Signal transduction mediated by Bid,a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro- apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.

Regulatory Effect of Bcl-2 Family Proteins in CPB-induced Cardiomyocyte Apoptosis in Dog Hearts

Summary: Whether conventional hypothermic CPB induces myocyte apoptosis in dog hearts and modulation of bcl-2, bcl-xl, bax, bad, and caspase-3 pathways in this setting was investigated. Ten healthy adult dogs were randomized into sham-operated and CPB groups. Samples of left ventricle were obtained before, during and 3 h after CPB. In situ TUNEL was used to detect apoptotic myocytes. Immunohistochemistry and flow cytometry were employed for detection of expressions of bcl-2, bcl-xl, bax and bad proteins. Z-DEVD-AMC substrate cleavage and TBARS methods were used to measure the activity of caspase-3 and the content of lipid peroxide in LV myocardium, respectively. After CPB, the number of apoptotic myocytes in CPB group was significantly increased. The results of immunohistichemistry demonstrated that bcl-2, bcl-xl, bax and bad proteins were constitutionally present on the sarcolemma of the LV myocytes. FACS results showed that, after CPB, expressions of bax and bad in CPB group were significantly upregulated, while the expressions of bcl-2 and bcl-xl were not significantly changed in both groups. The activity of caspase-3 and the content of lipid peroxide in LV myocardium in CPB group were also significantly increased after CPB. The present study shows that there exists myocardiocyte apoptosis in dog hearts undergoing conventional hypothermic CPB and the myocyte apoptosis is initiated by ischemia and performed during reperfusion. Moreover, the CPB-induced myocyte apoptosis was associated with upregulation of expressions of bax and bad proteins, activation of caspase-3 and increase of oxidative stress.

Dioscin-induced Apoptosis of Human LNCaP Prostate Carcinoma Cells through Activation of Caspase-3 and Modulation of Bcl-2 Protein Family

Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin（1, 2 and 4 μmol/L） could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.

低剂量CT结合SHOX2、RASSF1A甲基化在肺癌早期预警中的应用

目的探讨低剂量CT结合Ras相关区域家族蛋白1A(RASSF1A)、矮小同源盒基因2(SHOX2)甲基化在肺癌早期预测中的应用价值。方法选取2021年1月~2023年1月我院90例拟行肺结节手术患者,根据手术病理学分为肺良性结节组和肺癌组。2组均于术前行低剂量CT检查、SHOX2、RASSF1A甲基化检测,采用Kappa指数分析上述检查结果与手术病理学一致性,分析低剂量CT、SHOX2、RASSF1A甲基化与血清肿瘤标志物[癌胚抗原(CEA)、神经元特异性烯醇化酶(NSE)、鳞状细胞癌抗原(SCC-Ag)、细胞角蛋白19片段(CYFRA21)]对肺癌诊断效能,采用Spearman低剂量CT检查、SHOX2、RASSF1A甲基化与临床病理特征相关性。结果低剂量CT、SHOX2、RASSF1甲基化及三者联合分别确定40例、43例、46例、58例肺癌,三者联合与手术病理学诊断肺癌效能一致性Kappa值为0.951;三者联合诊断肺癌敏感度96.67%、准确度97.78%均高于三者单一诊断效能(P<0.05);肺癌患者血清CEA、SCC、NSE、CYFRA21水平均高于肺良性结节患者(P<0.05);低剂量CT联合SHOX2、RASSF1甲基化诊断肺癌效能的AUC为0.983,近似于四种血清肿瘤标志物诊断肺癌效能的AUC 0.933;不同肿瘤直径、临床分期、组织学分化肺癌患者低剂量CT检出率及SHOX2、RASSF1A甲基化阳性率比较差异有统计学意义(P<0.05);肺癌患者低剂量CT检出率、SHOX2及RASSF1A甲基化阳性率与肿瘤直径、临床分期呈正相关,与组织学分化呈负相关(P<0.05)。结论低剂量CT联合SHOX2及RASSF1A甲基化可用于肺癌早期预警中,临床可通过其进行早期诊断、评估病情进展程度,以针对性展开后续治疗,改善预后。

