Effect and Mechanism of Yiyi Fuzi Baijiangsan on Apoptosis of Human Colon Cancer HCT116 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of Yiyi Fuzi Baijiangsan （YYFZBJ） on the apoptosis of colon cancer cell line HCT116. MethodYYFZBJ at different concentrations （0.5， 1， 2， 4， 6， 8， 10， 12， 14， 16 g·L-1） was used to intervene in HCT116 cells for 24， 48， 72 h. The cell counting kit-8 （CCK-8） method was used to determine the effect of YYFZBJ on cell proliferation in vitro. The cells were divided into a blank group， a capecitabine group（1.8 g·L-1）， and low-， medium-， and high-dose YYFZBJ groups （6， 10， and 14 g·L-1） and treated for 48 hours. Flow cytometry was used to detect the apoptosis. Hoechst 33342 staining was used to observe the apoptotic morphology of cells. Mitochondrial membrane potential （MMP） was analyzed by a mitochondrial-targeted deep-red fluorescent probe （Mito-Tracker Red CMXRos）. The expression of proteins related to the mitochondrial apoptosis pathway， such as B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cytochrome C （Cyt C）， cysteinyl aspartate-specific protease （Caspase）-9， Caspase-3， cleaved Caspase-9， and cleaved Caspase-3 was detected by Western blot. The mRNA levels of Bcl-2， Bax， Cyt C， Caspase-9， and Caspase-3 were determined by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， YYFZBJ （8， 10， 12， 14， 16 g·L-1） significantly inhibited the proliferation of HCT116 cells in vitro （P<0.05） in a dose-dependent manner. Compared with the blank group， the medium- and high-dose YYFZBJ groups and the capecitabine group showed increased apoptosis rates of colon cancer cells （P<0.05）. The YYFZBJ groups and the capecitabine group showed reduced number of colon cancer cells with significantly changed cellular morphology and cell apoptosis manifestations， such as strong dark blue fluorescence， nucleus concentration， shrinkage， and fragmentation. With the increase in the mass concentration of YYFZBJ， the blue fluorescence intensity was significantly enhanced. Compared with the blank group， the YYFZBJ groups and the capecitabine group showed reduced MMP in a dose-dependent manner， decreased protein and mRNA levels of Bcl-2， and increased protein expression of Bax， Cyt C， Caspase-9， Caspase-3， cleaved Caspase-9， and cleaved Caspase-3 and mRNA expression of Bax， Cyt C， Caspase-9， and Caspase-3 （P<0.05）. ConclusionYYFZBJ can induce the apoptosis of colon cancer HCT116 cells through the mitochondrial apoptosis pathway.

BIX-01294 inhibits the proliferation of esophageal squamous cell carcinoma cells by inducing DNA damage and activating the mitochondrial apoptosis pathway / 中国胸心血管外科临床杂志

@#Objective    To explore the effects and molecular mechanisms of histone methylase G9a inhibitor BIX-01294 on apoptosis in esophageal squamous cell carcinoma (ESCC). Methods    MTT assay and Colony-forming Units were adopted to determine the effects of BIX-01294 on the growth and proliferation of ESCC cell lines EC109 and KYSE150. Flow cytometry was used to analyze the apoptosis status of ESCC cells after the treatment of BIX-01294. The effects of BIX-01294 treatment on the expressions of G9a catalytic product H3K9me2, DNA double-strand break (DSB) markers, and apoptosis-related proteins were detected by Western blotting. Results    BIX-01294 inhibited the growth of EC109 and KYSE150 cells in a dose-dependent manner (P<0.05), and BIX-01294 with the inhibitory concentration 50%(IC50) significantly inhibited the formation of colony (P<0.05). After 24 hours treatment of BIX-01294 (IC50), the apoptosis rate of EC109 cells increased from 11.5%±2.1% to 42.5%±5.4%, and KYSE150 cells from 7.5%±0.9% to 49.2%±5.2%(P<0.05). The expression level of the G9a catalytic product, H3K9me2, significantly decreased (P<0.05); while the expression of the DSB marker γH2AX was dramatically enhanced (P<0.05). We also found that the mitochondrial apoptosis pathway was activated and the expression levels of cleaved caspase3 and cleaved PARP were significantly elevated (P<0.05). Conclusion    BIX-01294, the inhibitor of methyltransferase G9a, prompted apoptosis in ESCC cells by inducing DSB damage and activating mitochondrial apoptosis pathway.

