Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Genome structure and variation of Reynoutria japonica Houtt. chloroplast genome / 生物工程学报

Reynoutria japonica Houtt., belonging to Polygoneae of Polygonaceae, is a Chinese medicinal herb with the functions of draining dampness and relieving jaundice, clearing heat and detoxifying, dispersing blood stasis and relieving pain, and relieving cough and resolving phlegm. In this study, we carried out high-throughput sequencing for the chloroplast genome sequences of five cultivars of R. japonica and analyzed the genome structure and variations. The chloroplast genomes of the five R. japonica cultivars had two sizes (163 376 bp and 163 371 bp) and a typical circular tetrad structure composed of a large single-copy (LSC) region of 85 784 bp, a small single-copy (SSC) region of 18 616 bp, and a pair of inverted repeat (IR) regions (IRa/IRb) which are spaced apart. A total of 161 genes were obtained by annotation, which consisted of 106 protein-coding genes, 10 rRNA-coding genes, and 45 tRNA-coding genes. The total GC content was 36.7%. Specifically, the GC content in the LSC, SSC, and IR regions were 34.8%, 30.7%, and 42.7%, respectively. Comparison of the whole chloroplast genome among the five cultivars showed that trnk-UUU, rpoC1, petD, rpl16, ndhA, and rpl12 in coding regions had sequence variations. In the phylogenetic tree constructed for the 11 samples of Polygoneae, the five cultivars of R. japonica clustered into one clade near the root and was a sister group of Fallopia multiflora (Thunb.).

Overexpression of a fusion protein of 4-coumaroyl-CoA ligase and polyketide synthase for raspberry ketone production in Chlamydomonas reinhardtii / 生物工程学报

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).

Microbial synthesis of salidroside / 生物工程学报

Salidroside, as one of the main active ingredients of Rhodiala plant, has the effects of anti-hypoxia, anti-radiation, anti-fatigue, anti-tumor, hypoglycemia and improving immunity. With the increasing demand for salidroside and the decreasing of plant resources, microbial production of salidroside has attracted much attention due to its advantages of short period and easy controlling. At present, microbial production of salidroside is still at the basic research stage. In order to make it easier for researchers to understand the advances of microbial synthesis of salidroside, the biosynthesis pathways, uridine diphosphate glucosyltransferases, wild strain/natural enzymes and engineered strain/recombinant enzymes were reviewed.

Effects of erythropoietin administrated by intranasal on nerve function and CD31 in cerebral ischemia reperfusion rats / 中华行为医学与脑科学杂志

Objective To explore the protective effect of erythropoietin(EPO) administrated by intranasal on cerebral ischemia reperfusion in rats with acute cerebral infarction reperfusion.Methods Total of 100 SD rats were divided into model control group,sham operation group,intraperitoneal administration group ([PEPO group),nasal saline group (INNS group) group,and nasal drug delivery group (INEPO group) with 20 in each group.The middle cerebral artery occlusion model of rat was established by thread embolism method and the NSS method was used to evaluate the neural behavior of rats.The expression of EPO in peripheral blood,cerebrospinal fluid and brain regions of rats were detected by Elisa.The vascular endothelial growth factor(VEGF) in brain was detected by immunofluorescence and then the density of newborn blood vessels in the brain was measured.Results Fifteen days after the operation,the NSS score of INEPO group(3.80± 1.61) was significantly lower than that of IPEPO group (11.53±2.11),and the difference was statistically significant(P＜0.01).And the levels of EPO in blood,cerebrospinal fluid and different brain regions of rats in INEPO group were higher than that of INNS group(all P＜0.01).Compared with IPE-PO group,the level of EPO cerebrospinal fluid and different brain regions of rats in INEPO group increased obviously,the difference was statistically significant (all P＜0.01),and the EPO concentration of the olfactory bulb was the most obvious (INEPO group:(1 456.90 ± 128.22) pg/ml,IPEPO group:(426.11 ± 36.68)pg/ml,P＜0.01).Seventy-two hours after operation,the expression of CD31 in ischemic penumbra of rats of model control group (18.21 ± 3.45),INNS group (18.54 ± 2.58),IPEPO group (27.01 ± 2.13) and INEPO group(35.52±2.79)was increased compared with sham operation group (5.14± 1.28),and the difference was statistically significant (all P＜0.05).The expression of CD31 in IPEPO group and INEPO group was significantly higher than that in INNS group (P＜0.05).In INEPO group,the expression of CD31 increased significantly compared with that of IPEPO group (P＜0.05).Conclusion Nasal administration of EPO can effectively improve the neurological function of rats with ischemia-reperfusion,and increase the expression of CD31 in the brain tissue of rats.The effect of nasal administration is better than that of intraperitoneal administration.

