. RESULTS:(1) According to the content of histamine in serum and thickness of airway mucosa and smooth muscle, the order was: the histamine group>continued asthma model group>the asthma model group>the normal control group (P<0.01), and the histamine receptor antagonist groupthe continued asthma model group (P<0.01), but for PaCO2, the order was conversed. Airway remodeling, increase in histamine in serum, respiratory acidosis and metabolic acidosis in asthmatic guinea pig were observed. Exogenous histamine accentuated the change, however, histamine receptor antagonist attenuated it. CONCLUSION:Histamine may take part in the airway remodeling of asthma. Histamine receptor antagonist can prevent and ameliorate airway remodeling and acid-base imbalance in asthma of guinea pig.     

Upregulation of neurokinin A levels by nerve growth factor in the experimental asthmatic guinea pig

AIM: To explore the regulatory mechanism of nerve growth factor (NGF) on neurokinin A in the experimental asthmatic guinea pig. METHODS: Radioimmunoassay was used to determine the alteration of neurokinin A levels in the lower respiratory tract and visceral sensory afferent sites while NGF was absent (inhalation of NGF antibody through nasal cavity) in the asthmatic guinea pig. RESULTS: The contents of neurokinin A in the trachea, bronchus, lung, C₇-T₅ spinal ganglia and the correspondent spinal dorsal horn, nodose ganglia and solitary nucleus area in the experimental asthmatic guinea pig with the absent of NGF in the respiratory tract were much lower than those in the asthmatic and control groups (P＜0.01). CONCLUSION: NGF upregulated the contents of neurokinin A in the lower respiratory tract and visceral sensory sites of the experimental asthmatic guinea pig, and both might be involved in the pathogenesis of asthma.     

Effects of hypoxia inhalation therapy on the number of eosinophil and CD4+T-lymphocyte in remission stage in asthmatic guinea pigs

AIM:To explore the mechanism underlying the therapeutic effects of hypoxia inhalation on asthma. METHODS:Guinea pigs were randomized into the normal group(NG), asthmatic group(AG) and the hypoxia inhalation-treated group(HITG). The model of asthma was established in the latter two groups through sensitization and induction with 10% ovalbumin(OA) and 1% OA, respectively. The animals in HITG were treated with hypoxia inhalation (13.0%±0.5% O₂/N₂ mixed gas). The content of serum cortisol, the number of eosinophils(EOS) and percentage of hypodense eosinophils(HEOS) in bronchoalveolar lavage fluid(BALF),the number of CD₄⁺T-lymphocyte in peripheral blood(PB) and the tension of airway muscle were determined. RESULTS:(1)The content of serum cortisol was significantly higher in NG and HITG than in AG(P＜0.01); (2)The number of EOS and percentage of HEOS in BALF was significantly lower in HITG than in AG(P＜0.01); (3) The number of CD₄⁺T-lymphocyte in PB was significantly higher in AG than in HITG(P＜0.01).CONCLUSION:After treatment with hypoxia inhalation, the content of serum cortisol in asthmatic guinea pigs was significantly increased to result in marked decreased of the number of EOS, the percentage of HEOS in BALF, and the number of CD₄⁺T-lymphocyte in PB, thus result in the tension of airway muscle and alleviation of the airway hyperresponsiveness. All these may be beneficial to preventing the relapse of asthma.     

Effect of airway remodeling on airway responsiveness in asthmatic guinea pigs

AIM: To establish a guinea pig asthma model and to evaluate the effect of airway remodeling on airway responsiveness. METHODS: The guinea pig asthma model was established by ovalbumin (OVA) sensitization and challenge repeatedly. Bronchial provocation tests were conducted through intravenous injection of acetylcholine. The airway morphologic parameters were measured by computer image analysis system. White blood cells and the differential count in bronchoalveolar lavage fluid (BALF) were examined. RESULTS: The resistance of airway was increased significantly after 4 weeks of OVA exposure, but the increase disappeared upon prolonged exposure. After 8 weeks of OVA exposure, fiber tissue in large airway was increased, and the thickness of smooth muscle layer of small airway was enlarged, as compared with that in control animals. CONCLUSION: Airway responsiveness has changed after prolonged OVA exposure in guinea pigs. This change is related to airway remodeling.     

