MiR-133b inhibits MPP+-induced apoptosis in Parkinson's disease model by inhibiting the ERK1/2 signaling pathway.

OBJECTIVE: The aim of this study was to explore the effect of micro ribonucleic acid (miR)-133b on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in the Parkinson's disease (PD) model. MATERIALS AND METHODS: PC12 cells were induced by different concentrations of MPP+ to establish the PD cell model. Subsequently, the survival rate of PC12 cells was detected using Cell Counting Kit-8 (CCK-8) assay. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of miR-133b in the PD model induced by different concentrations of MPP+. Next, PC12 cells were transfected with miR-133b mimic and miR-negative control (NC), and divided into MPP+ group, MPP+ + miR-NC group and MPP+ + miR-133b mimic group. Transfection efficiency was verified using qRT-PCR. The apoptosis of cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated (p)-ERK1/2 were determined using Western blotting. RESULTS: After MPP+ treatment, the survival rate of PC12 cells significantly declined (p<0.05). MPP+ exhibited toxicity against PC12 cells in a concentration-dependent manner. Meanwhile, cell survival rate decreased remarkably with the increase of MPP+ concentration (p<0.05). With increased concentration of MPP+, the expression of miR-133b in the PD cell model declined significantly (p<0.05). The apoptosis of PC12 cells was remarkably inhibited by overexpression of miR-133b in the PD cell model (p<0.05). In addition, the protein expression of p-ERK1/2 in PC12 cells was notably reduced after overexpression of miR-133b in the PD cell model (p<0.05). CONCLUSIONS: MiR-133b is lowly expressed in the PD cell model. Furthermore, overexpression of miR-133b inhibits cell apoptosis in the PD cell model by regulating the ERK1/2 signaling pathway.

EZH2-mediated H3K27me3 enrichment on the lncRNA MEG3 promoter regulates the growth and metastasis of glioma cells by regulating miR-21-3p.

OBJECTIVE: Glioma is one of the most common and invasive brain tumors worldwide. Long non-coding RNAs (LncRNAs) play an important role in the development of glioma. However, the regulatory mechanism of LncRNAs in glioma has not been fully elucidated. This study aimed to explore the interaction of lncRNA maternally expressed gene 3 (MEG3) and aberrant histone modification in glioma. MATERIALS AND METHODS: The expression levels of MEG3 and miR-21-3p in glioma cells were measured by quantitative polymerase chain reaction (qPCR). EZH2 (enhancer of zeste homolog 2) and H3K27me3 expression in glioma cells were detected by Western Blot (WB). The binding site of the promoter of MEG3 by H3K27me3 was confirmed by ChIP-Real-time PCR. The direct target of MEG3 and miR-21-3p in glioma cells was measured by a luciferase reporter assay. Cell proliferation was detected by Cell Counting Kit-8 (CCK8), and cell invasion and migration were measured by Transwell assays. RESULTS: EZH2 and miR-21-3p were upregulated and MEG3 was downregulated in glioma cells. Silencing of EZH2 inhibited cell proliferation, migration, and invasion in U87 and U251 cells. Meanwhile, the expression of H3K27me3 could be significantly inhibited by EZH2 interference. H3K27me3 protein can bind to MEG3 promoter directly. EZH2 inhibition and MEG3 down-expression in U87 cells reversed the effects of silencing of EZH2 on glioma cell growth and metastasis. However, EZH2 inhibition and MEG3 overexpression in U251 cells restricted cell proliferation, migration, and invasion. Furthermore, miR-21-3p was verified to interact with MEG3 by direct binding. Inhibition of MEG3 promoted U87 cell growth and metastasis, which was further strengthened following the co-transfection of si-MEG3 and miR-21-3p. Overexpressed MEG3 inhibited U251 cell growth and metastasis and a complete reversal of the results observed in the co-transfection of LV-MEG3 and miR-21-3p. CONCLUSIONS: EZH2 was highly expressed in glioma cells and EZH2-mediated H3K27me3 enrichment on the MEG3 promoter regulated the growth and metastasis of glioma cells by targeting miR-21-3p. It potentially provided a new therapeutic marker targeting glioma.

