Effects of Aspergillus fumigatus extract on expression of Muc5ac in human bronchial epithelial cells

AIM: To investigate the effects of Aspergillus fumigatus extract (AFE) on the expressions of Muc5ac in human bronchial epithelial cells and its possible mechanism. METHODS: Human bronchial epithelial cells (16HBE-14o) were cultured in vitro, which were exposed to different concentrations of AFE (0, 8, 16, 20 mg/L) for different times. In order to explore the mechanisms, heat-treated AFE, serine protease inhibitors (aprotinin) and protease-activated receptor-2 (PAR-2) antagonist (FSLLRY-NH2) were used. The production and release of Muc5ac in different intervals were tested by immunohistochemistry and ELISA. The expression of Muc5ac mRNA was measured by RT-PCR. RESULTS: In normal control group, only a few Muc5ac was detected. In the experimental groups with AFE exposure, cells produced more Muc5ac compared to normal control group (P＜0.01), which were positively related to the exposure time or the concentration of AFE. Aprotinin and PAR-2 antagonist (FSLLRY-NH2) inhibited the effect of AFE on Muc5ac production by 16HBE-14o. Heat-treated AFE, which lost protease activities, exerted no effect on Muc5ac production and mRNA expression. CONCLUSION: AFE, depending on its protease activity, activates PAR-2 and causes airway epithelial cells to produce and release more Muc5ac, which may contribute to deterioration of asthma.     

Effects of Retinervus luffae fructus on mRNA expression of low-density lipoprotein receptor in hyperlipidemia mice

AIM: To observe the effects of Retinervus luffae fructus (RLF) on mRNA expression of low-density lipoprotein receptor (LDL-R) in hyperlipidemia mice. METHODS: Mice were fed with high fat diet to induce a hyperlipidemia model. By using xuezikang, a Chinese medicine, as a positive control, the effect of RLF on serum total cholesterol (TC) and level of low density lipoprotein cholesterol (LDL-C) in mice were observed. The liver total RNA was extracted by Trizol method. The LDL-R mRNA expression was determined by RT-PCR. RESULTS: (1) The levels of TC ［(5.71±0.82) mmol/L］ and LDL-C ［(3.99±1.12) mmol/L］ in hyperlipidemia (HPL) group were higher than those in control (P<0.01). The levels of TC ［(3.65±0.28) mmol/L］ and LDL-C ［(2.74±0.54) mmol/L］ in RLF treatment group, and the levels of TC ［(3.94±0.65) mmol/L］ and LDL-C ［(3.00±0.23) mmol/L］ in positive control (PC) group were lower than those in HPL group (P<0.01). (2) The level of hepatic LDL-R mRNA expression was lower in HPL group than that in control group (P<0.01). Compared to HPL group, significant increases in hepatic LDL-R mRNA expression in RLF treatment group and PC group (P<0.01) were observed. CONCLUSION: Retinervus Luffae Fructus exerts obviously lipid-lowering effect and enhances the hepatic LDL-R mRNA expression in experimental hyperlipidemia mice.     

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.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.     

Inhibitory effect of ground dragon on the expression of α-SMA and FN in the lung tissue of mouse with asthma

AIM: To investigate the inhibitory effect of ground dragon on the expression of α-SMA and FN in the lung tissue with asthma. METHODS: The BALB/c mice were divided into four groups: control group (group A, n=20), asthmatic model group (group B, n=20), large-dose ground dragon treatment group (group C, n=20) and low-dose ground dragon treatment group (group D, n=20). To establish a mouse model of chronic asthma, we sensitized the mouse with 0.02% ovalbumin (OVA) by intraperitoneal injection, and stimulated the mice with 1% OVA by atomization. The treatment groups were given ground dragon before stimulation every time. After the last time of stimulation, the mice were subjected to laboratory tests. Inflammatory cells in bronchoalveolar lavage fluid were counted. Total level of IgE in serum was detected by ELISA. FN mRNA and α-SMA mRNA in the lung tissue were measured by RT-PCR and AlphaImager 2200 semi-quantitation analysis system. Expressions of FN and α-SMA were measured by the method of two-step immunohistochemistry and leica QWIN V3 analysis system. RESULTS: (1) Compared with those in group A, the expressions of α-SMA and FN in group B were significantly increased (P<0.01). Compared with group B, those in group C were significantly decreased (P<0.01), while those in group D were slightly decreased (P>0.05). (2) Compared with those in group A, the expression levels of α-SMA mRNA and FN mRNA in group B had a great increase (P<0.01). There was a notably decreases of α-SMA mRNA and FN mRNA levels in group C, compared with group B (P<0.01). However, α-SMA and FN mRNA level in group D was just a slightly decreased, compared with group B (P>0.05). CONCLUSION: The ground dragon inhibits α-SMA and FN expression in the lung tissue of mice with chronic asthma, indicating that ground dragon may inhibit airway remodeling in asthma through the inhibition of α-SMA and FN expressions.     

