Potential Role of Glycogen Synthase Kinase-3ß in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3ß, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3ß results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3ß regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3ß inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3ß inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3ß inhibitors were mimicked by transfection with GSK-3ß siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3ß mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3ß in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3ß reduces myocardin transcriptional activity, suggesting a role for GSK-3ß in myocardin transcriptional activity and smooth muscle differentiation.

[Expression of calcineurin and nuclear factor of activated T cells 1 in testis of rats with chronic fluorosis].

OBJECTIVE: To discuss the significance of calcineurin (CaN) and nuclear factor of active T cells 1 (NFATc1) in the damage mechanism of the testis of rats with chronic fluorosis. METHODS: Eighteen clear class SD male rats, aging 6 week-old, were randomly divided into 3 groups, 6 rats in each. The rats of control group were fed with tap water (NaF < 1 mg/L) and the experimental rats were exposed to NaF (lower group: 5 mg/L, higher group: 50 mg/L) to established the chronic fluorosis model. After 8 months, we observed the occurrence of dental fluorosis among rats in different groups, and the contents of urine fluoride were detected by fluorine ion selective electrode method. The body of the rats were weighted as well as their testis. The testis tissues were stained with hematoxylin-eosin and observed under light microscope to find the morphological changes. The expression of CaN and NFATc1's protein and mRNA in testis were detected by Immunocytochemistry (IHC) and In-situ hybridization (ISH). RESULTS: The number of rats which was found dental fluorosis were separately 0, 4 and 5 in control group, low dose group and high dose group (χ(2) = 10.60, P < 0.05). The contents of urine fluoride were gradually increased in control group, low group and high group, which were (1.26 ± 0.17), (2.06 ± 0.64) and (7.69 ± 1.96)mg/L, respectively (F = 36.57, P < 0.05). The body weight were significantly different in all three groups(629.00 ± 16.00), (585.17 ± 17.27), (560.50 ± 16.07)g, F = 26.67, P < 0.05) and the testis weight were without statistical difference ((2.58 ± 0.17), (2.43 ± 0.31), (2.35 ± 0.38)g, F = 0.91, P > 0.05). Compared with the control group, the testicular structures were damaged in the experimental groups and especially significant in high dose group. The expression of CaN (59.10 ± 5.62, 77.93 ± 4.16, 101.69 ± 6.31, F = 74.18, P < 0.05) and NFATc1's (76.11 ± 4.41, 93.42 ± 3.85, 120.42 ± 9.31, F = 92.4, P < 0.05) protein in testis tissues were increased by the fluorine concentration. The mRNA expression of CaN and NFATc1 were separately (CaN: 58.76 ± 7.70, 82.01 ± 6.88, 99.47 ± 8.33, F = 42.65, P < 0.05 and NFATc1: 59.39 ± 4.74, 90.02 ± 5.37, 121.15 ± 7.69, F = 155.47, P < 0.05). There were positive correlation between the expression of CaN and NFATc1's protein and mRNA expression (r = 0.899, r = 0.908). CONCLUSION: The changes in the signaling pathway of expression of CaN may be involved in the injury mechanism of testis tissues of rats with chronic fluorosis.

[Changed expression of mito-fusion 1 and mitochondrial fragmentation in the cortical neurons of rats with chronic fluorosis].

