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ObjectiveTo study the effect of Bushen Huoxuetang on the apoptosis and the expression of B-cell lymphoma （Bcl-2）-associated X protein （Bax）/ Bcl-2 and cleaved cysteine-containing aspartate proteolytic enzyme-3 （cleaved Caspase-3） in the nude mouse model of bone metastasis of breast cancer， and explore the mechanism of Bushen Huoxuetang in inhibiting bone destruction. MethodThirty BALB/c female nude mice were randomly assigned into blank group （n=6） and model group （n=24）. The suspension of 4T1 breast cancer cells was injected into the tibia of mouse right lower limb to establish model of bone metastasis of breast cancer. The successfully modeled nude mice were randomly assigned into model group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group， with 6 mice in each group. Bushen Huoxuetang was administrated at a dose of 36.67 g·kg-1， once a day， and zoledronic acid was administrated by subcutaneous injection at a dose of 100 μg·kg-1， twice a week. The combined drug group was administrated with the same doses of Bushen Huoxuetang group by gavage and zoledronic acid by subcutaneous injection. The mice in the blank group and the model group were administrated with the same volume of distilled water by gavage for 14 days. On the next day at the end of drug administration， the mice were sacrificed by cervical dislocation. The general situation and weight changes of the mice were examined. The right lower limb was collected， and X-ray scanning and hematoxylin-eosin （HE） staining methods were used for observation of pathological changes in the bone. The terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of bone tissue in nude mice， and Western blot to determine the expression of Bax/Bcl-2 and cleaved Caspase-3 in the bone tissue. ResultCompared with the blank group， the modeling reduced the body weight （P<0.01） and increased the right lower limb weight of the nude mice （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination increased the body weight （P<0.01） and decreased the right lower limb weight （P<0.01）. Compared with the blank group， the other groups showed obvious tumor cell atypia， deep nuclear staining， and clear bone metastasis， and the model group showed obvious osteolytic damage in right lower limb and loss of proximal tibia and knee joint. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination reduced the osteolytic lesions in the right lower limb and recovered part of the bone structure， demonstrating an inhibitory effect on bone destruction. The TUNEL assay showed that the model group had lower apoptosis rate of bone metastatic tumor cells than the blank group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group （P<0.01）. Compared with the blank group， the modeling down-regulated the expression of Bax and cleaved Caspase-3 （P<0.01） and up-regulated the expression of Bcl-2 （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination up-regulated the expression of Bax （P<0.01） and cleaved Caspase-3 （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 （P<0.05， P<0.01）. ConclusionBushen Huoxuetang may inhibit bone destruction in the nude mouse model of bone metastasis of breast cancer by up-regulating the expression of Bax， down-regulating the expression of Bcl-2， activating cleaved Caspase-3， and further inducing apoptosis.
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Objective:To investigate the effect of<italic> Stemona tuberosa</italic> alkaloids on the apoptosis of human hepatoma SMMC-7721 cells and the expression of apoptosis-related proteins including B lymphocytoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）. Method:SMMC-7721 cells were routinely cultured， passaged， and treated with various concentrations （50， 75， 112， 167， and 250 mg·L<sup>-1</sup>） of <italic>S. tuberosa </italic>alkaloids， while those in the blank control group were only treated with 10% fetal bovine serum. The cell proliferation was determined by tetrazolium bromide （MTT） colorimetry and colony assay and the cell apoptosis by Hoechst 33258 staining. The protein expression levels of Bcl-2， Bax， and cleaved Caspase-3 were detected by Western blot. Result:<italic>S. tuberosa</italic> alkaloids inhibited the proliferation of SMMC-7721 cells， and the inhibition rate was significantly increased in comparison with that in the blank control group （<italic>P</italic><0.01）， with the half maximal inhibitory concentrations （IC<sub>50</sub>） at 24 h， 48 h， and 72 h being （173.36±8.75）， （112.14±16.50）， and （96.41±2.60）mg·L<sup>-1</sup>， respectively. The cell colony-inhibitory activity was significantly increased in a dose-dependent manner （<italic>P</italic><0.01）. Compared with the blank control group， <italic>S. tuberosa</italic> alkaloids promoted the apoptosis of SMMC-7721 cells， manifested as increased number of apoptotic cells and elevated apoptotic rate （<italic>P</italic><0.01）. The typical morphological changes such as brightly blue-fluorescent condensed nuclei， cytoplasmic shrinking， and karyopyknosis were found under the upright fluorescence microscope. As revealed by comparison with the blank control group， the expression of Bcl-2 was significantly down-regulated （<italic>P</italic><0.01）， while the protein expression levels of pro-apoptotic protein Bax and cleaved Caspase-3 in the 75， 112， 167， and 250 mg·L<sup>-1</sup> <italic>S. tuberosa</italic> alkaloids groups were significantly up-regulated （<italic>P</italic><0.01）. Conclusion:<italic>S. tuberosa </italic>alkaloids inhibit the proliferation of SMMC-7721 cells and promote their apoptosis possibly by inhibiting Bcl-2 protein expression and promoting Bax and cleaved Caspase-3 protein expression.
