Objective To investigate the effect of grape seed proanthocyanidin extract (GSPE) on ultrastructure injury in hippocampous and cognition impairment in rat model of obstructive sleep apnea hypoxia. Methods 80 male Sprague-Dawley rats were randomly divided into control group, model group, high and low dose GSPE groups. The control group was exposed in air, while the model group was suffered from intermittent hypoxia conditions (50 ml/L, 8 h everyday, for 2 or 6 weeks), and the GSPE groups accepted GSPE 200 mg/kg or 100 mg/kg 2 weeks respectively before hypoxia. Pathology in hippocampal region was observed under electromicroscope. Malondialdehyde (MDA) contents and superoxide dismutase (SOD) activity were detected with colorimetry, and apoptotic cells were measured with TUNEL. The cognition function of rats was assessed with the Morris water maze (MWM). Results The ultrastructure in hippocampous was significantly injured,with the increase of MDA and decrease of SOD (P<0.001) in the model group. The apoptotic cells increased (P<0.001). The escaping latency prolonged (P<0.001) and the frequency of crossing the platform decreased (P<0.001) in MWM test in the model group. Compared with the model group, the GSPE groups decreased in MDA content, increased in SOD level, decreased in apoptotic cells and ultrastructure damages, shortened the escaping latency, and increased the frequency of crossing the platform (P<0.001), especially in the high dose group (P<0.05). Conclusion GSPE can relieve the damage of ultrastructure and improve cognition function after obstructive sleep apnea hypoxia in rats.

Objective To establish a diagnosis method for fungal infection using two-round PCR,and to evaluate its sensitivity in the detection of clinical specimens suspected to be infected with fungi.Methods A total of 29 specimens of clinical sputum and alveolar wash solution were collected from patients with suspicious fungal infection.All specimens uaderwent direct microscopy with 10%KOH,fungal culture,one-round PCR and two-round PCR.The fungal universal primer targeting ITS regions of rDNA was used in PCR.The detection rate for fungi was compared between these methods.Results The detection rate for fungi was 20.69%by direct microscopy,37.9%by fungal culture,17.2%by one-round PCR,and 48.3%by two-round PCR.More than one species of fungus were detected in 6.9%(2/29),3.4%(1/29)and 24.1%(7/29)of these specimens by fungal culture.one-round PCR and two-round PCR, respectively.There was a significant difference in the detection rate between two-and one-round PCR(x2=6.34,P＜0.05).With regard to the detection rate for more than one species of fungus,two-round PCR was significantly higher than one-round PCR and fungal culture(x2=4.09,6.30.bom P＜0.05).Conclusion Two-round PCR may help to improve the sensitivity of molecular diagnosis of fungus-infected specimens.

Objective To investigate the application value of CT/MR image fusing in gross tumor volume (GTV) delineation of esophageal squamous cell carcinoma.Methods Twenty-nine patients with esophageal squamous cell carcinoma to be treated with radical surgery underwent routine CT scanning,MR T2-weighted imaging (T2WI) and diffusion weighted imaging (DWI) before surgery.Diffusion-sensitive gradient b values were taken at 400,600,and 800 s/mm2.GTVs were delineated on the CT image,CT/ MR T2WI,and CT/MR DWI respectively.The MR T2W1 image was used as the intermediary for the fusion of the CT image and MR DWI image.The length of GTVs measured under different imaging conditions were compared with the length of the resected specimen of esophagus.Results The GTV length was (44.94 ± 18.46) and (45.05 ±21.47) mm on the CT images and CT/MR T2WI images respectively.When the b values were 400,600,and 800 s/mm2,the esophageal carcinoma GTV length on CT/MR DWI images was (42.12 ± 17.79),(41.18 ± 17.17) and (39.77 ± 17.66) mm,respectively.The coefficient between the esophageal carcinoma GTV lengths on CT/MR DWI images and the pathological lesion lengths was 0.928,0.926 and 0.927 respectively.Conclusions CT/MR DWI images displays esophageal carcinoma GTV length more accurately,thus guiding the delineation of GTV effectively.

