Effect of α7nAChR on learning and memory dysfunction in a rat model of diffuse axonal injury.

Diffuse axonal injury (DAI) is the predominant effect of severe traumatic brain injury and significantly contributes to cognitive deficits. The mechanisms that underlie these cognitive deficits are often associated with complex molecular alterations. α7nAChR, one of the abundant and widespread nicotinic acetylcholine receptors (nAChRs) in the brain, plays important physiological functions in the central nervous system. However, the relationship between temporospatial alterations in the α7nAChR and DAI-related learning and memory dysfunction are not completely understood. Our study detected temporospatial alterations of α7nAChR in vulnerable areas (hippocampus, internal capsule, corpus callosum and brain stem) of DAI rats and evaluated the development and progression of learning and memory dysfunction via the Morris water maze (MWM). We determined that α7nAChR expression in vulnerable areas was mainly reduced at the recovery of DAI in rats. Moreover, the escape latency of the injured group increased significantly and the percentages of the distance travelled and time spent in the target quadrant were significantly decreased after DAI. Furthermore, α7nAChR expression in the vulnerable area was significantly positively correlated with MWM performance after DAI according to regression analysis. In addition, we determined that a selective α7nAChR agonist significantly improved learning and memory dysfunction. Rats in the α7nAChR agonist group showed better learning and memory performance than those in the antagonist group. These results demonstrate that microstructural injury-induced alterations of α7nAChR in the vulnerable area are significantly correlated with learning and memory dysfunctions after DAI and that augmentation of the α7nAChR level by its agonist contributes to the improvement of learning and memory function.

Inhibition of miRNA-21 promotes retinal ganglion cell survival and visual function by modulating Müller cell gliosis after optic nerve crush.

BACKGROUND: Müller cell gliosis not only plays an important physiological role by maintaining retinal neuronal homeostasis but is also associated with multiple pathological events in the retina, including optic nerve crush (ONC) injury. Modulating Müller cell gliosis contributes to the creation of a permissive environment for neuronal survival. However, the underlying mechanism of Müller cell gliosis has remained elusive. OBJECTIVE: To investigate the underlying mechanism of Müller cell gliosis after ONC. METHODS: Rats with ONC injury were transfected with miRNA-21 (miR-21) agomir (overexpressing miR-21) or antagomir (inhibiting miR-21) via intravitreous injection. Immunofluorescence and western blotting were performed to confirm the effects of miR-21 on Müller cell gliosis. The retinal nerve fiber layer (RNFL) thickness was measured using optical coherence tomography and the positive scotopic threshold response (pSTR) was recorded using electroretinogram. RESULTS: In the acute phase (14 days) after ONC, compared with the crushed group, inhibiting miR-21 promoted Müller cell gliosis, exhibiting thicker processes and increased GFAP expression. In the chronic phase (35 days), inhibiting miR-21 ameliorated Müller cell gliosis, which exhibited thicker and denser processes and increased GFAP expression. Retinal ganglion cell (RGC) counts in retinas showed that the number of surviving RGCs increased significantly in the antagomir group. The thickness of the RNFL increased significantly, and pSTR showed significant preservation of the amplitudes in the antagomir group. CONCLUSIONS: Inhibition of miR-21 promotes RGC survival, RNFL thickness and the recovery of RGC function by modulating Müller cell gliosis after ONC.

Ara-C increases gastric cancer cell invasion by upregulating CD-147-MMP-2/MMP­9 via the ERK signaling pathway.

Gastric cancer cell are not particularly sensitive to Ara-C, a deoxycytidine analog that affects DNA synthesis. In the present study, AGS and MKN-45 gastric cancer cell lines were treated with Ara-C to determine its role in cell prolife-ration and apoptosis. The antiproliferative effect of Ara-C was assessed using the Cell Counting kit-8. Gelatinase zymography was utilized to detect the activity of MMP-2 and MMP-9, and an in vitro invasion assay was performed. Using RT-PCR, CD-147, MMP-2 and MPP-9 mRNA levels were assessed in AGS cells with various doses of Ara-C treatment. CD-147, MMP-2 and MMP-9 protein levels were analysed in Ara-C­treated AGS and MKN-45 cells. AGS cells were treated with or without U-0126 or siRNA-CD147 and/or Ara-C for 24 h, and an in vitro invasion assay was performed. Although low-dose Ara-C had no obvious effect on cell proliferation, it upregulated the expression of MMP-2, MMP-9 and CD-147 and ERK activation. Low-dose Ara-C increased gastric cancer cell invasion. U-0126 and siRNA-CD-147 inhibited the induction of Ara-C in gastric cancer cell invasion. Therefore, Ara-C enhances the invasiveness of gastric cancer cells by expression of CD-147 /MMP-2 and MMP-9 via the ERK signaling pathway. The results are therefore useful in the prevention of Ara-C collateral damage associated with standard, conventional protocols of chemotherapy administration.

The role of mitochondria in mTOR-regulated longevity.

