异丙酚对大鼠离体心脏缺血/再灌注损伤时Bcl-2蛋白表达和细胞凋亡的影响

为探讨异丙酚对大鼠离体心肌缺血再灌注损伤后心肌细胞凋亡的影响及与 Bcl-2蛋白表达的关系,将 Wistar 大鼠36只随机分为对照组和异丙酚处理组各18只;每组再分为缺血前、缺血30min 和复灌30min 3个亚组各6只。分别用 TUNEL 法测定凋亡细胞和用免疫组化法测定 Bcl-2蛋白的表达。结果表明,与缺血前相比,缺血30min 及再灌注30min 亚组的凋亡指数(AI)显著增加(P<0.01),Bcl-2蛋白的光密度值显著减少(P<0.01);与对照组相比,异丙酚组缺血30min 及再灌注30min 亚组的 AI 显著降低(P<0.01),Bcl-2 蛋白的光密度值显著增加(P<0.01)。结论:缺血30min 及再灌注30min 的心肌均发生了细胞凋亡,且再灌注组的细胞凋亡现象更为显著。异丙酚通过调控 Bcl-2表达而减少心肌缺血再灌注时心肌细胞凋亡的发生。

Regulation of Bcl-2 Family in TIP30-Induced Apoptosis in Hepatoblastoma Cells

Objective: To investigate the role of TIP30 in the apoptotic signal pathwayin HepG2, and Hep3B and Hu-7 hepatoblastoma cell lines. Methods: In order to confirm whether TIP30conducted Bcl-2 family was involved in apoptosis signal pathway, MTT assay, in situ 3' end labellingof DNA assay and Western blot were carried out to detect the diverse apoptotic function of TIP30and the regulation of Bcl-2 family. Results: TIP30 induced apoptosis as evidenced by morphologicalchanges in hepatoblastoma cells, which was accompanied by up-regulating Bax and Bad proteins andstimulating them from cytoplasm to mitochondria, and down-regulating Bcl-xl, while it had no effecton the level of Bak protein. Conclusion: TIP30 induced apoptosis partly by modulating the proteinlevels of members of Bcl-2 family in hepatoblastoma cells. Elucidating the mechanism by which TIP30induces cell death might establish it as an anticancer factor.

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.

Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2 (COX-2) inhibitor, on the proliferation and apoptosis of HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells were evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells. DNA ploidy and apoptotic cell percentage were calculated by flow cytornetry. The expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. Furthermore, expression level of Bcl-2 was detected using Western blot in HepG2 after treated with NS-398. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis of HepG2 cells in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with increase of NS-398 concentration. The quiescent GO/G1 phase was accumulated with decrease of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, and no correlations were found between COX-2 mRNA and HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). Bcl-2 protein level was inhibited after treated with NS-398. CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis of HepG2 cells. Mechanisms involved may be accumulation of quiescent GO/G1 phase and decrease of Bcl-2 expression.

Combined transfection of Bcl-2 siRNA and miR-15a oligonucleotides enhanced methotrexate-induced apoptosis in Raji cells

Objective: B-cell lymphoma 2 (Bcl-2) is an important member of the Bcl-2 family of proteins that regulate the induction of apoptosis. This study aims to investigate whether Bcl-2 small interfering RNA (siRNA) combined with miR-15a oligonucleotides (ODN) could enhance methotrexate (MTX)-induced apoptosis in Raji cells. Methods: Chemically synthesized miR-15a ODN and Bcl-2 siRNA were transfected in Raji cells by using a HiPerFect Transfection Reagent and then combined with MTX. Expression levels of Bcl-2 protein were detected by Western blot. Cell proliferation was determined by CCK8 assay. The rate of cell apoptosis was determined by Annexin V/PI double staining. The morphology of apoptotic cells was observed by Hoechst-33 258 staining. Results: After the cells were transfected with miR-15a ODN combined with Bcl-2 siRNA, Bcl-2 protein levels were evidently decreased. CCK8 assay showed that cell proliferation was significantly decreased and was significantly lower in miR-15a ODN combined with Bcl-2 siRNA plus MTX group than in miR-15a ODN with methotrexate group, Bcl- 2 siRNA with MTX group, and single MTX group (P<0.05). Hoechst 33258 staining revealed numerous apoptotic cells. AnnexinV/PI double staining showed that the apoptotic rates were (13.13±1.60)%, (34.47±2.96)%, (32.87±3.48)%, and (45.47±2.16)% in MTX, Bcl-2 siRNA plus MTX, miR-15a ODN plus MTX, and miR-15a ODN combined with Bcl- 2 siRNA plus MTX groups, respectively. Among these groups, the apoptotic rate of miR-15a ODN combined with Bcl-2 siRNA plus MTX group was the highest; this apoptotic rate was also significantly different from that of miR-15a ODN or Bcl-2 siRNA plus MTX (P<0.05). Conclusions: Bcl-2 siRNA combined with miR-15a ODN could enhance MTX-induced apoptosis in Raji cells. Bcl-2 siRNA and miR-15a combined with MTX may be a useful approach to improve the treatment effects on lymphoma.