OBJECTIVE: To observe the mitochondrial fragmentation and the expression of mito-fusion 1 gene in the cortical neurons of rats with chronic fluorosis, and to reveal their roles in mitochondria damage to neurons due to chronic fluorosis. METHODS: SD rats were divided randomly into three groups of 20 each (a half females and a half males housed individually in stainless-steel cages), and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride and high supplemented with 10 and 50 mg/L fluoride, respectively). After 3 or 6 months exposure, the mitochondrial morphology of the neurons in rat brains were observed by transmission electron microscopy (TEM), then the expression of mitochondrial fusion gene, Mfn1, were detected by immunohistochemistry and western-blotting, respectively. RESULTS: Dental fluorosis was obvious in the rats exposed to excessive fluoride in their drinking water, that is, (16 rats out of 20) numbers of I° detal fluorosis in the low-fluoride group, and (11 rats out of 20) numbers of I° and (9 rats out of 20) numbers of II° detal fluorosis in the high-fluoride group were observed after 3 months exposure. Moreover, (14 rats out of 20) numbers of I° and (6 rats out of 20) numbers of II° detal fluorosis in the low-fluoride group and (6 rats out of 20) numbers of Io, (13 rats out of 20) numbers of II°, and (1 rats out of 20) numbers of III° detal fluorosis in the high-fluoride group were observed after 6 months exposure. And both of untreated controls without detal fluorosis were also observed. The urinary level of fluoride in the low-fluoride group (3.30 ± 1.18) mg/L and in the high-fluoride group (5.10 ± 0.35) were observed after 3 months exposure (F = 3.18, P < 0.05). Moreover, the urinary level of fluoride in the low-fluoride group (4.16 ± 1.39) mg/L and in the high-fluoride group (5.70 ± 1.70) mg/L were also observed after 6 months exposure (F = 3.17, P < 0.05). The normal mitochondrial morphology of neurons in rats without fluorosis was observed after 3 and 6 months, while the abnormal mitochondrial morphology of neurons with fluorosis was shown, presenting mitochondrial fragmentation with swollen cristae and even the fragmented, shortened or stacked punctuate membranes (section observation of three bullous mitochondrial-mitochondrial fission process) by TEM. As compared with controls (53.0 ± 4.54 and 1.21 ± 0.18) at the experiment period of 3 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 51.09 ± 6.25) and western-blotting (1.22 ± 0.26) were no significant difference for low fluoride group (t = 1.7, 1.1, P > 0.05); Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 59.71 ± 5.64) and western-blotting (1.66 ± 0.20) were significantly increasing for high fluoride group (t = 2.1, 2.1, P < 0.05). As compared with controls (36.43 ± 4.04 and 1.00 ± 0.13) at the experiment period of 6 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells 20.05 ± 4.55 and 17.10 ± 3.86) and western-blotting (0.64 ± 0.08 and 0.39 ± 0.06) were significantly decreasing for the two fluoride group (t = 2.1, 2.2; 2.2, 2.2 respectively, all P value were < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the mitochondrial fragmentation for the changed expression of Mfn1, and the neurons damage from the chronic fluorosis might be associated with the dysfunction of mitochondrial fusion.

[Influence of chronic fluorosis on the expression of mitochondrial fission protein dynamin-related 1 in the cortical neurons of rats].

OBJECTIVE: To explore the changes of protein expression of mitochondrial fission gene dynamin-related 1(Drp 1) in the cortical neurons of rats with chronic fluorosis. METHODS: A total of 120 one-month-old SD rats (each weighing approximately 100-120 g at the beginning of the experiment) were randomly divided into three groups, and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride & high-fluoride supplemented with 10 and 50 mg/L fluoride,respectively). After 3 or 6 months exposure, 20 rats from each group were killed. Then the protein expression of mitochondrial fission gene, Drp1, was detected by immunohistochemistry and western-blotting method. RESULTS: Dental fluorosis and urinary fluorosis were obviously found in the rats exposed to fluoride. At the experiment period of 3 months, the numbers of positive cells of Drp1 detected by immunohistochemistry changed. Compared with the control group (36.3 ± 5.8), the changes in low-fluoride group (34.7 ± 4.1) showed no significant difference (t = 1.5, P > 0.05),but the increase in high-fluoride group (45.0 ± 4.7) had statistical significance (t = 8.8, P < 0.05). The western-blotting method had consistent results. Compared with the control group (0.59 ± 0.03), a significant increase of the average topical density in low- fluoride (0.62 ± 0.03) and high-fluoride (0.71 ± 0.02) groups were found (t = 0.02,0.11, P < 0.05). At the experiment period of 6 months, the numbers of positive cells of Drp1 detected by immunohistochemistry significantly changed. Compared with the control group (33.2 ± 4.4), the number in low- fluoride and high-fluoride groups were separately (36.6 ± 3.8) and (39.4 ± 4.2),both increased significantly (t = 3.5,6.3, P < 0.05). Same results could be found in western-blotting method,compared with the control group (0.65 ± 0.06), the average topical density in low- fluoride (0.80 ± 0.09) and high-fluoride (0.76 ± 0.08) groups both increased significantly (t = 0.1,0.1, P < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the changes of expression of Drp1, and the neurons damage from the chronic fluorosis might be associated with the hyperfunction of mitochondrial fusion.

