LncRNA ANRIL knockdown ameliorates retinopathy in diabetic rats by inhibiting the NF-κB pathway.

OBJECTIVE: To evaluate the impacts of long non-coding ribonucleic acid (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) on diabetic retinopathy (DR) in rats and the underlying mechanism. MATERIALS AND METHODS: A total of 60 adult male Sprague Dawley (SD) rats were randomly divided into 3 groups: the Sham group (n=20), DR group (n=20) and DR + lncRNA ANRIL knockdown group [DR + lncRNA ANRIL small interfering RNA (siRNA) group, n=20]. DR model in rats was established by intraperitoneal injection of streptozocin (STZ; 60 mg/kg). Meanwhile, a certain dose of lncRNA ANRIL siRNA was added dropwise into rat eyes of DR + lncRNA ANRIL siRNA group during model induction to downregulate lncRNA ANRIL expression in the retina. 16 weeks later, rat retinal tissues were taken to extract total RNA and protein. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression levels of lncRNA ANRIL, interleukin-1 (IL-1), IL-6 and monocyte chemotactic protein-1 (MCP-1) in each group of the rat retina. Pathological structure of rat retinal tissues in each group was observed via hematoxylin and eosin (H&E) staining. Immunohistochemistry was adopted to measure the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and P65 in each group of retinal tissues. In addition, the retinal vascular permeability in each group of rats was detected by fluorescent staining. Finally, Western blotting was utilized to determine the expressions of genes in the P65 signaling pathway. RESULTS: Compared with rats in the Sham group, lncRNA ANRIL was upregulated in rat retinal tissues harvested from the DR + lncRNA ANRIL siRNA group (p<0.05). After knockdown of lncRNA ANRIL in the retinal tissues of DR rats, pathologic damage was alleviated notably, and the levels of inflammatory markers (IL-1, IL-10 and MCP-1) were lowered markedly (p<0.05). The protein expressions of Bax and P65 decreased evidently, while the protein level of Bcl-2 increased markedly (p<0.05) in DR rats with ANRIL knockdown. Furthermore, Western blotting results revealed that inhibition of lncRNA ANRIL could prominently repress the phosphorylation level of P65 in the retinal tissues of DR rats (p<0.05). CONCLUSIONS: LncRNA ANRIL knockdown can significantly ameliorate the retinopathy in diabetic rats by blocking the nuclear factor-kappa B (NF-κB) signaling pathway.

Influence of lncRNA-MALAT1 on neuronal apoptosis in rats with cerebral infarction through regulating the ERK/MAPK signaling pathway.

OBJECTIVE: To investigate the regulatory effect of long non-coding ribonucleic acid (lncRNA)-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling pathway, and to explore its influence on neuronal apoptosis in rats with cerebral infarction. MATERIALS AND METHODS: A total of 45 adult male Sprague-Dawley rats were randomly divided into sham group (n=15), model group (n=15) and MALAT1 low-expression group (n=15). The model of cerebral infarction was successfully established in the model group and MALAT1 low-expression group via middle cerebral artery occlusion (MCAO). After 3 d, the nerve injury in each group was evaluated using Zea-Longa score. Meanwhile, the area of cerebral infarction in each group was detected via 2,3,5-triphenyl tetrazolium chloride (TTC) staining. After the cortical tissues were separated, the expression level of lncRNA-MALAT1 was detected via quantitative Polymerase Chain Reaction (qPCR). The apoptotic level of neurons in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The expression levels of inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, the expression levels of apoptosis-related proteins and ERK/MAPK signaling pathway-related proteins were detected via Western blotting. RESULTS: Compared with the sham group, the behavioral score and area of cerebral infarction in the model group were significantly increased (p<0.01). The low expression of MALAT1 could effectively reduce the behavioral score and area of cerebral infarction in the model group (p<0.01). The expression level of lncRNA-MALAT1 in cortical tissues of the model group was markedly higher than that of the sham group and MALAT1 low-expression group (p<0.01). Compared with the sham group, the content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cortical tissues was significantly increased (p<0.01). However, the content of IL-10 was remarkably decreased in the model group (p<0.01). Low expressed MALAT1 could markedly reduce the content of TNF-α and IL-6 and increase the content of IL-10 in cortical tissues (p<0.01). The level of apoptosis in cortical tissues was increased in the model group when compared with that of the sham group (p<0.01). Meanwhile, low expression of MALAT1 could effectively reduce the apoptosis level in cortical tissues in model group (p<0.01). In the model group, the expression levels of B-cell lymphoma-2/Bcl-2 associated X protein (Bcl-2/Bax), p-ERK and matrix metalloproteinase-2 (MMP-2) in cortical tissues were significantly declined than the sham group (p<0.01). However, the protein expression level of cleaved caspase-3 was markedly increased (p<0.01). Furthermore, the low expression of MALAT1 could remarkably increase the expressions of Bcl-2/Bax, p-ERK and MMP-2 (p<0.01), as well as decrease the expression of cleaved caspase-3 (p<0.01). CONCLUSIONS: LncRNA-MALAT1 may increase the release of inflammatory cytokines by inhibiting the ERK/MAPK signaling pathway, thereby up-regulating the level of neuronal apoptosis and aggravating the cerebral injury in rats with cerebral infarction.

