Serum myoglobin modulates kidney injury via inducing ferroptosis after exertional heatstroke.

Background and Objectives: Myoglobin released by rhabdomyolysis (RM) is considered to be involved in pathogenesis of kidney disease caused by crush injury, but whether high level of serum myoglobin predisposes patients to acute kidney injury (AKI) and its molecular mechanisms are still unclear in exertional heatstroke (EHS). We aimed to determine the association and potential mechanism of myoglobin and AKI, and further investigate the targeted therapeutic agents for myoglobinemia. Methods: Serum myoglobin concentrations in patients with EHS were measured at admission, 24 h and 48 h after admission and discharge. The risk of AKI at 48 h was the primary outcome; the secondary outcome was composite outcome events with myoglobin levels and AKI at discharge and death at 90 days. In experimental studies, we further investigated the mechanisms of human kidney proximal tubular (HK-2) cells that were exposed to human myoglobin under heat stress conditions and the effect of baicalein. Results: Our measurements showed that the highest myoglobin quartile (vs. the lowest) had an adjusted odds ratio (OR) of 18.95 (95% confidence interval [CI], 6.00-59.83) for AKI and that the OR (vs. quartile 2) was 7.92 (95% CI, 1.62-38.89) for the secondary outcome. The survival rate of HK-2 cells treated with myoglobin under heat stress was significantly decreased, and the production of Fe2+ and reactive oxygen species (ROS) was markedly increased, accompanied by changes in ferroptosis proteins, including increased p53, decreased SLC7A11 and GPX4, and alterations in endoplasmic reticulum stress (ERS) marker proteins. Treatment with baicalein attenuated HK-2 cell ferroptosis induced by myoglobin under heat stress through inhibition of ERS. Conclusions: High myoglobin was associated with AKI in the EHS, and its mechanisms involved ERS-associated ferroptosis. Baicalein may be a potential therapeutic drug for the treatment of AKI in patients with high myoglobin induced by rhabdomyolysis following EHS.

Fecal microbial signatures of healthy Han individuals from three bio-geographical zones in Guangdong.

Important forensic evidence traced from crime scenes, such as fecal materials, can help in the forensic investigation of criminal cases. Intestines are the largest microbial pool in the human body whose microbial community is considered to be the human "second fingerprint". The present study explored the potential for community characteristics of gut microbes in forensic medicine. Fecal microbiota profiles of healthy individuals from three representative Han populations (Guangzhou, Shantou and Meizhou) in Guangdong Province, China were evaluated using High-throughput sequencing of V3-V4 hypervariable regions of the 16SrRNA gene. Results of the present study showed that at the genus level, Shantou, Guangzhou, and Meizhou behaved as Enterotype1, Enterotype2, and Enterotype3, which were mainly composed of Bacteroides, Prevotella, and Blautia, respectively. Based on OTU abundance at the genus level, using the random forest prediction model, it was found that there might be potential for distinguishing individuals of Guangzhou, Meizhou, and Shantou according to their fecal microbial community. Moreover, the findings of the microbial community of fecal samples in the present study were significantly different from that of saliva samples reported in our previous study, and thus it is evident that the saliva and feces can be distinguished. In conclusion, this study reported the fecal microbial signature of three Han populations, which may provide basic data for the potential application in forensic practice, containing body fluid identification, and geographical inference.

Nupr1 mediates renal fibrosis via activating fibroblast and promoting epithelial-mesenchymal transition.

