【 Abstract 】 Objective To probe into the role of inositol 1, 4, 5-trisphosphate (IP3) and bax gene expression in apoptosis of HepG2 cells induced by genistein (Gen). Methods HepG2 cells were treated with different concentrations including 20, 40, 60 and 80 μ mol/L Gen as HepG2 cells cultured with 0 μmol/L Gen for 72 h was control; HepG2 cells were treated with 60 μmol/L Gen for 6, 12, 24, 48 and 72 h as HepG2 cells treated with 60 μmol/L Gen for 0 h was control. IP3 content, bax mRNA expression and apoptosis rate were assayed by IP3- ［ 3H ］ Birtrak assay, RT-PCR and flow cytometry, respectively. ResultsHepG2 cells incubated with each concentration of Gen for 72 h ， IP3 content was lower than that of control 〔 (17.7 ± 1.3), (11.2 ± 0.9), (4.9 ± 0.5), (4.8 ± 0.3) pmol/106 cells vs (29.4 ± 0.5) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression (RI which was the gray degree multiply area of bax/the gray degree multiply area of β -actin) was higher than that of control (0.26 ± 0.02, 0.33 ± 0.05, 0.35 ± 0.06, 0.38 ± 0.05 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate was higher than that of control 〔 (10.1 ± 0.9)%, (18.7 ± 1.6)%, (28.7 ± 2.5)%, (27.9 ± 2.0)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. HepG2 cells were incubated with 60 μ mol/L Gen for 6, 12, 24, 48 and 72 h ， IP3 content was lower than that of control 〔 (22.6 ± 0.9), (12.0 ± 1.4), (7.5 ± 0.8), (5.6 ± 0.5), (4.3 ± 0.6) pmol/106 cells vs (29.2 ± 0.6) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression was higher than that of control incubated with 60 μ mol/L Gen for above 12 h (0.25 ± 0.06, 0.29 ± 0.02, 0.30 ± 0.02, 0.35 ± 0.04 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate in groups incubated with 60 μ mol/L Gen for 24, 48 and 72 h was significantly higher than that in control 〔 (7.4 ± 0.5)%, (20.5 ± 2.0)%, (30.7 ± 1.6)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. ConclusionGen induces apoptosis of HepG2 cells by reducing IP3 production and increasing bax gene expression.
Objective To observe the outcomes of using different concentrations of arsenic trioxide at varying phases on the breast cancer cell line MCF-7 and to study the mechanism of this effect. Methods The effect of arsenic trioxide on the growth of breast cancer cell line MCF-7 was observed after applying arsenic trioxide of different concentrations （0.5-16 μmol/Ｌ）. The inhibitory effect of arsenic trioxide on the cell proliferation was investigated with 3-(4，5-dimethyl-thizazol-2-yl)-2， 5-diphenyl tetrazolium bromide (MTT) and the induction of arsenic trioxide on cell apoptosis was detected by DNA ladder and terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Results The effect of arsenic trioxide on breast cancer cell line MCF-7 depended on the phase and the dose. The number of cell decreased significantly and there were conspicuously typical morphological changes of apoptosis after the use of arsenic trioxide， including membrane blebbing， chromatin pyknosis, nuclear fragmentation and the formation of apoptotic body. The typical DNA ladders were observed in the MCF-7 cells after 48 h administration of arsenic trioxide at concentrations 1-8 μmol/Ｌ. Significant elevations of apoptosis index at 24 h, 48 h and 72 h were all detected by TUNEL after incubating with 4 μmol/L arsenic trioxide. Conclusion Arsenic trioxide may inhibit the growth of breast cancer cell line MCF-7 significantly by inducing the apoptosis of breast cancer cell.