Objective To study the effect of astragaloside on TGF-β1,SMAD2/3,and α-SMA expression in the kidney tissue of diabetic KKAy mice,and evaluate its potential role in renal interstitial fibrosis.Methods 20 type 2 diabetic KKAy mice were randomly divided into model group and astragaloside group,while 10 male C57BL/6J mice were selected as the control.Astragaloside at 40 mg · kg-1 · d-1 was given when the KKAy mice fed with high-fat diet to 14 weeks old.The mice in the control and model group received normal saline at 40 mg · kg-1 · d-1.Blood glucose meter was used to detect the blood glucose value of each mice at 16th,20th and 24th week.The mice were killed at 24 weeks old and the kidney tissue samples were collected.Pathology morphological changes were observed.Results (1) blood glucose value:cmpared with the control group,the blood glucose value of KKAy mice at 14 week increased significantly,and that of model group also increased significantly at 16th,20th and 24th week (P＜0.05);the blood glucose value of astragaloside group decreased compared with control group (P＜0.05).(2) Morphology of kidney:in the control group,the glomerular and tubular had clear structure,there was no renal interstitial fibrosis;in the model group,the renal glomerular mesangial matrix had broaden,mesangial cell had increased,renal tubular epithelial cell cytoplasm showed vacuole degeneration,renal interstitial inflammatory cell had increaised.In astragaloside group,there were few renal tubular epithelial cell cytoplasm,and there was no obvious fibrosis.(3)TGF-β1,SMAD2/3,and α-SMA expression levels of the kidney issuse:compared with control group,mice in model group up-regulated TGF-β1,SMAD2/3 and α-SMA expression (P＜ 0.05).TGF-β1,SMAD2/3,and α-SMA expression levels in astragaloside group were significantly lower than those in the model group (P＜0.05).There was few phosphorylated SMAD2/3 expression in renal tubular and glomerular nuclei,while that of model group increased (P＜0.01),and compared with model group,that of the astragaloside group decreased (P＜0.05).Conclusion Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1 and α-SMA expression,thus to relieve renal fibrosis in diabetic mice.

Objective To explore the mechanism of ischemic postconditioning in relieving cerebral ischemia reperfusion (IR) by regulating autophagy through P38MAPK pathway.Methods Cerebral ischemia reperfusion model was established by using modified Pulsinelli four-vessel occlusion (4-VO).Totally 128 male SD rats were divided into 4 groups randomly:control group (sham),cerebral ischemia reperfusion model group (CIR),cerebral ischemic postconditioning group (CIP),and cerebral ischemic postconditioning + P38MAPK inhibitor group (SB203580 group).Each group was subdivided into four time points:6 h,24 h,48 h,and 72 h.The morphological changes of the hippocampus CA1 area neurons at each time point and the number of surviving nerve cells were detected with HE staining.The expression of the hippocampus CA1 area phosphorylated P38MAPK and the autophagy-related genes of Beclin-1 and LC3-Ⅱ were detected with immunohistochemistry.The protein content of the hippocampus phosphorylated P38MAPK and autophagy-related genes of Beclin-1 and LC3-Ⅱ were detected with Western blotting.Results Compared with those in sham group,the damage of rats' hippocampal neuron structure and the survival rate of neurons at each time point decreased in CIR group,the expressions of p-P38MAPK,LC3-Ⅱ and Beclin-1 increased.Compared with those in CIR group,in CIP and SB203580 groups the structure of rats hippocampal neurons was improved,the survival rate of neurons increased,the expression of p-P38MAPK decreased and the expressions of LC3-Ⅱ and Beclin-1 increased at each time point.Compared with CIP group,SB203580 grouphad improved structure of rats' hippocampal neurons,increased survival rate of neurons,decreased expression of p-P38MAPK,and increased expressions of LC3-Ⅱ and Beclin-1 at each time point.Conclusion Cerebral ischemic postconditioning through inhibiting P38MAPK pathway can regulate autophagy and exert its nerve-protective effect.

Objective To explore the regulation of PI3K-mTOR signaling pathways on autophagy of hippocampus nerve cells after subarachnoid hemorrhage (SAH)in rats.Methods We randomly divided 72 male Sprague-Dawley rats into sham group,SAH model group and LY294002 group with 24 rats in each group.We established SAH model with the secondary injection of blood method while the sham group was not injected with blood.PI3K signaling pathways specific inhibitor LY294002 was injected with 500μmol per rat 30 minutes before modeling.After 6,24,72 and 144 h morphologic changes of hippocampus CA1 neural cells were observed by microscopy;the expression levels of PI3K,mTOR,Beclin-1 and LC3-Ⅱ were detected by immunohistochemical method.Results The density of survival neurons in the SAH group was significantly lower than that in the control group (P<0.05),PI3K-mTOR signaling pathways were activated obviously,and the expressions of Beclin 1 and LC3-Ⅱ were significantly higher than those in the control group (P<0.05 ).The number of survival neurons significantly decreased in the LY294002 group compared with the SAH group at each time point (P<0.05),PI3K-mTOR signaling pathways were suppressed.The expressions of Beclin-1 and LC3-Ⅱ were significantly lower than those in the SAH group (P<0.05).Conclusion PI3K-mTOR signaling pathways protect neurons by activating the autophagy of neurons after SAH.