Renal interstitial fibrosis (RIF) is a pathological process that fibrotic components are excessively deposited in the renal interstitial space due to kidney injury, resulting in impaired renal function and chronic kidney disease. The molecular mechanisms controlling renal fibrosis are not fully understood. In this present study, we identified Nuclear protein 1 (Nupr1), a transcription factor also called p8, as a novel regulator promoting renal fibrosis. Unilateral ureteral obstruction (UUO) time-dependently induced Nupr1 mRNA and protein expression in mouse kidneys while causing renal damage and fibrosis. Nupr1 deficiency (Nupr1-/- ) attenuated the renal tubule dilatation, tubular epithelial cell atrophy, and interstitial collagen accumulation caused by UUO. Consistently, Nupr1-/- significantly decreased the expression of type I collagen, myofibroblast markers smooth muscle α-actin (α-SMA), fibroblast-specific protein 1 (FSP-1), and vimentin in mouse kidney that were upregulated by UUO. These results suggest that Nupr1 protein was essential for fibroblast activation and/or epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Indeed, Nupr1 was indispensable for TGF-ß-induced myofibroblast activation of kidney interstitial NRK-49F fibroblasts, multipotent mesenchymal C3H10T1/2 cells, and the EMT of kidney epithelial NRK-52E cells. It appears that Nupr1 mediated TGF-ß-induced α-SMA expression and collagen synthesis by initiating Smad3 signaling pathway. Importantly, trifluoperazine (TFP), a Nupr1 inhibitor, alleviated UUO-induced renal fibrosis. Taken together, our results demonstrate that Nupr1 promotes renal fibrosis by activating myofibroblast transformation from both fibroblasts and tubular epithelial cells.

Involvement of C/EBPß-related signaling pathway in methamphetamine-induced neuronal autophagy and apoptosis.

Methamphetamine (METH) is a widely abused illicit psychoactive drug. Our previous study has shown that CCAAT-enhancer binding protein ß (C/EBPß) is an important regulator in METH-induced neuronal autophagy and apoptosis. However, the detailed molecular mechanisms underlying this process remain poorly understood. Previous studies have demonstrated that DNA damage-inducible transcript 4 (DDIT4), Trib3 (tribbles pseudo kinase 3), alpha-synuclein (α-syn) are involved in METH-induced dopaminergic neurotoxicity. We hypothesized that C/EBPß is involved in METH-induced DDIT4-mediated neuronal autophagy and Trib3-mediated neuronal apoptosis. We tested our hypothesis by examining the effects of silencing C/EBPß, DDIT4, Trib3 or α-syn with small interfering ribonucleic acid (siRNA) on METH-induced autophagy and apoptosis in the human neuroblastoma SH-SY5Y cells. We also measured the levels of phosphorylated tuberous sclerosis complex 2 (TSC2) protein and Parkin protein level in SH-SY5Y cells. Furthermore, we demonstrated the effect of silencing C/EBPß on METH-caused neurotoxicity in the striatum of rats by injecting LV-shC/EBPß lentivirus using a stereotaxic positioning system. The results showed that METH exposure increased C/EBPß, DDIT4 protein expression. Elevated DDIT4 expression raised up p-TSC2/TSC2 protein expression ratio, inhibited mTOR signaling pathway, activating cell autophagy. We also found that METH exposure increased the expression of Trib3, α-syn, decreased the Parkin protein expression. Lowering levels of Parkin raised up α-syn expression, which initiated mitochondrial apoptosis by down-regulating anti-apoptotic Bcl-2, followed by up-regulation of pro-apoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. These findings were supported by data showing METH-induced autophagy and apoptosis was significantly inhibited by silencing C/EBPß, DDIT4, Trib3 or α-syn, or by Parkin over-expression. Based on the present data, a novel of mechanism on METH-induced cell toxicity is proposed, METH exposure increased C/EBPß protein expression, triggered DDIT4/TSC2/mTOR signaling pathway, and evoked Trib3/Parkin/α-syn-related mitochondrial apoptotic signaling pathway. Collectively, these results suggest that C/EBPß plays an important role in METH-triggered autophagy and apoptosis and it may be a potential target for therapeutics in METH-caused neurotoxicity.

Genetic polymorphisms of 20 STR loci in a Chaoshan group from Jieyang, China.

We obtained allelic frequencies and forensic parameters of 20 short tandem repeat (STR) loci (D3S1358, FGA, D5S818, D7S820, CSF1PO, D16S539, D19S433, vWA, D8S1179, D18S51, D13S317, TPOX, TH01, D2S1338, D12S391, D1S1656, D21S11, D6S1043, Penta D, Penta E) from 529 unrelated individuals in Jieyang Han population using PowerPlex® 21 (Promega, Madison, Wi, USA). The relationship between the Jieyang Han group and other Han populations was studied and the results showed that the Jieyang Han population had the closest genetic relationship with the Fujian Han population.

Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPß-related signaling pathway.