Effect of atorvastatin on blood pressure in spontaneously hypertensive rats

AIM: To explore the mechanisms underlying the effect of atorvastatin on blood pressure in spontaneously hypertensive rats (SHR). METHODS: The effects of atorvastatin on plasma endothelin-1, aortic nitric oxide synthase, aortic smooth muscle cell (ASMC) apoptosis and p27 expression in SHR were evaluated. 12 eight-week-old SHR were randomized into atorvastatin group (ATV, n=6) and SHR group (n=6). 6 age-matched normotensive Wistar-Kyoto rats (WKY) were served as controls. 50 mg·kg-1·d-1 of atorvastatin was administered to ATV by gavage for 10 weeks. Serum cholesterol and triglycerides were measured, and systolic blood pressure of caudal artery was examined. Plasma endothelin-1 and nitric oxide synthase activity of aortic tissue were measured. ASMC apoptosis rate was detected by TUNEL technique, and positive expression rate of P27 in ASMC was analyzed. RESULTS: After 10 weeks, systolic blood pressure in ATV was significantly lower than that in SHR ［(134.17±3.60）mmHg vs （173.33±3.78）mmHg, P<0.01］. Compared with SHR, serum cholesterol and triglycerides were significantly lower (P<0.01, P<0.01) in ATV. Additionally，atorvastatin significantly decreased plasma endothelin-1 ［(130.04±40.07）ng/L vs （196.74±59.69）ng/L, P<0.05］ and increased nitric oxide synthase activity in aortic tissue ［(0.189±0.040）kU/g protein vs (0.124±0.057)kU/g protein, P<0.01］, compared with SHR. ASMC apoptosis rate was higher in ATV than that in SHR (16.94%±3.08% vs 9.01%±2.36%, P<0.01). Compared with WKY, positive expression rate of p27 in ASMC from ATV was higher (33.02%±5.01% vs 24.25%±4.41%, P<0.05), whereas that was lower in SHR (16.08%±7.09% vs 24.25%±4.41%, P<0.01). CONCLUSION: Atorvastatin may reduce the plasma endothelin-1， up-regulate nitric oxide synthase activity and ASMC P27 expression and facilitate ASMC apoptosis，which may effectively reduce blood pressure in SHR.     

Effects of Maxing-Shigan decoction on airway remodeling and expression of MMP-9 and TIMP-1 in lung tissues of asthmatic mice

AIM:To investigate the effects of Maxing-Shigan decoction on airway remodeling and expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the lung tissues of asthmatic mice, and to explore its possible mechanism in treatment of asthma. METHODS:The BALB/c mice were divided into blank control group, model group, low-dose Maxing-Shigan decoction group, middle-dose Maxing-Shigan decoction group, high-dose Maxing-Shigan decoction group and positive control group. The mice were sensitized and challenged with ovalbumin to establish asthma model. The mice in blank control group and model group were given saline by oral administration before 30 min of suscitation. The mice in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups were given Maxing-Shigan decoction at 5.0 g/kg, 10.0 g/kg and 20.0 g/kg, respectively, by oral administration before 30 min of suscitation. The mice in positive control group was given dexamethasone at 0.005 g/kg by oral administration before 30 min of suscitation. After consecutive administration for 7 d, the variations of airway responsiveness, the percentage of the goblet cells, the collagen deposition, and the eosinophil (EOS) counts in bronchoalveolar lavage fluid (BALF) of each group were observed. The protein levels of MMP-9 and TIMP-1 in the lung tissues were determined by ELISA and Western blot. The mRNA expression of MMP-9 and TIMP-1 was detected by RT-qPCR. RESULTS:Compared with blank control group, the airway responsiveness, the goblet cell percentage, the collagen deposition, the EOS counts in BALF, the protein levels of MMP-9 and TIMP-1, and the mRNA expression of MMP-9 and TIMP-1 were significantly increased in model group (P<0.01). Compared with model group, all of the indexes were reversed in low-dose, middle-dose and high-dose Maxing-Shigan decoction groups and positive control group (P<0.05 or P<0.01). CONCLUSION:Maxing-Shigan decoction improves airway remodeling in asthma model mice by down-regulating the expression of MMP-9 and TIMP-1.     

