Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

Toll-like receptor 4 expression on mast cells in human gingival tissues with chronic periodontitis

AIM: To observe the expression of Toll-like receptor 4 (TLR4) on mast cells in human gingival tissues with chronic periodontitis. METHODS: A total of 68 volunteers, including 23 cases of mild chronic periodontitis, 25 cases of severe chronic periodontitis and 20 healthy controls, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining. The expression of TLR4 in gingival tissues was detected by immunohistochemical staining, and TLR4 expression on mast cells was detected by immunofluorescence double staining. RESULTS: The expression of TLR4 in gingival tissues and on mast cells in chronic periodontitis groups was significantly higher than that in normal control group (P<005), and that in severe chronic periodontitis group was significantly higher than that in mild chronic periodontitis group (P<005). CONCLUSION: The expression of TLR4 in gingival tissues and on mast cells is increased with the severity of chronic periodontitis, suggesting that TLR4, especially TLR4 on mast cells, may play an important role in human chronic periodontitis.     

Expression of IL-1β and IL-17 on mast cells in human periapical diseases

AIM：To identify and quantify the expression of IL-1β and IL-17 in mast cells (MCs) in different types of human pericapical diseases using double immunofluorescence staining. METHODS：The specimens (n=102), including healthy control (n=35), periapical cyst (n=35) and periapical granuloma (n=32), were involved in the present study. The tissue samples were fixed in 10 % buffered formalin for at least 48 h and then embedded in paraffin. Serial 5-μm-thick sections were deposited onto SuperFrost/Plus microscope glasses. Routine staining of the sections using hematoxylin & eosin (HE) was performed for morphological evaluation. The number of IL-1β and IL-17 positive MCs was identified by double immunofluorescence staining. RESULTS：Compared with the healthy controls, the inflammation score of periapical lesions was significantly increased in the periapical patients (P<0.01). The density of IL-1β and IL-17 positive MCs in the periapical lesions were obviously higher than that in the healthy controls (P<0.01). However, no significant difference between periapical cyst and periapical granuloma was observed. The Pearson correlation analysis showed that there was a positive correlation between the density of IL-1β and IL-17 double positive MCs and inflammation score in different groups of specimens (P<0.01). CONCLUSION：There is significantly increased number of MCs, along with increased density of IL-1β and IL-17 positive MCs in human periapical lesions. The increased density of IL-1β and IL-17 positive MCs has the similar tendency as the severity of tissue inflammation in human periapical lesions, suggesting that IL-1β and IL-17 positive MCs may play an important role in the pathogenesis of human periapical diseases.     

Expression of TLR2 and TLR4 on mast cells in human chronic periapical diseases

AIM: To observe the expression of TLR2 and TLR4 on mast cells(MCs) in the periapical tissues from different types of human chronic periapical diseases, and to analyze the role of TLR2 and TLR4 on tryptase-positive MCs in the immunopathogenesis of human chronic periapical diseases. METHODS: A total of 60 donors, including healthy control group, periapical granuloma group and periapical cyst group, were enrolled in the study. The periapical tissue specimens were fixed in 10% buffered formalin and stained with hematoxylin and eosin for histopathology, or stained with double-immunofluorescence for identification of TLR2-tryptase and TLR4-tryptase double-positive MCs in the periapical tissues. RESULTS: Compared with the healthy control, the densities of TLR2-tryptase and TLR4-tryptase double-positive MCs in periapical tissues were significantly increased in human chronic periapical diseases(P<0.01). The densities of TLR2-tryptase and TLR4-tryptase double-positive MCs in periapical cyst group were significantly higher than those in periapical granuloma group(P<0.01). CONCLUSION: TLR2 and TLR4 were expressed on the MCs in the periapical tissues of human chronic periapical diseases. TLR2-tryptase and TLR4-tryptase double-positive MCs may participate in the pathogenesis of chronic periapical diseases.     