Methamphetamine (Meth) is a widely abused psychoactive drug that primarily damages the nervous system, notably causing dopaminergic neuronal apoptosis. CCAAT-enhancer binding protein (C/EBPß) is a transcription factor and an important regulator of cell apoptosis and autophagy. Insulin-like growth factor binding protein (IGFBP5) is a proapoptotic factor that mediates Meth-induced neuronal apoptosis, and Trib3 (tribbles pseudokinase 3) is an endoplasmic reticulum (ER) stress-inducible gene involved in autophagic cell death through the mammalian target of rapamycin (mTOR) signaling pathway. To test the hypothesis that C/EBPß is involved in Meth-induced IGFBP5-mediated neuronal apoptosis and Trib3-mediated neuronal autophagy, we measured the protein expression of C/EBPß after Meth exposure and evaluated the effects of silencing C/EBPß, IGFBP5, or Trib3 on Meth-induced apoptosis and autophagy in neuronal cells and in the rat striatum after intrastriatal Meth injection. We found that, at relatively high doses, Meth exposure increased C/EBPß protein expression, which was accompanied by increased neuronal apoptosis and autophagy; triggered the IGFBP5-mediated, p53-up-regulated modulator of apoptosis (PUMA)-related mitochondrial apoptotic signaling pathway; and stimulated the Trib3-mediated ER stress signaling pathway through the Akt-mTOR signaling axis. We also found that autophagy is an early response to Meth-induced stress upstream of apoptosis and plays a detrimental role in Meth-induced neuronal cell death. These results suggest that Meth exposure induces C/EBPß expression, which plays an essential role in the neuronal apoptosis and autophagy induced by relatively high doses of Meth; however, relatively low concentrations of Meth did not change the expression of C/EBPß in vitro. Further studies are needed to elucidate the role of C/EBPß in low-dose Meth-induced neurotoxicity.-Xu, X., Huang, E., Luo, B., Cai, D., Zhao, X., Luo, Q., Jin, Y., Chen, L., Wang, Q., Liu, C., Lin, Z., Xie, W.-B., Wang, H. Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPß-related signaling pathway.

Nupr1 Modulates Methamphetamine-Induced Dopaminergic Neuronal Apoptosis and Autophagy through CHOP-Trib3-Mediated Endoplasmic Reticulum Stress Signaling Pathway.

Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH exposure causes detrimental effects on multiple organ systems, primarily the nervous system, especially dopaminergic pathways, in both laboratory animals and humans. In this study, we hypothesized that Nuclear protein 1 (Nupr1/com1/p8) is involved in METH-induced neuronal apoptosis and autophagy through endoplasmic reticulum (ER) stress signaling pathway. To test this hypothesis, we measured the expression levels of Nupr1, ER stress protein markers CHOP and Trib3, apoptosis-related protein markers cleaved-caspase3 and PARP, as well as autophagy-related protein markers LC3 and Beclin-1 in brain tissues of adult male Sprague-Dawley (SD) rats, rat primary cultured neurons and the rat adrenal pheochromocytoma cells (PC12 cells) after METH exposure. We also determined the effects of METH exposure on the expression of these proteins after silencing Nupr1, CHOP, or Trib3 expression with synthetic small hairpin RNA (shRNA) or siRNA in vitro, and after silencing Nupr1 in the striatum of rats by injecting lentivirus containing shRNA sequence targeting Nupr1 gene to rat striatum. The results showed that METH exposure increased Nupr1 expression that was accompanied with increased expression of ER stress protein markers CHOP and Trib3, and also led to apoptosis and autophagy in rat primary neurons and in PC12 cells after 24 h exposure (3.0 mM), and in the prefrontal cortex and striatum of rats after repeated intraperitoneal injections (15 mg/kg × 8 injections at 12 h intervals). Silencing of Nupr1 expression partly reduced METH-induced apoptosis and autophagy in vitro and in vivo. These results suggest that Nupr1 plays an essential role in METH-caused neuronal apoptosis and autophagy at relatively higher doses and may be a potential therapeutic target in high-dose METH-induced neurotoxicity.

Effects of DDIT4 in Methamphetamine-Induced Autophagy and Apoptosis in Dopaminergic Neurons.

