[Effect of Bushen Qiangdu Recipe on Histomorphology and Wnt Pathway of DBA/1 Mice].

Objective To observe the effect of Bushen Qiangdu Recipe (BQR) on the entheses ossification histomorphology of articular ligament of DBA/1 mice with spontaneous ankylosing spondylitis (AS) , and to study its mechanism for prevention and treatment of AS. Methods Thirty 12-week old male DBA/1 mice were randomly divided into the model group, the positive drug group, low, medium, high dose BQR groups, 6 in each group. Another 6 C57BLE mice of the same age were recruited as a blank control group. BQR containing 11. 25, 22. 50, 45.00 g/kg crude drugs was respectively adminis- tered to mice in low, medium, high dose BQR groups by gastrogavage, 0. 2 mL for each mouse, once per day. Celecoxib Capsule (0. 2 mL/0. 8 mg for each mouse, once per day) was administered to mice in the positive drug group by gastrogavage. Equal volume of normal saline was administered to mice in the model group and the blank control group by gastrogavage. All mice were fed and intragastically adminis- tered for 12 successive weeks. Body weight, diet, stools, and hair were routinely observed. Signs of ar- thritis were evaluated once per two weeks. Mice were sacrifice, and then general observation of achilles tendon was performed. The achilles tendon tissue was HE stained. Protein expressions of alkaline phos- phatase (ALP) , bone gamma-carboxyglutamic-acid-containing proteins (BGP) , Dickkopfl (DKK1) , and Wnt5a in the achilles tendon were detected using immunohistochemical method. Results Compared with the blank control group, the scoring of arthritis obviously increased in the model group (P <0. 05). But the scoring of arthritis was obviously lower in the 3 BQR groups and the positive drug group than in the model group (P <0. 05). Histopathological results of achilles tendon tissue showed that no infiltration of inflammatory cells or fibroblasts occurred in the normal group. Their histomorphological structures were normal. Cartilage formation and bone formation at various degrees occurred in the model group. Filtration of fibroblast-like cells occurred in inflammatory cells and attachment points. Scattered lymphocyte infiltra- tion was often seen in the achilles tendon tissue of each medicated group. Cartilage formation and bone formation were rarely seen. Compared with the blank control group, the scoring of arthritis increased in the model group (P <0. 05). Compared with the model group, the scoring of arthritis was decreased in the 3 BQR groups and the positive drug group (P <0. 05). Compared with the blank control group, protein expression of DKK1 decreased and protein expression of Wnt5a increased in the model group (P <0. 05). Compared with the model group, protein expression of DKK1 increased and protein expression of Wnt5a decreased in middle and high dose BQR groups (P <0. 05). Conclusion BQR could delay the occur- rence and development of arthritis and ossification in DBA/1 mice of spontaneous AS model possibly by inhibiting classical Wnt pathway.

[Mechanism of p38 mitogen-activated protein kinase inhibitor SB203580 to glucocorticoid sensitivity].

OBJECTIVE: To establish a model of cigarette smoke exposure to asthmatic rats and glucocorticoid resistance induced by nicotine in alveolar epithelioid cells A549 and study the mechanism for the change of glucocorticoid sensitivity induced by p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580. METHODS: Sixty Wistar rats were randomly divided into 4 groups: normal group, asthmatic group, cigarette smoke exposure to asthmatic group and SB203580 group. The mRNA expressions of glucocorticoid receptor (GR), heat shock protein 90 (HSP90) and p38 MAPK were detected by real time-polymerase chain reaction (RT-PCR) while their protein expressions detected by Western blot in vivo. A549 cells were divided averagely into 4 groups: group A: normal; group B: 1 µmol/L dexamethasone (DEX); group C: 1 µmol/L DEX +1 µmol/L nicotine; group D: 1 µmol/L DEX +1 µmol/L nicotine+1 µmol/L SB203580. Immunofluorescence staining was used to study the in vitro colocalization of glucocorticoid receptor (GR) in A549 cells. RESULTS: The mRNA expression of GR was 0.671 ± 0.002 in cigarette smoke exposure to asthmatic group and 0.595 ± 0.061 in SB203580 group (P = 0.065). The protein expression of GR was 0.700 ± 0.033 in cigarette smoke exposure to asthmatic group and 0.628 ± 0.091 in SB203580 group (P = 0.148). The mRNA expression of HSP90 was 0.558 ± 0.009 in cigarette smoke exposure to asthmatic group and 0.377 ± 0.046 in SB203580 group (P = 0.000). The protein expression of HSP90 was 0.507 ± 0.030 in cigarette smoke exposure to asthmatic group and 0.402 ± 0.050 in SB203580 group (P = 0.005). The mRNA expression of p38 MAPK was 0.971 ± 0.012 in cigarette smoke exposure to asthmatic group and 0.278 ± 0.049 in SB203580 group (P = 0.000). The protein expression of p38 MAPK was 0.982 ± 0.038 in cigarette smoke exposure to asthmatic group and 0.338 ± 0.042 in SB203580 group (P = 0.000). The ratio of GR amount within A549 nucleus versus that in cytoplasm was 0.077 ± 0.047 in group C and 0.592 ± 0.249 in group D (P = 0.000). CONCLUSION: The mechanism of SB203580 enhancing the corticosteroid sensitivity may be improving nuclear translocation of GR to elevate corticosteroid sensitivity.