5-FU upregulates stem cell marker CD133 expression in colon cancer cells

AIM: To investigate the effect of 5-fluorouracil(5-FU)on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS: CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self-renew ability and MTS assay was used to detect the sensitivity to 5-FU after separation. After 5-FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20%, 82.00%, 0.34%, 91.80%, 85.30%, 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21%±5.33% and the negative purity was 84.30%±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay(46.33%±4.44% vs 31.00%±2.00%, P<0.05). CD133 positive cells were less sensitive to 5-FU compared to CD133 negative cells(20% less, P<0.01). 5-FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684±0.012(P<0.01)and 30.702±0.280 to 49.379±0.460(P<0.01)in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5-FU. 5-FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5-FU treatment.     

Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.     

Changes of calcium handling protein after acute myocardial infarction in rats and effect of carvedilol

AIM: To observe the changes of sarcoplasmic reticulum Ca2+-ATPase (SERCA)， phospholamban (PLB) during heart failure after acute myocardial infarction (AMI) in rats and the effect of carvedilol. METHODS: Rats were randomly assigned to normal control group, sham-operation group, AMI group and carvedilol (CAR) group. 6 weeks later, in vivo hemodynamic, morphometry and SERCA, PLB mRNA and protein expression of myocytes were measured in all animals. RESULTS: In comparison with sham-operation group, LV end diastolic pressure (LVEDP) and weight of ventricles were increased, while maximal rate of rise and fall (±dp/dt) of LV pressure were decreased in AMI group. After treatment with carvedilol, these parameters were all improved. The mRNA and protein expression of SERCA were downregulated (P<0.01). PLB mRNA and protein expression were upregulated (P<0.01) in AMI group relative to sham-operation group. Carvedilol restored the low expression of SERCA mRNA and protein (P<0.05), but was no effect on PLB mRNA and protein expression (P>0.05). CONCLUSIONS: The changes of SERCA and PLB may be the important mechanism of contractile dysfunction in heart failure after AMI. Carvedilol is effective in preventing LV dysfunction after AMI. The molecular mechanism may be related with normalization of SERCA expression.     

Premature response to luteinizing hormone of granulosa cells from women with polycystic ovary syndrome

AIM:To demonstrate the relationship between hormones in follicular fluid and the expression of LH receptor in granulosa cells (GC) in anovulatory women with polycystic ovary syndrome (PCOS). METHODS:Follicles were obtained from 12 women with PCOS and 15 women with normal menstrual period through surgery at time between day 7 and day 10 of menstrual cycle. The accumulations of estrogen (E2), progesterone (P), luteinizing hormone (LH), follicle stimulating hormone (FSH) and insulin in follicular fluid were determined by a automatism chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination. The accumulation of androstenediol (A) was determined by ELISA. The amounts of the mRNA expressions of LH receptors from GC and theca cells (TC) respectively were measured by RT-PCR using β-actin as intra-control simultaneously. RESULTS:The levels of LH ［(3.8±2.1 vs 1.7±0.8)IU/L, P<0.01］, A ［(600.0±373.4 vs 212.4±205.4)μg/L, P<0.05］ and expressions of LH receptor mRNA of GC (0.29±0.16 vs 0.12±0.13, P<0.01) and TC (0.46±0.14 vs 0.34±0.09，P<0.05) in the women of PCOS group were statically higher than those in control group. The expression of LH receptor mRNA was not detected by RT-PCR in control group when the diameter of an follicle was less than 7 mm, while it was detected in women with PCOS when it remained as small as 4 mm. Expression of LH receptor mRNA in granulose cells was positive related to the concentration of LH (r=0.67, P<0.01) and insulin (r=0.51, P<0.05) in follicular fluid, and that in theca cells (r=0.60, P<0.01). CONCLUSION:The high level of LH in follicular fluid occurs and GC responds to LH prematurely and more intensively in anovulatory women with PCOS. Larger amount of A and P was produced as a result. All of above may contribute to the mechanism of anovulatory.     