ObjectiveTo explore the influence of different radiation doses of 125Ⅰseed on human poorly differentiated gastric cancer cells (BGC823). MethodsSixty four male nude mice of BLAB nu/nu inoculated with human poorly differentiated gastric cancer cells (BGC823) were took as animal models. When tumors of nude mice grew to 0.7-1.2 cm, the nude mice were randomly divided into blank control group, low dose group, middle dose group, and high dose group (n=16). The tumors of nude mice in blank control group were implanted with blank seed, but tumors of nude mice in low dose group, middle dose group, and high dose group were implanted with 125Ⅰseed (the radiation doses of 3 groups were 1.48×10-7 Bq, 2.22×10-7 Bq, and 2.96×10-7 Bq respectively). Four nude mice of 4 groups were randomly collected to measure the tumor volume and weight before implantation, and on 7, 14, 21, and 28 days after implantation, but apoptosis rates and expression levels of cyclinE mRNA were measured on 14 days and 28 days after implantation, by using flow cytometer and semi quantitative RT-PCR method respectively. Results①Except for the blank control group, the tumor volume and weight decreased over time in low dose group, middle dose group, and high dose group. The tumor volume and weight in blank control group were higher than those of other 3 groups on 7, 14, 21, and 28 days after implantation (P < 0.05);in addition, on 28 days after implantation, tumor volume and weight in low dose group were lower than those of middle dose group and high dose group (P < 0.05).②On 14 days and 28 days after implantation, the apoptosis rates of blank control group were lower than those of other 3 groups (P < 0.05), but expression levels of cyclinE mRNA were all higher than those of other 3 groups (P < 0.05). In addition, on 28 days after implantation, apoptosis rate of low dose group was higher than both of middle dose group and high dose group (P < 0.05), but expression level of cyclinE mRNA was lower (P < 0.05). Compared with 14 days in the same group, except for the blank control group (P > 0.05), the apoptosis rates in other 3 groups on 28 days were higher (P < 0.05), with the lower expression levels of cyclinE mRNA (P < 0.05). Conclusions125Ⅰseed in organizational implantation can effectively inhibit the expression of cyclinE mRNA and the growth of human poorly differentiated gastric cancer tissue. Compared with doses of 2.22×10-7 Bq and 2.96×10-7 Bq of 125Ⅰ, low dose (1.48×10-7 Bq) contributes to the apoptosis of human poorly differentiated gastric cancer cells (BGC823).
Objective To investigate the pathogenesis of acute lung injury in rats induced by intra-peritoneally injection of perforative peritonitis ascitic fluids（PPAF） and the role of L-arginine （L-Arg） in acute lung injury in this model. Methods Perforative peritonitis （PP） models were established in 60 rats and PPAF were collected. Forty-eight rats were randomly divided equally into NS group，PPAF group, and L-Arg group. Rats were randomly subjected to death at 7 h and 12 h. Peripheral blood WBC were counted，levels of NO and malondialdehyde （MDA） in serum were examined. Lung injury score and wet/dry ratio were evaluated， and level of myeloperoxidase （MPO） in lung tissues and lung cell apoptosis were tested. Results WBC count of peripheral blood, levels of NO and MDA in serum， level of MPO in lung tissue, lung injury score， wet/dry ratio， and lung cell apoptosis rate in PPAF group were significantly higher than that in NS group at each time point（P＜0.01）. Level of NO in serum in L-Arg group was higher than that in PPAF group （P＜0.01）， but lower level of MDA in serum， lower level of MPO in lung tissue and lung injury score，lower wet/dry ratio， and lung cell apoptosis rate were observed in L-Arg group（P＜0.05）. In PPAF group and L-Arg group， level of NO in serum， wet/dry ratio, and lung cell apoptosis rate were higher at 12 h than that at 7 h（P=0.000）. Serum NO level was in negative correlation with serum MDA level （r=-0.257，P=0.021）， MPO level in lung tissue（r=-0.444， P=0.011），and lung cell apoptosis（r=-0.351， P =0.010） in PPAF group and L-Arg group， but serum MDA level was in positive correlation with cell apoptosis（r=0.969， P＜0.001） in each group. Conclusions Acute lung injury rats model can be established by intra-peritoneally injection of PPAF. Enhanced oxidizing reaction and cell apoptosis take part in the occurrence of acute lung injury. L-Arg plays a protective role in acute lung injury.
Objective To investigate the effect of renal cell apoptosis induced by obstructive jaundice on the expression of bcl-2 in rats, and to explore the mechanism of renal impairment induced by obstructive jaundice. Methods Thirty-two male SD rats were randomly divided into 2 groups: SO group and BDL group. The rats in SO group received sham operation. Bile ducts of rats in BDL group were ligated. Pathology of kidneys was observed under the microscope. The levels of D-Bil, TBA, GOT, GPT, Cr and BUN in serum and β2-MG in urine were measured. The apoptotic rate of renal cells was calculated by flow cytometry and the forms of DNA fragmentation in renal cells were detected by agarose gel electrophoresis. The expression of inhibitory gene bcl-2 in the renal tissues was detected by immunohistochemistry. Results The color of urine in BDL group became dark yellow in day 2 after operation; The ears, tails and the muscle of abdominal wall and splanchnic organs, such as liver and kidney, also became yellow and swollen in day 7. The D-Bil, TBA, GOT, GPT, BUN of serum and β2 -MG of urine in BDL group were higher than those in SO group (P＜0.05, P＜0.01), and each value (except β2 -MG) in BDL group of 14 d was higher than that in BDL group of 7 d (P＜0.05, P＜0.01), respectively. The result of flow cytometry showed that the apoptotic rate of SO group and BDL (7 d and 14 d) group were (2.10±0.75)%， (18.17±0.86)% and (36.39±2.23)% respectively, there were significantly difference among them (P＜0.05). The expression rate of bcl-2 of renal cell in BDL group of 7 d was higher than that in BDL group of 14 d. Conclusion Obstructive jaundice could induce apoptosis of the renal cells, and activate the expression of bcl-2 of the renal tubular epithelial cells in feedback, which may regulate the process of apoptosis.
