The early protective effect of NADPH on OGD/R injury of human umbilical vein endothelial cells / 天津医药

Objective To study the early protective effect of NADPH on human umbilical vein endothelial cells (HUVECs) and the expression of occludin and MMP9 induced by oxygen glucose deprivation and reoxygenation (OGD/R). Methods HUVECs were cultured and divided into blank control group, OGD/R group and OGD/R+NADPH 20 μmol/L group. The proliferation of HUVECs after treatment was detected by CCK-8 assay. The cytotoxicity was detected by LDH release assay. The morphological changes of HUVECs were observed by inverted microscope. Superoxide dismutase (SOD MDA) activity and malondialdehyde (MDA) were detected by commercially available kit. The expressions of occludin and MMP9 were detected by Western blot assay. Results Compared with the OGD/R, NADPH enhanced the cell viability significantly (P<0.05), reduced the release of LDH (P<0.05), promoted the maintance of HUVECs morphology, reduced MDA generation (P<0.05) and increased SOD activity (P<0.05). Following OGD/R,the treatment of NADPH can inhibit MMP9 level (P<0.05) and promote the recovery of occludin level (P<0.05). Conclusion NADPH can protect HUVECs from the damage induced by OGD/R by reducing oxidative stress and regulating the expressions of MMP9 and occludin.

Whole blood allele-specific PCR, a simple method to detect four ATP7B gene mutations in Wilson disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a simple method to detect four ATP7B gene mutations in Wilson disease using allele-specific PCR(AS-PCR) with whole blood polymerase chain reaction.</p><p><b>METHODS</b>Four allele-specific PCR primers specific for the mutations(G2333T, C2850T, G2855A, G2975T) were designed, and PCR was optimized to screen the whole blood samples. The amplified gene products with mutation were separated with agarose gel electrophoresis to detect the pattern of point mutation and allele types. Exons 8, 12 and 13 of the ATP7B gene were amplified with PCR, and the amplification products were sequenced to confirm the mutation.</p><p><b>RESULTS</b>The detection of four ATP7B gene mutations by AS-PCR with whole blood was accomplished with 100% accuracy. In the 27 healthy subjects, the mutation rate of G2855A was 51.8%. No mutation was detected for G2333T, C2850T and G2975T. Among the 22 patients, 11 were mutated for G2333T, C2850T or G2975T. The mutation rate was therefore 50%.</p><p><b>CONCLUSION</b>Our experiment has established an AS-PCR based method for detecting four ATP7B gene mutations using whole blood samples, which has provided a simple and effective means for the early diagnosis of Wilson disease. This method is rapid, convenient, accurate and economical for detecting point mutations of the ATP7B gene.</p>

The value of supplement of recombinational Tth-single strand binding protein in polymerase chain reaction for genotyping / 中华检验医学杂志

ObjectiveTo explore the value of Tth-single strand binding protein (SSB) used as an additive to improve the polymerase chain reaction (PCR) specificity for single nucleotide polymorphisms(SNP) alleles genotyping.MethodsTth-SSB plasmid was constructed and the protein was expressed,then the expressed Tth-SSB was added into PCR system detecting cytochrome P450 Protein ( CYP2C19 * 3,636G＞A)genotype to determine the optimal usage and condition.Then,the genotypes of 30 cerebral ischemia patients were tested with established methods and compared with direct sequencing to verify the accuracy of Tth-SSB as an additive into PCR for SNP genotyping.ResultsThe purity of Tth-SSB was 85％ and optimal dosage was 1 μg.The protein could improve the specificity and reduce the dimer when Tth-SSB was added into the PCR system.Thirty patients genotyping results as follow:26 patients belong to G/G homozygote,4 patients belong to G/A heterozygote,no body belong to A/A homozygote.The coincidence acquire 100％ with parallel sequencing.ConclusionAs an additive,Tth-SSB could significantly improve the accuracy of genotyping by eliminating non-specific bands.

