Isoflurane attenuates dynorphin-induced cytotoxicity and downregulation of Bcl-2 expression in differentiated neuroblastoma SH-SY5Y cells

Background: It has been proposed that the volatile anesthetic isoflurane induces neuroprotection and that the endogenous opioid peptide dynorphin induces neurocytotoxicity in cells. The levels of dynorphin are often significantly elevated in neuropathophysiological conditions, and dynorphin can directly induce toxicity. However, the neuroprotective effects of isoflurane on dynorphin-induced cytotoxicity are still unclear.
Methods: In order to determine the effect of isoflurane on dynorphin-induced cytotoxicity in neuronal cells, we have designed a device wherein cultured human neuroblastoma SH-SY5Y cells can be exposed to isoflurane. Fully differentiated SH-SY5Y cells were obtained by treating the cells with retinoic acid for 6 days. We examined SH-SY5Y cell survival, apoptosis, and antiapoptotic protein expression by cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling stain, and Western blot analysis, respectively.
Results: After 16 h of dynorphin (10 μM) treatment, the SH-SY5Y cells showed significant cytotoxicity, apoptosis, and downregulation of the antiapoptotic Bcl-2 protein expression. These effects of dynorphin were significantly inhibited by isoflurane exposure for 32 h [pretreatment for 16 h and posttreatment (after dynorphin treatment) for 16 h].
Conclusion: Thus, our results suggest that isoflurane exerts neuroprotective effects in the case of dynorphin-induced pathophysiological disruption.     

Clinical significance of telomerase activity in peritoneal lavage fluid from patients with gastric cancer and its relationship with cellular proliferation

AIM: To evaluate the efficacy of telomerase activity assay and peritoneal lavage cytology (PLC) examination in peritoneal lavage fluid for the prediction of peritoneal metastasis in gastric cancer patients, and to explore the relationship between telomerase activity and proliferating cell nuclear antigen expression. METHODS: Telomeric repeated amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA) was performed to measure the telomerase activity in 60 patients with gastric cancer and 50 with peptic ulcer. PLC analysis of the 60 patients with gastric cancer was used for comparison. The proliferating cell nuclear antigen (PCNA) in gastric carcinoma was immunohistochemically examined. RESULTS: The telomerase activity and PLC positive rate in peritoneal lavage fluid from patients with gastric cancer was 41.7/ (25/60), and 25.0/ (15/60), respectively. The positive rate of telomerase activity was significantly higher than that of PLC in the group of pT4 (15/16 vs 9/16, P < 0.05), P1-3 (13/13 vs 9/13, P < 0.05) and diffuse type (22/42 vs 13/42, P < 0.05). The patients with positive telomerase activity, peritoneal metastasis, and serosal invasion had signifi cantly higher levels of average PCNA proliferation index (PI), (55.00 ± 6.59 vs 27.43 ± 7.72, 57.26 ± 10.18 vs 29.15 ± 8.31, and 49.82 ± 6.74 vs 24.65 ± 7.33, respectively, P < 0.05).CONCLUSION: The TRAP assay for telomerase activity is a useful adjunct for cytologic method in the diagnosis of peritoneal micrometastasis and well related to higher proliferating activity of gastric cancer. The results of this study also suggest a promising future therapeutic strategy for treating peritoneal dissemination based on telomerase inhibition.     

预先给予酪氨酸激酶抑制剂IPTRK3对神经源性疼痛的影响

目的探讨预先给予酪氨酸激酶(tyrosine kinase,Tr-kA)抑制剂IPTRK3对神经源性疼痛的抑制作用及其可能作用机制。方法建立小鼠坐骨神经部分结扎(partial sciaticnerve ligation,PSNL)模型,术前10 min单次腹腔给予IPTRK3 10 mg·kg-1,测定给药前后不同时间点小鼠的热痛觉过敏阈值(paw withdrawal latency,PWL)和机械痛觉过敏阈值(paw withdrawal threshold,PWT),免疫印迹法测量左侧L4-5背根神经节瞬时感受器电位香草酸受体1(transient re-ceptor potential vanilloid 1,TRPV1)的表达情况,免疫组织化学染色法测定Fos蛋白在L4-5脊髓背角的表达变化。结果与假手术(sham)组相比,PSNL组小鼠出现明显热、机械痛觉过敏(P<0.05),背根节TRPV1蛋白表达及脊髓背角Fos蛋白阳性神经元数目明显增多(P<0.05)。与PSNL组相比,预先给予IPTRK3 10 mg·kg-1明显减轻小鼠热痛觉过敏,背根神经节TRPV1蛋白表达水平及脊髓背角Fos蛋白阳性神经元数目明显降低(P<0.05),但仍明显高于sham组(P<0.05)。结论预先给予IPTRK3可以明显减轻神经源性疼痛症状,抑制背根神经节TRPV1及脊髓背角Fos蛋白的表达可能部分参与其早期迅速减轻伤害性刺激信息传递。     

High-mobility group box 1 protein activates hepatic stellate cells in vitro

Objectives

Our previous study noticed remarkably elevated titers of anti-high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis.

Methods

The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure α-smooth muscle actin (α-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs).

Results

HMGB1 at concentrations >1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. α-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment.

Conclusion

Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.     

胰腺癌单克隆抗体重链可变区基因的扩增

目的 从分泌抗体的胰腺癌杂交瘤ＹＰＣ３细胞扩增单抗重链可变区基因。方法 以一步法提取杂交瘤细胞ＲＮＡ，通过逆转录及聚合酶链式反应，反应产物以琼脂糖凝胶电泳观察。结果 扩增的基因长度为３５０ｂｐ，对照骨髓瘤细胞ＳＰ２／０未见扩增。结论该基因的成功扩增将为胰腺癌基因工程抗体的制备奠定了基础。     

Effects of morphine withdrawal on the membrane properties of medium spiny neurons in the nucleus accumbens shell

Medium spiny neurons (MSNs) in the nucleus accumbens (NAc) undergo persistent alterations in their biological and physiological characteristics upon exposure to drugs of abuse. Previous studies demonstrated that the biochemical, morphological, and intrinsic physiological properties of MSNs are heterogeneous and provided new insights into the physiological and molecular roles of individual MSNs in addictive behaviors. However, it remains unclear whether MSNs in the NAc shell (NAcSh), an important region for mediating behavioral sensitization, are electrophysiologically heterogeneous and how such heterogeneity is relevant to neuroadaptation associated with drug addiction. Here, the membrane properties, i.e., the intrinsic excitability and spike adaptation, of MSNs in the NAcSh from saline- or morphine-treated rats were investigated in vitro by whole-cell recording. In saline-treated rats, three distinct cell types were identified by their membrane properties: type I neurons showed high levels of intrinsic excitability and rapid spike adaptation; type II neurons showed moderate levels of intrinsic excitability and relatively slow spike frequency adaptation; type III neurons showed low levels of intrinsic excitability and putative strong spike adaptation. MSNs in rats undergoing withdrawal from chronic morphine treatment (10–14 days after the last injection) also exhibited the typical firing behaviors of these three types of neurons. However, the membrane properties of the MSNs were differentially altered after withdrawal. There was an enhancement in intrinsic excitability in type II MSNs and a promotion of spike adaptation in type I MSNs. The apamin-sensitive afterhyperpolarization current (I_AHP) and the apamin-insensitive I_AHP of the NAcSh MSNs were attenuated after chronic morphine withdrawal. These findings suggest that individual MSNs in the NAcSh manifest unique electrophysiological properties, which might contribute to psychostimulant-induced neuroadaptation.