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:To investigate the effect of transient receptor potential melastatin 2 （TRPM2） inhibitor A10 on oxygen glucose deprivation/reperfusion （OGD/R） injury in SH-SY5Y cells．:Human neuroblastoma SH-SY5Y cells were subject to OGD/R injury，and then were divided into blank control group，model control group and A10 group randomly. The cell survival rate was detected by cell counting kit 8 （CCK-8）; the level of cellular reactive oxygen species （ROS） was detected by reactive oxygen detection kit; the mitochondrial membrane potential was detected by tetramethylrhodamine （TMRM） method; the number of apoptotic cells was detected by TUNEL apoptosis assay kit; the protein expression level of cleaved caspase 3 was detected by Western blot．:Compared with 3，20，30，50， has lower cytotoxicity and better inhibition effect on channel activity. Compared with the model control group，ROS level was reduced，the mitochondrial membrane potential was improved，the number of apoptosis cells was reduced ，and the expression of cleaved caspase 3 was significantly reduced in the A10 group（all <0.05）. : A10 can alleviate cell damage after OGD/R by inhibiting TRPM2 channel function，reducing extracellular calcium influx，reducing cell ROS levels，stabilizing mitochondrial membrane potential levels，and reducing apoptosis.
Subject(s)
Humans , Apoptosis , Benzeneacetamides , Cell Survival , Glucose , Oxygen/metabolism , Piperidones , Reactive Oxygen Species/metabolism , Reperfusion , TRPM Cation ChannelsABSTRACT
The aim of this study was to investigate the role of seminal plasma miR-210-3p in the impairment of semen quality caused by varicocele. This study included 102 patients whose semen quality was normal when they were diagnosed with varicocele. A 2-year follow-up for included patients was performed, and they were divided into Group A (semen quality became abnormal) and Group B (semen quality remained normal) according to the results of semen analysis during the follow-up. Semen parameters and seminal plasma miR-210-3p expression were investigated by semen analysis and quantitative real-time polymerase chain reaction, respectively. In vitro experiments with GC-2 cells were performed to explore the role of miR-210-3p in spermatogenic cells. The results of quantitative real-time polymerase chain reaction showed that the level of seminal plasma miR-210-3p in Group A was higher than that in Group B both after 2-year follow-up and when they were diagnosed with varicocele (both P < 0.01). Apoptosis and proliferation assays showed that miR-210-3p induces apoptosis of spermatogenic cells by promoting caspase-3 activation. In conclusion, our study indicated that seminal plasma miR-210-3p induces spermatogenic cell apoptosis by activating caspase-3 in patients with varicocele. Seminal plasma miR-210-3p may be a potential biomarker for predicting impaired semen quality caused by varicocele.
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Objective To investigate the effect of abdominal paracentesis drainage (APD) on pancreatic cell apoptosis in severe acute pancreatitis. Methods Male adult SD rats were randomized into the sham operation (SO) group, SAP group, and APD group, with 18 rats in each group. In the SAP group, 5% sodium sulfonate was pumped into the retrograde pancreatic bile duct to prepare the SAP model. On this basis, a gastric tube was introduced into the right lower abdomen for drainage, namely the APD group. Blood from the abdominal aorta and pancreatic tissues were collected at 6, 12, and 24 h time points in each group. The changes of serum amylase, inflammatory factor, and endotoxin were detected by ELISA. The HE staining was used to evaluate the pancreatic tissue injury. The apoptosis of pancreatic tissue was detected by TUNEL. Western blot and immunohistochemistry were used to detect the expression of apoptosis-related proteins and PI3K/AKT signaling pathway. Results Pancreatic tissue necrosis and edema were significantly lower in the APD group than in the SAP group, and the pathological score was decreased (P<0.05). Serum amylase, TNF-α, IL-1β, IL-6, and endotoxin levels in the APD group were significantly lower than those in the SAP group (P<0.05). The number of pancreatic cell apoptosis in the APD group was significantly higher than that in the SAP group (P<0.05), and the expression levels of pancreatic apoptotic proteins cleaved-caspase-3 and Bax were significantly increased in the APD group, while the expression levels of anti-apoptotic protein Bcl-2 were significantly decreased (P<0.05). Compared with the SAP group, the expression levels of PI3K/AKT signaling pathway key molecules p-PI3K, p-AKT, and NF-kB p65 were significantly decreased in the APD group (P<0.05). Conclusions Our data indicate that APD attenuates the severity of SAP by enhancing cell apoptosis via suppressing PI3K/AKT signaling pathway. This study provides a new theoretical basis for the treatment of severe acute pancreatitis with APD technology.