Expression and distribution of osteopontin in the development of pulmonary hypertension induced by hypoxia-hypercapnia

AIM: To study the expression and distribution of osteopontin (OPN) in lungs and pulmonary arteries in pulmonary hypertensive rats induced by hypoxia-hypercapnia, and to explore the role of OPN in pathogenesis of pulmonary hypertension. METHODS: Forty-eight male Sprague-Dawley rats (Weight 180 g-220 g) were randomly divided into four groups: normal control group (NC), hypoxic hypercapnia 1-week，2-week and 4-week group (1HH, 2HH and 4HH). The expressions of OPN mRNA and protein in lungs and pulmonary arteries were detected by RT-PCR and immunohistochemistry. ELISA was used to detect the concentration of OPN in lung homogenates. The content of OPN in pulmonary arteries was detected by Western blotting. RESULTS: ① The mean pulmonary artery pressure (mPAP) and weight ratio of right ventricle to left ventricle and septum ［RV/(LV+S)］ in all hypoxic hypercapniac groups were higher than those in normal control group (P<0.01), respectively. Differences of mean carotid artery pressure (mCAP) among these four groups were not significant (P>0.05). ② The expression of OPN mRNA was significantly increased in pulmonary arteries and lung tissues in hypoxic hypercapnic groups compared with normal control group (P<0.01). ③ The result of immunohistochemistry showed that OPN was only detected in bronchus and alveolar epithelium, but not detected in pulmonary arterioles of normal control group. In contrast，OPN expression was evident in pulmonary arterioles of 1HH rats，especially in media. Moreover, the expression of OPN was markedly increased in group 2HH and 4HH. ④ OPN levels in lung homogenates in 1HH, 2HH and 4HH were increased by 69%, 128% and 187% (P<0.01), respectively, compared with control rats. ⑤ Western blotting analysis showed that the contents of OPN were significantly higher in all hypoxic hypercapnic groups than those in NC group (P<0.01).CONCLUSION: The expressions of OPN in pulmonary arteioles and lung are increased in rats with pulmonary hypertension. OPN might play an important role in the pathogenesis of pulmonary hypertension induced by chronic hypoxia and hypercapnia.     

Angiotensin II-induced matrix metalloproteinase-9 expression mediated by NF-κB pathway in human THP-1 cells

AIM: The present study was undertaken to investigate the effect of angiotensin II (AngⅡ) on expression of MMP-9 in THP-1 macrophages. METHODS: Macrophages converted from THP-1 monocytes by incubating with PMA (0.1 μmol/L) for 48 h were divided into PMA group; PMA+AngⅡ group (10-7mol/L, 1 h); PMA+AngⅡ+PDTC group (10 μmol/L, 30 min) and PDTC group. Western blotting was used to detect the MMP-9 and phosphorylation of NF-κB p65, and the expression of MMP-9 mRNA in THP-1 macrophages was measured by RT-PCR.RESULTS: Compared to control group, the expression of MMP-9 (1.06±0.11, P<0.05) and phosphorylation of NF-κB p65 (1.02±0.10, P<0.05) in THP-1 macrophages were expressed when treated with AngⅡ (10-7mol/L); and the expression of MMP-9 mRNA were upregulated (1.22±0.08, P<0.05). However, NF-κB inhibitor PDTC reduced the NF-κB p65 (0.99±0.12, P<0.01) and MMP-9 (1.04±0.14, P<0.01) expressions and decreased the expression of MMP-9 mRNA (0.90±0.06，P<0.01). CONCLUSION: NF-κB signaling pathway contributes to the expression of MMP-9 in THP-1 macrophage induced by AngⅡ.     

Effects of panax notoginseng saponins on pneumocyte apoptosis and Fas/FasL expression in rabbits with lung ischemia/reperfusion injury

AIM: AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and observe effects of panax notoginseng saponins (PNS) on apoptosis in lung ischemia/reperfusion injury. METHODS: Single lung in situ ischemia/reperfusion animal model was used. Eighty four Japanese white rabbits were randomly divided into control group (control), ischemia/reperfusion 1 h group (IR1h), IR3h, IR5h, Panax Notoginseng Saponins 1 h group (PNS1h), PNS3h and PNS5h. TUNEL, immunocytochemistry and in situ hybridization techniques were used to observe apoptosis and Fas/FasL expression in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues were significantly high, Fas/FasL mRNA and its protein were up-regulated in lung tissues of lung ischemia/reperfusion injury compared with control (all of P<0.01). The PNS suppressed apoptosis as well as expression of Fas/FasL mRNA and its protein (P<0.05 or P<0.01, respectively). There was a significant correlation between expression of Fas/FasL protein, Fas/FasL mRNA and cell apoptosis (r=0.540，0.658，0.668，0.686；all P<0.01). CONCLUSIONS: Activation of Fas/FasL system and its initiating cell apoptosis of lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of PNS include suppressing the activation of Fas/FasL system and blocking apoptosis in lung tissues in lung ischemia/reperfusion injury.     

