BACKGROUND: Lung adenocarcinoma (LUAD) is the most common type of non-small cell lung cancer, and any change of miRNAs expression will affect the degree of target regulation, thus affecting intracellular homeostasis. This study verified that miR-186-5p could inhibit the proliferation, migration and invasion of LUAD cells by regulating PRKAA2. METHODS: Previous investigations found that the expression of miR-186-5p was markedly suppressed in LUAD. Bioinformatics method is used to predict the target protein related to ferroptosis downstream and inquire about its expression level in LUAD and its influence on the survival of patients. Double luciferase verified the binding site of PRKAA2 and miR-186-5p. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of PRKAA2. The effects of miR-186-5p of LUAD cells as well as the mechanism by which miR-186-5p inhibits Fer-1's sensitivity to ferroptosis were confirmed by EdU, Transwell, and scratch assays. The effect of miR-186-5p on the amount of reactive oxygen species (ROS) in LUAD cells was discovered using ROS experiment. Malondialdehyde (MDA) and glutathione (GSH) experiments were used to detect the effects of miR-186-5p and PRKAA2 on ferroptosis index of LUAD cells. The concentration of lipid ROS (L-ROS) in LUAD cells were measured using the L-ROS tests to determine the effects of miR-186-5p and PRKAA2. RESULTS: The expression of PRKAA2 is up-regulated, and a high level of PRKAA2 expression was associated with a poor prognosis for patients with LUAD. Overexpression of miR-186-5p decreased the gene and protein expression of PRKAA2. By promoting ferroptosis, miR-186-5p overexpression prevented lung cancer cells from proliferating, invading, and migrating. ROS could be produced in higher amounts in LUAD cells due to miR-186-5p. Overexpression of miR-186-5p and knockdown PRKAA2 up-regulated MDA content and reduced GSH content in LUAD cells, respectively. miR-186-5p could increase the content of L-ROS and promote the ferroptosis sensitivity of LUAD cells by targeting PRKAA2. CONCLUSIONS miR-186-5p promotes ferroptosis of LUAD cells through targeted regulation of PRKAA2, thus inhibiting the proliferation, invasion and migration of LUAD.
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Humans ; Lung Neoplasms/genetics* ; Carcinoma, Non-Small-Cell Lung ; Ferroptosis/genetics* ; Reactive Oxygen Species ; Adenocarcinoma of Lung/genetics* ; MicroRNAs/genetics* ; 3,4-Methylenedioxyamphetamine ; Cell Proliferation/genetics* ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; AMP-Activated Protein Kinases

Objective To establish a method to identify unknown samples based on combined use of gas chromatography-mass spectrometry （GC-MS）, high resolution mass spectrometry （HRMS） and nuclear magnetic resonance spectrum （NMR） technique. Methods The unknown samples were dissolved in methanol solution containing internal standard SKF525A and detected by GC-MS and HRMS. The mixed samples were separated and purified by silica gel column chromatography, and then dissolved in methanol-d4 solution for structural analysis of ¹H nuclear magnetic resonance spectroscopy （¹H NMR）. Results The characteristic fragment ions （m/z） were 86.1 （base peak）, 71.2, 121.1, and 149.0, and the accurate mass number of molecular ion peak was measured by HRMS to be 236.128 89. By combined use of data analysis and database comparison, a new psychoactive substance of the cathinone class, Dibutylone, was detected in the sample, and the sample also contained a small amount of caffeine. The sample was purified, then identified using ¹H NMR, and was further confirmed to be Dibutylone. In addition, the GC-MS retention time and characteristic fragment ions of the main components of the sample were consistent with those of Dibutylone reference material. Conclusion The method established in this study can be used for the identification of Dibutylone in mixed samples.
Chromatography, Liquid ; Gas Chromatography-Mass Spectrometry ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; N-Methyl-3,4-methylenedioxyamphetamine/isolation & purification* ; Psychotropic Drugs/chemistry*

OBJECTIVETo explore the relationship of gentamicin-induced cochlear damage with autophagy-related protein LC3, beclin1, Na(+-)K(+-)2Cl(-) cotransporter (NKCC1) mRNA and endothelin-1 (ET-1), and investigate the protective mechanism of PPTA against gentamicin-induced cochlear damage.