凋亡蛋白对Bcl-2家族几个基因表达影响

凋亡蛋白(Apoptin)只诱导肿瘤细胞或转化的细胞凋亡,但其作用机制还不十分清楚.从鸡贫血病毒总DNA中克隆出凋亡蛋白基因,构建pcDNA3.1/CT-Apoptin-GFP-TOPO融合表达的载体.用该载体转染HHCC单层传代细胞24 h后,观察到绿色荧光蛋白表达,同时观察到细胞形态呈凋亡特征变化.提取细胞总RNA,按SuperArray芯片操作方法检测到实验组Bcl-2家族中的BimL,Bcl-2,Bax和Bak基因上调表达.Bcl-2家族在细胞凋亡的过程中作用重要.凋亡蛋白诱导HHCC细胞凋亡过程中,促凋亡的BimL,Bax 和Bak基因上调,抗凋亡的Bcl-2基因也上调.可能BimL与Bcl-2竞争性结合导致Bax或Bak释放,最终诱导细胞凋亡.揭示Bcl-2家族的基因参与凋亡蛋白诱导的HHCC细胞凋亡过程.

肿瘤免疫治疗中Bcl-2家族蛋白与凋亡蛋白抑制因子IAP研究

阐述了T细胞抗原的研究结果,主要涉及到B细胞淋巴瘤/白血病-2基因(即Bcl-2)家族蛋白及凋亡蛋白抑制因子(即IAP)。

The Experimental and Clinical Study on the Effect of Curcumin on Cell Cycle Proteins and Regulating Proteins of Apoptosis in Acute Myelogenous Leukemia

To investigate whether the Bcl- 2 gene family is involved in m odulating mechanism of apoptosis and change of cell cycle protein induced by curcumin in acute myeloid leukemia HL - 6 0 cell line and primary acute m yelogenous leukem ic cells,the Bcl- 2 family member Mcl- 1,Bax and Bak and cell cycle proteins including P2 7kipl,P2 1wafl,cyclin D3and p Rbp- were selected and their ex- pression detected by SABC imm uno- histochem ical stain m ethod.The attitude of sub- G1 peak in DNA histogram was determined by FCM.The TU NEL positive cell percentage was identified by term inal deoxynucleotidyl transferase (Td T ) - m ediated Biotin d U NP end labeling technique.It was found that when HL - 6 0 cells were treated with 2 5μm ol/ L curcumin for 2 4 h,the expression level of Mcl- 1was down- regulated,but that of Bax and Bak up- regulated time- dependently.There was significant difference in the expression level of Mcl- 1,Bax and Bak between the curcumin- treated groups and control group(P<0 .0 5 - 0 .0 1) .At the sam e time,curcumin had no effect on progress of cell cycle in prim aty acute m yelogenous leukemia at newly diagnosis,but could in- crease the peak of Sub- G1 (P<0 .0 5 ) ,and down- regulate the expression of Mcl- 1and up- regulate the expression of Bax and Bak with the difference being statistically significant.The expression of P2 7kipl,P2 1wafl and p Rbp- were elevated and thatof cyclin D3decreased in the presence of curcum in. These findings suggested thatthe Bcl- 2 gene fam ily indeed participated in the regulatory process of apoptosis induced by curcumin in HL - 6 0 cells and AML cells.Curcumin can induce apoptosis of primary acute myelogenous leukemic cells and disturb cell cycle progression of HL - 6 0 cells.The m echanism appeared to be m ediated by perturbing G0 / G1 phases checkpoints which associated with up- regulation of P2 7kipl,P2 1wafl and p Rbp- expression,and down- regulation of cyclin D3.