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Objective: To investigate the inhibitory effect of nitidine chloride (NC) on human esophageal carcinoma cell line Eca109 and the molecular mechanism of its induction. Methods: CCK-8 method was used to detect the inhibition rate of human esophageal cancer Eca109 cells with different concentrations of NC and different intervention time. According to the result of CCK-8 method, the experiment was divided into four groups, and the concentrations of NC in each group were 0, 5, 10, and 15 μmol/L, respectively, and the drug action time was 48 h. The apoptotic rate was detected by flow cytometry with Annexin V-FITC/PI double staining. The mRNA expressions of Caspase-3, Caspase-9, and Noxa were detected by qRT-PCR. The expressions of cleaved Caspase-3 and Bcl-2, p53, and Noxa protein levels were detected by Western blotting. Results: NC had inhibitory effect on Eca109 cells in a certain range of time and dose-dependent manner. Flow cytometry showed that NC at 5 μmol/L mainly induced early apoptosis (P < 0.01); NCs at 10 and 15 μmol/L mainly induced late apoptosis (P < 0.01). qRT-PCR results showed that the mRNA expression of Caspase-3, Caspase-9 and Noxa was increased with the increase of NC concentration, of which 10 and 15 μmol/L group increased significantly. The results of Western blotting showed that the protein levels of cleaved Caspase-3, p53, and Noxa were both increased with the increase of NC concentration (P < 0.01), but the increase of Noxa was not significant in 5 μmol/L group (P < 0.01). The expression of Bcl-2 protein was decreased with the increase of NC concentration, and which was significantly higher in 10 and 15 μmol/L groups (P < 0.05, P < 0.01). Conclusion: The inhibitory effect of NC on human esophageal carcinoma Eca109 cells is mainly through apoptosis. The apoptosis of NC induced of Eca109 cells is associated with increased expression of p53 and Noxa, downregulation of Bcl-2, and activation of Caspase-3.
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Aim To investigate the effect of asiatic acid(AA) on the proliferation, apoptosis and cell cycle arrest of human tongue carcinoma TCA-8113 cells, and the possible mechanism. Methods The inhibitory effect of AA(0, 20, 30, 40, 50 pjnol • L"1) at different concentrations on TCA-8113 cells for 24 h was determined by MTT. The effect of AA on the colony formation rate of TCA-8113 cells was detected by colony formation test; The apoptosis of TCA-8113 cells by AA was detected by Hoechst 33342 staining and Annexin V-FITC/PI staining. Cell cycle changes were analyzed by flow cytometry. The expressions of protein in human tongue cancer cells, such as bcl-2, Bax, cleaved caspase-3, p53 and p21 proteins were detected by Western blot. Results The inhibitory rate of AA on TCA-8113 cells was concentration-dependent (P < 0.05), with the IC50 concentration of 42. 13 (xmol • L- 1, and the ability of cell colony formation decreased. The apoptosis rate increased with the increase of AA concentration (P < 0. 05). The concentration of AA 30 u.mol -L"1 gradually blocked the cell cycle in G2/M phase. Western blot analysis showed that the expressions of p53, p21 and Bax increased, while Bcl-2 decreased in a dose-dependent effect (P<0. 05). Conclusions AA can significantly inhibit the proliferation and apoptosis of human tongue carcinoma TCA-8113 cells, and its mechanism may be related to the regulation of p53 pathway related proteins p53, p21, Bax and the inhibition of Bcl-2 expression, and AA can arrest TCA-8113 cells cycle in G2/M phase.
