ABSTRACT
Aim To study the effect of butein on apop-tosis of PC12 cells induced by methylglyoxal (MG) and its mechanism. Methods Being pretreated with different concentrations of butein, PC12 cells were damaged by 1.5 mmol·L-1MG. Cell viability and cell toxicity were evaluated by MTT and LDH assay. Cell apoptosis and death were analyzed by PI and Ho-echst 33342. The antioxidant gene and proapoptotic gene expressions were determined by RT-PCR. The protein expression of p53 was detected by Western blot. Results Being pretreated with 2.5~10 μmol· L-1butein for 1 h significantly increased the cell via-bility,decreased LDH release,and protected from cell nuclei shrinkage, condensation and cleavage by MG. Meanwhile, butein increased the gene expression of SOD2, decreased the gene expression of proapoptotic genes p53 and caspase-9, and lowered the protein ex-pression of p53. Conclusion Butein can protect ap-optosis of PC12 cells from MG in a dose-dependent manner,which is linked with antioxidation and inhibi-ting p53 and caspase-9 gene expression.
ABSTRACT
Aim To investigate the peptides and its protection for vascular endothelial cells, derived from the absorbed components of rice α-globulin,which was shown to be effective in anti-atherosclerosis. Methods The amino acid sequence was purified by gel chro-matography and RP-HPLC, and determined by ESI/MS. Then the peptide was chemically synthesized. Hu-man umbilical vein endothelial cell injury model was induced by tumor necrosis factor-α. The cell viability was measured by cell counting kit to screen the appro-priate peptide intervention concentration. The apoptotic rate was detected by flow cytometry. Bcl-2, Bax, p-p38, vascular cell adhesion molecule and the protein expression level of NF-κB signaling pathway were de-tected by Western blot and immunofluorescent stai-ning. Results Apoptosis of HUVECs induced by TNF-α was significantly increased by YGEGSSEEG, which also regulated expression of Bcl-2/Bax proteins and inhibited phosphorylation of p38 protein. Besides, the peptide suppressed the production of VCAM-1, ICAM-1 and activation of NF-κB pathway. While it did not significantly improve the oxidative stress response in HUVECs. Conclusion Peptide YGEGSSEEG pro-tects vascular endothelial cells through suppressing ap-optosis and expression of adhesion molecules.
ABSTRACT
Aim To investigate the effect of sanguina-rine on regulating the pathway of cell apoptosis by in-ducing reactive oxygen species (ROS) in HepG2 cells. Methods MTT method was used to detect the cell viability of HepG2 cell after the treatment of san-guinarine. The changes of ROS were observed by indi-cator DCFH-DA and DHE staining. The apoptosis was detected by Hoechst 33342 and Annexin V/PI stai-ning;Rhodamine 123 staining was used to detect mito-chondrial membrane potential. Western blot was used to detect expressions of key cell-apoptotic protein. Re-sults The cell viability of HepG2 cells showed a de-creasing trend with the increasing concentration of san-guinarine. Sanguinarine could significantly increase cellular ROS,decrease mitochondrial membrane poten-tial in HepG2 cell, and promote apoptosis of HepG2 cells. The expression of Bax, cleaved-caspase-3 and cytoplasmic Cyt-C significantly increased after the treatment of sanguinarine, however, the expression of Bcl-2 was inhibited. Conclusion Sanguinarine could activate mitochondrial pathway of apoptosis mediated by cellular uncontrolled ROS and promote apoptosis of HepG2 cells.
ABSTRACT
Aim To investigate the neuroprotective effects of puerarin on H2O2-induced SH-SY5Y cell ap-optosis and the molecular mechanisms underlying the neuroprotective effects. Methods Neuron injury mod-el was established in vitro through H2O2-induced SH-SY5Y injury. MTT assay was performed to detect the effect of puerarin on H2O2-induced SH-SY5Y survival rates. Hoechst 33342 staining was used to observe the cell apoptosis. JC-1 staining was employed to detect the level of mitochondria membrane poential. Caspase-3 was determined by caspase-3 catalyze the substrate specificity Ac-DEVD-pNA. Caspase-9 was determined by caspase-9 catalyze the substrate specificity Ac-LE-HD-pNA. The effects of puerarin on the protein level of Bcl-2,Bax,p-Akt and Akt were determined by West-ern blot. Results The cell survival rate significantly increased after puerarin pretreatment compared with H2O2model group. Furthermore, puerarin pretreat-ment not only inhibited the decreasing of mitochondrial membrane potential,increasing of caspase-3, caspase-9 enzymatic activity and the expression of Bax,but also promoted the expression of p-Akt and Bcl-2, which was prevented by LY294002, an inhibitor of PI3K/Akt. Conclusion Puerarin can play a neuroprotective role for SH-SY5Y cell apoptosis induced by H2O2, maybe via activating PI3K/Akt signaling pathway.