Neuroprotective effects of kukoamine A against rotenone-induced neurotoxicity in PC12 cells / 中草药

Objective: To investigate the neuroprotective effects of kukoamine A (KuA) on rotenone-induced PC12 cells damage and to preliminary verify its potential action mechanisms. The present study may lay the foundation for finding leading compounds with anti-Parkinson's disease (PD) effects. Methods: A PD model induced by rotenone was established in vitro, and MTT, LDH, and Hoechst33342 staining were used for preliminary confirmation of KuA resistance to rotenone-induced PC12 cell injury in vitro. The effects of KuA on superoxide dismutase (SOD) activity, malondialdehyde (MDA) and reactive oxygen species (ROS) content, and mitochondrial membrane potential (MMP) were investigated by colorimetric method and fluorescence staining, respectively. Western blotting was applied to explore the underlying mechanisms of protective effects of KuA against rotenone-induced PC12 cells damage. Results: The PC12 cell viability was significantly decreased after exposure to 0.5 μmol/L rotenone, whereas pretreatment with different concentrations of KuA could attenuate the cell injury induced by rotenone. Compared with the rotenone-treated group, KuA could decrease the ROS production and MDA level, while increase the SOD activity. In addition, KuA could effectively increase the MMP, decrease the cytochrome c release and the Bax/Bcl-2 ratio as well as inhibit caspase-3, caspase-9, and α-synuclein protein expressions. Conclusion: KuA showed neuroprotective ability on rotenone-induced PC12 cells PD model and the potential protective mechanisms of KuA can be related with inhibition of ROS generation, protection of MMP, regulation of protein expressions involved in the mitochondrial apoptosis pathway and reduction of α-synuclein expression.

Effects of cinnamaldehyde on apoptosis of oesophageal squamous cell carcinoma Eca109 cells and its mechanism / 中草药

Objective: To investigate effects of cinnamaldehyde (CA) on the proliferation and apoptosis of oesophageal squamous cell carcinoma Eca109 cells and to explore its mechanism. Methods: The effects of different concentrations (1.88, 3.75, 7.50, 15.00, 30.00 μg/mL) of CA on the proliferation of Eca109 cells were detected by MTS assay; Morphological changes were observed by phase contrast microscope; Flow cytometry was used to detect cell cycle distribution and apoptosis rate of Eca109 at different concentrations of CA; The expression of apoptosis-related protein Caspase-3/Caspase-9, pro-apoptosis protein Bax and anti-apoptosis protein Bcl-2 and Mcl-1 in ECA109 cells treated with different concentrations of CA for 24 h was detected by Western blotting. Results: After treated by CA with a concentration of 15.00, 30.00 μg/mL for 24 h, compared with the control group, the cell proliferation of Eca109 cells was significantly inhibited [(42.91 ± 2.15)%, (36.04 ± 2.97)% vs (100.00 ± 0.00)%, P < 0.05]; After treated by CA, Eca109 cells showed obvious apoptotic morphological changes; After treated with different concentrations of CA for 24 h, the proportion of cells in G0/G1 phase was decreased significantly (P < 0.05), while the proportion of cells in G2/M phase was increased significantly (P < 0.05); After treated by CA for 24 h, the apoptosis rate of Eca109 cells was increased significantly [(4.3 ± 0.11)%, (4.8 ± 0.07)%, (9.1 ± 0.13)% vs (1.0 ± 0.03)%, P < 0.05]; Compared with the control group, the fragment of protein Caspase-3 and Caspase-9 was significantly cleaved in Eca109 cells treated with different concentrations of CA for 24 h (P < 0.05); The expression levels of anti-apoptotic protein Bcl-2 and Mcl-1 were significantly decreased (P < 0.05), while the pro-apoptotic protein Bax expression was significantly up-regulated (P < 0.05). Conclusion: Cinnamaldehyde can inhibit the proliferation of oesophageal squamous cell carcinoma Eca109 cells and promote its apoptosis. The mechanism may be associated with the up-regulation of Caspase-3, Caspase-9, pro-apoptotic protein Bax and down-regulation of anti-apoptotic protein Bcl-2 and Mcl-1.

Herbal cake-partitioned moxibustion improves intestinal epithelial barrier dysfunction by suppressing TNF-α-mediated apoptosis pathway of intestinal epithelium in rats with Crohn's disease / 针刺研究

OBJECTIVE: To observe the effect of herbal cake-partitioned moxibustion (Moxi) on tumor necrosis factor (TNF)-α/ TNF receptor 1 (TNFR1)-associated death domain (TRADD) / Fas-associated death domain (FADD) pathway-mediated apoptosis of intestinal epithelial cells in Crohn's disease (CD) rats, so as to explore its underlying mechanisms in the treatment of CD. METHODS: Forty-eight SD male rats were randomly divided into normal, model, Moxi and medication groups (n＝12 rats in each). The CD model was established by intra-annual perfusion of 2，4，6-trinitrobenzene sulfonic acid (TNBS) solution (TNBS∶50% alcohol＝2∶1, 3 mL/kg), once every 7 days, 4 times altogether. For rats of the Moxi group, moxibustion was given to "Tianshu" (ST25) and "Qihai" (CV6), two moxa-cones every time, once daily for 10 days. For rats of the medication group, intragastric perfusion of mesalazine solution was given twice daily for 10 days. After the treatment, the colonic epithelium tissue was sampled. The epithelial cells were purified and cultured to establish an in vitro intestinal epithelial barrier, and added with TNF-α (a pro-inflammatory factor, 100 ng/mL) in the culture medium for 24 h for making an increased epithelial permeability model. The permeability of intestinal epithelial cell barrier was evaluated by detecting the fluorescence yellow transmittance of the TNF-α-incubated cell medium. Western blot was used to detect the expression levels of TNFR1, TRADD, receptor-interacting protein 1 (RIP1), FADD and zinc finger protein A20 (A20, a ubiquitination enzyme for inhibiting activation of TRADD and RIP1) of the cultured intestinal epithelium cells. The apoptosis of the TNF-α-incubated intestinal epithelial cells was detected by flow cytometry. RESULTS: After modeling and compared with the normal group, the fluorescence yellow transmittance of intestinal epithelia cells, apoptosis rate, and expression levels of TNFR1, TRADD, and RIP1 proteins were significantly increased (P<0.001, P<0.01), and the expression of A20 was significantly decreased (P<0.01) in the model group. In comparison with the model group, the fluorescence yellow transmittance of intestinal epithelial cells, the apoptosis rate and expression levels of TRADD, RIP1 and FADD were remarkably down-regulated (P<0.001, P<0.01), and the expression of A20 was significantly up-regulated (P<0.01) in both the Moxi and medication groups. CONCLUSION: Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.