5-AZA-CdR联合EGCG对HL-60和K562的影响

目的探讨细胞因子INFα和甲基化抑制剂5-杂氮脱氧胞嘧啶核苷(5-AZA-CdR)能否诱导HL-60和K562 Xaf1表达,以及Xaf1表达诱导剂联合表没食子儿茶素没食子酸酯(EGCG)是否具有协同抗癌作用。方法(1)1000U/mlINFα和不同剂量的5-AZA-CdR作用于HL-60和K56248h,通过RT-PCR检测Xaf1和XIAP mRNA的表达并比较差异。(2)最佳Xaf1表达诱导剂联合EGCG作用于白血病细胞,流式细胞技术检测Bcl-2家族成员、线粒体膜电位和细胞凋亡的情况。结果(1)随5-AZA-CdR剂量增加,HL-60和K562Xaf1 mRNA表达增加,INFα也能诱导Xaf1表达,以5-AZA-CdR(5μmol/L)的作用最强;INFα和5-AZA-CdR均不影响XIAP mRNA的表达。(2)5-AZA-CdR和EGCG可改变HL-60和K562细胞Bcl-2(Bcl-xl)和Bax的表达,降低线粒体膜电位,诱导细胞凋亡,二者联合作用被加强。结论(1)INFα和5-AZA-CdR能诱导HL-60和K562Xaf1 mRNA的表达,且5-AZA-CdR的作用具有剂量依赖性。(2)5-AZA-CdR和EGCG在体外能诱导白血病细胞凋亡,并具有协同效应。

隔药饼灸调节大鼠免疫抑制机制的转录组测序分析

背景:免疫抑制会导致机体免疫功能受损,加重病情。隔药饼灸能有效调节机体免疫功能,提高机体免疫力,但其调节机制目前仍不清楚。目的:基于转录组学的生物信息学技术对隔药饼灸干预的免疫抑制模型大鼠进行测序,探究隔药饼灸调控免疫的机制。方法:将24只SD大鼠随机分为空白组、模型组和隔药饼灸组,每组8只,模型组与隔药饼灸组采用腹腔注射环磷酰胺制备免疫抑制模型,35 mg/kg,连续3 d,造模结束后空白组与模型组不做干预,隔药饼灸组在大鼠中脘、神阙、关元及足三里穴位进行艾炷与药饼结合的隔药饼灸干预,连续10 d,1次/d,干预结束后次日取材。取各组大鼠外周血进行白细胞数量检测;采用Illumina测序平台进行RNA-seq,筛选差异基因,结合GO与KEGG数据库,对差异基因进行生物信息学相关分析。结果与结论:①与空白组比较,模型组白细胞数量显著减少(P<0.001)。②RNA-seq共筛选出模型组与空白组间差异基因3026个,其中1565个上调、1461个下调,隔药饼灸组与模型组间差异基因535个,其中280个上调、255个下调。③韦恩图分析获得模型组与空白组159个下调基因经隔药饼灸干预后上调,蛋白互作网络分析获得Oasl,Oas2,Isg15,Herc6,Mx2,Helz2,Mx1,Syk,Hspa1a及Ret等10个核心靶点;④GO与KEGG分析隔药饼灸调节机体的机制有免疫应答途径、病毒相关、血管新生和自身免疫系统等通路。⑤上述结果证实,实验筛选出隔药饼灸干预免疫抑制与Oasl,Oas2,Isg15,Herc6,Mx2,Helz2及Mx1靶点有明显关联,并且在免疫应答、病毒相关及血管新生等通路中发挥调控作用。

Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G_(2)/M accumulation in cardiac fibroblasts

Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψ_m) reduction, indicative of mitochondrial permeability transition pore (PTP) opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψ_m reduction and apoptosis, suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψ_m reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B1 was downregulated only in cardiac fibroblasts, which may be associated with G_2/M accumulation in response to mechanical stretch.

Apoptosis of human colon carcinoma HT-29 cells induced by ceramide

AIM： To investigate the effect of exogenous ceramideinduced apoptosis on human colon carcinoma HT-29 cells. METHODS： Light microscope, transmission electron microscope and fluorescence microscope were used to observe the morphology change of apoptosis in HT-29 cells. Agarose gel electrophoresis was performed to detect the DNA fragment. Mitochondrial function was detected by MTT assay, mRNA expression of Bcl-2 family gene members was determined by reverse transcription polymerase chain reaction （RT-PCR） assay. RESULTS： After C2-ceramide treatment, typical characteristics of apoptosis, such as nuclear chromatin breakage, apoptotic body and DNA ladder, could be observed. After exposure to 50μmol/L C2-ceramide for 12 and 24 h, cell apoptosis was 64.1% and 81.3% respectively, which had a time-and dose-effect relationship. Mitochondrial function started to decrease from 6 h after exposure to ceramide. Meanwhile, ceramide up-regulated or down-regulated the mRNA expression of Bcl-2 family gene members. CONCLUSION： Ceramide induces apoptosis of human colon carcinoma HT-29 cells by affecting the expression of Bcl-2 family gene members and impacting the mitochondrial function.