Objective To investigate the effect of butylphthalide on brain edema and the expression of phosphorylated myosin light chain in peri-infarct tissue in focal cerebral ischemic rats.Methods A total of 212 adult male Sprague-Dawley rats were divided into a sham operation group (n =12),a cerebral ischemia group (n =100),and a butylphthalide group (n =100) (40 mg/kg,1/day,gavage) according to the random number table.Both the cerebral ischemia group and the butylphthalide group were redivided into 6 h,12 h,1 d,3 d,and 7 d groups (n =20 for each time point).A rat model of focal cerebral ischemic model was induced by photochemical method; 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used to detect infarct volume (n =5); wet-dry weight method was used to detect the water content in brain tissue (n =5);immunohistochemistry (n =5) and Western blot (n =5) were used to detect the protein expression of periinfarct cortical p-MLC.Results TTC staining showed that no infarcts were observed in the sham operation group.The infarct volumes at each time points in the butylphthalide group were significantly less than those at the same time points in the cerebral ischemia group (all P ＜0.05).Wet-dry weight method showed that the water contents in brain tissue in the cerebral ischemia group and the butylphthalide group were significantly higher than that in the sham operation group (all P ＜ 0.05),but the water contents in brain tissue at each time points in the butylphthalide group was significantly lower than those at the same time points in the cerebral ischemia group (all P ＜ 0.05).Both immunohistochemistry and Western blot showed that the expressions of peri-infarct cortical p-MLC in both the cerebral ischemia group and the butylphthalide group were upregulated significantly compared to the sham operation group (all P＜ 0.05),but the expressions of peri-infarct cortical p-MLC at each time points in the butylphthalide group was significantly lower than those at the same time points in the cerebral ischemia group (all P＜ 0.05).Conclusions Butylphthalide can be up-regulated by reducing the expression of p-MLC caused by cerebral ischemia and reduce cerebral edema,and then reduce the infarct volume,and thus play a neuroprotective effect.

Objective To investigate the effect of BQ-123 on the ability of learning and memory and nerve cell autophagy in hippocam-pus in rats with subarachnoid hemorrhage (SAH). Methods 72 male Sprague-Dawley rats were randomly divided into sham group, SAH model group (SAH group) and BQ-123 group with 24 rats in each group. SAH model was established by injecting the autologous blood into cisterna magna twice. The sham group was not injected blood. BQ-123 group received intracerebroventricular injection with BQ-123 18μg 30 minutes before modeling. 6, 24, 72 and 144 hours after modeling, the passive avoidance latency (PAL) and active avoidance reaction rate (AARR) were tested with Shutter Box Test, the nerve cell morphological changes of hippocampus were observed with HE staining, and the expressions of Beclin-1 and LC3-II were detected with immunohistochemical staining. Results Compared with the sham group, the PAL pro-longed, the AARR decreased (P<0.05), the nerve cells in the hippocampus reduced (P<0.05), and the expressions of Beclin-1 and LC3-II in-creased (P<0.05) in SAH group. Compared with SAH group, PAL shortened (P<0.05), AARR increased (P<0.05), the nerve cells in the hip-pocampus increased (P<0.05), and the expressions of Beclin-1 and LC3-II increased (P<0.05) in BQ-123 group. Conclusion BQ-123 can promote the recovery of learning and memory ability, which may relate to the activation of nerve cell autophagy in the hippocampus.

Objective To investigate the changes in the expression of phosphatidylinositol 3?kinase(PI3?K),mammalian target of rapamycin (mTOR)and Beclin?1 in the hippocampus of normal rats and intermittent hypoxia rats with cerebral ischemia/reperfusion ,so as to explore the role of PI3K/mTOR/autophagy pathway in global cerebral ischemia/reperfusion injury aggravated by intermittent hypoxia. Methods A total of 80 healthy male Wistar rats were randomly divided into sham operation group(SO group,n=20),merely ischemia/reperfusion group(I/R group,n=20),intermittent hypoxia for 7?day ischemia/reperfusion group(IH7+I/R group,n=20),and intermittent hypoxia for 21?day ischemia/reperfusion group(IH21+I/R group,n=20). IH7+I/R group and IH21+I/R group were respectively given intermittent hypoxia for 7 days and 21 days before ischemia/reperfusion. The cerebral ischemia/reperfusion model was established by modified Pulsinelli four?vessel occlusion method. The morpholog?ical changes of nerve cells in hippocampal CA1 region were observed by HE staining and electron microscope. The protein expressions of PI3?K, mTOR and Beclin?1 of nerve cells in hippocampal CA1 region were detected by immunohistochemical staining and RT?PCR. The learning memory capacity of rats were assessed by the Morris water maze test. Results Compared with SO group,I/R group increased the never cells morphology damages,reduced the number of survival neurons,and declined the ability of learning and memory(P<0.05). Immunohistochemistry showed that the number of PI3?K immunoreactive cell,mTOR immunoreactive cell and Beclin?1 immunoreactive cell increased in I/R group compared with S0 group(P<0.05). RT?PCR showed that the expressions of PI3?K,mTOR and Beclin?1 increased in I/R group compared with S0 group(P<0.05). Compared with I/R group,intermittent hypoxia groups increased the never cells morphology damages,decreased the number of survival neu?rons,and declined the ability of learning and memory(P<0.05). Immunohistochemistry showed that the number of PI3?K immunoreactive cell, mTOR immunoreactive cell and Beclin?1 immunoreactive cell increased in IH7+I/R and IH21+I/R groups compared with I/R group(P<0.05). RT?PCR showed that the expressions of PI3?K,mTOR and Beclin?1 increased in IH7+I/R and IH21+I/R groups compared with I/R group(P<0.05),and the changes were more significant in IH21+I/R group(P<0.05). Conclusion Intermittent hypoxia can aggravate neurological injury after ischemia,which is related to PI3K/mTOR/autophagy pathway activation.