Expression of transforming growth factor-β on mast cells in human chronic periapical diseases

AIM: To investigate the distribution of mast cells (MCs) and the expression of transforming growth factor-β (TGF-β) on tryptase positive MCs in different types of human periapical diseases. METHODS: Total 78 cases of specimens were involved in this study, including healthy control, periapical cyst and periapical granuloma. The tissue samples were fixed in 10% formalin for at least 48 h, stained with hematoxylin and eosin for histopathological examination, stained with toluidine blue staining for identifying MCs and MCs degranulation, and stained with double immunofluorescence for identification of tryptase-TGF-β double positive MCs. RESULTS: The density of tryptase-TGF-β double positive MCs in the periapical lesions was significantly higher than that in the healthy controls (P<0.01). The number of TGF-β positive MCs in the periapical cyst was significantly higher than that in the periapical granuloma (P<0.01). Compared with toluidine blue staining, the number of MCs with double immunofluorescence staining significantly increased (P<0.01). CONCLUSION: The TGF-β positive MCs may play an important role in the pathogenesis of human chronic periapical diseases, particularly in the formation of fibrous tissue in periapical cyst. Double immunofluorescence staining is more sensitive than the traditional toluidine blue staining for identifying MCs.     

Effect of hyperbaric oxygen on periodontitis with psychological stress in rats

AIM: To investigate the role of chronic psychological stress on periodontitis and the effects of hyperbaric oxygen (HBO) on periodontitis with psychological stress in rats. METHODS: Male special pathogen-free Wistar rats (n=80) were randomly divided into 4 groups: (1)normal control group; (2)experimental periodontitis group: the periodontitis model was induced by wrapping 3/0 silk ligature inoculated with Porphyromonas gingivalis around the left maxillary second molar of the rats; (3)psychological stress stimulation group; (4)periodontitis model with stress stimulation group. Psychological stress was removed at the 9th week after ligature, and 4 rats from each experimental group were randomly chosen for HBO treatment. The rats were sacrificed at the 2nd, 4th, 8th and 10th weeks after ligature. The levels of blood glucose, adrenocorticotropic hormone (ACTH), corticosterone and adrenaline were measured as the stress markers. The histological changes of periodontal tissues were observed under microscope with HE staining. RESULTS: The levels of blood glucose, ACTH, corticosterone and adrenaline in psychological stress stimulation group and periodontitis with stress group were significantly higher than those in control group and experimental periodontitis group at the 2nd and 4th weeks after ligature (P<0.05). The levels of the stress markers were significantly lower than those in untreated groups in the 10th week after HBO (P<0.01). The sites of gingival attachment were normal in control group and psychological stress stimulation group. Periodontal pocket, and periodontal attachment loss (AL) were observed in experimental periodontitis group. The tissue damage was much heavier in periodontitis model with stress stimulation group as the furcation of tooth was exposed and the tissue damage was observed on both sides of the adjacent teeth. No significant difference of AL between psychological stress stimulation group and normal control group during the experiment was observed. The AL in periodontal model with stress stimulating group was significantly higher than that in experimental periodontitis group at the 2nd, 4th and 8th weeks (P<0.01). The level of AL was attenuated at the 10th week after HBO (P<0.01). No difference of histological change in periodontal tissues was observed between control group and psychological stress stimulation group. Severer inflammatory changes and alveolar bone destruction were observed in periodontitis with stress group than those in experimental periodontitis group. The levels of inflammation reduced at the 10th week after HBO. CONCLUSION: Stress stimulation is one of the inducing factors of periodontitis in rats, which aggravates periodontitis. HBO may represent a useful way in treating psychological stress periodontitis.     

Relationship between TNF-α gene polymorphism-308 and type 2 diabetes with periodontitis

AIM: To investigate the gene polymorphism-308 of tumor necrosis factor alpha (TNF-α) in the relation with the susceptibility to periodontitis combined with type 2 diabetes mellitus (DM) and its severity.METHODS: Human DNA samples were obtained from 240 DM patients with periodontitis (periodontitis group, n=120) and without periodontitis (control group, n=120). All patients were genotyped by PCR-RFLP analysis. The frequencies of genotypes and alleles were analyzed. Sulcus bleeding index (SBI) and probing depth (PD) in all patients were measured. The polymorphism-308 of TNF-α gene in the relation with the susceptibility to periodontitis combined with DM and its severity was analyzed.RESULTS: No significant difference in the frequency of genotype and allele was found between DM patients with mild periodontitis and DM patients without periodontitis (P>0.05). However, the frequencies of these genotypes and alleles in DM patients with moderate and severe periodontitis were significantly higher than those in DM patients without periodontitis (P<0.01). The findings showed that the level of TNF-α was associated with SBI and PD (P<0.01).CONCLUSION: TNF-α -308 G/A polymorphism is not associated with DM patients with mild periodontitis, whereas it may have a role in pathogenesis and prognosis of moderate and severe periodontitis combined with DM through TNF-α level.     