Study on the Effect Mechanism of Iridoid Glycosides of Scrophularia ningpoensis Inhibiting Cardiomyocytes Apoptosis in Myo- cardial Infarction Model Rats Based on Different Caspase Apoptosis Pathways / 中国药房

OBJECTIVE： To study the effect mechanism of iridoid glycosides extracted from Scrophularia ningpoensis inhibiting cardiomyocytes apoptosis in myocardial infarction model rats. METHODS： The male Wistar rats were randomly divided into sham operation group， model group and S. ningpoensis iridoid glycosides low-dose， medium-dose and high-dose groups， with 10 rats in each group. Myocardial infarction models were established by ligating the left anterior descending coronary artery of the rats， and sham operation group was only threaded without ligation. After the model was established， each administration group was given S. ningpoensis iridoid glycosides suspension intragastrically at three different doses of 50，100，200 mg/kg （by the amount of total glycosides extract） with 10 mL/time， twice a day， for consecutive 7 days. Sham operation group and model group were given constant volume of normal saline intragastrically with same method. The changes of S-T segment of lead ECG Ⅱ were recorded before， after and during 7 days of administration. Cardiac function of rats was examined. The serum levels of LDH， CK-MB， cTnⅠ， NT-pro BNP and TNF-α were determined by colorimetry， immunosuppression or ELISA. The apoptosis of myocardial cells was observed by TUNEL method. SOD activity and MDA content in cardiac myocytes were detected by colorimetry. The expressions of Bcl-2， Bax， Cyt C， Caspase-8， Caspase-9， Caspase-12， Caspase-3 and Calpain in cardiac myocytes were detected by ELISA， enzymolysis colorimetry or enzymatic fluorescence assay. RESULTS： Compared with sham operation， electrocardiogram S-T segment was significantly elevated and the left ventricular end-diastolic diameter and left ventricular end-systolic diameter were significantly increased in the model group； left ventricular ejection fraction and short axis shortening rate decreased significantly； serum levels of LDH， CK-MB， cTnⅠ， NT-pro BNP and TNF-α were increased significantly； there were a large number of yellow-brown apoptotic cells in myocardial tissue； the activity of SOD in myocardial tissue was significantly decreased while the content of MDA was significantly increased； the protein expression level of Bcl-2 and Bcl-2/Bax were significantly decreased， while the levels of Bax， Cyt C， Caspase-3， Caspase-8， Caspase-9， Caspase-12 and Calpain were significantly increased （P＜0.05 or P＜0.01）. Compared with model group， above indexes and pathological changes of myocardial tissue were improved significantly in administration group； the level of Bcl-2 and Bcl-2/Bax in cardiomyocytes increased significantly， while the levels of Bax， Cyt C， Caspase-3， Caspase-8， Caspase-9， Caspase-12 and Calpain decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： S. ningpoensis iridoid glycosides can inhibit the activation of Caspase-3 by inhibiting three apoptotic pathways related to Caspase-8， Caspase-9 and Caspase-12， and then inhibit the apoptosis of cardiomyocytes.

The hepatoprotective effect of Aesculus hippocastanum seed extract against concanavalin A-induced acute liver injury in mice via inhibition of ROS and JNK pathway / 中国药理学通报

Aim To investigate the effect of Aesculus hippocastanum seed extract(AH) on concanavalin A (ConA)-induced acute liver injury in mice,and to ex-plore whether the mechanism was related to the inhibi-tory effect of AH on oxidative stress and c-Jun N-termi-nal kinase (JNK). Methods ConA(20 mg·kg-1) was administered via tail vein injecting to induce he-patic damage in mice. The groups of AH were given at 12.5,25,50 mg·kg-1by oral gavage separately for 20 days. The serum levels of AST,ALT,TP,and Alb were determined by automatic biochemical analyzer and the A/G ratio was calculated. TNF-α and IFN-γ levels were assayed by ELISA. The liver tissue was attained by HE and the histopathological changes were calculat-ed. The MDA, SOD, GSH contents of liver tissues were assayed by related kits. The activity of caspase-3 was detected by spectrophotometry. The expressions of cytochrome C and Bax, Bcl-2, p-JNK and p-Akt were detected by Western blot. Results The serum levels of ALT, AST, IFN-γ and TNF-α in AH groups were significantly lower than those in ConA-injured group, while the levels of TP,Alb and A/G were significantly higher. The SOD and GSH levels of liver tissues signif-icantly increased and MDA level decreased; liver his-topathological changes were consistent with those of the serological indicators, and AH treatment significantly reduced the pathological damage induced by ConA. In AH group,the expression of cytochrome C,caspase-3, Bax/Bcl-2 ratio and p-JNK markedly decreased, while the expression of p-Akt protein increased compared with ConA model group. Conclusion AH could sig-nificantly protect the ConA-induced acute liver injury in mice via inhibition of ROS and JNK pathway.