Expression of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3

AIM: To observe the dynamic variation of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3.METHODS: A total 72 inbred male BALB/c mice of 4-6 weeks were divided randomly into 2 groups as follows: 32 mice were inoculated intraperitoneally (ip) with virus free 1640 culture solution 0.1 mL on day 0 as blank group (C); 40 mice were ip 0.1 mL tissue culture infectious dose 50 (TCID50 is 10-4.36/mL) coxsackie viruses B3 (CVB3) on day 0 as VMC group (V), then each mouse in both groups was ip 0.1 mL NS every day. 8 mice in each of C group and V group were sacrificed on 4, 8, 15, and 21 d respectively after infections. The blood specimens gathered by taking out the eyeballs of mice were tested for the content of carboxyhemoglobin (COHb) using spectrophotometer method.The heart tissue slides were also stained by immunohistochemistry (IHC) for HO-1 and in situ hybridization (ISH) for HO-1 mRNA. The histological and ultrastructure changes were observed under light microscope and electron microscope.RESULTS: (1)The histopathological changes of myocardial cells: many inflammatory cells were found in the heart and large area myocardial cells necrosis was observed under light microscope. The inflammatory area was reduced at late stage in the heart of mouse in group V, while the myocardium in group C was normal. (2) The myocardial observation by electron microscopy: the myofibril in group V was dissolved and mitochondrial membrane disappeared, mononuclear cell infiltration was also observed under electron microscopy, which contained many lysosomes. The myocardial cells in group C were normal. (3) The changes of blood COHb level: compared with group C, the group V COHb level showed significantly higher on the day 8 and day 15 after CVB3 innoculation (0.047±0.005 vs 0.031±0.004; 0.076±0.006 vs 0.030±0.005, P<0.01). No obvious change in group C was observed. (4) The result of HO-1 IHC staining: myocardial cells had positive expression in group V, group C was negative. The absorbance (A) values in group V was significantly higher than that in group C (P<0.01) at different time points. (5) The result of HO-1 ISH was similar to HO-1 IHC. The A values in group V was all higher than that in group C (P<0.01).CONCLUSION: Viral myocarditis caused by coxsackie viruses B3 induces the expression of HO-1 mRNA and protein, and these expressions may play self-protection in inflammation injury in myocardial cells.     

Effects of histamine and hypoxia on eNOS gene expression in cultured porcine pulmonary and aorta endothelial cells

AIM: To investigate the effect of histamine and hypoxia on the expression of eNOS mRNA and protein in cultured porcine pulmonary artery and aorta endothelial cells. METHODS: Semi-quantitative RT-PCR and immuno-cytochemistry were used. RESULTS: (1) Histamine increased eNOS mRNA expression in a dose-and time dependent manner. For pulmonary endothelial cells, the effect reached peak when exposed to 10-5 mol/L histamine in 24 h. eNOS mRNA level was increased to 178.2%±7.7% (P<0.01) compared with control. eNOS protein was also enhanced to 173%±47% (P<0.01) compared with control. For aorta endothelial cells, the effect reach peak when exposed to 10-6 mol/L histamine in 24 h. The eNOS mRNA level was increased to 177.4%±14.3% (P<0.01) compared with control. The eNOS protein was also enhanced to 165%±54% (P<0.01). (2) The eNOS mRNA was enhanced in pulmonary endothelial cells after exposed to hypoxia for 12 h and reached peak in 24 h, increasing to 151.0%±9.1% (P<0.01). The protein expression was also enhanced to 216%±44% (P<0.01) compared with control. But there was no significant change in eNOS mRNA and protein expression in aorta endothelial cells during hypoxia. CONCLUSION: The experiments show that histamine increases the endothelial eNOS expression in both pulmonary and aorta endothelial cells, whereas hypoxia only increases eNOS expression in pulmonary endothelial cells. This may account partly for the different responses of pulmonary circulation and systemic circulation to hypoxia.     

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.