Up/down-regulation of miR-21 changes biological function of colon can-cer cells and sensitivity to cetuximab

AIM: To explore the effects of miR-21 on biological behavior of colon cancer cells and their sensitivity to epidermal growth factor receptor monoclonal antibody cetuximab. METHODS: Lentiviral vectors were constructed to generate up- and down-regulations of miR-21 lentiviruses (LV-miR-21 and LV-anti-miR-21, respectively), and the corresponding negative control viruses (LV-miR-21 NC and LV-anti-miR-21 NC, respectively) were also constructed. The viruses were used to infect human colon cancer RKO cells. The changes of the miR-21 expression level, the cell proliferation, the colony-forming ability, the cell apoptosis and the sensitivity of the cells to cetuximab were detected by real-time PCR, MTT assay, soft agar colony assay, flow cytometry and CCK-8 assay. RESULTS: The lentivirus titers of LV-miR-21, LV-miR-2 NC, LV-anti-miR-21 and LV-anti-miR-21 NC were 3.0×1012 TU/L, 6.0×1011 TU/L, 2.0×1012 TU/L and 8.0×1011 TU/L, respectively. The infection efficiency was over 80% by the observation of green fluorescence. The miR-21 expression level, the cell proliferation, and the colony-forming ability in LV-miR-21 group were significantly higher than those in LV-anti-miR-21 group. The early apoptotic rate and the inhibitory rate of cetuximab for the cells in LV-anti-miR-21 group were higher than those in LV-miR-21 group. CONCLUSION: miR-21 promotes the proliferation of colon cancer cells. Down-regulation of miR-21 enhances the sensitivity of the colon cancer cells to the targeted therapy drug cetuximab.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Effects of synthetic kainic acid on microfilament expression and gap junction in astrocytes

AIM：To study the effects of synthetic kainic acid (SKA) and 1-heptanol (1-Hep), a gap junction blocker, on the cytoskeletal filament expression in the astrocytes. METHODS：The neonatal rat brain was obtained from the Wistar rats (1 day old) and primary purified astrocytes were obtained by differential attachment for removing filamentoblasts and orbital shaker for removing the oligodendrocytes. The effects of SKA and other interventions on the morphologic changes and expression levels of skeleton protein filamentous actin (F-actin) were observed in the astrocytes after 24 h of the exposures by laser scanning confocal microscopy. The effect of 1-Hep on the expression of F-actin was also explored. RESULTS：Compared with control group, the fluorescence intensities in KCl group and KCl+SKA group were increased, and highly increased in KCl+SKA group. The F-actin filaments in the above 2 groups were more intensive, thickened and concentrated than those in control group, and more obvious in KCl+SKA group. l-Hep significantly decreased the expression of F-actin in KCl group and KCl+SKA group as compared with control group, and parts of the filamentous fracture were seen in the astrocytes in all 1-Hep-treated groups, in which some of the filamentous lines were crosscut. CONCLUSION：Increase in the expression of F-actin in the astrocytes affects the structure and function of the intercellular gap junctions, which may be involved in the mechanism of SKA-induced epilepsy.     

Inflammatory response in rat kidney with contrast-induced nephropathy

AIM：To observe the expression of tumor necrosis factor α (TNF-α) and nuclear factor κB (NF-κB) in the renal tissue of the rats with contrast-induced nephropathy (CIN). METHODS：Male Sprague-Dawley rats (n=96) were randomly divided into control group (n=48) and CIN group (n=48). The model rats in CIN group were intravenously injected with iodinated contrast media (76% compound diatrizoate injection,10 mL/kg), while the rats in control group were injected with the same volume of saline. Six rats in each group were sacrificed at 6 h, 12 h, 24 h, 48 h, 72 h, 5 d, 10 d and 15 d after intravenous injection, respectively, and the blood and kidney samples of the rats were obtained. The renal tubular injury was assessed by histological examination (HE staining). The expression of kidney injury molecule-1 (KIM-1), TNF-α and NF-κB at mRNA and protein levels in the renal tissues were semiquantitatively measured by the methods of RT-PCR and immunohistochemistry, respectively. The correlations between the expression of TNF-α, NF-κB and tubular injury score, KIM-1 expression in renal tissue of CIN group were analyzed. RESULTS：The levels of serum creatinine (SCr) and blood urea nitrogen (BUN) in control group were not changed between different time points (P>0.05). The levels of SCr and BUN in CIN group displayed significant increases at different time points (except 15 d) compared with control group (P<0.05). The renal tubular injury score in CIN group was significantly higher at all time points than that in control group (P<0.05). The expression of KIM-1, TNF-α and NF-κB at mRNA and protein levels up-regulated significantly at 6 h and the peaking of KIM-1 expression was at 24 h, while the peaking of TNF-α and NF-κB expression was at 48 h in CIN group. The expression of KIM-1,TNF-α and NF-κB was significantly increased in CIN group compared with control group except at 15 d (P<0.05). The expression of TNF-α and NF-κB at mRNA and protein levels showed close correlations with renal tubular injury score (r=0.843, 0.758, 0.743 and 0.707, P<0.05). The expression of TNF-α and NF-κB at mRNA and protein levels was also positively correlated with KIM-1 expression (r=0.863, 0.807, 0.839 and 0.855, P<0.05). CONCLUSION：The expression of TNF-α and NF-κB at mRNA and protein levels in the renal tissues of CIN group is up-regulated and is closely related with renal tubular injury, indicating that the inflammatory response is involved in the pathogenesis of CIN.     

