Association between polymorphism and haplotype of ATP2B1 gene and skeletal fluorosis in Han population.

To evaluate the association between ATP2B1 gene polymorphisms and skeletal fluorosis, a cross-sectional study was conducted. In China, 962 individuals were recruited, including 342 cases of skeletal fluorosis. Four TP2BA1 polymorphisms (rs2070759, rs12817819, rs17249754, and rs7136259) were analysed. The results suggested that rs17249754 and rs7136259 were associated with skeletal fluorosis. After controlling confounders, the protective effect of GG genotype in rs17249754 was apparent in individuals over 45 years old, female, with urine fluoride concentration below 1.6 mg/L, serum calcium above 2.25 mmol/L or serum phosphorus between 1.1 and 1.3. Heterozygote TC in rs7136259 increased the risk of skeletal fluorosis in subjects who are elderly, female, with urinary fluoride more than 1.6 mg/L, serum calcium more than 2.25 mmol/L and blood phosphorus between 1.1 and 1.3 mmol/L. Four loci were found to be tightly related by linkage disequilibrium analysis, and the frequency of distribution of haplotype GCGT was lower in the skeletal fluorosis group.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat.

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

Membrane permeability-guided identification of neuroprotective components from Polygonum cuspidatun.

CONTEXT: Polygonum cuspidatum Sieb et Zucc. (Polygonaceae) possesses various pharmacological activities and has been widely using as one of the most popular and valuable Chinese herbal medicines in clinics. Its usage has increasingly attracted much of our attention and urges investigation on its bioactive components. OBJECTIVE: To establish a rapid and valid approach for screening potential neuroprotective components from P. cuspidatum. MATERIALS AND METHODS: Potential neuroprotective components from P. cuspidatum were screened utilizing liposome equilibrium dialysis followed by high-performance liquid chromatography (HPLC) analysis. Their neuroprotective effects on modulation of protein expression of α7 nAChR, α3 nAChR and synaptophysin (SPY) on SH-SY5Y human neuroblastoma cell line (SH-SY5Y) were evaluated by means of Western blotting. RESULTS: Two potential compounds, polydatin (C1) and emodin-8-O-ß-D-glucoside (C2), were detected and identified in our study. The biological tests showed that both compounds C1 and C2, respectively, at concentrations of 0.1 and 0.25 mg/mL significantly increased protein expression of α7 and α3 nicotinic acetylcholine receptors (nAChRs) in SH-SY5Y cells. Moreover, C1 and C2 at 0.1 mg/mL significantly reversed the Aß1₋42-induced decrease of α7 and α3 nAChRs protein expression in SH-SY5Y cells. In addition, C2 at 0.1 mg/mL significantly increased protein expression of SPY in SH-SY5Y cells and Aß11₋42-induced SH-SY5Y cells whereas C1 did not provide any positive effects. DISCUSSION AND CONCLUSION: In conclusion, our approach utilizing liposome equilibrium dialysis combined with HPLC analysis and cell-based assays is a prompt and useful method for screening neuroprotective agents.

Pretreatment of SH-SY5Y cells with dicaffeoylquinic acids attenuates the reduced expression of nicotinic receptors, elevated level of oxidative stress and enhanced apoptosis caused by ß-amyloid peptide.