[Studies on the role of chromobox protein homolog 2 in the inhibition of progression of hepatoma].

Objective: To explore chromobox protein homolog 2 (CBX2) expressions in relation to clinical features of patients and elucidate its role in the progression of hepatocellular carcinoma. Methods: Using the Cancer Genome Atlas (TCGA) database, R language was used to analyze the distribution of differentially expressed mRNA in hepatocellular carcinoma. The different expression of CBX2 in HCC and adjacent tissues and its relationship with survival and clinical characteristics of patients were further analyzed. The expression of CBX2 in liver tissues, liver cancer tissue, and L02, HepG2 and SMMC-7721 cell lines was detected by real time-PCR and western blot. The expression of CBX2 was interfered by siRNA in hepatoma cell line. MTT, colony formation, transwell assays, and flow cytometry were used to identify the proliferation, apoptosis, invasion and clone-formation ability of HepG2 and SMMC-7721 cells after CBX2 down-regulation. According to the different data, t-test, ANOVA, chi-square test, and COX regression model were used for statistical analysis. Survival curve was plotted through Kaplan-Meier method. Results: TCGA public database analysis showed that the expression of CBX2 mRNA in hepatocellular carcinoma tissues (7.296 ± 1.6115) was significantly higher than normal liver tissues (4.706 ± 0.940) (P = 0.000). In addition, the overall survival time of patients with low CBX2 mRNA expression was significantly longer than that of patients with high CBX2 mRNA expression [(5.971 ± 0.411) years vs. (4.650 ± 0.503) years, P = 0.001]. The expression level of CBX2 mRNA was correlated with the pathological TNM stage (P = 0.025) and differentiation degree (P < 0.001) of liver cancer. COX regression analysis showed that CBX2 mRNA expression was an independent predictor of patient survival (P = 0.013). siRNA was transfected and compared with the blank control group. The transgenic ability of HepG2 and SMMC-77221 cells decreased significantly at 72h (P < 0.05) and 96h (P < 0.05), and the apoptosis rate (11.430% ± 0.215%) was higher than blank control group (6.6 00% ± 0.170%) (P = 0.003). The number of invasive cells ((both P < 0.05) and relative colony forming cells ((both P < 0.001) were significantly decreased. In 20 cases of tissue samples, the expression of CBX2 protein (relative expression level 3.020 ± 0.269) in liver cancer was higher than that in adjacent tissues (relative expression level 0.886±0.065) (P < 0.001). The overall survival time of patients with low CBX2 expression in liver cancer was longer than that of patients with high expression [(3.670 + 0.576) years vs. (0.834 + 0.153) years, P = 0.004]. Conclusion: An evident high expression of CBX2 is an independent poor prognostic factor in hepatoma. Down-regulation of CBX2 expression can inhibit the progression of liver cancer. Therefore, CBX2 may be a prognostic biomarker and a new target for HCC treatment.

G-quadruplex antibody attenuates human gastric cancer cell proliferation and promotes apoptosis through hTERT/telomerase pathway.

OBJECTIVE: To investigate the expression of G-quadruplex antibody BG4 in human gastric cancer AGS cells and assess its functions in attenuating proliferation and promoting apoptosis in gastric cancer. MATERIALS AND METHODS: BG4 high-expression gastric cancer AGS cell line was established by pEGFP-N1-BG4 transient transfection. AGS cells transfected with pEGFP-N1 plasmids were included into the pEGFP-N1 group and those not transfected with plasmids were included into the negative control group. Cell counting kit-8 (CCK8) assay was performed to examine the AGS cell proliferation ability, while flow cytometry was used to detect the cell cycle distribution and cell apoptosis. Cell migration was measured using Transwell migration and wound healing assay. Then the expression levels of cell apoptosis associated factors were determined. The mRNA and protein expressions of human telomerase reverse transcriptase (hTERT), B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) were examined with real-time quantitative polymerase chain reaction (PCR) and Western blotting, respectively. RESULTS: The results revealed that pEGFP-N1-BG4 group exhibited reduced proliferation and migration, induction of apoptosis. hTERT and Bcl-2 mRNA and protein levels in pEGFP-N1-BG4 group were down-regulated compared with those in the pEGFP-N1 group and control group, but there were no significant differences in Bax mRNA and protein levels compared with those in the pEGFP-N1 group and control group. CONCLUSIONS: We showed that the expression of BG4 in the gastric cancer cell line AGS inhibits cell proliferation and promotes apoptosis though inducing telomere to form G-quadruplex structure and attenuating telomerase activity, thus resulting in reduced expression of hTERT and Bcl-2.