Clinical diagnosis of atlanto-axial joint involvement in patients with rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the clinical manifestations,laboratory tests,and image modalities on rheumatoid arthritis (RA) accompanied with atlanto-axial joint involvement.Methods Sixty-four cases of RA accompanied with cervical vertebra involvement were divided into 2groups by lesion location.Twenty-two cases were RA accompanied with atlanto-axial joint involvement,while 42 cases were RA with cervical vertebra other than atlanto-axial joint involvement.The age,course,clinical manifestations and the lab results were compared between the two groups by t test,Chi-square test and Fisher's exact probability.The X-ray,CT and magnetic resonance imaging (MRI) for cervical vertebra were analyzed in RA with atlanto-axial joint involvement.Results Compared with non-atlantoaxial cervical group,the disease course [(15±10) years vs (8±9) years,t=3.030,P=0.004],upper cervical vertebra pain (73％ vs 7％,x2=29.75,P＜0.01),lower cervical vertebra pain (9％ vs 40％,x2=6.813,P=0.009),cervical activity limitation (68％ vs 14％,x2=19.023,P＜0.01),upper cervical vertebra pressing pain (100％ vs 7％,22=52.297,P＜0.01),lower cervical vertebra pressing pain (9％ vs 60％,x 2=15.056,P＜0.01) and erythrocyte sedimentation rate (ESR) [(73±34) mm/1 h vs (53±37) am/1 h,t=2.039,P=0.046)] were significantly different in atlantoaxial joint lesions group.CT combined with MRI exams had high diagnostic..value in RA with atlanto-axial joint involvement.CT scan had the conformed diagnostic value.Sixteen cases were positive (73％) by MRI scan,while 3 cases (14％) by X-ray.Conclusion Timely CT scan and/or MRI scan for RA patients with neck pain in upper cervical vertebra,long disease course,cervical activity limitation and high ESR are helpful for early diagnosis of atlanto-axial joint involvement.

Metformin regulate the balance of Th17/regulatory T cells and expression of serum cytokines through AMPK-mTOR signaling pathway in collagen-induced arthritis / 中华风湿病学杂志

Objective To evaluate therapeutic effects of metformin on the expression of serum cytokines,balance of splenic Th17/regulatory T cells (Treg) and expression of splenic AMPK-mTOR in collagen-induced arthritis (CIA) rats,and the mechanism thereof.Methods The rat model of CIA was established by injecting bovine type Ⅱ collagen.Forty rats were randomly divided into five groups:the CIA model group(sterile water by gavage),Met-30 mg/kg group (metformin 30 mg ·kg-1 ·d-1 by gavage),Met100 mg/kg group(metformin 100 mg ·kg-1 ·d-1 by gavage),Met-300 mg/kg group(metformin 300 mg ·kg-1 ·d-1 by gavage) and control group (sterile water by gavage).The hind paw volume was recorded once a week.The serum level of cytokines,tumor necrosis factor (TNF)-α,interleukin (IL)-1β,IL-6,and IL-17,were measured by enzyme linked immunosorbent assay (ELISA) 35 days after the initial immunization.The quantity of Th17 and Treg cells were examined by flow cytometry.The expression of AMPK,p-AMPK,mTOR and p-mTOR were examined by western blotting.One-way analysis of variance was used to evaluate the experimental data.Results The values of hind paw volume were decreased on the 35 d of the initial immunization in the Met100 mg/kg [(2.43±0.37) ml,t=2.97,P＜0.05] and Met-300 mg/kg groups [(2.58±0.21) ml,t=2.96,P＜0.05] than those of CIA model group (2.97±0.23) ml.The serum levels of TNF-α,IL-1β,IL-6 and IL-17 on the 35-d after the initial immunization were significantly lower in the metformin therapeutic groups [Met-300 mg/kg group TNF-α (104±8) pg/ml,t=42.77,P＜0.05;IL-1β (183±24) pg/ml,t=60.457,P＜0.05;IL-6 (19.3±2.8) pg/ml,t=53.62,P＜0.05;IL-17 (64.5±6.7) pg/ml,t=47.92,P＜0.05] than those of the CIA model group [TNF-α (246±8) pg/ml;IL-1β (1 336±40) pg/ml;IL-6 (87.0±5.1) pg/ml;IL-17 (282.3±6.8) pg/ml].The quantity of Th17 and Treg cells were significantly different in the Met-300 mg/kg group than those of the CIA model group 35 d after the initial immunization [Th17(6.57±0.39) vs (9.89±0.53),t=8.74,P＜0.05;Treg (7.60±0.45) vs (3.94±0.61),t =8.37,P＜0.05].The expression of p-AMPK on the 35 d after the initial immunization in the metformin therapeutic groups was significantly higher than in the CIA model group(P＜0.05),and the expression of p-mTOR was significantly lower (P＜0.05).Conclusion Metformin can significantly inhibit the serum levels of TNF-α,IL-1β,IL-6 and IL-17 in CIA model rats,and regulate the balance of Th17/Treg through AMPK-mTOR signaling pathway.

Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase / 生物工程学报

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.

Effec ts of hypoxic preconditioning on learning and memory in mice with cerebral sichemia-reperfu-sion injury and underlyin g mechanisms / 中华行为医学与脑科学杂志

Objective To investigate the effects of hypoxic preconditioning on learning and memory and the possible protective mechanism in mice with cerebral ischemia-reperfusion injury.Methods Healthy adult male Kunming mice were randomly divided into five groups by Random number table:normal group( N group),hypoxic preconditioning group (HPC group),sham operation group (C group),ischemia-reperfusion group(O group),hypoxic preconditioning and ischemia-reperfusion group(HPC+O group).HPC+O group were given hypoxic preconditioning before 24h of ischemia-reperfusion.The escape latency was detected by Morris water maze and the neuron apoptosis of CA 1 area of hippocampal was determined by immunofluores-cence techniqueR.e sults The escape latency in HPC+O group on the second,third and fourth day of MWM was (39.92±4.52)s,(30.98±2.44)s,(19.69±4.27)s,and significantly lower than that in O group((54.35± 3.66)s,(46.31±4.81)s,(36.81±3.86)s).Mice in HPC+O spent longer time in the target quadrant than that in O group((36.44±5.33)%and(24.5±2.59)%,respectively, P<0.05).Immunofluorescence showed that the apoptotic ration of nerve cells in hippocampal CA 1 was significantly lower than that in O group ( 11.7 ± 0.14 and 1.35±0.14, P<0.05).Conclusion Hypoxic preconditioning can increase hippocampal CA1 neurons hypoxia tolerance of ischemia reperfusion injury in mice,and reduce the incidence of neural cell apoptosis.

Pathways of flowering regulation in plants / 生物工程学报

Flowering, the floral transition from vegetative growth to reproductive growth, is induced by diverse endogenous and exogenous cues, such as photoperiod, temperature, hormones and age. Precise flowering time is critical to plant growth and evolution of species. The numerous renewal molecular and genetic results have revealed five flowering time pathways, including classical photoperiod pathway, vernalization pathway, autonomous pathway, gibberellins (GA) pathway and newly identified age pathway. These pathways take on relatively independent role, and involve extensive crosstalks and feedback loops. This review describes the complicated regulatory network of this floral transition to understand the molecular mechanism of flowering and provide references for further research in more plants.

Comparison of stilbene synthase from different plant sources for resveratrol biosynthesis / 生物工程学报

Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.

Crystal structures of plant uridine diphosphate-dependent glycosyltransferases / 生物工程学报

Glycosyltransferases (GTs) catalyze the transfer of a sugar residue of an activated sugar donor to an acceptor molecule. Many families 1 GTs utilize an uridine diphosphate (UDP) activated sugar as donor in the glycosylation reaction, and most of these belong to a group of GTs referred to as the UGTs. The relationship between the degree of amino acid sequence identity and substrate specificity of the plant UGTs is highly complicated, and the prediction of substrate specificity based on phylogenetic analyses need to be improved by more biochemical characterization. This review summarizes the three dimensional structures of plant UGTs published in the Protein Data Bank (PDB), including the detailed substrate interactions with the sugar and receptor binding pockets and mutational analyses of some critical amino acids. It will be helpful for biochemical characterization the substrate specificity of the individual UGT, and lay the foundation for the enzymatic and genetic manipulation of plant UGTs in the future.

