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.

Effect of Icariin on apoptosis and expression of Fas,Fas ligand,B cell lymphoma,and Bcl-2-associated X protein in CD4＋ T lymphocytes from patients with ankylosing spondylitis

OBJECTIVE:To investigate the effects of icariin on apoptosis and the expression of Fas, Fas ligand(Fas L), B cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) in CD4+ T lymphocytes from patients with ankylosing spondylitis.METHODS:Primary cultures of peripheral blood CD4+ T lymphocytes were established and treated with icariin at high, medium, and low doses(0.5,0.25, and 0.125 mg/mL).Sulfasalazine treated and helthy cells were used as controls.Apoptosis of treated cells was determined by flow cytometry.Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine the effects of icariin on the expression of Fas, Fas L, Bcl-2, and Bax.The activity of caspase 8 and caspase 3 was determined by a colorimetric assay.RESULTS:The m RNA and protein expression of Fas,and activity of caspase 8 and caspase 3 in CD4+ T lymphocytes were increased by icariin(P < 0.05).Conversely, the m RNA and protein expression of Bcl-2 was decreased(P < 0.05).The expression of Fas L and Bax were not significantly different between groups.The proapoptotic effects of icariin were dose-dependent.CONCLUSION:Icariin induces the apoptosis of CD4 + T cells from patients with AS comparing to normal control.Therefore, the induction of apoptosis may be the likely mechanism of action of icariin's antirheumatics activities.

哮喘平对哮喘模型大鼠肺组织细胞凋亡相关调控基因蛋白Bcl-2、Bax水平的影响

目的:观察哮喘平对哮喘大鼠模型肺组织细胞凋亡相关调控基因蛋白Bcl-2、Bax水平的影响,研究哮喘平治疗哮喘的作用机制。方法:将60只SD雄性大鼠随机分为6组,每组10只,即空白对照组、哮喘模型组、桂龙咳喘宁组、哮喘平低剂量组、哮喘平中剂量组、哮喘平高剂量组。以卵蛋白致敏复制大鼠哮喘模型,成功复制模型21 d后,空白对照组和模型组每天给予等量的氯化钠溶液灌胃,连续给药2周后处死大鼠,解剖大鼠并取出肺组织。采用免疫组化法测定大鼠肺组织中细胞凋亡相关调控基因蛋白Bcl-2及Bax的表达。结果:与空白对照组比较,模型组大鼠Bcl-2表达升高,Bax表达降低(P<0.01),与模型组比较,给药组大鼠Bcl-2表达降低,Bax表达升高(P<0.05,P<0.01);与桂龙咳喘宁组比较,哮喘平中高剂量组Bcl-2表达显著降低,Bax表达显著升高(P<0.01)。结论:哮喘平可以降低抑凋亡基因蛋白Bcl-2的表达,升高促凋亡基因蛋白Bax的表达。

Effects of erythropoietin on the expression of tumor necrosis factor-alpha and Bax after facial nerve axotomy in rats

This study sought to evaluate the effect of high-dose erythropoietin （EPO; 5 000 IU/kg） on the expression of tumor necrosis factor-alpha （TNF-α） and Bax in the facial nucleus after facial nerve transection in rats. A total of 42 Wistar rats of both genders were used in this study, and 40 rats were randomly divided into 2 groups： EPO group and model group. The EPO group was treated with EPO once a day for 5 days at a dose of 5 000 IU/kg body weight. The model group was treated with saline of the same amount. At day 3 after EPO （or saline） treatment, the right facial nerves of the 40 rats were transected at the level of the stylomastoid foramen, with the left sides untreated. The remaining 2 rats that did not undergo axotomy served as the control group. The surviving motor neurons in operated rats were counted in coronal paraffin sections of the facial nucleus. The expression of TNF-a and Bax in the facial nucleus was detected by immunohistochemical staining at days 3, 7, 14, 21, and 28 after axotomy. At days 14, 21, and 28 after facial nerve axotomy, a significantly greater proportion of facial motor neurons survived in the EPO group than in the model group. After axotomy, the expression of TNF-a and Bax increased in motor neurons in both the EPO and the model groups. TNF-o expression reached its peak level at day 14 after axotomy, while Bax expression reached its peak level at day 21. TNF-α expression was much lower in the EPO group than in the model group at all time points. No significant difference in Bax expression was found between the EPO and the model groups. These results indicate that high-dose EPO treatment attenuates the increase in TNF-α expression in the facial nucleus and reduces the loss of motor neurons after facial nerve transection in rats. However, high-dose EPO treatment has little effect on Bax expression.