[Effects of robot-assisted minimally invasive transforaminal lumbar interbody fusion and traditional open surgery in the treatment of lumbar spondylolisthesis].

Objective: To compare the clinical effects of robot-assisted minimally invasive transforaminal lumbar interbody fusion (TLIF) and traditional open TLIF in the treatment of lumbar spondylolisthesis. Methods: A total of 41 patients with lumbar spondylolisthesis accepted surgical treatment in Department of Spinal Surgery of Beijing Jishuitan Hospital From July 2015 to April 2016 were retrospectively analyzed. There were 16 cases accepted robot-assisted minimally invasive TLIF and 25 accepted traditional open TLIF. The operation time, X-ray radiation exposure time, perioperative bleeding, drainage volume, time of hospitalization, time for pain relief, time for ambulatory recovery, visual analogue scale (VAS), Oswestry disability index (ODI) and complications were compared. T test and χ(2) were used to analyze data. Results: There were no significant difference in gender, age, numbers, degrees, pre-operative VAS and ODI in spondylolisthesis (all P>0.05). Compared with traditional open TLIF group, the robot-assisted minimally invasive TLIF group had less perioperative bleeding ((187.5±18.4) ml vs. (332.1±23.5) ml), less drainage volume ((103.1±15.6) ml vs. (261.3±19.8) ml), shorter hospitalization ((7.8±1.9) days vs. (10.0±1.6) days), shorter time for pain relief ((2.8±1.0) days vs. (5.2±1.1) days), shorter time for ambulatory recovery ((1.7±0.9) days vs. (2.9±1.3) days) and less VAS of the third day postoperatively (2.2±0.9 vs. 4.2±2.4) (t=2.762-16.738, all P<0.05), but need more operation time ((151.3±12.3) minutes vs. (102.2±7.1) minutes) and more X-ray radiation exposure ((26.1±3.3) seconds vs. (5.5±2.1) seconds) (t=6.125, 15.168, both P<0.01). In both groups ODI was significantly lower in final follow-up than that of the pre-operation (t=12.215, 14.036, P<0.01). Intervertebral disc height of the final follow-up in both groups were significantly larger than that of the preoperation (robot-assisted minimally invasive TLIF group: (11.8 ± 2.8) mm vs. (7.5 ± 1.9) mm, traditional open TLIF group: (12.7 ± 2.5) mm vs. (7.9±2.0) mm), and so was the lumbar lordosis angle (robot-assisted minimally invasive TLIF group: (48.7±9.2)°vs. (39.6±7.9)°, traditional open TLIF group: (50.1±10.8)°vs. (41.4±8.8)°), the lordosis angle of the slippage segment (robot-assisted minimally invasive TLIF group: (18.7±5.6)°vs. (10.9±3.8)°, traditional open TLIF group: (17.6±6.1)°vs.(8.7±3.2)°) (t=4.128-16.738, all P<0.01). Slippage rate of the final follow-up in both groups were significantly smaller than those of the pre-operation (robot-assisted minimally invasive TLIF group: (5.3±2.3) % vs. (27.8±7.2) %, traditional open TLIF group: (6.6±2.8) % vs. (29.1±9.5) %) (t=11.410, 18.504, both P<0.01). There was no difference of the upper data between two groups (t=0.106-1.227, P>0.05). The results of the post-operative CT showed that the pedicle screws in the robot-assisted minimally invasive TLIF group were more precisely placed than traditional open TLIF group (χ(2)=4.247, P=0.039). The mean follow-up time was 8 months (ranging from 3 to 12 months). There were no significant difference in outcomes between the two groups (χ(2)=0.366, P=0.545). Conclusions: In the treatment of lumbar spondylolisthesis, Robot-assisted minimally invasive TLIF can lead to less perioperative bleeding, less post-operative pain, and quicker recovery than traditional open TLIF surgery, but it needs more operation time and radiation exposure.