Autophagic pathway and probable mechanism in degradation of mutant α-synuclein in PC12 cells / 中华神经科杂志

Objective To observe the effect of mutant α-synuclein(A30P)in autophagic programmed cell death by transfected PC12 cells and explore its probable role and pathway in PD.Methods The definite PC12 cells which were transfected mutant α-synuclein(A30P)were constructed at first and MPP+,Rapamycin and Wortmanin were administrated to transfected PC12 cells with mutant α-synuclein. Not only the proliferative activity of cells was detected with MTT method but also the ultrastructttre changes of cells and expression of α-synuclein in different circumstance were observed by transmission electron microscopy(TEM),Western Blot and the level of SOD.Results (1)The expression of α-synuclein in groups A30P+Wortmannin and A30P+MPP+was higher than that in group A30P(P＜0.01), particularly.there was more significant expression of α-synuclein in group A30P+Wortmannin.The expression of α-synuclein in group A30P+Rapamycin was weaker than that in group A30P(P＜0.01); (2)The results showed that the SOD level(group A30P+MPP+:3 h:97.49±13.8;12 h:102.7±12.7; 24 h:101.5±11.8;48 h:104.3±12.4)was significantly decreased at various time points after MPP+ treatment compared that of group A30P(t=3.7721,P=0.0017).SOD level gradually increased in A30P +Rapamycin 12 h and showed significant difference at 24 h(121.2±13.0),48 h(124.3±14.1)and 72 h(127.7±13.7)after drug treatment compared with that in group A30P+Wortmannin(t:2.9746, P=0.0083);(3)Mutant α-synuclein(A30P)leading to PC12 cells death by means of autophagy involved α-synuclein accumulation,membrane lipid oxidation,and loss of plasma membrane integrity.Mutant α- synuclein(A30P)mediated the toxicity of MPP+.Rapamycin,an inducer of autophagy,reduced the aggregation of α-synuclein in transfected cells.Meanwhile,Wortmanin,an inhibitor of autophagy,promoted the aggregation of α-synuclein in transfected cells and induced cells to die.Conclusions The abnormal aggregation of α-synuclein induces autophagic programmed cell death in PC12 cells and mutant α-synuclein (A30P)mediates the toxicity of MPP+.Meanwhile,Rapamycin may reduce the aggregation of α-synuclein in transfeeted cells by activation of autophagic pathway.

Chronic subcutaneous injection of rotenone produces rodent Parkinsonian models / 中国药理学通报

Aim To establish a rat model of Parkinsons disease by chronic subcutaneous injection of low-dose rotenone.Methods Lewis rats were randomly divided into two groups: vehicle-treated group and rotenone-treated group.Rotenone(1.0 mg?kg~(-1)?d~(-1)) was subcutaneously injected at 08:00 and 20:00 for 30 days with drug holidays every 7 days.Following 30 days of rotenone administration,neuronal loss in the substantia nigra(SN) was determined with tyrosine hydroxylase(TH) immunohistochemistry and Nissl staining.Aggregation of ?-synuclein in SN neurons was observed with a laser cofocal microscopy.Result Three rotenone-treated rats showed resting tremor.The number of TH-positive neurons in SN significantly reduced(P

The research progress of cobra venom secretory phospholipase A_2 / 中国药理学通报

Cobra venom secretory phospholipase A_2 (sPLA_2) is an important component of cobra venom which has a variety of biological activities. Recent studies are mainly focusing on each pharmacological active component of venom, SPLA_2 is one of them. This review summarized the structure, purification and biological activities of cobra venom sPLA_2 with emphasizing its diverse pharmacological effects and toxicity. In addition, some mechanisms of actions of sPLA_2 and possible applications of sPLA_2 were also discussed.

