Proteomics for Studying the Effects of Ketogenic Diet Against Lithium Chloride/Pilocarpine Induced Epilepsy in Rats.

The ketogenic diet (KD) demonstrates antiepileptogenic and neuroprotective efficacy, but the precise mechanisms are unclear. Here we explored the mechanism through systematic proteomics analysis of the lithium chloride-pilocarpine rat model. Sprague-Dawley rats (postnatal day 21, P21) were randomly divided into control (Ctr), seizure (SE), and KD treatment after seizure (SE + KD) groups. Tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectroscopy (LC-MS/MS) were utilized to assess changes in protein abundance in the hippocampus. A total of 5,564 proteins were identified, of which 110 showed a significant change in abundance between the SE and Ctr groups (18 upregulated and 92 downregulated), 278 between SE + KD and SE groups (218 upregulated and 60 downregulated), and 180 between Ctr and SE + KD groups (121 upregulated and 59 downregulated) (all p < 0.05). Seventy-nine proteins showing a significant change in abundance between SE and Ctr groups were reciprocally regulated in the SD + KD group compared to the SE group (i.e., the seizure-induced change was reversed by KD). Of these, five (dystrobrevin, centromere protein V, oxysterol-binding protein, tetraspanin-2, and progesterone receptor membrane component 2) were verified by parallel reaction monitoring. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that proteins of the synaptic vesicle cycle pathway were enriched both among proteins differing in abundance between SE and Ctr groups as well as between SE + KD and SE groups. This comprehensive proteomics analyze of KD-treated epilepsy by quantitative proteomics revealed novel molecular mechanisms of KD antiepileptogenic efficacy and potential treatment targets.

Long-Term Effects of Zinc Deficiency and Zinc Supplementation on Developmental Seizure-Induced Brain Damage and the Underlying GPR39/ZnT-3 and MBP Expression in the Hippocampus.

We previously illustrated that long-term upregulated expression of ZnT-3 in the hippocampus of rats that underwent neonatal seizures was restored by pretreatment with a ketogenic diet. It was recently demonstrated that upregulated expression of ZnT-3 was associated with increased concentrations of intracellular free zinc ions in an in vitro model of glutamate-induced hippocampal neuronal excitotoxic damage. However, there is still a lack of research on the effects of different concentrations of zinc in the diet on developmental convulsive brain injury. The aim of this study was to investigate the effects of different zinc concentrations in the diet on long-term neurobehavioral and seizure thresholds following lithium chloride-pilocarpine-induced developmental seizures. Sprague-Dawley rats (postnatal day 27, P27) were randomly assigned to one of six dietary groups for 4 weeks: normal zinc control group (Control group, 44 mg/kg Zn), Zn-deficient control group (ZD group, 2.7 mg/kg Zn), Zn supplemented control group (ZS group, 246 mg/kg Zn), pilocarpine-induced seizure plus regular zinc diet group (SE group, 44 mg/kg Zn), seizure plus low-zinc diet group (SE + ZD group, 2.7 mg/kg Zn), and seizure plus high-zinc diet group (SE + ZS group, 246 mg/kg Zn). Novel object recognition and passive avoidance tests were performed on rats at P42 and P56. After routine seizure threshold detection and Timm staining procedures at P57, expression of GPR39, ZnT-3, and MBP were detected in the hippocampus by Western blot analysis. The results revealed that the Zinc-deficient diet for 4 weeks aggravated the long-term adverse effects of developmental seizures, evidenced by weight, cognition, seizure threshold and serum zinc concentrations, which were paralleled by expression changes in hippocampal GPR39 and ZnT-3. In contrast, zinc supplementation for 4 weeks significantly improved damage-related changes described above and rescued the abnormal expression of GPR39, ZnT-3, and MBP in the hippocampus. Similar alterations between the expression pattern of MBP and aberrant sprouting of mossy fibers in the hippocampus may indicate that sprouting is a secondary pathological change caused by developmental brain damage rather than the cause of epileptogenesis. Up-regulation of MBP protein levels in the high zinc diet-treated seizure group as well as the corresponding improvement of cognitive impairment and reduced hippocampal mossy fiber regenerative sprouting, may represent a compensatory mechanism for neuronal membrane damage and repair.

Neuro-Behavioral Status and the Hippocampal Expression of Metabolic Associated Genes in Wild-Type Rat Following a Ketogenic Diet.

While a ketogenic diet (KD) is a well-established therapy for medically intractable epilepsy, clinical evidence of relevant adverse events of a KD has also been reported. We asked whether this kind of diet would have deleterious effects on wild-type brain function by evaluating KD-induced biochemical changes in the hippocampus as well as neurobehavioral changes occurring in wild-type rats. Fifty-four Sprague-Dawley rats were randomly assigned to three groups on postnatal day 28 (P28): wild-type rats fed with a KD qd (daily for 4 weeks, KD) or qod (every other day for 4 weeks, KOD), and wild-type rats fed with standard normal laboratory diet (ND). Neurobehavioral changes were observed on P35, P42, and P49. The hippocampal mossy fiber sprouting, the expression levels of zinc transporters (ZnTs) and lipid metabolism related genes were detected by Timm staining, RT-qPCR and western blot analysis, respectively, on P58. The KD-treated KOD and KD groups showed a significant delay of negative geotaxis reflex on P35, but not on P42 or P49. In the open field test, daily KD treatment only led to a reduction in exploratory activity and increased grooming times but induced no significant changes in the scores of vertical activity or delay time. KD qod treated rats (KOD) displayed a slight delay in the place navigation test on P35 compared with the KD group. There were no significant differences in Timm staining among the three groups. In parallel with these changes, KD treatment (both KD and KOD) induced significantly downregulated mRNA levels of Apoa1, Pdk4, and upregulated expression of ApoE, ANXN7, and cPLA2 in the hippocampus when compared with the ND group (except in the case of ApoE in the KOD group). Notably, both the mRNA and protein levels of cPLA2 in the KOD rats were significantly downregulated compared with the KD group but still markedly higher than in the ND group. No significant difference was found in ZnTs among the three groups. Our data suggest that early-life KD can provoke minor neurobehavioral effects in particular a delay in negative geotaxis reflex and an increase in grooming activity. The hippocampal lipid metabolism signaling pathway, especially cPLA2, may be the target of the protective effect of KD on long-term brain injury after developmental seizures.