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Allicin is one of the main bioactive substances in garlic, with antibacterial, hypolipidemic and other pharmacological effects. In this study, apoptosis-related indicators were detected to explore the molecular mechanism of allicin on KG-1 cell proliferation inhibition. The apoptosis rate of KG-1 cells induced by allicin was detected by flow cytometry. The effect of allicin on the expressions of Bax, Bcl-2, survivin and ERK mRNA in KG-1 cells was detected by RT-qPCR. Western blot was used to detect the expressions of caspase 3, cleaved caspase 3, ERK1/2, p-ERK1/2 and survivin protein in KG-1 cells. According to the findings, compared with the control group, allicin could significantly inhibit the proliferation activity of KG-1 cells in a concentration-dependent and time-dependent manner. Flow cytometry showed that allicin could induce the apoptosis of KG-1 cells, which was mainly late apoptosis. The results of RT-qPCR showed that the expressions of Bax mRNA, Bcl-2, survivin and ERK mRNA in KG-1 cells increased after treatment with allicin. The results of Western-blot showed that after KG-1 cells were treated with allicin, the expressions of caspase 3 and its active form cleaved caspase 3 increased, the expressions of survivin, ERK1/2 and its active form p-ERK1/2 were decreased, of which p-ERK1/2 was down-regulated in a dose-dependent manner. The above results suggest that allicin inhibited the proliferation of KG-1 cells primarily by inducing late apoptosis; the execution of apoptosis involved cleaved caspase 3; the induction of apoptosis involved the protein expression, the decrease of ERK1/2andexpression of survivin and the dose-dependent decrease of p-ERK1/2; the mRNA expression involved the increase of Bax, and the down-regulation of survivin, Bcl-2 and ERK1/2.
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Objective To study the role of Cx43 in X-ray induced apoptosis of HUVEC cells and its mechanism.Methods Flow cytometry was used to detect the apoptosis of HUVEC cells at 48-96 h after 10 Gy X-ray irradiation and at 72 h after irradiation of different doses.Western blot was used to detect the protein expressions of Cx43 and cleaved caspase-3 in HUVEC cells at 72 h after 0,5,10 and 20 Gy irradiation.Small interfering RNA was transfected into HUVEC cells to silence Cx43 expression,the Cx43 bearing plasmid was transfected into cells to overexpress Cx43.The effect of Cx43 knockdown or overexpression on apoptosis induction and cleaved caspase-3 protein expression were detected by flow cytometry and Western blot,respectively.Results The apoptosis of HUVEC increased significantly from 48 h to 96 h after X-ray irradiation and in a dose-dependent manner at 72 h after irradiation.The expression of Cx43 protein was negatively correlated with the dose but the expression of cleaved caspase-3 was positively correlated with the dose in the range of 0-20 Gy.After Cx43 silencing,the proportion of early apoptosis and apoptosis combined with dead cells were significantly higher than that of the siRNA control group(t =3.674,6.375,P ＜ 0.05).After Cx43 overexpression,the proportion of early apoptosis and apoptosis combined with dead cells were significantly lower than that of vector control group(t =9.399,11.190,P ＜ 0.05).The expression of cleaved caspase-3 in the Cx43 silencing group was higher than that in the siRNA control group,but this protein in the Cx43 overexpressed group was lower than that in the vector control group.Conclusions Cx43 may protect X-ray irradiated HUVEC cells from apoptosis by down-regulating the activation of caspase-3.
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Purpose To investigate the effects and mechanism of 17β-estradiol on the apoptosis and inflammation of renal tubular cells in rats with renal ischemia/reperfusion injury. Methods All the female Sprague-Dawley rats were ovariectomized and randomly divided into four groups: Control group, Sham group, I/R group and estrogen plus I/R (E2 + I/R) group (n = 8). Right kidney of the rat was excised and artery of the left kidney was blockaded for 45 min.24 h after the reperfusion, we collected the blood and nephridial tissue of each group. An automatic biochemical analyzer was used to measure the expression level of BUN and Cr in blood. Hematoxylin-eosin (HE) staining was used to observe the pathological changes and the degree of inflammatory reaction of the ischemia/reperfusion injury kidney. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used to detect the apoptosis of renal tubular cells. The expression levels of Cleaved-Caspase-3 protein were measured by Western blot, while the numbers of CD4+ T lymphocyte infiltration in each group were tested by immunofluorescence (IF). Results Compared with the Sham group, expression level of BUN, Cr and Cleaved-Caspase-3 in I/R group significantly increased (P＜0.05) as well as the number of apoptotic cells (P＜0.05). In the meantime, inflammatory reaction significantly aggravated (P＜0.05) and the number of CD4 + T lymphocytes increased remarkably (P＜0.05). However, expression level of BUN, Cr and Gleaved-Caspase-3 in E2 + I/R group decreased significantly (P＜0.05) and the pathological damage in the kidney was alleviated (P＜0.05) compared with I/R group, furthermore, the number of apoptotic cells decreased (P＜0.05) compared with I/R group. The inflammatory reaction significantly blunted (P＜0.05) and the infiltration of CD4 + T lymphocytes decreased remarkably (P＜0.05) compared with I/R group. Conclusion Estrogen can inhibit the expression of Cleaved-Caspase-3 in renal tissue during ischemia/reperfusion injury and reduce the apoptosis of renal tubular cells. It can also reduce the infiltration of CD4 + T lymphocytes, thus playing a protective role on renal ischemia/reperfusion injury.