Fluoride induces osteoblast autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in vivo and in vitro.

Fluorosis primarily manifests as bone damage in the form of dental fluorosis and skeletal fluorosis and represents a critical global public health challenge. However, few studies have examined autophagy-related signaling pathways in skeletal fluorosis. This study aimed to investigate the effect of fluoride on autophagy in osteoblasts using comprehensive methods and to explore the role of the PI3K/AKT/mTOR signaling pathway in regulating fluoride-induced autophagy in osteoblasts. Sprague-Dawley (SD) rats were exposed to different concentrations of fluoride (NaF: 5, 50, and 100 mg/L) for six months. Primary osteoblasts were treated with 0.5, 1.0, or 3.0 mM NaF. Hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence staining, and western blotting were performed to evaluate morphological changes in bone tissues and autophagosomes and to detect the protein expression of autophagy-related markers and PI3K/AKT/mTOR signaling pathway-related molecules both in vivo and in vitro. The bone tissues of fluoride-exposed rats showed osteosclerosis, autophagosomes and autolysosomes. LC3B immunofluorescence staining revealed an increase in autophagosomes in the primary osteoblasts treated with fluoride. The LC3Ⅱ/Ⅰ ratio and levels of autophagy-related markers (Beclin 1 and Atg7) were increased, whereas P62 levels were decreased in bone tissues and primary osteoblasts in the fluoride groups. Simultaneously, p-AKT and p-mTOR levels were reduced in bone tissues and primary osteoblasts in the fluoride groups. Moreover, a PI3K inhibitor (LY294002) further downregulated p-AKT and p-mTOR protein expression but slightly increased the LC3Ⅱ/Ⅰ ratio in primary osteoblasts. These results demonstrate that fluoride induces autophagy in osteoblasts by inhibiting the PI3K/AKT/mTOR signaling pathway, which deepens our understanding of the molecular mechanisms underlying fluoride-induced bone damage and provides a theoretical basis for the prevention and treatment of skeletal fluorosis.

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.

[Effect of fluoride on expression of CaN mRNA and protein in bone tissue of rats].

OBJECTIVE: To investigate the changes of mRNA and protein expression of CaN in the bone of rats with chronic fluorosis, and the mechanism of skeletal fluorosis. METHODS: Thirty-six SD rats were divided into three groups (12 in each group, half male and half female selected according to body weight): control, low-dose and high-dose fluorosis groups. Controls were fed tap water (NaF < 0.5 mg/L), experimental animals in the low- or high-dose groups were fed water containing NaF of 5.0 and 50.0 mg/L, respectively. The rats were sacrificed after 6 months of treatment with fluoride. The serum was kept for testing bone metabolic marker bone gla protein (BGP) by enzyme-linked immunosorbent assay (ELISA), the protein and mRNA levels of CaN in distal femur of the rats with chronic flurosis were assessed by immunohistochemistry and in-situ hybridization. RESULTS: The levels of BGP (1.99 ± 0.62, 2.38 ± 0.16)µg/L in the low- or high-dose fluorosis groups were higher than that in the control group (0.15 ± 0.03) µg/L; and the high fluorosis group showed higher level than the low fluorosis group (all P < 0.05). Compared to the control group (131.11 ± 1.95, 111.82 ± 2.39), the protein and mRNA levels of CaN were higher in the low- or high-dose fluorosis groups (142.69 ± 1.17, 157.54 ± 1.88 and 121.28 ± 3.27, 134.63 ± 3.19, respectively), and the high fluorosis group showed higher levels than the low fluorosis group (all P < 0.05). CONCLUSIONS: BGP content could be used as a bone metabolic index in endemic fluorosis disease. Fluoride might up-regulate the mRNA and protein expression of CaN, and the changes in CaN level may be involved in the increase of the bone turnover and could be one of the pathogenetic factors in fluorosis.