Thoracolumbar kyphosis is associated with compressive vertebral fracture in postmenopausal women.

The main aim of this retrospective cross-sectional study was to examine the relationship between vertebral compression fracture and thoracolumbar Cobb angles. Fracture prevalence was found to be significantly higher for patients with moderate [odds ratio (OR) = 4.78 (2.88-7.95)] or severe kyphosis [OR = 10.7 (5.11-22.40)] than for patients with mild kyphosis. The relationship between degree of thoracolumbar kyphosis and vertebral compression fracture was analyzed. INTRODUCTION: The hypothesis that vertebral compression fracture in women is related to thoracolumbar kyphosis severity was tested, and a clinically important cutoff degree of sagittal thoracolumbar Cobb angle (TLCobb) was determined. METHODS: Demographic data, clinical data, and quantitative computed tomography (QCT) findings were compiled for 212 postmenopausal women with thoracolumbar fracture (study group) and 150 postmenopausal women with degenerative lumbar disease (control group). Group proportions and characteristics were compared with chi-squared tests and unpaired t tests, respectively. RESULTS: In this retrospective cross-sectional study cohort, 17 patients had T11 fractures, 79 had T12 fractures, 89 had L1 fractures, and 27 had L2 fractures. QCT findings and TLCobb differed between the study and control groups (both p < 0.001). No significant differences were found in body mass index (BMI), disk height, or coronal TLCobb. After adjustment for age, BMI, and QCT findings, fracture prevalence was found to be higher in the thoracolumbar kyphosis study group than in the control group [OR = 6.16, 95% confidence interval (CI) 3.88-9.78]. Sagittal TLCobbs of 7.5-15° and >15° were associated with an increased fracture prevalence, with ORs of 4.78 (2.88-7.95) and 10.7 (5.11-22.40), respectively. CONCLUSION: Vertebral fracture prevalence in postmenopausal women was found to be associated with thoracolumbar kyphosis. A TLCobb sagittal angle >15° should be considered an indicator for vertebral fracture assessment.

Hyperbaric oxygen preconditioning protects against traumatic brain injury at high altitude.

BACKGROUND: Recent studies have shown that preconditioning with hyperbaric oxygen (HBO) can reduce ischemic and hemorrhagic brain injury. We investigated effects of HBO preconditioning on traumatic brain injury (TBI) at high altitude and examined the role of matrix metalloproteinase-9 (MMP-9) in such protection. METHODS: Rats were randomly divided into 3 groups: HBO preconditioning group (HBOP; n = 13), high-altitude group (HA; n = 13), and high-altitude sham operation group (HASO; n = 13). All groups were subjected to head trauma by weight-drop device, except for HASO group. HBOP rats received 5 sessions of HBO preconditioning (2.5 ATA, 100% oxygen, 1 h daily) and then were kept in hypobaric chamber at 0.6 ATA (to simulate pressure at 4000m altitude) for 3 days before operation. HA rats received control pretreatment (1 ATA, room air, 1 h daily), then followed the same procedures as HBOP group. HASO rats were subjected to skull opening only without brain injury. Twenty-four hours after TBI, 7 rats from each group were examined for neurological function and brain water content; 6 rats from each group were killed for analysis by H&E staining and immunohistochemistry. RESULTS: Neurological outcome in HBOP group (0.71 +/- 0.49) was better than HA group (1.57 +/- 0.53; p < 0.05). Preconditioning with HBO significantly reduced percentage of brain water content (86.24 +/- 0.52 vs. 84.60 +/- 0.37; p < 0.01). Brain morphology and structure seen by light microscopy was diminished in HA group, while fewer pathological injuries occurred in HBOP group. Compared to HA group, pretreatment with HBO significantly reduced the number of MMP-9-positive cells (92.25 +/- 8.85 vs. 74.42 +/- 6.27; p < 0.01). CONCLUSIONS: HBO preconditioning attenuates TBI in rats at high altitude. Decline in MMP-9 expression may contribute to HBO preconditioning-induced protection of brain tissue against TBI.

