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.     

Nrf2 regulates the expression of γ-glutamylcysteine synthetase in the inflammatory cells of bronchial asthma guinea pig

AIM:To investigate Nrf2 that regulates the expression of γ-glutamylcysteine synthetase (γ-GCS) in the inflammatory cells in bronchial asthma guinea pig bronchoalveolar lavage fluid (BALF). METHODS:Adult male guinea pigs were randomly divided into control group (group A), asthmatic group (group B) and dexamethasone group (group C). Asthmatic model was established by the method of ovalbumin challenge. MDA concentration in the lung tissue homogenate was detected. The total cell count and the proportion of inflammatory cells in BALF were measured. The methods of immunohistochemistry and in situ hybridization were used for detection of the protein and mRNA expressions of Nrf2, Bach1 and γ-GCS. RESULTS:(1) The proportion of eosinophils (EOS) in BALF and the MDA concentration of the lung tissue in group B were higher than those in group A and group C. (2) The result of in situ hybridization indicated that the A value of γ-GCS was the highest in group A compared to group B and group C, but the A value of Nrf2 and Bach1 in 3 groups has no statistical significance. (3) Immunohistochemistry indicated that the A value of γ-GCS in group B was lower than that in group A. The positive rate in cell nucleus of Nrf2 in group B was lower than that in group A and group C. The positive rate in cell nucleus of Bach1 in group B was higher than that in group A and group C. (4) The mRNA expression of γ-GCS (A value) showed positive correlation with the positive rate in cell nucleus of Nrf2 and negative correlation with the positive rate in cell nucleus. A negative correlation between the proportion of EOS in BALF of group B and the γ-GCS mRNA was observed. CONCLUSION:There is disequilibrium between oxidation and anti-oxidation in bronchial asthma guinea pig. Inflammatory reaction decreases the expression of γ-GCS in the inflammatory cells in bronchial asthma guinea pig. Dexamethasone regulates the nuclear translocation of Nrf2/Bach1 and increases the expression of γ-GCS.     

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.     

Role of calcineurin in airway remodeling in guinea pig model of asthma

AIM:To investigate the role of calcineurin (CaN) in airway remodeling in guinea pig model of asthma.METHODS:Male guinea pigs were randomly divided into three groups: control, asthma group and CsA group. The following parameters were measured: 1. The protein content, cell count and differential count of BALF; 2. The amount of [³H]-TdR incorporation into central airway smooth muscle; 3. The mean thickness of airway wall and airway smooth muscle of small airwaysl; 4.CaN activity of trachea and lung tissue.RESULTS:1. The protein content, cell count and eosinophil of BALF in CsA group were 46%, 51% and 60% lower than those in asthma group, respectively (P＜0.01); 2. [³H]-TdR incorporation in CsA group was 22% lower than that in asthma group (P＜0.05);3. The mean thickness of airway wall and airway smooth muscle were 34% and 37% less in CsA group than those in asthma group, respectively (P＜0.01); 4. CaN activity of lung tissue and trachea were 52% and 44% lower in CsA group than those in asthma group, respectively (P＜0.01).CONCLUSION:CsA reduced airway remodeling in guinea pig model of asthma, indicating the role of CaN in the airway remodeling.     

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.     

Effects of mitogen activated protein kinase on γ-glutamylcysteine synthase in lung of guinea pigs with bronchial asthma

