Expression of the zinc importer protein ZIP9/SLC39A9 in glioblastoma cells affects phosphorylation states of p53 and GSK-3β and causes increased cell migration

Zinc importer proteins (ZIPs) have been proven as important molecular regulators in different cancers. As a member of the solute carrier family, ZIP9/SLC39A9 is overexpressed in prostate and breast cancer and affects B-cell receptor signaling. Here, we present data indicating that changes in intracellular zinc levels in glioblastoma cells can cause enhanced cell survival and cell migration, both hallmarks of the disease process. In particular, treatment of human glioblastoma cells with sublethal doses of cell-permeable heavy metal (Zn²⁺ > Fe²⁺ > Mn²⁺) chelator (N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN)) induced ZIP9 expression. Either TPEN treatment or expression of ZIP9 cDNA causes enhanced migration behavior of glioblastoma cells. Compared to untreated glioblastoma cells TPEN treatment or expression of ZIP9 results in activation of the tumor suppressor p53 by phosphorylation at serine residue 46 (Ser46) and in inactivation of the migration relevant glycogen synthase kinase 3 beta (GSK-3β) by phosphorylation at serine residue 9 (Ser9). Whilst p53 activation affects cell survival in response to TPEN, GSK-3β inactivation directly affects glioblastoma cell migration. Therefore, ZIP9 expression could regulate the migratory behavior of glioblastoma cells, so that ZIP9 may be of biological, but not of clinical relevance for glioblastomas, since in GBM tumor tissues, ZIP9 expression is not significantly increased compared to normal brain.     

Manganese elevates manganese superoxide dismutase protein level through protein kinase C and protein tyrosine kinase

Three experiments were conducted to investigate the effects of inorganic and organic Mn sources on MnSOD mRNA, protein and enzymatic activity and the possible signal pathways. The primary broiler myocardial cells were treated with MnCl₂ (I) or one of organic chelates of Mn and amino acids with weak, moderate (M) or strong (S) chelation strength for 12 and 48 h. Cells were preincubated with superoxide radical anions scavenger N-acetylcysteine (NAC) or specific inhibitors for MAPKs and protein tyrosine kinase (PTK) or protein kinase C (PKC) for 30 min before treatments of I and M. The MnSOD mRNA, protein and enzymatic activity, phosphorylated MAPKs or protein kinases activations were examined. The results showed that additions of Mn increased (P < 0.05) MnSOD mRNA levels and M was more effective than I. Additions of Mn elevated (P < 0.05) MnSOD protein levels and enzymatic activities, and no differences were found among I and M. Addition of NAC did not decrease (P > 0.05) Mn-induced MnSOD mRNA and protein levels. None of the three MAPKs was phosphorylated (P > 0.05) by Mn. Additions of Mn decreased (P < 0.05) the PTK activities and increased (P < 0.05) the membrane PKC contents. Inhibitors for PTK or PKC decreased (P < 0.05) Mn-induced MnSOD protein levels. The results suggested that Mn-induced MnSOD mRNA and protein expressions be not related with NAC, and MAPK pathways might not involve in Mn-induced MnSOD mRNA expression. PKC and PTK mediated the Mn-induced MnSOD protein expression.     

Phosphorylation of JNK Increases in the Cortex of Rat Subjected to Diabetic Cerebral Ischemia

Previous studies have demonstrated that the c-Jun N-terminal kinase (JNK) pathway plays an important role in inducing neuronal apoptosis following cerebral ischemic injury. JNK signaling pathway in activated during cerebral ischemic injury. It participates in ischemia-induced neuronal apoptosis. However, whether JNK signaling is involved in the process of neuronal apoptosis of diabetes-induced cerebral ischemia is largely unknown. This study was undertaken to evaluate the influence of cerebral ischemia–reperfusion injury on phosphorylation of JNK in diabetic rats. Twenty-four adult streptozotocin induced diabetic and 24 adult non-diabetic rats were randomly subjected to 15 min of forebrain ischemia followed by reperfusion for 0, 1, 3, and 6 h. Sixteen sham-operated diabetic and non-diabetic rats were used as controls. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Protein expression of phospho-JNK was examined by immunohistochemistry and Western blot. The numbers of TUNEL-positive cells and phospho-JNK protein expression in the cerebral cortices after 1, 3 and 6 h reperfusion was significantly higher in diabetic rats compared to non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). Western blot analysis showed significantly higher phospho-JNK protein expression in the cerebral cortices of the diabetic rats after 1 and 3 h reperfusion than that was presented in non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). These findings suggest that increased phosphorylation of JNK may be associated with diabetes-enhanced ischemic brain damage.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