Therapeutic Mechanism of Simvastatin on Collagen Induced Arthritis in Rats / 天津医药

Objective To evaluate therapeutic effects of simvastatin on serum expressions of cytokines and synovial tissue aspartic protease-3 (Caspase-3) in collagen induced arthritis (CIA) in rats, and the mechanism thereof. Methods The rat model of CIA was established by injecting bovine Ⅱ collagen. Sixteen model rats were randomly divided into two groups:CIA model group (sterile water 5 mL·kg-1·d-1 by gavage) and simvastatin group (2.0 mg·kg-1·d-1 by gavage). Seven normal rats were included in control group (sterile water 5 mL·kg-1·d-1 by gavage). The arthritis index (AI) and hind paw vol-umes were recorded once a week. The serum levels of cytokine, tumor necrosis factor (TNF)-αand interleukin (IL)-6 were measured by ELISA 42 days after the initial immunization. The expression of Caspase-3 in ankle synovial tissue was detect-ed by immunohistochemical method, and pathological results of HE staining in rat ankle were compared between three groups. Results Values of AI were decreased on the 24-d of the initial immunization in simvastatin group and CIA model group, which was significantly decreased on the 35-d of the initial immunization in simvastatin group than that of CIA model group (P＜0.05). The values of hind paw volumes were decreased on the 14-d of the initial immunization in simvastatin group and CIA model group, which was still significantly higher than those of control group (P＜0.05). The values of hind paw volumes were decreased on the 35-d and 42-d of the initial immunization in simvastatin group than those of CIA model group (P＜0.05). The serum levels of TNF-αand IL-6 on the 42-d of the initial immunization were significantly lower in simvastatin group than those of CIA model group, but which were significantly higher than those of control group ( P＜0.05). There were more synovial hyperplasia in simvastatin group than those of CIA model group. Only a small amount of inflamma-tory cell infiltration was found in simvastatin group. The expression of Caspase-3 was significantly higher in simvastatin group than that of CIA model group. Conclusion Simvastatin can significantly inhibit the serum levels of TNF-αand IL-6 in CIA model rats, and can up-regulate the expression of Caspase-3 in ankle of model rats.

Effect of protein kinase C inhibitor on the level of phosphralated extracellular regulated protein kinases in the spinal trigeminal nucleus of migraine model rats / 中华行为医学与脑科学杂志

Objective To explore the effect of protein kinase C inhibitor on the level of phosphralated extracellular regulated protein kinases in the spinal trigeminal nucleus of migraine model rats.Methods Healthy adult male SD rats were randomly divided into four groups:normal group (group C),sham operation group (group C),migraine model group(group M),and H-7group(H-7group),with 18 rats in each group.Dural blood flow and the extracellular discharge frequency in the spinal trigeminal nucleus was recorded.ERK1/2 phosphorylation was tested.Results (1) Dural blood flow:compared with group C((3.8± 1.0)％),the dural blood flow in M group ((78.0±4.2) ％)increased obviously(P＜0.01) ; compared with M group((78.0±4.2)％),the dural blood flow in H-7 group((-24.8±4.9) ％) decreased obviously(P＜0.01).(2) The percentage of extracellular discharge frequency change:two hours after treatment,the percentage of extracellula discharge frequency change in group M ((325.9 ±47.3)％)was higher than that in group C((107.3±16.4)％).The percentage of discharge frequency change in group H-7((136.0±26.5)％) was lower than that in group M((325.9±47.3)％).There was no significant difference in the percentage of discharge frequency change between group H-7((136.0±26.5) ％) and group C((107.3 ± 16.4)％).(2) ERK1/2 phosphorylation:the ERK1/2 phosphorylation in group M was higher than that in group C.The ERK1/2 phosphorylation in group H-7 was lower than than that in group C and group M.Conclusion ERK1/2 is a downstream PKC signal path and PKC may have indirect activation of ERK1/2.