高通量测序技术检测乳腺癌患者BRCA1、BRCA2基因的意义

目的采用高通量测序技术检测乳腺癌易感基因(BRCA)1和BRCA2,并探讨BRCA1、BRCA2在家族性乳腺癌筛查中的意义。方法选取7例女性乳腺癌患者及12名健康女性,采用高通量测序技术对BRCA1、BRCA2基因进行测序分析,用Sanger测序法验证检出的位点并对新发BRCA1基因突变位点携带者的家庭成员进行检测。结果7例乳腺癌患者中,检测出1例致病性突变BRCA2(c.5073dupA),1例可能致病的突变BRCA1(c.3343G>T)及1例临床未明意义的突变BRCA2(c.1211A>T);12名健康女性中均未检测出BRCA1、BRCA2基因的可疑致病突变位点;携带BRCA1(c.3343G>T)突变的家系中有2名乳腺癌患者。结论BRCA1(c.3343G>T)是首次发现的遗传性乳腺癌的可能致病性变异位点,其携带者家系中乳腺癌发病率明显升高,建议对其他携带者加强随访,尽早进行手术或药物干预。

Ndfip2表达对肝癌细胞增殖和凋亡的影响及机制

目的探究Nedd4家族交互作用蛋白2(Ndfip2)对人肝癌细胞SMMC-7721和HepG2增殖和凋亡能力的影响。方法选择正常肝细胞LO2、肝癌细胞SMMC-7721和HepG2进行培养,采用蛋白印迹法检测3组细胞中的Ndfip2的蛋白表达;对培养的SMMC-7721和HepG2细胞转染靶向Ndfip2小干扰RNA(siRNA)构建Ndfip2基因沉默组(si-Ndifip2组),转染无义siRNA序列构建阴性对照组(NC组),采用蛋白印迹检测两组细胞的转染效率,CCK-8、平板克隆形成和流式细胞术检测两组细胞的增殖率和凋亡率,蛋白免疫印迹检测相关细胞内磷酸酶及张力蛋白同源的基因(PTEN)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、磷酸化Akt(Ser473)[p-Akt(S473)]、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、Bcl2关联X蛋白(Bax)、B淋巴细胞瘤-2(Bcl2)的蛋白表达。结果与正常肝细胞LO2组比较,肝癌细胞SMMC-7721组和HepG2组中Ndfip2表达升高(P<0.05);与NC组比较,si-Ndfip2组SMMC-7721和HepG2细胞Ndfip2蛋白表达明显下调(P<0.01);与NC组相比,si-Ndifip2组肝癌细胞的增殖速率明显降低(P<0.01),凋亡率增加(P<0.05);si-Ndifip2组肝癌细胞中PI3K、p-Akt(S473)和Bcl2的蛋白表达水平均下降(P<0.05);而PTEN、E-cadherin、Caspase-3和Bax蛋白的表达则增加(P<0.05)。结论沉默Ndfip2抑制肝癌细胞的增殖能力,促进肝癌细胞凋亡,其可能机制与PTEN-PI3K/Akt信号通路的失活有关。

Effect of Lichong decoction on expression of Bcl-2 and Bcl-2-associated X protein mRNAs in hysteromyoma model rat

OBJECTIVE:To study on effects of Lichong decoction on expression of apoptosis-controlling genes,Bcl-2 and Bcl-2-associated X protein(Bax) mRNAs in hysteromyoma tissue of the hysteromyoma model rat.METHODS:Fifty Wistar female rats were randomly divided into a normal group,a model group,a Lichong decoction group,a Guizifuling capsule group and a Mifepristone group.The hysteromyoma rat model was established by intraperitoneal injection of exogenous estrin and progestogens.Pathological examination of uterine tissue,uterine coefficient and uterine transverse diameter were made under optic microscope and expressions of Bcl-2 and Bax mRNAs in uterine tissue in the groups were detected with real-time fluorescent quantitative polymerase chain reaction(PCR) technique.RESULTS:After treatment,under microscope it was found that in the Lichong decoction group myometrium thinned,muscle fiber slightly overgrowth or long and thin,regular arrangement,inserting phenomenon of inner circular muscle and external longitudinal muscle was occasionally or not seen in the Lichong decoction group.The uterine coefficient and the uterine transverse diameter significantly decreased(P<0.01),and Bcl-2 mRNA expression significantly decreased(P<0.01) and Bax mRNA expression significantly increased in hysteromyoma tissue(P<0.01) in the Lichong decoction group as compared with the model group.CONCLUSION:Therapeutic effects of Lichong decoction on hysteromyoma is related with decrease of Bcl-2 mRNA expression and increase of Bax mRNA expression.

