OBJECTIVETo study the effect of hypoxia on cofilin activation in intestinal epithelial cells and its relation with distribution of tight junction protein zonula occludens 1 (ZO-1).

METHODSThe human intestinal epithelial cell line Caco-2 was used to reproduce monolayer cells. The monolayer-cell specimens were divided into control group (no treatment), hypoxic group ( exposed to hypoxia), and normoxic group (exposed to normoxia) according to the random number table. Western blotting was used to detect the protein expressions of cofilin and phosphorylatedl cofilin (p-cofilin) of cells in normoxic group and hypoxic group exposed to normoxia or hypoxia for 1, 2, 6, 12, and 24 h and control group, with 9 samples in control group and 9 samples at each time point in the other two groups. The other monolayer-cell specimens were divided into hypoxic group (exposed to hypoxia) and control group (no treatment) according to the random number table. Cells in hypoxic group exposed to hypoxia for 1, 2, 6, 12, and 24 h and control group were obtained. Morphology and distribution of F-actin was observd with laser scanning confocal microscopy, the ratio of F-actin to G-actin was determined by fluorescence method, and distribution of ZO-l and cellular morphology were observed with laser scanning confocal microscopy. The sample number of last 3 experiments was respectively 3, 6, and 3 in both hypoxic group (at each time point) and control group. Data were processed with paired ttest, analysis of variance of repeated measurement, and LSD-t test.

RESULTSThe protein expressions of cofilin and p-cofilin of cells between normoxic group exposed to normoxia for 1 to 24 h and control group showed no significant changes (with values from -0.385 to 1.701, t(p-cofilin)values from 0. 040 to 1.538, P values above 0.05). There were no obvious differences in protein expressions of en filmn of cells between hypoxic group exposed to hypoxia for 1 to 24 h and control group ( with values from 1.032 to 2.390, P values above 0.05). Compared with that in control group, the protein expressions of p-cofilin of cells were greatly reduced in hypoxic group exposed to hypoxia for 1 to 24 h (with values from 4.563 to 22.678, P values below 0.01), especially exposed to hypoxia for 24 h. The protein expressions of cofilin of cells between normoxic group and hypoxic group at each time point were close ( with t values from -0.904 to 1.433, P values above 0.05). In hypoxic group, the protein expressions of p-cofilin of cells exposed to hypoxia for 1, 2, 6, 12, and 24 h were 0.87 +/- 08, 0.780 .05, 0.89 +/- 0.07, 0.68+0. 07, and 0.57 +/- 0.06, respectively, significantly lower than those in normoxic group (0.90 +/- 0.07, 0.97 +/- 0.06, 1.00 +/- 0.06, 1.00 +/- 0.05, and 0.99 +/- 0.05, with t values from 3.193 to 16.434, P values below 0.01). In control group, F-actin in the cytoplasm was abundant, most of it was in bunches. The trend of F-actin was disorderly in hypoxic group from being exposed to hypoxia for 1 h, shortened in length or even dissipated. The ratios of F-actin to G-actin of cells in hypoxic group exposed to hypoxia for 12 and 24 h (0.89 +/- 0.12 and 0.84 +/- 0.19) were obviously decreased as compared with that in control group (1. 00, with t values respectively 3. 622 and 3. 577, P values below 0.01). There were no obvious differences in the ratios of F-actin to G-actin of cells between hypoxic group exposed to hypoxia for 1, 2, and 6 h and control group ( with values from 0.447 to 1.526, P values above 0.05). In control group, cells were compact in arrangement, and ZO-1 was distributed continuously along the cytomnembrane. From being exposed to hypoxia for 2 h, cells became irregular in shape in hypoxic group. ZO-1 was distributed in discontinuous fashion along the cytomembrane with breakage in hypoxic group exposed to hypoxia for 24 h.

CONCLUSIONSHypoxia may cause the disorder of dynamic balance between F-actin and G-actin by inducing cofilin activation, which in turn leads to the changes in distribution of tight junction protein ZO-1 in intestinal epithelial cells.

