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Background At present, radiation therapy is widely used in clinical treatment of tumors. However, while radiation therapy damages tumor cells, it also injures surrounding normal tissues. Studies have shown that hydrogen is a potential radiation-protective agent. Objective To investigate the neuroprotective mechanisms of hydrogen-rich water activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/cysteinyl aspartate specificproteinase-9 (Caspase-9) signaling pathway in acute radiation-induced brain injury. Methods Forty male SD rats were randomly divided into four groups: control group, irradiation only group (IR), high-dose hydrogen-rich water intervention group (IR+HHRW), and low-dose hydrogen-rich water intervention group (IR+LHRW), 10 rats in each group. Except for the control group, animals in each group received a single 20 Gy whole brain irradiation. Animals in all groups were gavaged once a day from 3 d before irradiation to 7 d after irradiation, pure water (20 mL·kg−1) was given to the control and the IR groups, and hydrogen-rich water (20 mL·kg−1, 10 mL·kg−1) was given to the IR+HHRW and the IR+LHRW groups. After 7 d of intervention, 5 rats in each group were selected for the Morris water maze experiment for behavioral evaluation. Autopsies were conducted after anesthesia for the remaining animals and blood samples were collected for hematological analysis. Rat brains were harvested for TUNEL staining to observe neuronal apoptosis. HE staining was performed to observe histopathological changes, enzyme-linked immunosorbent assay was adopted to detect oxidative stress-related indicators, and real-time PCR and Western blotting were used to measure the expressions of PI3K/AKT/Caspase-9 pathway-related genes and proteins. Results The body weight of rats receiving irradiation decreased after 7 d of irradiation compared with the control group (P<0.05), and the symptoms such as arched back and malaise occurred to varying degrees, and the symptoms of rats in the IR+HHRW group were significantly milder than those in the IR group. The behavioral test results showed that the escape latency of rats in the IR+HHRW group or the IR+LHRW group was shorter than that in the IR group from day 2 to day 5 (P<0.05), and it took less time for rats in the IR+HHRW group to reach the original position after removing the platform on day 6 (P<0.05). The hematological test results showed that red blood cell (RBC) count, hemoglobin (HGB) level, and white blood cell (WBC) count were significantly decreased in the IR group (P<0.05), and the changes in the IR+HHRW group were improved (P<0.05). The HE staining results showed that the number of abnormal nerve cells, broken and dissolved nuclei, and the degree of damage in the IR+HHRW group were significantly reduced than those in the IR group. The results of oxidative stress evaluation showed that the ability of the IR group to inhibit free radicals decreased, the level of malondialdehyde (MDA) increased (P<0.01); the MDA level decreased after LHRW intervention (P<0.05); the SOD activity was elevated after HHRW intervention (P<0.05). The TUNEL staining results showed that the apoptosis signals in the IR+HHRW group were sparser than those in the IR group (P<0.05). The real-time PCR results showed that compared with the IR group, the mRNA expression levels of PI3K and AKT in the IR+HHRW group and the IR+LHRW group increased (P<0.05), while the mRNA expression levels of Cytc and Caspase-9 decreased (P<0.05). The Western blotting results showed that compared with the IR group, the phospho-AKT (pAKT) protein expression level in the IR+HHRW group increased significantly (P<0.05), while the expression of Caspase-9 and Cytc proteins decreased significantly (P<0.05). Conclusion Hydrogen-rich water can significantly reduce inflammation and oxidative stress caused by acute irradiation-induced brain injury, and decrease neuronal apoptosis. The mechanism may be related to the PI3K/AKT/Caspase-9 signaling pathway.
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In order to explore the antitumor effect and mechanism of different extracts of cultivated Phellinus vaninii fruit body on H22 tumor bearing mice, 150 ICR mice were randomly divided into blank group, model group, CTX group, P. vaninii water extract group, ethanol extract group, petroleum ether extract group and crude polysaccharide group. H22 liver cancer cells were used to establish a solid tumor model and the mice were sacrificed on the 10th day after administration. The spleen and thymus organ index and tumor inhibition rate were calculated, the serum levels of TNF-α, INF-γ, VEGF, and hematoxylin-eosin were detected, and the immunohistochemical staining method was used to observe the pathological changes of tumor tissues, while Western blotting was used to detect the expression of tumor-related proteins. The high-dose petroleum ether extract group showed the best tumor inhibition rate (73.21%), increased serum levels of TNF-α, IFN-γ, and VEGF, as well as significantly promoted tumor necrosis and ablation. The immunohistochemistry of the water extract group showed negative regulation, indicating an insignificant tumor suppression. Western blotting showed the apoptosis genes Caspase-3, Caspase-9 and pathway genes NF-κB and JAK were all highly expressed in each administration group compared with the model group, and their expression levels gradually decreased with increasing doses. In summary, the petroleum ether extract of P. vaninii fruit body showed a significant anti-tumor effect which is presumably mediated through the mitochondrial pathway. The metabolism of drug in the body induces activation of Caspase-3 and Caspase-9 apoptotic proteins by Bax, Bcl-2, and TNF, which further caused nuclear chromatin or DNA to condense or degrade, and subsequently destroy the normal proliferation of tumor cells, thereby inducing their apoptosis and inhibiting tumor growth.