[Effect of fluoride on oxidative stress and Mn-SOD expression in rats with endemic fluorosis of coal burning].

OBJECTIVE: To study the effect of fluoride on the oxidative stress of the rats in endemic fluorosis of coal burning and Mn-SOD expression at mRNA and protein levels. METHODS: SD rats were divided into 2 groups (the number of female and male in each group was the same): control group and fluorosis group. All rats of the fluorosis group were fed corn dried by burning coal from endemic fluorosis areas with high fluoride content (fluoride 17 mg/kg in feed) to establish an animal model of fluorosis. In these rats, dental fluorosis was evaluated. The fluoride content in the urine was measured by fluorine ion-elective electrode method. The hepatic tissue and serum level of malonaldehyde (MDA) and the activities of superoxide dismutase (SOD) and glutathion reductase (GR) were measured by biochemical methods. The index signs of liver function were also measured from the serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the alterations of Mn-SOD expression in the liver at mRNA and protein levels. RESULTS: The dental fluorosis was observed in the fluorosis group, and the incidence was 11/11. The fluoride contents [(3.50 ± 2.58) mg/L] in the urine of fluorosis rats were increased as compared with the control [(1.42 ± 0.38) mg/L] (P < 0.05). AST [(223.74 ± 71.51) U/L] and total protein [(72.43 ± 5.59) g/L] of the hepatic function index in fluorosis rats showed obviously abnormal as compared with the control [(169.28 ± 53.74) U/L and (82.36 ± 7.31) g/L], respectively (P < 0.05). In the liver the content of MDA [(10.41 ± 0.59) µmol/g protein] increased as compared to the control [(5.80 ± 1.31) µmol/g protein, P < 0.01], and the activities of SOD [(62.60 ± 8.65) U/mg protein] and GR [ (1.17 ± 0.66) U/g protein] markedly decreased in the fluorosis group compared to the control [SOD (117.28 ± 8.64) U/mg protein and GR [(8.80 ± 1.59) U/g protein; P < 0.05, P < 0.01]. The level of Mn-SOD in the liver was markedly decreased in the fluorosis group [(14.83 ± 2.50) U/mg protein] as compared with the control [(34.05 ± 5.22) U/mg protein, P < 0.01]. The levels of mRNA (0.64 ± 0.15) and protein (0.84 ± 0.13) of Mn-SOD were markedly decreased in the fluorosis group as compared with the control [(0.86 ± 0.21) and (1.04 ± 0.14)], respectively (P < 0.05, P < 0.01). CONCLUSIONS: Fluorosis can decrease the activities of Mn-SOD, which is associated with decreased levels of mRNA and protein of Mn-SOD. Down-regulation of Mn-SOD expression may play an important role in the aggravation of oxidative stress in endemic fluorosis.

[Expression of mRNA and protein of p38, Osx, PI3K and Akt1 in rat bone with chronic fluorosis].