ABSTRACT
AIM:To study whether cordycepin (Cordy) plays a role in myocardial protection by regulating the expression of microRNA-455 ( miR-455) and reducing apoptosis induced by endoplasmic reticulum stress in the ischemia -reperfusion (IR) rats.METHODS: SD rats (250 ~300 g) were randomly divided into 3 groups: control group, the chests of the rats were only opened;IR group, the rats were given myocardial ischemia for 30 min, and then reperfusion for 120 min;IR+Cordy group:before IR, the rats were given Cordy (10 mg/kg) through femoral vein injection once a day for one week, and the last injection was given 30 min before ischemia.Automated biochemical analysis was used to detect rat serum activity of LDH and CK-MB.The myocardial endothelial cell ( EC) apoptotic rate was measured by TUNEL , and the ultrastructural changes of the myocardial EC were observed under transmission electron microscope .RT-qPCR was used to detect the expression of miR-455 and the mRNA levels of glucose-regulated protein 78 ( Grp78) and caspase-12 in the myo-cardium.RESULTS:Compared with control group, EC apoptosis in IR group was significantly increased (P<0.05). Compared with IR group , EC apoptosis in IR +Cordy group decreased significantly ( P<0.05 ) .Compared with control group, swelling of mitochondria , irregular membrane , loose wrinkles with vacuoles , disappeared matrix granules , irregular nuclear membrane , chromatin condensation , disappeared nucleoli and even small apoptosis body in the EC were found in IR group.Compared with IR group , the symptoms in IR +Cordy group were greatly improved .Compared with control group, the mRNA expression of Grp78 and caspase-12 as well as the miR-455 level in IR group was increased (P<0.05). Compared with IR group , the mRNA expression of Grp78 and caspase-12, as well as the miR-455 level in IR+Cordy group was decreased (P<0.05).CONCLUSION:Cordycepin attenuates myocardial EC apoptosis , down-regulates the expres-sion of miR-455, and inhibits endoplasmic reticulum stress in the myocardium .
ABSTRACT
Aim To investigate the protective effects of Diterpene Ginkgolides Meglumine Injection(DGMI)on SY5 Y cells damaged by oxygen-glucose deprivation and its functional mechanisms.Methods After 4 h of OGD,the cells were treated with 25 mg·L-1 drugs for 1 h.Subsequently,cell viabilities were measured by cell counting kit-8(CCK-8 kit)and cell apoptosis was measured by flow cytometric analysis.Furthermore, the mitochondrial membrane potential was detected by rhodamine123 staining.The levels of phospho-p38, phospho-p53,Bcl-2,Bax and cleaved caspase-9/3 were evaluated by western blot.Results DGMI signif-icantly increased the cell viabilities of SY5 Y cells dam-aged by OGD,and reduced OGD-elicited dissipation of mitochondrial membrane potential and cell apoptosis. Furthermore,DGMI also reduced p-p38,p-p53,Bax/Bcl-2 ratio,cleaved caspase-9 and cleaved caspase-3. Conclusion DGMI shows good neuroprotective effects on SY5 Y cells after oxygen-glucose deprivation.The underlying mechanisms may be associated with the sup-pression of p38/p53/Bcl-2 /caspase-9/caspase-3 sig-naling pathway.