Methamphetamine (METH) is an illicit psychoactive drug that can cause a variety of detrimental effects to the nervous system, especially dopaminergic pathways. We hypothesized that DNA damage-inducible transcript 4 (DDIT4) is involved in METH-induced dopaminergic neuronal autophagy and apoptosis. To test the hypothesis, we determined changes of DDIT4 protein expression and the level of autophagy in rat catecholaminergic PC12 cells and human dopaminergic SH-SY5Y cells, and in the hippocampus, prefrontal cortex, and striatum of Sprague Dawley rats exposed to METH. We also examined the effects of silencing DDIT4 expression on METH-induced dopaminergic neuronal autophagy using fluorescence microscopy and electron microscopy. Flow cytometry and Western blot were used to determine apoptosis and the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) after blocking DDIT4 expression in PC12 cells and SH-SY5Y cells with synthetic siRNA, as well as in the striatum of rats by injecting LV-shDDIT4 lentivirus using a stereotaxic positioning system. Our results showed that METH exposure increased DDIT4 expression that was accompanied with increased autophagy and apoptosis in PC12 cells (3 mM) and SH-SY5Y cells (2 mM), and in the hippocampus, prefrontal cortex, and striatum of rats. Inhibition of DDIT4 expression reduced METH-induced autophagy and apoptosis in vitro and in vivo. However, DDIT4-related effects were not observed at a low concentration of METH (1 µM). These results suggest that DDIT4 plays an essential role in METH-induced dopaminergic neuronal autophagy and apoptosis at higher doses and may be a potential gene target for therapeutics in high-dose METH-induced neurotoxicity.

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes.

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes.

Role of PUMA in methamphetamine-induced neuronal apoptosis.

Exposure to methamphetamine (METH), a widely used illicit drug, has been shown to cause neuron apoptosis. p53 upregulated modulator of apoptosis (PUMA) is a key mediator in neuronal apoptosis. This study aimed to examine the effects of PUMA in METH-induced neuronal apoptosis. We determined PUMA protein expression in PC12 cells and SH-SY5Y cells after METH exposure using western blot. We also observed the effect of METH on neuronal apoptosis after silencing PUMA expression with siRNA using TUNEL staining and flow cytometry. Additionally, to investigate possible mechanisms of METH-induced PUMA-mediated neuronal apoptosis, we measured the protein expression of apoptotic markers, including cleaved caspase-3, cleaved PARP, Bax, B-cell leukemia/lymphoma-2 (Bcl-2) and cytochrome c (cyto c), after METH treatment with or without PUMA knockdown. Results showed that METH exposure induced cell apoptosis, increased PUMA protein levels, activated caspase-3 and PARP, elevated Bax and reduced Bcl-2 expression, as well as increased the release of cyto c from mitochondria to the cytoplasm in both PC12 and SH-SY5Y cells. All these effects were attenuated or reversed after silencing PUMA. A schematic depicting the role of PUMA in METH-induced mitochondrial apoptotic pathway was proposed. Our results suggest that PUMA plays an important role in METH-triggered apoptosis and it may be a potential target for ameliorating neuronal injury and apoptosis caused by METH.

Caspase-11 plays an essential role in methamphetamine-induced dopaminergic neuron apoptosis.

Methamphetamine (METH) is an extremely addictive stimulant drug that is widely used with high potential of abuse. Previous studies have shown that METH exposure damages the nervous system, especially dopaminergic neurons. However, the exact molecular mechanisms of METH-induced neurotoxicity remain unclear. We hypothesized that caspase-11 is involved in METH-induced neuronal apoptosis. We tested our hypothesis by examining the change of caspase-11 protein expression in dopaminergic neurons (PC12 and SH-SY5Y) and in the midbrain of rats exposed to METH with Western blotting. We also determined the effects of blocking caspase-11 expression with wedelolactone (a specific inhibitor of caspase-11) or siRNA on METH-induced apoptosis in PC12 cells and SH-SY5Y cells using Annexin V and TUNEL staining. Furthermore, we observed the protein expression changes of the apoptotic markers, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1 (PARP), after silencing the caspase-11 expression in rat midbrain by injecting LV-shcasp11 lentivirus using a stereotaxic positioning system. Results showed that METH exposure increased caspase-11 expression both in vitro and in vivo, with the effects in vitro being dose- and time-dependent. Inhibition of caspase-11 expression with either wedelolactone or siRNAs reduced the number of METH-induced apoptotic cells. In addition, blocking caspase-11 expression inhibited METH-induced activation of caspase-3 and PARP in vitro and in vivo, suggesting that caspase-11/caspase-3 signal pathway is involved in METH-induced neurotoxicity. These results indicate that caspase-11 plays an essential role in METH-induced neuronal apoptosis and may be a potential gene target for therapeutics in METH-caused neurotoxicity.