[Aortic artery elastic lamina degradation, collagen remodeling, oxides stress and inflammation in the apolipoprotein E deficient mice with or without aortic banding].

OBJECTIVE: To explore the elastic lamina degradation and the collagen remodeling of aortic artery as well as oxides stress and inflammation of the apolipoprotein (Apo E) deficient mice with or without experimental hypertension. METHODS: Eighty male Apo E deficient mice were fed with high-fat diet beginning at six weeks of age. At 8-week old, they were randomly divided into hypertension group and control group (n=40 each), the mice in hypertension group were subjected the suprarenal aortic constriction operation and then randomly divided into two subgroups: 15 weeks age and 30 weeks age groups. At the end of experiment, the vascular elastic lamina degradation and the content of collagen were determined by morphological method, plasma ICAM-1 level was measured by ELISA, and the rennin activity measured by radioimmunoassay, the superoxide anion detected by fluorescence, the MOMA-2 observed by immunofluorescence in all animals. mRNA expression of NF-κB P65 and MMP9 was detected by real-time PCR. RESULT: In 15-week old group, the elastic lamina degradation Grade II and the intima-media thickness in the hypertension group were significantly higher than in the control group [(5.4±3.3)% vs. (8.9±2.5)%, P<0.05; (98.66±18.90) µm vs. (70.08±11.71) µm, P<0.05]. In 30-week old group, the elastic lamina degradation Grade III, the III type of collagen and the intima-media thickness were also significantly higher than in the control group [(15.2±3.7)% vs. (8.1±3.3)%, P<0.01; (23.00±7.73)% vs. (11.00±3.82)%, P<0.05; (147.31±22.60) µm vs. (103.98±17.21) µm, P<0.01]. The level of ICAM-1 in hypertension group was significantly higher than that of control group in both 15-week old and in 30-week old mice [(46.3±3.7) µg/ml vs. (40.6±5.7) µg/ml, P<0.05; (56.0±3.1) µg/ml vs. (45.2±2.8) µg/ml, P<0.05]. The superoxide anion, the MOMA-2, mRNA expression of NF-κB P65 and MMP9 in the hypertension group were significantly higher than in the control group in both 15-week old and in the 30-week old mice. The increase in hypertension group was more pronounced in the 30-week old mice than in the 15-week old mice. CONCLUSION: The elastic lamina degradation and the collagen remodeling of aortic artery as well as oxides stress and inflammation are more significant in the Apo E deficient mice with hypertension than in control Apo E deficient mice.

Total Flavonoids of Rhizoma Drynariae Restore the MMP/TIMP Balance in Models of Osteoarthritis by Inhibiting the Activation of the NF-κB and PI3K/AKT Pathways.

Total flavonoids of Rhizoma Drynariae (TFRD) have been shown to have beneficial effects on osteoarthritis (OA) clinically, but the mechanisms have not been elucidated. In this study, we investigated the effect of TFRD on articular cartilage in an OA rat model established by the Hulth method and in SW1353 chondrocytes induced by the proinflammatory factor interleukin-1ß (IL-1ß). The results showed that TFRD could alleviate the pathological changes in knee cartilage in OA model rats. In vivo, the qPCR analysis indicated that the mRNA levels of matrix metalloproteinases, MMP-1, MMP-3, and MMP-13, were decreased, while tissue inhibitor of matrix metalloproteinases- (TIMP-) 4 was increased in cartilage, and these changes could be partially prevented by TFRD. In vitro experiments showed that IL-1ß could significantly increase the expression of MMP-1, MMP-3, and MMP-13 and decrease the expression of TIMP-4 in SW1353 cells at the mRNA and protein levels. TFRD could increase the expression of MMP-3 and MMP-13 and decrease the expression of TIMP-4. Transfection of siRNA and addition of pathway inhibitors were used to clarify that inhibition of NF-κB and PI3K/AKT pathway decreased MMP-1, MMP-3, and MMP-13 and increased TIMP-4 expression. We also found that in IL-1ß-induced SW1353 cells, TFRD pretreatment had a modest inhibitory effect on p-AKT (Ser473) and reversed the increase of nuclear factor kappa-B (NF-κB) p65 in nuclear fraction and the decrease of inhibitor of NF-κB(IκB)-α in the cytosolic fraction. Further immunofluorescence confirmed that TFRD can inhibit IL-1ß-induced NF-κB p65 translocation to the nucleus to some extent. In conclusion, TFRD showed chondroprotective effects by restoring the MMP/TIMP balance in OA models by suppressing the activation of the NF-κB and PI3K/AKT pathways.