Several unbiased genome-wide RNA interference (RNAi) screens have pointed to mitochondrial metabolism as the major factor for lifespan regulation. However, conflicting data remain to be clarified concerning the mitochondrial free radical theory of aging (MFRTA). Recently, mTOR (mechanistic target of rapamycin) has been proposed to be the central regulator of aging although how mTOR modulates lifespan is poorly understood. Interestingly, mTOR has been shown to regulate many aspects of mitochondrial function, such as mitochondrial biogenesis, apoptosis, mitophagy and mitochondrial hormesis (mitohormesis) including the retrograde response and mitochondrial unfolded protein response (mito-UPR). Here we discuss the data linking mitochondrial metabolism to mTOR regulation of lifespan, suggesting that hormetic effects may be key to explaining some controversial results regarding the MFRTA. We also discuss the possibility that dysfunction of mitochondrial adaptive responses rather than free radicals per se contributes to the aging process.

Abnormal activation of the EGFR signaling pathway mediates the downregulation of miR­145 through the ERK1/2 in non-small cell lung cancer.

The expression of miR-145 with tumor suppressor function is decreased in lung cancer cells. Epidermal growth factor receptor (EGFR) signaling pathway is abnormally activated in lung cancer cells. It is not clear whether the EGFR signaling pathway is involved in the regulation of miR-145 expression in lung cancer. In the present study, we found that the reduction of miR-145 was associated with EGFR abnormal activation in lung cancer cells. AG1478, an inhibitor of EGFR, may restore the expression of miR-145, indicating that EGFR activation is involved in the downregulation of miR-145 in lung cancer cells. Then, the application of STAT3, AKT and ERK1/2 inhibitors and siRNA against these signaling molecules indicated that ERK1/2 or AKT instead of STAT3 was involved in the process of miR-145 downregulation by EGFR. It was confirmed that AKT through activation of the ERK1/2 signaling molecules mediated the effect of EGFR on miR-145. Furthermore, we found that EGFR downregulated miR-145 through ERK1/2 in lung cancer cells. These findings establish EGFR and miR-145 links in lung cancer cells and therefore contribute to a better understanding of the role of EGFR in lung cancer cells, and provide clues for in-depth study of miR-145 expression and a possible direction for the further increase of miR-145 in lung cancer cells.

Tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts by the inhibition of superoxide anion production and glutathione depletion.

BACKGROUND/OBJECTIVES: Free radicals and reactive oxygen species (ROS), which are generated by UV irradiation, may induce an irreversible growth arrest similar to senescence. Tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid, is a widely used antioxidant to rescue ROS-evoked cell death. The aim of the article was to explore the effects of tiron on skin photoaging and associated mechanisms. METHODS: The effects of tiron on cell proliferation were determined using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide. Senescent cells were determined by morphology and senescence-associated ß-galactosidase activity analysis. Intracellular hydrogen peroxide, superoxide anion and glutathione concentration were analysed by a fluorescent probe. The concomitant changes of protein expression were analysed with Western blot. RESULTS: Human dermal fibroblasts were induced to premature senescence by sub-cytotoxic doses of irradiated UVB. Strong senescence-associated ß-galactosidase activity and increased intracellular superoxide anion were observed in human dermal fibroblasts irradiated by UVB. Tiron blocks UVB-induced glutathione depletion and increase of superoxide anion and protects against UVB-induced senescence-like characteristics in human dermal fibroblasts. Compared with normal fibroblasts, UVB-irradiated human dermal fibroblasts showed a higher ratio of active (hypophosphorylated) to inactive (phosphorylated) forms of Rb and p38, upregulation of p53 or p16 and c-Myc and insulin-like growth factor 1 (IGF-1) downregulation. After treatment with tiron, p53, p16 c-Myc and IGF-1 as well as phosphorylation Rb and p38 could partially recover. CONCLUSION: These results indicate that tiron protects against UVB-induced senescence-like characteristics in human dermal fibroblasts via the inhibition of production of superoxide anion and glutathione depletion, and modulation of related senescence proteins.

Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells.

We recently demonstrated that oridonin could induce apoptosis and senescence of colon cancer cells in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the involvement of reactive oxygen species in oridonin-induced cell death and senescence was investigated in colon adenocarcinoma-derived SW1116 cells. Oridonin increased intracellular hydrogen peroxide levels and reduced the glutathione content in a dose-dependent manner. N-acetylcysteine, a reactive oxygen species scavenger, not only blocked the oridonin-induced increase in hydrogen peroxide and glutathione depletion, but also blocked apoptosis and senescence induced by oridonin, as evidenced by the decrease in Annexin V and senescence-associated ß-galactosidase- positive cells and the inhibition of oridonin-induced upregulation of p53 and p16 and downregulation of c-Myc. Moreover, exogenous catalase could inhibit the increase in hydrogen peroxide and apoptosis induced by oridonin, but not the glutathione depletion and senescence. Furthermore, thioredoxin reductase (TrxR) activity was reduced by oridonin in vitro and in cells, which may cause the increase in hydrogen peroxide. In conclusion, the increase in hydrogen peroxide and glutathione depletion account for oridonin-induced apoptosis and senescence in colorectal cancer cells, and TrxR inhibition is involved in this process. Given the importance of TrxR as a novel cancer target in colon cancer, oridonin would be a promising clinical candidate. The mechanism of oridonin-induced inhibition of TrxR warrants further investigation.

Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc.

BACKGROUND: Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. METHODS: Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated ß-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. RESULTS: Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. CONCLUSION: Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

[Protective effects of rhu TNFR: Fc against the lipopolysaccharide induced intestinal damage of rats and its underlying mechanism].

To investigate the protective effects of recombinant human tumor necrosis factor receptor II: IgG Fc fusion protein (rhu TNFR: Fc) against the lipopolysaccharide (LPS) induced intestinal damage of rats and its underlying mechanism. SD rats were randomly divided into four groups: control group, rhuTNFR: Fc group, LPS group and rhu TNFR: Fc + LPS group. Mean arterial pressure (MAP) was continuously monitored and the mortality rates were assessed. The levels of TNF-alpha and its bioactivity in the serum were assessed by ELISA and flow cytometry respectively. Pathologic changes of intestinal tissue were observed by HE staining. The rats of control and rhu TNFR: Fc group all survived with stable MAP, and the low level and bioactivity of TNF-alpha in the serum were maintained. While 83% of the rats in LPS group died by 6 h with the levels and bioactivity of TNF-alpha increasing significantly. In rhu TNFR: Fc + LPS group, the mortality rate of rats dropped to 33%. The TNF-alpha level increased compared with control group but its bioactivity decreased significantly compared with LPS group. The MPO activity and content of MDA decreased significantly. The status of pathological manifestation in the intestine was also ameliorated. These data suggest that rhu TNFR: Fc could protect rats from the acute intestine injury induced by LPS through ablating the rise in serum TNF-alpha level and bioactivity as well as anti-oxidation.

mTOR signaling pathway is a target for the treatment of colorectal cancer.

BACKGROUND: mTOR signaling has been suggested to be an important factor involved in tumorigenesis, but its role in human colorectal cancer (CRC) has not been completely elucidated. Herein, the purpose of this study was to analyze the distribution pattern of mTOR signaling components in CRC and adenoma and to determine whether targeted inhibition of mTOR could be a potential therapeutic strategy for CRC. METHODS: Immunohistochemical analysis was performed on human CRC and adenoma for mTOR signaling components, including mTOR, p70s6 K, and 4EBP1. HCT116 and SW480 human CRC cell lines were treated with siRNA directed against mTOR, and cell viability, cell cycle, and apoptosis were assessed. HCT116 and SW480 cells were injected into athymic nude mice to establish a CRC xenograft model. Mice were randomly transfected with either nontargeting control or mTOR siRNA, and tumor volume, mTOR signaling activity, and apoptosis were evaluated. RESULTS: mTOR signaling components, including mTOR, p70s6 K, and 4EBP1, were highly activated in glandular elements of CRC and colorectal adenomas with high-grade intraepithelial neoplasia (HIN), with a correlation between staining intensity and depth of infiltration in CRC. Inhibition of mTOR expression using a specific mTOR siRNA resulted in considerably decreased in vitro and in vivo cell growth. CONCLUSIONS: mTOR signaling is associated with the clinical pathological parameters of human CRC. siRNA-mediated gene silencing of mTOR may be a novel therapeutic strategy for CRC.

[Effect of ryanodine receptor 2 gene silencing on ischemia-reperfusion injury of rat myocardial cells].

OBJECTIVES: To block the synthesis of ryanodine receptor 2 (RyR2) in myocardial cells by RNA interference and to investigate its biological impact on ischemia-reperfusion (I/R) in rat myocardial cells. METHODS: Rat myocardial cells were isolated and cultured for an I/R model in vitro. RNA interference technique was used to block the synthesis of RyR2 in myocardial cells. Changes of LDH level, apoptosis, RyR2 mRNA expression and cytosolic Ca(2+) concentration were analyzed accordingly. RESULTS: Myocardial cells after I/R manipolation were severely injuried (LDH leakage, 125 IU/L vs 12 IU/L, P < 0.05), apoptosis (60.1% vs 5.5%, P < 0.05), significant cytosolic Ca(2+) overload (21.2 vs 7.6, P < 0.05) and remarkable mitochondrial membrane potential loss (37.2 vs 85.1, P < 0.05). However, no visible change of RyR2 was observed (20.1 vs 22.7, P > 0.05). Pre-treatment with RyR2 specified siRNA demonstrated suppressed expression of RyR2 (6.8 vs 20.1, P < 0.05), increased mitochondrial membrane potential (55.8 vs 37.2, P < 0.05), attenuated cytosolic Ca(2+) overload (8.6 vs 21.2) and cellular apoptosis (31.2% vs 60.1%, P < 0.05). CONCLUSION: RyR2 gene silencing enables to protect myocardial cells from I/R injury in vitro.