Protective effect of alpha-synuclein knockdown on methamphetamine-induced neurotoxicity in dopaminergic neurons.

The over-expression of α-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson's disease. In the present study, α-synuclein knockdown rats were created by injecting α-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraperitoneally with methamphetamine to establish the model of Parkinson's disease. Expression of α-synuclein mRNA and protein in the right striatum of the injected rats was significantly downregulated. Food intake and body weight were greater in α-synuclein knockdown rats, and water intake and stereotyped behavior score were lower than in model rats. Striatal dopamine and tyrosine hydroxylase levels were significantly elevated in α-synuclein knockdown rats. Moreover, superoxide dismutase activity was greater in α-synuclein knockdown rat striatum, but the levels of reactive oxygen species, malondialdehyde, nitric oxide synthase and nitrogen monoxide were lower compared with model rats. We also found that α-synuclein knockdown inhibited methamphetamine-induced neuronal apoptosis. These results suggest that α-synuclein has the capacity to reverse methamphetamine-induced apoptosis of dopaminergic neurons in the rat striatum by inhibiting oxidative stress and improving dopaminergic system function.

Insulin-like growth factor binding protein 5 (IGFBP5) mediates methamphetamine-induced dopaminergic neuron apoptosis.

Overexposure to methamphetamine (METH), a psychoactive drug, induces a variety of adverse effects to the nervous system, including apoptosis of dopaminergic neurons. Insulin-like growth factor binding protein 5 (IGFBP5), a member of insulin-like growth factor (IGF) system, is a pro-apoptotic factor that plays important roles in neuronal apoptosis. To test the hypothesis that IGFBP5 can mediate METH-induced neuronal apoptosis, we examined IGFBP5 mRNA and protein expression changes in PC12 cells exposed to METH (3.0mM) for 24h and in the striatum of rats following 15 mg/kg × 8 intraperitoneal injections of METH at 12h interval. We also checked the effect on neuronal apoptosis after silencing IGFBP5 expression with TUNEL staining and flow cytometry; Western blot was used for detecting the expression of apoptotic markers active-caspase3 and PARP. To elucidate the mechanisms underlying IGFBP5-mediated neuronal apoptosis, we determined the release of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria after METH treatment with or without IGFBP5 knockdown. Our results showed that IGFBP5 expression was increased significantly after METH exposure in PC12 cells and in the METH-treated rats' striatum. Further, METH-exposed PC12 cells exhibited higher apoptosis-positive cell number and activity of caspase3 and PARP compared with control cells, while these changes can be blocked by silencing IGFBP5 expression. In addition, a significant increase of cyto c release from mitochondria after METH exposure was observed and it was inhibited after silencing IGFBP5 expression in PC12 cells. These results indicate that IGFBP5 plays key roles in METH-induced neuronal apoptosis and may be a potential gene target for therapeutics in METH-caused neurotoxicity.

S-Nitrosylating protein disulphide isomerase mediates α-synuclein aggregation caused by methamphetamine exposure in PC12 cells.

Methamphetamine (METH) belongs to Amphetamine-type stimulants, METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). However, there are still no effective treatments to METH-induced neurodegeneration because its mechanism remains unknown. In order to investigate METH's neurotoxic mechanism, we established an in vitro PD pathology model by exposing PC12 cells to METH. We found the expression of nitric oxide synthase (NOS), nitric oxide (NO) and α-synuclein (α-syn) was significantly increased after METH treatment for 24h, in addition, the aggregattion of α-syn and the S-nitrosylation of protein disulphideisomerase(PDI) were also obviously enhanced. When we exposed PC12 cells to the NOS inhibitor N-nitro-L-arginine(L-NNA) with METH together, the L-NNA obviously inhibited these changes induced by METH. While when we exposed PC12 cells to the precursor of NO L-Arginine together with METH, the L-Arginine resulted in the opposite effect compared to L-NNA. And when we knocked down the PDI gene, the L-NNA did not have this effect. Therefore, PDI plays a significant role in neurological disorders related to α-syn aggregation, and it suggests that PDI could be as a potential target to prevent METH-induced neurodegeneration.