RASSF1A expression mediated by lentivirus inhibits growth of small-cell lung cancer cell line H446

AIM：To explore the inhibitory effect of Ras-association domain family 1A (RASSF1A) on the small-cell lung cancer cell growth. METHODS：The lentiviral expression vector containing RASSF1A gene was constructed and used to infect the small-cell lung cell line H446. The growth curve and cell cycle were detected by MTT assay and flow cytometry. The mRNA and protein levels of cell cycle-associated proteins were determined by real-time PCR and Western blotting. RESULTS：We obtained the H446 cells in which RASSF1A was stably expressed (named RASSF1A-H446). Compared with normal cell group and negative cell group, RASSF1A inhibited the proliferation of H446 cells, and arrested H446 cells in G1 phase. The expression of p21 and p27 was significantly increased, and E2F1 was significantly decreased in RASSF1A-H446 cells. CONCLUSION：RASSF1A inhibits the H446 cell growth by increasing the expressions of p21 and p27, and decreasing the expression of E2F1.     

紫色马铃薯新品种桂彩薯1 号的选育

桂彩薯1 号是以日本北海道岛系571 号为母本,以日本北海道岛系568 号为父本经有性杂交系统选育而成。早中熟,生育期（出苗至成熟）85～95 d（天）。块茎长椭圆形,中等大小,紫皮紫肉,表皮光滑,芽眼少而浅,结薯集中,单薯质量100 g 左右,鲜薯产量1 000～1 500 kg· （ 667 m²）^-1,最高可达2 000 kg（ 667 m²）^-1。块茎淀粉含量9.07%,粗蛋白质2.45%,还原糖0.67%,VC 137.0 mg·kg^-1（FW）,花色苷1 786.0 mg·kg^-1（FW）。适宜广西、广东、福建、云南、湖南等地秋冬种植。     

Vasonatrin peptide attenuates injuries of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium

AIM：To investigate the neuroprotective effects of vasonatrin peptide (VNP) on the injury of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium (MPP+). METHODS：Cultured dopaminergic neurons from the mouse ventral mesencephalon were exposed to MPP+, and the effects of VNP on the neurotoxicity of MPP+ were eva-luated by cell viability analysis and immunofluorescence staining. Various kinds of agonists and antagonists were used to clarify the mechanism underlying the effects of VNP. RESULTS：MPP+ caused injuries in the dopaminergic neurons. VNP significantly increased the viability, axon number and axon length of the dopaminergic neurons. The MPP+-induced depolymerization of β-tubulin Ⅲ was also attenuated by the treatment with VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-Br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 ［the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors (NPR)］, or KT-5823 ［a cGMP-dependent protein kinase (PKG) inhibitor］. CONCLUSION：VNP attenuates the neurotoxicity of MPP+ via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neural degeneration of dopaminergic neurons in Parkinson disease.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Quantification of tryptase and T-cell immunoglobulin mucin 1 double-positive mast cells in different degrees of human periodontitis

AIM：To examine the expression of T-cell immunoglobulin mucin 1 (TIM-1) on tryptase-positive mast cells (MCs) in different severities of human chronic periodontitis. METHODS：Human gingival specimens (n=92) were involved in this study, including healthy control (n=27), mild chronic periodontitis (n=34) and severe chronic periodontitis (n=31). The gingival specimens were fixed in 4% formaldehyde. Paraffin embedding and serial sectioning with hematoxylin and eosin staining were performed for histopathological examination, and double-immunofluorescence staining was conducted for identification of tryptase-TIM-1 double-positive MCs in the gingival tissues. RESULTS：Compared with the healthy controls, the densities (cells/mm2) of tryptase-TIM-1 double-positive MCs were significantly increased in both mild chronic periodontitis group (P<0.05) and severe chronic periodontitis group (P<0.01). However, compared with mild chronic periodontitis group, both the score of gingival tissue inflammation and the density of tryptase-TIM-1 double-positive MCs in the gingival tissues were significantly increased in severe periodontitis group (P<0.05). CONCLUSION：Significantly increased number of tryptase-TIM-1 double-positive MCs has the similar tendency as the severity of periodontitis inflammation in human chronic periodontitis, suggesting that tryptase-TIM-1 double-positive MCs may play an important role in human chronic periodotitis.     

Influence of high-mobility group box 1 on proliferation of neural stem cell in pre-infarction cortex of focal cerebral ischemia/reperfusion model rats

AIM：To investigate the influence of high-mobility group box 1 (HMGB1) on the proliferation of neural stem cells in peri-infarction cortex of focal cerebral ischemia/reperfusion model rats. METHODS：Male SD rats (n=48) were randomly divided into sham group, ischemia/reperfusion (I/R) group, RNA interference group and negative interference group. The rat middle cerebral artery was blocked to establish focal cerebral I/R model (ischemia for 1 h and reperfusion for 7 d). Lentivirus vector of HMGB1 shRNA was used to suppress the HMGB1 protein expression in the rat brain. The effect of RNA interference was evaluated by the methods of double-immunofluorescence labeling of HMGB1/GFAP and Western blotting. The proliferation of neural stem cells in the peri-infarction cortex was assessed by double labeling of BrdU/nestin. RESULTS：The protein expression of HMGB1 in I/R group was much higher than those in sham group (P<0.05). RNA interference effectively inhibited the HMGB1 expression (P<0.05). Double labeled BrdU/nestin positive cells in I/R group were more than that in sham group (P<0.05). The double labeled BrdU/nestin positive cells were significantly decreased in RNA interference group (P<0.05). CONCLUSION：Focal cerebral ischemia/reperfusion injury promotes the proliferation of neural stem cells in peri-infarction cortex by increasing HMGB1 protein level.