Antagonistic effect of vitamin E on di-2-ethylhexyl phthalate-induced reproductive toxicity in male rats / 中华男科学杂志

<p><b>Objective</b>To explore the antagonistic effect of vitamin E (VE) on male reproductive toxicity induced by di-2-ethylhexyl phthalate (DEHP) in pubertal SD rats and its underlying mechanisms.</p><p><b>METHODS</b>Thirty 5-week-old male SD rats were randomly divided into five groups of equal number, corn oil control, low-dose (10 mg/kg/d), medium-dose (100 mg/kg/d) and high-dose DEHP exposure (500 mg/kg/d), and VE intervention (high-dose DEHP + VE ［100 mg/kg/d］), and treated respectively for 30 successive days. At 3 days after treatment, the testes of the animals were harvested for determination of the oxidative stress index, serum reproductive hormone levels, cauda epididymal sperm parameters, and expressions of cell apoptosis-related genes and proteins.</p><p><b>RESULTS</b>Compared with the control group, the rats of the medium- and high-dose DEHP groups showed significant decreases in the levels of such serum reproductive hormones as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), sperm parameters as average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), linearity (LIN) and wobble (WOB), and the activities of superoxide dismutase (SOD) and glutathione peroxide (GSH-Px), but significant increases were observed in the latter two groups in the content of malondialdehyde (MDA)(［3.32±0.87］ nmol/mg pro vs ［2.13±0.49］ nmol/ mg pro), mRNA expressions of Bad, Bax, Cytochrome C, Caspase-3 and the Bax/Bcl-2 ratio, and protein expressions of Cytochrome C and Caspase-3. In comparison with the high-dose DEHP group, the VE intervention group exhibited remarkably increased serum LH and T levels, sperm VAP, VSL, VCL, STR and WOB, and activities of SOD and GSH-Px, but markedly decreased mRNA expressions of Bad, Bax, Cytochrome C, Caspase-3 and the Bax/Bcl-2 ratio as well as the protein expressions of Cytochrome C and Caspase-3 in the testis tissue (P<0.05).</p><p><b>CONCLUSIONS</b>Exposure to DEHP induces androgen secretion disorders, causes oxidative damage to the testicular tissue, activates the mitochondrial apoptosis pathway in the testis, and ultimately reduces the quality of epididymal sperm, while VE can protect the rat testis from DEHP-induced reproductive toxicity.</p>

Ethyl acetate extract of Pleione bulbocodioides(Franch.)Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mi-tochondrial apoptosis pathway / 中国病理生理杂志

AIM:To investigate the effects of ethyl acetate(EtOAc)extract of Pleione bulbocodioides (Franch.)Rolfe on proliferation and apoptosis of human leukemia K 562 and HL-60 cells and the possible apoptosis path-way.METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different con-centrations.XTT method was used to evaluate the viability of K 562 cells and HL-60 cells.The cell growth inhibition was calculated by Trypan blue exclusion test.The percentage of apoptotic cells was determined by flow cytometry,and 4,,6-dia-midino-2-phenylindole(DAPI)was used to observe morphological changes of the cells.The cell cycle was observed by pro-pidium iodide(PI)staining.The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose)polymerase(PARP), cleaved caspase-3,cytochrome C and apoptosis-inducing factor(AIF)wase determined by Western blot.RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC50of(42.14 ±2.54)mg/L for HL-60 cells and(51.28 ±3.12)mg/L for K562 cells at 24 h.The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner.The apoptotic rate was increased compared with control group(P<0.05).The G2phase increased with typical cell apoptosis-induced mor-phological changes.The levels of pro-apoptotic proteins Bax,cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated(P<0.05).Cytochrome C and AIF in cytosol,characteristic proteins of intrinsic mitochondrial apoptosis pathway,also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing(P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.

