OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemia- reperfusion injury in rats. METHODS The model of middle cerebral artery occlusion （MCAO） was established by using the improved thread occlusion method. The experiment was divided into six groups： sham surgery group （only separating blood vessels without inserting thread plugs， given the same volume of normal saline）， model group （modeling， given the same volume of normal saline）， nimodipine group （positive control， modeling， dose of 20 mg/kg）， and low-dose， medium-dose， and high-dose groups of Longshengzhi capsules （modeling， doses of 0.72， 1.44 and 2.88 g/kg， respectively）， with 10 mice in each group. Each group was given corresponding medication solution/normal saline by gavage， once a day， for 7 consecutive days. One hour after the last administration， the Zea Longa scoring method was used to score the neurological deficits in each group of rats， and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rats； TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio. Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats. Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats. Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the brain tissue of rats. Western blot assay was adopted to detect the relative expressions of TLR4， NLRP3 and phosphorylated NF-κB （p-NF-κB） protein in the brain tissue of rats and its intracellular NF-κB protein. RESULTS Compared with the sham surgery group， the neural dysfunction score， serum levels of TNF-α and IL-6， cerebral infarction volume ratio， relative expression levels of NF-κB and TLR4 mRNA， as well as protein relative expressions of TLR4， NLRP3 and p-NF-κB in the brain tissue， and relative protein expression of intracellular NF-κB were increased significantly in the model group （P＜0.01）； the enlarged gap and significant edema were observed in cortical nerve cells of brain tissue in rats， with a large amount of inflammatory cell infiltration； the positive expression of NLRP3 protein in brain tissue of rats obviously increased. Compared with the model group， the levels of the above indicators in the medium-dose and high-dose groups of Longshengzhi capsules， as well as the Nimodipine group， were reversed to varying degrees， and most differences were statistically significant （P＜0.05 or P＜0.01）； the pathological morphology observation showed a significant improvement， and the positive expression of NLRP3 protein in the brain tissue of rats was obviously reduced. CONCLUSIONS Longshengzhi capsules may inhibit TLR4/NF-κB/NLRP3 signaling pathway and neuroinflammatory response， thereby achieving a protective effect against cerebral ischemia-reperfusion injury in rats.

OBJECTIVE：To st udy the effects of α7 nicotinic acetylcholine receptor agonists （PNU282987）on improving cardiac remodeling of mice and Janus kinase 2/signal transducer and activator of transcription 3（JAK2/STAT3）signaling pathway. METHODS：Male Kunming mice were randomly divided into normal control group ，model group ，propranolol group （positive control，i.g. 40 mg/kg）and PNU 282987 low-dose，medium-close and high-dose groups （intraperitoneal injection of 0.5，1.0，3.0 mg/kg），with 10 mice in each group. Except for the normal control group ，mice in the other groups were given isoproterenol （ISO，30 mg/kg） subcutaneously for 7 days to induce the cardiac remodeling model. After 30 minutes of ISO injection ， administration groups were given relevant liquid ，once a day ，for 7 consecutive days. Twelve hours after last administration ，the left ventricular ejection fraction （EF）and left ventricular short axis shortening rate （FS）of mice in each group were measured ，and the whole heart mass index （HMI）was calculated ；the pathological changes of myocardium were observed. The serum contents of lactate dehydrogenase （LDH），creatine kinase （CK），tumor necrosis factor α（TNF-α），interleukin 6（IL-6），the protein expression of intercellular adhesion molecule 1（ICAM-1）and adhesion molecule 1（VCAM-1）were also determined. The ratios of p-JAK2/JAK2，p-STAT3/STAT3 in myocardial tissue were detected. RESULTS ：Compared with normal control group ，EF and FS of model group were significantly reduced ，HMI，the contents of LDH，CK，TNF-α and IL-6，the protein expression of ICAM- 1 and VCAM- 1，the ratio of p-JAK 2/JAK2 and p-STAT 3/STAT3 were increased significantly （P＜0.05 or P＜0.01）； blue collagen deposition in the interstitium of myocardium was obvious，and the degree of fibrosis was severe. Compared with model group ， the EF and FS of the mice in the medium-dose and high-dose groups were increased significantly ， HMI （except for PNU 282987 medium-dose group ），the contents of LDH （except for PNU 282987 medium-dose group ），CK，TNF-α and IL-6，the protein expression of ICAM- 1 and VCAM- 1，the ratio of p-JAK 2/JAK2 and p-STAT 3/STAT3 were decreased significantly （P＜0.05 or P＜ 0.01）；blue collagen deposition in the myocardial interstitium was significantly reduced ，and the degree of myocardial fibrosis was significantly reduced. There was no significant difference in the comparison of the above indicators in PNU 282987 low-dose group （P＞0.05）. CONCLUSIONS ：PNU282987 can improve cardiac remodeling of mice ，the mechanism of which may be associated with inhibiting JAK 2/STAT3 signaling pathway.

Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6%, and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6%. Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.

Objective Changes of thyroid stimulating antibody(TSAb) and thyroid stimulating blocking antibody(TSBAb) in the treatment of anti-thyroid drugs(ATDs), and the effect of ATDs combining with levothyrocine(LT4) on TSAb and TSBAb were analyzed. Methods Using recombinant Trxfus. TSHRn protein and Trxfus. TSHRc protein as antigens, and TSH receptor antibody(TRAb)-N(TSAb binding hot spots), TRAb-C(TSBAb binding hot spots)in the serum of thyroid disease patients were measured with ELISA. The changes of TRAb-N, TRAb-C over 36 months in 117 TRAb-N positive Graves′ patients with hyperthyroidism were analyzed retrospectively. In the course of treatment, 41 cases as A group with ATDs and LT4 treatment, 76 cases as B group with only ATDs, The changes of TRAb-N and TRAb-C were observed in the two groups. Results (1)According to the change of TRAb-N, 117 TRAb-N positive Graves′ patients with hyperthyroidism were different. In group Ⅰ, 10 patients continued to have persistently positive TSAb and continued to have hyperthyroidism, remission rate 0%. In group Ⅱ, 17 patients showed complicated TRAb-N changes, 12 of 17 patients got relapse, 5 of 17 patients got remission, remission rate 29.4%. And in group Ⅲ, with TRAb-N dropping gradually, 15 of 89 patients got relapse, 74 of the 89 patients got remission, remission rate 83.1%. Three groups were significantly different with x2 test(P<0.01). One of the 117 TRAb-N positive Graves′ patients with hyperthyroidism developed TRAb-C positive hypothyroidism. (2)According to combining with and without LT4 during the treatment of ATDs,the patients were divided into 2 groups(Group A: ATDs combined with LT4; Group B: only ATDs). These 2 groups were significantly different in TRAb-N at baseline and 3 months(P<0.01), TRAb-C between two groups were not significantly different in all times(P>0.05). Conclusion TSAb and TSBAb can be used to document TRAb-function, which is significant for us to predict the changes of thyroid function. During ATDs treatment, the temporary early low-dose application of LT4 did not significantly affect TSAb and TSBAb.

