Colorectal cancer is a common malignant tumor with high mortality, for which chemotherapy resistance is one of the main reasons. The high expression of ABCG2 in the cancer cells and expulsion of anticancer drugs directly cause multidrug resistance (MDR). Therefore, the development of new ABCG2 inhibitors that block the active causes of MDR may provide a strategy for the treatment of colorectal cancer. In this study, we find that dorsomorphin (also known as compound C or BML-275) potently inhibits the transporter activity of ABCG2, thereby preserving the chemotherapeutic agents mitoxantrone and doxorubicin to antagonize MDR in ABCG2-overexpressing colorectal cancer cells. Additionally, dorsomorphin does not alter ABCG2 protein expression. The results of molecular docking studies show that dorsomorphin is bound stably to the ABCG2-binding pocket, suggesting that dorsomorphin is a potent ABCG2 inhibitor that attenuates ABCG2-mediated MDR in colorectal cancer.

[Objective]To investigate the effect and mechanism of Shuxuetong and its main component hirudin on the apoptosis of cerebellar granule neurons(CGNs)in Sprague-Dawley(SD)rats.[Methods]CGNs incubated in vitro for 7 days were divided into survival control group or 25 K group(cultured in medium containing 25 mmol/L KCL)and apopto-sis group or 5 K group(cultured in medium containing 5 mmol/L KCL).CGNs were separately treated with proportionally diluted and different concentrations of Shuxuetong(1/50,1/40,1/30,1/20 and 1/10)and the corresponding different con-centrations of hirudin(2,2.5,3.34,5 and 10 U/mL).Hoechst staining was performed to analyze the apoptosis.Western blot was used to detect the expression levels of Cleaved Caspase-3,Bim and VEGF.[Results]Hoechst staining showed that 5 K group had a higher apoptosis rate than 25 K group.In 25 K group,there was no significant change in the apoptosis rate between neurons treated with different concentrations of Shuxuetong and hirudin,but significant changes was found in 5 K group and the higher the concentration,the lower the apoptosis rate.Western blot results revealed that,compared with control neurons in 5 K group,Shuxuetong injection and hirudin treatments resulted in a decrease of Cleaved Caspase-3 and Bim expression,but an increase of VEGF protein.[Conclusions]Shuxuetong and its main component hirudin inhibits the apoptosis of CGNs through suppressing proapoptotic BH3-only protein Bim.

Glioma is the most common primary malignant brain tumour, and survival is poor. Hirudin has anticancer pharmacological effects through suppression of glioma cell progression, but the molecular target and mechanism are poorly understood. In this study, we observed that hirudin dose- and time-dependently inhibited glioma invasion, migration and proliferation. Mechanistically, hirudin activated LC3-II but not Caspase-3 to induce the autophagic death of glioma cells by decreasing the phosphorylation of mTOR and its downstream substrates ULK1, P70S6K and 4EBP1. Furthermore, hirudin inhibited glioma growth and induced changes in autophagy in cell-derived xenograft (CDX) nude mice, with a decrease in mTOR activity and activation of LC3-II. Collectively, our results highlight a new anticancer mechanism of hirudin in which hirudin-induced inhibition of glioma progression through autophagy activation is likely achieved by inhibition of the mTOR signalling pathway, thus providing a molecular basis for hirudin as a potential and effective clinical drug for glioma therapy.