METHODSSixty guinea pigs were randomly divided into control group (with saline and artificial perilymph injections), model group (with gentamicin and artificial perilymph injections), concurrent treatment group (with gentamicin and PPTA injections), model control group (with artificial perilymph injection 7 days after gentamicin injection) and delayed treatment group (with PPTA injection 7 days after gentamicin injection). Saline and gentamicin (160 mg/kg) were injected intraperitoneally, and artificial perilymph and PPTA were injected into the otocysts on a daily basis for 7 consecutive days. Hearing impairment of the guinea pigs was analyzed with ABR, and the protein expressions of beclin1 and LC3 in cochlear tissue were tested. The expression of NKCC1 mRNA was detected with RT-PCR, and the expression of ET-1 was detected immunohistochemically.

RESULTSThe ABR thresholds in the model group and model control group were similar (P>0.05) , but significantly higher than those in the other 3 groups (P<0.05); the threshold was significantly lower in concurrent treatment group than in delayed treatment group (P<0.05). Compared with those in the other 4 groups, the expressions of LC3 II, beclin1, and NKCC1 mRNA were significantly increased in the model group (P<0.05); and those in delayed treatment group were significantly lower than those in the model control group (P<0.05). The expressions of ET-1 in the Corti organ, striavascularis and spiral ganglion were significantly higher in the model group but significantly lower in the control group than those in the other 4 groups; ET-1 expression was significantly lower in delayed treatment group than in the model control group.

CONCLUSIONPPTA offers protection against gantamicin-induced cochlear damage in guinea pigs by inhibiting cell autophagy and suppressing of NKCC1 and ET-1 expressions. Early intervention with PPTA produces better therapeutic effect, suggesting that gantamicin causes irreversible injury of the auditory cells.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; metabolism ; Beclin-1 ; Cochlea ; drug effects ; Endothelin-1 ; metabolism ; Gentamicins ; adverse effects ; Guinea Pigs ; Hearing Loss ; chemically induced ; prevention & control ; Microtubule-Associated Proteins ; metabolism ; Solute Carrier Family 12, Member 2 ; metabolism

OBJECTIVETo study the protective effect of peperphentonamine hydrochloride (PPTA) against gentamicin-induced cochlear damage and its mechanism to inhibit cell apoptosis.

METHODSGuinea pigs with normal hearing were randomized into control, gentamicin, and PPTA treatment groups, and the guinea pigs models of gentamicin-induced cochlear damage received intraperitoneal injection of PPTA. The changes of hearing of the guinea pigs were evaluated with auditory brainstem response (ABR) test, and the protein expression of caspase-3 in the cochlear tissue was detected using Western blotting. TUNEL staining, scanning and transmission electron microscopy were performed to observe the morphological changes of the cochlea.

RESULTSThe threshold in ABR in PPTA treatment group was significantly higher than that in the control group (P<0.05) but significantly lower than that in gentamicin group. Western blotting showed a significantly increased caspase-3 expression in gentamicin group (P<0.001); caspase-3 expression in PPTA group was obviously higher than that in the control group but much lower than that in gentamicin group (P<0.001). TUNEL assay and electron microscopy revealed serious damages of the hair cells in gentamicin group with numerous apoptotic cells in the organ of Corti, stria vascularis and spiral ganglion, and such cochlear damages were obviously alleviated in PPTA group.

CONCLUSIONPPTA can protect against gentamicin-induced cochlear damage in guinea pigs by decreasing the protein expression of caspase-3 to inhibit cell apoptosis.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cochlea ; drug effects ; pathology ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; adverse effects ; Guinea Pigs

Adult ; Brain ; drug effects ; metabolism ; Female ; Hallucinogens ; adverse effects ; Humans ; Magnetic Resonance Imaging ; Motion Perception ; drug effects ; N-Methyl-3,4-methylenedioxyamphetamine ; adverse effects

OBJECTIVETo investigate the relationship between IL-1β and TNF-α mRNA and Fas protein expressions and cochlear ischemia reperfusion injury and investigate the protective mechanism of PPTA against cochlear reperfusion injury.