The role of autophagy in cell survival and tumor development / 中国药理学通报

Autophagy occurs in all types of eukaryotic cells, which has a rigid connection with the normal or abnormal development of cells and is associated with many diseases. There're lots of molecular control elements and multiple signaling pathways involved in regulating autophagy. As a form of type Ⅱ programmed cell death, autophagy participates in maintaining cell homeostasis and pathogenesis of various of diseases through interacting with apoptotic pathway. Recent studies show that autophagy has effects on the occurrence and development of tumor cells through influencing on cell cycle, apoptosis-associated factors and angiogenesis.

Neuroprotective Actions and Possible Underlying Molecular Mechanisms of Valproate / 中国药理学通报

Valproate (VPA) has long been used for the treatment of bipolar mood disorder. VPA is effective in control of mania and depression. Recent studies have demonstrated that VPA has profound neuroprotective effects in against various apoptotic stimuli. Moreover, VPA can promote neurogenesis, neuronal proliferation and differentiation. Although intensive research has been dedicated to VPA′s neuroprotection, the molecular mechanisms by which VPA protects neurons are still not fully understood. In this paper, recent progresses in the study of VPA′s neuroprotection and underlying mechanism are reviewed.

Matrix metalloproteinases and nervous diseases / 中国药理学通报

Matrix metalloproteinases (MMPs) are a family of Zn~ 2+ -dependent endopeptidases targeting extracellular matrix (ECM) compounds as well as a number of other proteins. Their proteolytic activity acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. Recently, it has been reported that MMPs play an important role in nervous diseases including cerebral ischemia, Alzheimers disease,multiple sclerosis and Parkinson′s disease.

Research progress in molecular pathology of Huntington's disease / 中国药理学通报

Huntington's disease is a fetal neurological disorder manifested as movement disorder accompanied by cognitive and psychological impairments. The disease is inherited as autosomal dominant. Huntington's disease is caused by an expansion of a polyglutamine tract in a protein named huntingtin. The length of polyglutamine tract in huntingtin in normal individual is less than 35 glutamines. In Huntington's disease patients the length of polyglutamine tract increases to more than 37 glutamines. The pathogenic mechanisms by which mutant huntingtin causes Huntington's disease have not been fully understood. This paper reviews main progresses in studying the pathogenic mechanisms of mutant huntingtin.

Peroxisome prolifreator-activated receptors and neurodegenerative diseases / 中国药理学通报

Peroxisome prolifreator-activated receptors (PPARs) are ligand-activated nuclear transcription factors, members of nuclear hormone receptor superfamily. PPARs play critical roles in growth, proliferation and apoptosis of variety of cells. Recently, PPAR ligands have been reported to ameliorate neuronal damage in neurodegenerative diseases including Alzheim- ers disease, Parkinsons disease,cerebral ischemia and multiple sclerosis. PPAR agonists may have potential values in treatment of neurodegenerative diseases. In this paper, we reviewed recent findings on PPARs′ neuroprotective actions and their underlying mechanisms.

A lipophilic fraction of Ganoderma lucidum promotes PC12 differentiation / 中国药理学通报

Aim To investigate the effects of lipophilic fraction of Ganoderma lucidum on PC12 differentiation. Methods Rat PC12 cells were cultured in DMEM medium. Differentiation of PC12 cells was evaluated by monitoring neurite outgrouth of PC12 cells with phase-contrast microscopy after treatment with lipophilic extracts of Ganoderma lucidum or it's effective component B_2. Expression of neuronal marker,microtubule-associated protein-2,was determined with immonofluorescence and western blot analysis. Results Lipophilic extracts of Ganoderma at the concentrations between 125 and 500 mg?L -1 induced neurite outgrowth of PC12 cells in a dose-dependent manner. Nearly all cells showed morphological differentiation in response to lipophilic extracts of Ganoderma. Lipophilic extracts of Ganoderma also potentiated NGF-induced differenti-ation of PC12 cells. Lipophilic extracts of Ganoderma increased the expression of MAP-2. Conclusion Lipophilic extracts of Ganoderma lucidum can induce differentiation of PC12 cells and potentiate NGFs actions.