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Animals , Mice , Apoptosis , Basidiomycota , Mice, Inbred ICR , Neoplasms/metabolismABSTRACT
Abstract Introduction: Blood cardioplegia (BC) and Custodiol cardioplegia (CC) have been used for a long time in open heart surgery and are highly effective solutions. The most controversial issue among these two is whether there is any difference between them regarding myocardial damage after ischemia surgery. In this study, autophagy, apoptosis, and hypoxia markers were investigated and that way we evaluated the differences between BC and CC patients. Methods: A total of 30 patients were included in this study, using two different cardioplegic solutions. Three different whole blood samples of the patients were taken from a central vein (preoperatively, immediately postoperatively, and one day after surgery). Total ribonucleic acid was extracted from these samples. Quantitative real-time polymerase chain reaction was performed, and changes in gene expression were determined by the 2-∆∆Ct method of relative quantification. Results: In the CC group, Beclin gene expression level was found to be higher and this difference was statistically significant (P=0.0024). Similarly, cysteine-aspartic acid protease (caspase) 9 and hypoxia-inducible factor 1α messenger ribonucleic acid (mRNA) gene expression level increased and were significantly different in the CC group. In the BC group, Beclin and microtubule-associated protein light chain 3 expressions were higher in the samples taken one day after surgery. Caspases 3 and 8 gene expressions were significantly different in the BC group. Conclusion: As a result of the analysis performed between the two cardioplegia groups, it has been shown that CC harms the myocardium more than BC at the level of mRNA expression of related markers.
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Humans , Cardioplegic Solutions/therapeutic use , Heart Arrest, Induced , Autophagy , RNA, Messenger , Apoptosis , Hypoxia/drug therapyABSTRACT
@#[Abstract] Objective: To investigate whether AP1903, a small-molecule chemical inducer, can terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vivo and in vitro. Methods: CD19CAR-T cells over-expressing iCasp9 (iCasp9-CD19CAR-T) were constructed and co-incubated with AP1903. Then, the cell phenotype and apoptosis were detected by Flow cytometry, and the iCasp9/CID suicide gene system was verified on K562 and T cells, respectively. The cytotoxicity of iCasp9-CD19CAR-T cells was detected in vivo (survival rate of NCG mice bearing Raji cell transplanted xenograft) and in vitro (cell killing function was detected by Flow cytometry) under the administration of AP1903. Results: Compared with CD19CAR-T cells, iCasp9-CD19CAR-T cells showed in significant difference in proliferation, phenotype and cytotoxicity both in vitro and in vivo (all P>0.05). At 2 h after AP1903 administration, the apoptosis rates of K562 and T cells co-expressing iCasp9 and CD19CAR were (33.8±0.9)% and (27.95±0.35)%, respectively; and at 24 h after AP1903 administration, the apoptosis rates reached 100% in both cell lines. The in vitro cytotoxicity of iCasp9-CD19CAR-T cells induced by AP1903 was significantly lower than that without AP1903 treatment (P<0.01); the 60-day survival rate of mice bearing Raji cell transplanted tumor treated with AP1903-induced iCasp9-CD19CAR-T cells was also significantly lower than those treated with iCasp9-CD19CAR-T cells alone (P<0.01). Conclusion: AP1903 can effectively terminate the cytotoxicity of CD19CAR-T cells over-expressing iCasp9 suicide gene in vitro and in vivo.