[Clinical multicenter study of carboprost methylate suppository for cervical ripening prior to diagnostic hysteroscopy].

Objective: To evaluate the effectiveness of carboprost methylate suppository for cervical ripening before diagnostic hysteroscopy in premenopausal women. Methods: From July 2014 to July 2015, 1 614 women who were undergone diagnostic hysteroscopy in 12 hospitals were randomly assigned into study group (n=1 209) and control group (n=405) . The cases in study group were given 1 mg carboprost methylate suppository in vagina before hysteroscopy, the cases in control group were given 1 mg placebo. The extent of cervical ripening, the time of dilated cervix, pain scoring, incidence of drug side reactions after 24, 48, 72 hours, satisfaction degree of operators and patients, the time of hysteroscopy, incidence of complications between the two groups were observed and compared. Results: (1) Mean cervical widths in the study and control groups were 6.11±1.11 and 5.95±1.11, and showed a significant difference (P=0.034) ; the percentage of women requiring cervical dilatation in study group was lower than the percentage in control group significantly [28.3% (342/1 209) versus 34.6% (140/405) , P=0.020]. (2) The time of dilated cervix in study group was shorter than the time in control group significantly [ (34±25) versus (52±49) s, P=0.028] for the patients whose mean cervical widths≤4. (3) There was no significant difference in pain scores between the two groups (P>0.05) . (4) The incidence of side reactions 24, 48, 72 hours after operation were no significant difference between the two groups (P>0.05) . (5) The satisfaction degree of operators and patients, the time of hysteroscopy, incidence of complications between the two groups were no singnifcant difference between the two groups (all P>0.05) . Conclusion: Application of carboprost methylate suppository by vagina before hysteroscopy is an effective and safe method of cervical ripening.

Tanshinone IIA protects dopaminergic neurons against 6-hydroxydopamine-induced neurotoxicity through miR-153/NF-E2-related factor 2/antioxidant response element signaling pathway.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder with increased oxidative stress, the underlying vital process contributing to cell death. Tanshinone IIA (Tan IIA), a major bioactive diterpene quinone of Salva miltiorrhiza, had been proved effective in the MPTP model through its anti-inflammatory activity. Here in this research, we found that Tan IIA prevented the loss of nigrostriatal dopaminergic neurons by activating the NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. The cytotoxicity of 6-hydroxydopamine (6-OHDA) was attenuated by the treatment of Tan IIA in SH-SY5Y cells, which significantly reduced 6-OHDA-induced lactic dehydrogenase release and reactive oxygen species production. Further study indicated that Tan IIA contributed to the nuclear accumulation of Nrf2, which bound to the ARE sequence, and activated ARE-regulated genes, including heme oxygenase-1, glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM). Tan IIA also protected against damage to mitochondrial membrane potential, reduced the translocation of cytochrome c from the mitochondria to the cytoplasm and the activation of Caspase-9 and Caspase-3. Moreover, we demonstrated the above effects were performed in Nrf2-dependent manner. Further studies revealed that Tan IIA reduced the enhancement of miR-153 by 6-OHDA, which targeted the 3'-UTR of Nrf2, and suppressed its expression and activation. Additionally, neurodegeneration caused by in vivo stereotaxic injection of 6-OHDA could also be ameliorated by the administration of Tan IIA. Taken together, our results strongly suggest that Tan IIA may be beneficial for the treatment of PD, and also confirm that targeting the Nrf2/ARE pathway is a promising strategy for therapeutic intervention in PD.

Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-X(L)/p53-mediated amplification mechanisms.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X(L) overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X(L) downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X(L) and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X(L) and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.