Change of nitric oxide concentration in ligature-induced periodontitis in rats

AIM: To study the possible role of nitric oxide (NO) in the development of periodontitis and the relationship between the NO concentration and the attachment loss. METHODS: Seventy-two Sprague-Dawley rats were randomly assigned to two groups, the control group and periodontitis group. Experimental periodontitis in rats was produced by a ligature of braided silk. The nitric oxide concentration was indirectly ascertained by the concentration of nitrite (NO2-) and nitrate (NO3-) in the gingival tissue, which was assayed by spectrophotometry. The attachment loss (AL) was measured by the technology of the cellular graphics engineering research. The histopathologic change in periodontium was observed under a light microscope by using the histotomy. RESULTS: Compared to control group, the NO2-/NO3- concentration in gingival tissue was significantly higher in periodontitis group at four weeks and eight weeks following ligation (P<0.01). In periodontitis group, the NO2-/NO3- concentration in gingival tissue was higher at eight weeks than that at four weeks following ligation (P<0.01). At four weeks and eight weeks, the AL in experimental periodontitis in rats was significantly increased than that at one week after ligation (P<0.01); and the AL was also much higher at eight weeks than that at four weeks (P<0.01). CONCLUSIONS: The NO2-/NO3- concentration in the gingival tissue in periodontitis group was significantly higher than that in control group. These results demonstrate that the NO2-/NO3- concentration is related to the severity of AL, and NO synthesis is very important to the process of inflammation and lesion in periodontium. Reducing NO production may be of great therapeutic value in the treatment of periodontitis.     

Expression of stem cell factor in mast cells isolated from patients with chronic periapical diseases

AIM:To investigate the expression of stem cell factor (SCF) in tryptase -positive mast cells (MCs) in different types of human periapical diseases for determining the role of SCF and MCs in the pathogenesis of periapical diseases. METHODS:A total 50 cases of specimens were involved in this study, including healthy control (n=20), periapical cyst (n=15) and periapical granuloma (n=15). The tissue material was fixed in 10% formalin for at least 48 h, stained with hematoxylin and eosin for the observation of histopathology, stained with immunohistochemistry for identifying MCs and MCs degranulation, and stained with double immunofluorescence for identification of tryptase-SCF double positive MCs. RESULTS:Compared with healthy control, significantly higher densities of both total and degranulated MCs in human periapical lesions were observed. The densities of both total and degranulated MCs in the periapical cyst were significantly higher than that in the periapical granuloma. The density of tryptase-SCF double positive MCs in the periapical lesions was significantly higher than that in the healthy controls. The density oftryptase-SCF double positive MCs in the periapical cyst was significantly higher than that in periapical granuloma. No significant difference in the density of MCs between immunohistochemistry staining and double immunofluorescence staining was observed. CONCLUSION: The tryptase-SCF double positive MCs play an active role in the pathogenesis of the periapical inflammatory lesions, particularly in the formation of fibrous tissue in periapical cyst. The potential role of the tryptase-SCF double positive MCs relates with the initiation, development, and persistence of the periapical inflammatory process.     

Effect of nimesulide on ligature-induced periodontitis in rats

AIM: The purpose of this study was to observe the effect of nimesulide on periodontitis. METHODS: The gingival index (GI) was measured before the rats were sacrificed at the ends of week 4, 5 and 8. The periodontal tissues were stained with hematoxylin and eosin. Histological changes were observed by microscope. The periodontal attachment loss (AL) was measured by Tiger cell image analyzer. RESULTS: (1) Experimental periodontitis was successfully induced in rats by placing a piece of 3/0 braided silk around the cervix of the lower incisors at week 4 after the ligature. (2) In ligature-induced periodontitis group, at week 4 after the ligature, the GI and AL were significantly higher than those in control group (P<0.01). The histopathologic changes of periodontium in periodontitis group showed obvious inflammation, and the severity of destruction for periodontium was increased as time passed. (3) In the nimesulide prevention group, the GI and AL were significantly lower than those in periodontitis group (P<0.01). The histopathologic examination showed less inflammatory responses, and no obvious alveolar bone resorption was observed. (4) In the nimesulide treatment group, the GI and AL were significantly lower than those in periodontitis group at the end of week 5 and 8 after the ligature (P<0.01).CONCLUSION: (1) In ligature-induced periodontitis, nimesulide inhibits effectively its progress. (2) In the developing periodontitis, a significant improvement is observed in GI and AL following the treatment with nimesulide.     