[目的]探究共济失调毛细血管扩张突变(ATM)激酶失活诱导神经元凋亡的具体分子机制.[方法]体外成熟7 d的小脑颗粒神经元(CGNs)分别用含25 mmol/L KCl(25 K)培养基、5 mmol/L KCl(5 K)培养基和含ATM特异性抑制剂(Ku55933,10 μmol/L;Ku60019,15 μmol/L)的25 K培养基后进行免疫印迹检测ATM、Caspase3、Cleaved Caspase-3的蛋白表达水平或培养8 h后进行Hoechst染色分析.在C6细胞和CGNs中转染ATM和GADD45α特异性siRNA,q-PCR和免疫印迹验证其干扰效率.体外成熟5 d的CGNs通过磷酸钙转染ATM特异性siRNA和pCMV-EGFP 48 h后进行Hoechst染色和凋亡分析.体外成熟7 d的CGNs用含ATM特异性抑制剂的25 K培养基培养8 h后进行全转录组测序、差异表达基因鉴定和通路富集分析.体外成熟5 d的CGNs通过磷酸钙转染GADD45α特异性siRNA和pCMV-EGFP 48 h,用含15 μmol/L Ku6的25 K培养基处理8 h或5 K培养基处理 8 h后进行Hoechst染色和凋亡分析.[结果]与25 K组相比,5 K处理组和ATM特异性抑制剂处理组的CGNs ATM蛋白表达降低、Cleaved Caspase-3蛋白表达增加、细胞核固缩率增加.C6细胞和CGNs中转染ATM和GADD45α特异性siRNA后有效降低ATM 和GADD45α 的mRNA和蛋白的表达.与对照相比,CGNs转染ATM特异性siRNA 后,细胞核固缩率升高.在Ku55933处理组中鉴定出835个基因表达上调,848个基因表达下调;在Ku60019处理组中鉴定出454个基因表达上调,314个基因表达下调;274个基因在Ku5处理组和Ku6处理组中共同上调,179个基因在Ku5处理组和Ku6处理组中共同下调,其中ATM下游靶标GADD45α表达上调;通路富集结果显示TNF信号通路、NF-κB信号通路和凋亡信号通路等被显著富集.与对照相比,抑制剂处理组和5K组GADD45α的mRNA和蛋白表达均升高.与对照相比,转染GADD45α特异性siRNA后用含15 μmol/L Ku60019的25 K培养基处理8 h或5 K培养基处理后的CGNs细胞核固缩率降低.[结论]ATM活性降低诱导GADD45α依赖的神经元凋亡.

目的 探索他喹莫德(Tasquinimod,Tasq)对胶质母细胞瘤(glioblastoma multiforme,GBM)细胞增殖、迁移、上皮-间质转化(epithelial-mesenchymal transition,EMT)、干细胞特性等恶性表型的影响及相关机制,为开发Tasq作为潜在治疗GBM的药物提供研究基础.方法 通过CCK-8法、细胞凋亡染色、Transwell实验、克隆形成实验评价Tasq对不同GBM细胞系(U87、U251)增殖、凋亡、迁移和集落形成的影响;Western blot检测凋亡、EMT等相关蛋白的表达情况.利用肿瘤干细胞成球实验检测Tasq对胶质瘤干细胞(glioma stem cell,GSC)球体形成能力的影响,免疫荧光染色和Western blot检测GSC中干细胞特性相关蛋白的表达情况.结果 Tasq呈浓度依赖性和时间依赖性抑制U87和U251的增殖;Tasq能抑制U87和U251迁移和集落形成,诱导其发生凋亡;Tasq促进凋亡蛋白Cleaved Caspase-3表达的同时下调抗凋亡蛋白BCL-2和EMT相关蛋白(Snail、Vimentin)的表达;Tasq能显著降低GSC的成球能力及干细胞特性相关蛋白(SOX2、Nestin)的表达,与对照组相比差异均有统计学意义(P<0.01).结论 Tasq能在体外抑制GBM细胞增殖、迁移、集落形成,诱导其凋亡,Tasq可能通过抑制GBM细胞的EMT和干细胞特性抑制其恶性进展.

Background: P2X 4 receptor (P2X 4 R)-mediated spinal microglial activation makes a critical contribution to pathologically enhanced pain processing in the dorsal horn. It can be upregulated under conditions of neuropathic pain. However, the specific mechanism of pathogenesis and potential molecular targets has not yet been made explicit. MicroRNAs (miRNAs) are commonly recognized as indicators in neuropathic pain pathophysiology. Methods: We established the pain model of spared nerve injury (SNI), and the 50% paw withdrawal thresholds (PWMTs) were used to assess behavior of mouse. MiRNA expression profiling was performed to detect differential expressed miRNA. The western-bolt and quantitative real time PCR to examine P2X4R and miRNA expression in the mouse. Dual-luciferase reporter assays confirmed the correlation between P2X4R and miRNA. Fluorescence in situ hybridization was used to show location between P2X4R and miRNA. Results: In the present study, we found that P2X 4 R was up-regulated in the spinal dorsal horn of mice following spared nerve injury (SNI), and we identified 69 miRNAs (46 up-regulated and 23 down-regulated miRNAs) were differently expressed (fold change > 2, P < 0.05). P2X 4 R was a major target of miR-106b-5p (one of down-regulated miRNAs in SNI) with bioinformatics technology and quantitative real time PCR analysis validated the expressed change of miR-106b-5p, and dual-luciferase reporter assays confirmed the correlation between them. Fluorescence in situ hybridization showed that miR-106b-5p was co-localized with P2X 4 R in the spinal cord. Transfection with miR-106b-5p mimic on BV2 cells reversed the up-regulation of P2X 4 R induced by LPS. Moreover, miR-106b-5p overexpression significantly attenuated neuropathic pain induced by SNI, with decreased expression of P2X 4 R mRNA and protein in the spinal cord. Conclusion: Taken together, our results suggest that miR-106b-5p can serve as an important regulator of neuropathic pain development by targeting P2X 4 R.