Lutein protects against β-amyloid peptide-induced oxidative stress in cerebrovascular endothelial cells through modulation of Nrf-2 and NF-κb

In the present study, we determined the protective role of lutein against Aβ _25–35 peptide-induced oxidative stress and apoptosis in bEND.3 cells. Cell viability was determined through MTT assay. Reactive oxygen species, lipid peroxides, and antioxidant enzyme activities were evaluated to analyze the oxidative stress status. NF-κB and Nrf-2 downstream target protein expressions were determined through western blot. Apoptosis was analyzed through caspase activities and subG1 accumulation. The results showed that Aβ _25–35 significantly increased (p < 0.001) oxidative stress biomarkers. Aβ _25–35 significantly up-regulated NF-κB nuclear expression and down-regulated Nrf-2 levels and HO-1 and, NQO-1 expressions. Aβ _25–35 induced apoptosis through decreasing mitochondrial membrane potential and increasing caspase 9 and 3 activities. Lutein pre-treatment significantly (p < 0.001) improved cell viability and decreased ROS levels (p < 0.001) and lipid peroxidation (p < 0.01). Lutein prevented Aβ _25–35-induced NF-κB nuclear expressions and up-regulated Nrf-2 expressions. Further, lutein also improved mitochondrial membrane potential and down-regulated caspase activities and subG1 accumulation. The present study shows the protective role of lutein against Aβ _25–35-induced toxicity by modulating Nrf-2 and NF-κB expressions in cerebrovascular endothelial cells.     

Accumulation of Heavy Metals in Some Marine Fisheries Resources Collected from Gulf of Mannar Marine Biosphere Reserve,Southeast Coast of India

Concentrations of toxic metals viz. mercury (Hg), cadmium (Cd) and lead (Pb) were evaluated in four species of fishes (Sardinella longiceps, Selaroides leptolepis, Epinephelus quoyanus and Lethrinus lentjan), one species of shrimp (Penaeus semisulcatus) and one species of crab (Portunus sanguinolentus) sampled from Thoothukudi, Keelakarai and Veerapandian pattinam of Gulf of Mannar, Southeast coast of India. Results revealed accumulation of these metals in the following order Hg > Cd > Pb. Hg concentration was found to be higher in Po. sanguinolentus followed by E. quoyanus, Pe. semisulcatus and L. lentjan however, the same was absent in Sa. longiceps and Se. leptolepis. Cd concentration was recorded in decreasing order in Po. sanguinolentus > Pe. semisulcatus > L. lentjen > E. quoyanus > Sa. longiceps > Se. leptolepis. Pb was detectable only in four species. Results of One-way ANOVA revealed significant variations (p < 0.05) in accumulation of Cd in Sa. longiceps, Se. leptolepis and Pe. semisulcatus and Hg in E. quoyanus, L. lentjan and Po. sanguinolentus. Variations noted in Pb were not statistically significant throughout.     

Heavy metal accumulation in the leaves of <Emphasis Type="Italic">Potamogeton natans</Emphasis> and <Emphasis Type="Italic">Ceratophyllum demersum</Emphasis> in a Himalayan RAMSAR site: management implications

Heavy metals have detrimental impacts on the health of organisms including human beings. Wetlands are economical, natural alternatives for the removal of heavy metals from the environment and macrophytes play a pivotal role in this direction, though they vary in their potential to do so. Heavy metal accumulation capability of two dominant species (Ceratophyllum demersum and Potamogeton natans) in a Kashmir Himalayan Ramsar site was studied. The accumulation of the different metals in P. natans was in the order of Al > Mn > Pb > Cu > Zn > Ni > Co > Cr > Cd, while in C. demersum it was Al > Mn > Zn > Co > Cu > Pb > Cr > Ni > Cd. In C. demersum the highest bioconcentration factor (BCF) was obtained for Co (3616) and Mn (3589) while in P. natans the highest BCF corresponded to Cd (1027). Overall Potamogeton–Ceratophyllum combination may provide a useful mix for Co, Mn and Cd removal from contaminated sites. The management implications of these results are briefly discussed.     

