|
ObjectiveThis study was to investigate the proliferative inhibition and mechanisms of crocin on human Leukemia Jurkat cells.MethodThe experiment was divided into control and experimental groups According to a final concentration of crocin the experimental groups were respectively divided into five subgroups, group A (0.625 mg/ml)、group B (1.25 mg/ml)、group C (2.5 mg/ml)、group D (5.0 mg/ml)and group E(10 mg/ml). Under the role of different concentrations of crocin, the number of different groups’cells was counted by blood cell count plate artificial count method, and the growth curve of Jurkat cells was made. Cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) method. After being cultured with different concentrations of crocin for 24 and 48 hours, the apoptosis rates of Jurkat cells were tested by Annexin V/PI through flow cytometry, and bcl-2 and bax gene expression were detected by reverse transcription-polymerase chain reaction (RT-PCR.).Result1. proliferation of Jurkat cell:①Cell morphology: In control group, cell body was round like eggs, cytoplasm was Uniform and transparent, with good refraction, suspended growth like grapes. In experimental groups, cell body was shrinkaged, particles increased in cytoplasm, with reduced refraction, single suspended growth when the concentration of crocin was 5.0mg/ml and 10mg/ml.②Cell number: In the 24 hours, the number of cells of control group was (7.5±0.62)×105, which of the experimental groups were (7.2±0.41)×105、(6.7±0.45)×105、(5.5±0.34)×105、(4.1±0.23)×105、(3.8±0.35)×105, groups B、C、Dand E were significantly lower than the control group and group A (F=108.76,p<0.01),group D and E were with the smallest number(p<0.05),but no statistical significance between the two(p>0.05). In the 48 hours, the number of cells of control group was (14.0±1.72)×105, which of the experimental groups were (9.5±1.15)×105(8.2±0.94)×105 (7.0±0.63)×105、(2.5±0.27)×105、(2.2±0.22)×105, all of them were significantly lower than the control group (F=151.97. p<0.01), group D and E were with the smallest number(p<0.05),but no statistical significance between the two (p>0.05)③Inhibition rate of proliferation:In the 24 hours, the inhibition rate of control group was (1.826±0.025)%, which of the experimental groups were (3.566±0.025)% (34.174±1.042)%、(43.582±1.745)%、(70.116±1.603)%、(71.086±1.756)%, groups B、C、D and E were significantly higher than the control group and group A (F=2586.52, p<0.01), group D and E were with the highest inhibition rate(p<0.05),but no statistical significance between the two (p>0.05). In the 48 hours, the inhibition rate of control group was (2.002±0.011)%, which of the experimental groups were (16.082±2.236)%、(53.080±2.503)%、(67.346±1.678)%、(84.932±1.337)%、(86.041±2.067)%, all of them were significantly higher than the control group (F=1755.66、p<0.01), group D and E were with the highest inhibition rate(p<0.05),but no statistical significance between the two(p>0.05). Under the same drug concentration, the role of 48h were significantly higher than 24h (p<0.01).2.Apoptosis of Jurkat cell:In the 24 hours, the apoptosis o of control group was (0.347±0.208)%, which of the experimental groups were (2.107±0.417)%、(6.830±1.080)%、(9.167±1.478)%、(21.950±1.725)%、(22.850±1.541)%, groups B、C、D and E were significantly higher than the control group and group A(F=190.69, p<0.01), group D and E were with the highest apoptosis(p<0.05),but no statistical significance between the two (p>0.05). In the 48 hours, the apoptosis o of control group was (0.580±0.300)%,which of the experimental groups were (9.150±1.010)%、(12.540±1.490)%、(19.060±1.601)%、(38.980±1.712)%、(40.220±1.243)%, all of them were significantly higher than the control group (F=1755.66> p<0.01), group D and E were with the highest apoptosis(p<0.05),but no statistical significance between the two (p>0.05). Under the same drug concentration, the role of 48h were significantly higher than 24h(p<0.01).3.expression of bcl-2 mRNA and bax mRNA:①bcl-2 mRNA:In the 24 hours, the grayscale scan ratio of control group was (0.802±0.017), which of the experimental groups were (0.782±0.015) (0.702±0.017)、(0.636±0.020)、(0.536±0.022)、(0.515±0.021), groups B、C D and E were significantly lower than the control group and group A(F=124.78,p<0.01), group D and E were with the most obviously decreased (p<0.05),but no statistical significance between the two (p>0.05). In the 48 hours, the grayscale scan ratio of control group was(0.794±0.026), which of the experimental groups were(0.685±0.020)、(0.646±0.018)、(0.547±0.023)、(0.420±0.016)、(0.398±0.017), all of them were significantly lower than the control group (F=176.08, p<0.01), group D and E were with the most obviously decreased (p<0.05),but no statistical significance between the two (p >0.05). Under the same drug concentration, the role of 48h were significantly lower than 24h (p<0.05). ②bax mRNA:In the 24 hours, the grayscale scan ratio of control group was (0.634±0.019), which of the experimental groups were (0.664±0.016)、(0.767±0.020)、(0.837±0.020)、(0.920±0.017)、(0.931±0.015), groups B、C、D and E were significantly higher than the control group and group A(F=148.59, p<0.01), group D and E were with the most obviously increased (p<0.05),but no statistical significance between the two (p>0.05). In the 48 hours, the grayscale scan ratio of control group was (0.634±0.019), which of the experimental groups were(0.792±0.022)、(0.827±0.016)、(0.926±0.014)、(1.047±0.017)、(1.071±0.019), all of them were significantly higher than the control group (F=221.67, p<0.01), group D and E were with the most obviously increased (p<0.05),but no statistical significance between the two (p>0.05). Under the same drug concentration, the role of 48h were significantly higher than 24h (p<0.05).4. Apoptosis and gene expression:①bcl-2 and apoptosis:Pearson correlation coefficient was-0.964, p=0.00, the difference was significant, bcl-2 gene expression and apoptosis showed a strong negative linear correlation.(2)bax and apoptosis:Pearson correlation coefficient was 0.957, p=0.00, the difference was significant, bax gene expression and apoptosis showed a strong positive linear correlation.③bcl-2 and bax: Pearson correlation coefficient was-0.983, p=0.00, the difference was significant, bcl-2 and bax gene expression showed a strong negative linear correlation.Conclusion1.within the concentration of crocin 0.625-10mg/ml, the growth of Jurkat cells was inhibited remarkably in the dosage dependence and time way, concentration of 5.0 mg/ ml, in the 48 hours, effect of inhibition was the most obvious.2.Crocin can promote Jurkat cells apoptosis, and the apoptosis rate was gradually increased within the concentration 0.625-10mg/ml, concentration of 5.0 mg/ml, in the 48 hours, apoptosis was the highest.within the concentration of crocin 0.625-10mg/ml,crocin can inhibite the expression of bcl-2 gene, concentration of 5.0 mg/ml, in the 48 hours, effect of inhibition was the most obvious, bcl-2 gene expression and apoptosis showed a strong negative linear correlation.4. within the concentration of crocin 0.625-10mg/ml,crocin can increase the expression of bax gene, concentration of 5.0 mg/ml, in the 48 hours, effect of promotion was the most obvious, bax gene expression and apoptosis showed a strong positive linear correlation.5.Crocin can promote Jurkat cells apoptosis and inhibit cell growth. The mechanism may be related to the inhibition of bcl-2 gene expression and the promotion of the bax gene expression.
|