METHODSSixty-four guinea pigs were randomly divided into normal control group, blank control group, ischemia/reperfusion (by clamping the bilateral vertebral artery and right common carotid artery for 1 h) control group, and ischemia/reperfusion with PPTA treatment group. In PPTA group, PPTA was injected via the femoral vein immediately after reperfusion, and ischemia/reperfusion control group received saline injection. In 6 guinea pigs from each group, the cochlear tissues were removed after 24 h of reperfusion for examination of expressions of IL-1β and TNF-α mRNA by real-time PCR, and the rest animals were used for immunohistochemical detection of Fas protein.

RESULTSCompared with those of normal group and blank control group, the expressions of IL-1β and TNF-β mRNA increased significantly after cochlear ischemia/reperfusion (P<0.001), but were lowered significantly by PPTA (P<0.001). Positive expression of Fas protein expression was detected in the Corti organ, spiral ganglion and stria vascularis in ischemia/reperfusion control group with significantly higher IOD values than those of the other 3 groups (P<0.05). The IOD value showed no significant difference between PPTA-treated group, normal control group, and blank control group (P>0.05).

CONCLUSIONSPPTA can suppress the expression of Fas protein and IL-1β and TNF-β mRNAs in the cochlea of guinea pigs with cochlear ischemia/reperfusion. The protective effect of PPTA against cochlear ischemia/reperfusion is mediated probably by inhibition of inflammatory responses and cell apoptosis.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; pharmacology ; Animals ; Cochlea ; blood supply ; metabolism ; pathology ; Female ; Guinea Pigs ; Interleukin-1beta ; genetics ; metabolism ; Male ; Neuroprotective Agents ; pharmacology ; Organ of Corti ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Reperfusion Injury ; metabolism ; Spiral Ganglion ; metabolism ; Stria Vascularis ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the effect of piperphentonamine hydrochloride (PPTA) on cognitive deficits induced by ischemia-reperfusion and explore the possible mechanisms.

METHODSSD rats were randomly divided into sham-operated group, ischemia-reperfusion group (with saline injection), PPTA-treated groups (2.5, 5, 10 mg/kg) and edaravone-treated group (6 mg/kg). Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion, and the agents were administrated 1 h after ischemia. At 24 h after ischemia, step-through passive avoidance test was carried out, and 24 h later IL-1β, TNF-α, caspase-3 and HSP-70 mRNA expressions in the ischemic brain tissues were measured with RT-PCR.

RESULTSIn the step-through passive avoidance test, the rats in the ischemia-reperfusion group showed significantly shorter latency and more error times than those in the sham group, and these behavioral changes were improved significantly by treatments with PPTA and edaravone. Cerebral ischemia-reperfusion caused significantly increased expressions of IL-1β, TNF-α, caspase-3 and HSP-70 mRNA, and these changes were obviously reversed by PPTA, but not by edaravone.

CONCLUSIONSPPTA can reverse cognitive deficits induced by cerebral ischemia-reperfusion probably by decreasing the inflammatory responses and cell apoptosis in the brain, suggesting its potential as a new therapeutic agent for improving the cognitive function following cerebral ischemia-reperfusion.

3,4-Methylenedioxyamphetamine ; analogs & derivatives ; therapeutic use ; Animals ; Brain Ischemia ; drug therapy ; Cognition Disorders ; prevention & control ; Infarction, Middle Cerebral Artery ; drug therapy ; Male ; Neuroprotective Agents ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; prevention & control

3,4-Methylenedioxymethamphetamine (MDMA) is a substituted amphetamine with stimulating and hallucinogenic properties. Since MDMA induces "ecstasy" it is extensively used as a "recreational" drug. It has been well established that MDMA is neurotoxic and can result in long-term degeneration of cerebral 5-hydroxytryptamine (5-HT) nerve terminals in many species. The present study was undertaken to investigate the long-term neurotoxic effects of MDMA on cortical and hippocampal structures, by repeatedly administering MDMA in short time. Male Wistar rats were randomly assigned to control group and MDMA-treated group. MDMA (10 mg/kg) was administered to rats of MDMA-treated group, once per hour, total 40 mg/kg; rats of control group were treated with the same volume of saline. Thirty-two weeks after administering MDMA, the expression of serotonin transporter (SERT) mRNA and diazepam binding inhibitor (DBI) mRNA was detected by in situ hybridization. The expression of glial fibrillary acidic protein (GFAP) was detected by immunohistochemistry, and the degeneration of nerve terminals was demonstrated by Bielschowsky and Glee Marsland silver staining. The results showed that the expression of SERT mRNA in hippocampus decreased by 31.96%, while expression of DBI mRNA in neocortex increased by 40.51%, compared with the control group (P<0.05). The expression of GFAP in the brain tissue increased (P<0.05), while significant reduction of the nerve terminals in neocortex was demonstrated by silver staining, compared with the control group. These results suggest that the neurotoxicity of MDMA results in sustained cortical and hippocampal structural changes, which in turn result in disorder of the brain functions.
Animals ; Cerebral Cortex ; pathology ; physiopathology ; Diazepam Binding Inhibitor ; genetics ; metabolism ; Hippocampus ; pathology ; physiopathology ; Male ; N-Methyl-3,4-methylenedioxyamphetamine ; toxicity ; Neurotoxicity Syndromes ; etiology ; pathology ; physiopathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Serotonin Plasma Membrane Transport Proteins ; genetics ; metabolism