ABSTRACT
Background and purpose:Insulin-like growth factor-1 (IGF-1) is a peptide that participates in many biological processes by stimulating the downstream signaling pathways through their interaction with IGF-1 re-ceptor (IGF-1R) and insulin receptor (IR). Bone morphogenetic proteins (BMPs) are a group of functional proteins which participate in the biological processes of proliferation and migration in many kinds of cancers and have become a hot area of cancer research. The study aimed to investigate the effects of silencingIGF-1R gene on the expression level ofBMP2 gene, and the cell proliferation and apoptosis of SMMC7721 cells.Methods:The RNAi plasmid targetingIGF-1R gene was constructed and transfected into SMMC7721 cells. Then the inhibition effect on the expression level of IGF-1R and BMP2 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The SMMC7721 growth curve and cell apoptosis were detected by MTT assay and flow cytometry after they were transfected with RNAi plasmid.Results:The RNAi plasmid targetingIGF-1R gene was constructed successfully. The inhibition efficiencies at mRNA expression levels were 68.9% and 80.7% (IGF-1R gene), 79.5% and 83.3% (BMP2 gene), respectively, after transfection with IGF-1R-siRNA-1 and IGF-1R-siRNA-2 plasmid (P<0.05). The inhibition efficiencies at protein levels were 46.1% and 62.1% (IGF-1R gene,P<0.05), 42.5% and 60.9% (BMP2 gene,P<0.05), respectively. The results of MTT growth curve showed that the proliferation rate in the transfected SMMC7721 cells was significantly slower than that in the control group (P<0.05). The proportion of apoptotic cells in transfected groups was significantly higher than that in the control group (P<0.05).Conclusion:SilencingIGF-1R gene can downregulate the expression ofBMP2 gene at different levels that results in inhibition of cell proliferation and promotion of apoptosis in SMMC7721 cells.
ABSTRACT
Aim To investigate the effects of panax nonstaining saponins ( PNS ) on the apoptosis of renal cells induced by cisplatin through the path of mitochon-drion . Methods Male Sprague-Dawley( SD) rats were randomized divided into normal control group, cisplatin model group and the cisplatin+PNS group,with 12 rats of each group. Animals were sacrificed to determine the N-acetyl-β-D-Glucosaminidase ( NAG ) in urine, blood urea nitrogen ( BUN) and serum creatinine ( Cr) concentrations after 8d of intraperitoneal injection. HE-staining was employed to observe renal pathologies. Transmission electron microscopy ( TEM ) was em-ployed to observe the mitochondria of the rats′ injured kidney region. TUNEL staining was employed to detect the distribution of apoptotic cells. Immunnohistochem-istry was used to detect Bax and caspase-9 expression, and expression of Bcl-2 protein was detected by West-ern blot. Results Compared with the normal control group, contents of BUN, serum Cr and urinary NAG levels of rat in the cisplatin model group were increased ( P <0. 01 ) , and some mitochondria of the epithelial cells of renal tubules got injured seriously. The apopto-sis rate of kidney cell was increased ( P<0. 01 ) . The expression of Bax,caspase-9 and Bcl-2 proteins was in-creased ( P < 0. 01 ) . Compared with the cisplatin model group, contents of BUN, serum Cr and urinary NAG levels of rats in the cisplatin model group were decreased ( P <0. 01 ) , and some mitochondria of the epithelial cells of renal tubules were significantly im-proved. The apoptosis rate of kidney cell was decreased ( P<0. 01 ) . The expression of Bax and caspase-9 pro-tein was decreased (P<0. 01),but Bcl-2 protein was increased ( P < 0. 01 ) . Conclusion PNS may in-crease the expression of Bcl-2 protein and decrease the expression of Bax and caspase-9 proteins, which may play a protective role in cisplatin nephritic damage.
ABSTRACT
Objective To investigate the effects of brain-derived neurotrophic factor (BDNF) pretreatment on H9c2 myocardial hypoxia/reoxygenation (H/R) injury, and explore its mechanism. Methods The H9c2 myocardial cells were cul?tured in vitro and (95%O2+5%CO2) oxygen cultured 12 h after (95%N2+5%CO2) hypoxia cultured 4 h to establish the H/R model. The cells were divided into normal control group, H/R group, different concentrations (1, 10, 100μg/L) BDNF pre?treatment in H/R groups and TrkB-inhibitor group (with 100μg/L BDNF and 1∶1 000 TrkB inhibitor pre-treatment in H/R group). The cell survival rate was measured by MTT method in different groups. The lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA) and superoxide dismutase (SOD) content and activity were detected after H/R injury. The apoptotic rate of H9c2 myocardial cells were detected by flow cytometry, and the expressions of TrkB, Bcl-2 and Bax protein were detected by Western blot assay. Results Compared with the normal control group, the survival rate of H9c2 myocardial cells was decreased significantly in H/R model group (P < 0.05), LDH, CK and MDA contents were increased and SOD activity was decreased (P<0.05). The cell apoptosis rate was increased significantly (P<0.05). The anti-apoptosis Bcl-2 protein expression was decreased, pro-apoptosis Bax protein expression was increased in H/R model group (P<0.05). Compared with the H/R model group, the cell survival rates of H9c2 myocardial cells were increased after pre-treatment with different concentrations of BDNF (P<0.05);LDH, CK and MDA contents were decreased and SOD activity were in?creased respectively (P < 0.05). The cell apoptotic rates were decreased (P < 0.05). The expressions of TrkB receptor and Bcl-2 protein gradually increased, while the expression of Bax protein was gradually decreased (P<0.05). The role of BDNF was inhibited by TrkB inhibitor. Conclusion BDNF pre-treatment can promote the cell survival rate of H9c2 myocardial cells after H/R injury, which plays a protective role by inhibiting the cell apoptotic rate and maintaining antioxidant capacity, and associates with BDNF-TrkB signaling pathways.