Correlation of serum PDCD5 and Bax contents with the pathological features of tumor lesions in patients with lung cancer

Objective:To study the correlation of serum PDCD5 and Bax contents with the pathological features of tumor lesions in patients with lung cancer.Methods:Patients with lung cancer who underwent surgical treatment in West China Hospital between June 2014 and March 2017 were selected as the lung cancer group for the study, the serum specimens were collected before surgery, and the lung cancer lesion and adjacent lesion were taken after surgery;the healthy subjects who underwent physical examination in West China Hospital during the same period were selected as the control group, and the serum samples were taken during the physical examination;the contents of PDCD5 and Bax in serum as well as the contents of PDCD5 and Bax, and the expression of proliferation genes and invasion genes in the lung cancer lesion and adjacent lesion were measured.Results: PDCD5 and Bax contents in serum of lung cancer group were significantly lower than those of control group, PDCD5 and Bax contents in lung cancer lesion were significantly lower than those in adjacent lesion, and the PDCD5 and Bax contents in serum of patients with lung cancer were positively correlated with the PDCD5 and Bax contents in lung cancer lesion;C-myc, RACK1, CatL, MMP2 and N-cadherin mRNA expression in lung cancer lesion were significantly higher than those in adjacent lesion and negatively correlated with PDCD5 and Bax contents in serum whereas LAST1, PAQR3, TCF21, E-cadherin, TIMP1 and TIMP2 mRNA expression were significantly lower than those in adjacent lesion and positively correlated with PDCD5 and Bax contents in serum. Conclusion:The decrease of PDCD5 and Bax in serum of patients with lung cancer is closely related to the aggravation of cancer cell proliferation and invasion in tumor lesions.

Overexpression of Bcl-2 partly inhibits apoptosis of human cervical cancer SiHa cells induced by arsenic trioxide

OBJECTIVE: To study the biological effect of arsenic trioxide (As2O3) on human cervical cancer SiHa cells and SiHa cells overexpressing bcl-2 gene. METHODS: SiHa cells with overexpression of Bcl-2 (SiHa-Bcl2 cells) were established by transfecting SiHa cells with Bcl-2 expression vector. The sensitivities of SiHa and SiHa-Bcl2 cells to As2O3 were determined using MTT (Thiazolyl blue) reduction and colony forming ability assay, morphological analysis, flow cytometric analysis, DNA agarose gel electrophoresis, in situ cell death detection (TUNEL), Northern blot, RT-PCR and Western blot. RESULTS: As2O3 inhibited the growth of SiHa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR and Western blot analysis revealed that As2O3 induced SiHa cell apoptosis possibly via inhibiting the expression of HPV16 E7 and decreasing the expression of c-myc. However, we found that SiHa-Bcl2 cells partly resisted As2O3 induced apoptosis, which might be related to the prevention of the down-regulation of HPV16 E7 and c-myc gene expression. Nevertheless, As2O3 at a high concentration could still induce apoptosis of SiHa-Bcl2 cells mainly via decreasing Bcl-2 expression and slightly inhibiting viral gene expression. CONCLUSION: As2O3 is an inducer of the apoptosis of human cervical carcinoma cells and the cells overexpressing Bcl-2 can partly resist As2O3 induced apoptosis, but the exact mechanism is unclear.

大黄酚脂质体对脑缺血再灌注损伤小鼠海马神经元凋亡的影响

目的:观察脑缺血再灌注损伤后,大黄酚脂质体对小鼠海马神经元凋亡的影响,探讨大黄酚脂质体对脑缺血再灌注损伤小鼠海马的保护作用及其机制。方法:采用暂时性阻断颈总动脉的方法建立小鼠脑缺血再灌注损伤模型,腹腔注射(ip)大黄酚脂质体10.0,1.0,0.1 mg.kg-1,观察小鼠脑缺血再灌注后神经功能学和病理形态学的改变,并采用免疫组化方法检测海马神经元凋亡相关蛋白半胱氨酰天冬氨酸蛋白酶-3(caspase-3),Bcl-2和Bcl-2-Associated X(Bax)的表达。结果:大黄酚脂质体可明显抑制脑缺血再灌注引起的神经元的丢失,提高Bcl-2的表达,降低caspase-3和Bax的表达,提高神经功能评分,减少病理形态改变,减轻海马神经元损伤,其中以10.0 mg.kg-1组大黄酚脂质体的作用最为明显(P<0.05)。结论:脑缺血再灌注损伤后应用大黄酚脂质体对海马神经元有明显的保护作用,其机制可能与上调Bcl-2的表达,下调caspase-3和Bax的表达有关。