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Quinacrine (QC), an FDA-approved anti-malarial drug, has shown to have anticancer activities. Due to its‘shotgun’ nature, QC has become an inevitable candidate for combination chemotherapy. There is lack of studyof the molecular interplay between colorectal cancer (CRC) microenvironment and its metastasis. In this study,we focused on the differential anti-cancerous effect of QC on two different human cancer cell lines, HCT 116and INT 407. Results suggest that cytotoxicity increased in both the cell lines with an increase in QCconcentration. The expression patterns of small-GTPases and caspases were altered significantly in QC-treatedcells compared to non-treated cells. HSP70 and p53 showed comparable differences in the expression pattern.The wound-healing assay showed an increase in the denuded zone, with an increase in the concentration ofQC. The formation of apoptotic nuclei increased with a rise in the concentration of QC in both the cell lines.The decrease and increase in caspase 9 and caspase 3 expression respectively were studied, confirmingapoptosis by the extrinsic pathway
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BACKGROUND: Steroid-induced avascular necrosis of the femoral head has a complex biological process, and its pathogenesis is unknown. To date, there is no effective treatment in clinical practice. Therefore, exploring the etiology of steroid-induced avascular necrosis of the femoral head is still an important content of research in this field. OBJECTIVE: To explore the effect of grape seed proanthocyanidins on osteocyte apoptosis due to steroid-induced osteonecrosis of the femoral head. METHODS: Twenty-seven Japanese white rabbits were randomly divided into three groups. The model group was intravenously injected with E. coli endotoxin, 100 μg/kg, twice at an interval of 24 hours. Two injections of E. coli endotoxin were followed by intramuscular injection of methylprednisolone 20 mg/kg, for 3 times. The interval was 24 hours. The treatment group was intravenously injected with E. coli endotoxin, 100 μg/kg, twice at an interval of 24 hours. After the second injection of E. coli endotoxin, methylprednisolone 20 mg/kg and grape seed proanthocyanidin extract 200 μg/kg were injected intramuscularly three times at an interval of 24 hours. The control group was treated with the same dose of saline intravenously. The animals were killed by air embolization at the 4th week after the last injection. Under the relatively aseptic condition, the bilateral femoral heads were routinely fixed, decalcified, embedded and sliced. Histomorphological observation and hematoxylin-eosin staining were performed to count empty bone lacunae under light microscope. Hoechst staining was used to detect cell apoptosis. The expressions of Caspase-9 and Bcl-2 in the femoral head were detected by immunohistochemical staining.
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OBJECTIVE@#To observe the impacts of electroacupuncture (EA) on neurological function, the pathological morphology in brain tissue, apoptosis level and the protein expressions of apoptosis-related cytochrome C (Cyt-C) and cysteine aspartic acid protease-9 (Caspase-9) in the rats with traumatic brain injury (TBI) and explore the potential mechanism of EA in treatment of TBI.@*METHODS@#A total of 70 clean-grade SD mice were randomized into a blank group (8 rats), a sham-operation group (8 rats), a model group (27 rats) and an EA group (27 rats). In terms of interventions of 3, 7 and 14 days, 3 subgroups were divided in the model group and the EA group successively, 9 rats in each subgroup. The modified Feeney free-fall percussion method was adopted to establish TBI models of rats. In the sham-operation group, only the skull was exposed and drilled and no free-fall percussion was exerted. One day after modeling, EA was given in the rats of EA group at "Shuigou" (GV 26), "Baihui" (GV 20) and "Neiguan" (PC 6) and "Zusanli" (ST 36) on the affected side, with intermittent wave, 2 Hz in frequency, once daily, 10 min each time, for 3, 7 and 14 days successively. Separately, on the day 3, 7 and 14 of intervention, the modified neurological severity scale (mNSS) was used to evaluate the degree of neurological function injury in the rats, HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue, TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry (IHC) method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue.@*RESULTS@#Compared with the sham-operation group, on the day 3, 7 and 14 of intervention, mNSS scores were increased obviously in the rats of the model group respectively (<0.01). Compared with the model group, on the day 3, 7 and 14 of intervention, mNSS scores were reduced in the rats of the EA group respectively (<0.05). On day 3 of intervention, in brain injury region of the rats in the model group and the EA group, gross tissue necrosis, nuclear fragmentation, consolidation and obvious vacuolar changes, reduced Nissl bodies and scattered arrangement were found. On day 7 and 14 of intervention, in the model group and the EA group, the new connective tissue filling and normal cells were visible and Nissl bodies increased. The overall repair and Nissl body quantity in the EA group were better than the model group. Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the numbers of apoptotic cells were increased obviously in the model group (<0.01) and they were reduced in the EA group as compared with the model group (<0.05). Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group (<0.01) and they were all reduced in the EA group as compared with the model group successively (<0.05).@*CONCLUSION@#Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats, which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.