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@#Objective To investigate the effect of acupuncture-rehabilitation therapy on the neurological function and the expression of cleaved-caspase-8, cleaved-caspase-3 and cellular inhibitor of apoptosis 1 (cIAP1) in ischemic penumbra of rats with cerebral ischemia. Methods A total of 90 male Sprague-Dawley rats were randomly divided into sham group, model group, acupuncture group, rehabilitation group and acupuncture-rehabilitation group. Each group was divided into three days, seven days and 14 days subgroups (n=6). The cerebral ischemia model was established with the modified Koizumi suture method. The sham group and the model group received no treatment. The acupuncture group received cluster needling of scalp acupuncture, the rehabilitation group received treadmill training, and the acupuncture-rehabilitation group received both acupuncture and treadmill training. Three days, seven days and 14 days after modeling, their neurological function was assessed with modified Neurological Severity Score (mNSS) and Rota-rod test, and the expression of cleaved-caspase-8, cleaved-caspase-3 and cIAP1 protein in cerebral ischemic penumbra were detected with Western blotting. Results Compared with the model group, the mNSS scores decreased, the retention time of Rota-rod test increased, the expression of cleaved-caspase-8, cleaved-caspase-3 protein decreased and the expression of cIAP1 protein increased in each treatment group at each time point (P<0.05). Compared with the other two treatment groups, the mNSS scores further decreased, the retention time further increased, the expression of cleaved-caspase-8, cleaved-caspase-3 protein further decreased, and the expression of cIAP1 protein further increased (P<0.05) seven days and 14 days after modeling in the acupuncture-rehabilitation group.Conclusion Acupuncture-rehabilitation therapy can improve the neurological function in rats with cerebral ischemia, that is better than the simple acupuncture or exercise, which may relate to the inhibition of caspase-8 and caspase-3 protein activation, and promotion of cIAP1 protein expression, to inhibit the apoptotic caspases cascade reaction.
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Aim To observe the effect of icariside Ⅱ(ICS Ⅱ)on cardiomyocyte apoptosis in spontaneously hypertensive rats (SHR)and to explore its possible mechanism. Methods Thirty male 13-week-old SHRs were randomly divided into model group,ICS Ⅱ low, medium,high and positive drug group (n = 6),ho-mologous male Wistar-Kyoto rats as control group (n =6). After a week of adaptive feeding,ICS Ⅱ low,me-dium and high dose groups were given ICS Ⅱ 4,8,16 mg · kg - 1 (ig,qd),and the positive drug group was given losartan 20 mg·kg - 1 . At the same time,the WKY and SHR group were given equal volume double distilled water. After 12 weeks of administration,the blood pressure was measured in rats. Then,the rats were sacrificed and the left ventricles were separated in order to calculate the left ventricular mass index. HE staining was used to observe the pathological changes of the left ventricle,and the apoptosis of the left ventricu-lar myocardium was detected by TUNEL staining. The expressions of Bcl-2 and Bax mRNA in left ventricle were detected by real time RT-PCR,and Bcl-2,Bax and cleaved-caspase-3 protein expressions were detec-ted by Western blot. Results Compared with WKY group,the blood pressure and left ventricular mass in-dex increased in SHR group (P < 0. 05),and the my-ocardial cell arrangement was disordered and the cell hypertrophy and apoptosis were obvious,accompanied by rupture of filament ;the level of Bax mRNA was up-regulated (P < 0. 05),and Bcl-2 mRNA was down-regulated (P < 0. 05 );the expressions of Bax and cleaved-caspase-3 protein were up-regulated (P <0. 05),and the level of Bcl-2 protein was down-regu-lated (P < 0. 05 ),and the ratio of Bax / Bcl-2 in-creased (P < 0. 05). Compared with SHR group,the blood pressure and left ventricular mass index de-creased in ICS Ⅱ middle,high group and the positive drug group (P < 0. 05);moreover,the arrangement of myocardial cells became more orderly,the cell hyper-trophy and the apoptosis of myocardial cells were im-proved;the level of Bax mRNA was down-regulated and Bcl-2 mRNA was up-regulated (P < 0. 05);the expression of Bax and cleaved-caspase3 protein were down-regulated and the level of Bcl-2 protein was up-regulated (P < 0. 05 );the ratio of Bax / Bcl-2 de-creased (P < 0. 05). Conclusions ICS Ⅱ can im-prove the left ventricular cardiomyocytes apoptosis in SHR,and its mechanism is related to the decrease of blood pressure and the inhibition of mitochondrial ap-optosis pathway.