EGCG诱导人视网膜色素上皮细胞凋亡作用的研究

目的:探讨表没食子儿茶素没食子酸酯(EGCG)对人视网膜色素上皮细胞(ARPE-19)凋亡的影响及其机制。方法:体外培养ARPE-19,分别采用0、40、80、160μg/mL EGCG处理。处理预定时间后分别用hoechst 33258染色法检测细胞凋亡形态学变化;流式细胞仪检测细胞凋亡率;实时荧光定量RT-PCR和Western blotting检测细胞凋亡相关因子B淋巴细胞瘤-2基因(bcl-2)、BCL2-Associated X的蛋白质(Bax)、胱天蛋白酶-3(caspase-3)和p53的表达。结果:hoechst 33258染色结果发现ARPE-19随着EGCG药物浓度的增加,凋亡细胞数量逐渐增多,可见明显的凋亡小体;流式细胞仪结果显示随着EGCG药物浓度的升高,凋亡率逐渐增高,40、80、160μg/mL凋亡率分别为4.95%±0.071%、11.75%±0.075%和21.25%±0.919%与对照组(2.8%±1.556%)相比有差异(P<0.01),呈现出药物浓度依赖性;实时荧光定量PCR和Western blot结果表明EGCG能明显上调凋亡促进因子Bax、caspase-3和p53的mRNA和蛋白表达,同时下调凋亡抑制因子bcl-2的表达,均呈现浓度依赖性。结论:EGCG能明显诱导ARPE-19发生凋亡,其机制与抑制bcl-2的表达,增强Bax、caspase-3和p53的表达有关。

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

Synergistic impacts of rifampicin and doxorubicin against thioacetamide-induced hepatocellular carcinoma in rats

Background and aims:Combination therapy is a promising new strategy that has been proposed to increase the efficacy of cancer treatment.We aimed to investigate the anti-cancer activity of rifampicin monotherapy and its combination with doxorubicin against hepatocellular carcinoma(HCC).Materials and methods:The in vitro half maximal inhibitory concentration(IC50)and selectivity index(SI)of the drugs under investigation against HepG2 and human lung fibroblast(WI38)cell lines were determined.For the in vivo experiment,male Sprague-Dawley albino rats were injected with thioacetamide at 200 mg/kg twice a week for 90 days;HCC development was confirmed histopathologically.Following HCC induction,the rats were treated with intraperitoneal doxorubicin,rifampicin,or their combination for 45 or 90 days.After sacrifice,the livers were examined histopathologically.The levels of aminotransferases,albumin,bilirubin,malondialdehyde,superoxide dismutase(SOD),catalase(CAT),total antioxidant capacity(TAC),and nitric oxide were measured by spectrophotometry.Alphafetoprotein,cancer antigen 19-9,tumor necrosis factor-alpha,interleukin-6,Bcl-2-associated X protein,caspase 3,caspase 8,and p53 were estimated using ELISA.Results:In vitro,the combination of doxorubicin and rifampicin showed the highest SI of 3.43.In vivo,among the measured markers,the levels of TAC,CAT,SOD,and p53 decreased(P<0.001)and the rest of the measured marker levels increased(P<0.001)in the HCC-bearing rats;after treatment in all groups,all these changes improved toward normal in a time-dependent manner.The combination of doxorubicin and rifampicin optimized the effects of the two individual drugs and exerted the best antioxidant effects.Conclusions:In general,compared with rifampicin or doxorubicin alone,combination therapy has favorable outcomes.Based on our results,the combination of rifampicin and doxorubicin might be applicable for HCC chemotherapy.

Effect of Qiangxin Huoli decoction on rats with adriamycin-induced chronic heart failure