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Animals , Rats , Apoptosis , Brain Injuries, Traumatic , Therapeutics , Caspase 9 , Metabolism , Cytochromes c , Metabolism , Electroacupuncture , Random Allocation , Rats, Sprague-DawleyABSTRACT
OBJECTIVE@#To observe the effect of electroacupuncture (EA) on neuronal apoptosis in rats with traumatic brain injury (TBI), and to explore the action mechanism of EA on improving the brain nerve function of TBI.@*METHODS@#A total of 88 6-week-old SD rats were randomly divided into a sham operation group, a model group, an EA group and a LY294002+EA group, 22 rats in each group. The TBI model on the left side was established by the improved Feeney's free fall method. After modeling for 24 h, the rats in the EA group and LY294002+EA group were treated with acupuncture at "Baihui" (GV 20) for 10 min and pricking acupuncture at "Shuigou" (GV 26) for 20 s; EA was applied at "Neiguan" (PC 6) and "Zusanli" (ST 36) on the right side (discontinuous wave, 2 Hz of frequency, 1 mA of intensity) for 10 min, once a day for 3 days. After 3 days of intervention, the TUNEL method was used to detect the level of neuron apoptosis in left cerebral cortex; the Western blot method was used to detect the expression of Akt, p-Akt, Bcl-2, Bax, Cyt-C and Caspase-9 in the left cerebral cortex.@*RESULTS@#After 3-day treatment, compared with the sham group, the number of neuronal apoptosis in the left cortex was increased in the model group (<0.01), and the expression of Bax, Cyt-C and Caspase-9 protein was increased (<0.01), and the expression of p-Akt/Akt, Bcl-2 was decreased (<0.01). Compared with the model group, the number of neuronal apoptosis in the left cortex was decreased in the EA group (<0.01), and the expression of Bax, Cyt-C and Caspase-9 was decreased (<0.01), and the expression of p-Akt/Akt and Bcl-2 was increased (<0.01). Compared with the LY294002+EA group, the number of neuronal apoptosis in the left cortex was decreased in the EA group (<0.01), and the expression of Bax, Caspase-9 and Cyt-C was decreased (<0.01, <0.05), and the expression of p-Akt/Akt and Bcl-2 was increased (<0.01).@*CONCLUSION@#EA could significantly reduce the neuronal apoptosis in rats with TBI, and its mechanism may be related to the activation of PI3K/Akt signaling pathway.
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BACKGROUND AND OBJECTIVES: To reveal the detail mechanism of miR-484 on myocardial ischemia-reperfusion (MI/R) injury.METHODS: Rats model of MI/R injury was established based on control (Con; sham operate) group, ischemia-reperfusion (I/R) group, miR-484 treatment (miR) group, and I/R-negative control (IR-C) group, followed by pathological and interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β expression evaluation. Then the myocardial apoptosis, as well as the expression of miR-484, caspase-3, and caspase-9 in myocardium were examined. Finally, the regulatory relation between miR-484 and SMAD family member 7 (SMAD7) was predicated, followed by verification analysis.RESULTS: Compared with Con group, the expression of miR-484 in I/R and IR-C group was decreased. Compared with I/R and IR-C group, the expression of miR-484 was increased in miR group. Compared with Con group, the expression levels of IL-6, TNF-α, and IL-1β in cardiac myocytes of I/R group and IR-C group were increased. Compared with Con group, the apoptotic index, membrane potential of I/R, and the expression of caspase-3/9 were increased in IR-C group. Compared with the I/R and IR-C groups, the apoptotic index of myocardial cells in the ischemic region was decreased, the membrane potential was increased, and the expression of caspase-3/9 was decreased significantly in the miR group. SMAD7 was the target gene of miR-484.CONCLUSIONS: MiR-484 protected myocardial cells from I/R injury by suppressing caspase-3 and caspase-9 expression during cardiomyocyte apoptosis. MiR-484 reduced the expression of IL-6, TNF-α, and IL-1β in MI/R. MiR-484 might alleviate the decreasing of mitochondrial membrane potential in MI/R cells.