ABSTRACT
Myocardial ischemia/reperfusion injury ( MI/RI) is a pathophysiological phenomenon commonly seen during thromboly-sis, percutaneous transluminal coronary angioplasty ( PTCA ) , and coronary artery bypass grafting ( CABG ) . It is defined as restoration of blood flow to a previously ischemic region followed by complex pathological events leading to tissue injury greater than the original ischemic insult. Many experimental interven-tions have been reported to protect the ischemic myocardium in experimental animals; however, with the exception of early reperfusion, none has been translated into clinical practice. The root of Panax notoginseng ( Burk. ) F. H. Chen ( PN) is one of the iconic herbs in traditional Chinese medicine. Traditional pharmacopeia recommended it among the most efficacious herbs for‘promoting blood circulation ’ and hemostasis. Inspired by this, in the last decade, a large number of modern investigators made substantial efforts to search the PN activities against a vari-ety of MIRI. The systematic review was performed according to the protecting drug of the MIRI development guidelines.
ABSTRACT
Aim To explore the possible protective effect of ginsenoside Rg1 on oligomeric Aβ1-42 induced apoptosis and its possible mechanism. Methods The damage was induced by oligomeric Aβ1-42 in primary cortical neurons. Cells were incubated in the absence or presence of Aβ, or co-incubated in sp600125 with Aβ, or pre-incubated in ginsenoside Rg1 then co-incu-bated in Aβ. The p-JNK, JNK, caspase-3 activity and TUNEL-positive cells were detected. Results In Aβ1-42 treated group, the ratio of p-JNK/JNK level was increased more than that in non-treated group for 15 min. However, in neurons preincubated with (2. 5, 5, 10 μmol·L-1 ) ginsenoside Rg1 and then co-incuba-ted with 5 μmol·L-1 oligomeric Aβ1-42 , the p-JNK/JNK ratio, caspase-3 activity and TUNEL positive neu-rons were significantly decreased compared with those of Aβ1-42 treated group. Conclusion Ginsenoside Rg1 can attenuate the oligomeric Aβ1-42-induced apop-tosis by JNK pathway.
ABSTRACT
Objective To investigate the tumor-inhibitory effect of cytokine-induced killer cells(CIK)co-cul-tured with dendritic cells (DC)pulsed by breast cancer stem cell antigen on the same tumor-bearing mice. Methods Breast cancer stem cells were isolated from the cell line of MCF-7/ADR and extract lyses antigen of the stem cell was saved. DC and CIK derived from peripheral blood mononuclear cells of healthy individuals were co-cultured and pulsed or un-pulsed by the above antigen lyses. This DC+CIK were injected to breast tumorbearing mice (BCSC-AP-DC+CIK group), and were used to compared with the common breast cancer cell antigen (rather than breast cancer stem cell antigen) pulsed DC+CIK group(AP-DC+CIK group), DC+CIK group, CIK CIK group and normal saline group(NS group). The tumor-inhibitory effect were evaluated and compared among all 5 groups through the tumor size, TdT-mediated dUTP nick end labeling test (TUNEL), examining expression level of bcl-2 and bax by immunohistochemistry. Results The tumor size in each group before and after therapy and the tumor size after therapy between each group was of significant difference(P＜0.05). The maximum size is NS group(3.625±0.093)cm3 and BCSC-AP-DC+CIK group is minimum,which is (1.234±0.131)cm3. BC-SC-AP-DC+CIK group is of highest expression of bax and apoptotic index value, lowest bcl-2 expression in all 5 groups. Conclusion The CIK co-cultured with DC pulsed breast cancer stem cell antigen was more effective to induce apoptosis of breast cancer cells than those of CIK cells co-cultured with DC pulsed breast cancer cell antigen,CIK cells co-cultured with DC and CIK cells.