Objective To analyze the hot research spots of MOOC research of higher medical edu-cation in China. Methods We searched the database of China National Knowledge Infrastrucure (CNKI), VIP database of Chinese journal (VIP), Wanfang Database for published studies on MOOC research of higher medical education from 2005 to April 2015. The high frequency words were counted by an analysis program-Bibliographic Item Co-occurrence Matrix Builder (BICOMB). According to the ranking word frequency threshold formula by Donohue, we collected the high-frequency keywords. The corresponding co-word ma-trixes were constructed by Bibexcel, then the data was input into SPSS 19.0 software for hierarchical clus-tering analysis. Results Finally 2 459 articles were included. 11 177 keywords and 13 high frequency words (the number of the real selected was 24) were taken into account. The keywords “stanford” clus-tered with “education module”, “online education”, “online courses”, “distance education”, “learning pro-cess”, “self-regulated learning”, “information technology”, “classroom teaching”. The keywords “network course” clustered with “teaching design”. The keywords “online learning” clustered with itself. The key-words “challenge” clustered with “colleges and universities”. The keywords “flipped classroom” clustered with“micro-lecture”,“MOOC”,“higher education”, “library”, “teaching module”, “teaching reform”, “big data”,“open education”. The keywords“SPOC”clustered with itself. Conclusion The research of MOOC research of higher medical education in our country has formed the basic characteristics with six hot spots involving teaching design, the model of online learning, dispassion thinking, teaching reform, and develop-ment. And there are many potential areas.

To explore the long-term effects of L-T4 treatment on physical and intellectual development in neonatal congenital hypothyroidism(CH) patients during adolescence and adulthood.14 out of 15 patients,aged 15-31 years old,including 9 males and 6 females,were diagnosed by neonatal screening for congenital hypothyroidism.(1) By treatment for 10 years until adolescence,return visit ages were 12.6-18.1 years old,the height of patients were normal in 14 cases,weight normal in 8 cases,and overweight in 6 cases.Only the first patient of neonatal screening for congenital hypothyroidism height 154 cm,weight of 43.5 kg,which were below the standard.Bone age by X-ray showed 9 normal,1 case of rapid development,4 cases with left wrist bone age retardation.IQ combined Raven's test(CRT) showed 3 cases excellent,7 cases normal,3 cases borderline,and 2 cases low.(2)Treatment for more than 20 years to adulthood,9 cases of return visits (8 cases were screened out) by 23-31 years of age,with 5 males and 4 females,height and bone age were all normal,normal weight,only 2 cases BMI slightly overweight.As to IQ,good were in 7 cases,mild retardation in 2 cases.7 patients received above average education;they were all employed except one.The employed patients were all capable for their jobs.In summary,screening out of 8 patients,1 case were of mild mental retardation (12.5％);while 7 patients(87.5％),both in physical and intellectual levels were as those of the normal population.

Objective To explore the value and methods of echocardiographic application in percutaneous left atrial appendage(LAA) closure for stroke prevention in patients with nonvalvular atrial fibrillation.Methods 6 male patients with nonvalvular atrial fibrillation were enrolled for percutaneous LAA closure,the mean age was (68.7 ± 5.6) years old,the mean CHADS2 (congestive heart failure,hypertension,age≥75 years,diabetes mellitus,and prior stroke or transient ischemic attacks) score was 3.2 ± 1.0.Rheumatic valvular diseases were excluded by transthoracic eehocardiography(TTE) before closure procedure.Transesophageal echocardiography(TEE) was performed to guide the punctures of the atrial septum and then monitored the operation all through the closure procedure.Diameter of LAA orifice was measure by TEE to help choosing the closure device.Immediate results of closure and complications were inspected by TEE simultaneously.24 hours,7 days,3 months,6 months and 1 year follow-up were performed using TTE.Results All the 6 patients underwent LAA closure successfully.3 LAmbre(Lifetech Scientific,Shenzhen) devices and 3 Watchman(Boston Scientific,Natick,Massachusetts) devices were implanted respectively in the 6 patients.Mean diameter of the LAA orifice was (22.4 ± 3.3)mm,and mean size of the closure devices was (28.0 ± 2.9) mm.2 mm in width residual flow at the inferior edge of closure device existed in 1 ease.No complication was observed.Post-procedure 24 hours and 7 days post-procedure followup showed optimal results in all cases.Conclusions Implantation of both LAA closure devices can be performed with high success rates in patients with nonvalvula ratrial fibrillation,with high risk for stroke,and who either had contraindication or were not willing to accept oral antieoagulation.Echocardiography plays a core role all through the closure procedure and can make it safer and more efficient.