Subject(s)
Animals , Humans , Rats , Apoptosis , Caspase 3 , Caspase 9 , Interleukin-6 , Interleukins , Membrane Potential, Mitochondrial , Membrane Potentials , Myocardium , Myocytes, Cardiac , Reperfusion Injury , Tumor Necrosis Factor-alphaABSTRACT
Aim To study the effects of salidroside on the caspase-9, GSK-30, NMDAR1, GluR2 in MCAO ratsand to further explore the mechanism of neuroprotection of salidroside. Methods For the first part, 36 healthy male Sprague-Dawley rats were randomly divided into sham operation (Sham) group, model (MCAO) group, and salidroside (MCAO + Sal) group. Rats were administered salidroside, or vehicle, daily for 1 day, after middle cerebral artery occlusion (MCAO) 2 h and reperfusion 1 h. The protein expression of GSK-3β, NMDAR1, GluR2 and caspase-9 was detected by Western blot. For the second part, rats were randomly divided into Sham group, SB216763 + Shamgroup, MCAO group, SB216763 + MCAO group, MCAO + Sal group, and SB216763 + MCAO + Sal group. After 30 minutes of injection of GSK-3βinhibitor SB216763 or artificial cerebrospinal fluid into the lateral ventricle, the other groups were subjected to MCAO modeling except for the sham operation group. After the modeling, the administration group was given salidroside, and the material was taken after 1 day. The protein expression of GSK-3β, NMDAR1, GluR2 andcaspase-9 was detected by Western blot. Results Compared with MCAO group, salidroside could reduce the protein expression of cleaved caspase-9 protein in mitochondria and promote the expression of pGSK-3β, NMDAR1, GluR2 protein after 1 day salidroside treatment. And the treatment of salidroside and GSK-3β inhibitor did not show remarkable additive effects. Conclusions Salidroside has a neuroprotective effect on MCAO rats, mainly by promoting GSK-3β phosphorylation, thereby inhibiting caspase-9 and promoting protein expression of NMDAR1 and GluR2.
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Objective: To observe the antitumor effect of triptolide in the human pancreatic cancer tumor-bearing nude mice, and to explore its molecular mechanism. Methods: The healthy BALB/ c nude mice were selected and transplanted with the human pancreatic cancer SW1990 cells into the skin of the right hind limb to establish the tumor-bearing model of nude mice. The successful modeling nude mice were divided into model group, gemcitabine group and triptolide group, 10 in each group; another 10 healthy nude mice were selected as control group. The tumor tissue of SW1990 pancreatic cancer tumor-bearing nude mice were weighed and the inhibitory rate of tumor was calculated. The expressions of apoptosis-related proteins Bax and bcl-2 in the pancreas tissue of the nude mice in each group were detected by rmmunohistochemistry. The expression levels of Bax, bcl-2, Caspase-9 and Caspase-3 in the pancreas tissue of the nude mice in each group were detected by Western blotting method. Results: Compared with gemcitabine group, the inhibitory rate of tumor of SW1990 pancreatic cancer in triptolide group was significantly increased (P < 0. 05). The HE staining results showed that compared with model group, the proliferation of pancreatic cancer tumor cells in triptolide group was inhibited. The immunohistochemistry results showed that the expression level of Bcl-2 in the pancreas tissue and the Bcl-2 /Bax ratio of the nude mice in tripptolide group were lower than those in model group (P < 0 . 05). The Western blotting results showed that compared with model group, the expression levels of Bax, Caspase-9 and Caspase-3 in the pancreas tissue of the nude mice in triptolide group were significantly increased (P < 0 . 05), while the expression level of Bcl-2 was significantly decreased (P < 0 . 05). Conclusion: Triptolide can inhibit the growth of tumor tissue in the nude mice; its mechanism may be related to inhibiting the activation of Bcl-2 in pancreatic tumor tissue, promoting the expression of Bax, reducing the ratio of Bcl-2 /Bax, promoting the activation of Caspase-9 and Caspase-3, and inducing the apoptosis of pancreatic cancer transplanted tumor cells.
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Objective: To observe the effects of ginsenoside Rg1 pretreatment on the expression of survivin protein and apoptosis after spinal cord ischemia-reperfusion injury (SCII) in rats so as to explore the possible mechanism of ginsenoside Rg1 on motor function recovery after SCII in rats. Methods: We selected 120 adult healthy SD rats to construct the model of SCII and randomly divided them into four groups: sham operation group, ischemia group, ischemia-reperfusion group, and drug group. Basso Beattie and Bresnahan score (BBB score) was used to evaluate the motor function of the hind limbs of the rats. The expressions of survivin protein and apoptosis-inducing factor (AIF) was observed by immunohistochemistry. The expression and activity of survivin protein and Caspase-9 in each group were observed and analyzed by Western blot and RT-PCR. Results: The intervention of ginsenoside Rg1 could increase the score of the motor function of the rat hind limbs. It could decrease the number of AIF positive cells, but increase the number of survivin protein positive cells. Ginsenoside Rg1 could decrease the expressions of survivin and Caspase-9, and decrease the apoptosis of nerve cells in SCII. Conclusion: Ginsenoside Rg1 could inhibit the expression of Caspase-9 by promoting the expression of survivin protein and decrease the apoptosis of rat SCII induced by the level of cytoplasmic AIF.