AIM: To investigate the effects of mitogen activated protein kinase on γ-glutamylcysteine synthase (γ-GCS) in lung of guinea pigs with bronchial asthma.METHODS: Twenty adult male guinea pigs were divided into asthmatic group and control group (10 in each group).Asthmatic model was established by ovalbumin intraperitoneal injection combined with inhalation.The numbers of total and inflammation cells in bronchoalveolar lavage fluid (BALF) were measured.The γ-GCS-h mRNA in lung tissue was examined by in situ hybridization and RT-PCR.Immunohistochemistry was used to detecte the expression of γ-GCS,phosphorylated extracellular signal regulated kinase (p-ERK),phosphrylated c-Jun amino terminal kinase (p-JNK) and phosphorylated p38 (p-p38) in lung tissues.Western blotting was conducted to determine the expressions of p-ERK,p-JNK and p-p38 in lung tissue.The activity of γ-GCS was measured by coupled enzyme assay.RESULTS: (1) The total cell number and number of eosinophils in BALF of asthmatic group were significantly higher than those in control group (P<0.01).(2) Immunohistochemistry indicated that the p-ERK,p-p38,p-JNK and γ-GCS were stronger expressed in asthmatic group than those in control group (P<0.01).Western blotting also discovered that the expressions of p-ERK,p-JNK and p-p38 in lung tissue of asthmatic group were stronger than those in control group.(3) Both in situ hybridization and RT-PCR analysis showed that the expression of γ-GCS-h mRNA was more positive in asthmatic group compared with control group (P <0.01).(4) The activity of γ-GCS of asthmatic group was significantly higher than that in control group (P<0.01).(5) Linear correlation analysis indicated that in lung tissue of guinea pig with asthma,p-ERK and p-p38 markedly positive correlated with γ-GCS-h mRAN and γ-GCS protein.No relationship between p-JNK and γ-GCS-h mRAN,γ-GCS protein was observed.CONCLUSION: The expressions of p-ERK,p-p38,p-JNK and γ-GCS increase in lung of guinea pigs with bronchial asthma.p-ERK and p-p38 may positively regulate the expression of γ-GCS.     

Expression of PPARγ, Nrf2 and γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid of guinea pigs with bronchial asthma

AIM: To investigate the expression of PPARγ and Nrf2/γ-GCS-h in inflammatory cells in bronchoalveolar lavage fluid(BALF) of guinea pig with bronchial asthma of acute episode, and to explore the roles of PPARγ on Nrf2/γ-GCS-h expression. METHODS: Forty adult male guinea pigs were randomly divided into 4 groups (10 guinea pigs in each group): control group (group A), asthmatic group (group B), dexamethasone treatment group (group C) and rogridone treatment group (group D). The asthmatic model was established by an ovalbumin challenge method. BALF was collected, and the total cell count and the proportion of the inflammatory cells were measured. After centrifugation, the concentrations of ROS and MDA in the clear supernatant were detected. The methods of in situ hybridization and immunohistochemistry were used for detecting the expression of PPARγ and Nrf2/γ-GCS-h at mRNA and protein levels. RESULTS: The proportion of eosinophils (EOS) in BALF in group B was significantly higher than that in groups A, C and D (P<0.01). The concentrations of ROS and MDA in BALF of group B was the highest. The difference of ROS and MDA was statistically significant (all P<0.05) as compared to the control. The results of immunohistochemistry and in situ hybridization indicated that the A value was the lowest in group B as compared to that in groups A, C and D (all P<0.01). In group B, the positive correlations were observed between PPARγ and Nrf2/γ-GCS-h, between γ-GCS-h and Nrf2. A negative correlation between the proportion of EOS in BALF and the expression of PPARγ and Nrf2/γ-GCS-h was also observed (all P<0.05). CONCLUSION: In acute asthmatic models induced by ovalbumin, the expression of PPARγ and Nrf2/γ-GCS-h is decreased, and PPARγ may up-regulate the expression of Nrf2/γ-GCS-h to inhibit the inflammatory and oxidative reactions, indicating a new way for prevention and treatment of bronchial 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.     