[目的]探讨组蛋白去乙酰化酶抑制剂(HDACIs)抑制神经胶质瘤细胞增殖的机制.[方法]体外培养U251和H4细胞,用组蛋白去乙酰化酶抑制剂:包括帕比司他(LBH589)和M344、伏立诺他(SAHA)处理后,MTT比色法检测细胞存活率,流式细胞术检测细胞周期进程改变,Western blotting和RT-qPCR法分别检测minichromo-some maintenance(MCM)蛋白家族成员 MCM2、MCM3、MCM4、MCM5、MCM6 和 MCM7 的 mRNA和蛋白表达水平.用组蛋白去乙酰化酶抑制剂:包括帕比司他和曲古霉素(TSA)处理U251细胞后,BrdU掺入实验检测细胞增殖和DNA复制能力.用MCM抑制剂环丙沙星(CPX)处理U251和H4细胞,MTT法检测细胞存活率,流式细胞术检测细胞周期进程改变.[结果]与对照组相比,HDACIs降低了U251和H4活细胞数量(P＜0.05);HDACIs组BrdU的掺入率降低(P＜0.05);HDACIs减少细胞周期S期的比率(P＜0.05);Western blotting和qPCR结果显示,HDACIs降低MCM2-7 mRNA和蛋白表达水平(P＜0.05);与对照组相比,CPX降低了U251和H4活细胞数量(P＜0.05),减少细胞周期S期的比率(P＜0.05).[结论]HDACIs通过下调MCM2-7表达抑制神经胶质瘤细胞增殖.

[目的]探讨荜茇酰胺(PPLGM)对口腔癌存活的影响及机制.[方法]体外培养口腔鳞状细胞癌Cal27及UM1细胞株,经不同浓度的荜茇酰胺处理后,MTT比色法检测荜茇酰胺对增殖的影响,蛋白质印迹法检测胞内微管相关蛋白轻链3-I、3-Ⅱ(LC3-I、LC3-Ⅱ)、Beclin1、p62、雷帕霉素靶蛋白(mTOR)及ribosomal S6(S6)的表达或活性变化;用自噬抑制剂3-Methyladenine(3-MA)或者Bafilomycin A1(BAFA1)和荜茇酰胺共处理细胞后,检测LC3-Ⅱ、p62表达和细胞活力.[结果]与对照组比较,荜茇酰胺显著抑制Cal27及UM1细胞株增殖能力,呈浓度及时间依赖性(P<0.05);Western blot结果显示,不同浓度(1.0、3.0和5.0μmol/L)荜茇酰胺处理细胞均使LC-I、LC3-Ⅱ表达水平显著高于对照组(P<0.05),而Beclin1和p62表达低于对照组(P<0.05);荜茇酰胺处理组中p-mTOR和p-S6磷酸化水平显著低于对照组(P<0.05);与单独荜茇酰胺处理比较,3-MA和荜茇酰胺共处理使胞内LC3-I和LC3-Ⅱ表达水平明显下降,p62表达升高(P<0.05),但BAFA1和荜茇酰胺共处理使细胞内p62表达升高(P<0.05),LC3-Ⅱ表达水平无明显改变;3-MA和B AFA1处理都能拮抗荜茇酰胺对细胞活力的抑制效应.[结论]荜茇酰胺通过抑制mTOR/S6通路活性诱导口腔鳞癌细胞发生自噬,进而抑制增殖.