Insulin Attenuates Beta-Amyloid-Associated Insulin/Akt/EAAT Signaling Perturbations in Human Astrocytes

The excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), mostly located on astrocytes, are the main mediators for glutamate clearance in humans. Malfunctions of these transporters may lead to excessive glutamate accumulation and subsequent excitotoxicity to neurons, which has been implicated in many kinds of neurodegenerative disorders including Alzheimer’s disease (AD). Yet, the specific mechanism of the glutamate system dysregulation remains vague. To explore whether the insulin/protein kinase B (Akt)/EAAT signaling in human astrocytes could be disturbed by beta-amyloid protein (Aβ) and be protected by insulin, we incubated HA-1800 cells with varying concentrations of Aβ_1–42 oligomers and insulin. Then the alterations of several key substrates in this signal transduction pathway were determined. Our results showed that expressions of insulin receptor, phospho-insulin receptor, phospho-protein kinase B, phospho-mammalian target of rapamycin, and EAAT1 and EAAT2 were decreased by the Aβ_1–42 oligomers in a dose-dependent manner (p < 0.05) and this trend could be recovered by insulin treatment (p < 0.05). However, the expressions of total Akt and mTOR were invariant (p > 0.05), and the mRNA levels of EAAT1 and EAAT2 were also unchanged (p > 0.05). Taken together, this study indicates that Aβ_1–42 oligomers could cause disturbances in insulin/Akt/EAAT signaling in astrocytes, which might be responsible for AD onset and progression. Additionally, insulin can exert protective functions to the brain by modulating protein modifications or expressions.     

GL261 glioma tumor cells respond to ATP with an intracellular calcium rise and glutamate release

Necrotizing enterocolitis (NEC) is one of the most severe and unpredictable complications of prematurity. There are two possible mechanisms involved in the pathogenesis of NEC: individual inflammatory response and impaired blood flow in mesenteric vessels with secondary ischemia of the intestine. The aim of this study was to evaluate the possible relationship between polymorphisms: Il-1β 3953C>T, Il-6 ?174G>C and ?596G>A, TNFα ?308G>A, and 86 bp variable number tandem repeat polymorphism of interleukin-1 receptor antagonist (Il-1RN VNTR 86 bp) and three polymorphisms that may participate in arteries tension regulation and in consequence in intestine blood flow impairment: eNOS (894G>T and ?786T>C) and END-1 (5665G>T) and NEC in 100 infants born from singleton pregnancy, before 32 + 0 weeks of gestation, exposed to antenatal steroids therapy, and without congenital abnormalities. In study population, 22 (22%) newborns developed NEC. Surgery-requiring NEC was present in 7 children. Statistical analysis showed 20-fold higher prevalence of NEC in infants with the genotype TT [OR 20 (3.71–208.7); p = 0.0004] of eNOS 894G>T gene polymorphism. There was a higher prevalence of allele C carriers of eNOS 786T>C in patients with surgery-requiring NEC [OR 4.881 (1.33–21.99); p = 0.013]. Our investigation did not confirm any significant prevalence for NEC development in another studied genotypes/alleles. This study confirms the significant role of polymorphisms that play role in intestine blood flow. Identifying gene variants that increase the risk for NEC development may be useful in screening infants with inherent vulnerability and creating strategies for individualized care.     

Fibroblast growth factor-8 inhibits oxidative stress-induced apoptosis in H9c2 cells

Fibroblast growth factors (FGFs) comprise a large family of signaling molecules that involve cell patterning, mobilization, differentiation, and proliferation. Various FGFs, including FGF-1, FGF-2, and FGF-5, have been shown to play a role in cytoprotection during adverse cardiac events; however, whether FGF-8 is a cytoprotective remains unclear. The current study was designed to evaluate the effect of FGF-8 treatment on oxidative stress-induced apoptosis in H9c2 cells. Cells were divided into three groups: control, H₂O₂ (400 µm H₂O₂), and H₂O₂ + FGF-8 (4 ng/ml FGF-8). Our results suggest apoptosis was significantly (p < 0.05) enhanced in the H₂O₂ group relative to control. Moreover, a significant (p < 0.05) decline in apoptosis was observed in the H₂O₂ + FGF-8 group compared to H₂O₂-treated cells as evidenced by TUNEL staining, a cell death detection ELISA, and cell viability. Levels of downstream apoptotic mediators, caspase-3 and caspase-9, were significantly (p < 0.05) upregulated following H₂O₂ treatment but were abrogated following FGF-8 application. Expression levels of Forkhead box protein O1 (FoxO-1), MnSOD, catalase, pAKT, and p-mTOR were significantly (p < 0.05) reduced in the H₂O₂ group (p < 0.05). Notably, these levels were significantly (p < 0.05) reversed following FGF-8 treatment. Our data, for the first time, suggest FGF-8 is an anti-apoptotic mediator in oxidative-stressed H9c2 cells. Furthermore, our data demonstrate that apoptotic inhibition by FGF-8 is consequent to FoxO-1 oxidative detoxification as well as augmentation to the PI3K/AKT cell survival pathway.     