The pre-styloid compartment of the parapharyngeal space: a three-dimensional digitized model based on the Chinese Visible Human.

To build a digitized visible model of the parapharyngeal space of the Chinese Visible Human and to provide a sectional anatomic basis for radiological and clinical diagnosis of the parapharyngeal space, sectional anatomy data of the parapharyngeal space were selected from the Chinese Visible Human male and female to compare with MR imaging findings in the axial planes. From these data the parapharyngeal space and surrounding structures were segmented. They were then reconstructed in three dimensions on PC. In the axial planes of the sectional anatomy and MR imaging, the shape, content and relations of the parapharyngeal space were clearly displayed and the dominant plane for showing the parapharyngeal space was elicited. The three-dimensional reconstructed images displayed perfectly the anatomic relationships of the parapharyngeal space, parotid, muscles, mandible and vessels. All reconstructed structures can be displayed singly, in groups or as a whole; any diameter or angle of the reconstructed structures can be easily measured. The Chinese Visible Human male and female data set can provide complete and accurate data. The digitized model of the parapharyngeal space and its surroundings offers unique insights into the complex anatomy of the area, providing morphologic data for imaging diagnosis and surgery of the parapharyngeal space.

Pinelloside, an antimicrobial cerebroside from Pinellia ternata.

An antimicrobial cerebroside, pinelloside, was isolated from the air-dried tubers of Pinellia ternata (Thunb.) Breit. Its structure was determined as 1-O-beta-D-glucopyranosyl-(2S,3R,4E,11E)-2-(2'R-hydroxyhexadecenoylamino)-4,11-octadecadiene-1,3-diol by chemical transformation and extensive spectroscopic analyses (IR, MS, 1H and 13C NMR, DEPT as well as 2D NMR techniques HMBC, HMQC, 1H-1H COSY and NOESY). The antimicrobial assay showed that this compound was inhibitory to the growth of Bacillus subtilis, Staphylococcus aureus, Aspergillus niger and Candida albicans, with minimum inhibitory concentrations (MICs) of 20, 50, 30 and 10 microg/ml, respectively. The MICs of penicillin G against bacteria B. subtilis, S. aureus, E. coli, P. fluorescens and H. pylori were 0.80, 0.34, 0.56, 1.34 and 0.92, and those of ketoconazole against fungi A. niger, C. albicans and T. rubrum 0.90, 0.65 and 1.0 microg/ml, respectively.

Analgesic domains of interferon-alpha.

Interferon-alpha (IFNalpha) is not only an immunoregulatory factor, but is also an analgesic molecule. We ever reported that there exist distinct domains in IFNalpha molecule that mediate immune and analgesic effects respectively and inferred that the analgesic domain locates around the 122nd Tyr residue of IFNalpha molecule in the tertiary structure. After the 36th Phe residue, which was located closely to the 122nd Tyr residue in the tertiary structure, was mutated to Ser using site-directed mutagenesis, the analgesic activity of this mutant lost completely, but the antiviral activity of IFNalpha still maintained 40.5% of wild type IFNalpha. The results suggest that the 36th Phe residue is one of the constituent for the analgesic domain of IFNalpha and inferred that the analgesic domain of IFNalpha consists of the 122nd Tyr and the residues around the 122nd in the tertiary structure, which include the 36th Phe.

[Effect of injectio Salvia Miltiorrhiza on gentamicin ototoxicity-induced activity of nitric oxide synthase in cochlear stria vascularis of guinea pig].

