ObjectiveTo investigate the effect of puerarin on the expression of pigment epithelium-derived factor (PEDF) in the retina of diabetic rats.MethodsThe diabetic retinopathy rat model was established.Rats were randomly divided into three groups (normal,diabetic,and diabetic with puerarin injection).The pathological changes of the retinopathy were examined.PEDF was analyzed with PCR in different time point.Results Body weight of diabetic group [ ( 216.9 ± 8.37 ) g,and(179.1 ±7.56)g] was compared with normal group[ (298.2 -±6.78)g,and(323.5 -±6.45)g] in the 3 month and 5 month ( q =12.39,P ＜0.01 ; q =15.47,P ＜0.01 ).Blood glucose of diabetic group and diabetic with puerarin injection group was compared with normal group at each time point ( P ＜ 0.01,P ＜ 0.05 ).HE staining of the retina of diabetic rats indicated a diabetic retinopathy occurred in the early - stage in diabetic group,but changed slightly in the diabetic model with an injection of puerarin.The PEDF expression at the 5 month time point in normal group,diabetic group,and diabetic group with puerarin injection was 8.833 (1.200),1.650(3.300),and 8.350( 1.100),respectively.Compared with normal group and diabetic group with puerarin injection,the level of PEDF was significantly decreased in diabetic group (x2 =48.57,P ＜ 0.01 ;x2=48.46,P ＜ 0.01 ).ConclusionsPuerarin can alleviate the retinal pathological damage of diabetic rats,early intervention with puerarin can prevent from the expression of PEDF and delay the diabetic retinopathy process.

Objective To study the relationship between Xsp Ⅰ restriction fregment length polymorphism (RFLP) in exon 4 of low-density lipoprotein receptor (LDLR) gene and hypercholesterolemia. Methods PCR-RFLP method was applied to determine the polymorphism of LDLR gene in 446 cases of hypercholesterolemia, 284 of borderline hypercholesterolemia and 187 normal control subjects. Results Three genotypes, X+X+, X+X- and X-X-, were found in the population. (1) The frequencies of the X+X+ genotype and X+ allele in the group with hypercholesterolemia were higher than those of the other 2 groups (P<0.05). (2) From X-X- to X+X+ genotype, cholesterol and low-density lipoprotein-cholesterol increased gradually, while high-density lipoprotein-cholesterol decreased (all P < 0.05). (3) With logistic regression analysis, LDLR genotype was associated with hypercholesterolemia. Conclusion Polymorphism in Xsp I restriction site at the exon 4 of LDLR (X+ X+ genotype and X+ allele) may be a risk factor of hypercholesterolemia in Chinese population.

The primary neural stem cells were isolated from SD rat and formed the neuropheres, the neuropheres were passaged and planted on the dish coated with 0.1% gelatin, the colony was picked up under the microscope, then dispersed and cultured, to obtain the clone proliferated from one cell, passaging and picking up the cells 5～6 times at least. The NSC and its differentiated cells were identified with the marker genes respectively. The results showed that the neural stem cells were isolated from the SD rat embryos and the real clone were obtained by picking up the cells again and again, and then cultured in the form of monolayer. The marker genes of the neural stem cells and its differentiated cells could be detected at last. It will provide the rat model the resource of the cells for the treatment and the basic research for the morphology standard.

Objective To investigate the characteristics of executive function in patients with brain injury. Methods From March 1st, to June 30th, 2015, 44 patients with brain injury were investigated with Wisconsin Card Sorting Test (WCST), the indexes including Responses Answer, Categories Completed, Correct Responses, Errors Responses, Trials to Complete First Category, Percent Conceptual Level Respons-es Percentage, Perseverative Responses Errors, Nonperseverative Responses Errors, Failure to Maintain Set, and Learning to Learn. Results The abnormal rates were the most in Nonperseverative Responses Errors and Percent Conceptual Level Responses Percentage (61.36%), and then in Responses Answer/Categories Completed/Correct Responses (59.09%), Correct Responses (43.18%), Trials to Complete First Category (38.64%), Perseverative Errors (29.51%), Learning to Learn (25.00%), and Failure to Maintain Set (9.09%). The patients with trau-matic brain injury were different from those with stroke in Responses Answer, Errors Responses, Perseverative Responses Errors, Catego-ries Completed, Percent Conceptual Level Responses Percentage, and Learning to Learn (Z>2.444, t>2.156, P<0.05). The patients injured in frontal lobe were different from those in other areas in Perseverative Responses Errors (t=2.595, P=0.015). Conclusion Executive function damaged generally in patients with brain injury, which related to concentration, abstract, shifting attention, working memory, etc. The frontal lobe damage may associate with the disorder of shifting attention.