Actin Depolymerizing Factors ; Actins ; Blotting, Western ; Caco-2 Cells ; drug effects ; physiology ; Epithelial Cells ; cytology ; drug effects ; Humans ; Hypoxia ; metabolism ; Intestinal Mucosa ; drug effects ; metabolism ; pathology ; Intestines ; Oxygen ; pharmacology ; Tight Junctions ; drug effects ; metabolism ; Zonula Occludens-1 Protein ; metabolism

Objective To investigate audiological characteristics of patients with intact tympanic membranes and conductive or mixed hearing loss .Methods A retrospective study was carried out among 30 patients (42 ears) with intact tympanic membranes and conductive or mixed hearing loss who underwent exploratory tympanotomy . The preoperative outcomes of pure tone audiometry ,tympanometry ,resonant frequency of middle ear and temporal bone CT scan were analyzed .Results Among 42 ears ,30 ears with otosclerosis and 12 ears with ossicular chain dis-ruption were confirmed in exploratory tympanotomy ,but only 5 ears showed positive findings in CT scan .The mean thresholds of bone conduction ,air conduction and air -bone gap at frequencies of 0 .5 ,1 and 2 kHz were 27 .5 ± 1 .3 dB HL ,67 .0 ± 1 .8 dB HL ,39 .5 ± 1 .1 dB HL ,respectively .An analysis of tympanometric data of all patients re-vealed that 50% of all ears (21/42) were type A tympanograms ,42 .9% (18/42) were type As tympanograms ,and 7 .1% (3/42) were type Ad tympanograms .The mean of the resonant frequency of the middle ear in otosclerositic patients (1 079 .0 ± 67 .4 Hz) was significantly higher than ossicular chain disruption patients (633 .3 ± 43 .6 Hz) . Conclusion Otosclerosis is the most common in the patients with intact tympanic membranes and conductive or mixed hearing loss .The middle ear resonant frequency of otosclerositic patients is significantly higher than that of ossicular chain disruption patients .

OBJECTIVE： To evaluate the clinical therapeutic effect of L-glutamine granules on intestinal damage of severe burn patients and the safty of the drug.METHODS： Thirty-nine severe burn patients were randomly divided into two groups： control group（C group， nineteen patients） and L-glutamine treatment group（GLN group， twenty patients） .GLN group patients were given L-glutamine in a dose of 30g per day for 7 days， and C group patients were given the same dosage of placebo for 7 days.The plasma L-glutamine concentration， the degree of intestinal mucosa damage， blood biochemistry and complication were observed and wound healing rate of burn area was determined， then the length of hospital stay was recorded.RESULTS： After 7 days of taking L-glutamine orally， plasma L-glutamine concentration in GLN group was significant higher than that in C group（P<）. The degree of intestine damage and intestinal mucosal permeability in GLN group were lower than those in C group. In addition, the wound healing rate was faster and the length of hospital stay was shorter in GLN group than those in C group. CONCLUSION: Administration of L-glutamine could abate the degree of intestine damage obviously, lessen intestinal mucosal permeability, ameliorate wound healing rate and reduce the length of hospital stay.

This paper was aimed to study effects of different preparation methods on the content of ginsenosides Rg1,Re and Rb1 in Yi-Xin-Shu (YXS) tablets by HPLC-ELSD.HPLC-ELSD was used as the detection method.The separation and content of ginsenosides Rg1,Re and Rb1 were used as indexes.The influences of three different preparation methods (i.e.,defatted alcohol extraction and butanol extraction,alcohol extraction and butanol extraction,alcohol extraction and butanol extraction ammonia solution washing) on the effect of YXS tablets were studied.Then,the same content determination method was used to compare the influence of alkali washing treatment to ginsenosides Rg1,Re and Rb1 among different batches of Panax ginseng.The results showed that a good separation of ginsenosides Rg1,Re and Rb1 component peak of YXS tablets was achieved by three kinds of separation methods.The separation degree was greater than 1.5.Ammonia solution washing had some effect on ginsenosides Rg1,Re and Rb1 content,which made the content of ginsenosides Rg1,Re and Rb1 be 1.5-1.8 times to those without alkali washing.No effect was shown on the content of ginsenosides Rg1,Re and Rb1 during ammonia solution washing.It was concluded that some other ginsenosides can be transferred into ginsenosides Rg1,Re and Rb1 in YXS tablets solution after ammonia solution washing.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is an important transcription factor for cells to resist oxidative stress. It interacts with antioxidant response elements to induce the expression of downstream protective phase II detoxification enzymes and antioxidant enzymes and thus exerts a protective effect on cells. Oxidative stress is the common pathogenesis of many liver diseases, and the Nrf2-ARE antioxidant pathway is an important anti-oxidative stress pathway in vivo. It can induce the expression of downstream target genes and thus plays an important role in the development, progression, and prevention of liver diseases. This article briefly describes the mechanism of action of the Nrf2 antioxidant pathway in the development and progression of liver diseases and points out that Nrf2 may be a potential target for the prevention or treatment of liver diseases.