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Aim To investigate the effect of Ginsen-oside Rh2 on apoptosis in human colorectal cancer cell SW480,and to explore the possible mechanism of it. Methods The proliferation activity of SW480 treated with Ginsenoside Rh2 was measured CCK-8 assay.Ap-optosis rates were evaluated by FCM.Hoechst 33258 staining was used to observe cell nucleus morphologi-cal;change SW480 cells were treated with Ginsenoside Rh2,and the protein expressions of Bcl-2,Bax,p53, cleaved caspase-3 ,PI3 K,AKT,P-AKT,GSK-3β,P-GSK-3βwere detected by Western blot;SW480 cells were treated with LY294002,Rh2,LY294002+Rh2, the expressions of PI3 K,AKT,P-AKT,GSK-3β,P-GSK-3βwere detected by Western blot.Results The proliferation of SW480 cells was significantly inhibited by Ginsenoside Rh2 in dose-dependent and time-de-pendent manner.FCM showed the inducing apoptosis effect of Ginsenoside Rh2 was significantly different from that of control group.Hoechst 33258 staining in-dicated clearly cell apoptosis in Ginsenoside Rh2 treat-ment groups.Western blot showed Ginsenoside Rh2 decreased expression of Bcl-2,increased expression of Bax,p53 and cleaved caspase-3,PI3K/AKT/GSK-3βpathway proteins PI3 K,P-AKT,P-GSK-3βdecreased obviously,AKT and GSK-3βwere not changed signifi-cantly in SW480.SW480 cells were separately treated with LY294002,Rh2,LY294002 +Rh2,there were no significant difference in AKT and GSK-3βprotein a-mong all groups,and the expression of PI3 K,P-AKT, P-GSK-3βdecreased more obviously in LY294002 +Rh2 group compared with LY294002 and Rh2 alone. Conclusion Rh2 induces colorectal cancer cell apop-tosis through PI3 K/AKT/GSK-3βpathway,which ac-tivates p53 and cleaved caspase-3,and destroys the balance of Bcl-2/Bax.
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AIM:To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3 (LC3) at various stages of follicular development and atresia in the mice.METHODS:On 0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG),expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the method of immunohistochemical staining.The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH.LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence.RESULTS:The LC3 protein expressed in granulosa cells during all developmental stages mainly.Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3.The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG (P ＜ 0.05).The protein levels of cleaved caspase3 and LC3-Ⅱ in the granulosa cells increased in turn on 3,4 and 5 day after intraperitoneal injection of PMSG.The positive correlation between LC3-Ⅱ and cleaved caspase-3 protein levels was observed (r2 =0.8299,P ＜ 0.05).The LC3-Ⅱ protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH.CONCLUSION:LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity.Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis.Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.
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AIM:To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3 (LC3) at various stages of follicular development and atresia in the mice.METHODS:On 0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG),expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the method of immunohistochemical staining.The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH.LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence.RESULTS:The LC3 protein expressed in granulosa cells during all developmental stages mainly.Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3.The protein levels of cleaved caspase-3 and LC3-Ⅱ in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG (P ＜ 0.05).The protein levels of cleaved caspase3 and LC3-Ⅱ in the granulosa cells increased in turn on 3,4 and 5 day after intraperitoneal injection of PMSG.The positive correlation between LC3-Ⅱ and cleaved caspase-3 protein levels was observed (r2 =0.8299,P ＜ 0.05).The LC3-Ⅱ protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH.CONCLUSION:LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity.Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis.Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.