ObjectiveAfter establishing the rabbit brain death model, TUNEL, western blotting, and immuno-histochemical methods were used to detect hepatocyte apoptosis to study hepatocyte apoptosis level from rabbit donors after brain death. MethodsSixty healthy male New Zealand rabbits were divided into brain death group (n=30) and sham group (n=30). The rabbits of brain death group were established by increasing intracranial pressure in a modified, slow, and intermittent way, collecting liver tissues after corresponding treatment respectively, using TUNEL to detect apoptosis rate, western blotting and immunohistochemical methods to detect the expression of Cleaved-caspase 3. ResultsThe hepatocyte apoptosis rate at each time point of brain death group were higher than those of the corresponding time point of sham group (P<0.05), and the rate of hepatocyte apoptosis increased gradually with the extension of brain death time (P<0.05). The results of Western blot assay and immunohistochemistry assay showed that the relative expression amount of Cleaved-caspase 3 protein increased gradually with the extension of brain death time (P<0.05), and relative expression amount of Cleaved-caspase 3 protein at each time point of brain death group were higher than those of the corresponding time point of sham group (P<0.05). ConclusionsThe relationship between brain death donor liver and cell apoptosis is closely related. Along with the extension of the brain death time in rabbits, the level of apoptosis of liver cells gradually increased, which affects the quality of liver donors after brain death.
ObjectiveTo evaluate the expression of miR-338-5p in colorectal cancer tissues and study its role in colon cancer cell proliferation, apoptosis, and cell cycle. MethodsThe expression of miR-338-5p was detected by real-time PCR in the colorectal cancer tissues and corresponding adjacent to cancer tissue samples. The miR-338-5p-mimics was transfected into the colon cancer cell lines HCT116 and SW620 to investigate its role in cell proliferation, apoptosis, and cell cycle. The cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. The cell cycle was also analyzed by flow cytometry. Results①miR-338-5p expression was significantly downregulated in the colorectal cancer tissues as compared with corresponding adjacent to cancer tissue samples(P < 0.01). 2 Compared with the transfected negative control cells, the proliferation ability of colon cancer cell HCT116 or SW620 was significantly decreased(P < 0.01), cell apoptosis was significantly increased[HCT116 cell:(11.43±0.67)% versus(7.98±0.36)%, P < 0.01;SW620 cell:(10.5±0.2)% versus(7.93±0.5)%, P < 0.01), and cell G1 was arrested[HCT116 cell:(80.41±1.34)% versus (64.87±1.83)%, P < 0.01;SW620 cell:(68.76±0.41)% versus(54.89±0.78)%, P < 0.01) after transfecting miR-338-5p-mimics cells. ConclusionmiR-338-5p may act as an anti-oncogene in colorectal cancer through regulation of cell proliferation, apoptosis, and cell cycle.
ObjectiveTo explore apoptosis of acinar cells during pancreatic allograft rejection in rats.MethodsGroups of Wistar rats underwent heterotopic pancreaticoduodenal transplantation from syngenic Wistar of allogenic SD rats. The grafts were harvested on postoperative day 3, 5 and 7. All graft samples were subjected to histological examination and apoptotic cells of graft acinar cells using in situ terminal deoxynucleotidy1 transferasemediated dUTP nickend labeling (TUNEL). Histopathological rejection score and apoptotic index (AI) were analyzed. ResultsThe incidence of apoptotic cells was increased steadily over time in allografts, in contrast with syngenic grafts. The apoptotic cells in allografts were mainly acinar cells and few infiltrating lymphocytes. A significant correlation between apoptotic index and histopathological rejection score was noted.ConclusionTUNEL can display apoptosis of single cell in situ. Apoptosis is an important mechanism of tissue injury in acute pancreatic allograft rejection in rats. Acinar cell apoptosis can be used as a valuble index to estimate the injury of grafts and to monitor the acute rejection.