Expression of bcl-2 protein in chronic hepatitis C:Effect of interferon alpha 2b with ribavirin therapy

AIM: Mechanisms responsible for persistence of HCV infection and liver damage in chronic hepatitis C are not clear. Apoptosis is an important form of host immune response against viral infections. Anti-apoptotic protein bcl-2 expression on liver tissue as well as the influence of interferon alpha 2b (IFNa2b) and ribavirin (RBV) were analyzed in patients with chronic hepatitis C. METHODS: In 30 patients with chronic hepatitis C (responders - R and non-responders - NR) treated with IFNα2b+RBV, protein bcl-2 was determined in hepatocytes and in liver associated lymphocytes before and after the treatment. RESULTS: The treatment diminished bcl-2 protein accumulation in liver cells in_patients with hepatitis C (P<0.05). Before and after the therapy, we detected bcl-2 protein in R in 87±15% and 83±20% of hepatocytes and in 28±18% and 26±10% of liver-associated lymphocytes, respectively. In NR, the values before treatment decreased from 94±32% to 88±21% of hepatocytes and 39±29% to 28±12% of lymphocytes with bcl-2 expression. There was no statistical correlation between bcl-2 expression on liver tissue with inflammatory activity, fibrosis and biochemical parameters before and after the treatment. CONCLUSION: IFNα2b+RBV treatment, by bcl-2 protein expression decrease, enables apoptosis of hepatocytes and associated liver lymphocytes, which in turn eliminate hepatitis C viruses.

Down-Regulation of Bcl-2 Protein Sensitizes NCI-H460 Cells to Radiotherapy-Induced Apoptosis

OBJECTIVE To determine whether Bc1-2 protein down-regulation can render NCI-460 cells more susceptible to gamma radiation-induced apoptosis by treatment with antisense oligonucleotide （ASODN） against the coding region of Bcl-2 mRNA. METHODS Cell survival was determined using the trypan blue dye exclusion. Expression of the Bcl-2 protein was assayed using immunofluo- rescence labeling with fluoresce isothiocyanate. Apoptosis was determined by Giemsa staining and flow cytomertry. RESULTS It was found that Bcl-2 ASODN combined with radiation sig- nificantly reduced the number of viable cells （P〈0.05）. There was no difference in cell survival between a nonsense oligodeoxynucleotide/radiation combination and cells treated with radiation alone. Bcl-2 ASODN combined with radiation significantly inhibited expression of the Bcl-2 protein in the NCI-H460 cells （P〈0.05）. Using Giemsa staining, cells treated with Bcl-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptotic rates of the NCI-H460 cells treated with Bcl-2 ASODN combined with radiation significantly increased （P〈 0.05）, compared with either a nonsense oligodeoxynucleotide/radiation combination or radiation-treatment cells alone. CONCLUSION ASODN against the coding region of Bcl-2 mRNA increases radiation-induced apoptosis in NCI-H460 cells.