OBJECTIVES: This in vitro investigation was designed to examine potential neuroprotection by dicaffeoylquinic acids (diCQAs) extracted from a traditional Chinese medicinal herb herba erigerontis and their effects against the toxicity induced by ß-amyloid peptide (Aß25-35 ). METHODS: The neuroblastoma SH-SY5Y cell line was treated with Aß or 3, 4-diCQA, 3, 5-diCQA or 4, 5-diCQA. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction was assayed by spectrophotometrical method, lipid peroxidation (malondialdehyde) on the basis of the level of thiobarbituric acid-reactive substance, the activity of superoxide dismutase by the xanthine oxidase procedure, the frequency of apoptosis by flow cytometry, and the levels of α3 and α7 nicotinic acetylcholine receptor (nAChR) subunit proteins by Western blotting. KEY FINDINGS: When the cells were exposed to Aß25-35 , MTT reduction declined, oxidative stress and apoptosis were enhanced, and the expression of α3 and α7 nAChR subunit proteins was lowered. Expression of the α7 nAChR subunit protein was increased by all three diCQAs, and the level of α3 was increased by 3, 5-diCQA and 4, 5-diCQA. Significantly, pretreatment with diCQAs attenuated the neurotoxic effects of Aß25-35 , a neuroprotective effect in which the upregulation of α7 and α3 nAChR may be involved. CONCLUSION: The diCQAs exert a protective effect on Aß-induced neurotoxicity in SH-SY5Y cells and a potential underlying mechanism involving stimulation of nAChRs.

The neurotoxicity of ß-amyloid peptide toward rat brain is associated with enhanced oxidative stress, inflammation and apoptosis, all of which can be attenuated by scutellarin.

This study was designed to investigate the processes underlying the neurotoxicity induced by ß-amyloid peptide (Aß) in the rat brain, as well as to examine whether scutellarin (Scu) can prevent this neurotoxicity. Thirty Wistar rats were randomly divided into 3 groups, i.e., untreated (control), treated with Aß and treated with both Aß and Scu. The treated rats were subjected to bilateral intracerebroventricular injection of Aß(25-35) with or without subsequent dietary exposure to Scu. Learning and memory were assessed with the Morris water maze test; the activities of superoxide dismutase (SOD) and monoamine oxidase (MAO) were assayed biochemically; expression of the interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins was determined by immunohistochemistry; and neuronal apoptosis was detected with Annexin staining followed by flow cytometry. The animals treated with Aß exhibited impaired learning and memory; reduced SOD and elevated MAO activity, elevated protein levels of IL-1ß, IL-6 and TNF-α; and a higher percentage of apoptotic neurons in the brain. Interestingly, all of these effects were ameliorated by administration of Scu. These findings indicate that the deficits in learning and memory demonstrated by the rats receiving Aß are due to elevated oxidative stress and inflammation, which result in apoptosis and that Scu may prevent these deleterious effects.

Corn baked by burning coal triggered overexpression of osteopontin in hepatocytes of rats following fluorosis.

OBJECTIVE: To observe the expression of osteopontin (OPN) in hepatocytes of rats fed with corn baked by burning coal from fluorosis areas and a deficiency of calcium/protein intake following fluorosis. METHODS: A total of 48 Wistar rats as objects were randomly assorted into four groups: dose-free fluorine group, which were mainly fed with fluorine-free corn (56% structurally), dose-free fluorine with biased dietary group, which were fed with lower contents of protein (119.41 g/kg) and calcium (0.68 g/kg), high-dose fluorine group (fluorine contents: 104.2 mg/kg), and high-dose fluorine with biased dietary group. After 180 days of cultivation, the contents of fluorine in the bones of rats were tested for the assessment of construction of fluorosis animal model. And the expression of OPN in hepatocytes of rats in different groups was detected with immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: The present study validated the result that OPN was overexpressed in hepatocytes following fluorosis after oral intake of burning coal-baked corn. OPN was expressed most significantly in high fluorine with biased dietary group, and the high-fluorine group ranked the second most; and dose-free fluorine with biased dietary group ranked the third. The dose-free fluorine group expressed the least OPN. CONCLUSION: Overexpression of OPN in hepatocytes following fluorosis after excess fluorine intake was involved in liver damage process, which was enhanced by deficiency of calcium and protein intake. The results also demonstrated that the development of fluorosis in Guizhou province was correlated with local baking staple corn as a way of excess intake of fluorine and deficiency of calcium/protein intake.

Correlations between cholinesterase activity and cognitive scores in post-ischemic rats and patients with vascular dementia.