OBJECTIVE: To investigate the expressions of mRNA and protein of p38, Osx, PI3K, Akt1 in the rats bone with chronic fluorosis. METHODS: Dental fluorosis were observed and the fluoride contents in the urine and bone were detected by fluorin-ion selective electrode. The morphologic changes and ultrastructure of rats' bone were observed by light and electronic microscopy. The expressions of protein and mRNA of p38, Osx, PI3K and Akt1 were detected by immunohistochemistry and real-time PCR, respectively. The contents of BALP and BGP in serum were detected by ELISA. RESULTS: The rates of dental fluorosis in the fluorosis rats were increased, and the fluoride contents in bone and urine of the fluorosis rats were increased compared to the control group, the difference was statistically significant (P < 0.05). The bone trabeculae thickness and density and the thickness of bone cortex in fluorosis rats were remarkably increased, the space of bone trabeculae was reduced, and in accordance with the matching morphometrical indices, the difference was statistically significant (P < 0.05) as compared with the control rats. The contents of BALP [(54.61 ± 2.27) U/L] and BGP [(2.38 ± 0.16) µg/L]in the fluoride groups were higher than those in the control group, the difference was statistically significant (P < 0.05). Ultrastructurally, the broadening of the osseouslacuna was observed. The reduced protuberances of the osteocytes, the unclear organelle structure, pyknosis, karyotheca increasation and edged chromatin were also observed. Compared to the control group, the expressions of protein and its mRNA of p38, Osx, PI3K and Akt1 were higher in the fluorosis rats than those in the control rats, and the difference was statistically significant (P < 0.05). There is no any expression of p38, Osx, PI3K and Akt1 in the osteocytes in fluorosis rats. CONCLUSIONS: The over-expression of p38, Osx, PI3K and Akt1 in bone tissue of fluorosis rats may relate to the accumulation of fluorine in the body. The bone injury mainly occur in the stage of the differentiation and proliferation. The upregulation of P38MARK signal path and PI3K/Akt1 signal path may be involved in the pathogenesis of bone injury caused by fluoride.

[Alteration of mitochondrial distribution and gene expression of fission 1 protein in cortical neurons of rats with chronic fluorosis].

OBJECTIVE: To investigate the changes of mitochondrial distribution in axon/soma and the expression of mitochondrial fission 1 (Fis1) protein in the cortical neurons of rats with chronic fluorosis. METHODS: Sixty SD rats were divided into 3 groups (20 each) according to weight hierarchy and fed with different concentrations of fluoride in drinking water (0, 10 and 50 mg/L, respectively) for 6 months. Images of mitochondria and tubulin labeled by immunofluorescence COXIV and tubulin-α were captured with fluorescence microscope. Fis1 protein expression in cortical neurons was analyzed with immunohistochemistry and Western blot. The expression of Fis1 mRNA was detected with real-time PCR. RESULTS: Varying degrees of dental fluorosis and increased fluoride contents in urine were observed in the rats receiving additional fluoride in drinking water. In the cortical neurons of rats fed with 10 mg/L and 50 mg/L fluoride, the numbers of neuronal soma stained with COXIV(34.8 ± 4.7 and 39.3 ± 3.0, respectively), and the expression of Fis1 protein (immunohistochemistry: 54.0 ± 3.6 and 51.3 ± 4.1, respectively; Western blot: 2.9 ± 0.4 and 2.6 ± 0.6, respectively) and mRNA (3773 ± 1292 and 1274 ± 162, respectively) was markedly increased as compared with controls (4.4 ± 2.3, 25.2 ± 2.5, 1.8 ± 0.2 and 277 ± 73) over the experimental period of 6 months. CONCLUSIONS: Excessive intake of fluoride results in an altered mitochondrial distribution in axon and soma in cortical neurons (i.e., the increase in soma and the decrease in axon), increased expression of Fis1 gene and enhanced mitochondrial fission. The altered mitochondrial distribution may be related to the high expression level of Fis1 and a functional disorder of mitochondria.

Increased Stiffness of the Superficial Cervical Extensor Muscles in Patients With Cervicogenic Headache: A Study Using Shear Wave Elastography.