Immunohistochemical localization of CD14, IL-4 and TNF-α in periapical tissue of healthy individuals and patients with chronic periapical diseases

AIM: To observe the expression of cluser of differentiation 14 (CD14), tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in periapical tissues of patients with chronic periapical diseases, and to analyze the role of immunopathogenesis of CD14, TNF-α and IL-4 expression in human chronic periapical diseases. METHODS: A total of 88 samples were divided into 3 groups: healthy control group (n=45), chronic periapical abscess group (n=23), and periapical cyst group (n=20). All samples were fixed in 10% buffered formalin and stained with double immunofluorescence for identification of TNF-α-CD14 and IL-4-CD14 double-positive cells in periapical tissues. RESULTS: Compared with healthy control group, the densities of TNF-α-CD14 and IL-4-CD14 double-positive cells in the 2 groups of chronic periapical diseases were increased significantly (P<0.05). Compared with periapical cyst group, the density of TNF-α-CD14 double-positive cells in chronic periapical abscess was increased significantly (P<0.05). The density of IL-4-CD14 double-positive cells in periapical cyst group was significantly higher than that in chronic periapical abscess group (P<0.05). CONCLUSION: There is high expression of TNF-α-CD14 and IL-4-CD14 double-positive cells in the periapical tissues of the patients with chronic periapical abscess and perapical cyst, suggesting that the Th cytokines participate in the immune regulation of periapical diseases, which may be one of the immune mechanisms of the interaction between Th1 and Th2 cytokines.     

Correlation between Helicobacter pylori infection and apoptosis in gingival tissues

AIM:To observe the effects of Helicobacter pylori(Hp) on the apoptosis of human gingival tissue.METHODS:Gingival tissue samples were taken from 30 patients without chronic periodontitis,and Hp was detected by conventional PCR.The apoptosis of the gingivival cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to analyze the correlation between Hp infection and apoptosis of the gingival tissues.RESULTS:The Hp positive detections were 12 in the 30 patients without periodontitis,so the positive rate of Hp in the gingival tissue samples was 40%.The gingival tissue showed a large number of apoptotic cells in Hp positive group,and less apoptotic cells in Hp negative group.The apoptotic index in Hp positive group (0.498±0.092) was significantly higher than that in normal group (0.207±0.053)(P<0.05).CONCLUSION:Hp might play a role in the apoptosis of gingival tissues.     

Antagonistic effects of aminophylline on airway inflammation and oxidative lung tissue damage in chronic hypoxic rats

AIM: To investigate the effects of chronic hypoxia and antagonistic effects of aminophylline on airway inflammation and oxidative lung damage in rats. METHODS: Thirty-four male Wistar rats were randomly divided into three groups: normal control group (n=10); hypoxia group (n=12); aminophlline-treated group (n=12). The last two groups were both exposed to hypoxia 7 hours per day for 21 days. The third group was treated with aminophlline (100 mg·kg-1·d-1) before exposed to hypoxia. The level of tumor necrosis factor (TNF) -α, interleukin (IL)-10, lipid peroxide (LPO) and the activity of superoxide dismutase (SOD) were determined in blood and homogenates of lung tissue. RESULTS: Compared to the control group, the levels of TNF-α, IL-10 and LPO were significantly increased (P<0.01), the activity of SOD was significantly decreased (P<0.01) both in blood and homogenate of lung tissue. The ratio of TNF-α and IL-10 was significantly increased (P<0.01) in homogenate of lung tissue in hypoxia group. Compared to the hypoxia group, the levels of TNF-α, LPO and the ratio of TNF-α and IL-10 were significantly decreased (P<0.01) in blood and homogenate of lung tissue in aminophylline treated group, while the level of IL-10 and activity of SOD was significantly increased (P<0.05, P<0.01, respectively). CONCLUSIONS: Chronic hypoxia induces airway inflammation. Aminophylline produces anti-inflammatory effects on airway and anti-oxidantive effects on lung.     

