Objective To observe the structural changes of urinary center and the expression of Bcl-2 after conus medullaris injury in rats brain so as to explore the possible influence factors of degeneration in brain. Methods Thirty-six adult Sprague-Dawley rats were randomly divided into experimental group (n=30) and control group (n=6). In the experimental group, the conus medullaris injury model was established by cutting off the spinal nerve below L4, and no treatment was done in the control group. The modeling operations in the experimental group were successful, and 2 rats died at 3 months and 5 months after modeling operation respectively, which may be caused by renal failure or urinary tract infection. In the experimental group, 6, 6, 6, 5, and 5 rats were killed at 1 day, 1 week, and 1, 3, 6 months after operation respectively, and 1 rat was killed at each time point in the control group. The dorsolateral tissue of the pontine tegmentum was harvested to perform HE staining and Bcl-2 immunohistochemical SP staining. Results HE staining showed that there was no obvious difference between the experimental group and the control group at 1 day after operation, the neurons were densely packed, arranged neatly, and the nucleoli were clear; at 1 week, the space between the neurons in the experimental group were slightly widened; at 1 month, nucleus retraction in some neurons happened in the experimental group; at 3 and 6 months, the nuclei in the experimental group were more and more condensed, and even some cells disappeared. Bcl-2 immunohistochemical SP staining showed that the expression of Bcl-2 in the control group was weakly positive. The positive expression of Bcl-2 was found at 1 day after operation in the experimental group; the positive expression of Bcl-2 at 7 days after operation was significantly higher than that in the control group, and reached the peak; the positive expression of Bcl-2 decreased gradually at 1, 3, and 6 months after modeling operation, but it was still higher than that of the control group. Conclusion The urinary center appears structure degeneration and necrocytosis after conus medullaris injury in rats brain. The elevated expression of Bcl-2 may be associated with brain tissue repair and function remodeling.
ObjectiveTo investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells.MethodsThe MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.ResultsAfter 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance (IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) (t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups (t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) (t=23.420, P=0.002).ConclusionFTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.
HtrA serine peptidase 2 (HTRA2) is a serine protease existing in the mitochondrial gap. Among the four members of the human HtrA serine peptidase family, HTRA2 is the only protease with clear localization in the cell. It plays a dual role in the maintenance of mitochondrial homeostasis and the promotion of cell apoptosis. HTRA2 has been found to be associated with a variety of tumors. Meanwhile, the expression of HTRA2 can enhance the sensitivity of chemotherapy and radiotherapy, and can be used as a diagnostic and prognostic marker for malignant tumors and a target for combined therapy. This article reviews the structure, biological function and role of HTRA2 in malignant tumors, in order to provide clues and basis for early diagnosis and individualized treatment of tumor patients.
Objective To determine the anti-apoptosis effects of heme oxygenase-1 (HO-1) on lung injury after cardiopulmonary bypass (CPB), and to investigate its probable mechanisms. Methods A total of 144 male Wistar rats with wight of 250-350 g were divided into 3 groups: group A (control group), group B (cobalt protoporphyrin, CoPP), and group C [CoPP and zinc protoporphyrin (ZnPP)] randomly. A modified rat model of CPB-induced lung injury was established. And then the lung tissues were taken at different times for the relevant indicators test: before CPB (T0), immediately after CPB (T1), 2 h after CPB (T2), 6 h after CPB (T3), 12 h after CPB (T4), and 24 h after CPB (T5). The expression of HO-1 and Bcl-2 protein in each group was tested by immunohistochemistry, and cell apoptosis by TUNEL. Results The HO-1 protein expression in group B was significantly higher than that in groups A and C at any given time point, so was the HO-1 activity (P<0.05). There was no significant difference in Bcl-2 expression of lung tissue before CPB among each group (P>0.05). The Bcl-2 protein reduced gradually after CPB. The expressions of Bcl-2 protein in group B at all time points after bypass were significantly higher than that in groups A and C (P<0.05). The apoptosis index (AI) showed no significant difference before CPB in each group (P>0.05), and increased gradually after CPB. AI in group B at any time point after bypass was lower than that in groups A and C (P<0.05). The HE staining results showed that the damage of lung tissue in group B obviously reduced compared with groups A and C. Conclusion CoPP can induce a large amount of HO-1 expression in the lung tissue, and it is still highly expressed after CPB. So it plays an important role in anti-apoptosis through the up-regulation of Bcl-2 protein expression.
This paper aims to study the effects of traditional Chinese medicine Euphorbia esula on multidrug resistant human gastric cancer cells in the cell proliferation, migration, invasion and apoptosis, and to study the apoptosis-inducing pathway. Different dilutions of Euphorbia esula extract were used to process human multidrug resistant gastric cancer SGC7901/ADR cells. Cell proliferation inhibition phenomenon was determined by MTT experiment. Nuclear morphological changes of apoptotic cells and apoptotic indexes were observed and determined by Hochest33528 staining followed with fluorescence microscope observing. Flow cytometry was used to detect cell apoptosis rate. Cell migration and invasion ability were observed and determined by Transwell method. Spectrophotometry was used to detect caspase-3 and caspase-9 enzyme activity. Western blotting was used to detect subcellular distribution of cytochrome c. The results showed that Euphorbia esula extract had obvious inhibition effect on proliferation of gastric cancer multidrug resistant SGC7901/ADR cells, which was time- and concentration-dependent. After processing multidrug resistant gastric cancer SGC7901/ADR cells with Euphorbia esula extract, the apoptotic index and apoptosis rate were significantly increased than those in the control group, which showed a time- and dose-dependent mode; but if a caspase inhibitor was added, apoptosis index was not obviously increased. Transwell method showed that migration and invasion ability of the Euphorbia esula extract-processed SGC7901/ADR cells dropped significantly. Spectrophotometry showed that in Euphorbia esula extract-processed SGC7901/ADR cells, caspase-3 and caspase-9 expression were increased, which had significant differences with the control group. Western blotting test showed that the distribution of cytochrome c decreased in mitochondria, while increased in the cytoplasm (i.e., cytochrome c escaped from mitochondria to the cytoplasm). In conclusion, Euphorbia esula extract could inhibit the proliferation, migration and invasion, and induce apoptosis in human gastric cancer multidrug resistant SGC7901/ADR cells; and cytochrome c, caspase-9 and caspase-3 might be involved in cell apoptosis induced by Euphorbia esula extract, suggesting endogenous or mitochondrial apoptotic pathway.