Background: Cervicogenic headache (CEH) is a secondary headache caused by lesions of the cervical spine and surrounding soft tissues. Cervical muscle dysfunction may be related to the onset of CEH. However, whether cervical muscle stiffness changes in patients with CEH has not been well studied. The purpose of this study was to explore changes in superficial cervical extensor muscle stiffness in patients with CEH using shear wave elastography (SWE). Methods: In this study, 19 patients with CEH and 20 healthy controls were recruited. Superficial cervical extensor muscle stiffness was obtained from SWE, and the SuperLinear SL10-2 MHz linear array probe in the musculoskeletal muscle mode was chosen as the transducer. Regions of interest in the trapezius (TRAP), splenius capitis (SPL), semispinalis capitis (SCap), and semispinalis cervicis (SCer) were manually segmented. Correlations between superficial cervical extensor muscle stiffness and visual analog scale (VAS) scores, age, and body mass index (BMI) were analyzed using Pearson's correlation. Receiver operating characteristic (ROC) curve was used to investigate the diagnostic ability of superficial cervical extensor stiffness for CEH. Results: Superficial cervical extensor muscle stiffness on the headache side of patients with CEH was higher than that on the non-headache side and in healthy controls (p < 0.05). Increased stiffness was also observed in SCer on the non-headache side of patients with CEH compared to healthy controls (p < 0.01). In patients with CEH, SCer stiffness was positively correlated with VAS scores (r = 0.481, p = 0.037), but no correlation was found between other muscles and VAS scores (p > 0.05). The areas under the curve of TRAP, SPL, SCap, and SCer in diagnosing CEH were 0.766, 0.759, 0.964, and 1.000, respectively. Conclusions: Increased stiffness was observed in the superficial cervical extensor muscles on the headache side of patients with CEH. SCer stiffness was correlated with headache intensity in patients with CEH and may provide clues for the diagnosis of CEH.

[Effect of endemic fluoride poisoning caused by coal burning on the oxidative stress in rat testis].

OBJECTIVE: To explore the effect of endemic fluoride poisoning caused by coal burning on the oxidative stress in rat testis. METHODS: Totally 40 male SD rats were equally randomized into four groups control group, low fluorosis group, middle fluorosis group, and high fluorosis group. Rats in all three fluorosis groups were fed with corn dried by burning coal obtained from endemic fluorosis areas with high fluoride, and thus the animal models of fluorosis were established. After 120 and 180 days, all the rats were sacrificed. Testis tissues were stained with hematoxylin eosin and observed under light microscope. The malonaldehyde (MDA) content, superoxide dismutase (SOD) activity, total nitric oxide synthase (TNOS), and inducible nitric oxidase synthase (iNOS) were measured by biochemical methods in the testis tissues. The content of NaF in testis was measured by fluorine selective electrode. RESULTS: The rat fluorosis models were successfully established. The fluoride content in testis was significantly increased in all the fluorosis groups(P<0.01). Testicular structures were damaged in all of fluoride groups. The TNOS, iNOS activities, and MDA content of each fluoride group were significantly higher than that of the control group on day 120 and 180 (P<0.05 or 0.01 ). The TNOS, iNOS activities, and MDA content significantly increased in a dose dependent manner (P<0.05 or 0.01). The SOD activities significantly decreased in all the fluoride groups (P<0.05 or 0.01). CONCLUSIONS: Endemic fluoride poisoning caused by coal burning can cause disorders in the oxidative system and antioxidative system in rat testis. The oxidative stress may play an important role in the fluorides induced reproductive toxicity in male rats.

[The influence of fluoride on expression of OPGL and M-CSF genes and their proteins in rats with experimental fluorosis and the therapeutic effect of Danlan Xianpeng Liaofu caspule].

