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.

Protective effect of total triterpenoids from Chaenomeles speciosa against Helicobacter pylori-induced gastritis in mice / 中国中药杂志

This study investigated the protective effect of total triterpenoids from Chaenomeles speciosa against Helicobacter pylori(Hp)-induced gastritis in mice and explored its possible mechanism. The chronic atrophic gastritis(CAG) model mice were randomly divided into four groups of model, total triterpenoids from C. speciosa(50 and 100 mg·kg~(-1)) and triple therapy, with C57 BL/6 J mice without Hp infection taken as the normal group. Mice in the treatment groups were given corresponding drugs once a day for 4 weeks. Then the following indexes were detected: the contents of reactive oxygen species(ROS), monocyte chemotactic protein 1(MCP-1), keratinocyte chemokines(KC), TNF-α, IL-1β, IL-6, IL-18, IL-4 and IL-10 in blood and gastric tissue, the activities and contents of LDH, MPO, SOD, GSH-Px, CAT and MDA in gastric tissue and the activities of β-glucuronidase, β-galactosidase, cathepsins B and D in blood, gastric tissue and lysosome. Besides, the mRNA expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), Bcl-2, Bcl-xl, Bax and Bad in gastric tissue were determined by quantitative real-time PCR. Western blot was employed to detect the protein expression levels of TLR4, MyD88, p-IKKβ, p-IκBα, NOD-like receptor 3(NLRP3), apoptosis-associated speck-like protein(ASC), pro-caspase-1, caspase-1, thioredoxin-interacting protein(TXNIP), pro-IL-1β, pro-IL-18, Bcl-2, Bcl-xl, Bax, Bad, cytochrome C, apoptotic protease-activating factor-1(Apaf-1), pro-caspase-9, pro-caspase-3, cleaved-caspase-9, cleaved-caspase-3, poly(ADP-ribose) polymerase 1(PARP-1), cleaved-PARP-1 and cytosol and nucleus NF-κB p65 in gastric tissue. The results indicated that the total triterpenoids from C. speciosa significantly suppressed Hp proliferation, alleviated the damage to gastric mucosa and improved lymphocyte infiltration and gland atrophy. They were also effective in reducing the activities of β-glucuronidase, β-galactosidase, cathepsins B and D in blood and gastric tissue, elevating the activities of β-glucuronidase and cathepsin D in lysosomal organelles, decreasing the contents of ROS, MCP-1, KC, TNF-α, IL-1β, IL-6, IL-18 in blood, MDA content and MPO and LDH activities in gastric tissue and increasing the contents of IL-4 and IL-10 in blood and activities of SOD, CAT and GSH-Px in gastric tissue. Other phenomena were also observed after the treatment with total triterpenoids from C. speciosa, including the down-regulation of the mRNA and protein expression levels of TLR4, MyD88, Bax and Bad, the protein expression levels of p-IKKβ, p-IκBα, NLRP3, ASC, pro-caspase-1, caspase-1, TXNIP, pro-IL-1β, pro-IL-18, cytochrome C, Apaf-1, cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 and nuclear NF-κB p65, reduction of p-IKKβ/IKKβ and p-IκBα/IκBα ratios and up-regulation of the mRNA and protein expression levels of Bcl-2 and Bcl-xl, up-regulation of pro-caspase-9, pro-caspace-3, cytosol NF-κB p65 protein expression levels and Bcl-2/Bax and Bcl-xl/Bad ratios in gastric tissue. These aforementioned results suggest that the total triterpenoids from C. speciosa have significant protective effects against CAG induced by Hp, and its mechanism may be related to enhancing the function of endogenous antioxidant system, suppressing the oxidative stress and inflammatory reaction induced by Hp, correcting lysosomal dysfunction and inflammatory activation of TLR4/NF-κB/NLRP3 inflammasome signaling pathway and thus inhibiting mitochondria-mediated apoptosis.