ObjectiveTo investigate the mechanism of lignans-1inhibiting the proliferation of human gastric cancer cell line SGC-7901. MethodsThe morphological changes of the cells were observed by the inverted phase contrast microscope. The cell surviving ratio was determined by methylthiazoly tetrazolium (MTT) assay after lignans-1 added to the cells at different concentrations on human gastric cancer SGC-7901 cell line in vitro, and half maximal (50%) inhibitory concentration (IC50) values were calculated. The cell cycle phase distribution and apoptosis were measured by flow cytometry. The expressions of apoptosis associated proteins of Caspase3, Bcl-2 and Bax were determined by Western blot. ResultsMorphological examination showed that lignans-1 could destroy the SGC-7901 cells with the increasing concentration of lignans-1. The inhibitory effect of lignans-1 on SGC-7901 cell was associated with time-and dose-dependent manner at the different concentration (2.5-20 μg/mL), P < 0.05. The IC50 of lignans-1 on the SGC-7901 cells was 4.19 μg/mL. The rate of the apoptosis cells and G2/M phase cells raised significantly after 48 hours' treatment with lignans-1, as same as the expression of Caspase3 and Bax (P < 0.05). G0/G1 phase cells and Bcl-2 decreased significantly with the increasing concentration of lignans-1 (P < 0.05). ConclusionsThe lignans-1 could inhibit the proliferation of SGC-7901 cells and induce apoptosis by arresting cells at G2/M phase in vitro. The mechanism is associated with activation of Caspase3 and Bax and inhibition of Bcl-2.
Objective To observe the effects of SARS-CoV-2 infection on the morphology, proliferation, apoptosis, cell cycle, and immune response function of mouse retinal photoreceptor cells (661w cells). MethodsA cell experiment. Logarithmic growth phase 661w cells were cultured in vitro and transfected with angiotensin-converting enzyme 2 (ACE2) overexpressing lentivirus to construct ACE2 overexpressing 661w cells that could be infected with SARS-CoV-2 pseudovirus (hereafter referred to as ‘pseudovirus’). The 661w cells were divided into three groups: the normal group (untreated), the siACE2 group (overexpressing ACE2 and not infected with the pseudovirus) and the infected group (overexpressing ACE2 and infected with the pseudovirus), in which the infected group was 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, and the cells were infected with the pseudovirus for 12, 24, 48 and 72 h, respectively. The infected group was infected with 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, respectively, for 12, 24, 48 and 72 h. Fluorescence microscopy was used to observe the transfection efficiency of ACE2; protein immunoblotting (Western blot) was used to detect the relative expression level of ACE2 in the cells; light microscope was used to observe the morphology of the cells in the normal and the infected groups; cell proliferation was detected by Cell Counting Kit-8 (CCK8) assay; flow cytometry was used to detect the cell cycle; Western blot and real-time quantitative polymerase chain reaction (qPCR) were used to detect the relative expression of interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), B lymphocytoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax) proteins and mRNA in the cells of siACE2 group, infected group (30 TU/ml pseudovirus group); qPCR was used to detect the relative expression of nuclear factor (NF)- κB1 and NF-κB2, as well as NF- kB enhancer (P65) and precursor protein (P100) in cells of the siACE2 group and the infected group (30 TU/ml pseudovirus group). One-way ANOVA was used for comparison between multiple groups; t-test was used for comparison between two groups. Results Compared with the siACE2 group, the cells in the infected group showed different degrees of crumpling, and with the increase of the concentration and time of pseudovirus induction, the crumpling of the cells worsened, and the number of cells decreased. Compared with the normal group, the cells in the infected group showed a gradual decrease in cell viability with the prolongation of pseudovirus induction time, and the difference was no statistically significant (F=0.840, 0.412, 1.498, 1.138; P＞0.05), and the apoptotic index of the cells induced in the 30 and 50 TU/ml pseudovirus group was significantly elevated, and the difference was statistically significant (F=2.523, 6.716, 3.477, 3.421; P＜0.05). At 72 h of pseudovirus induction, compared with the siACE2 group, the G1 phase cells in the 30 TU/ml pseudovirus group were significantly increased, and the difference was statistically significant (t=3.812, P＜0.05); the relative expression of IL-6, TNF-α, Bax protein and mRNA in the cells was up-regulated (t=7.601, 6.039, 3.088, 5.193, 6.427, 7.667; P＜0.05), the relative expression of Bcl-2 protein and mRNA was down-regulated (t=3.614, 6.777; P＜0.05), and the relative expression of NF-κB1, NF-κB2, P65, and P100 mRNA was significantly up-regulated with statistically significant differences (t=3.550, 3.074, 3.307, 4.218; P＜0.05). ConclusionSARS-CoV-2 infection may inhibit photoreceptor cell proliferation, promote apoptosis and cycle blockade by activating the NF-κB signalling pathway.