BACKGROUND AND OBJECTIVE:We aimed to report the pathological features of T lymphocytes in autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy (GFAP-A).METHODS:A retrospective pathological analysis of patients with GFAP-A was performed.RESULTS:Eight patients with GFAP-immunoglobulin G (IgG) and pathological data were included. Their biopsy findings were similar, and all showed marked lymphocytic infiltration in the white matter, with perivascular predominance. The lymphocytic infiltration was predominantly composed of CD8+ T lymphocytes rather than CD4+ T lymphocytes, except in one patient who had overlapping positive myelin oligodendrocyte glycoprotein-IgG. Unlike CD4+ T cells, CD8+ T cells were frequently observed adjacent to dystrophic neurons and astrocytes. There was also diffuse infiltration by CD68+ and CD163+ macrophages. CD8+ astrocytes were identified in two samples, but no CD4+ astrocytes were observed.CONCLUSIONS:A predominance of CD8+ T cells may be an important pathological and diagnostic feature in GFAP-A.

Histone deacetylase 6 (HDAC6) activity contributes to the malignant proliferation, invasion, and migration of glioma cells (GCs), but the molecular mechanisms underlying the processes remains elusive. Here, we reported that HDAC6 inhibition by Ricolinostat (ACY-1215) or CAY10603 led to a remarkable decrease in the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, which preceded its suppressive effects on glioma cell growth. Further investigation showed that these effects resulted from HDAC6 inhibitor-induced suppression of MAPK kinase 7 (MKK7), which was identified to be critical for JNK activation and exerts the oncogenic roles in GCs. Selectively silencing HDAC6 by siRNAs had the same responses, whereas transient transfections expressing HDAC6 promoted MKK7 expression. Interestingly, by performing Q-PCR, HDAC6 inhibition did not cause a down-regulation of MKK7 mRNA level, whereas the suppressive effects on MKK7 protein can be efficiently blocked by the proteasomal inhibitor MG132. As a further test, elevating MKK7-JNK activity was sufficient to rescue HDAC6 inhibitor-mediated-suppressive effects on c-Jun activation and the malignant features. The suppression of both MKK7 expression and JNK/c-Jun activities was involved in the tumor-growth inhibitory effects induced by CAY10603 in U87-xenograft mice. Collectively, our findings provide new insights into the molecular mechanism of glioma malignancy regarding HDAC6 in the selective regulation of MKK7 expression and JNK/c-Jun activity. MKK7 protein stability critically depends on HDAC6 activity, and inhibition of HDAC6 probably presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c-Jun axis in GCs.

[目的]探讨口腔鳞状细胞癌(OSCC)分化程度与Tip60(KAT5)表达相关性.[方法]选取口腔鳞状细胞共49例及癌旁正常口腔组织36例,采用免疫组化方法检测Tip60(KAT5)的表达状态.体外培养口腔鳞状细胞癌Cal27及UM1细胞株,Tip60(KAT5)抑制剂Nu9056处理Cal27及UM1细胞株后,MTT比色法检测Nu9056对Cal27及UM1细胞株增殖的影响.[结果]口腔鳞状细胞癌中Tip60(KAT5)的表达均明显高于癌旁正常组织中的表达(P=0.000);随着组织学分化程度的降低,Tip60(KAT5)的表达增高,且其表达与组织学分化程度的降低呈明显正相关(r=0.461,P=0.001);随着浓度及时间的增加,Nu9056对Cal27及UM1细胞株的增殖抑制率逐渐增加,呈浓度及时间依赖关系(P<0.05).[结论]Tip60(KAT5)蛋白在口腔鳞状细胞癌组织中表达上调,且与其的组织学分化程度相关,Tip60(KAT5)可作为预测口腔鳞状细胞癌进展和预后的生物学指标.