ObjectiveTo observe the changes in TSH receptor antibody (TRAb) and soluble intercellular adhesion molecule-1 ( sICAM-1 ) levels after treatment of propylthiouracil ( PTU ) and methimazole( MMI ) in patients with Graves' disease (GD). MethodsOne hundred and six cases of clinically diagnosed patients with GD were divided into PTU and MMI groups( each group, n =53 ). The patients in two groups were regularly followed for 36 months. TRAb and sICAM-1 were measured with ELISA method. Results( 1 ) The general data of two groups were not significantly different before treatment( all P＞0. 05 ). ( 2 ) There was no difference in TRAb positive rates between two groups before treatment. The clinical remission rates between PTU and MMI groups showed no difference (83.02％ vs88.68％ ). The cure rate was higher in MMI group than in PTU group( 58.49％ vs 37.74％, P＜ 0. 05 ). (3) There existed significant differences in TRAb levels determined before and 6, 12, 24,30, and 36 months after treatment( all P＜0. 01 ), being gradually decreased with time( F=275.48 ,P＜0.01 ). TRAb levels between two groups were significantly different( F=5.86, P＜0. 05 ). (4) sICAM-1 levels at 36 months after treatment compared with the baseline in both groups were statistically different (P＜0. 01 ), but no difference was found between two groups.ConclusionsBoth PTU and MMI improve the immune status of patients with GD,and the immunosuppressive effect of methimazole is more evident.

Objective To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms. Methods According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group( incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group( incubated with 100 μ mol/L atorvastatin), the fluvastatin group (incubated with 100 μ mol/L fluvastatin)and the pravastatin group (incubated with 100 μ mol/L pravastatin). Stimulated by 2. 8,5. 5,11.1,16. 7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 μ mol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37℃ bath incubation method and ATP content was measured by chemiluminescence method. Results Incubated with 100 μ mol/L atorvastatin for 30 minutes, in the presence of 16. 7 mmol/L glucose, the ATP content [(9. 54 ± 1. 64) pmol/islet vs ( 12. 33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0. 21 vs 2. 39 ± 0. 30) were significantly reduced in comparison with the control group (P＜0. 05). Cultured with 100 μmol/L fluvastatin for 24 hours, the ATP content [( 10. 24 ±2.01 )pmol/islet vs (12. 31 ±2. 16) pmol/islet] and GSIS (3. 12 ± 0. 32 vs 4. 17 ±0. 37 ) were all significantly decreased at the higher glucose concentration of 16. 7 mmol/L ( P ＜ 0. 05). Conclusion Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.

Objective Using recombinant TrxTSHRc protein which contains TSBAb antigen epitope as antigen, an enzyme linked immunosorbent assay (ELISA) was established. The cut-off of positive values of TSH receptor antibody (TRAb) was established. Methods Using recombinant TrxTSHRc protein as antigen, the optimum condition was determined, and the indirect ELISA for detecting TRAb (TRAb-C) in the serum of normal control subjects and thyroid disease patients was established. A total of 1 085 patients and healthy controls were tested for this antibody. Results TRAb-C ELISA mainly detected TSBAb. The A405((-x)±s) value in healthy group was 0. 319±0. 107 with a cut-off value ((-x)±2 s)of 0. 533. The positive rate in thyroid disease patients showed significant deviation, with 66. 01 % in patients with Hashimoto's thyroiditis, 41.68% in newly-diagnosed Graves'disease, 6.98% in simple goiter, 11.1% in subacute thyroiditis, 8.7% in nontoxic thyroid nodular goiter, and 3.2% in thyroid adenoma. Conclusion The TRAb-C ELISA mainly detects TSBAb, which is very valuable for autoimmune thyroid disease, especially for the diagnosis, prognosis and therapy evaluation in Hashimoto' s thyroiditis.