[Expression of severe acute respiratory syndrome coronavirus receptors, ACE2 and CD209L in different organ derived microvascular endothelial cells].

OBJECTIVE: To investigate the expression of the 2 severe acute respiratory syndrome coronavirus (SARS-CoV) receptors, angiotensin-converting enzyme 2 (ACE2) and CD209L in different human organ/tissue derived microvascular endothelial cells. METHODS: Endothelial cells from the microvessels in human brain, lung, hepatic sennoside, fat adipose tissue, adrenal gland, esophagus, lymph nodes, and bone were culture. RT-PCR, Western blotting and immunocytochemistry were used to detect the expression of ACE2 and CD209L receptors. RESULTS: Both SARS-CoV receptors of ACE2 and CD209L were expressed in the 8 organ/tissue-derived endothelial cells. The expression of ACE2 receptor was the highest in the human lung microvascular endothelial cells, and lowest in the lymphatic endothelial cells. The expression of CD209L was relatively higher in the human lymphatic endothelial cells. CONCLUSION: The organ derived microvascular endothelial cells are the important target of SARS-CoV. The pathological injury of lung and lymph system induced by SARS-CoV may be mediated respectively by different receptors of SARS-CoV.

[The effect of a vascular endothelial growth factor receptor inhibitor on airway inflammation and airway remodeling in a murine model of asthma].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor (VEGF) and the effect of a VEGF receptor inhibitor on airway inflammation and remodeling in a murine model of asthma. METHODS: BALB/c mice were randomly divided into three groups: group A (control group, n = 10), group B (asthmatic model group, n = 10) and group C (VEGF receptor inhibitor group, n = 10). The asthmatic model was established by OVA sensitization and challenge. Bronchoalveolar lavage fluid (BALF) cell count and differential were carried out, VEGF levels in BALF and serum were measured by enzyme-linked-immunosorbent assay (ELISA), the pathologic changes of bronchi and the lung tissue were evaluated, and the expression of VEGF was analyzed with immunohistochemistry (ISH). The airway wall thickness (WAt/Pi), the bronchial smooth muscle thickness (WAm/Pi), the bronchial smooth nucleus counts (N/Pi) and vascular counts were measured by using image analysis system. RESULTS: There was a significant increase in the numbers of total cells and eosinophils (EOS) in the BALF of group B [(142 +/- 63) x 10(7)/L, 98.0 +/- 46.9 compared to those of group A (30 +/- 14) x 10(7)/L, 0.7 +/- 1.1, all P < 0.01]. After administration of the VEGF receptor inhibitor, the numbers of total cells and EOS were significantly decreased [(41 +/- 17) x 10(7)/L, 4.9 +/- 3.5, all P < 0.01]. BALF and serum levels of VEGF in group B were [(55 +/- 26) pg/ml, and (72 +/- 26) pg/ml] respectively. They were significantly higher than those in group A [(37 +/- 9) pg/ml, (49 +/- 18) pg/ml, respectively] and in group C [(34 +/- 3) pg/ml, (43 +/- 19) pg/ml, respectively]. ISH showed that expression of VEGF in group B was increased compared with group A and C. WAt/Pi, WAm/Pi, and N/Pi in group B [(17 +/- 5) microm(2)/microm, (6.3 +/- 2.2) microm(2)/microm, (0.050 +/- 0.020)/microm, respectively] were all significantly higher than those in group A [(8 +/- 3) microm(2)/microm, (3.2 +/- 0.8) microm(2)/microm, (0.027 +/- 0.017)/microm respectively, P < 0.01 and P < 0.05]. The vascular count of the airways in group B (19 +/- 3) was significantly increased compared to that of group A (10 +/- 5, P < 0.01). Administration of the VEGF receptor inhibitor reduced WAm/Pi (4.5 +/- 1.3) microm(2)/microm, P < 0.05) and the vascular count (11 +/- 3, P < 0.01). CONCLUSIONS: VEGF was over-expressed in the murine model of asthma, and involved in angiogenesis and airway remodeling. VEGF receptor inhibitors may be effective in reducing allergic airway inflammation and airway remodeling.