Influence of Moxibustion Serum on the Expression of Fas bcl-2 mRNA and Protein of EL-4 Lymphoma Cells

Objective： To observe the influence of moxibustion serum of mice on the expression of Fas, bcl-2 mRNA and protein of EL-4 lymphoma cells in vitro. Method： The EL-4 lymphoma cells were cultivated for 24 h in the moxibustion serum of mice. The expression of Fas and bcl-2 mRNA of EL-4 lymphoma cells were detected by in-situ hybridization method, and the protein expression of Fas and bcl-2 were observed by the immuocytochemistry method. Results： The expression of bcl-2 mRNA and protein decreased , and the expression of Fas mRNA and protein increased significantly in EL-4 cells, which were cultivated in the moxibustion serum compared those cultivated in normal mice serum （P〈0.05）. Conclusion： Moxibustion serum could down-regulate the bcl-2 mRNA and protein and up-regulate the Fas mRNA and protein of EL-4 cells.

小桐子CDPK及相关激酶基因家族的全基因组鉴定及表达分析

基于小桐子基因组数据库,在全基因组水平对小桐子CDPK及相关激酶基因家族进行鉴定,并对其系统进化、基因结构、保守结构域、染色体定位以及器官与低温胁迫表达进行分析,为深入开展小桐子CDPK及相关激酶基因的功能鉴定及其在小桐子遗传改良中的应用提供依据。利用CDPK结构域的隐马尔可夫模型对小桐子蛋白质数据库进行相似性检索,经过Pfam与CDD在线工具的验证获得小桐子CDPK及相关激酶基因家族成员。利用不同的工具对CDPK及相关激酶基因进行生物信息学对比分析。通过小桐子器官与低温表达谱数据进行CDPK及相关激酶基因的表达分析。小桐子基因组中共检索到17个CDPK基因、5个CRK基因、2个PPCK基因、4个PEPRK基因以及1个CCaMK基因,各基因家族成员的基因长度、蛋白质等电点及亚细胞定位等理化参数具有家族特异性。序列比对与系统进化分析显示,5个基因家族都单独聚类,而CDPK与PEPRK基因家族又分别聚类为4个与2个亚族,且鉴定到小桐子CDPK家族成员的N-端可变区、激酶结构区、自抑区及EF-hand调控区4个保守结构域以及其他激酶成员的激酶结构域。同时,分别有21个与24个CDPK及相关激酶可能发生N-豆蔻酰化与N-十六烷酰化的修饰。染色体定位表明,小桐子CDPK及相关激酶基因不均匀地分布于10条染色体上,且存在JcCDPK7/JcCDPK9串联复制现象。转录组表达分析表明,29个CDPK及相关激酶基因中,有24个基因在叶片、根及种子中都有表达,而JcCDPK3、JcCDPK11、JcCRK3及JcCCaMK属于低温诱导高表达基因,与小桐子的抗冷性直接相关。

Ginsenoside Rg_3 inhibit hepatocellular carcinoma growth via intrinsic apoptotic pathway