CO-EXPRESSIONS OF SURVIVIN GENE, BCL-2 AND BAX PROTEINS IN OVARIAN CARCINOMA

Objective To characterize the cellular properties of ovarian cancer, we examined the correlation between the expression of apoptosis-related gene survivin and those of Bcl-2 and Bax proteins. Methods Expressions of survivin mRNA, and Bcl-2 and Bax proteins in 35 cases of ovarian carcinoma, 10 cases of borderline carcinoma, 10 cases of benign tumors and 10 cases of normal tissue were evaluated by reverse transcription polymer-ase chain reaction (RT-PCR) and immunohistochemistry SABC method, respectively. Results Expression of survivin gene was detected in a significantly greater proportion in ovarian carcinoma and borderline carcinoma than those in benign tumors and normal tissues. Although there was no relationship between expression of survivin gene and FIGO stage, histologic grade, pathological type and lymphatic metastasis, expressions of Bcl-2 and Bax proteins were positively and negatively correlated with that of survivin gene, respectively. Conclusion Survivin may play an important role in pathogenesis of ovarian carcinoma, with a synergistic role of apoptosis-related gene Bcl-2 protein and an antagonistic role of Bax protein in formation and progression of ovarian carcinoma.

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.

The Variety Selection and Testing for Effect on HepG-2 Cell Line of Dioscorea membranacea Pierre （Hua Khao Yen Tai） Extract Using Molecular Genetics

In this research, Dioscorea membranacea Pierre was studied by using 30 samples from Khao Ruak Sub-district, Chai Badan District, Lop Buri Province, in Thailand. In this research, some morphology was studied including shapes, leaves, stem colors, epidermal cells, stomata sizes and stipules. To study the genetic relationships, the AFLP technique and computer program were used. The Dioscorea membranacea Pierre was classified into 2 groups according to its phylogenetic type： the first group was ＂Hua Khao Yen Tai-Nuea＂ （Smilax corbularia Kunth）, and the second group included 30 further samples of Hua Khan Yen Tai （Dioscorea membranacea Pierre）. The ethanolic crude extract was also applied to test the anti-proliferative activity in the liver hepatocellular carcinoma （HepG2） cell lines which illustrates the characteristics of apoptosis： cell shrinkage, membrane blebbing and nuclear condensation. The expression ofBax gene is increased more than that of the control group while Bcl-2 gene which is anti-apoptotic is decreased. Furthermore, the result of western blot analysis reveals the up-regulation of Bax protein and down-regulation of Bcl-2 protein when compared with untreated cells. This might indicate that ethanolic crude extracts of Hua Khao Yen Tai could induce apoptosis and anti-proliferative on HepG2 cell lines, The results also revealed that some morphology cannot be used to predict which Dioscorea membranacea Pierre plants would be most effective.

氯沙坦在损伤血管重构中的作用及其机制的探讨

目的 ::探讨氯沙坦在大鼠血管内膜剥脱术后血管重塑中的作用及其内在机制。方法 :将 Wistar大鼠随机分为假手术组、氯沙坦组及单纯 PTCA组。后两组大鼠行胸主动脉球囊损伤术 ,术后不同时间段处死。对主动脉的组织切片分别进行 HE染色及计算机图像分析、Tunel标记检测细胞凋亡率、免疫组化方法检测血管壁凋亡相关基因表达物 Fas、Fas-L、Bcl-2、Bax的变化。结果 :单纯 PTCA组术后 7、14 d管壁面积明显增加 ,与假手术组比较有显著性差异 (P<0 .0 5)。术后 14 d,氯沙坦组与单纯 PTCA组比较 ,管壁增生程度较轻。术后 7d,管壁面积增生比与细胞凋亡率之间有负相关性 (r=-0 .586,P<0 .0 5) ,氯沙坦组的细胞凋亡率显著高于单纯 PTCA组。氯沙坦组与单纯PTCA组比较 ,术后 7d管壁组织内 Fas-L表达增加 ,Bcl-2表达降低 (P<0 .0 5)。结论 :AT1受体拮抗剂氯沙坦可有效防止大鼠动脉损伤后的血管增生性重塑。其机制可能与促进 Fas的表达、抑制 Bcl-2的表达 。

凋亡相关基因bcl-2和bax在小儿神经母细胞瘤中的表达及其意义

神经母细胞瘤（neuroblastoma,NB）是一种发展迅速、恶性程度及死亡率高、预后极差的小儿常见的恶性肿瘤.为探讨细胞凋亡调控基因bax、bcl-2蛋白及bcl-2mRNA在小儿神经母细胞瘤中的表达,我们进行了以下研究.