Apoptosis mechanism of gastric cancer cellSGC-7901 induced by Omphalia lapidescens protein pPeOp / 中国药理学通报

Aim To investigate the apoptosis mechanism of human gastric cancer cell SGC-7901 induced by Omphalia lapidescens protein pPeOp.Methods CCK-8 and flow cytometry were used to detect the inhibitory effect of different concentrations of pPeOp(30, 60, 90 mg·L-1) on SGC-7901.The mRNA and protein expression of TNF-R1, Fas/FasL, Bcl-2, caspase-3 and caspase-8 were detected by qRT-PCR and Western blot.Results SGC-7901 cells were treated with different concentrations of pPeOp(30, 60, 90 mg·L-1) for 24 h.CCK-8 test showed that there was no significant difference between PVP group and the control group.The survival rate of the 5-Fu group was(53.71±7.34)% (P<0.05).The survival rates of pPeOp group(30, 60, 90 mg·L-1) were(80.95±6.25)%, (53.48±5.70)% and(44.61±6.50)%(r=0.984,P=0.016),respectively.Flow cytometry showed that the apoptosis rate of PVP group had no significant difference with control group, and the apoptosis rate of 5-Fu group was about(39.30±3.34)%(P<0.05).The apoptotic rates of pPeOp group(30, 60, 90 mg·L-1) were(10.90±1.25)%, (28.80±2.70)% and (32.00±3.50)%,respectively(P<0.05).The mRNA and protein expression levels of Bcl-2 were down-regulated,whereas the expression of TNF-R1, Fas/FasL, caspase-3 and caspase-8 were significantly up-regulated(P<0.05).Conclusions pPeOp can significantly inhibit the proliferation of gastric cancer cell line SGC-7901 and induce apoptosis in a dose-dependent manner.Death receptor pathway and mitochondrial pathway may be related to pPeOp-induced apoptosis of gastric cancer SGC-7901.

HSP70 inhibitor combined with cisplatin suppresses the cervical cancer proliferation in vitro and transplanted tumor growth: An experimental study

Objective To study the regulating effect of HSP70 inhibitor (PES) combined with cisplatin on cervical cancer proliferation in vitro and transplanted tumor growth. Methods Cervical cancer Hela cell lines were cultured and divided into control group, cisplatin group, PES group and cisplatin + PES group that were treated with serum-free DMEM, cisplatin with final concentration of 10 μmol/L, PES 20 μmol/L and cisplatin 10 μmol/L combined with PES with 20 μmol/L, respectively; animal models with cervical cancer xenografts were established and divided into control group, cisplatin group, PES group and cisplatin + PES group who received intra-tumor injection of normal saline, 10 μmol/L cisplatin, 20 μmol/L PES as well as 10 μmol/L cisplatin + 20 μmol/L PES, respectively. Cell proliferation activity, transplanted tumor volume and mitochondria apoptosis molecule expression were detected. Results Cell viability value and Bcl-2 mRNA expression in cells of cisplatin group, PES group and cisplatin + PES group were significantly lower than those of control group while Bax, Caspase-3 and Caspase-9 mRNA expression in cells were significantly higher than those of control group; transplanted tumor volume and the Bcl-2 mRNA expression in transplanted tumor tissue of cisplatin group, PES group and cisplatin + PES group were significantly lower than those of control group while Bax, Caspase-3 and Caspase-9 mRNA expression in transplanted tumor tissue were significantly higher than those of control group. Conclusions HSP70 inhibitor combined with cisplatin can inhibit cervical cancer cell proliferation in vitro and transplanted tumor growth through mitochondrial apoptosis pathway.

The Killing Effects and Preliminary Mechanism of Natural Plant-derived Antimicrobial Solution(PAMs)to Human Liver Cancer HEPG-2 Cells / 现代生物医学进展

Objective:In this study,a series of experiments were conducted to research the mechanism of anticancer and preliminary molecular effects of PAMs on the HEPG-2 cancer cells.Methods:Morphological observation and MTT assay were used to explore the inhibition and killing effect of PAMs acting on HEPG-2.AO/EB staining and Annexin V-FITC/PI staining were employed to observe the apoptosis of HEPG-2 treated with PAMs.The expression level of Foxm1,bcl-2 and others genes in HEPG-2 cells were detected by using qRT-PCR and western blot.Wound healing and transwell experiments determined if PAMs can inhibit the migration of HEPG-2.Results:PAMs can inhibit and kill HEPG-2 cells in time and dose-dependent manners,and the cytotoxic effects were closely related to the cell apoptosis.The mRNA expression of foxm1,bcl-2 and surviving gene were remarkably decreased in HEPG-2 cells after the treatment of PAMs.PAMs decreased the FoXM1 protein expression in HEPG-2 cells,while up-regulating thep53 protein expression.,and it could also inhibit the migration of cancer cells.Conclusions:The possible molecular mechanism for the killing of HEPG-2 cancer cells by PAMs was proposed.By down-regulating the expression of foxm1 and up-regulating the expression of p53,the transcriptional expression of their downstream target genes survivin and bcl-2 was inhibited or reduced,hence enhancing the cancer cell apoptosis.This study provides an important foundation for the development of anti-cancer Chinese folk medicine based on PAMs.