Effects of antihistamine treatment on mast cell infiltration and c-Kit and SCF expression in liver tissues of rats with experimental hepatitis

AIM：To study the infiltration of mast cells and the expression of c-Kit and stem cell factor (SCF) in liver tissues of rats with experimental hepatitis and their changes after antihistamine (AH) treatment. METHODS：Thirty Wistar rats were divided into 3 groups at random: normal control (NC) group, chronic hepatitis (CH) group and AH group. The rat model of CH was established by composite factors (subcutaneous injection of carbon tetrachloride, accompanied by a diet containing high cholesterol, high alcohol, low protein and low choline). The rats in AH group were treated with ketotifen based on CH. At the end of the 4th week, blood samples were taken to determine plasma tryptase (TS) and histamine (HA) levels. Liver tissues were taken to detect HA content, observe the histological changes with HE staining and count the number of mast cells with toluidine blue (TB) staining. The mRNA and protein expression of c-Kit and SCF in liver tissues was detected by RT-PCR and immunohistochemistry. RESULTS：(1) The plasma TS and HA levels and liver HA content in CH group were significantly increased compared with NC group (P<0.05), while those in AH group were obviously decreased compared with CH group (P<0.05). (2) Fatty degeneration and fibrosis were observed in CH group under light microscope, but the hepatic injury was obviously attenuated in AH group. TB staining showed there were many degranulating and degranulated mast cells filled with purple granules around liver blood vessels and in fiber interval in CH group, and there were few purple granules in the cytoplasm of mast cells in AH group. The number of mast cells in CH group was increased compared with NC group (P<0.05), and that in AH group was reduced compared with CH group (P<0.05). (3) The results of RT-PCR showed that AH down-regulated the expression of c-Kit and SCF mRNA (P<0.05). The expression of c-Kit and SCF proteins in liver tissues increased in CH rats (P<0.05 vs NC group), decreased after AH treatment (P<0.05 vs CH group) and was positively correlated with liver HA content (P<0.05). CONCLUSION：These data suggest that an inflammatory pathway mediated by mast cell activation is involved in experimental hepatitis. Ketotifen can reduce mast cell degranulation by down-regulating the expression of mast cell membrane receptor c-Kit and its ligand SCF, thereby attenuating the liver inflammation.     

Effect of hyperbaric oxygen on HIF-1α expression in rat experimental periodontitis with psychological stress

AIM: To investigate the effect of hyperbaric oxygen (HBO) on hypoxia-inducible factor-1α (HIF-1α) expression in rat experimental periodontitis with psychological stress. METHODS: Male special pathogen-free Wistar rats (n=120) were randomly divided into 4 groups: normal control group; psychological stress stimulation group; experimental periodontitis group: the periodontitis model was induced by wrapping 3/0 silk ligature inoculated with Porphyromonas gingivalis around the left maxillary second molar of the rats; periodontitis model with stress stimulation group. Psychological stress was removed at the 9th weeks after ligature, 6 rats from each experiment group were randomly chosen to HBO treatment. The rats were sacrificed at the 2nd, 4th, 8th and 10th weeks after ligature. Gingival index (GI) and attachment loss (AL) were measured before sacrifice. The histological changes of periodontal tissues were observed under microscope with HE staining. The expression of HIF-1α was observed by the method of immunohistochemistry. RESULTS: The sites of gingival attachment were normal in control group and psychological stress stimulation group. Periodontal pocket, and periodontal attachment loss were observed in experimental periodontitis group. The tissue damage was much serious in periodontitis model with stress stimulation group. No significant difference of GI and AL among psychological stress stimulation group and normal control group during the experiment was observed. GI and AL in periodonitis model with stress stimulating group were significantly higher than those in experimental periodontitis group at the 4th and 8th weeks (P < 0.01). The levels of GI and AL were significantly lower at the 10th weeks after HBO treatmnt than those in untreated groups (P < 0.05). No significant difference of HIF-1α expression scores among psychological stress stimulation group and normal control group was found. HIF-1α expression scores in periodonitis model with stress stimulating group was significantly higher than that in experimental periodontitis group at the 4th and 8th weeks (P < 0.01). At the 10th weeks after HBO treatment the levels of HIF-1α were significantly lower than that in untreated groups (P < 0.01). CONCLUSION: Stress stimulation may aggravate periodontitis by decreasing tissue oxygenation in rats. HBO may represent a useful way in psychological stress periodontitis therapy.     