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OBJECTIVE@#To investigate the effect of inner-heating acupuncture on apoptosis of chondrocytes and expression of Caspase-3 and Caspase-9 in rats with knee osteoarthritis (KOA).@*METHODS@#A total of 32 rats were divided into a normal group, a model group, a control treatment group and a treatment group by random number grouping method, 8 rats in each one. The rats in the normal group received no intervention. The rats in the remaining three groups adopted modified Videman method to develop KOA model, the ankle joint of left posterior leg was fully extended and fixed with a resin bandage for 6 weeks. After successful modeling, the rats in the model group received no intervention. The rats in the control treatment group were treated with medium-frequency pulse electrotherapy. The rats in the treatment group were treated with inner- heating acupuncture, 30 min each treatment, once a day, five days per week, and totally 3-week treatment was given. After 3 weeks, the damaged cartilage tissue was collected, and HE staining was used to observe the pathological changes of the cartilage tissue of the knee joint. ELISA was used to detect the content of cytochrome-C in the tissue homogenate supernatant. The chondrocytes in damaged cartilage tissue were isolated, flow cytometer was used to detect the changes of apoptosis and mitochondrial membrane potential. The mRNA and protein expression of Caspase-3 and Caspase-9 in chondrocytes were detected by real-time quantitative PCR (qRT-PCR) and Western blot (WB), respectively.@*RESULTS@#Compared with the normal group, the damage of cartilage tissue in the model group was significant, and the expression level of Cyt-C in the homogenate supernatant of damaged cartilage tissue was increased (<0.01); the chondrocyte apoptosis was increased significantly (<0.01); the chondrocyte mitochondrial membrane potential was decreased significantly (<0.01); the mRNA and protein expression of Caspase-3 and Caspase-9 was increased significantly (all <0.01). Compared with the model group, the cartilage injury in the control treatment group and the treatment group was significantly relieved; the expression level of Cyt-C in the supernatant of damaged cartilage tissue homogenate was decreased (both <0.01); the chondrocyte apoptosis was significantly reduced (both <0.01); the chondrocyte mitochondrial membrane potential was increased significantly (both <0.01). Moreover, the mRNA and protein expression of Caspase-3 and Caspase-9 was significantly reduced (all <0.01). Compared with the control treatment group, the treatment group was more effective in the treatment of KOA.@*CONCLUSION@#The inner-heating acupuncture could significantly improve the pathological changes of KOA rats, inhibit the apoptosis of chondrocytes, which may be closely related to the suppression of Caspase-3 and Caspase-9 expression.
Subject(s)
Animals , Rats , Apoptosis , Cartilage, Articular , Caspase 3 , Caspase 9 , Chondrocytes , Heating , Osteoarthritis, KneeABSTRACT
Aim: To investigate the effect of salvianolic acid B (Sal B) on liver fibrotic cells in vivo and in vitro from die perspective of apoptosis, as well as the effect on cleaved caspase-9. Methods: Diethylnitrosamine (DEN) was used to induce liver fibrosis in mice for 12 weeks. The padiological changes were detected by HE staining, and the fibrotic lesion area was determined. The cell apoptosis in the fibrotic area was observed by Hoechst 33258 fluorescence staining. The expression of cleaved caspase-9 in fibrotic tissues was detected by Western blot. The apoptotic rate of each group was detected by double standard method AnnexinV-FITC/PI, and the expression of apoptotic protein cleaved caspase-9 in HSC-T6 was detected by Western blot. Results: HE staining suggested that 12 weeks were the period of liver fibrosis in mice. No pseudoplobular structure was formed in group with low and high dose of Sal B, and the degree of fibrosis was lower than that in model group. In the fibrotic lesion area, the fluorescence staining of Hoechst 33258 showed that apoptotic cells significantly increased in group with low Sal B and high dose compared with model group. The results of the AnnexinV-FITC/PI method showed that TGF-β1 inhibited the apoptosis of HSC-T6, and Sal B promoted the apoptosis of HSC-T6 after TGF-β1 intervention and showed a concentration dependence (P <0. 01). Western blot results showed that in fibrotic liver tissues, Sal B increased the expression of cleaved caspase-9 in HSC-T6 cells compared with model group. Compared with TGF-β1 group, Sal B increased cleaved caspase-9 protein expression (P < 0. 01). Conclusions: Sal B can significantly promote apoptosis of liver fibrotic cells in vitro and in vivo, and its pro-apoptosis mechanism may be related to the up-regulation of cleaved caspase-9.