Protopine inhibits the growth of hepatocellular carcinoma through a mitochondrially mediated apoptosis pathway / 药学学报

Hepatocellular carcinoma (HCC) is a serious threat for human health, the incidence of HCC in China accounts for more than 50% worldwide. There is an urgent need to develop novel anticancer agents for the treatment of HCC patients. Here we characterized the inhibitory effect and the molecular mechanism of protopine on HCC cancer cells. The results of a CCK-8 assay indicated that protopine displays anticancer activities on HCC cells. Flow cytometry and JC-1 staining confirmed that treatment with protopine decreased the mitochondrial membrane potential and induced apoptosis in HCC cells. Western blot analysis showed that protopine was able to increase protein expression in the mitochondrial apoptotic pathway; the level of cytochrome C, apoptotic protease activating factor-1 (Apaf-1), Bax, cleaved-poly ADP-ribose polymerase (cleaved-PARP), cleaved-caspase-3, and cleaved-caspase-9 were increased while the expression of Bcl-2 was suppressed significantly. An in vivo study revealed that protopine significantly suppressed the growth of tumors in nude mice bearing HepG2 cells. Administration of protopine intraperitoneally at a concentration of 50 mg·kg-1 inhibited tumor growth by 72.46%. Animal experiments were carried out according to the Regulation of the Animal Ethics Committee of Southwest Medical University. This study provides preliminary evidence that there is potential to develop protopine as a lead compound for the treatment of HCC.

Effect of Ranae Oviductus Protein Hydrolysate on Ethanol-induced L-02 Cell Injury / 中国实验方剂学杂志

Objective:To study the protective effect and mechanism of Ranae Oviductus protein hydrolysate （ROPH） on the expression of pathway-related proteins in ethanol-induced L-02 cell injury. Method:The ROPH was prepared by compound enzymatic hydrolysis. L-02 cell injury model was induced with 400 mmol·L^-1ethanol. Cell viability was detected by cell counting kit-8 （CCK-8） assay. Cell cycle and apoptosis were examined by flow cytometry. JC-1/Hochest staining was employed for qualitative investigation. The expression of related proteins in apoptosis， mitogen-activated protein kinase （MAPK） signaling pathway， and pyroptosis in L-02 cells was detected by Western blot. Result:The results of the CCK-8 assay showed that 400 mmol·L^-1ethanol could induce L-02 cell injury within 12 hours. Compared with the blank group， the model group showed decreased viability of L-02 cells （<italic>P</italic><0.01）， elevated percentage of the cell cycle in the G₀/G₁ phase （<italic>P</italic><0.01）， increased total cell apoptosis rate （<italic>P</italic><0.01）， reduced mitochondrial membrane potential （<italic>P</italic><0.01）， up-regulated expression of apoptosis-related proteins ［B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， Cytochrome C （Cyt C）， and cysteine-dependent aspartate specific protease-3 （Caspase-3）］ （<italic>P</italic><0.05， <italic>P</italic><0.01） and MAPK signaling pathway-related proteins ［C-Jun amino-terminal kinase （JNK） and p38 MAPK］ （<italic>P</italic><0.05， <italic>P</italic><0.01）， and potentiated expression of pyrolysis-related proteins Caspase-1 and interleukin-1<italic>β </italic>（IL-1<italic>β</italic>） （<italic>P</italic><0.05）. Compared with the model group， the ROPH treatment group exhibited improved cell cycle arrest （<italic>P</italic><0.05， <italic>P</italic><0.01）， diminished total cell apoptosis rate （<italic>P</italic><0.01）， elevated mitochondrial membrane potential in a dose-dependent manner， down-regulated expression of Bax， Cyt C， and Caspase-3 proteins （<italic>P</italic><0.05， <italic>P</italic><0.01）， up-regulated expression of Bcl-2 protein （<italic>P</italic><0.05， <italic>P</italic><0.01）， and a downward trend in expression of proteins related to MAPK signaling pathway and pyrolysis （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:ROPH could inhibit oxidative stress-triggered liver injury in ethanol-induced cells by improving mitochondrial membrane potential， reducing the expression of proteins in the mitochondria-mediated apoptosis pathway， and inhibiting the expression of proteins related to the MAPK signaling pathway and pyrolysis pathway to reduce the mitochondrial dysfunction and inflammatory response in ethanol-induced L-02 liver cells and inhibit oxidative stress， thereby exerting a therapeutic role in alcoholic liver injury.