Expression of P16 protein and Bcl-2 protein in malignant eyelid tumors

Objective To investigate the relationship between P16 gene (the tumor suppressor gene) and the bcl-2 gene (the apoptosis inhibitor gene) and the incidence and development of malignant eyelid tumors. Methods The streptavidin-biotin-peroxidase complex immunohistochemistry method was used to study the expression of P16 gene and the bcl-2 gene in 96 cases of malignant eyelid tumors. [WTF5絉esults [WTFZ]Among the 96 cases, there were 40 basal cell carcinomas (BCCs), 33 squamous carcinomas and 23 sebaceous carcinoma, with P16 protein positive (nuclear staining) rates 70%, 54.6% and 56.5%, respectively. The P16 positive rate was negatively correlated with the degree of tumor histological differentiation, and the rate difference between the high differentiated carcinomas was significant (P【0.05). Positive Bcl-2 protein expression was detected in the cytoplasm. All 40 BCC cases were Bcl-2 positive, and nearly all of the tumor cells showed positive cytoplasmic expression, while in the 33 squamous cell carcinoma cases only one showed positive focal reaction, and the staining in the other 32 cases was relatively faint. None of the 23 sebaceous carcinomas expressed Bcl-2. Conclusions The expression of the P16 protein was related to the occurrence and degree of differentiation of malignant eyelid tumors. The overexpression of the Bcl-2 protein suggests that suppression of apoptosis might play a role in the tumorigenesis of BCC.

Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats

Objective: To study the relationship between the expression of Bax, Bcl 2 proteins, and apoptosis in radiation compound wound healing of rats. Methods: Apoptosis, Bax and Bcl 2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. Results: (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl 2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively. Conclusions: Bax and Bcl 2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.

Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer

AIM:To investigate the mechanisms of the biological roles of Dickkopf-3(Dkk-3) in cell invasion,survival and apoptosis in colon cancer cells.METHODS:Three human colon cancer cell lines,i.e.,HT-29,LoVo and SW480,were used.Overexpression of Dkk-3 induced by pEGFP-N1-Dkk-3-GFP plasmid in LoVo cells was performed using Lipofectamine 2000 reagent.Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of Dkk-3,respectively.Cell proliferation assay,cell cycle analysis,hoechst 33258 assay and Matrigel invasion assay were performed on Dkk-3 overexpressing transfectants.RESULTS:The mRNA and protein expressions of Dkk-3 in HT-29(mRNA:0.06 ± 0.02,protein:0.06 ± 0.01) and LoVo(mRNA:0.07 ± 0.02,protein:0.07 ± 0.02) cells were significantly lower than that in SW480 cells(mRNA:0.92 ± 0.04,protein:0.69 ± 0.13;all P < 0.05),and the greatest levels of invasiveness wasin LoVo cells.Dkk-3 overexpression inhibited the proliferation and invasion of LoVo cells and induced cell cycle arrest at G0/G1 phase and subsequent apoptosis,as indicated by increased chromatin condensation and fragments,upregulated Bax and cytochrome c protein,downregulated survivin and Bcl-2 protein,and the activation of caspase-3 and caspase-9.Furthermore,Dkk-3 overexpression reduced the accumulation of cytosolic fraction of β-catenin.CONCLUSION:Dkk-3 overexpression induced apoptosis in human colon cancer possibly through the mitochondrial pathway.Dkk-3 may be involved in the Wnt/β-catenin signaling pathways in colon cancer.