The biochemical changes such as the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were investigated in rats with global cerebral ischemia and in vascular dementia (VaD) subjects in this study. The AChE activity showed a significant decrease in plasma and a significant increase in the hippocampus but not in the cerebral cortices in the post-ischemic rats as compared to the controls. The learning abilities and spatial memory were impaired in the post-ischemic rats as compared to controls. Furthermore, the AChE activity in plasma was significantly reduced in VaD subjects as compared to normal control subjects. The BuChE activity did not show any change in both post-ischemic rats and VaD patients. Interestingly, the decreased AChE activity in plasma from the post-ischemic rats and the VaD subjects showed a significant correlation with the declined learning and memory ability, and the Mini-Mental State Examination score, respectively. These data suggest that the AChE activity is involved in the cognitive recovery after ischemia, and the plasma level of AChE might be a reliable supplementary peripheral biomarker to evaluate the cognitive recovery degree of VaD patients.

Rapid screening and identification of caffeic acid and its esters in Erigeron breviscapus by ultra-performance liquid chromatography/tandem mass spectrometry.

Caffeic acid and its esters (CAEs) are widely distributed in the plant kingdom and have been reported to elicit a wide range of exceptional biological activities. Present methods for screening and characterization of CAEs normally need the use of liquid chromatography diode-array detection/multistage mass spectrometry (LC-DAD/MS(n)). In this report, a rapid and efficient method coupling ultra-performance liquid chromatography (UPLC) with fragment-targeted multi-reaction monitoring (MRM) has been developed for screening CAEs in a crude extract of Erigeron breviscapus, while a UPLC/quasi-MS(n) method has been applied in the structural identification of these compounds. Furthermore, a simple quasi-UPLC/MS/MS method based on in-source collision-induced dissociation (CID) has been proposed for rapid identification of the CAEs. As a result, a total of more than 34 CAEs were detected and their structures characterized. Nine of them were reported from E. breviscapus for the first time. Applications of these strategies in the chemical investigation of an injection of E. breviscapus resulted in the identifications of 16 CAEs. These strategies, if appropriate modifications are made, will be very useful in screening and characterization not only of CAEs, but of other structural types of compounds in various complex matrices.

[Study on fingerprints correlated with pharmacodynamic of constituents in Herba Erigerontis against neurotoxicity induced by beta-amyloid peptide].

OBJECTIVE: To investigate the neuroprotective effects of the constituents in Herba Erigerontis on neuroblastoma SH-SY5Y cells, and find out its possible material foundation in treating Alzheimer's disease (AD). METHOD: Different combinations of the three constituents in Herba Erigerontis were prepared according to the orthogonality experiment, and the indexes (MTT reduction assay, lipid peroxidation and expressions of nAChR alpha7 protein)were observed upon the SH-SY5Y cells followed by treatment of these combinations and beta-amyloid peptide (AP). The pharmacology data thus obtained and peak data in UPLC fingerprint were analyzed through ANOVA and correlationship by SPSS to give the information of active possible material foundation. RESULT: Constituents B and C showed clear activity and peaks of 4, 7-12 did positive correlationship according to the correlation of fingerprints and pharmacology. CONCLUSION: This study makes a valid approach for deducing the active constituents even the exact compounds against neurotoxicity induced by Abeta by correlation of fingerprints and pharmacology.

[Protection of Tianshen Yizhi Recipe against low expression of nicotinic receptor and neurotoxicity induced by beta-amyloid peptide].