AIM:To investigate the anti-tumor function of ginsenoside Rg3 on hepatocellular carcinoma(HCC) in vitro and in vivo,and its mechanism.METHODS:Hep1-6 and HepG2 cells were treated by Rg3 in different concentrations(0,50,100 and 200 μg/mL) in vitro.After incubation for 0,6,12,24 and 48 h,cell viability was measured by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis was identified by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling.Caspase-3 activity was measured by chromophore p-nitroanilide and flow cytometry.Bcl-2 family proteins were ascertained by Western-blotting.Mitochondria membrane potentialwas detected by 5,5',6' 6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide.Forty liver tumor-bearing C57Bl6 mice were divided randomly into 4 groups for intra-tumor injection of saline,ginsenoside Rg3,cyclophosphamide(CTX) and ginsenoside Rg3 + CTX combination.RESULTS:The survival time was followed up to 102 d.The mice in the Rg3 + CTX group showed significant increased survival time compared with those in the control group(P < 0.05).Rg3 could inhibit HCC cell proliferation and induce cell apoptosis in vitro in the concentration and time dependent manner.It also induced mitochondria membrane potential to decrease.Caspase-3 activation can be blocked by the inhibitor z-DEVD-FMK.Bax was up-regulated while Bcl-2 and Bcl-XL were down-regulated after Rg3 treatment.CONCLUSION:Our data suggested that Rg3 alone or combined with CTX inhibited tumor growth in vivo and prolonged mouse survival time by inducing HCC cell apoptosis via intrinsic pathway by expression alterations of Bcl-2 family proteins.

香砂六君子汤调控RhoA/ROCK2/MYPT1信号通路改善功能性消化不良大鼠胃动力的机制

目的:基于RAS同源基因家族成员A(RhoA)/Rho关联卷曲螺旋蛋白激酶2(ROCK2)/肌球蛋白轻链磷酸酶靶向亚基1(MYPT1)信号通路探讨香砂六君子汤对功能性消化不良(FD)大鼠胃动力障碍的干预机制。方法:将60只SPF级雄性SD乳鼠随机分为正常组(n=10)和造模组(n=50),造模组大鼠采用综合造模法复制FD大鼠模型。模型复制成功后将造模组大鼠随机分为模型组、莫沙必利组和香砂六君子汤高、中、低剂量组,共5组,每组10只。正常组和模型组大鼠给予10 mL·kg^(-1)·d^(-1)生理盐水灌胃,莫沙必利组给予1.35 mg·kg^(-1)·d^(-1)莫沙必利灌胃,香砂六君子汤高、中、低剂量组分别给予12、6、3 g·kg^(-1)·d^(-1)香砂六君子汤汤剂灌胃,持续干预14 d。造模和给药前后观察大鼠一般生存状况,测定大鼠体质量、3 h平均进食量。给药干预结束后,测定大鼠肠推进率,采用苏木素-伊红(HE)染色观察胃组织病理改变,采用酶联免疫吸附测定法(ELISA)测定各组大鼠延髓和胃组织匀浆液中胆碱乙酰转移酶(ChAT)、血管活性肠肽(VIP)含量,采用冰冻切片染色技术观察各组大鼠胃窦平滑肌中腺嘌呤核苷三磷酸(ATP)酶分布,采用蛋白免疫印迹法检测胃组织中RhoA、ROCK2、磷酸化(p)-MYPT1蛋白表达水平。结果:与正常组比较,模型组大鼠毛发枯乱、倦懒怠动、行动缓迟,一般生存状况较差,体质量、3 h平均进食量以及肠推进率均明显下降(P<0.05);胃组织病理未见明显异常;延髓和胃组织中ChAT含量减少,胃组织中VIP含量增多,胃窦平滑肌中ATP酶分布明显减少,胃组织中RhoA、ROCK2、p-MYPT1蛋白表达水平均明显降低(P<0.05)。与模型组比较,各给药组大鼠一般生存状况明显好转,体质量、3 h平均进食量及肠推进率明显增加(P<0.05);胃组织病理未见明显差异;延髓和胃组织中ChAT含量明显增加,胃组织中VIP含量减少,胃窦平滑肌中ATP酶分布明显增加,胃组织中RhoA、ROCK2、p-MYPT1蛋白表达水平明显升高(P<0.05),上述指标香砂六君子汤组干预作用呈剂量依赖性。结论:香砂六君子汤能够有效改善FD大鼠一般生存状况及胃动力,其具体机制可能与香砂六君子汤激活胃组织中RhoA/ROCK2/MYPT1信号通路调控平滑肌舒缩,促进胃动力有关。