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.

Cis-3-O-p-hydroxycinnamoyl Ursolic Acid Induced ROS-Dependent p53-Mediated Mitochondrial Apoptosis in Oral Cancer Cells

Cis-3-O-p-hydroxycinnamoyl ursolic acid (HCUA), a triterpenoid compound, was purified from Elaeagnus oldhamii Maxim. This traditional medicinal plant has been used for treating rheumatoid arthritis and lung disorders as well as for its anti-inflammation and anticancer activities. This study aimed to investigate the anti-proliferative and apoptotic-inducing activities of HCUA in oral cancer cells. HCUA exhibited anti-proliferative activity in oral cancer cell lines (Ca9-22 and SAS cells), but not in normal oral fibroblasts. The inhibitory concentration of HCUA that resulted in 50% viability was 24.0 µM and 17.8 µM for Ca9-22 and SAS cells, respectively. Moreover, HCUA increased the number of cells in the sub-G1 arrest phase and apoptosis in a concentration-dependent manner in both oral cancer cell lines, but not in normal oral fibroblasts. Importantly, HCUA induced p53-mediated transcriptional regulation of pro-apoptotic proteins (Bax, Bak, Bim, Noxa, and PUMA), which are associated with mitochondrial apoptosis in oral cancer cells via the loss of mitochondrial membrane potential. HCUA triggered the production of intracellular reactive oxygen species (ROS) that was ascertained to be involved in HCUA-induced apoptosis by the ROS inhibitors YCG063 and N-acetyl-L-cysteine. As a result, HCUA had potential antitumor activity to oral cancer cells through eliciting ROS-dependent and p53-mediated mitochondrial apoptosis. Overall, HCUA could be applicable for the development of anticancer agents against human oral cancer.

Porcine deltacoronavirus induces mitochondrial apoptosis in ST cells / 生物工程学报

Porcine deltacoronavirus (PDCoV) is a newly emerging enteropathogenic swine coronavirus causing acute diarrhea and vomiting in pigs. The apoptosis of ST cells induced by PDCoV infection was studied in this research. In ST cells, caspase activity assay showed that the activity of caspase 3, caspase 8 and caspase 9 increased significantly with the infection of PDCoV, but not observed in UV irradiated PDCoV-infected cells, indicating that PDCoV infection activated both endogenous and exogenous apoptotic pathways in ST cells, and the induction of apoptosis depended on viral replication. To further investigate the endogenous apoptosis induced by PDCoV, cytochrome C and apoptosis-inducing factors in cytoplasm and mitochondria were detected. Compared with normal cells, the amount of cytochrome C released from mitochondria to cytoplasm increased significantly in PDCoV-infected cells, and the release increased with the prolongation of infection, while the apoptosis-inducing factor was always localized to mitochondria, suggesting that PDCoV induced apoptosis was initiated through caspase-dependent mitochondrial apoptosis pathway by promoting cytochrome C in the mitochondrial membrane gap into the cytosol. In conclusion, this study reveals the mechanism of PDCoV inducing apoptosis.

A new double MEK/PDK1 inhibitor 9za retards cell cycle progression at G0/G1 phase and induces mitochondrial apoptosis in non-small cell lung cancer cells