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Purpose To explore the expression,significance and relationship of apoptosis related gene Apollon and Caspase 9 in gastric carcinoma. Methods The SP immunohistochemical method was used to detect the expression of Apollon and Caspase 9 in 105 cases of gastric carcinoma,38 adjacent tissues and 29 normal tissues,and the expression of Apollon and Caspase 9 was analyzed with relation to clinicopathologic factors. Results The numbers of positive expression of Apollon gene in gastric carcinoma tissues,adjacent tissues and normal tissues were 82(78. 10％) ,8(21. 05％) and 2(6. 90％) respectively, there was significant difference between gastric carcinoma tissues, adjacent tissues and normal tissues (P < 0. 01) . The expression of Apollon in gastric carcinoma was positively correlated with degree of tumor differentiation,TNM staging and lymph node metastasis (P < 0. 05) ,but not with other clinicopathologic factors (P > 0. 05) . The numbers of positive expression of Caspase 9 gene in gastric carcinoma tissues,adjacent tissues and normal tissues were 21 (20. 00％) ,23 (60. 53％) and 21 (72. 41％) ,respectively,and there was significant difference between gastric carcinoma tissues,adjacent tissues and normal tissues (P < 0. 01) . The expression of Caspase 9 in gastric carcinoma was positively correlated with degree of tumor differentiation, TNM staging and lymph node metastasis (P < 0. 01) ,but not with other clinicopathologic factors (P > 0. 05) . The expression of Apollon was negatively correlated to Caspase 9 in gastric carcinoma with statistical significance (r = - 0. 541 1,P < 0. 01) . Conclusions The interaction of Apollon and Caspase 9 may be involved in the gastric carcinogenesis and progression. Apollon is closely related with invasion and metastasis of gastric carcinoma,and it may be a potential treatment target.
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Objective: To investigate the effects of rosmarinic acid (RosA) on the proliferation and apoptosis of human colon cancer HCT-8 cells, and explore the related mechanisms. Methods: The proliferation of HCT-8 cells was detected by CCK-8 assay at different concentrations of RosA for different time periods. The apoptosis rates of HCT-8 cells and the expression of related proteins were investigated after the treatment of RosA at 15, 45, and 75 μmol/L for 72 h according to the CCK-8 results. The apoptosis rates of HCT-8 cells were detected by FCM. The mRNA expressions of p53, Bax, and Puma were detected by RT-qPCR. The protein levels of p53, Bax, Puma, and active Caspase-9 were detected by Western blotting. Results: RosA could inhibit the proliferation of human colon cancer cells HCT-8 in a time- and dose-dependent manner. RosA at 15 and 45 μmol/L mainly induced early apoptosis (P < 0.01), RosA at 75 μmol/L mainly induced late apoptosis (P < 0.01). RosA could up-regulate the mRNA expression of Puma in a dose-dependent manner. RosA at 45 and 75 μmol/L increased the mRNA expression of p53 and Bax (P < 0.05). RosA could up-regulate the protein levels of Puma, Bax, and active Caspase-9 in a dose-dependent manner. RosA at 75 μmol/L could significantly increase the protein expression of p53 (P < 0.05). Conclusion: RosA can significantly inhibit the proliferation of HCT-8 cells by inducing the apoptosis. The apoptosis-inducing proteins of p53, Puma, Bax, and active Caspase-9 induce the apoptosis of cells.
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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.