目的 探讨建立C57BL/6J小鼠急性前庭损伤动物模型的方法及其行为学评估.方法 健康成年雄性C57BL/6J小鼠(20～30g)共21只,随机分成单侧迷路切除术组(n=15)及假手术组(n=6)两组.单侧迷路切除术组小鼠,在麻醉后在显微镜下进行其左耳的单侧迷路切除术(UL),行耳前切口,钝性分离组织,勾除鼓膜、锤骨、砧骨,电凝镫骨动脉,勾除镫骨,打开镫骨底板,暴露前庭池,用吸引器吸除流出的内淋巴液,用精细的勾针分离椭圆囊及球囊,并用勾针破坏该区域剩余的前庭神经上皮细胞及半规管,最后往前庭内塞人含有无水乙醇的明胶海绵,缝合皮肤.而假手术组小鼠,通过耳前切口打开其外耳道,缝合皮肤.对所有术后小鼠进行行为学观察,并在术后第10天进行爬杆试验、平衡木试验的行为学检测.结果 所有单侧迷路切除术后小鼠在清醒后均开始出现一种特殊的偏斜姿势,头右偏,走路时向右转圈,身体筒状翻滚,提尾时出现以身体为轴线的旋转.以上体征在一周内逐渐缓解.术后第10天,手术组跟假手术组相比,爬杆试验(P=0.000)及平衡术试验(P=0.002)的结果均具有统计学差异(P＜均0.01).结论 使用单侧迷路切除术(UL)配合化学切除的方法能建立稳定、有效、造模成功率高的急性前庭损伤小鼠模型.在术后一周时,可观察到小鼠的部分静态症状明显缓解.而行为学检测结果提示在术后第10天,前庭损伤小鼠模型尚未完全完成前庭代偿.

The latest perspective indicates that apoptotic dysregulation is an important mechanism in male infertility induced by varicocele. To elucidate the molecular mechanism of apoptosis caused by varicocele, we used proteomics (2D-MALDI-TOF MS) to identify the altered proteins in the testes of experimental varicocele rats compared with the control. Here, 21 significantly different protein spots were detected by proteomics technology. 14-3-3 epsilon (14-3-3ε) was our subsequent research target because of its function in apoptosis. The expression of 14-3-3ε in rat testes was confirmed by Western blot and immunohistochemistry, and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method was used to analyse the apoptosis of germ cells. GC-1 spg cells transfected with small interfering RNA were used to confirm the function of 14-3-3ε in vitro. 14-3-3ε protein expression decreased, accompanied by a higher apoptosis index in rat testes of the varicocele group. Furthermore, 14-3-3ε siRNA-treated GC-1 spg cells caused the upregulation of the apoptotic rate detected by flow cytometry. The expression of Bax and Bcl-2 was found to be regulated by 14-3-3ε in vitro. Our investigation demonstrated the pro-apoptotic function of the downregulation of 14-3-3ε, which may play an important role in germ cell apoptosis induced by varicocele.

Objective To investigate the expression of phospho-c-Jun and MKK7 in gliomas and their correlation. Methods We collected and analyzed retrospectively 92 cases of gliomas, including 15 cases of diffuse astrocytoma, 5 cases of oligodendroglioma, 11 cases of anaplastic astrocytoma, 8 cases of anaplastic oligodendroglioma 53 cases of glioblastoma and 25 normal brain tissues adjacent to glioblastoma. The expression of c-Jun, phospho-c-Jun and MKK7 were detected by immunohistochemical staining. Glioma U87 cell line cultured in vitro was transfected with MKK4-siRNA, MKK7-siRNA and control siRNA for 48h, respectively. Western blot was performed to test the expression of c-Jun, phospho-c-Jun and MKK7. Results The expression of p-c-Jun and MKK7 in glioblastoma were significantly higher than those in other glioma and normal brain tissues adjacent to glioblastomas(P=0.000, P=0.000). The expression of MKK7 and phospho-c-Jun were positively correlated with WHO grading of gliomas (r＝0.494, P＝0.000; r=0.606, P=0.000). There was positive correlation between the expression of MKK7 and p-c-Jun (r=0.387, P=0.000). The knockdown of MKK7 suppressed c-Jun activities. Conclusion MKK7 promotes the development of glioblastoma by regulating the activity of c-Jun.