Objective: To identify the molecule mechanism of potential cytotoxicity and proapoptosis of 9za in non-small cell lung cancer (NSCLC) cells. Methods: MTT assay, cell cycle detection kit, JC-1 staining assay were adopted to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential, respectively. Cell apoptosis was measured by Annexin V-FITC/propidium iodide apoptosis detection and the morphology was observed under a light microscope and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosisrelated proteins and some proteins involved in the pathways. Results: MTT assay showed that 9za significantly weakened the viability of NSCLC cells. Cell cycle analysis demonstrated that the low concentrations of 9za arrested the cell cycle at G0/G1 phase, which was further confirmed by the down-regulated levels of Cyclin D1, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, Annexin V-FITC/propidium iodide apoptosis analysis, morphological observations and TUNEL assays revealed that the high concentrations of 9za could induce cell apoptosis. Furthermore, the JC-1 staining assay indicated that the mitochondrial membrane potential was reduced after 9za treatment. Western blot also manifested that 9za dramatically decreased the protein levels of the total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X in the cytoplasm. However, the levels of BCL2 associated X in the mitochondria, Cytochrome C in the cytoplasm, cleaved caspase-9, cleaved caspase-3 and the ratio of cleaved- PARP1/PARP1 showed the opposite trends. Moreover, the dose-dependently decreased phosphorylation levels of PDK1, protein kinase B, MEK and extracellular signal regulated kinase 1/2 following 9za treatment confirmed that 9za was indeed a double MEK/PDK1 inhibitor as we expected. Coadministration with PD0325901 or BX517 strengthened the cytotoxic and pro-apoptotic effect of 9za, verifying that 9za inhibited cell proliferation and induced cell apoptosis through the double MEK/PDK1 signaling pathways in NSCLC cells. Conclusions: This study demonstrates that 9za could play the effect of cytotoxicity and pro-apoptosis via the dual MEK/PDK1 signaling pathways in NSCLC cells and provides certain experimental foundation for 9za as a new type of drug candidate against non-small cell lung cancer.

Protective Effect and Mechanism of Aqueous Extract of Perillae Folium on Adriamycin-induced Oxidative Injury in HK-2 Cell / 中国实验方剂学杂志

Objective: To investigate the protective effect of Perillae Folium with aqueous extract (PFAE) on some key factors of Adriamycin (ADR)-induced oxidative injury in human renal tubular epithelial cells(HK-2), including the survival rate, oxidative injury indexes and cell apoptosis,in order to define the underlying mechanism. Method: A model of ADR-induced HK-2 cells oxidative injury was established in vitro, then cell viability was detected by cell counting kit-8 (CCK-8) after intervention with positive reference N-acetylcysteine (NAC) or PFAE (5,15,45 g·L-1) at different concentrations. According to the morphological changes under microscopy, the optimum concentration of PFAE was screened out for the follow-up experiments. Then, the experiments were divided into six groups:blank group, ADR (0.05 g·L-1) group, PFAE (15 g·L-1) group, ADR+PFAE (0.05+15) g·L-1 group, NAC (0.81 g·L-1) group, and ADR+NAC (0.05+81) g·L-1 group. After that, malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity(TAC) were measured in the cell homogenate after 24 h administration. The level of reactive oxygen species (ROS) was detected by 2',7'-dichloroflurescin diacetate (DCFH-DA) fluorescence probe. Flow cytometry and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to monitor the cell apoptosis. Western blot was used to observed the expressions of mitochondrial apoptosis-associated proteins, like B lymphocyte tumor-2 gene (Bcl-2), Bcl-2 related X protein (Bax), cysteine aspartate protease-9 (Caspase-9), cysteine aspartate protease-3 (Caspase-3) and poly ADP-ribose polymerase (PARP), as well as their shear bodies. In addition, the phosphorylation protein expressions of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signaling transduction pathway were detected by Western blot. Result: Compared with blank group, ADR group showed a decreased cell viability (PPPPPPPP-1. The ATC and SOD levels were increased in ADR+PFAE group and ADR+NAC group (PPConclusion: PFAE could alleviate the oxidative injury of HK-2 cells induced by ADR, and have an antioxidant effect, which inhibited cell apoptosis through mitochondrial apoptotic pathway and ERK/p38 MAPK signaling pathway.

High fatty acid induced H9c2 cardiomyocytes injury through mitochondrial pathways / 中国药理学通报