Promotive effect of neovascularization on rats with cerebral infarction by intranasal administration of granulocyte colony-stimulating factor / 中华行为医学与脑科学杂志

Objective To study the promotive effect of neovascularization on rats with cerebral infarction by nasal administration of granulocyte colony-stimulating factor.Methods A blinded,vehicle-controlled study of ING-CSF and IHG-CSF administration was performed by intraluminal middle cerebral artery occlusion (MCAO) model.All Sprague-Dawley rats were randomly divided into sham-operation group,model group,INNS group,IHGCSF group and ING-CSF group.The neurologic behavioral tests were assessed after reperfusion 72 h.Mter 72 h of MCAO,the brains of rats were stainned with TTC and the infarcted volume was calculated by computer image analysis.The expression of vascular endothelial growth factor (VEGF) in the brain was determined by immune-histochemistry.The density of angiogenesis in the brain was counted under fluorescence microscope.Results The score of neurological function of ING-CSF group(3.90± 1.65)was improved significantly compared with the IHG-CSF group (10.55±2.19) at the point of 72 h after cerebral infarction (P＜0.01).The cerebral infarct volume of ING-CSF group((20.01±3.29) ％) was reduced evidently compared with the IHG-CSF group((33.48±4.49) ％) at 72 h (P＜ 0.01);while the cerebral infarct volume of INNS group ((60.20±7.72) ％)was not markedly different compared with the model group((61.49±6.41)％) at 72 h (P＞0.05).The expression of VEGF in the brains of ING-CSF group was significantly higher than other groups at 72 h.Conclusion Intranasal administration G-CSF can improve neurological function and vascular angiogenesis in rats following MCAO.

Progress in molecular biology of Jatropha curcas / 生物工程学报

Jatropha curcas L., has been widely recognized as a potential source of biodiesel. In this review, we presented several aspects about the recent progress in molecular biology of J. curcas. First, molecular markers were used to assess its genetic diversity. Second, large-scale genome, transcriptome and proteome analyses were applied for decoding its molecular network. Third, functional characterization of key genes involved in metabolism and regulation of plant development was performed to breed lines with higher quality or higher resistance. Finally, we discussed the limitation of current progress and then proposed the future molecular biology research on J. curcas.

The effects of Penehyclidine Hydrochloride on [ Ca2+ ]i and the expression of cysteine-containing aspartare-specific proteases-3 in neural cells of neonatal rats with hypoxic-ischemic encephalopathy / 中国小儿急救医学

ObjectiveTo explore the influences of Penehyclidine Hydrochloride on [ Ca2 + ] i and the expression of cysteine-containing aspartate-specific proteases-3 (Caspase-3) in neural cells of neonatal rats after hypoxic-ischemic injury within 12 h.MethodsSeven-day-old Sprage-Dowley rats ( n =128) were randomly assigned into four groups:Sham group ( sham operation group,n =32),hypoxic-ischemic encephalopathy (HIE) group ( HIE models,n =32 ),N group ( HIE models treated with Nimodipine,n =32 ),and P group (HIE models treated with Penehyclidine Hydrocloride,n =32).Brain tissues were collected at different time points (2 h,4 h,6 h,and 12 h) after modeling.We utilized fluorescent microscope to detect the expression of Caspase-3 via making frozen slices of rat brain tissue,while [ Ca2+ ]iof neural cells in live brain slices of rats was measured by laser scanning confocal microscope.ResultsCompared with Sham group,both [ Ca2 + ] i and the expression of Caspase-3 of neural cells increased significantly ( P ＜ 0.01 ) in brain cortex in HIE group.[ Ca2 + ] i and Caspase-3 activity increased gradually with time since 2 h after making HIE models and reached a peak at the time points of 12 h ( P ＜0.05).In N group and P group,both of [ Ca2+ ]i and Caspase-3 decreased compared with HIE group (P ＜0.01 ),however,there was no significant difference between N group and P group ( P ＞ 0.05 ).In addition,[ Ca2 + ] i and Caspase-3 activity had no significant difference between 6 h and 12 h (P ＞ 0.05 ).ConclusionPenehyclidine Hydrocloride can protect the brain tissue from hypoxic-ischemic injury via relief of calcium overload and inhibition of Caspase-3 activity.