补阳还五汤对糖尿病性勃起功能障碍大鼠阴茎海绵体纤维化及RhoA、ROCK2蛋白表达的影响

目的观察补阳还五汤(BHD)对糖尿病性勃起功能障碍(DMED)的作用,并探讨其作用机制。方法经腹腔注射链脲佐菌素制备糖尿病DMED大鼠模型,通过阿扑吗啡实验筛选DMED大鼠。将36只DMED大鼠分为模型组、低、中、高剂量BHD组,每组9只。另取9只同月龄SD大鼠作为正常组。低、中、高剂量BHD组分别给予12.8、25.6、51.2 g/(kg·d)BHD灌胃,正常组和模型组给予等量生理盐水灌胃。测量阴茎海绵体内压(ICP)及ICP曲线下面积(AUC)/平均动脉压(MAP)观察勃起功能;Masson染色检测海绵体纤维化程度;免疫组化技术检测阴茎海绵体Ras同源基因家族成员A(RhoA)、Rho相关卷曲螺旋蛋白激酶2(ROCK2)蛋白表达;Western Blot检测阴茎海绵体α-平滑肌肌动蛋白(α-SMA)、转化生长因子-β1(TGF-β1)、RhoA、ROCK2蛋白表达。结果与正常组比较,模型组ICP峰值、AUC/MAP、阴茎海绵体平滑肌面积、平滑肌/胶原纤维、α-SMA蛋白表达降低(P<0.05),TGF-β1、RhoA、ROCK2蛋白表达升高(P<0.05)。与模型组比较,中、高剂量BHD干预后ICP峰值、AUC/MAP、阴茎海绵体平滑肌面积、平滑肌/胶原纤维、α-SMA蛋白表达升高(P<0.05),TGF-β1、RhoA、ROCK2蛋白表达降低(P<0.05)。结论BHD可有效改善DMED大鼠勃起功能,其作用机制可能与抑制阴茎海绵体纤维化、维持其平滑肌含量以及抑制RhoA/ROCK2信号蛋白表达有关。

牛膝-白芍配伍对帕金森病小鼠神经炎症及RhoA/ROCK2信号通路的影响

目的:观察牛膝和白芍不同剂量对肝阳上亢证帕金森病小鼠神经炎症及多巴胺能神经元的保护作用,并探究其作用机制。方法:将C57BL/6小鼠随机分为正常组、模型组、牛膝-白芍低、中、高剂量组(3.25、6.5、13 g·kg-1)、司来吉兰组(0.01 g·kg-1)。采用附子汤灌胃联合1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)腹腔注射的方法制备肝阳上亢证帕金森病模型。通过小鼠转棒实验和爬杆实验评估其行为学改变;蛋白免疫印迹法(Western blot)测定小鼠脑黑质中Ras同源基因家族蛋白A(RhoA)、Rho相关螺旋卷曲蛋白激酶2(ROCK2)、肌球蛋白轻链1(MLC1)、α-突触核蛋白(α-synuclein)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测脑黑质中RhoA、ROCK2、MLC1 mRNA表达水平;采用酶联免疫吸附测定法(ELISA)检测各组中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)促炎细胞因子含量;透射电镜观察小鼠神经元超微结构变化。结果:与正常组比较,模型组跌落潜伏期显著缩短及爬杆时长显著增加(P<0.01),脑黑质内RhoA、ROCK2、MLC1、α-synuclein、TNF-α、IL-1β水平均明显升高(P<0.05);与模型组比较,牛膝-白芍低、中、高剂量组、司来吉兰组跌落潜伏期明显增加及爬杆时长明显降低(P<0.05,P<0.01),且ROCK2、MLC1、α-synuclein、TNF-α、IL-1β水平均降低(P<0.05)。电镜下与正常组比较,模型组神经元胞质内细胞器数量明显减少,线粒体肿胀且内质网形态异常,而牛膝-白芍高剂量组和司来吉兰组经元胞核结构完整,细胞轮廓清晰,内质网形态正常。结论:牛膝-白芍配伍改善肝阳上亢证PD小鼠运动能力,下调RhoA/ROCK2信号通路相关蛋白表达,抑制炎性因子表达,减轻多巴胺能神经元损伤,其机制可能与抑制脑内RhoA/ROCK2信号通路介导的神经炎症反应有关。