[Protective effect of hydrophilic Salvia monomer on liver ischemia/reperfusion injury induced by pro-inflammatory cytokines].

OBJECTIVE: To observe the changes of tumor necrosis factor alpha (TNF alpha), interleukin 8 (IL-8) in liver ischemia/reperfusion injury and the protective effect of hydrophilic Salvia monomer MP-1 on them. METHODS: Hypothermic hypoxia reoxygenation model of human liver sinusoidal endothelial cell line and ischemia/reperfusion model of isolated rat liver were used. TNF alpha and IL-8 were measured with ELISA kits. Cell injury was excluded by trypan blue stain, sinusoidal endothelial cell function was assessed by hyaluronic acid uptake rate through RIA. Liver function was assayed by alanine transaminase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) release as well as output of bile flow. RESULTS: During hypoxia reoxygenation, sinusoidal endothelial cell injury, TNF alpha and IL-8 increased significantly in time-dependent manner, while sinusoidal cell function decreased. Cell injury was positively correlated to the released amount of TNF alpha (r = 0.949, P < 0.05) and IL-8 (r = 0.892, P < 0.05), respectively, the mortality could be reduced within 6 hrs by adding anti-TNF alpha monoclonal antibody and increased by treating with recombinant human TNF alpha. Function of isolated rat liver lowered alone the increasing of low temperature ischemia/reperfusion time. MP-1 could markedly lower the mortality of endothelial cells and TNF alpha and IL-8 release, it also could alleviate ischemia/reperfusion injury to isolated rat liver. CONCLUSION: TNF alpha participated in liver ischemia/reperfusion injury directly, and MP-1 might alleviate the injury through inhibiting TNF alpha and IL-8.

[Covert infection of severe acute respiratory syndrome in health-care professionals and its relation to the workload and the type of work].

OBJECTIVE: To study severe acute respiratory syndrome (SARS) covert infection in health-care professionals and its relation to work intensity and type of work. METHODS: Specific antibodies to SARS virus in 1 127 serum specimens from health-care professionals who had a close contact history with patients of SARS and 92 healthy persons were assayed with ELISA. RESULTS: The health-care professionals consisted of 287 men and 840 women, aged 21 - 55 years (mean 34 +/- 9 years). The overall positive rate of specific IgG and IgM was 2.57% (29/1 127); the positive rates in nurses, doctors and laboratory workers, and supervisors were 3.46%, 1.41%, 2.86%, respectively. 34.5% of the infected worked in the ICU, and 65.5% in the general wards. None of them presented clinical SARS. Medical examination was also carried out for 313 people who had close contact with the 29 antibody positive health care professionals, but no clinical SARS was found. CONCLUSIONS: Covert infection can be found in the health-care professionals and it showed no contagiousness. The positive rate of specific antibodies was higher in ICU where the work intensity was higher. The positive rate in nurses was higher than in doctors.

Nicotine inhibits histone deacetylase 6 activity and chaperone-dependent activation of the glucocorticoid receptor in A549 cells.

BACKGROUND: Nicotine, a major component of tobacco, is the main cause of smoking addiction. It was found that asthmatic patients who smoke were insensitive to glucocorticoid treatment. In this paper, we investigated whether nicotine could inhibit histone deacetylase 6 activity (HDAC6) and chaperone-dependent activation of the glucocorticoid receptor (GR) in A549 cells. Furthermore, the expression level of heat shock protein 90 (HSP90) was determined. METHODS: Quantitative real-time polymerase chain reaction was used to detect the levels of RNA transcription, and Western blotting was applied to analyze the levels of protein expression of HDAC6, GR, and HSP90 in A549 cells. Moreover, the effects of dexamethasone and trichostatin A were observed in A549 cells. RESULTS: A549 cell proliferation was inhibited in the presence of nicotine, and the level of RNA and protein expression of HDAC6 and GR were down-regulated. CONCLUSIONS: Nicotine could inhibit HDAC6 activity and chaperone-dependent activation of GR. This might be the main reason why asthmatic patients who smoke show insensitivity to the glucocorticoid treatment.