Edaravone Attenuates the Proinflammatory Response in Amyloid-ß-Treated Microglia by Inhibiting NLRP3 Inflammasome-Mediated IL-1ß Secretion.

BACKGROUND/AIMS: Microglial activation is an important pathological feature in the brains of patients with Alzheimer's disease (AD), and amyloid-ß (Aß) peptides play a crucial role in microglial activation. In addition, edaravone (EDA) was recently shown to suppress oxidative stress and proinflammatory cytokine production in APPswePS1dE9 (APP/PS1) mice. However, the mechanism by which EDA inhibits the Aß-induced proinflammatory response in microglia is poorly understood. METHODS: The mitochondrial membrane potential (∆ψm) was evaluated using JC-1 staining. Intracellular reactive oxygen species (ROS) and mitochondrial ROS levels were detected using CM-H2DCFDA and MitoSOXTM Red, respectively. The levels of CD11b, NLRP3, pro-caspase-1 and manganese superoxide dismutase (SOD-2) were observed by western blotting, and the levels of interleukin-1beta (IL-1ß) in culture supernatants were quantified using an ELISA kit. RESULTS: Aß induced microglia activation and mitochondrial dysfunction. In addition, mitochondrial dysfunction was associated with ROS accumulation and activation of the NLRP3 inflammasome. Importantly, Aß induced activation of the NLRP3 inflammasome, leading to caspase-1 activation and IL-1ß release in microglia. Moreover, EDA obviously attenuated the depolarization of ∆ψm, reduced mitochondria-derived ROS production and increased SOD-2 activity, resulting in the suppression of NLRP3 inflammasome-mediated IL-1ß secretion in Aß-treated microglia. CONCLUSION: EDA is a mitochondria-targeted antioxidant and exhibits anti-inflammatory effects on Aß-treated microglia.

LncRNA-MALAT1 Promotes Angiogenesis of Thyroid Cancer by Modulating Tumor-Associated Macrophage FGF2 Protein Secretion.

Tumor-associated macrophages (TAMs) in the tumor microenvironment have been associated with enhanced tumor progression. In this study, we investigated the role and molecular mechanisms of MALAT1 in TAMs derived from thyroid cancer. The expression of MALAT1 and FGF2 in thyroid cancer tissues and cells were measured by quantitative real-time PCR and Western blot. TAMs were transfected with indicated constructs. Then the culture medium (CM) from TAMs was harvested for assay. Secreted FGF2 protein levels and TNF-α, IL-12, and IL-10 levels were detected by ELISA. The cell proliferation, migration, and invasion of FTC133 cells were determined with a CCK-8 assay and a Transwell assay, respectively. In addition, HUVEC vasculature formation was measured by matrigel angiogenesis assay. The higher levels of MALAT-1 and FGF2 were observed in thyroid cancer tissues and in thyroid cancer cells compared to that in the control. Besides, in the presence of si-MALAT1, the levels of TNF-α and IL-12 were significantly up-regulated whereas IL-10 was down-regulated in the CM from TAMs. Moreover, down-regulation of MALAT1 in TAMs reduced proliferation, migration, and invasion of FTC133 cells and inhibited angiogenesis. However, overexpression of FGF2 blocked the effects of MALAT1 siRNAs on cell migration, invasion, and angiogenesis. Our results suggest that MALAT1-mediated FGF2 protein secretion from TAMs inhibits inflammatory cytokines release, promotes proliferation, migration, and invasion of FTC133 cells and induces vasculature formation. J. Cell. Biochem. 118: 4821-4830, 2017. © 2017 Wiley Periodicals, Inc.

Quality of life and cosmetic result of single-port access endoscopic thyroidectomy via axillary approach in patients with papillary thyroid carcinoma.

BACKGROUND: Endoscopic thyroidectomy for minimally invasive thyroid surgery has been widely applied in the past decade. The present study aimed to evaluate the effects of single-port access transaxillary totally endoscopic thyroidectomy on the postoperative outcomes and functional parameters, including quality of life and cosmetic result in patients with papillary thyroid carcinoma (PTC). PATIENTS AND METHODS: Seventy-five patients with PTC who underwent endoscopic thyroidectomy via a single-port access transaxillary approach were included (experimental group). A total of 123 patients with PTC who were subjected to conventional open total thyroidectomy served as the control group. The health-related quality of life and cosmetic and satisfaction outcomes were assessed postoperatively. RESULTS: The mean operation time was significantly increased in the experimental group. The physiological functions and social functions in the two groups were remarkably augmented after 6 months of surgery. However, there was no significant difference in the scores of speech and taste between the two groups at the indicated time of 1 month and 6 months. In addition, the scores for appearance, satisfaction with appearance, role-physical, bodily pain, and general health in the experimental group were better than those in the control group at 1 month and 6 months after surgery. CONCLUSION: The single-port access transaxillary totally endoscopic thyroidectomy is safe and feasible for the treatment of patients with PTC. The subjects who underwent this technique have a good perception of their general state of health and are likely to participate in social activities. It is worthy of being clinically used for patients with PTC.

