| Objectives: This research is to induce esophageal precancerous lesion in animal models, the C57BL/6 mice, with the chemical carcinogen 4-nitroquinoline-1-oxide(4-nitro-quinoline-1-oxide,4NQO). By observing the effect of Qigehuacai prescription on esophageal precancerous lesions differentiationrelated genes NDRG1 and c-myc expression in mice, part of mechanism action about Qigehuacai prescription reversing esophageal precancerous lesions will be explored in order to provide an experimental basis for clinical application.Methods: 130 SPF C57BL/6 mice, aged 4 weeks, half male and half female, weighing 18±2g were sampled. The mice were randomly divided into five groups:(1)Normal control group:20 mice,regular feeding;(2)Model group:30 mice drank 4NQO aqueous solution(at a concentration of 100?g /ml) for 14 weeks, 25ml/100 g of body weight per day and intragastric infusion with water was administered after modeling;(3) Positive control group: 20 mice drank 4NQO aqueous solution for 14 weeks and then the intragastric infusion with all-transretinoic acid was delivered;(4) Prevention group: 30 mice drank 4NQO water and at the same time Qigehuacai prescription(Mianjian granule 240 mg/ml inside) was administered. After 14 weeks the intragastric infusion with Qigehuacai prescription only was delivered;(5)Treatment group:30 mice drank water 4 NQO. After 14 weeks the intragastric infusion with Qigehuacai prescription solution was administered, three times a week, 0.1 ml/20 g of body weight for each group of mice. The intragastric infusion volume was adjusted with the changes of the body weight of the mice weekly. At the end of 14 th weeks, the modeling of esophageal precancerous lesions was successful. Up to the end of 24 weeks, all the mice were killed.The esophageal tissue in mice at the end of 14 weeks and 24 weeks were sampled for HE staining respectively. The pathological morphological changes of the esophageal tissue were observed by light microscopy in normal control group, model group and prevention group. At the end of the 24 th Week, the ultrastructural changes of esophageal tissue under TEM were observed. the NDRG1 protein expression of each groups of mice was determined with the western blot method; the type of NDRG1, c-myc gene expression of each groups of mice were determined with the reverse transcription-polymerase chain reaction method.Results:1 General condition in each group In the process of the experiment, the mice in the normal control group showed no abnormalities while the mice in other groups showed no abnormalities within 2 weeks. After 2 weeks, the mice in the model group had some changes, such as messy fur, dry and lackluster, the neck hair loss,lassitude, curled and arched back, clustering, eye closed, inactive, loss of appetite, and a drop in body weight. After 14 weeks, the positive control group,the prevention group and the treatment group were in good spirits with good appetite than the model group. The condition of the prevention group was better than that of the treatment group.2 Test results2.1 The pathological morphological changes of the esophageal tissue of the mice in each group under the light microscope.At the end of 14 and 24 week, there is no abnormal hyperplasia in the esophageal squamous epithelium of the mice in the normal control group. At the 14 th week, the esophageal epithelial cell layers increased in the model group and the atypical cells involved the 2/3 under the esophageal epithelium.At the end of the 24 th week, the dysplasia in the model group became serious and most atypical cells involved the full-thickness of esophageal epithelium.The nuclei were deeply stained and the size of them increased and showed visible pathological mitotic. At the end of the 24 th week, the esophageal mucosa epithelial of the mice in the positive control group became thickening while the esophageal epithelium atypical cells were between the epithelium of lower 1/3 to 2/3 in the prevention group. At the end of the 14 th week, the esophageal epithelium atypical cells was between the epithelium of lower1/3 to 2/3 in the prevention group. At the end of the 24 th week, abnormal cells were confined to the lower 1/3 of the all-epithelial-layer. At the end of the 24 th week, the majority of abnormal cells in the treated mice were confined to the lower two-thirds of the skin and a small number of them between 1/3 and 2/3.2.2 The ultrastructural changes of esophageal epithelial cells of the mice in each group by TEM There were no abnormal changes on the esophageal mucosa in Normal control group. The esophageal epithelial cells in the mice in the model group were gap-widened. The desmosomes were reducing. The mitochondrial had edema. Part of the vacuolar became degenerated, necrosis. The epithelial got shedding and the nucleus irregular while the perinuclear increased and the nucleoli got bigger. The nuclear notch was visible and the nuclear chromatin marginated. Compared with the model group, the prevention group, the positive control and the treated groups of the esophageal epithelial lesions in the mice were relieved, cell gap smaller, desmosomes increased. The intercellular space were smaller between the prevention group and the treatment group, desmosomes were relatively abundant.2.3 Determination of the expression of NDRG1 protein with the Westernblotting method at the end of the 24 th week in each group.In the control group, there was low protein expression level of NDRG1.Compared with the normal control group, the NDRG1 protein expression level in the model group was significantly increased(P<0.01). Compared with the model group, the NDRG1 protein levels in the positive control group, the prevention group and the treatment group significantly decreased(P<0.01).The prevention group was better than the positive control group and the treatment group(P <0.05).2.4 Determination of the NDRG1, c-myc gene expression 2.4 in mice with RT-PCR method Total RNA of each group used the same amount of Olig(d T) 15 to conduct the reverse transcription and then used NDRG1, c-myc primers to do PCR amplification, which appeared in the 483 bp, 263 bp specific zone respectively. NDRG1 m RNA, c-mycm RNA expression levels were represend by the ratio of NDRG1, c-myc gene amplification fragment integrated absorbance and GAPDH the amplified fragment integrated absorbance.Statistical analysis found that NDRG1 si RNA/GAPDH m RNA in the model group was significantly higher than those in the control group(P<0.01).NDRG1 m RNA/GAPDHm RNA in the positive control group, the treatment group and the prevention group were significantly lower than the model group and there was a significant difference(P<0.01); The prevention group and the positive control group were not statistically significant( P>0.05); The prevention group was significantly lower than the model group(P<0.05); The positive control group was significantly lower than the treatment group(P<0.01). c-mycm RNA/GAPDHm RNA in the model group were significantly higher than those in the normal control group and there were significant differences(P<0.01). The positive control group, the treatment group and the prevention group and the treatment group significantly reduced(P<0.05); The prevention group and the positive control group were not statistically significant(P >0.05); The positive control group w was significantly lower than the treatment group and there were significantly differences(P<0.01).Conclusions:1 In the process of the esophageal precancerous lesions, NDRG1 protein,NDRG1 and c-myc gene expression significantly increased. Qigehuacription can lower NDRG1 protein, NDRG1 and c-myc gene expression in the esophageal tissue of esophageal precancerous lesions in mice, delay the development of the esophageal precancerous lesions, Prophylaxis has certain adva- ntages.2 Qigehuacai prescription can delay the development of the esophageal precancerous lesions and play a role in prevention and treatment. Its mechanism may be related to regulate the differentiation-associated gene NDRG1 and the expression of proto-oncogene expression c-myc. |
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