Expression of nuclear factor-κB in asthmatic guinea pigs and the effect of erigeron breviscapus on it

AIM:To explore the expression of nuclear factor-κB(NF-κB)in asthmatic guinea pigs,and the effect of erigeron breviscapus,a protein kinase C(PKC)inhibitor,on the expression of nuclear factor-κB(NF-κB).METHODS:48 guinea pigs were randomly divided into 6 groups(n=8).Airway resistance and eosinophilic inflammation of airway wall were examined,the expression of NF-κB in the lung tissue was detected by immunohistochemical staining.RESULTS:The expression of NF-κB was mainly found in airway epithelium,all the asthmatic animals showed significantly higher optical densities than that of the normal control group(P＜0.01),and the rats subjected therapeutic treatment for two weeks showed significantly lower NF-κB expression than those of the asthmatic groups(P＜0.01).Positive correlation exist between the airway resistance and the percentage of cells expressing NF-κB in epithelium,and between the amount of eosinophil in airway wall and the percentage of cells expressing NF-κB in epithelium(P＜0101).CONCLUSION:The increased expression of NF-κB in airway epithelium of the asthmatic guinea pigs suggested that NF-κB may be involved in asthma.And result that the increased expression of NF-κB was inhibited significantly by the treatment of the erigeron breviscapus suggested that PKC may play a significant role in the pathogenesis of asthma through NF-κB.     

Effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs

AIM: To study the effects of M. vaccae on eosinophil apoptosis and Bcl-2 protein expression in lung tissues of asthmatic guinea pigs. METHODS: 30 guinea pigs were divided into normal saline (NS) group, asthma group and M. vaccae treatment group at random, every group included 10 guinea pigs. Guinea pigs in M. vaccae treatment group were injected intramuscularly with 22.5 μg M. vaccae 10 days before OVA immunization. TdT-mediated dUTP nick end labeling (TUNEL) technique was used to investigate the apoptosis of eosinophils and immunohistochemistry method was used to study the expression of Bcl-2 protein in lung tissues. RESULTS: The apoptosis index (AI) of eosinophils in lung tissues in M. vaccae treatment group was significant higher than that in asthma group ［(23.78±5.42)% vs (4.56±0.68)%, P<0.01］. The mean optical density value of Bcl-2 protein in lung tissues of M. vaccae treatment group was significant lower than that of asthma group ［(1 556.3±492.4) vs (2 321.9±751.2), P<0.05］. CONCLUSION: The apoptosis of eosinophils induced by M. vaccae in lung tissues of asthmatic guinea pigs may be due to the inhibition of Bcl-2 protein expression.     

Study on the damage of auditory path of the experimental autoimmune encephalomyelitis

AIM: To explore if there is lesion in the auditory path in the brainstem of the Wistar rat with experimental autoimmune encephalomyelitis（EAE）.METHODS: EAE was induced by guinea pig spinal cord homogenate (GPSCH),then the evoked potential in both EAE and control groups was examined.The HE staining and myelin staining of the auditory nucleus were conducted in order to corroborate the damage of the auditory path in the brainstem.Through studying the pathological changes using light microscopy and behavior changes of rats,we defined that the model was successful.RESULTS: The latency period of auditory evoked potentials of EAE rats was much longer than that in control group.The pathological pictures manifested that auditory nucleus were invaded by a great number of lymphocytes,demyelinations were discovered in it.CONCLUSION: The auditory path in the brainstem of the Wistar rat with EAE is suffered from inflammation and demyelination.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Security assessment of complex peptide vaccine VBP3 for anti-tumor angiogenesis: analysis of local irritation and systemic anaphylaxis

AIM: To evaluate the security of an anti-tumor angiogenesis complex peptide vaccine VBP3.METHODS: Three rabbits were tested for local irritation by local subcutaneous injection of the vaccine with a self comparison method on the left/right sides of the same body. The test of systemic anaphylaxis was performed in 28 guinea pigs, in which the animals were divided into 4 groups: normal saline (NS) group, bovine serum albumin (BSA) group, low-dose vaccine group and high-dose vaccine group. The other 4 guinea pigs, 2 received BSA and 2 received vaccine at a high dose, were activated directly without sensitization.RESULTS: No irritable and allergic reaction was observed in the test of all rabbits with local subcutaneous injection and most of the guinea pigs with systemic anaphylactic test. Only one guinea pig with slightly allergic response was found and spontaneously recovered soon.CONCLUSION: The complex peptide vaccine VBP3 for anti-tumor angiogenesis is safe under the experimental conditions.     