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Objective To investigate the effect of propofol exposure on neuroapoptosis in pri-mary cultured cortical neurons and its mechanisms.Methods Cortical neurons were primarily cultured for seven days,then divided into two groups:control group (treated with equal volume of 20% in-tralipid),propofol-treated group (treated with 500 μmol/L propofol).The neurons were treated for 12 h.The neuron viability was determined by MTT.Neuroapoptosis was identified by Hoechest 33 258 dying.Mitochondrial membrane potential was measured by the fluorescent dye rhodamine 123 (Rh123).Western blot was performed to detect the level of cyt-c and cleaved-caspase-3.Results Neu-rons survival rate (54.4%±6.4%)in the propofol group was significantly lower than that of control group (99.8% ± 4.1%) (P < 0.05 ), the rate of neuronal apoptosis (46.5% ± 5.3%) was significantly higher than that of control group (7.2%±0.9%)(P <0.05),mitochondrial membrane potential (59.6%±4.3%)was significantly lower than that of the control group (99.9% ± 5.7%) (P <0.05 ),cyt-C protein level (0.38 ± 0.03 )was significantly higher than that of control group (0.1 5±0.02)(P < 0.05 ),level of cleaved-caspase-3 protein level (0.46 ± 0.04)was significantly higher than that of control group (0.13±0.02)(P <0.05).Conclusion Propofol induces neuroapo-tosis in primary cultured cortical neurons,which is associated with the decreased level of MPP and the increase levels of cyt-c and cleaved-caspase-3.
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ObjectiveTo investigate the effects of caffeine citrate (CC) on the neuronal proliferation and apoptosis and long-term learning ability in neonatal rats with hypoxia-ischemic brain damage (HIBD).MethodsForty-eight 7-day-old Sprague-Dawley neonatal rats were randomly divided into 3 groups: sham operation group (n=16), HIBD group (n=16), HIBD + caffeine citrate group (CC group,n=16). Rats in HIBD and CC groups received ligation of left common carotid artery followed by 2 hours of hypoxia to establish HIBD model. Rats in CC group were injected intraperitoneally with CC (20 mg/kg) before and at 0 min, 24 h, 48 h, and 72 h after hypoxia-ischemic (HI), and rats in the other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time. Meanwhile, from postnatal day 10, each rat was injected intraperitoneally with 5-bromo-2’-de-oxyuridine (BrdU) (50 mg/kg) for 5 consecutive times, once every 12 h. On postnatal day 12, BrdU in the hippocampal dentate gyrus and cleaved caspase-3 in the hippocampal CA1 area were detected by immunohistochemistry, and neuronal apoptosis in hippocampal CA1 area were detected by TUNEL staining. On postnatal day 28, long-term learning and memory ability of rats was tested by Y maze.ResultsThere was signiifcant difference in the number of BrdU-positive cells in brain tissues of rats among three groups (F=101.38,P<0.01). The BrdU-positive cells in HIBD group and CC group were signiifcantly more than those in sham operation group (P<0.05). There was signiifcant difference in the number of cleaved caspase-3-positive cells in hippocampal CA1 area among three groups (F=379.77,P<0.01). The cleaved caspase-3-positive cells in CC group were signiif-cantly fewer than those in HIBD group but signiifcantly more than those in sham operation group (P<0.05). The TUNEL-pos-itive cells in hippocampal CA1 area were signiifcantly different among three groups (F=505.92,P<0.01) which was most in HIBD group and fewest in sham operation group and signiifcant difference was found through multiple comparison (P<0.05). The total learning number of avoiding electric shock tested by Y maze was signiifcantly different among three groups (F=32.05, P<0.01) which was most in HIBD group. Correct response rate was significantly different among three groups (F=24.99, P<0.01) which was lowest in HIBD group.ConclusionsCaffeine citrate can improve the ability of long-term learning and memory in neonatal rats with hypoxia-ischemic brain damage, the mechanism of which may be related to reducing the neuronal apoptosis after hypoxia ischemia.
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This study was aimed to explore the mechanism of baicalin on high altitude cerebral hypoxia-ischemia on mice and its influence on related target protein expressions. Morris water maze was used to screen 50 Kunming mice, which were randomly divided into the model group, control group, the low dose (0.05 mg·kg-1), middle dose (0.20 mg·kg-1) and high dose (0.60 mg·kg-1) baicalin group, with 10 rats in each group. The space memory and learning ability of mice were tested. The animal cabin with low oxygen (simulating at 4 000 m altitude) was used to establish the stable high altitude cerebral hypoxia-ischemia mouse model. Changes on SOD content, GSH-PX activities and MDA content in hippocampal tissues of mice were detected. The expressions of different target proteins, including cleaved-caspase 3, P-AKT, GFAP, Bax and Bcl-2 in brain stem of mice were detected by western blot. The results showed that the latent period of the model group was obviously longer than that of the control group (P < 0.05). The latent period of high dose baicalin group was shorter than the model group with significant difference (P< 0.05). Therefore, the best effective dose of baicalin was 0.60 mg·kg-1. Compared with the control group, the content of MDA in the hippocampal tissues of mice in the model group was significantly increased; the SOD and GSH-PX activity were obviously reduced (P < 0.05). Compared with the model group, the SOD and GSH-PX activity were obviously increased in the brain tissues of mice in the high dose baicalin group; and the content of MDA was obviously reduced (P < 0.05). From the level of protein changes, the stripes of cleaved-caspase 3, P-AKT, GFAP protein expressions in the model group were strengthened compared to the control group; the ratio of Bax/Bcl-2 was also obviously increased (P < 0.05). The expression of the baicalin group was lower than that of the model group (P < 0.05). Among them, the expression of the high dose baicalin group was the lowest. It had certain dose-response relationship. It was concluded that baicalin had protective effect on high altitude cerebral hypoxia-ischemia. Its mechanism may be related to its powerful oxidation resistance and its inhibition on expression of different target proteins, including cleaved-caspase 3, P-AKT, GFAP, Bax, Bcl-2 for the change of apoptotic pathway.