Role of transforming growth factor-β in the airway inflammation and remodeling in asthma

Transforming growth factor-β (TGF-β) was reported to be increased in asthma in some studies. Accumulation of TGF-β in airway promotes smooth muscle cell mitogenesis and hyperplasia, and induces fibroblast and myofibroblast and smooth muscle proliferation as well as increase in protein synthesis in connective tissue (such as collagen deposition on the reticular basement membrane). The autocrine induction of collagen expression by smooth muscle may contribute to the thickening of the reticular basement membrane, irreversible fibrosis and remodeling seen in the airways in some asthmatics. TGF-β is considered to be a major fibrogenic cytokine. It can increase smooth muscle mass and lead to severe bronchial obstruction in an asthma attack.     

Effects of electrolyzed free radicals on endothelium of isolated guinea pig coronary and epithelium of airway tube

AIM and METHODS:To study the damage effects of free radicals from electrolyzed krebs solution(direct current,10 mA,1-2 min) on isolated guinea pig coronary and airway tube. RESULTS:In Langendorff’s perfused guinea pig hearts,the electrolyzed free radicals increased coronary perfusion pressure(4.4±1.2) kPa,inhibited myocyte contractility [(0.8±0.8) g vs (2 9±0 6) g, P< 0.05],increased TBARS level and decreased SOD activity.In isolated perfused lungs of guinia pig,electrolyzed Krebs solution promoted significantly the airway perfusion pressure [(0.03±0.01) kPa vs (2.20±0.29) kPa, P< 0.01] and histamine reactivity [(0.65±0.37) kPa vs (2.05±0.25) kPa, P< 0.01]. Hydroxyl radicals scavenger DMSO and natural medicine gypenosides prevented the effects of oxygen free radicals. CONCLUSION: These results indicated that the free radicals by electrolyzation could induce damages of coronary endothelium and airway epithelium.     

Up-regulatory effect of nerve growth factor on substance P in the experimental asthma

AIM: To explore the regulatory mechanism of nerve growth factor (NGF) on substance P in the experimental asthmatic guinea pig. METHODS: Radioimmunoassay was used to determine the alteration of substance P levels in the lower respiratory tract and visceral sensory afferent sites while NGF was absent (inhalation of NGF antibody through nasal cavity) in the asthmatic guinea pig. RESULTS: The contents of substance P in the trachea, bronchus, lung, C7-T5 spinal ganglia and the correspondent spinal dorsal horn, nodose ganglia and solitary nucleus area in the experimental asthmatic guinea pig with the absent of NGF in the respiratory tract, were much lower than those in the asthmatic and control groups (P<0.01). CONCLUSION: NGF upregulates the contents of substance P in the lower respiratory tract and visceral sensory sites of the experimental asthmatic guinea pig, which might be involved in the pathogenesis of asthma.     

Role of GATA-3 in the pathogenesis of airway inflammation in a rat asthma model

AIM: To investigate the role of GATA-3 in the pathogenesis of airway inflammation in a Wistar rat asthma model. METHODS: The Wistar rat asthma model was made with conventional method and animals were divided into five groups (10 rats in each group): asthma group (A group), dexamethasone group (D group), antisense oligonucleotide group (AS group), nonsense oligonucleotide group (NS group) and normal control group (N group). Antisense, nonsense oligonucleotide were administered intranasally, and the dexamethasone was injected intraperitoneally. The airway inflammation was observed with HE staining method. The GATA-3 positive cells were stained immunohistochemically. The GATA-3 mRNA expression in pulmonary tissue was investigated with RT-PCR. The GATA-3 protein in pulmonary tissue was detected by Western blotting. RESULTS: In contrast to N group, the expression of GATA-3 mRNA, protein and the amount of inflammatory cells in pulmonary tissue in group A were increased significantly (P<0.01) and were decreased evidently in group AS and D (P<0.01). The expression of GATA-3 mRNA, protein and the amount of inflammatory cells in NS group were obviously increased compared with those in gropu AS and D (P<0.01). The expression of GATA-3 was related to the amount of eosinophils (r=0.995). CONCLUSION: GATA-3 antisense oligonucleotide blocks the expression of GATA-3 gene and the infiltration of eosinophils. GATA-3 plays an important role in the effector phase of allergic airway inflammation in a Wistar rat asthma model.     