目的 观察在SD大鼠蛛网膜下腔出血动物模型中,抑制HDAC4对其早期脑损伤的影响.方法 40只SD大鼠随机分为空白注射组和药物注射组各20只,于SAH造模前24 h分别行侧脑室立体定位注射给药HDAC4抑制剂LMK235,在术前12 h、 术后12 h和术后24 h进行神经生物学评分,在术后24～36 h行灌注,取出脑组织的额叶和颞叶皮层,利用Western Blot检测额颞叶组织中H3及Ac-H4K5的表达来反映脑组织的乙酰化水平.结果 在14例成功SAH造模的SD大鼠动物模型中(对照组8只,给药组6只),行神经功能缺损评分后进行统计,给药组的分值低于对照组;额叶及颞叶的皮层蛋白经Western Blot检测及分析,给药组的Ac-H4K5较Vehicle组增高,提示组蛋白的乙酰化水平增高;相关性分析提示组蛋白的乙酰化水平与其神经缺损评分分值的大小呈负相关.结论 在SD大鼠蛛网膜下腔出血的早期脑损伤动物模型中,抑制HDAC4可改善其神经行为.

[目的]研究抑制共济失调毛细血管扩张突变(ATM)蛋白对SD大鼠小脑颗粒神经元(CGN)凋亡的影响及机制.[方法]将原代培养7～8 d的CGN分为25 K组、5K组和ATM抑制组.分别应用含有25和5 mmol/LKCl的培养基处理25 K组和5K组,建立体外神经元存活和凋亡模型.在25 K神经元存活模型中,应用ATM抑制剂KU-55933(10 μmol/L,ATM抑制组)、KU-55933联合MithramycinA(1μmol/L,MMA组)或Chromomycin A3(0.3 μmol/L,CMA3组)处理,用Western Blot方法检测p-ATM、ATM、Bim、Caspase 3的表达水平,用Hoechst染色法观察并统计核固缩情况.[结果]Western Blot结果显示,与25 K组比较,5K组和ATM抑制组的Bim、Caspase 3蛋白表达均上调(P＜0.05).核染色结果显示,与25K组比较,5K组和ATM抑制组核固缩率均明显增多(P＜0.05).与ATM抑制组比较,MMA组和CMA3组中Bim表达、Caspase3活性及核固缩率均明显减少.[结论]抑制ATM上调Bim活性,促进小脑颗粒神经元凋亡.

The c-Jun N-terminal kinase (JNK)/c-Jun cascade-dependent neuronal apoptosis has been identified as a central element for early brain injury (EBI) following subarachnoid hemorrhage (SAH), but the molecular mechanisms underlying this process are still thoroughly undefined to date. In this study, we found that pan-histone deacetylase (HDAC) inhibition by TSA, SAHA, VPA, and M344 led to a remarkable decrease in the phosphorylation of JNK and c-Jun, concomitant with a significant abrogation of apoptosis caused by potassium deprivation in cultured cerebellar granule neurons (CGNs). Further investigation showed that these effects resulted from HDAC inhibition-induced transcriptional suppression of MKK7, a well-known upstream kinase of JNK. Using small interference RNAs (siRNAs) to silence the respective HDAC members, HDAC4 was screened to be required for MKK7 transcription and JNK/c-Jun activation. LMK235, a specific HDAC4 inhibitor, dose-dependently suppressed MKK7 transcription and JNK/c-Jun activity. Functionally, HDAC4 inhibition via knockdown or LMK235 significantly rescued CGN apoptosis induced by potassium deprivation. Moreover, administration of LMK235 remarkably ameliorated the EBI process in SAH rats, associated with an obvious reduction in MKK7 transcription, JNK/c-Jun activity, and neuronal apoptosis. Collectively, the findings provide new insights into the molecular mechanism of neuronal apoptosis regarding HDAC4 in the selective regulation of MKK7 transcription and JNK/c-Jun activity. HDAC4 inhibition could be a potential alternative to prevent MKK7/JNK/c-Jun axis-mediated nervous disorders, including SAH-caused EBI.