OBJECTIVE: To investigate the effect of Qiangxin Huoli decoction on rats with chronic heart failure(CHF) induced by adriamycin(ADR), and to investigate the underlying mechanism of this effect.METHODS: Ninety-six healthy Wistar rats were divided into six groups: control, CHF model, CHF treated by Shenfu injection, and three CHF groups treated with Qiangxin Huoli decoction at high, medium, and low doses, respectively. Qiangxin Huoli decoction was administered orally to protect the stomach in the three Qiangxin Huoli decoction groups, while the control group and the CHF model group were administered the same volume of 0.9% physiological saline, and the Shenfu group wereadministered the same volume of Shenfu injection. Ten days later, the CHF model was then induced in all groups except the control group by intraperitoneal injection of ADR at gradient dose intervals. The bodyweights were recorded on days 10, 20, 30, and 40. Hemodynamic indices were recorded, including left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), maximum increase in left ventricular pressure(+dp/dt_(max)), maximum decrease in left ventricular pressure(-dp/dt_(max)), heart rate(HR), and electrocardiogram using an eight-channel physiological recorder with LabChart software monitoring. The plasma brain natriuretic peptide(BNP) concentration was determined by enzyme-linked immunosorbent adsorption. The expressions of B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein(Bax) were detected by immunohistochemical methods.RESULTS: The CHF model group were in poor condition, and the mean bodyweight was significantly decreased compared with the control group. Furthermore, compared with the control group, the CHF groups had significantly decreased LVSP, +dp/dt_(max), and-dp/dt_(max), and significantly increased LVEDP. The CHF groups also showed significant increases in HR, S-T segment elevation, and plasma BNP levels compared with the control group. Compared with the CHF model group, the treatment groups had significantly increased Bax expression(P < 0.05) and significantly decreased Bcl-2 expression(P < 0.01), indicating less apoptosis. The high dose Qiangxin Huoli decoction group and the Shenfu group showed the most significant improvements.CONCLUSION: In the rat model of CHF, Qiangxin Huoli decoction significantly reduces the abnormal hemodynamics, improves cardiac function, reduces plasma BNP concentration, regulates the expression of apoptosis proteins, inhibits the apoptosis of myocardial cells, and plays a protective role.

白术多糖抗神经细胞缺氧性凋亡的机制研究

目的:探讨白术多糖抗神经细胞缺氧性凋亡的机制。方法:研究分为正常对照组、凋亡阳性组、白术多糖0.025g/L组、白术多糖0.05g/L组、白术多糖0.1g/L组、白术多糖0.25g/L组。采用"Neurobasal+B27"体外神经细胞无血清培养,免疫细胞化学鉴定神经细胞,MTT测定药物毒性,Rh-123染色流式细胞仪检测线粒体损伤,RT-PCR及免疫细胞化学测定Caspase-3、Bax、Bcl-2 mRNA及蛋白表达。结果:与凋亡阳性组比较,白术多糖在0.025g/L^0.1g/L降低缺氧的神经细胞线粒体损伤,0.025g/L^0.05g/L下调缺氧的神经细胞Caspase-3 mRNA的表达,0.05g/L下调缺氧的神经细胞Bax mRNA的表达,0.025g/L^0.25g/L下调缺氧的神经细胞Caspase-3和Bax蛋白表达,0.025g/L^0.25g/L上调缺氧的神经细胞Bcl-2蛋白表达,0.025g/L^0.05g/L提高Bcl-2/Bax mRNA及0.025g/L^0.1g/L提高Bcl-2/Bax蛋白的比例(P<0.05)。结论:白术多糖在一定发范围内能有效地抑制神经细胞缺氧性凋亡,其机理是降低凋亡基因及蛋白产生,上调抗凋亡蛋白产生,提高Bcl-2/Bax比例。

木犀草素对小鼠T淋巴细胞体外增殖及凋亡的作用

目的：研究木犀草素对小鼠T淋巴细胞体外增殖及凋亡作用的影响。方法：设置刀豆蛋白A（Con A）组和空白组,设置3个木犀草素药物组,终质量浓度分别为2.5,5,10 mg·L-1,细胞毒性活性检测-8（CCK-8）法检测各组细胞增殖情况,末端脱氧核苷酸转移酶介导的d UTP缺口末端标记测定法（TUNEL）和Annexin V-FITC/PI双染法检测各组细胞凋亡情况,蛋白免疫印迹法（Western blot）检测各组细胞凋亡通路蛋白B淋巴细胞瘤-2（B-cell lymphoma-2,Bcl-2）,Bcl-2相关X蛋白（Bcl-2-associated X protein,Bax）表达水平。结果：CCK-8实验结果显示3个木犀草素药物组细胞数明显低于Con A组和空白组（P〈0.01）,差异有统计学意义。TUNEL实验结果显示,Con A组的凋亡细胞数量显著低于3个木犀草素药物组（P〈0.01）,差异有统计学意义。流式细胞仪检测结果显示3个木犀草素药物组的细胞凋亡率显著高于Con A组和空白组（P〈0.01）,细胞凋亡率随着药物浓度增加而增高,呈明显的量-效关系。Western blot检测结果显示,2.5,5 mg·L-1木犀草素药物组中Bcl-2表达量高于对照组,Bax表达量低于对照组;10 mg·L-1药物组中Bcl-2表达量低于对照组,Bax表达量高于对照组。结论：2.5,5,10 mg·L-1木犀草素能够抑制T淋巴细胞增殖,能够诱导T淋巴细胞凋亡,10 mg·L-1木犀草素可能主要通过下调Bcl-2,上调Bax影响线粒体凋亡通路诱导细胞凋亡,2.5,5 mg·L-1木犀草素可能通过其他通路诱导细胞凋亡。