Aim: To explore the role of mitochondrial apoptosis pathway in high fatty acid induced injury in H9c2 cardiomyocytes. Methods: Cardiomyocytes were exposed to different concentrations of palmitic acid (PA) at 0 ∼0. 4 mmol · L-1 for 24 h and different time points (0 ∼48 h) of PA 0.2 mmol · L-1. Cell viability was measured by MTT; the intracellular reactive oxygen species (ROS) was detected by ROS kit; the cells were detected by apoptosis kit; the cell mitochondrial membrane potential changes were detected by mitochondrial membrane potential kit, and the protein expressions of mitochondrial apoptosis pathway such as Cyt-C, Cleaved caspase-3, Bax, and Bcl-2 were detected by Western blot. Results: When the cells were stimulated with PA for 24 h, the cell proliferation rates of 0. 2 and 0. 4 mmol · L-1 PA groups significantly decreased. The level of ROS increased gradually, the cell mitochondrial membrane potential decreased and the cell apoptosis increased. When the cells were stimulated with PA (0. 2 mmol · L-1) for 24 h, 36 h and 48 h, and all of the cell proliferation rates showed significant decline. Cardiomyocytes exposed to PA (0. 4 mmol · L-1) for 24 h showed an increase in the expression of mitochondrial related proteins (Cyt-C, Cleaved caspase-3 and Bax) (P < 0. 05), while Bcl-2 expression was significantly reduced (P < 0. 05). Conclusion: Mitochondrial apoptosis signaling pathway might play an important role in high fatty acid induced H9c2 myocardial injury.

Role of mitochondrial apoptosis pathway in palmitic acid-induced cell apoptosis in vascular endothelial cells / 中国药理学通报

Aim: To explore the role of mitochondrial apoptosis pathway in palmitic acid (PA) -induced cell apoptosis in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were exposed to different concentrations of PA (0. 1, 0. 2, 0. 4, 0. 8 mmol · L-1) for 24 h and different time points of 0.4 mmol · L-1 PA (0, 12, 24, 48 h). Cell viability was measured by MTT. The expression of reactive oxygen species (ROS) in HUVECs was detected by immunofluorescence. The level of intracellular apoptosis was detected by TUNEL assay. The protein expressions of mitochondrial apoptosis pathway proteins such as AIF, Cyt-C, cleaved caspase-3, Bcl-2, Bax were determined by Western blot. Results: HUVECs exposed to PA at 0. 4 mmol · L-1 for 24 h showed a decrease in their viability and an increase in the level of AIF, Cyt- C, cleaved caspase-3, Bax/Bcl-2 (P < 0. 05). Cell apoptosis level was significantly up-regulated (P < 0. 05). The intracellular apoptosis levels in the vascular endothelial cells treatment with mitochondrial apoptosis pathway inhibitor ciclosporine A (CsA) were significantly lower than those of PA group (P < 0. 05). Conclusion: Activated mitochondrial apoptosis pathway might play an important role in PA-induced cell apoptosis in vascular endothelial cells.

Effect of Descurainia sophia on H2O2-induced myocardial injury via suppressing oxidative stress and autophagy pathway / 中草药

Objective To study the protective effect of aqueous extract from Descurainia Sophia (DS) on H2O2-induced H9c2 cardiomyocyte injury and to initially explore the potential mechanism. Methods The peaks of main components in DS were analyzed and identified by HPLC-MS. H9c2 cell injury model was established by H2O2. H9c2 cells were cultured in vitro and divided into control group, model group, probucol group, and DS at 100, 200, 400 μg/mL groups. In order to reveal the possible molecular mechanisms, the viability of H9c2 cells was measured by MTT assay; The apoptosis rate, autophagy rate, mitochondrial membrane potential, and reactive oxygen species (ROS) level were detected by flow cytometry; The relative indicators of cell oxidative stress were determined by biochemical kit; The expression levels of apoptosis-related protein and the autophagy-related protein were evaluated by Incell-western method. Results Seven components with the highest content were identified in DS through the results of mass spectrometry. Compared with the model group, DS can improve the cell viability (P < 0.05, 0.01) and survival rate of H9c2 cells (P < 0.01); At the same time, apoptosis was attenuated (P < 0.01), mitochondrial membrane potential was upregulated (P < 0.01), apoptosis related proteins Caspase-3, Bax/Bcl-2 were obviously downregulated (P < 0.01), autophagy phenomenon was attenuated (P < 0.01), autophagy related proteins LC3B and p62 were upregulated (P < 0.01). In addition, ROS level was decreased (P < 0.01), T-SOD and GSH-PX were upregulated and the levels of LDH and MDA were significantly decreased (P < 0.01). Conclusion This study suggests that DS can effectively protect H2O2-induced H9c2 cells injury, and the mechanism may be associated with improving oxidative stress in cells, inhibiting cell apoptosis and autophagy, which may be related to flavonoid glycosides.