Calcium-dependent protein kinases (CDPKs) play an important regulatory role in the plantarbuscular mycorrhiza/rhizobium nodule symbiosis. However, the biological action of CDPKs in orchid mycorrhiza (OM) symbiosis remains unclear. In the present study, a CDPK encoding gene, designated as DoCPK1 (GenBank accession No. JX193703), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoCPK1 was 2137 bp in length and encoded a 534 aa protein with a molecular weight of 59.61 kD and an isoelectric point (pI) of 6.03. The deduced DoCPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain and four Ca2+ binding EF hand motifs. Multiple sequence alignment demonstrated that DoCPK1 was highly homologous (85%) to the Panax ginseng PgCPK1 (ACY78680), followed by CDPKs genes from wheat, rice, and Arabidopsis (ABD98803, ADM14342, Q9ZSA2, respectively). Phylogenetic analysis showed that DoCPK1 was closely related to CDPKs genes from monocots, such as wheat, maize and rice. Real time quantitative PCR (qPCR) analysis revealed that DoCPK1 was constitutively expressed in the included tissues and the transcript levels were in the order of roots > stems > seeds > leaves. Furthermore, DoCPK1 transcripts were significantly accumulated in roots 30 d after fungal infection, with 5.16 fold compared to that of the mock roots, indicating involvement of DoCPK1 during the early interaction between D. officinale and Mycena sp., and a possible role in the symbiosis process. This study firstly provided important clues of a CDPK gene associated with OM symbiosis, and will be useful for further functional determination of the gene involving in D. officinale and Mycena sp. symbiosis.

The mitogen-activated protein kinase (MAPK) cascade, composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK, is abundantly conserved in all eukaryotes. MAPK along with MAPK cascade plays a vital regulatory role in the plant-arbuscular mycorrhiza/rhizobium nodule symbioses. However, the biological function of MAPK in orchid mycorrhiza (OM) symbiosis remains elusive. In the present study, a MAPK gene, designated as DoMPK1 (GenBank accession No. JX297594), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK1 was 1 263 bp and encoded a 372 aa protein with a molecular weight of 42.61 kD and an isoelectric point (pI) of 6.07. The deduced DoMPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain (39-325) and MAP kinase signature (77-177). Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK1 was highly homologous (71%-85%) to MAPK genes from various plant species and was closely related to those from monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK1 was constitutively expressed in leaves, stems, roots and seeds, and the transcript abundance was not significantly different in the four included tissues. Furthermore, DoMPK1 transcript was markedly induced in roots at 30 d after fungal infection, with 7.91 fold compared to that of the mock inoculated roots, suggesting implication of DoMPK1 in the early D. officinale and Mycena sp. interaction and an essential role in the symbiosis. Our study characterized a MAPK gene associated with OM symbiosis for the first time, and will be helpful for further functional elucidation of DoMPK1 involving in D. officinale and Mycena sp. symbiotic interaction.