OBJECTIVETo study the effect of hypoxia on Slingshot protein expression in human intestinal epithelial cell and its relation with changes in barrier function of the cells.

METHODSThe human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. One portion of the monolayer-cell specimens were divided into six parts according to the random number table, and they were respectively exposed to hypoxia for 0 (without hypoxia), 1, 2, 6, 12, and 24 h. Transepithelial electrical resistance (TER) was determined with an ohmmeter. Another portion of the monolayer-cell specimens were exposed to hypoxia as above. Western blotting was used to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, claudin-1, Slingshot-1, Slingshot-2, and Slingshot-3. The remaining portion of the monolayer-cell specimens were also exposed to hypoxia as above. The content of fibrous actin (F-actin) and globular actin (G-actin) was determined by fluorescence method. The sample number of above-mentioned 3 experiments was respectively 10, 10, and 18 at each time point. Data were processed with one-way analysis of variance and Dunnett test.

RESULTS(1) Compared with that of cells exposed to hypoxia for 0 h, TER of cells exposed to hypoxia for 1 to 24 h was significantly reduced (P values below 0.01). (2) Compared with those of cells exposed to hypoxia for 0 h (all were 1.00), the protein expressions of ZO-1, occludin, and claudin-1 of cells exposed to hypoxia for 1 to 24 h were generally lower, especially those of cells exposed to hypoxia for 12 h or 24 h (respectively 0.69 ± 0.20, 0.47 ± 0.15, and 0.47 ± 0.22, P<0.05 or P<0.01). Compared with those of cells exposed to hypoxia for 0 h, the protein expressions of Slingshot-1 and Slingshot-3 of cells exposed to hypoxia for 1 to 24 h were not obviously changed (P values above 0.05). The protein expression of Slingshot-2 of cells was decreased at first and then gradually increased from hypoxia hour 1 to 24. The protein expression of Slingshot-2 of cells exposed to hypoxia for 24 h (1.54 ± 0.57) was significantly higher than that of cells exposed to hypoxia for 0 h (1.00, P<0.05). (3) Compared with those of cells exposed to hypoxia for 0 h, the content of F-actin of cells exposed to hypoxia for 1, 6, 12, and 24 h was significantly decreased, whereas the content of G-actin of cells exposed to hypoxia for 6-24 h was significantly increased, P<0.05 or P<0.01; the content of F-actin and G-actin of cells exposed to hypoxia for the other time points was not obviously changed (P values above 0.05).

CONCLUSIONSHypoxia may cause cofilin activation after dephosphorylation and the depolymerization of F-actin by inducing Slingshot-2 protein expression, which in turn affects the tight junction of human intestinal epithelial cells, thus leading to deterioration of barrier function of these cells.

Actins ; metabolism ; Blotting, Western ; Caco-2 Cells ; Cell Hypoxia ; Claudin-1 ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestines ; cytology ; Occludin ; metabolism ; Phosphoprotein Phosphatases ; metabolism ; Tight Junctions ; metabolism ; Zonula Occludens-1 Protein ; metabolism