OBJECTIVETo explore the changes of methylenedioxyamphetamine (MDA), total antioxidants content (TAC) and sialic acid (SA) from the unilateral epididymis of experimental varicocele in adolescent rats, and to illuminate the effects of varicocele on unilateral epididymis epithelium.

METHODSExperimental left varicocele model of 16 adult male Sprague-Dawley rats were established by partial ligation of left renal vein. The epididymis were collected for detecting the content of MDA, TAC and SA by using spectrophotometry.

RESULTSThere was statistically significant differences in the contents of three substances between experimental varicocele and sham-operated groups.

CONCLUSIONThe content of MDA, TAC and SA will change and the sialic acid-secreting-function of unilateral epididymis will be injured because of varicocele.

Animals ; Antioxidants ; metabolism ; Epididymis ; metabolism ; Male ; N-Acetylneuraminic Acid ; metabolism ; N-Methyl-3,4-methylenedioxyamphetamine ; metabolism ; Rats ; Rats, Sprague-Dawley ; Varicocele ; metabolism

OBJECTIVETo investigate whether 3,4-methylenedioxymethamphetamine (MDMA) abuse produces another neurotoxicity which may significantly inhibit the acetylcholinesterase activity and result in severe oxidative damage and liperoxidative damage to MDMA abusers.

METHODS120 MDMA abusers (MA) and 120 healthy volunteers (HV) were enrolled in an independent sample control design, in which the levels of lipoperoxide (LPO) in plasma and erythrocytes as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and acetylcholinesterase (AChE) in erythrocytes were determined by spectrophotometric methods.

RESULTSCompared with the average values of biochemical parameters in the HV group, those of LPO in plasma and erythrocytes in the MA group were significantly increased (P < 0.0001), while those of SOD, CAT, GPX and AChE in erythrocytes in the MA group were significantly decreased (P < 0.0001). The Pearson product-moment correlation analysis between the values of AChE and biochemical parameters in 120 MDMA abusers showed that significant linear negative correlation was present between the activity of AChE and the levels of LPO in plasma and erythrocytes (P < 0.0005-0.0001), while significant linear positive correlation was observed between the activity of AchE and the activities of SOD, CAT and GPX (P < 0.0001). The reliability analysis for the above biochemical parameters reflecting oxidative and lipoperoxidative damages in MDMA abusers suggested that the reliability coefficient (alpha) was 0.8124, and that the standardized item alpha was 0.9453.

CONCLUSIONThe findings in the present study suggest that MDMA abuse can induce another neurotoxicity that significantly inhibits acetylcholinesterase activity and aggravates a series of free radical chain reactions and oxidative stress in the bodies of MDMA abusers, thereby resulting in severe neural, oxidative and lipoperoxidative damages in MDMA abusers.

Acetylcholinesterase ; metabolism ; Adolescent ; Adult ; Amphetamine-Related Disorders ; blood ; enzymology ; metabolism ; Catalase ; blood ; Cholinesterase Inhibitors ; adverse effects ; urine ; Erythrocytes ; enzymology ; Female ; Humans ; Lipid Peroxidation ; drug effects ; Lipid Peroxides ; blood ; Male ; N-Methyl-3,4-methylenedioxyamphetamine ; adverse effects ; urine ; Oxidative Stress ; drug effects ; Superoxide Dismutase ; blood