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Aim To explore the effect of Klotho (KL) gene transfection on the apoptosis of MC3T3-E1 osteo-blasts induced by dexamethasone(DEX). Methods MC3T3-E1 osteoblasts were transfected by recombinant adenovirus containing KL gene(Ad-KL) and recombi-nant adenovirus containing green fluorescent protein (GFP) gene(Ad-GFP). The apoptosis model was con-structed. The transfection efficiency of Ad-KL and Ad-GFP in cells were observed using inverted fluorescent microscope, and the level of KL mRNA and protein was detected by qPCR and Western blot,respectively. The cell viability after different concentrations of DEX acting on the cells and the viability of every research group were determined by cell counting kit-8 (CCK-8) assay. The apoptotic rate was evaluated by flow cytom-etry. The level of mRNA and protein was analyzed by qPCR and Western blot, respectively. The level of caspase-9 protein was detected by immunofluorens-cence assay. Results Cells were transfected by Ad-KL and Ad-GFP successfully. KL group and KL +DEX group had higher level of KL mRNA and protein than that in other groups. The optimum concentration of DEX was 2.0 mmol·L-1. When DEX acting on the cells, the cells viability decreased and apoptotic rate increased obviously in DEX group and GFP + DEX group. The level of Bax mRNA and protein presented a upward trend in DEX group and GFP +DEX group, while the level of Bcl-2 mRNA and protein was oppo-site. But after KL transfecting MC3T3-E1 osteoblasts, the markers described above in KL group had more dramatic improvement than in DEX group and KL +DEX group. Conclusions High-dosage DEX can in-duce the apoptosis of MC3T3-E1 osteoblasts, and the pro-apoptosis effect of high-dosage DEX in MC3T3-E1 osteoblasts can be suppressed by up-regulating KL gene expression level, suggesting that the glucocorticoid-in-duced osteoporosis might be improved by up-regulating KL gene expression level, and it may be a new target for the treatment of latrogenic osteoporosis induced by high-dosage glucocorticoid in clinic.
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Objective To detect the expression of Survivin and Caspase-9 gene in Uygur breast cancer pa-tients with different HER-2 phenotypes,to find out the difference and association of the two genes and to find out the potential roles of the two genes breast cancer pathogenesis. Methods We selected 72 Uygur patients diag-nosed as breast cancer initially and they were divided into group A with HER-2 positive(n = 39)and group B with HER-2 negative(n = 33). Another 40 Uygur patients with benign breast were involved as the controls. Immunohis-tochemistry and real-time RT-PCR were used to detect the two genes,and analyze the differences and association of each gene between the groups. Results (1)The expression of Survivin gene in group A and B were higher than that in the control group. Further analysis showed that the expression of Survivin gene was enhanced in group A when compared with that in group B(P < 0.05);while even the expression of Survivin gene in group B was higher than that in the control group but no statistical difference was found(P > 0.05).(2)The expression of Caspase-9 gene in group A and B were lower than that in the control group. Real-time RT-PCR showed that the expression of Caspase-9 gene of group A was decreased when compared with that in group B(P < 0.05);While the expression of Caspase-9 gene of group B was slightly lower that of the control group but it showed no statistical significance (P > 0.05). Immunohistochemical results showed there were no statistical differences of expression of Caspase- 9 gene in group A and B and control group(all P > 0.05). The expression of Survivin and Caspase-9 gene was nega-tively associated in group A and B(P < 0.01;P < 0.05). Conclusions In Uygur patients with HER-2 positive breast cancer,the expression of Survivin gene is enhanced but that of caspase-9 gene is decreased,and they are negatively associated. Through inhabiting caspase-9 gene,Survivin gene may potentially lead to the occurrence of HER-2 positive breast cancer.
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Objective To explore the relationship between apoptosis promoting effector molecules caspase-3,caspase-9 and lubar intcrvertebral disc protrusion.Methods 99 of operation patients with lubar intervertebral disc protrusion in their mater nity ward were recruited.Among them,single segment of lubar intervertehral disc protrusion were 70 (Group A),more than one segments of lubar intervertebral disc protrusion were 29(Group B).In addition,40 unrelated healthy people from physi cal examination center were enrolled as controls.Enzyme linked immunosorbent assay (ELISA) was used to examine serum caspase-3 and caspase-9 levels in lubar intervertebral disc protrusion patients.Results The level of caspase-3 in control group,group A and group B,respectively were 11.24±0.41,14.31±0.67 and 17.43±1.86 pmol/L.The caspase-3 activity in each group was statistically significant difference (F=8.47,P＜0.01).The level of caspase-9 in control group,group A and group B respectively were 18.54±2.19,30.57±3.63 and 43.68±5.15 pmol/L.The caspase-9 activity in each group was statistically significant difference (F=7.85,P=0.001).Compared with control group,the caspasc 3 and caspase-9 ac tivity in group A (q=3.08.3.29,all P＜0.05),group B (q=5.78,4.50,all P=0.014) was statistically significant differ ence.The caspase-3 and caspase-9 activity in group A,group B was statistically significant difference (q=3.21,3.22,all P＜0.05).Conclusion The augment of caspase-3 and caspase-9 promoted apoptosis of lubar intervertebral disc protrusion.It was connected with quantity of protrusive segments.The more segments of protrusion,the higher caspase-3 and caspase-9 levels of examination would be.