MALAT1 promotes the proliferation and invasion of thyroid cancer cells via regulating the expression of IQGAP1.

BACKGROUND: Increasing evidence indicated that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acted as a key regulator in the proliferation and invasion of several cancers. However, the function of MALAT1 in the development of thyroid cancer has not been experimentally established. METHODS: The expression of MALAT1 and IQGAP1 in thyroid cancer tissues and cells were detected by quantitative real-time PCR and western blot. The effects of MALAT1 and IQGAP1 on the cell proliferation and invasion of thyroid cancer cells were detected with a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium 4 (MTT) assay and a Transwell assay, respectively. FTC-133 or SW1736 transfected with si-MALAT1 or pcDNA-MALAT1 were injected subcutaneously into 4-week-olds BALB/c mice to examine the impact of MALAT1 on the tumor development of thyroid cancer in vivo. RESULTS: In this study, we discovered the higher level of MALAT-1 and expression of IQGAP1 in thyroid cancer tissues and in thyroid cancer cells compared to that in the control. MTT and Transwell assay showed that the proliferation and invasion of FTC-133 cells with MALAT-1 knockdown were inhibited. Moreover, MALAT-1 could upregulate the expression of IQGAP1 in thyroid cancer cells. In addition, IQGAP1 knockdown reversed the decreasing cell proliferation and invasion of thyroid cancer induced by MALAT-1 overexpression. Finally, the study in vivo verified that MALAT-1 promoted the tumor growth of thyroid cancer. CONCLUSION: Our study indicated that MALAT1 promoted the proliferation and invasion of thyroid cancer cells via regulating the expression of IQGAP1.

[Expressions of MACC1, Snail, and KISS-1 Proteins in Infiltrating Breast Carcinoma and Its Clinicopathological Features].

OBJECTIVES: To investigate the protein expressions of metastasis-associated in colon cancer 1 (MACC1), KISS-1 (a cancer ruppressor gene) and Snail (the marker of epithelial-mesenchymal transition) in infiltrating breast carcinoma (IBC) and explore the role of them in invasion, metastasis and prognosis in IBC. METHODS: Expressions of MACC1, Snail and KISS-1 were examined by immunohistochemistry in 250 specimens of IBC and 80 specimens of normal breast tissues. Their clinicopathological features were analyzed, and their influence on patients' survival was identified. RESULTS: The positive rate of MACC1, Snail and KISS-1 in normal breast tissues and IBC tissues was 7.5%, 5.0%, 87.5% and 63.6%, 58.8%, 38.0%, respectively. And there was a significant difference between the IBC group and control group ( P<0.05). The positive expressions of MACC1, Snail and KISS-1 were significantly different in different TNM stages, lymph node metastasis, types and grades of tumor ( P<0.05). The survival time of positive KISS-1 group was significantly longer than that of negative group ( P<0.001); the survival time was significantly shorter in positive MACC1 group or Snail group than that of negative groups (both P<0.001). Cox regression analysis indicated that the positive expressions of MACC1, Snail and negative expression of KISS-1 were independent risk factors of IBC (P<0.05). CONCLUSIONS: Abnormal expression of MACC1, Snail and KISS-1 should be involved in the invasion and metastasis of IBC. The combined detection in the expressions of MACC1, Snail and KISS-1 at the early stage may play an important role in predicting the progression and prognosis of IBC.

3-N-butylphthalide (NBP) attenuates the amyloid-ß-induced inflammatory responses in cultured astrocytes via the nuclear factor-κB signaling pathway.

BACKGROUND/AIMS: Activation of astrocytes is a common feature of Alzheimer's disease (AD). Proinflammatory molecules produced by activated astrocytes contribute to neuronal damage in AD. Moreover, dl-3-n-butylphthalide (NBP) has been reported to attenuate astroglial activation and exert neuroprotective effects in AD transgenic mice. However, the mechanism by which NBP inhibits activated astrocytes is poorly understood. METHODS: In this study, the primary astrocytes were obtained from the cerebral cortices of 1-day-old Sprague-Dawley rats. The levels of GFAP, COX-2, NF-κB, and IκBα were examined by Western blotting and the levels of TNF-α and IL-6 were determined by ELISA. RESULTS: NBP inhibited the amyloid-ß (Aß)-induced activation of astrocytes and the up-regulation of proinflammatory molecules. Importantly, NBP markedly suppressed Aß-induced IκBα degradation and nuclear factor-κB (NF-κB) translocation. CONCLUSION: Our results suggest that NBP attenuates Aß-induced activation of astrocytes and neuroinflammation via inhibition of the NF-κB signaling pathway.