OBJECTIVE: To investigate mRNA and protein expressions of OPGL and M-CSF mRNA in bones of rats with experimental fluorosis induced by intake of fluoride in the drinking water, and to study the antagonizing effects of Danlan Xianpeng Liaofu capsules on the gene expression. METHODS: Totally 72 SD rats were randomly assorted into 6 groups including the control group, the fluoride group, the high-dosage (0.8 g/kg×d), mid-dosage (0.4 g/kg×d) and low dosage (0.2 g/kg×d) medication groups and the borax group (borax, 0.8 g/kg×d). The distribution of female and male rats in each group was divided up on a fifty-fifty basis. Except the control group, a NaF containing water (NaF 50 mg/L in concentration) was supplied as the drinking water for all the experimental rats in order to establish experimental fluorosis. The thickness and density of trabecula and the thickness of bone cortex were measured by light microscopy. The fluoride content in urine and bone were analyzed by using fluoride ion selective electrode method. Expressions of OPGL and M-CSF mRNA and protein were studied using RT-PCR and immuno-histochemistry, respectively. RESULTS: (1) 10/12 of the experimental fluorosis rats developed dental fluorosis, and 2/12 of dental fluorosis rats occurred in the low-dosage medication group. Fluoride content in urine and bone of the fluorosis rats increased (P<0.05). (2) Compared with that of the control rats, the bone trabecular depth, cortical thickness and trabecular density in experimental fluorosis rats were remarkably reduced. (3) Compared with that of the control group, mRNA expression of both OPGL and M-CSF was increased in the fluoride group rats. The difference was statistically significant (P<0.05). (4) Compared with that of the fluoride group animals, the expression intensity of OPGL mRNA decreased in animals of the control group, the high, mid- and low- dosage medication groups and the borax group. Among them, except the low-dosage group, the difference between all the other groups and the fluoride group was statistically significant, respectively (P<0.05). There was also a decrease of M-CSF mRNA in all the 3 medication groups and the borax group animals in comparing with that of the fluoride group and the difference was also statistically significant (P<0.05), respectively. (5) Compared with that of the control group. There were an increase of OPGL and a decrease of M-CSF protein expression; and in addition, there were a decrease of OPGL and an increase of M-CSF protein expression in all 3 medication groups and the borax group in comparing with that of the fluoride group anima (P < 0.05). CONCLUSIONS: Excessive fluoride induces an accelerated bone turnover and may promote the absorption activity of osteoclasts by increasing the expression of OPGL and M-CSF. Danlan Xianpeng Liaofu capsule may be capable of regulating bone remodeling through a down-regulation on OPGL and M-CSF expression.

[Cyclin E overexpression and centrosome amplification in squamous cell carcinoma of oral cavity].

OBJECTIVE: To study the correlation between cyclin E protein overexpression and centrosome amplification in oral squamous cell carcinoma (OSCC). METHODS: Formalin-fixed, paraffin-embedded tissues from 12 normal oral epithelium cases and 46 cases of OSCC were studied. Their centrosome status was analyzed by indirect immunofluorescence double staining with antibodies to centrosome protein gamma-tubulin and cytokeratin. The expression of cyclin E protein was studied by immunohistochemical methods. The correlation between cyclin E protein expression and centrosome amplification in OSCC was statistically analyzed by SPSS 12.0. RESULTS: Thirty-seven of the 46 OSCC cases (80.4%) studied showed evidence of centrosome amplification, as signified by enlargement and/or increase in number of centrosomes, while normal oral epithelium possessed centromeres of normal size and number. Positive staining for cyclin E protein was observed in 30 of the 46 OSCC cases (65.2%), while all the normal oral epithelium cases were cyclin E protein-negative. The percentage of centrosome amplification in OSCC with positive cyclin E protein staining (90.0%, 27/30) was higher than that in OSCC with negative cyclin E protein staining (62.5%, 10/16) (chi(2) = 5.014, P < 0.05). Centrosome amplification showed positive correlation with cyclin E protein overexpression (r = 0.330, P < 0.05). CONCLUSION: Up-regulation of cyclin E protein may represent one of the possible mechanisms for centrosome amplification in OSCC.

Effects of a GSK-3ß inhibitor on the renal expression levels of RANK, RANKL and NF-κB in a rat model of diabetic nephropathy.