Effect of CoCl2 on induction hypoxia inducible factor-1α in mice hippocampus depend on time delay / 中华行为医学与脑科学杂志

Objective To observe the effects of CoC12 treatment on the expression of Hypoxia-inducible factor-1(HIF-1α) in mice hippocampus at different time point.Methods Balb/c mice were injected with CoCl2 and the change of HIF-1 α was detected by western blot and immunofluorescence and confocal laser scanning microscope at different time point(0h,1h,2h,3h,4h,5h and 6h) after injection.Results The relative protein level of HIF-1α was 0.135 ±0.01,0.572 ±0.01,0.595 ±0.03,1.09 ±0.03,1.30 +0.04,1.275 ±0.03,0.947 ±0.03respectively at different time point after the injection.The HIF-1α protein level reached its peak value at 4 h and decreased at 5h and 6h.Fluorescence intensity of HIF-1α was 13.33 ± 3.42,30.95 ± 7.86,46.50 ± 9.65,61.50± 10.02,88.30 + 15.69,71.39 ± 11.28,67.41 ± 10.78 respectively at different time point after the injection.The HIF-1α fluorescence intensity also reached its peak value at 4 h and decreased at 5h and 6h.Conclusion Time dependent HIF-1α accumulation was in close correlation with the CoCl2.

A method to evaluate lymphatic drainage of macromolecular tracer in cerebrospinal fluid / 中国药理学通报

Aim To establish a method to evaluate lymphatic drainage of macromolecular tracer in cerebrospinal fluid in rats.Methods Rat cervical lymphatic blockade(CLB)models were established by occlusion of cervical lymphatic tubes and removal of cervical lymphatic nodes.The rats were divided into non CLB(normal controls) and CLB groups.~(125)I-labeled human serum albumin(~(125)I-HSA)was injected into the left lateral cerebral ventricle,and blood samples were collected and ~(125)I-HSA concentrations were detected continually within 24 hours.Concentration-time curve was drawn according to the single compartment model in pharmacokinetics.Parameters of pharmacokinetics such as area under curve(AUC),maximum concentration(C_(max)),transfer rate constant K_a and peak time(T_(max))were derived.The AUC,C_(max),K_a,and T_(max) regarding the lymphatic drainage of ~(125)I-HSA were calculated based on the differences between the two groups.Results AUC,C_(max),K_a of ~(125)I-HSA by lymphatic drainage were 51.97 mg·L~(-1)·h~(-1),2.91 mg·L~(-1),and 0.64 h~(-1),respectively.The proportion of AUC,C_(max),K_a of ~(125)I-HSA by lymphatic drainage to those of drained by both arachnoid granulations and lymphatics was 71.53%,44.02%,58.18%,respectively.T_(max) in CLB group(8.36±0.82 h)was much longer than that in non CLB group(3.57±0.54 h).Conclusions A method to evaluate lymphatic drainage of macromolecular tracer in cerebrospinal fluid in rats is successfully established.The lymphatic drainage pathway plays an important role in eliminating macromolecular substances in cerebrospinal fluid.

Injured effect of cerebrospinal fluid from experimental subarachnoid hemorrhage after cerebral lymphatic blockage on PC12 cells / 中国病理生理杂志

AIM:To determine the injured effect of cerebrospinal fluid(CSF) from subarachnoid hemorrhage(SAH) after cerebral lymphatic blockage(CLB) on PC12 cells. METHODS:SAH and CLB models of adult New Zealand rabbits were used. CSF was obtained from experimental animals after 5 d of modeling and was added into cultured PC12 cells. The cells were randomly divided into blank control(F12 Ham's),normal CSF,SAH CSF,and SAH+CLB CSF groups. At different time points,the survival rate of PC12 cells was measured by MTT assay. LDH leakage was detected. Expression of Bax and heat-shock protein 70(HSP70) was determined by immunohistochemical staining. RESULTS:MTT assay and detection of LDH leakage revealed that the survival rate of PC12 cells was obviously inhibited and the leakage of LDH increased in SAH CSF group and SAH+CLB CSF group. CSF from normal rabbit did not damage the PC12,as compared to blank controls. Above effects were more obvious in SAH+CLB CSF group than those in SAH CSF group. Bax and HSP70 protein expression was found in both SAH CSF group and SAH+CLB CSF group. Expression of Bax protein in SAH+CLB CSF group was stronger than that in SAH CSF group in a time dependent manner. At 0.5 h and 1 h,the expression of HSP70 protein in SAH+CLB CSF group was stronger than that in SAH CSF group,whereas the expression became weaker at 2 h and 4 h in that group. CONCLUSION:Blockage of cerebral lymphatic drainage pathway deteriorates the damage of CSF from SAH on PC12 cells,indicating this pathway may acts as an endogenous protective mechanism in SAH.