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.

Zuo Jin Wan formula inhibits cisplatin-resistance of gastric cancer cells via mitochondrial translocation of cofilin-1 / 中国药理学与毒理学杂志

OBJECTIVE Despite the status of cisplatin (DDP) as a classical chemotherapeutic agent in the treatment of cancer, the development of multidrug resistance often leads to a failure of DDP therapy.Traditional Chinese medicine(TCM)as adjuvant chemotherapy of cancer drugs in China has been widely used in cancer treatment.ZuoJin WAN (ZJW),a TCM formula,was proved reversing drug resistance in gastric cancer,but its exact mechanism was still unclear. METHODS CCK-8 assay was used to detect the cell viability. The levels of proteins and mRNA were evaluated using Western blot and q-PCR. Mitochondrial membrane potential was measured by fl ow cytometry. Depolymerisa-tion of F-actin and translocation of G-actin(gamma-actin)from the cytoplasm to the mitochondria was detected using an immuno fl uorescence assay. RESULTS phosphorylated coflin-1 (p-coflin-1) was overexpressed in the DDP-resistant human gastric cancer cell lines SGC7901/DDP and BGC823/DDP, relative to the respective parent cell lines(SGC7901 and BGC823),and DDP induced the dephosphory-lation of p-coflin-1 in both parent lines but not in the DDP-resistant lines. However, ZJW could induce the dephosphorylation of pcoflin-1 and promote coflin-1 translocation from the cytoplasm into the mito-chondria in both SGC7901/DDP and BGC823/DDP cells. This mitochondrial translocation of coflin-1 was found to induce the conversion of flamentous actin to globular-actin, activate mitochondrial dam-age and calcium overloading, and induce the mitochondrial apoptosis pathway. These effects of ZJW on DDP-resistant human gastric cancer cell lines could be reversed via transfection with coflin-1-specifc siRNA,or treatment with a PP1 and PP2A inhibitor.CONCLUSION ZJW can be used as an inhibitor of chemoresistance in gastric cancer, which may partly be due to dephosphorylation of p-coflin-1 via the activation of PP1 and PP2A.

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.

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.

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>

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>

Dihydroartemisinin inhibits proliferation of pancreatic cancer JF-305 cells by regulating expression of apoptosis related proteins and production of reactive oxygen species / 中国中药杂志

To investigate the effect of dihydroartemisinin on apoptosis of human pancreatic cancer cell line JF-305 and the role of reactive oxygen species(ROS) in the apoptosis of JF-305 cells induced by dihydroartemisinin. MTT assays were used to detect effect of different concentrations of dihydroartemisinin on cells proliferation of JF-305 lines. Cell cycle was detected by flow cytometry, and the apoptotic morphology was observed by Hoechst 333258 fluorescence staining. Annexin V fluorescence staining was used to detect the apoptosis changes of JF-305 cells, while DCFH-DA was used to detect the changes of ROS during apoptosis process. Western blot was used to detect the protein expression changes of Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9 and Cyto C. As compared with the control group, the JF-305 cells proliferation was inhibited significantly(P<0.05) after treatment with different concentrations of dihydroartemisimin for 48 h; cell cycle was blocked in the G2/M phase; apoptotic morphology of nuclear condensation, aggregation, and fragmentation was found, and the apoptosis ratio was increased(P<0.05). DCFH-DA detection showed that the cell ROS was increased significantly after dihydroartemisinin treatment(P<0.05). Western blot results showed that the expression of Bcl-2 protein was down-regulated; the expression of Bax protein was up-regulated; the ration of Bax/Bcl-2 was increased and the protein expression levels of Cleaved caspase-3, Cleaved caspase-9 and Cyto C were increased after dihydroartemisinin treatment. Therefore, dihydroartemisinin could induce apoptosis of JF-305 cells, and the possible mechanism may be related to the formation and increasing of ROS.