Combination of total Astragalus extract and total Panax notoginseng saponins strengthened the protective effects on brain damage through improving energy metabolism and inhibiting apoptosis after cerebral ischemia-reperfusion in mice / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects and molecular mechanisms of the combination between total Astragalus extract (TAE) and total Panax notoginseng saponins (TPNS) against cerebral ischemia-reperfusion injury.</p><p><b>METHODS</b>C57BL/6 mice were randomly divided into sham-operated group, model group, TAE (110 mg/kg) group, TPNS (115 mg/kg) group, TAE-TPNS combination group and Edaravone (4 mg/kg) group, treated for 4 days, then, cerebral ischemia-reperfusion injury was established by bilateral common carotid artery (CCA) ligation for 20 min followed by reperfusion for 1 and 24 h.</p><p><b>RESULTS</b>TPNS could increase adenosine triphosphate (ATP) level, TAE and TAE-TPNS combination increased ATP, adenosine diphosphate (ADP) contents and Na-K-ATPase activity, and the effects of TAE-TPNS combination were stronger than those of TAE or TPNS alone after reperfusion for 1 h. After reperfusion for 24 h, TAE, TPNS and TAE-TPNS combination significantly increased neurocyte survival rate and decreased the apoptosis rate as well as down-regulated the expression of phosphorylated c-June N-terminal kinase1/2 (p-JNK1/2), cytochrome C (Cyt C), cysteine aspartic acid-specific protease (Caspase)-9 and Caspase-3. Furthermore, the effects in TAE-TPNS combination were better than those in TAE or TPNS alone.</p><p><b>CONCLUSION</b>The combination of TAE 110 mg/kg and TPNS 115 mg/kg could strengthen protective effects on cerebral ischemia injury, the mechanism underlying might be related to improving jointly the early energy metabolism, and relieving the delayed apoptosis via inhibiting the mitochondrial apoptosis pathway of JNK signal transduction.</p>

HSP70 inhibitor combined with cisplatin suppresses the cervical cancer proliferation in vitro and transplanted tumor growth: An experimental study

OBJECTIVE@#To study the regulating effect of HSP70 inhibitor (PES) combined with cisplatin on cervical cancer proliferation in vitro and transplanted tumor growth.@*METHODS@#Cervical cancer Hela cell lines were cultured and divided into control group, cisplatin group, PES group and cisplatin + PES group that were treated with serum-free DMEM, cisplatin with final concentration of 10 μmol/L, PES 20 μmol/L and cisplatin 10 μmol/L combined with PES with 20 μmol/L, respectively; animal models with cervical cancer xenografts were established and divided into control group, cisplatin group, PES group and cisplatin + PES group who received intra-tumor injection of normal saline, 10 μmol/L cisplatin, 20 μmol/L PES as well as 10 μmol/L cisplatin + 20 μmol/L PES, respectively. Cell proliferation activity, transplanted tumor volume and mitochondria apoptosis molecule expression were detected.@*RESULTS@#Cell viability value and Bcl-2 mRNA expression in cells of cisplatin group, PES group and cisplatin + PES group were significantly lower than those of control group while Bax, Caspase-3 and Caspase-9 mRNA expression in cells were significantly higher than those of control group; transplanted tumor volume and the Bcl-2 mRNA expression in transplanted tumor tissue of cisplatin group, PES group and cisplatin + PES group were significantly lower than those of control group while Bax, Caspase-3 and Caspase-9 mRNA expression in transplanted tumor tissue were significantly higher than those of control group.@*CONCLUSIONS@#HSP70 inhibitor combined with cisplatin can inhibit cervical cancer cell proliferation in vitro and transplanted tumor growth through mitochondrial apoptosis pathway.

Mechanism of SARI gene promoting apoptosis in acute myeloid leukemia cells / 中国免疫学杂志

Objective:To explore the apoptotic mechanisms of acute myeloid leukemia(AML) cells by a lentivirus mediated-SARI(suppressor of AP-1,regulated by IFN) overexpression system.Methods: The lentiviral vectors of overexpression SARI were transfected into AML cell HL-60 and NB4.Real-time quantitative PCR and Western blot were employed in the following evaluation tests.Both the cells were all divided into blank control group(CON group),empty vector control group(NC group)and SARI overexpression group(SARI group).Western blot was used to detect the expressions of apoptosis-related proteins Bcl-2,Bcl-xl,Bax and apoptotic pathway related proteins CytoC,Caspase8,Caspase9,Caspase3,Actived-Caspase3,PARP.Results:The SARI groups exhibited a high level of SARI mRNA and SARI protein when compared with CON group and NC group(P<0.05).In both the SARI groups,the expression of anti-apoptotic proteins Bcl-2,Bcl-xl were down-regulated and the pro-apoptotic protein Bax was up-regulated.CytoC increased,Caspase8,Caspase9 and Caspase3 decreased,Actived-Caspase3 up-regulated.PARP down-regulated,and PARP spliced variant up-regulated.Conclusion: SARI might promote the apoptosis of AML cells through activing the exogenous and endogenous apoptotic pathways which crossed to exogenous.