ABSTRACT
Objective To study improvement of neural function by stereotaxic transplantation of adipose-derived stem cells (ADSC) into lateral cerebral ventricle after intracerebral hemorrhage in rats and its mechanism. Methods ADSC were cultured and proliferated in vitro, which had been marked with Brdu for 48 h before transplantation. The rat caudate nucleus hemorrhage (ICH) models were divided into 2 groups. ADSC were stereotaxically transplanted into the right lateral ventricles in ADSC group, and equal volume of saline was transplanted into control group. The score of neurological behavior were evaluated at modeling and 1, 3, 7, 14, 28 days after transplantation respectively.Double-staining immunofluorescence technique was used to detect Brdu-positive cells and the differentiation of neurons and astrocytes. In accordance with the instructions of TUNEL kit, cell apoptosis, and the expression of VEGF and angiogenesis were assayed. Results In vitro ADSC expressed undergo osteogenic and adipogenic differentiation. Compared with the control group, ADSC group had better motor function at 3, 7, and 14 days (P＜0. 05). Double-staining immunofluorescence showed mostly grafted Brdu-reactive ADSC had migrated to the hematoma zone, and some survivedand expressed Neun of Gfap. TUNEL analysis revealed that, 3 days after transplantation, the number of apoptotic cells in ADSC group was significantly less than in the control group (P＜0. 05). Three days after transplantation, VEGF expression levels in ADSC group were significantly higher than in the control group (P＜0. 05). Conclusion ADSC stereotaxially transplanted into the lateral ventricle can survive and differentiate into neuron-like cells. ADSC transplantation may reduce apoptosis and secret VEGF to promote the angiogenesis, and improve neural functional in intracerebral hemorrhage rats.
ABSTRACT
Akabane, Aino and Chuzan virus are arthropod-borne (arbo)viruses mainly associated with reproductive failures in cattle. We investigated apoptosis in Vero cells (C-1586) infected with Akabane, Aino and Chuzan virus. The fragmentation of chromosomal DNA was simultaneously detected with the progress of cytopathic effect from 48 hr to 72 hr post infection, depending on viruses. Although the treatment of cycloheximide blocked apoptosis in Vero cells infected with three viruses, actinomycin D did not prevent DNA oligomerization, thus indicating that de novo viral protein synthesis is critical for viral apoptosis. In addition, the activation of caspase-3 was also detected in Vero cells by indirect fluorescent assay. From the present results, it is of future interest whether apoptotic characteristics of these viruses are related to pathogenecity in vivo.
Subject(s)
Animals , Apoptosis/physiology , Bunyaviridae/physiology , Caspase 3 , Caspases/metabolism , Chlorocebus aethiops , Cytopathogenic Effect, Viral/physiology , DNA Fragmentation/physiology , Dactinomycin , Enzyme Activation , Orbivirus/physiology , Vero CellsABSTRACT
AIM: To explore the role of calcineurin i n th e expression of NF-?B and the neurotoxicity in cultured cortical neurons treate d with interleukin-1? (IL-1?) and NMDA. METHODS: The cultured rat cortical neurons were used in the expe riment, damage of neurons was induced by interleukin-1?(IL-1?) or excitator y amino acid (NMDA). The degree of neuron damage was examined with the methods o f MTT assay and LDH releasing rate assay, as well as the Annexin V and PI immuno fluorescence. The expression of NF-?B p65 on the neurons was tested by the West ern blot analysis. RESULTS: Viability of neurons was obviously lower in the IL-1? group and NMDA group respectively than that in control group (P0.05). Annexin V and PI immunofluoresc ence showed that IL-1? mainly induced the neuron apoptosis, and NMDA induced th e neuron necrosis. CONCLUSIONS: The calcineurin mediates the higher expression of N F -?B p65 and neuron damage induced by IL-1?, but not play a critical role in th e necrosis induced by NMDA in the cultured cortical neurons. These results indic ate that calcineurin is the key molecule in the apoptotic signaling pathway.