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.     

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling

AIM: To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS: The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured. The protein expression of β-catenin, glycogen synthase kinase-3β (GSK-3β), c-Myc and cyclin D1 in the ASMC was determined by Western blot. After depressing the interaction between β-catenin and p300/CBP, the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry. Meanwhile, the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS: The protein levels of β-catenin, c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P<0.05). After depressing the interaction between β-catenin and p300/CBP, the cell activity of ASMC was decreased in asthma group compared with control group (P<0.05), and the change of the cell cycle distribution in asthma group was also more obvious (P<0.05). After inhibiting P38 MAPK activity, the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P<0.05).CONCLUSION: Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1, reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.     

Effects of 5-HT and electrolytes on the airway remodeling of bronchial asthma in guinea pigs

AIM: To explore the effects of 5-HT and electrolytes on the airway remodeling in guinea pigs with bronchial asthma.METHODS: 70 guinea pigs were divided into 7 groups: control group, model group, continued model group, 5-HT group, anti-5-HT group, high Mg2+ group, low Mg2+ group.Remodeling model was established with ovalbumin.RESULTS: ① In model group, 5-HT of serum and thickness of airway walls were significantly increased compared with control group (P<0.05,P<0.01).② In continued model group, 5-HT and thickness of airway walls were significantly increased compared with model group (P<0.05, P<0.01).③ In 5-HT group, thicknesses of airway walls were significantly increased compared with continued model group (P<0.05).④ In anti-5-HT group, thicknesses of airway walls were significantly decreased compared with continued model group (P<0.05).⑤ In high Mg2+ group, thicknesses of airway walls were significantly decreased compared with continued model group (P<0.05).⑥ In low Mg2+ group, thickness of airway smooth muscle was significantly increased compared with continued model group (P<0.05).⑦ With the aggravation or alleviation of airway remodeling, concentration of Ca2+ in serum was upward or downward.However, concentration of PO3-4 was downward or upward.⑧ Veriety of Na+, K+, Cl- was no significant difference among groups.⑨ Concentration of 5-HT in serum was corrected with that of Ca2+.CONCLUSIONS: ① 5-HT mediates airway remodeling of asthma in guinea pigs whereas Mg2+ may play a regulatory role.② Increase in Ca2+ and decrease in PO3+4 may promote the airway remodeling of asthma whereas Na+, K+, Cl- may not play any role.③ Concentration of 5-HT in serum was corrected with that of Ca2+ in airway remodeling of asthma.     

Changes of intracellular potassium activity and membrane potential in guinea pig ventricular myocardium during superfusion with low sodium solutions and effects of low pH

AIM:To investigate the changes in intracellular potassium activity(aⁱK) and membrane potential(V_m) induced by low external sodium infusion (Low [Na]_o) and to detect the mechanisms involved and the relationship between aⁱK and V_m. METHODS:aⁱK and V_m were measured in infusion with different sodium concentrations using methods of convenient and ion selective microelectrodes in guinea pig ventricular myocardium. RESULTS:Low [Na]_o resulted in a decrease in aⁱK and an increase in V_m in a Na⁺ concentration-dependent manner.At the same time,contraction and resting potential increased, and action potential duration decreased markedly,but action potential amplitude was not affected. A change of the pH from 7.4 to 7.0 in low [Na]_o solution reduced the decrease in aⁱK, but did not affect the increase in V_m.CONCLUSION:A better linear relationship appeared between the changes in aⁱk and [Na]_o or in V_m and [Na]_o,while during each low [Na]_o the change in both aⁱK or V_m may reach a new balance level.     

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.     