JNK activity has been implicated in the malignant proliferation, invasion and drug‐resistance of glioma cells (GCs), but the molecular mechanisms underlying JNK activation are currently unknown. Here, we reported that MKK7, not MKK4, directly activates JNK in GCs and exerts oncogenic effects on tumor formation. Notably, MKK7 expression in glioma tissues was closely correlated with the grade of the glioma and JNK/c‐Jun activation. Mechanistically, MKK7 transcription critically depends on the complexes formed by HDAC4 and the transcriptional factors SP1 and Krüppel‐like factor‐5 (KLF5), wherein HDAC4 directly deacetylates both SP1 and KLF5 and synergistically upregulates MKK7 transcription through two SP1 sites located on its promoter. In contrast, the increases in acetylated‐SP1 and acetylated‐KLF5 after HDAC4 inhibition switched to transcriptionally suppress MKK7. Selective inhibition of HDAC4 by LMK235, siRNAs or blockage of SP1 and KLF5 by the ectopic dominant‐negative SP1 greatly reduced the malignant capacity of GCs. Furthermore, suppression of both MKK7 expression and JNK/c‐Jun activities was involved in the tumor‐growth inhibitory effects induced by LMK235 in U87‐xenograft mice. Interestingly, HDAC4 is highly expressed in glioma tissues, and the rate of HDAC4 nuclear import is closely correlated with glioma grade, as well as with MKK7 expression. Collectively, these findings demonstrated that highly expressed MKK7 contributes to JNK/c‐Jun signaling‐mediated glioma formation. MKK7 transcription, regulated by SP1 and KLF5, critically depends on HDAC4 activity, and inhibition of HDAC4 presents a potential strategy for suppressing the oncogenic roles of MKK7/JNK/c‐Jun signaling in GCs.

[目的]研究抑制组蛋白去乙酰化酶成员HDAC4对SD大鼠小脑颗粒神经元(CGNs)凋亡的影响及机制.[方法]将原代培养6～9d的CGN分为存活对照组(用含K+浓度为25 mmol/L的培养基,即25 K组)、凋亡组(用含K+浓度为5 mmol/L的培养基,即5K组)和LMK-235处理组(5K合并LMK-235处理组),在5K条件下用不同浓度HDAC4抑制剂LMK-235处理细胞.在转染实验中,将CGN分为25 K+siNC组、5 K+siNC组、5 K+siH-DAC4-1组和5 K+siHDAC4-2组,共转染GFP标记目的细胞.用Western blot法检测p-JNK、JNK、p-c-Jun、c-Jun 的表达水平,用Hoeehst染色法观察并统计核固缩情况,用免疫荧光检测c-Jun磷酸化水平.[结果]Western blot结果显示,与5K组比较,LMK-235处理细胞后JNK、c-Jun磷酸化水平下降.核染色结果显示,与25 K组比较,5K组核固缩率明显增多(P＜0.05);与5K组比较,LMK-235处理细胞后核固缩率明显减少(P＜0.05).与5K+siNC组比较,小分子干扰HDAC4后,HDAC4的表达下调,c-Jun的磷酸化水平降低,CGNs核固缩率明显减少(P＜0.05).[结论]抑制HDAC4通过下调JNK/c-Jun活性使神经元核固缩率减少,从而抑制神经元凋亡.

Objective To study the effect of artificial cold wave on endothelin (ET) and calcitonin gene-related peptide (CGRP) in stroke-prone rats with renovascular hypertension.Methods Seventy-two male SD rats were divided into stroke-prone renovascular hypertension group as a model group (n=36) and control group (n=36).The animals in two groups were exposed to artificial cold wave.Brain tissue samples were taken brfore exposure to artificial cold wave,at 3,6 and 12 h during exposure to artificial cold wave,at 12 and 24 h after exposure to artificial cold wave respectively and were then cut into 1.5 mm-thick sections.The expressions of ET,CGRP and ET mRNA were detected by in situ hybridization.Results The expression level of ET and ET mRNA was significantly higher in model group than in control group before exposure to cold wave,at 3,6,12 h during exposure to cold wave,at 12 and 24 h after exposure to cold wave while that of CGRP was significantly lower in model group than in control group before exposure to cold wave and at 24 h after exposure to cold wave (P＜0.05).The expression level of ET and CGRP was significantly higher in model group at 3 and 6 h after exposure to cold wave (P＜0.05).The expression level of ET mRNA was significantly higher in model group at 3,6 and 12 h after exposure to cold wave than before exposure to cold wave (28.00±6.02,27.06±5.46,26.40±4.60 vs 22.81± 4.35,P＜0.05).Conclusion Elevated expression of ET and CGRP in stroke-prone rats with renovascular hypertension is related with stroke when they are exposed to cold wave.