Comparative analysis of molecular activity in dermal mesenchymal stem cells from different passages

Mesenchymal stem cells (MSCs) are used for tissue regeneration in several pathological conditions, including autoimmune diseases. However, the optimal sources and culture requirements for these cells are still under investigation. Here, we compared mRNA expression in dermal MSCs (DMSCs) at passage (P) 3 and P5 to provide a reference for future studies related to DMSCs expansion. In normal DMSCs, the expression of three of eight genes associated with basic cellular activity were different at P5 compared to that at P3: PLCB4 and SYTL2 were upregulated by 4.30- and 6.42-fold, respectively (P < 0.05), whereas SATB2 was downregulated by 39.25-fold (P < 0.05). At the same time, genes associated with proliferation, differentiation, inflammation, and apoptosis were expressed at similar levels at P3 and P5 (P > 0.05). In contrast, in DMSCs isolated from psoriatic patients we observed differential expression of three inflammation-associated genes at P5 compared to P3; thus IL6, IL8, and CXCL6 mRNA levels were upregulated by 16.02-, 31.15-, and 15.04-fold, respectively. Our results indicate that normal and psoriatic DMSCs showed different expression patterns for genes related to inflammation and basic cell activity at P3 and P5, whereas those for genes linked to proliferation, differentiation, and apoptosis were mostly similar.     

Association between <Emphasis Type="Italic">TNF,IL1B,IL6, IL10</Emphasis> and <Emphasis Type="Italic">IFNG</Emphasis> polymorphisms and recurrent miscarriage: a case control study

Background

Approximately half of recurrent miscarriages have unexplained etiology. Recent evidences suggest that cytokines are important determinants in pregnancy maintenance and as such, cytokine gene polymorphisms, which can affect cytokine production and/or functionality, could play a role in the disorder. Thus, we aimed to investigate the association of selected cytokine gene polymorphisms with risk of recurrent miscarriage among Chinese.

Methods

TNF -238G > A, TNF -308G > A, IL1B -511 T > C, IL1B 3954C > T, IL6 -174G > C, IL6 -634C > G, IL10 -1082A > G and IFNG 874A > T polymorphisms were genotyped on 775 women with idiopathic recurrent miscarriage and 805 healthy parous control women. Logistic regression analysis was performed to determine the odds ratios (ORs) of the association between the polymorphisms and recurrent miscarriage risk.

Results

Among the eight polymorphisms studied, only the IL1B -511 T > C and IL6 -634C > G polymorphisms showed statistically significant associations with recurrent miscarriage risk. For the former, a significantly increased risk of recurrent miscarriage was observed for the mutant (CC) genotype (OR: 1.377; 95% CI: 1.039–1.824; P?=?0.026). However, for the IL6 -634C?>?G polymorphism, a decreased recurrent miscarriage risk was observed for the heterozygous (CG) genotype (OR: 0.614; 95% CI: 0.493–0.765; P < 0.001) and the mutant (GG) genotype (OR: 0.414; 95% CI: 0.251–0.684; P?=?0.001).

Conclusions

The IL1B -511 T > C polymorphism may serve as important risk factor for recurrent miscarriage while the IL6 -634C > G polymorphism may protect against the risk of recurrent miscarriage.

     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨ_m), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨ_m loss, and apoptosis were all significantly reduced in TLR4^?/? macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.     