BACKGROUND: At present, there is few reports about using middl ecerebral artery obstraction (MCAO) model to determine the repair course of cerebral infarction during functional training.OBJECTIVE: To determine the effect of electro-stimulating therapy on promoting the rehabilitation of cerebral infarction and its mechanism.DESIGN: Randomized controlled study.SETTING: Animal Center and Electron Microscope Laboratory of Zhongshan University.MATERIALS: The experiment was carried out in the Animal Center of Zhongshan Medical College and Neurological Laboratory of the First Affiliated Hospital of Zhongshang University from January 2002 to December2004. A total of 200 healthy males SD rats, aged 3 months and weighing 90-110 g, were selected. According to the following criteria: SBP＞180mmHg (1 mmHg=0.133 kPa), BWT score of MCAO models which were reproduced by RHRSP was 1, totally 180 RHRSP were admitted to the research and divided into electro-stimulating therapy group (n=90) and control group (n=90).METHODS: Electro-stimulating was given to four accupuncture points of the paralyzed limbs of rats. The electro-stimulating treatment was given about 30 minutes once a day. And a therapy course was 6 days, and between two therapy courses there was one-day break. At the end of 1st, 3rd,6th and 9th therapy courses, the brain of motor function and tissue in marginal zone of cerebral infarction were assayed as follow: [1] The beam walking test (BWT, 1 as severe disorder and 7 as normal). [2] Electron microscope. [3] Astrpcyte glial fibriliary acidic protein, neurofilament protein and microtubule-associated protein-2 were assayed with immunohistochemistry. Five fields of each slice in the two groups were randomly selected to add up the positive cell number. Totally 30 positive cells of glial fibriliary acidic protein was selected to assay average absorbency (A) of positive cellular plasm. [4] Apoptosis of neurons were observed with in situ end-labeling (ISEL). [5] Brain-micro vasodilatatio was observed according to the criteria of one complete microvessel account under the field.MAIN OUTCOME MEASURES: [1] Scores of motor function; [2] Ultramicrostructure of cranial neurons and astrocyte; [3] Cranial glial fibriliary acidic protein, neurofilament protein and microtubule-associated protein-2;[4] Apoptosis of neurons; [5] Diastole of cerebral microvessel.RESULTS: Totally 180 rats were eligible while 20 rats were excluded because of their BWT score＞1 after MCAO operation. [1] Results of beam walking test (BWT): Functional recovery of paralysis limbs in electric stimulation group was better than that in control group from the third to the ninth course. In the ninth course, 6 points of rats in electric stimulation group was more than that in control group (42, 46, χ2=15.4, P ＜ 0.01). [2]Positive absorbency of cerebral glial fibriliary acidic protein: That in electric stimulation group was higher than that in control group in the 3rd, 6th,and 9th [(52.97±0.59)% vs (46.40±0.56)%; (49.44±0.80)% vs (46.40±0.56)%;(43.25±0.48)% vs (34.20±0.50)%, P ＜ 0.05]. [3] Assay of neurofilament protein: That in electric stimulation group was higher than that in control group in the 6th and 9th course [(22.9±2.7)% vs (11.9±2.3)%; (26.5±1.7)%vs (11.7±1.5)%, P ＜ 0.05]. [4] Assay of microtubule-associated protein-2:That in electric stimulation group was higher than that in control group in the 6th and 9th course [(21.7±1.3)% vs (11.3±1.1)%; (24.4±2.1)% vs(11.9±2.3)%, P ＜ 0.05]. [5] Apoptosis of neurons: There was not significantly different between the two groups. [6] Results of open number of cerebral microvessel: That in electric stimulation group was higher than that in control group in the 1st, 3rd, 6th and 9th course (33 vs 19; 48 vs 31;45 vs 25; 46 vs 23, Z=-2.309, P ＜ 0.05).CONCLUSION: Electro-stimulating treatment can promote motor function of paralyzed limbs, which was due to that electro-stimulating treatment may promote extinction of the swollen feet of astrocytes, reinforce neurons activity and arouse the dilatation of cerebral capillary which promote the microvascular dilatation in order to improve cerebral blood circulation.

Objective To observe the effects of high-fat diet on liver steatosis and liver endoplasm reticulum stress in mice and to investigate the interventional effect of metfomin on them.Methods Forth-five male C57BL/J6 mice were divided into healthy control group,high-fat group and metformin group.High fat group and metformin group were fed with high-fat diet.Mice in metformin group were given metformin since the fourth week of high-fat feeding.After feeding for eight weeks,subperitoneal glucose tolerance test was performed in mice.After mice were sacrificed,liver triglyceride (TG) content and the expression of endoplasmic reticulum stress related factors at gene and protein levels were measured.One-way ANOVA was applied for analysis between groups.Results Compared with healthy control group,area under curve (AUC) of glucose tolerance and TG contents in liver tissues significantly increased in high-fat group [998±87 vs 1409±106,(10.05±0.29) μmol/g vs (27.11 ±4.76) μmol/g].Glucose tolerance and liver steatosis were improved in metformin group,AUC and TG of metformin group were significantly lower than those of high-fat group in metformin group [1178±90,(15.12±2.11) μmol/g,F=55.328,89.212,both P ＜0.01].Compared with healthy control group,the expression of glucose-regulated protein 94 (GRP94)at mRNA level significantly increased in high-fat group.Meanwhile,the expression of phosphorylated eukaryotic translation initiation factor 2α at protein level,which indicated endoplasmic reticulum stress,significantly increased.However,the expression of those endoplasmic reticulum stress markers at mRNA and protein level of metformin group were both lower than those of high fat group (F=84.002,137.321,both P＜0.05).Conclusions High fat diet caused liver steatosis in mice and accompanied by endoplasmic reticulum stress.Fatty liver was significantly improved by metformin treatment in high-fat-fed mice.The mechanism may be related with the improvement of endoplasmic reticulum stress by metformin.

Twenty healthy subjects in each of 3 groups were fed with monounsaturated fatty-acid diet,polyunsaturated fatty-acid diet, or saturated fatty-acid diet separately for 3 days. It suggested that monounsaturated fatty-acids may ameliorate the oxidative stress and improve insulin sensitivity.