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Background Fluorine accumulates in the brain tissue after long-term excessive intake and subsequently cause nerve damage and decline of learning and memory ability. Receptor of advanced glycation end-products (RAGE)/p38 mitogen-activated protein kinase (p38MAPK)/nuclear factor kappa-B (NF-κB) signaling pathway is considered to be involved in the associated mechanism. Objective To study the changes of RAGE/ p38MAPK/ NF-κB signaling pathway in rats with subchronic fluorosis, and to explore the protective effects of extract of Ginkgo biloba 761 (EGb761) and RAGE antagonist (FPS-ZM1) on neuromemory ability. Methods Ninety male clean SD rats were divided into 9 groups with 10 rats in each group. The modeling period was 6 months. Control group (C group): free drinking tap water (fluoride content <0.5 mg·L−1), low- and high-dose fluoride groups (LF group, HF group): free drinking tap water with 10 or 50 mg·L−1 fluoride; intervention group of Ginkgo biloba extract (CE, LFE, and HFE groups): on the basis of the C group, LF group, and HF group, 100 mg·kg−1·d−1 EGb761 was given daily via intragastric administration; FPS-ZM1 intervention groups (CF, LFF, and HFF groups): 7 d before the end of modeling, 1 mg·kg−1·d−1 FPS-ZM1 was injected intraperitoneally daily on the basis of the C group, LF group, and HF group. The contents of fluoride in brain and blood of each group were detected. The learning and memory ability was tested by water maze experiment. The histopathologic changes of the hippocampus were detected by Nissl staining. The protein expression levels of RAGE and its ligand high mobility group protein B1 (HMGB1), NF-κB, p38MAPK, phospho-p38MAPK (p-p38MAPK), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) in brain tissue were detected by Western blotting. The mRNA expression levels of RAGE, HMGB1, and p38MAPK were detected by quantitative real-time PCR. Results Compared with the C group, the contents of blood fluoride and brain fluoride in the LF and the HF groups were increased (P<0.05). The results of the water maze experiment showed that, compared with the C group, the escape latency time of the LF group and the HF group was longer and the crossing times were reduced; compared with the HF group, the escape latency time of the HFE group and the HFF group was shortened, and the crossing times were increased (P<0.05). The Nissl staining results showed that the number of Nissl body in the HF group decreased compared with the C group; compared with the HF group, the number of Nissl body in the HFE group and the HFF group increased. The Western blotting results showed that compared with the relative protein expression levels of RAGE, HMGB1, NF-κB, p38MAPK, p-p38MAPK, IL-6, and TNF-α in the C group , the levels of above indicators in the HF group and the levels of RAGE, HMGB1, NF-κB, p-p38MAPK, and IL-6 in the LF group were up-regulated (P<0.05); compared with the HF group, the levels of above indicators in the HFE group and the HFF group were all down-regulated (P<0.05); compared with the relative protein expression levels of RAGE and HMGB1 in the LF group, the levels in the LFE group and the LFF group were all down-regulated (P<0.05). The quantitative real-time PCR results showed that compared with the C group, the mRNA expression levels of RAGE and HMGB1 in the LF group and the HF group were up-regulated; compared with the LF group, the mRNA expression levels of RAGE in the LFE group and the LFF group were down-regulated ; compared with the HF group, the mRNA expression levels of RAGE and HMGB1 in the HFE group and the HFF group were down-regulated (P<0.05). Conclusion The central nervous system injury caused by subchronic fluorosis may be related to the activation of RAGE/p38-MAPK/NF-κB signaling pathway, which can impair the learning and memory ability of rats, while EGb761 and FPS-ZM1 may have certain protective effects on the nerve injury.
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Objective To investigate the influence of inhibited gene expression of fisson 1 (Fis1) gene on the level of Fis1,mitofusin 1 (Mfn1) and mitochondrial membrane potential in SH-SY5Y cells with fluorine,to study the role of mitochondrial dynamic balance in the pathogenesis of chronic fluorosis.Methods SH-SY5Y cells were cultured in vitro,when adherent cells entered the logarithmic phase,using a group design,they were divided into four groups:blank control group (control),fluoride group [2 mmol/L sodium fluoride (NaF)],fluoride negative control group (2 mmol/L NaF + non-specific siRNA) and the gene-silencing group (2 mmol/L NaF + specific siRNA-Fis1).The protein expression levels of Fis1 and Mfn1 were measured by Western blotting;the mRNA expression levels of Fis1 and Mfn1 were measured by Real-time PCR;and the levels of the mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit.Results Compared with control (1.37 ± 0.18,1.00 ± 0.04;1.57 ± 0.19,1.00 ± 0.04;1.00 ± 0.10),the expression levels of Fisl protein (1.72 ± 0.04) and mRNA (1.48 ± 0.13) in fluoride group were increased,the expression levels of Mfn1 protein (0.87 ± 0.02) and mRNA (0.69 ± 0.07) in fluoride group were decreased,the level of mitochondrial membrane potential (0.76 ± 0.13) was decreased (P ＜ 0.05).Compared with control,the expression levels of Fis1 protein (0.79 ± 0.07) and mRNA (0.06 ± 0.03) in gene-silencing group were decreased,the expression levels of Mfn1 protein (1.71 ± 0.04) and mRNA (1.52 ± 0.05) in gene-silencing group were increased (P ＜ 0.05),the level of mitochondrial membrane potential (0.94 ± 0.01) was decreased.Compared with fluoride group,the expression levels of Fis1 protein and mRNA in gene-silencing group were decreased,the expression levels of Mfn1 protein and mRNA in gene-silencing group were increased,the level of mitochondrial membrane potential in gene-silencing group was increased (P ＜ 0.05).Conclusion Gene expression inhibition of Fis1 gene can reduce the mitochondrial division and damage of mitochondrial membrane potential in SH-SY5Y cells induced by fluoride.