Effect of Bushenyisui Formula on brain tissue apoptosis and Bcl-2 in beta-amyloid protein-induced Alzheimer's disease rat models

OBJECTIVE： To investigate the effect of Bushenyisui Formula on cell apoptosis and positive B cell lym- phoma （Bcl-2） in the Brain of rat models of Alzheim- er＇s disease （AD） induced by beta-amyloid protein （All3） and the mechanism underlying the effect. METHODS： Total of 40 SD rats, 20 females and 20 males, were randomly assigned to 4 groups, con- trolled group （A）, model group （B）, conventional treatment group （C） and Bushenyisui Formula treat- ment （BYFT） group （D）, 10 rats in each group. AI3 1-42 was injected into the bilateral hippocampus of the rats in group B, C and D to create the models of AD. Sham operation was performed on the rats of group A in the same way by injecting equal volume of 0.9% sodium chloride solution into their bilateral hippocampus. 5 days after operation, Bushenyisui Formula was intraperitoneally administered at a dose of 450 mg/kg to the rats of group D （QD） for 20 days. Equal volume of 0.9% sodium chloride solution was intraperitoneally injected into the rats of group B with the same procedure. C suspension （20 mg/mL） was intraperitoneally injected into the rats of group B with the same procedure. The number of apoptot- ic cells in Brain and the positive Bcl-2 were count- ed. The changes of learning and memory abilities were evaluated using Y-maze. RESULTS： Right after the establishment of the mod- els, group B, C and D compared to group A respec- tively, the outcomes of Y-maze were significantly different from that of group A, which suggested ob- vious learning and memory disorder in those groups （P〈0.01）. After treatment, the times of elec- tronic shocks of group C and D were significantly less than that of group B （P〈0.05）, and the numbers of apoptotic cells and positive Bcl-2 were signifi- cantly different from those of group B, apoptotic sells＇ number of group C and D smaller than that of group B and the number of positive Bcl-2 greater than that of group B. CONCLUSION： Bushenyisui Formula could increase the number of Bcl-2 in brain, which improved the function of nervous system pertaining to learning and memory abilities.

Casticin combination with Cisplatin in sub-toxic concentration induced apoptosis of human ovarian cancer HO-8910 cells in vitro

Objective: The aim of the study was to investigate the effect of Casticin (CAS) combination with Cisplatin (DDP) in sub-toxic concentration on apoptosis of human ovarian cancer HO-8910 cells in vitro and unravel the associated mechanisms. Methods: Human ovarian cancer HO-8910 cells were cultured in vitro. The inhibitory effect of CAS combination with DDP in sub-toxic concentration on viability of human ovarian cancer HO-8910 cells was evaluated by the MTT assay. Morphological changes of cell apoptosis were detected by Hoechst 33258 staining assay. Cell apoptosis rate was analyzed by flow cytometry. The protein expression level was analyzed by Western blot. Results: CAS in sub-toxic concentration and DDP in sub-toxic concentration could slightly inhibit Human ovarian cancer HO-8910 cells, but CAS combination with DDP in sub-toxic concentration significantly inhibited the growth of HO-8910 cells, and growth inhibition rate was increased drastically compared with the control group (P﹤0.01), and the inhibiting effect showed synergistic action. Human ovarian cancer HO-8910 cells showed the typical morphological changes of apoptosis and apoptosis rate markedly increased when they were exposed to CAS combination with DDP in sub-toxic concentration for 48 h. Western blot showed that the expression of bcl-2 protein was down-regulated and protein level of caspase-3 was activated by CAS combination with DDP in sub-toxic concentration. Conclusion: CAS combination with DDP in sub-toxic concentration could inhibit the cells growth and lead to cell apoptosis in human ovarian cancer HO-8910 cells. And the down-regulation of bcl-2 protein expression and activation of caspase-3 protein might contribute to CAS combination with DDP in sub-toxic concentration in human cancer HO-8910 cells.