Cytotoxic effect of <Emphasis Type="Italic">Semialarium mexicanum</Emphasis> (Miers) Mennega root bark extracts and fractions against breast cancer cells

The root bark of Semialarium mexicanum (Miers) Mennega (cancerina) is traditionally used in Mexico to treat cancer. However, there are no studies supporting its use. We evaluated whether S. mexicanum root bark induces cytotoxicity in breast cancer cells to determine if it has potential applications in the treatment of this disease. Extracts of S. mexicanum root bark in petroleum ether, ethanol, and water were obtained by ultrasound-assisted extraction. MTT and WST-1 assays were used to evaluate the cytotoxicity of the extracts toward breast cancer cells (MDA-MB-231 and MCF7), non-tumorigenic breast-derived cells (MCF 10A), and peripheral blood mononuclear cells (PBMCs). For the extract with greatest cytotoxicity, induction of apoptosis and oxidative stress were determined using flow cytometry. The extract was fractionated, and the cytotoxicity of its fractions was evaluated with the four cell types. The fractions were also analyzed by HPLC. Only the petroleum ether extract was cytotoxic for all cell types (MDA-MB-231?>?MCF 10A/MCF7?>?PBMCs). Cell death occurred by apoptosis, which could be associated with the induction of oxidative stress. Two fractions that were highly cytotoxic for breast cancer cells were obtained from this extract (IC₅₀?≤?4.15 µg/mL for the most active fraction at 72 h). The MCF 10A cells were less affected, while PBMCs were not affected after 72 h of treatment. Pristimerin was identified in both fractions and may be partially responsible for the cytotoxic effect. These results suggest that S. mexicanum root bark has a potential application in breast cancer treatment.     

The effect of mechanical stretch stress on the differentiation and apoptosis of human growth plate chondrocytes

The study is aimed to investigate the effect of stretch stress with different intensities on the differentiation and apoptosis of human plate chondrocytes. In the present study, the human epiphyseal plate chondrocytes were isolated and cultured in vitro. Toluidine blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes. Mechanical stretch stresses with different intensities were applied to intervene cells at 0-, 2000-, and 4000-μ strain for 6 h via a four-point bending system. The expression levels of COL2, COL10, Bax, Bcl-2, and PTHrp were detected by quantitative RT-PCR. Under the intervention of 2000-μ strain, the expression levels of COL2, COL10, and PTHrp increased significantly compared with the control group (P < 0.05), and the expression level of PCNA was also increased, but the difference was not statistically significant (P > 0.05). Under 4000-μ strain, however, the expression levels of PCNA, COL2, and PTHrp decreased significantly compared with the control group (P < 0.05), and the expression level of COL10 decreased slightly (P > 0.05). The ratio of Bcl-2/Bax gradually increased with the increase of stimulus intensity; both of the differences were detected to be statistically significant (P < 0.05). In conclusion, the apoptosis of growth plate chondrocytes is regulated by mechanical stretch stress. Appropriate stretch stress can effectively promote the cells’ proliferation and differentiation, while excessive stretch stress inhibits the cells’ proliferation and differentiation, even promotes their apoptosis. PTHrp may play an important role in this process.     

The Effect of Sodium Fluoride on Cell Apoptosis and the Mechanism of Human Lung BEAS-2B Cells In Vitro

Sodium fluoride (NaF) is a source of fluoride ions used in many applications. Previous studies found that NaF suppressed the proliferation of osteoblast MC3T3 E1 cells and induced the apoptosis of chondrocytes. However, little is known about the effects of NaF on human lung BEAS-2B cells. Therefore, we investigated the mode of cell death induced by NaF and its underlying molecular mechanisms. BEAS-2B cells were treated with NaF at concentrations of 0, 0.25, 0.5, 1.0, 2.0, and 4.0 mmol/L. Cell viability decreased and apoptotic cells significantly increased as concentrations of NaF increased over specific periods of time. The IC₅₀ of NaF was 1.9 and 0.9 mM after 24 and 48 h, respectively. The rates of apoptosis increased from 4.8 to 37.7% after NaF exposure. HE staining, electron microscopy, and single cell gel electrophoresis revealed that morphological changes of apoptosis increased with exposure concentrations. RT-PCR and Western blotting were used to detect the apoptotic pathways. The expressions of bax, caspase-3, caspase-9, p53, and the cytoplasmic CytC of the NaF groups increased, while bcl-2 and mitochondrial CytC decreased compared with that of the control group (P < 0.05). Further, the fluorescence intensities of ROS in the NaF groups were higher than those in the control group, and the membrane potential of mitochondria in the NaF group was significantly lower than that of the control group (P < 0.05). These findings suggested that NaF induced apoptosis in the BEAS-2B cells through mitochondria-mediated signal pathways. Our study provides the theoretical foundation and experimental basis for exploring the mechanisms of human lung epithelial cell damage and cytotoxicity induced by fluorine.