Methyl pyropheophorbide-a-mediated photodynamic therapy induces apoptosis in human ovarian cancer cells / 第二军医大学学报

Objective To investigate the mechanism by which methyl pyropheophorbide-a-mediated photodynamic therapy (Mppa-PDT) inhibit cell viability and induce apoptosis in human ovarian cancer cell line SKOV3. Methods Human ovarian cancer cells SKOV3 at the logarithmic growth phase were divided into Mppa-PDT treated group (both Mppa and PDT treated group) and control groups (the blank group, the only Mppa treated group and only PDT treated group). After Mppa-PDT treatment, the cell viability was examined with CCK-8 assay; cell apoptosis was detected by flow cytometry with Annexin -FITC/PI; and nuclear morphological changes during cell apoptosis was detected by DAPI staning. Moreover, the celluar reactive oxygen species (ROS) were detected by DCFH-DA staining; DNA damage was observed by single cell gel electrophoresis; and the protein expression of p53, Caspase-3, Bax, and Bcl-2 were assessed by Western blotting analysis. Results (1) Mppa-PDT could greatly suppress the cell viability of human ovarian cancer cells SKOV3 in a dose-dependent manner. (2) The cell apoptosis rate of Mppa-PDT treated group was significanlty higher than those of three control groups (blank group, Mppa group and PDT group) (P0.05). (3) After treating with Mppa-PDT, DAPI staining showed strongly stained nuclei of the apoptotic cells; DCFH-DA staining displayed higher level of ROS than those of the three control groups; single cell gel electrophoresis showed greater DNA damage than those of the three control groups; and Western blotting analysis showed that the expression of p53, Caspase-3 and Bax protein was increased and Bcl-2 protein was decreased (P<.05). Conclusion Mppa-PDT can significantly suppress cell viability and induce apoptosis in human ovarian cancer cell SKOV3, accompanied by DNA damage and the activation of mitochondrial apoptosis pathway.

Protective effect of PPAR-αagonist on PAN-induced podocyte injury / 中国药理学通报

Aim To investigate the function of fenofi-brate on PAN ( puromycin aminonucleoside )-induced podocyte injury. Methods SD female rats of 18-week-old were randomly assigned into 3 groups ( n =6 ) . Mice in PAN group and fenofibrate treated group received a single intravenous injection of PAN ( 65 mg ·kg-1 ) , while those in control group received equal volume of saline. Mice in fenofibrate treated group re-ceived 40 mg · kg-1 · d-1 of fenofibrate ( intragastric administration ) on day 1 after PAN injection , while those in PAN group and control group received equal volume of vehicle. 24 hours urine samples from all group were collected on day 0(1 day before PAN injec-tion), day 6, day 10. The 24 hours urine protein was detected by Bradford assay. All the rats were sacrificed 10 days after the induction of podocyte injury, and glo-merulus sample were collected. The expression of podocyte injury marker and transcription level in apop-tosis, podocyte cytoskeleton protein, slit diaphragm protein were evaluated by Western blot and real-time PCR. Results Compared with the control group, 10 days after injection of PAN, 24 hours urine protein was obviously increased, and the expression and transcrip-tion level of podocyte injury marker desmin, apoptosis, podocyte cytoskeleton protein, slit diaphragm protein were upregulated greatly, however, those were signifi-cantly lower in fenofibrate treated group as compared with those in PAN group. Conclusions PPAR-α ago-nist fenofibrate can ameliorate PAN-induced glomerulus podocyte injury, and the mechanism involved may be associated with inhibition of the mitochondria apopto-sis, TGF-β/Smad pathway and p38 pathway.

Hydroxycamptothecin-induced apoptosis of human Tenon capsule fibroblast and its mechanism / 中华实验眼科杂志

Background The hyperplasia of human Tenon capsule fibroblasts (HTFs) is a common cause of filtering surgery failure in glaucomous eye.Researches demonstrated that hydroxycamptothecin is a cell cycle arresting drug and induce apoptosis of cancer and fibroblasts.However,its mechanism is currently less understood.Objective This study was to investigate whether hydroxycamptothecin induce the apoptosis of HTFs and explore the possible mechanism.Methods Human Tenon capsule tissue was obtained from EyeBank of Jiangsu Province Hospital.HTFs were cultured using explant method in vivo and passaged in DMEM containing 10％ FBS.The cells were identified using vimentin and keratin by immunochemistry,and the cells of generation 3-6 in the logarithmic growth phase were used in the experiment.The cells were incubated with 0.01,0.05 or 0.10 g/L hydroxycamptothecin for 5 minutes respectively,and the cells without any hydroxycamptothecin were served as the control group.Cell viability then was assessed by cell counting kit-8 (CCK8) for the optimal inhibition concentration.The cells were treated by 0.10 g/L hydroxycamptothecin for 24 hours,and the apoptotic rate of the cells were assayed with annexin V/PI double-staining.Mitochondrial membrane potential of HTFs was assessed using JC-1 staining.The expressions of caspase-3,caspase-9 and cytochrome C (cyt C) in mitochondria and cytoplasm of HTFs were detected by Western blot.Results The proliferative value (A450) of the HTFs 0,0.01,0.05,0.10 g/L was 0.9716±0.0608,0.8035 ± 0.0346,0.7048 ±0.0446,0.6265 ±0.0286,with a significant difference (F =26.372,P =0.002).A450 of HTFs in the 0.01,0.05,0.10 g/L groups was significantly lower than the control group (P＜0.05),with the lowest A450 value in the 0.10 g/L group.The apoptotic percentage of HTFs was (18.72±1.41)％,in the 0.10 g/L hydroxycamptothecin group and that of the control group was (3.67 ±0.36)％,showing a significant difference between them (t =-10.374,P=0.001).The expression intensity of caspase-3 and caspase-9 protein in HTFs was higher in the 0.10 g/L hydroxycamptothecin group than that in the control group.JC-1 staining showed that the green fluorescence of the monomer JC-1 in cytoplasm was stronger in the 0.10 g/L hydroxycamptothecin group than that in the control group,but the red fluorescence of the polymer JC-1 in the 0.10 g/L hydroxycamptothecin group was weaker than that in the control group.The grey scale of cyt C protein in HTFs in mitochondrion was 0.0605±0.0022 in the 0.10 g/L hydroxycamptothecin group,showing a significant increase in comparison with 0.0301 ±0.0016 of the control group (t=4.865,P=0.014).However,the grey scale of cyt C protein in cytoplasm was declined in the 0.10 g/L hydroxycamptothecin group than that in the control group (0.0605 ±0.0022 vs.0.0301 ±0.0016) (t =-11.177,P =0.001).Conclusions Hydroxycamptothecin can induce the apoptosis of HTFs through activating the mitochondrial apoptosis pathway.