Therapeutic effects of hypobaric hypoxia on asthma in guinea pigs

AIM:To explore the mechanismof the therapeutic effect of hypobaric hypoxia on allergic asthmas guinea pigs.METHODS:After the model of asthma was established with oval bumin(OA)challenge i n OA sensitized guinea pigs,the ani mals were randomized i nto the asthmas group(AG),the alleviative group(ALG)and hypobaric hypoxiatreated group(HHTG).The levels of plasma cortisol and endothelin(ET),ET in bronchoalveolar lavage fl ui d(BALF)were deter mi ned by radioi mmunoassay.The pul monary pathological changes were observed with optical microscope.RESULTS:(1)Infiltration of eosinophils(EOS)in alveolar septumwas found in AGand ALG,while it was re-duced in HHTG.(2)The level of plasma cortisol was significantly higher in AGthanin normal control group(NCG)(P<0.01).But it was significantly lower in ALGthani n NCG(P<0.01),there was no difference in plasma cortisol level between HHTG and NCG.(3)The content of ET in plasma was significantly higher in ALGthanthat i n NCG,AG and HHTG(P<0.01),and ET level in BALF was significantly higher in AG than that in NCG,ALG and HHTG(P<0.05),however,no significant difference was found among the latter three groups, respectively.CONCLUSION:After the treatment with hypobaric hypoxia,the ET levels of the plasma and BALF were decreaced and the content of plasma cortisol was increaced,and infiltration of EOS in alveolar septum was decreaced in asthmatic gui nea pigs.That may be one of the mechanisms by which hypobavic hypoxia prevents and cures asthma.     

The relationship between Proto-oncogenes expression and airway inflammatory cell infiltration in asthma

AIM:To investigate the relationship between inflammatory cell infiltration and proto-oncogenes expression in asthma. METHODS:Guinea pigs were used as asthma models challenged by ovoglobulin. Dot-blot, Northern-blot and immunochemical techniques were used to detect the expression of c-fos, c-myc, c-jun and c-sis. Inflammatory cell infiltration was showed by pathologic study.RESULTS:c-fos and c-myc mRNA could not be detected or expressed at very low level in control group. Those were greatly increased after the animals are challenged by ovoglobulin. Immunochemical study showed that Fos, Myc, Jun and Sis expressed at low level in control group, and those were increased after the challenge. There was little inflammatory cell infiltration in control group. Lymphocyte, neutrophil and eosinophil were detected immediately after the challenge, a great number of inflammation cells could be seen after 12-24 h of the challenge. Majority of neutrophil and eosinophil were under mucosa or in epithelium in airway. CONCLUSION:Oncogenes expression had strong relationship with airway inflammation.     

Expression of calcium handling protein in diastolic heart failure

AIM:To elucidate molecular mechanisms underlying calcium handling protein in diastolic heart failure (DHF) from mRNA level and protein expression, including calcium adenosine triphosphatase (Ca²⁺-ATPase), phospholamban, ryanodine receptor, calsequestrin and L-type calcium channel.METHODS:The mRNA of these calcium handling genes were detected by RT-PCR, and the protein levels were analyzed by Western blot analysis.RESULTS:Compared with sham-operated rabbits, the steady-state levels of mRNA encoding the SR Ca²⁺-ATPase and cardiac L-type calcium channel were decreased significantly in rabbits with DHF, and protein level of SR Ca²⁺-ATPase was greatly reduced, whereas the mRNA and protein levels of other calcium handling protein were unchanged.CONCLUSION:L-type calcium channel and the sarcoplasmic reticular Ca²⁺-ATPase were down regulated in DHF. These changes may be a contributory factor for DHF.     

Distribution and activity of calcineurin in different tissues of rat

AIM: To investigate the activity and distribution of calcineurin (CaN) in different tissues of rat. METHODS: Using western blot and immunohistochemical staining methods to measure the amount and location of CnAα, isoform of catalytic subunit of CaN in different tissues of rat. CaN activity was measured by [³²P] labelled substrate peptide. RESULTS: 1. Western blot showed that CnAα expression was detected in brain, heart, skeletal muscle and lung tissues. There was no detectable CnAα expression in kidney and aorta. 2. In immunohistochemical staining study, there was strong immunostaining of CnAα in brain. CnAα was located in cytoplasm of cardiac cell, macrophage and connective tissue of peribronchiolar in lung tissue, aorta adventitia, connective tissue around small vessels and outer wall of renal tube. 3. CaN activity was highest in brain, the following was skeletal muscle, myocardium and lung tissue. CaN activity was lowest in aorta and kidney tissue. CONCLUSION: CaN is widely distributed in rats and might be involved in functional regulation of various organs and tissues.