Inhibition of ROCK2 expression protects against methamphetamine-induced neurotoxicity in PC12 cells.

Methamphetamine is a type of psychoactive drug. It is well known that neurotoxicity caused by Methamphetamine(METH) can damage the nervous system and lead to apoptosis and cell loss of dopaminergic neurons. ROCK2 is a prominent target for gene therapy because its inhibition has proved to have a protective effect in various cell lines and pathophysiological conditions. Although several of the negative effects of METH on the dopaminergic system have been studied, the protective molecular mechanisms and the effective treatment of METH-induced apoptosis remain to be clarified. We hypothesized that ROCK2 is involved in METH-induced apoptosis. We tested our hypothesis using RT-PCR and western blotting to analyze whether silencing of ROCK2 with small interfering RNA (siROCK2) could reduce damage and apoptosis in PC12 cells after METH exposure. Increases in viability and cytomorphological changes were detected by MTT assay and bright field microscopy after pretreatment of METH-treated PC12 cells with 100 nM siROCK2. Apoptosis decreased significantly after ROCK2 silencing, as shown by Annexin V and TUNEL staining. The results show that ROCK2 is a possible gene target for therapeutics in METH-induced neurotoxicity in vitro, providing a foundation for future in vivo research.

RNA interference targeting α-synuclein attenuates methamphetamine-induced neurotoxicity in SH-SY5Y cells.

The protein α-synuclein (α-syn) is abundant in neurons and has been claimed to play critical roles in the pathophysiology of Parkinson's disease. Overexpression of α-syn has been shown to be toxicity in methamphetamine (METH)-induced model in vivo and in vitro which has Parkinson's-like pathology. However, the exact mechanisms underlying toxicity of α-syn mediated METH-induced neuron remain unknown. In the present study, human dopaminergic-like neuroblastoma SH-SY5Y cells were used as METH-induced model in vitro. Cell viability was found to be dramatically increased after silencing α-syn expression followed by METH treatment compared with a-syn wild-type cells and the morphological damage to cells after METH treatment was abated through knockdown of α-syn expression in this model. The expression levels of tyrosine hydroxylase (TH), dopamine transporter (DAT) and vesicular monoamine transporter 2(VMAT-2) were significantly decreased and the activity/levels of reactive oxygen species (ROS), nitric oxide synthase (NOS) and nitrogen (NO) were notably increased after METH treatment. However, the changes of these expression levels were reversed in cells transfected with α-syn-shRNA. These results suggested that TH, DAT, VMAT-2, ROS and NOS maybe involved in α-syn mediated METH-induced neuronal toxicity.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on nuclear factor-kappa B and cytokine expression in the intestine of septic mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the expression of nuclear factor-kappa B (NF-κB) and cytokines in the intestinal tissue of septic mice. METHODS: Male BALB/c mice were randomly divided into 4 groups, namely the sham-operated group, model group, TSP-2 treatment group and rabbit IgG treatment group. Sepsis was induced by cecal ligation and puncture (CLP), and at 6, 12 or 24 h after the operation, the ileal tissues were harvested from the mice for HE staining. NF-κB expression was detected with immunohistochemistry. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expressions were detected with qRT-PCR and their protein expressions by ELISA. RESULTS: The NF-κB expression in the intestinal tissue significantly increased in the model group as compared with that in the sham- operated group, and decreased after TSP-2 treatment. The model group also showed significantly increased expression levels of TNF-α and IL-6 mRNA and protein in the intestinal tissue (P<0.05), which were lowered by TSP-2 (P<0.05) but not by rabbit IgG treatment (P>0.05). CONCLUSION: The TSP-2 antibody can protect the intestine and delay the development of sepsis by inhibiting NF-κB activation and down-regulating TNF-α and IL-6 expressions in mice.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis. METHODS: In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80. CONCLUSION: TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.