The present study aimed to investigate the effects of glycogen synthase kinase-3ß (GSK-3ß) on the expression levels of receptor activator of nuclear factor (NF)-κB (RANK), RANK ligand (RANKL) and NF-κB in the renal tissues of rats modeling diabetic nephropathy (DN). The rats were allocated at random into three groups, as follows: Normal control group (NC), the DN model group (DNM group) and the DN model lithium chloride (LiCl) intervention group (DNI group). Urinary proteins were examined by staining with the Coomassie Brilliant Blue dye for 24 h. Histochemical analyses of kidney tissue sections were conducted using hematoxylin and eosin staining, after which the kidney pathology of the rats was observed. In addition, the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB in the renal tissues were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. As compared with the NC group, the level of urinary protein was significantly increased in the DNM group (P<0.05); however, as compared with the DNM Group, the level of urinary protein at 12 weeks was significantly decreased in the DNI group (P<0.05). As compared with the NC group, marked pathological changes were detected, and the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB were significantly increased, in the renal tissues of the DNM group. Conversely, pathological alterations in the renal tissues were attenuated, and the mRNA and protein expression levels of GSK-3ß, RANK, RANKL and NF-κB were significantly decreased (P<0.05), in the DNI group, as compared with the DNM group. The results of the present study suggested that GSK-3ß, RANK, RANKL and NF-κB may be crucially involved in the development of DN, and that LiCl may effectively attenuate DN by reducing the expression levels of GSK-3ß, RANK, RANKL and NF-κB.

[The over-expression of serine/threonine kinase 15 protein in oral carcinogenesis].

OBJECTIVE: To investigate the expression of STK15 and P53 proteins in oral precancerous lesions and oral squamous cell carcinoma (OSCC) and elucidate the possible role of P53/STK15 switch activation-independent pathway in oral carcinogenesis. METHODS: Formalin-fixed, paraffin-embedded tissues of 8 cases of normal oral epithelium, 27 cases of dysplasia with different degree epithelium dysplasia and 43 cases of OSCC with different differentiation were investigated for the expression of STK15 and P53 proteins by using immunohistochemistry. The clinical and pathological significance of STK15 over-expression in oral carcinogenesis were statistically analyzed by SPSS 12.0. RESULTS: STK15 protein was not detectable in normal oral epithelium and significantly altered from mild-dysplasia to OSCC. The percentage of STK15 over-expression were 40.74% (11/27) in dysplasia and 67.44% (29/43) in OSCC (P < 0.05). The percentage of STK15 over-expression in OSCC with positive P53 staining was significantly higher than that in OSCC with negative P53 staining (P < 0.05). STK15 over-expression was significantly associated with regional lymph node involvement (P < 0.05), while no correlation was found for STK15 over-expression and tumor differentiation, as well as TNM stages. CONCLUSION: STK15 up-regulation was an early event in oral carcinogenesis. The up-regulation of STK15 protein in OSCC may partly result from p53 mutations, which probably contribute a role in lymph node metastasis of OSCC as well. P53/STK15 switch activation-independent pathway may play some roles in oral carcinogenesis.

[Centrosome amplification and chromosome instability in oral squamous cell carcinomas].

OBJECTIVE: To investigate the relationship between centrosome abnormalities and aneuploidy in oral squamous cell carcinoma (OSCC) and elucidate the possible underlying mechanisms of chromosome instability (CIN) in OSCC. METHODS: Formalin-fixed, paraffin-embedded tissues of 8 cases of normal oral epithelium and 32 cases of OSCC were examined for centrosome status by using indirect immunofluorescence staining, and chromosome instability (aneuploidy) in some tissues were detected by flow cytometry. The correlation between centrosome abnormalities and aneuploidy in OSCC was statistically analyzed by SPSS12.0. RESULTS: Normal oral epithelium showed normal size and number of centrosomes in epithelium cells, while 25 out of 32 cases of OSCC showed the evident centrosome amplification characterized by huge size and/or supernumerary centrosomes in a fraction of tumor cells, and 21 out of 32 cases were aneuploidy. The percentage of cases with abnormal centrosomes in aneuploid OSCC (19/21) was significantly higher than that in diploid OSCC(6/11) (P =0.032). Centrosome abnormality was significantly correlated with aneuploidy (Spearman r = 0.413, P = 0.047), and a positive correlation was found between the degree of centrosome amplification and the degree of DNA ploidy abnormality (Pearson r = 0.364, P = 0.041). CONCLUSIONS: Centrosome abnormality may be a contributing factor for chromosome instability in OSCC.