Objective To preliminarily explore the effects of human microRNA-181a on migration of gastric cancer cells and its mechanism.Methods The expression of miRNA-181a-5p in gastric cancer cell line GC9811 and peritoneal high metastasis gastric cancer cell line GC9811-P were tested by quantitative real-time polymerase chain reaction (qRT-PCR ). GC9811 cell line was transfected byendogenous synthetic analog mimic and its homologous negative control of miRNA-181a-5p,which were considered as up-regulated group and its control group respectively;GC9811-P were transfected by miRNA inhibitor and its homologous negative control of miRNA-181a-5p,which were considered as down-regulated group and its control group respectively.After miRNA-181a-5p was up or down-regulated,cell proliferation,migration and apoptosis capabilities of gastric cells were detected by matrix thiazolyl tetrazollium (MTT)assay,cloning-forming assay,Transwell migration test,wound healing assays and apoptosis test.After miRNA-181a-5p was up or down regulated,the changes of matrix metalloproteinase (MMP)14 expression were determined by Western blot.Independent sample t test was performed for mean comparison between samples and chi square test was used for rate comparison.Results The results of qRT-PCR showed the relative expression quantity of miRNA-181a-5p in GC9811 was 1 .000 00 ± 0.021 26 and in GC9811-P was 3.175 61 ±0.106 76,and the difference was statistically significant (t =34.620,P <0.01 ).The results of MTT assay indicated that the cell proliferation rate of up-regulated group was higher than that of up-regulated control group,and that of down-regulated group was lower than down-regulated control group.The cloning-forming assay demonstrated that the number of clone forming and clone forming rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 234.00±10.12 and 46.8%,93.00±9.61 and 18.6%,51 .00 ±7.96 and 10.2%,99.00±8.05 and 19.8%,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t = 17.500,7.344,χ2 = 12.27, 9.51 ,all P <0.01).The results of Transwell migration experiment showed the number of cells migrated through membrane hole of up-regulated group was 164.00±19.31 ,and which was higher than that of up-regulated control group (87.00±23.04,t=4.436,P <0.05);that of down-regulated group was 157.00± 11 .50,and which was lower than that of down-regulated control group (234.00 ±12.12,t =7.982,P <0.05).The result of wound healing assays indicated that the rate of migration distance of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were 2.09 ± 0.18,1 .27 ±0.23,1 .15 ±0.15 and 1 .67 ±0.12,respectively.The differences between up-regulated group,down-regulated group and their control group were statistically significant (t =4.863 and 4.689, both P <0.05).The results of apoptosis experiments demonstrated that the apoptosis rate of up-regulated group,up-regulated control group,down-regulated group and down-regulated control group were (6.10± 1 .02 )%,(9.10 ± 2.13 )%,(12.70 ± 1 .23 )%,(8.70 ± 2.54 )%,respectively,and there was no statistically significant difference between up-regulated group,down-regulated group and their control group (both P *0.05).The results of Western blot showed that the grey value of up-regulated group was 561 .881 ±35 .740,which was higher than that of up-regulated control group (275 .784±23.520);that of down-regulated group was 579.565 ±37.950,which was lower than that of down-regulated control group (1 312.760±51 .270),and the differnces were statistically significant (t =11 .580 and 19.910,both P <0.01).Conclusion miRNA-181a-5p highly expresses in peritoneal high metastasis gastric cancer cell line GC9811-P and promoted the proliferation and migration of gastric cancer cell line GC9811 and GC9811-P with a tendency to suppress apoptosis.