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Aim To investigate the effect of isoflurane on the phosphorylation of p38 mitogen-activated protein kinase (MAPK)in the hippocampus of neonatal rats, and the effect of p38 MAPK pathway on isoflurane-in-duced neuronal apoptosis.Methods Forty-eight neo-natal rats on postnatal day 7 were assigned randomly into four groups:DMSO group (group Air +DMSO), p38 MAPK inhibitor SB203580 group (group Air +SB20 ),isoflurane +DMSO group (group Iso +DM-SO),and isoflurane +SB203580 group (group Iso +SB20 ).Rats were exposed to air or isoflurane (volume fraction of 0.01 1 )for 4h.The p38 inhibitor SB203580 (20 nmol)or DMSO (volume fraction of 0.1 )5μl was intraventricularly administered 30 min before the expo-sure.The brains of some rats in each group were per-fused and embedded by paraffin 6h after the exposure. Neuronal apoptosis in the hippocampal CA1 area was detected by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL)(n =6). The hippocampal tissues of the other rats in each group were dissected 6h after the exposure,and the protein expressions of phospho-p38 (p-p38 ),p38,cleaved caspase-3,phospho-NF-κB (p-NF-κB ),Bcl-2 and Bax were detected by Westem blot (n =6).Results The number of TUNEL positive cells in the hippocam-pal CA1 region in group Iso +DMSO increased by 4.8 fold compared with that in group Air +DMSO (P <0.01 ),while the number of TUNEL positive cells in group Iso +SB20 decreased by 3 /5 compared with that in group Iso +DMSO (P <0.01 ).The protein expres-sion of cleaved caspase-3 in group Iso +DMSO signifi-cantly increasd (P =0.003)compared to that in group Air +DMSO,which was significantly decreasd in group Iso +SB20 (P =0.007 ).In addition,isoflurane also increased the protein expression of p-p38,p-NF-κB and Bax,decreased the level of Bcl-2,and reduced the ratio of Bcl-2 /Bax compared with control animals (P <0.01 ,P =0.004,P <0.01 ,P <0.01 ,P <0.01 ,respectively).Howerver,SB203580 partly at-tenuated the isoflurane-induced protein change above. Conclusion Isoflurane induces neuroapoptosis in neo-natal rat hippocampus by the activation of p38 MAPK pathway.
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Objective: To investigate the effect of peiminine (PMI) on reversing the multi-drug resistance (MDR) of gastric cancer cell and enhancing the antitumor activity of adriamycin (ADR) on SGC7901/VCR cell xenograft and to explore its functional mechanisms. Methods: The model of SGC7901/VCR cell line xenograft on athymic mice was established, and the mice were randomized to groups with ip injection of different drugs once every other day, for six times in all. The tumor volume and the body weight of mice were measured during the drug therapy. The mice were sacrificed after the last administration. The tumors were weighed and then the inhibitory rate was calculated. Western blotting was employed to detect the expression of P-glycoprotein (P-gp) and Cleaved Caspase-3. Results: During the treatment, the tumor volume of mice in PMI + ADR group was decreased significantly compared with the control group (P < 0.01). At the end of the treatment the mice were sacrificed and the tumor weight was measured. The tumor weight and inhibitory rate of mice in PMI + ADR group were obviously decreased compared with the control group (P < 0.05). The expression of P-gp in tumor of mice in ADR + PMI group was decreased. On the contrary, the expression of Cleaved Caspase-3 was increased. Conclusion: The PMI is able to enhance the sensitivity of drug resistance on gastric cancer cell to adriamycin and its mechanism may be related to decreasing the expression of P-gp and increasing the expression of Cleaved Caspase-3 in tumor tissues.