AIM: to investigate the change of nitric oxide synthase (NOS) activity in cochlear stria vascularis (SV) of guinea pig after gentamicin (GM) and Salvia Miltiorrhiza (SM) injection, and to explore the protective role of injectio SM on GM ototoxicity. METHODS: NADPH-diaphorase histochemistry staining and image quantitative analysis technique, combined with auditory brainstem response (ABR) measurement. RESULTS: SM + GM significantly reduced NOS activity in cochlear SV and ABR threshold as compared with CM along (P < 0.01); and ABR threshold shift was in high correlation with NOS activity (rControl = -0.9464; rGM = -0.9117; rSM + GM = -0.8958; P < 0.01). CONCLUSION: SM can reduce NOS activity in cochlear SV so as to alleviate GM ototoxicity, thus ameliorate hearing function.

Effects of calcium channel blockers on calcium release-activated calcium currents in rat hepatocytes.

AIM: To study the influences of calcium channel blockers on calcium release-activated calcium currents (ICRAC) in rat hepatocytes. METHODS: Whole-cell patch-clamp technique was used. RESULTS: The peak amplitude of ICRAC was -0.41 nA +/- 0.09 nA (n = 15), its reversal potential was about 0 mV. Verapamil (Ver), diltiazem (Dil), and nifedipine (Nif) decreased ICRAC strikingly, without affecting its reversal potential. The inhibitory rate of Ver 5 mumol.L-1 was 40% +/- 12% (n = 3), Ver 50 mumol.L-1 reduced the peak amplitude of ICRAC from -0.49 nA +/- 0.12 nA to -0.20 nA +/- 0.09 nA (P < 0.01 vs control, n = 5). The inhibitory rate was 57% +/- 15%. Dil 50 mumol.L-1 and Nif reduced ICRAC from -0.43 nA +/- 0.10 nA to -0.29 nA +/- 0.07 nA (P < 0.01 vs control, n = 5), from -0.32 nA +/- 0.08 nA to -0.27 nA +/- 0.08 nA (P < 0.01 vs control, n = 5). The inhibitory rate was 31% +/- 11%, 19% +/- 7%, respectively. The amplitude of ICRAC was dependent on extracellular Ca2+ concentration. The peak amplitude of ICRAC was -0.21 nA +/- 0.08 nA (n = 3) in Tyrode's solution with Ca2+ 1.8 mmol.L-1 (P < 0.01 vs the peak amplitude of ICRAC in external solution with Ca2+ 10 mmol.L-1). CONCLUSION: The three calcium antagonists inhibited ICRAC effectively and protected hepatocytes from calcium overload via the inhibition of ICRAC.

[Effects of noradrenaline on delayed outward potassium current in isolated rat hepatocytes].

Heretofore no reports are available on the outward potassium currents in rat hepatocytes. Using whole-cell patch clamp technique, the effects of noradrenaline and other reagents on the delayed outward potassium current (IK) in isolated rat hepatocytes were investigated. The experimental results showed that the magnitude of IK was 2.85 +/- 1.21 nA at a command potential of +140 mV from a holding potential of -50 mV. Noradrenaline decreased IK distinctly. Isoprenaline and acetylcholine showed no effect on IK in isolated rat hepatocytes.

Effects of N-methyl berbamine on delayed outward potassium current in isolated rat hepatocytes.

AIM: To study the effects of N-methyl berbamine (NMB) on the delayed outward potassium currents (Ik) in isolated rat hepatocytes. METHODS: With patch-clamp techniques and whole-cell recording method, holding potential -50 mV, command potential +30 to +140 mV, duration 900 ms. RESULTS: NMB reduced Ik in a concentration-dependent manner. When the concentrations of NMB were 20, 50, 400 nmol.L-1 and 50 mumol.L-1, the amplitude values of Ik were decreased to 3.6 +/- 0.4 (P > 0.05), 2.1 +/- 1.6 (P > 0.05), 3.7 +/- 1.6 (P < 0.05), 2.3 +/- 1.3 nA (P < 0.01) from 4.4 +/- 1.0 (n = 4), 2.5 +/- 1.8 (n = 4), 5.8 +/- 2.1 (n = 5), 4.6 +/- 1.3 (n = 6) nA of control, respectively. The inhibitory rates were 10%, 15%, 37%, and 51%, respectively. CONCLUSION: NMB was a K+ channel inhibitor.