OBJECTIVE: To investigate the inhibition effects of Tianshen Yizhi Recipe (TSYZR), a compound traditional Chinese herbal medicine, on decreased expression of nicotinic acetylcholine receptor (nAChR) and the neurotoxicity as well as lipid peroxidation induced by beta-amyloid peptide (Abeta) in human SH-SY5Y neuroblastoma cells. METHODS: The SH-SY5Y cells were treated by a certain concentration of TSYZR, and then exposed to Abeta(25-35). Methyl thiazolyl tetrazolium reduction assay was carried out to understand the influences of the drugs on cellular viability. Expressions of nAChR subunits (alpha3 and alpha7) at protein and mRNA levels were detected by Western-blotting and reverse transcription polymerase chain reaction, respectively. Lipid peroxidation was measured by thiobarbituric acid to observe the capacity of antioxidant of the drugs. RESULTS: TSYZR at a safe concentration could increase alpha7 protein in the cells, inhibit decreased expressions of alpha3 and alpha7 nAChR subunit proteins, prevent lower expression of alpha7 mRNA in SH-SY5Y cells induced by Abeta, reduce the neurotoxicity and lipid peroxidation resulting from Abeta, but had no significant effect on the lower expression of alpha3 mRNA. CONCLUSIONS: TSYZR can up-regulate the expression of alpha7 nAChR subunit protein and prevent decreased expressions of nAChRs and neurotoxicity as well as lipid peroxidation induced by Abeta. This drug may play an important therapeutic role in treatment of Alzheimer disease.

[Influence of beta-amyloid peptide on cell membrane lipids and cholinergic receptors in human neuroblastoma SH-SY5Y cells].

OBJECTIVE: To study the effects of beta-amyloid peptide (Abeta) on cell membrane lipids and cholinergic receptors of human neuroblastoma cells. METHODS: Human SH-SY5Y neuroblastoma cells were treated with different concentrations of Abeta(1-42) with and without pretreatment of vitamin E. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction, lipid peroxidation, protein oxidation and phospholipids were measured by spectrophotometry. Levels of cholesterol and unbiquinone were determined by high-performance liquid chromatography (HPLC). The numbers of cholinergic receptor binding sites were determined by receptor binding assay and the protein levels of nicotinic receptor alpha3 and alpha7 subunits were studied by Western blotting. RESULTS: SH-SY5Y cells showed decreased reduction rates of MMT and phospholipids, and increased lipid peroxidation and protein oxidation after exposure to Abeta (0.1 micromol/L) as compared to the control. The number of cholinergic receptor binding sites, the protein level of nicotinic receptor alpha3 and alpha7 subunits and the content of ubiquinone decreased in cells treated with high dose of Abeta (1 micromol/L). Although the level of cholesterol was not changed in any way, vitamin E partially prevented the neurotoxic effects of Abeta. CONCLUSION: beta-amyloid peptide reduces the level of cell membrane lipids and cholinergic receptors in human SH-SY5Y neuroblastoma cells, likely through the induction of an enhanced oxidative stress.

Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells.

In order to identify the properties of nicotine in relation to oxidative stress or neuroprotection, differentiated PC12 cells were treated with nicotine, beta-amyloid peptide (Abeta(25-35)), free radical inducer and antioxidant by a separate addition or a combination way. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lipid peroxidation, [3H]epibatidine binding sites for nicotinic receptor and [3H]quinuclidinyl benzilate (QNB) for muscarinic receptor have been detected. The significant decrease of MTT reduction and increase of lipid peroxidation in PC12 cells were only observed at treatments with high concentrations of nicotine (1 and 10 mM), while Vitamin E (VitE), an antioxidant, can prevent the neurotoxic effects. In addition, nicotine in low dosage (10 microM) rescued the decreased rates of cell viability and inhibited the production of lipid peroxidation resulted from H(2)O(2) and Abeta in the cultured cells. Significant increases in [3H]epibatidine binding sites were observed in PC12 cells exposed to nicotine, while no change was detected in [3H]QNB. The decreased number of nicotinic receptor binding sites due to the toxicity of Abeta was prevented by the addition of nicotine with low concentration. It is plausible that nicotine treatment may play dual effects on oxidative stress and neuroprotection, in which the effects are dependent on the differences in dosage of the drug used and their mechanisms of action. Generally, high dose of nicotine may induce neurotoxicity and stimulate oxidative stress, while reasonably low concentration may act as an antioxidant and play an important role for neuroprotective effect.