Effects of PPARγ agonist on calpain expression in model of acute experimental autoimmune encephalomyelitis

AIM: To explore the effects of peroxisome proliferator-activated receptor γ （PPARγ） agonist on calcium-activated neutral proteinase, calpain, expression in the brain of rats with acute experimental autoimmune encephalomyelitis (EAE). METHODS: EAE model was established in rats by inoculating the homogenate contained spinal cord of guinea pig and complete Freunds adjuvant. Respectively, the PPARγ agonist rosiglitazone and non-steroid anti-inflammatory drug (NSAID) ibuprofen were used to treat the EAE rats. Outcome measures (Kohs scale) were applied at baseline and after treatment to assess the improvement of clinical symptoms. Calpain expression levels were detected by RT-PCR and Western blotting. RESULTS: All the groups showed significant improvements of scales scores after treatment with rosiglitazone and ibuprofen. No significant difference of the expression of calpain mRNA was found among EAE group, rosiglitazone and ibuprofen groups (P>0.05), but the expression of calpain reduced markedly in rosiglitazone and ibuprofen groups compared with that in EAE group (P<0.05). CONCLUSION: Rosiglitazone and ibuprofen inhibit the expression of calpain and improve the clinical symptoms of EAE rats. PPARγ agonist plays a neuroprotective role in EAE rats.     

Determination of T cell cycle and the expression of bcl- 2 in asthmatic mice and its significance

AIM: To investigate the changes of T cell cycle, the expression of bcl- 2 in allergic asthmatic mice and the effects of dexamethasone on them. METHODS: An animal model with asthma was established by means of ovalbumin sensitizing-challenging. CD3 expression in spleen and lymphocytes in bronchoalveolar lavage fluid (BALF), T cell cycle and Bcl-2 expression in spleen were detected by flow cytometry. RESULTS: In BALF lymphocytes and spleen lymphocytes, CD3 expression rate in the asthmatic group was significantly higher than that of control group. In BALF lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly lower than that of the asthmatic group. However, in spleen lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly higher than that of the asthmatic group. In spleen lymphocytes, the cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmatic group were significantly higher than that from the control group. Cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmaplus dexamethasone group was significantly lower than that from the asthmatic group. The Bcl-2 expression rate of T cell from the asthmatic group was significantly higher than that from the control group. CONCLUSIONS: In the allergic asthmatic mice model, T cell count, proliferation and activation of T cells, apoptosis rate of T cells in spleen lymphocytes increase, meanwhile bcl- 2 expression also increases significantly. There was no significant effect of dexamethasone on the bcl- 2 expression. The therapeutic effects of dexamethasone on asthma may be not due to the inhibition of the bcl- 2 expression in T cells.     

Effect of poly(ADP-ribose) polymerase on the response of guinea pig tracheas induced by peroxynitrite in vitro

AIM and METHODS: to elucidater the effect of poly(ADP-ribose) polymerase(PARP) on tracheal hyperreactivity of guinea - pig induced by peroxynitrite, the responses of guinea pig tracheas to histamine af- ter incubation with peroxynitrite in the absence and presence of 3 - aminobenzamide(3 - AB), a highly selective inhibitor for PARP, were observed in vitro. RESULTS: The exposure of tracheal strips to peroxynitrite led to epithelial damage and hyperreacitivity to histamine, both of which were reversed by 3 - AB(lmmol/L or 5mmol/L), whereas incubation of tracheal strips with 3 - AB(5mmol/L) had no effect on the reponses. CONCLUSION: PARP is involved in the epithelial damage and hyperreactivity of guinea - pig tracheas induced by peroxynitrite. The results suggested that inhibition of excessive activation of PARP may represent a novel strategy for the prevention and therapy of airway hyperreactivity in asthma.