ObjectiveTo investigate the influence of endoplasmic reticulum stress (ERS) on smoking-induced nucleus pulposus cells apoptosis and inflammatory response.MethodsBetween October 2016 and October 2018, 25 patients with cervical disc herniation receiving discectomy were collected and divided into smoking group (14 cases) and non-smoking group (11 cases). The baseline data of age, gender, herniated segment, and Pfirrmann grading showed no significant difference between the two groups (P>0.05). The obtained nucelus pulposus tissues were harvested to observe the cell apoptosis via detecting the apoptosis-related proteins (Caspase-3 and PRAP) by TUNEL staining and Western blot test. The nucleus pulposus cells were isolated and cultured with enzyme digestion, of which the third generation cells were used in follow-up experiments. Then, the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by ELISA; the nuclear translocation of P65 was monitored by cell immunofluorescence staining. Furthermore, ERS-related proteins (GRP78 and CHOP) were detected by Western blot; and endoplasmic reticulum ultrastructure was observed under transmission electron microscope. To verify the regulatory effect of ERS, cells were pretreated by ERS specific inhibitor (4-PBA), then cell apoptosis and inflammatory response were tested.ResultsThe nucleus pulposus tissue observation showed that the cell apoptotic rate and the expressions of apoptosis-related proteins (Caspase-3 and PARP) were obviously higher in smoking group than in non-smoking group (P<0.05). The nucleus pulposus cells observation indicated that the expressions of the inflammatory factors (IL-1β and TNF-α) and the ERS-related proteins (GRP78 and CHOP) were also higher in smoking group than in non-smoking group (P<0.05). The results of cell immunofluorescence staining further confirmed that smoking stimulated nuclear translocation of P65 in nucleus pulposus cells. The ERS injury was much more serious in smoking group than in non-smoking group. Furthermore, after 4-PBA inhibiting ERS, the expressions of GRP78, CHOP, IL-1β, TNF-α, and P65 were significantly decreased (P<0.05), and flow cytometry results showed that cell apoptotic rate in smoking group was decreased, showing significant difference compared with the non-smoking group (P<0.05).ConclusionSomking can stimulate cell apoptosis and inflammatory response in nucleus pulposus cells via ESR pathway. Suppressing ESR may be a novel target to suspend smoking-induced intervertebral disc degeneration.
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
ObjectiveTo explore the effect of DDX46 silencing on growth and apoptosis in esophageal squamous cell carcinoma cell TE-1 by the shRNA. MethodsThe relative expression of DDX46 mRNA in TE-1 cells was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and compared with immortalized human esophageal squamous cell Het-1A. DDX46 shRNA-expressing lentivirus was applied to silence DDX46 (experimental group), and non-silencing control lentivirus was added (control group) with a multiplicity of infection of 5 in TE-1 cells. In both groups, cell growth was monitored using high content screening, cell colony-forming capacity was measured by colony formation assay, cell apoptosis were determined by flow cytometry. Further, the Stress and Apoptosis Signaling Antibody Array Kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. ResultsCompared with the control group, cell counting after DDX46 silencing showed that TE-1 cell growth was significantly inhibited (P<0.001). Colony formation assay showed that cell colony-forming capacity was significantly inhibited (P<0.01). Annexin V-APC flow cytometry showed a significant increase in apoptosis (P<0.001). In PathScan® Antibody Array, the expression levels of Akt (Ser473, phosphorylation) and IκBα (Total, N/A) significantly decreased (P<0.01), and the expression of Caspase-3 (Asp175, cleaved) increased (P<0.05). ConclusionDDX46 is overexpressed in TE-1 cells. Targeted gene silencing of DDX46 inhibits cell growth, and induces cell apoptosis. DDX46 silencing probably by negative regulation of Akt/NF-κB signaling pathway, to play a role in inhibiting TE-1 cells growth and inducing apoptosis.
ObjectiveTo understand the mechanisms of gastric carcinogenesis relevant to Helicobacter pylori (H. pylori)-related cell apoptosis and explore potential causes of gastric cancer development through cell apoptosis. MethodThe literature of recently domestic and international research on the mechanisms of H. pylori-related cell apoptosis in the gastric carcinogenesis was searched and reviewed. ResultsThe H. pylori infection was one of the important risk factors in the occurrence and development of gastric cancer, which was characterized by the imbalance of the interaction between gastric epithelial cells and various cell components in the gastric microenvironment, and which promoted or inhibited the process of apoptosis, and thus interfered with the process of gastric cancer. ConclusionsH. pylori, through the regulation of various cellular components and molecular pathways, increases the sensitivity of gastric epithelial cells to apoptosis, actively participates in the progression of gastric cancer. With the advent of the era of precision medicine, research on the mechanisms of H. pylori-related cell apoptosis in gastric carcinogenesis is transitioning to clinical applications, offering promising new treatment strategies for gastric cancer patients.