Inhibition of glycogen synthase kinase-3ß by lithium chloride suppresses 6-hydroxydopamine-induced inflammatory response in primary cultured astrocytes.

An increasing amount of evidence has emerged to suggest that neuroinflammatory process is involved in the pathogenesis of Parkinson's disease (PD). Activated microglia and astrocytes are found in the substantia nigra (SN) of Parkinson's disease brains as well as in animal models of Parkinson's disease. Although reactive astrocytes are involved in the progression of PD, the role of reactive astrocytes in neuroinflammation of PD has received limited attention to date. Recently, Glycogen synthase kinase-3ß (GSK-3ß) was identified as a crucial regulator of the inflammatory response. The purpose of this study was to explore the mechanism by which 6-hydroxydopamine (6-OHDA) induces inflammatory response in astrocytes and observe the anti-inflammatory effect of lithium chloride (LiCl) on 6-OHDA-treated astrocytes. In the present study, we found that glial fibrillary acidic protein (GFAP) was markedly upregulated in the presence of 6-OHDA. Moreover, our results revealed that proinflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2(COX-2), prostaglandins E2 (PGE2), and tumor necrosis factor-α (TNF-α) were obviously increased in astrocytes exposed to 6-OHDA. Western blot analysis revealed that 6-OHDA significantly increased dephosphorylation/activation of GSK-3ß as well as the nuclear translocation of nuclear factor-κB (NF-κB) p65. Besides, GSK-3ß inhibitor LiCl and SB415286 inhibited the GSK-3ß/NF-κB signaling pathway, leading to the reduction of proinflammatory molecules in 6-OHDA-activated astrocytes. These results confirmed that GSK-3ß inhibitor LiCl and SB415286 provide protection against neuroinflammation in 6-OHDA-treated astrocytes. Therefore, GSK-3ß may be a potential therapeutic target for the treatment of PD.

Age-dependent alterations in the presynaptic active zone in a Drosophila model of Alzheimer's disease.

The accumulation of beta amyloid (Aß) can cause synaptic impairments, but the characteristics and mechanisms of the synaptic impairment induced by the accumulation of Aß in Alzheimer's disease (AD) remain unclear. In identified single neurons in a newly developed Drosophila AD model, in which Aß accumulates intraneuronally, we found an age-dependent reduction in the synaptic vesicle release probability that was associated with a decrease in the density of presynaptic calcium channel clusters and an increase in the presynaptic and postsynaptic contact length. Moreover, these alterations occurred in the absence of presynaptic bouton loss. In addition, we found that Aß expression also produced an age-dependent decrease in the amount of Bruchpilot (Brp), which plays an important role in controlling Ca(2+) channel clustering and synaptic vesicle release in the presynaptic active zone. Our study indicates that the chronic accumulation of intraneuronal Aß can induce functional and structural changes in the presynaptic active zone prior to a loss of presynaptic buttons in the same neuron.

[Study on alternative methods for the acute oral toxicity in detection of chemicals].

OBJECTIVE: To evaluate three alternative methods for LD50 test-Fixed Dose Procedure (FDP), the Acute Toxic Class Method (ATC) and Up and Down Procedure (UDP). METHODS: Female SD rats (8-12 weeks of age, 160-200 g) were used. Three alternative methods from OECD were applied to assess 22 chemicals (10 cosmetic raw materials and 12 raw materials of personal and home care products). The toxicity ranking for tested chemicals was established according to Globally Harmonized System (GSH). The results LD50 test were compared for the consistency and correlation between alternative methods and traditional test. RESULTS: For cosmetic raw materials, the concordance rate of the three alternative methods was 80% (8/10); for raw material of personal and home care products, the concordance rates of FDP, ATC and UDP was 91.7% (11/12), 75.0% (9/12) and 83.0% (10/12), respectively. The number of animals required in three alternative methods was significantly lower than that in traditional test (P < 0.05), but the time required in three alternative methods was significantly higher than that in traditional test (P < 0.05). CONCLUSIONS: High consistency and correlation were found between each alternative method and LD50 test. FDP may be more potential when applied to assess acute oral toxicity of cosmetic raw materials.

Overexpression of human MRP1 in neurons causes resistance to antiepileptic drugs in Drosophila seizure mutants.