Sodium current in NG108-15 cell inhibited by scorpion toxin BmKAS-1 and restored by its specific monoclonal antibodies.

Effect on Na+ current of BmKAS-1, a novel polypeptide purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK), has been investigated in differentiated NG108-15 cells by using patch-clamp whole-cell recording. Neutralizing effect of four monoclonal antibodies of BmKAS-1 (mAb #2, #3, #4, and #5) has also been observed. The results showed that Na+ current was irreversibly inhibited by BmKAS-1 and the inhibitory effect was abolished by mAb #2 and #5, but not by mAb #3 and #4.

Beta-agkistrodotoxin inhibits fast and Ca2+-activated K+ currents recorded from mouse motor nerve terminals.

Beta-agkistrodotoxin (beta-AgTx), a polypeptide purified from the venom of Agkistrodon blomhoffii brevicaudus, is a presynaptic blocker acting on neurotransmitter release. In this work, perineural recording technique was employed to study the effects of beta-AgTx on sodium, potassium and calcium currents of mouse motor nerve terminals. The results showed that beta-AgTx selectively inhibited Ca2+-dependent (I(K,Ca)) and fast (I(K,f) K+ currents, but did not affect slow K+ current (I(K,s)), sodium and calcium currents. However there are other components in A. blomhoffii brevicaudus venom which inhibit perineural sodium current. The present data have provided additional evidence that the site of action of beta-AgTx is different from that of beta-bungarotoxin.

[The inhibitory effects of artemisinin-derivatives on Na+ and K+ channels in comparison with those of procaine].

Some derivatives of artemisinin have been shown to have local anesthesia action. By using patch-clamp whole cell recording configuration, the effects of five artemisinin-derivatives on the voltage-gated INa and IK have been studied on differentiated NG108-15 cells with reference to procaine. The results showed that all the five artemisinin-derivatives clearly inhibited the voltage-gated sodium current (INa) of the cells in a dose-dependent manner and the effect was partially reversibly. Among the five artemisinin-derivatives, SM541 has been shown to be most potent, approaching that of procaine. However, 300 mumol/L procaine showed only a slight inhibition of IK, whereas all the five derivatives at the same concentration not only decreased IK clearly, but also accelerated its inactivation. Except for SM541, the inhibitory effects on IK decreased rapidly with perfusion of the rest 4 derivatives.

Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma x glioma cells.

By using the whole-cell patch-clamp technique, an inwardly rectifying potassium current, which resembled the "classic" inward-rectifying potassium current (IKIR) of other cells in terms of electrophysiological and pharmacological properties, was identified in db-cAMP-differentiated NG108-15 cells. First, the current was dependent on voltage and time. It could be elicited by applying an initial depolarizing prepulse and a subsequent hyperpolarizing command pulse to the cell. The amplitude of the current depended on both the prepulse and the command pulse and increased with the hyperpolarization of the command pulse as well as the depolarization and the prolongation of the prepulse. The activation and inactivation of the current could be fitted well by single-exponential functions and increased with the hyperpolarization of the membrane. Second, the current was dependent on the extracellular potassium concentration ([K+]o). Elevation of [K+]o resulted in a marked increase in the current amplitude and a positive shift of the peak-current/voltage curve as well as the reversal potential. A tenfold increase of [K+]o introduced an approximately 43-mV shift of the reversal potential, indicating that the current was carried mainly by K+. The conductance (g/gMax) of the current was also dependent on the [K+]o and increased with increases in [K+]o in a manner approximately proportional to the square-root of [K+]o. Finally, the current was sensitive to Cs+ (1 mmol/l), Ba2+ (1 mmol/l) and quinidine (0.2 mmol/l); whereas, two typical potassium channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), were weak blockers and reduced the current at high concentration (>10 mmol/l). It was also observed that the current was depressed by Cd2+ (1 mmol/l) and Co2+ (1 mmol/l) and increased by perfusing the cell with Ca2+-free solution. Thus, except for the sensitivity to Cd2+, Co2+ and Ca2+, the current displayed most of the hallmarks described for the "classic" IKIR. In conclusion, there appears to be a voltage-dependent IKIR-type inward rectifier in db-cAMP-differentiated NG108-15 cells.

Effects of ADP, DTT, and Mg2+ on the ion-conductive property of chloroplast H+-ATPase(CF0-CF1) reconstituted into bilayer membrane.