BCL2 antagonist of cell death kinases, phosphatases, and ovarian cancer sensitivity to cisplatin / 부인종양

OBJECTIVE: The BCL2 family proteins are critical mediators of cellular apoptosis and, as such, have been implicated as determinants of cancer cell chemo-sensitivity. Recently, it has been demonstrated that the phosphorylation status of the BCL2 antagonist of cell death (BAD) protein may influence ovarian cancer (OVCA) cell sensitivity to cisplatin. Here, we sought to evaluate how kinase and phosphatase components of the BAD apoptosis pathway influence OVCA chemo-sensitivity. METHODS: Protein levels of cyclin-dependent kinase 1 (CDK1) and protein phosphatase 2C (PP2C) were measured by immunofluorescence in a series of 64 primary advanced-stage serous OVCA patient samples. In parallel, levels of cAMP-dependent protein kinase (PKA), AKT, and PP2C were quantified by Western blot analysis in paired mother/daughter platinum-sensitive/resistant OVCA cell lines (A2008/C13, A2780S/A2780CP, Chi/ChiR). BAD pathway kinase CDK1 was depleted using siRNA transfection, and the influence on BAD phosphorylation and cisplatin-induced apoptosis was evaluated. RESULTS: OVCA patient samples that demonstrated complete responses to primary platinum-based therapy demonstrated 4-fold higher CDK1 (p<0.0001) and 2-fold lower PP2C (p=0.14) protein levels than samples that demonstrated incomplete responses. Protein levels of PP2C were lower in the platinum-resistant versus that shown in the platinum-sensitive OVCA cell line sub-clones. Levels of PKA were higher in all platinum-resistant than in platinum-sensitive OVCA cell line sub-clones. Selective siRNA depletion of CDK1 increased sensitivity to cisplatin-induced apoptosis (p<0.002). CONCLUSION: BAD pathway kinases and phosphatases, including CDK1 and PP2C, are associated with OVCA sensitivity to platinum and may represent therapeutic opportunities to enhance cytotoxic efficacy.

Mechanism of HAG priming protocols inducing apoptosis of leukemia cell lines in vitro / 白血病·淋巴瘤

Objective To investigate the effect and the mechanism of different G-CSF-priming protocols on leukemia cell lines (HL-60 and U937) in vitro and provide the clinical guidance to clinical treatment of acute leukemia.Methods The leukemia cell lines HL-60 and U937 were used as model to detect the effects of three drugs alone and combined two drugs (HA) or three drugs (HAG) respectively.Cell viability and cell growth inhibition were performed by cell count kit-8 (CCK-8) assay.Apoptotic marker AnnexinV/PI,cell membrane surface antigen CD11b,cell cycle,mitochondrial membrane potential (JC-1) and Caspase-3 were determined by flow cytometry.Results After using of HAG for 48 h,HL-60 and U937 cells counts were decreased significantly and the apoptotic marker Annexin V was significantly increased. To compare the single drug group with two drug combination group,the result was significantly different (P ＜0.05),and the apoptosis of U937 cells was higher than HL-60 cell line.CD11b expression among the three groups did not change (P ＞ 0.05).Using of CAG and MAG,the mitochondrial nembrane potential of HL-60 and U937cells was increased,the three-drug combination group was significantly higher than single-drug group and control group (P ＜0.05); Caspase-3 was activated,the fluorescence intensities of Caspase-3 of the three-drug combination group and single drug group were significantly higher (P ＜0.05) comparing with the control group.Conclusion HAG regimen could induce leukemia cells to apoptosis through the reduction of mitochondrial membrane potential and the activation of Caspase-3 to induce apoptosis of leukemia cells.