Multidrug resistance-associated protein 1 (MRP1), an efflux multidrug transporter, was shown to be elevated in both glia and neurons in seizure focus in refractory epilepsy patients. Up-regulation of MRP1 and other multidrug transporters in perivascular astrocytes was suggested to cause resistance to antiepileptic drugs (AEDs) by reducing the concentration of AEDs at the epileptogenic areas. However, it is not known whether the up-regulation of MRP1 in neurons can cause resistance to AEDs, such as sodium phenytoin (PHT) and valproic acid (VPA). PHT inhibits voltage-gated sodium channel (VGSC) by occluding it, but whether PHT enters the channel through its inner or outer pore is not known. The authors overexpressed human MRP1 protein only in neurons in a Drosophila genetic seizure model, bang senseless (bss) mutants. The authors found that overexpression of MRP1 blocked the attenuation of the seizure behavior of bss mutants by acute and chronic application of PHT, and by chronic application of VPA. Conversely, overexpression of MRP1 in neurons increased the tolerance of bss flies to high-dosage PHT and VPA. Thus, up-regulation of MRP1 expression only in neurons causes resistance to AED in seizure flies. Moreover, the current data suggest that PHT enters VGSC through its inner pore.

Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila.

Alzheimer's disease (AD) is attributable to synapse dysfunction and loss, but the nature and progression of the presynaptic structural and functional changes in AD are essentially unknown. We expressed wild-type or arctic form of beta amyloid(1-42) (Abeta) in a small group of neurons in the adult fly and performed extensive time course analysis of the function and structure of both axon and presynaptic terminals at the identified single-neuron level. Abeta accumulated intracellularly and induced a range of age-dependent changes, including depletion of presynaptic mitochondria, slowdown of bi-directional transports of axonal mitochondria, decreased synaptic vesicles, increased large vacuoles, and elevated synaptic fatigue. These structural and functional synaptic changes correlated with age-dependent deficit in motor behavior. All these alterations were accelerated in flies expressing the arctic form of Abeta. The depletion of presynaptic mitochondria was the earliest detected phenotype and was not caused by the change in axonal transport of mitochondria. Moreover, axonal mitochondria exhibited a dramatic reduction in number but a significant increase in size in aged Abeta-expressing flies, indicating a global depletion of mitochondria in the neuron and an impairment of mitochondria fission. These results suggest that Abeta accumulation depletes presynaptic and axonal mitochondria, leading to other presynaptic deficits.

[Application of flow cytometry on functional assessment of health food].

OBJECTIVE: To establish flow cytometry (FCM) methods and evaluate their application value for measuring the index for enhancing immune function of health food. METHODS: In mice experiment model, the dosage groups were respectively oral fed with three test substances according to 5, 10, 30 times of the recommended dose for human body; both the negative and positive control groups were fed with equivalence purified water once a day. The positive control was fed with 25 mg/kg body weight levamisole for 3 days before finishing the administration, and the immune two percent of sheep erythrocytes were administrated at the last day. In rats experiment model, the test substance was given by mixing feed according to 25 and 50 times of the recommended dose for human body. At the end of the experiment, indices below were simultaneously detected. (1) The classical indices included: spleen lymphocyte transformation test by using ConA (MTT assay); spleen NK cell activity test (LDH assay); delayed-type hypersensitivity test by using sheep erythrocyte (foot palm thickening) method and phagocytosis activity tested by mice peritoneal macrophages. (2) FCM indices included: T and B lymphocytes quantitating in mice peripheral blood, activated antigen expression level in the surface of T lymphocytes and NK cells and phagocytosis activity for fluospheres in mice peritoneal macrophages. RESULTS: (1) Compared with the negative control group, there were no significant differences in T and B lymphocytes proportion and the number of lymphocytes in mice peripheral blood after given 0.83, 1.67, 5.01 g/kg protein powder; (2) mice peripheral blood T lymphocyte sub-cluster CD(69)(+)/CD(3)(+) of 3.75, 7.50, 15.0 ml/kg bw Cen-Rong Cream groups were all significantly increased (P < 0.05), which were shown a good coherence with the classic test index; (3) mice peripheral blood NK cell sub-cluster CD(69)(+)/NKG2D(+) of 0.83, 1.67 g/kg protein powder groups were both significantly increased (P < 0.05), which was kept in good coherence with those of NK cell activity test (LDH assay); rats peripheral blood NK cell sub-cluster CD(161a+)/CD(25)(+) of 1.50 g/kg ganoderma lucidum and cordycepicmycelia group was significantly increased (P < 0.05); (4) the phagocytosis activity in mice peritoneal macrophages: there were no significant difference found between the controls and the dosage groups in the classic test. However, in the FCM test, the percentage of phagocytic cells of 0.15, 0.30, 0.90 g/kg ganoderma lucidum and cordycepicmycelia groups and the phagocytic index of 0.30, 0.90 g/kg were enhanced. CONCLUSION: It suggests that was shown in detecting and assessing enhancing immune function of health food the results tested by FCM were fairly consistent with those by using traditional methods, most of them would have higher sensitivity. It should be valuable to applying FCM in the measurement and assessment of enhancing immune function of health food and worth while to further study as to enlarging its application.