Objective To explore the effect of the total saponins of panax notoginseng ( TSPN) on depression-like behavior following global cerebral ischemia depression in rats and its mechanism. Method-s Using four-vessel occlusion method to build the global cerebral ischemia model,then the cerebral ischemi-a rats were given solitary breeding with chronic unpredictable mild stress ( CUMS) to prepare depression model. Seventy rats were divided into sham group (n=10),model group ( n=20),PSD group ( n=20) and TSPN group (n=20). The rats in the TSPN group were administered TSPN intraperitoneally 30 min post-brain ischemia. The dose of TSPN (75mg/kg) was suspended in 0. 9% saline 10g/L,once per day for 30 days after reperfusion. While rats in the vehicle group and PSD group was treated with equal volume of 0. 9% saline,one injection per day until the rats were sacrificed at 30 days after brain ischemia. The BrdU,dou-blecortin (DCX) expression in the hippocampus was assessed by immunohistochemistry. Results In com-parison with the model group,the sucrose preference percentage in the PSD group was significantly lower ((46. 2±9. 2)%,(61. 2±7. 6)%;t=3. 18,P<0. 05),then the PSD rats were administered TSPN intraperito-neally,the sucrose preference percentage increased significantly ((62. 4±3. 4)%,(46. 2±9. 2)%;t=3. 43, P<0. 05). During the forced swimming test,the immobility time of PSD group was significantly increased compared with the model group ((119. 4±9. 7)s,(88. 0±15. 6)s ;t=4. 30,P<0. 01),while after PSD rats administering TSPN intraperitoneally,the immobility time was shorten remarkably ((97. 4±6. 7)s,(119. 4± 9. 7)s;t=3. 01,P<0. 05). Compared with the Model group( BrdU+:( 12. 6± 2. 2)/mm2,DCX+:( 38. 6± 4. 2)/mm2),the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in PSD group decreased (BrdU+:(8. 8±1. 5)/mm2,DCX+:(27. 2±2. 8)/mm2;t=3. 25,4. 29,both P<0. 01). And compared with PSD group,the number of BrdU+ and DCX+ cells in the SGZ of hippocampus in TSPN group increased sig-nificantly (BrdU+:(14. 8±2. 8)/mm2,DCX+:(37. 0±3. 3)/mm2;t=4. 68,3. 69,both P<0. 05). Conclu-sion TSPN can improve the depression-like behavior of rats following global cerebral ischemia,which may be related with promoting hippocampal nerve regeneration.

Objective:To investigate the effect of total saponins of Panax notoginseng (TSPN) on learning and memory of post-stroke depression (PSD) rats and its mechanism.Methods:Four-vessel occlusion method was used to build the rat stroke model and 7 days later these rats were given solitary breeding with chronic unpredictable mild stress (CUMS) to make depression model. Rats were randomly divided into Sham group ( n=10), Model group ( n=10), PSD group ( n=10) and TSPN group ( n=10). The rats in the Model group and PSD group were injected administered with equal volume of 0.9% saline 30 min post-brain ischemia, one injection per day for 30 days. while TSPN group were treated with TSPN. The dose of TSPN (75 mg/kg) was dissolved in 0.9% saline 10 g/L, once per day for 30 days. Then the learning and memory of rats were tested by Morris water maze.The protein levels of DCX and Nestin in the hippocampus were detected by Western blot. Furthermore, the DCX/Ki67 co-labeled cells in the SGZ of hippocampus were observed by the immunofluorescence. Results:The escape latency at the fifth day of PSD group((31.8±3.8)s) was longer than that in the Sham group((10.4±3.2)s) and Model group((19.8±3.7)s) ( t=9.23, 5.15; both P<0.05). The escape latency ((14.2±2.8)s) of TSPN group was shortened significantly than PSD group ( t=8.56, P<0.05). The times across the platform in the Sham group was (10.3±1.7), and the PSD group was (4.1±1.1), difference was statistically significant between two groups( t=11.24, P<0.05). The times across the platform (8.4±1.6) of TSPN group statistically increased compared with PSD group ( t=5.77, P<0.05). The protein levels of DCX and Nestin in the PSD group were (0.60±0.02), (0.58±0.03) respectively, and in the TSPN group were (1.07±0.07), (0.95±0.11) correspondingly, there were significant differences of the DCX, Nestin protein level between the two groups( t=20.22, 7.68, both P<0.01). Moreover, there was significant difference in the number of the DCX/Ki67cells in the hippocampus SGZ between the PSD group((16.2±2.8) /mm 2) and TSPN group ((21.2±3.1) /mm 2)( t=2.42, P<0.05). Conclusion:TSPN could improve the learning and memory of the rats with post-stroke depression through enhancing the hippocampus neurogenesis.