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Objective To evaluate the influence of fluoride on mitochondrial membrane potential of neuroblastoma SH-SY5Y cells,and on the expression levels of mitochondrial proteins mitofusion 1 (Mfn1) and fission 1 (Fis1).Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentration of sodium fluoride [0.0 (control),0.4,2.0 and 4.0 mmol/L],and various periods exposure of 6,12,24,48 h;the mitochondrial membrane potential of SH-SY5Y cells was detected by mitochondrial membrane potential assay kit (JC-1);and the expression levels of Mfn1 and Fis1 proteins were detected by Western blotting.Results Compared with the control group (1.63 ± 0.18,1.13 ± 0.15,1.30 ± 0.02) for various periods exposure (6,12,48 h),the red/green fluorescence ratios of the mitochondrial membrane potential of SH-SY5Y cells exposed to 2.0 and 4.0 mmol/L of sodium fluoride were decreased significantly (1.01 ± 0.10,0.80 ± 0.04;0.75 ± 0.13,0.62 ± 0.10;0.82 ± 0.01,0.56 ± 0.04,P ＜ 0.05);compared with the control group (0.93 ± 0.03,1.05 ± 0.07,1.17 ± 0.04) for various periods exposure,the expression levels of mitochondrial Mfn1 protein were decreased significantly in 0.4,2.0,4.0 mmol/L sodium fluoride groups (6,12,48 h:0.75 ± 0.02,0.65 ± 0.05,0.57 ± 0.06;0.83 ± 0.06,0.79 ± 0.06,0.69 ±0.06;0.98 ± 0.05,0.73 ± 0.07,0.62 ± 0.09,P ＜ 0.05).Compared with the control group (0.90 ± 0.05) for exposure time 12 h,the expression levels of Fis1 protein were increased significantly in 2.0,4.0 mmol/L sodium fluoride groups (1.14 ± 0.06,1.23 ± 0.06,P ＜ 0.05).Conclusions The mitochondrial membrane potential and the expression levels of mitofusion 1 and fission 1 of SH-SY5Y cells treated with fluoride are abnormal,which might be associated with the theory of nerve cell damage from high oxidative stress.
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Genetic generalized epilepsies (GGEs) are a group of epilepsy syndromes caused by genetic factors. A few of GGEs conform to the Mendelian patterns, while most of them show polygene inheritance. Researchers initially found that most of the genes associated with GGEs are related to ion channels including voltage-gated sodium channels, potassium channels, calcium channels and chloride channels, and ligand-gated gamma-aminobutyric acid receptor channels. Further researches have shown that certain non-ion channel genes are also related to GGEs, and that de novo mutations and copy number variants also play an important role in the pathogenesis of GGEs. Application of next- and third-generation sequencing promoted delineation of the molecular genetics of the GGEs, but also brought more challenges. Genetic findings have provided an important basis for the elucidation of the pathogenesis, clinical diagnosis and precise treatment of GGEs. This paper provided a review for recent progress made in molecular genetics of GGEs.
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Humanos , Epilepsia Generalizada , Genética , Pesquisa em Genética , Canais Iônicos , GenéticaRESUMO
Objective To establish a method of quality control for compound Cornu cervi degelatinatum tablets. Methods Thin layer chromatography ( TLC ) was used for qualitative identification of Lonicerae Japonicae Caulis and Pyrola Herba in the preparations. High performance liquid chromatography was used to determine the content of loganin and chiratin in Lonicerae Japonicae Caulis and monotropein in Pyrola Herba. Results The TLC spots were clear and specific in the qualitative identification.HPLC determined the content. In the concentration range, monotropein, loganin and chiratin had a good linear relationship with peak area (r>0.999).The mean recovery was 97.2%, 98.4% and 96.0%;RSD was 1.4%, 0.8% and 0.8%. Conclusion The present study employs TLC for quality control and HPLC for quantity control. The methods are simple and accurate, have good reproducibility, and can effectively control the quality of compound Cornu cervi degelatinatum tablets.