The purified CF0-CF1 complex of spinach was incorporated into planar lipid bilayer membranes (LBMs) formed with soybean phospholipid, and the transmembrane ion-transmission properties were studied under voltage-clamp mode. The results showed that the presence of both ADP and Pi decreased the membrane current while Dithiothreitol could evoke a stronger conductive change of CF0-CF1 containing LBMs when Ca2+ or Mg2+ exists. Mg2+ can dramatically increase the CF0-CF1 conductance in various conditions. These results indicated that the H(+)-transitive function of CF0-CF1 reconstituted in bilayer is sensitive to those factors which can affect its ATP synthase activity in vivo.

[Inhibition of voltage-gated K+, Na+ and Ca2+ currents in neuroblastoma x glioma hybrid cells by paeonol].

The effects of Paeonol (25 micrograms/ml to 400 micrograms/ml) on the membrane currents of NG108-15 cells were observed by means of whole cell voltage-clamp technique. The results showed that PA inhibited delayed outward K+ current (IK), and to a less extend, that the Na+ current (INa). Two Ca2+ currents (T and L type) were also inhibited significantly. All of the effects mentioned above were dose-dependent and reversible.

[Effect of hypoxia on the growth and differentiation of NG108-15 cells].

The neuroblastoma x glioma hybrid clone NG108-15 which were derived by fusion of mouse neuroblastoma clone N18TG6 with rat glioma clone C6BU1 proliferates in the presence of fetal calf serum, but differentiates when intracellular cAMP concentration is made to increase by addition of dBcAMP to the cultural medium. After differentiation, the cells express many neural properties. In this experiment, by means of an automatic colorimetric microassay for quantifying the growth and Brilliant Cresyl Violett stain for revealing Nissl substance, the effect of hypoxia (2% O2) on the hybrid cells was investigated. The results obtained were as follows: (1) Hypoxia depressed proliferation of non-differentiated cells and induced death of differentiated ones. (2) Hypoxia inhibited differentiation, either non-differentiated or differentiating cells, in the direction to be a neuro-like cell: some cells became large flat with short processes and had no Nissl substance. Whether hypoxia would enable non-differentiated NG108-15 cells to differentiate in a direction to express more properties of glial cell is an interesting problem.

[Channel-forming activity at planar lipid bilayer of the membrane active polypeptide B form venom of Bungarus fasciatus].

Using planar lipid bilayer formed by lecithin and cholesterol (20 and 5 mg/ml respectively in N-decane) the channel-forming activity of the membrane active polypeptide B(BMAP B) from the venom of Bungarus fasciatus was investigated. Under the existence of a voltage or a salt concentration gradient between two sides of the bilayer, unit conductance fluctuation and a decrease in steady state resistance accompanying BMAP B incorporation and channel formation were observed. By measuring the reversal potential in an asymmetric solution, the selectivity of the BMAP B-channel was estimated having a value of PK/PC1 = 1.4. Divalent cations, such as Ba2+, Ca2+ inhibited the channel activity as they did in biomembranes. These data might provide an explanation for the depolarizing effect of the membrane active polypeptide on the native membranes.

[Ionic channels formed in the lipid bilayer membranes by aureofuscin, a polyene antibiotics].

A chamber filled with salt solution and separated into two compartments by a Teflon partition with a pore of 700 microns diameter was used to investigate the action of aureofuscin, a polyene antibiotics, on a planar lipid bilayer. The pore was covered with a bilayer formed by a N-decane solution of lecithin and cholesterol (20 mg/ml and 5 mg/ml). The electrical property and ionic permeability of the bilayer were studied by using voltage clamp method. Decrease of the bilayer resistance and the channel-like activity could be observed 20 min after the addition of aureofuscin (10-20 micrograms/ml) to one compartment. The existence of a transmembrane voltage and ionic gradient were not necessary for the channel-forming activity of aureofuscin. Discrete current steps were recorded at a concentration of 1.4 micrograms/ml aureofuscin, with a predominant unit conductance of 4-6 pS in a symmetric KCl, solution of 100 mmol/L. By using Goldmann-Hodgkin-Katz voltage equation the ionic selectivity of the channel formed by aureofuscin was estimated by the reversal potential measured in the asymmetrical solution system. The results showed that aureofuscin channels were more permeable to potassium ion than to chloride ion (PK/PCl approximately 5.2). These data may be used to explain the action of aureofuscin on neurotransmitter release and muscle membrane potential in addition to providing some explanation on its curing effect in clinical use.