Association between PPAP2B gene polymorphisms and coronary heart disease susceptibility in Chinese Han males and females.

Little is known about gender-related differences in the association between PPAP2B single nucleotide polymorphisms (SNPs) and coronary heart disease (CHD) in Chinese Han males and females. We therefore conducted a case-control study with 456 cases and 685 healthy controls divided into male and female subgroups. Five PPAP2B polymorphisms (SNPs) were selected and genotyped using Sequenom Mass-ARRAY technology. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression adjusting for age and gender. Allelic model analysis revealed that for PPAP2B rs1759752, allele frequency distributions differed between cases and controls in the male subgroup (p = 0.015, OR: 1.401, 95%CI: 1.066-1.481). Genetic model analysis revealed that in the male subgroup, rs1759752 was associated with increased CHD risk in the dominant model (p = 0.035) and overdominant model (p = 0.045). In the female subgroup, rs12566304 was associated with a decreased CHD risk in the codominant model (p = 0.038) and overdominant model (p = 0.031). Additionally, the "GC" haplotypes of rs1759752 and rs1930760 were protective against CHD in males. These observations shed new light on gender-related differences in the association between PPAP2B gene polymorphisms and CHD susceptibility in the Chinese Han population.

[Two-dimensional gel electrophoresis map of serum proteins in patients with chronic Keshan disease].

OBJECTIVE: To identify the different serum proteins expressed in patients with Keshan disease (KD). METHODS: Two-dimensional gel electrophoresis (2-DE) was performed with serum samples from the patients with chronic KD and healthy controls to separate serum proteins. The gels were stained by sliver and scanned by Umax scanner. The data were analyzed by ImageMaster 2D software. KD related proteins were identified through searching the ExPASy SWISS-2DPAGE database. RESULTS: Stable two-dimensional gel electrophoresis maps were established for serum samples of KD patients and healthy controls. A total of 808 and 814 protein spots were observed in KD patients and healthy controls, respectively. The two maps had 96.5475% identical protein spots and 44 differentially expressed protein spots. Eleven protein spots were expressed exclusively in KD patients and 12 protein spots only appeared in healthy controls. About 21 proteins were expressed in both groups but varied in quantities (14 proteins were over-expressed by more than 3 times and 7 proteins were under-expressed by more than 3 times in KD patients, P < 0.01). Among the 353 protein spots matched with the ExPASy-SWISS-2DPAGE databank, No. 1177 protein appeared in the KD patient was found to have the closest match with P02774 2-D0004T6 known as vitamin D binding protein (VDBP). CONCLUSION: There is a significant difference in serum protein expression between KD patients and normal people. VDBP might play a role in cardiac muscle damage via inflammatory immune reactions.

[Effects of hVEGF cDNA on random skin flap via a replication-deficient adenovirus vector].

OBJECTIVE: The purpose of this study was to determine the effects of local delivery of vascular endothelial growth factor( VEGF) transferred with adenovirus-mediated gene on the survival of ischemic random skin flap in rats. METHODS: The animals were divided into three groups randomly (n = 10) . A 2 cm x 8 cm dorsal skin flap was designed with the pedicle at the level of the iliac crest. In group A (AdCMV-VEGF), each animal received 10(12) PR replication-deficient recombinant adenovirus (AdCMV-VEGF) in the distal two-thirds of the proposed flap by means of the subdermal injection at ten different locations. In group B (AdCMV-GaI), each received 1012 PR AdCMV-Gal. In Group C (Saline), each received 1 ml saline. Three days after the treatment, the flap was elevated as planed way and re-sutured back to its donor site. All the animals were evaluated 7 days after the operation. RESULTS: The mean percentage of surviving flap area was (85.91 +/- 2.54)% in group A, (59.56 +/- l.18)% in group B, and (61.48 +/- l.09)% in group C. There was a significant increase in the percentage of the survival area in the flaps of the group A, compared with the group B and group C (Group B vs. Group A, P < 0.01; Group C vs.Group A, P < 0.01, Group B vs. Group C, P >0.05). Hybridization in the situ, the immunohistochemical stain showed that the VEGF was expressed in the survival tissue of the flap treated with the AdCMV-VEGF, but it was not found in the control groups. Histological analysis demonstrated qualitatively greater amount of granulation tissue and angiogenesis was found in the group treated with the AdCMV-VEGF than the controls. CONCLUSIONS: The results may indicate that Ad vector carrying VEGF cDNA could be useful in enhancing the survival of the skin flap due to the effect of the local delivery of the VEGF.

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi.

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.