The relationship between cough response and airway inflammation in guinea pigs sensitized and challenged with ovalbumin

AIM: To investigate the effects of intensity of eosinophilic airway inflammation on cough response in guinea pigs sensitized and challenged with ovalbumin. METHODS: 34 sensitized guinea pigs were challenged with the aerosol of either saline (group A, n= 7) or 0.04% (group B, n= 7), 0.2% (group C, n= 8) and 1% (group D, n= 12) of ovalbumin. 24 hours later, cough response to inhaled capsaicin and airway responsiveness to inhaled methacholine (PC₁₅₀) were measured. Total cell number and differentials in bronchoalveolar lavage were analyzed. RESULTS: After challenge, one animal in group C and five animals in group D died from severe wheezing. With ascending concentration of ovalbumin, cough frequency induced by inhaled capsaicin increased from (6±2) times/3 min in group A to (22±4) times/3 min in group D( P< 0.05). PC₁₅₀, which did not change in group B, increased in both group C and group D significantly in addition to an increase in total cell number and eosinophils in bronchoalveolar lavage. Cough response to inhaled capsaicin was positively correlated with total cell number ( r= 0.84, P< 0.01) and eosinophils ( r= 0.78, P< 0.01) in bronchoalveolar lavage, and negatively correlated with PC₁₅₀ ( r= -0.78, P< 0.01). There was a negative correlation between PC₁₅₀ and total cell number ( r= -0.80, P< 0.01) or eosinophils ( r= -0.85, P< 0.01). CONCLUSION: Cough response in sensitized guinea pigs is enhanced and finally develops into wheezing with a progress in eosinophilic airway inflammation.     

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

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

Expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage in neonatal A2AR knockout mice

AIM： To study the expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage (HIBD) in the neonatal adenosine A2A receptor knockout (A2AR-/-) mice. METHODS：Base on the modified Rice method, the model of HIBD was established. The total 64 C57/BL6 neonatal mice (7 days old) of A2AR-/-(KO) and corresponding wild type (A2AR+/+, WT) were randomized into sham-operated group and model group. The mice in model group were divided into 3 subgroups： 1 d after HIBD, 3 d after HIBD and 7 d after HIBD (n=8 for each group). The cortex and hippocampal CA1 region were used as the study areas. The neuronal apoptosis was detected using TUNEL assay combined with Nissl staining. The expression of p-p38 MAPK and activated caspase-3 was determined by the method of immunohistochemistry. The KO mice and WT mice were also taken from sham-operated group (SKO and SWT, n=10) and model group (MKO and MWT, n=30) 1 d after HIBD to assess the early neurological behavior. RESULTS：The apoptotic neurons, activated caspase-3 and p-p38 MAPK increased after HIBD and peaked at 1 d after HIBD in the cortex and the hippocampal CA1 region. The apoptotic neurons and the expression of activated caspase-3 in KO mice were significantly higher than those in WT mice at the same time point after HIBD. The expression level of p-p38 MAPK in KO mice were significantly higher than that in WT mice at 1 d and 3 d after HIBD. The expression of activated caspase-3 was positively correlated with the expression of p-p38 MAPK in neonatal mice after HIBD (in the cortex：r=0.957, P<0.01; in the hippocampal CA1 region： r=0.939, P<0.01). CONCLUSION：p-p38 MAPK might be involved in the aggravated neuron apoptosis and brain damage induced by A2AR knockout after neonatal HIBO.     

Effects of Shenmai injection on myocardial fibrosis in rat model of diabe-tic cardiomyopathy

AIM：To explore the effects of Shenmai injection on myocardial fibrosis in a rat model of diabetic cardiomyopathy (DCM). METHODS：Wistar rats (n=30) were randomly divided into control group, diabetes group and treatment group. Single intraperitoneal injection of streptozotocin was utilized to establish a rat model of DCM. The rats with DCM in treatment group were intraperitoneally injected with Shenmai injection. Ventricular cannulation was applied to assess the cardiac functions. The formation of collagen in the cardiac tissues was assessed by Masson staining. The generation of reactive oxygen species (ROS) in the cardiac tissues was detected by dihydroethidium staining. The expression levels of matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase 2 (TIMP-2) and collagen I in the cardiac tissues were determined by Western blotting. RESULTS：Compared with control group, the cardiac functions were deteriorated in diabetes group (P<0.05), which was improved in treatment group as compared with diabetes group (P<0.05). Compared with control group, the formation of collagen and ROS increased significantly in diabetes group (P<0.05), which was decreased in treatment group as compared with diabetes group (P<0.05). Compared with control group, the expression level of MMP-2 in the cardiac tissues was deceased and TIMP-2 was increased significantly in diabetes group (P<0.05), but reversed significantly in treatment group (P<0.05).CONCLUSION:Shenmai injection attenuates cardiac fibrosis in the rats with DCM by inhibiting the generation of ROS.