Baicalin extracted from Huangqin(Radix Scutellariae Baicalensis) induces apoptosis in gastric cancer cells by regulating B cell lymphoma(Bcl-2)/Bcl-2-associated X protein and activating caspase-3 and caspase-9

OBJECTIVE:To evaluate the effects of baicalin in human gastric cancer cells, including apoptosis-inducing effects, and to investigate its underlying mechanisms of action.METHODS:Cell proliferation and apoptosis assays were performed to investigate the anti-proliferation effects of baicalin in human gastric cancer BGC-823 and MGC-803 cells.Real time-quantitative polymerase chain reaction and Western blotting analysis were performed to elucidate the molecular mechanisms underlying the anti-tumor properties of baicalin.RESULTS:In BGC-823 and MGC-803 gastric cancer cells treated with 80, 120, and 160 μmol/L baicalin for 48 h, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay showed that baicalin significantly inhibited cell proliferation in a dose-dependent manner, while flow cytometric analysis demonstrated that baicalin could induce apoptosis, also in a dose-dependent manner.Moreover, baicalin up-regulated the expression of caspase-3, caspase-9, and B cell lymphoma(Bcl-2)-associated X protein and down-regulated the expression of Bcl-2 at both the m RNA and protein level.CONCLUSION:Baicalin has potential as a therapeutic agent for gastric cancer by inducing apoptosis in cancer cells.

c-Jun N-terminal kinase is required for thermotherapyinduced apoptosis in human gastric cancer cells

AIM:To investigate the role of c-Jun N-terminal kinase(JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ for 0,0.5,1,2 or 3 h,the cells were cultured for a further 24 h with or without the JNK specific inhibitor,SP600125 for 2 h.Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)] and flow cytometry(Annexin vs propidium iodide).Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The production of p-JNK,Bcl-2,Bax and caspase-3 proteins was evaluated by Western blotting.The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.RESULTS:The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy,and was 32.7%,30.6%,43.8% and 52.9% at 0.5,1,2 and 3 h post-thermotherapy,respectively.Flow cytometry analysis revealed an increased population of SGC790l cells in G0/G1 phase,but a reduced population in S phase following thermotherapy for 1 or 2 h,compared to untreated cells(P < 0.05).The increased number of SGC-790l cells in G0/G1 phase was consistent with induced apoptosis(flow cytometry) following thermotherapy for 0.5,1,2 or 3 h,compared to the untreated group(46.5% ± 0.23%,39.9% ± 0.53%,56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%,P < 0.01),respectively.This was supported by the TUNEL assay(48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment(P < 0.01),and peaked at 2 h.A similar pattern was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased.Furthermore,the JNK specific inhibitor,SP600125,suppressed p-JNK,Bax and caspase-3 at the protein level in SGC790l cells following thermotherapy,compared to mock-inhibitor treatment,which was in line with the decreased rate of apoptosis.The expression of Bcl-2 was consistent with thermotherapy alone.CONCLUSION:Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels,and up-regulated the expression of Bax and caspase-3 proteins.Bcl-2 may play a protective role during thermotherapy.Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

Effects of Electroacupuncture on bcl-2 and bcl-XL in the Degenerative Cervical Intervertebral Disc of Rats

Objective： To observe the effects of electroacupuncutre at Tianzhu point on the expressions ofbcl-2 and bcl-XL in the degenerative cervical intervertebral disc of rats. Methods Forty SD male rats were randomly divided into 4 groups, sham surgery group, model group, Western medicine group and electroacuptmcture group, there were 10 rats in each group. Except for rats in the sham surgery group, rats in the other groups were made to be of cervical intervertebral disc degeneration by unbalanced dynamic and static forces. Rats in the electroacupunture group were treated with electroacupuncture at Tianzhu （BL 10） and Dazhu （BL 11） points, while rats in the Western medicine group with Fenbid, and rats in the model group were not treated. After 30-day treatments, the expressions of bcl-2 and bcl-XL were observed with immunohisto-chemical method in the cervical intervertebral disc. Results： The expression of bcl-2 was higher in the electroacupuncture, sham surgery, and western medicine groups than that in the model group （P〈0.01）, whereas, there was no difference among these three groups. The expression of bcl-XL in the electroacupuncture group was lower than that in the sham surgery group （P〈0.01）, and higher than those in the Western medicine group and model group （P〈0.01）. Conclusion： Electroacupuncture treatment can enhance the expressions of bcl-2 and bcl-XL, which may be the mechanism of delaying the cell apoptosis in the intervertebral disc to treat diseases.