| Object: In this experiment, we use 4-Nitroquinoline 1-oxide(4NQO) to establish esophageal precancerous lesions model, and took YIGE formula as observation object, all-trans-retinoicacid(ATRA) as positive control, to observe the interference effect of YG on esophageal precancerous lesions related indicators, and to investigate partial mechanism of YIGE formula.Methods:Grouping:①Normal group(20 mouse), with conventional breeding. ②Model group(30 mouse), 4NQO was used to induce cancer, at the end of 14 week, removed 4NQO when model was built successfully. ③ positive group(20 mouse), removed 4NQO when model was built successfully, and lavaged with 0.1ml(0.15mg)ATRA solutions. ④Prevention group(30 mouse), 4NQO was used to induce cancer, at the same time, lavaged with 0.1ml(contain 0.084 g avoid Fried particles) YIGE formula solutions, removed 4NQO when model was built successfully,continue lavage. ⑤ Treatment group(30 mouse), removed 4NQO when model was built successfully, lavaged with YIGE formula, the dosage was same to prevention group. Took notes of the weight of mice once a week, adjusted the dosage of lavage according to the weight.Model group mouse appeared to cancerization at the end of 24 week, then executed all mouse, took the esophageal tissue of mouse for hematoxylin and eosin staining at the end of 14 and 24 week, took the esophageal tissue of mouse for transmission electron microscope, immunohistochemistry, RT-PCR detection at the end of 24 week.Results:1 The observation of the general situation of live mouseAfter 2 weeks, in model group, the diet and actibity of individual was decreased, at the end of 10 week, the hair is no more luster, the cervical hair was depilated, sometimes met with trembling, curled. The diet, activity, lustrousness of hair in prevention group and treatment group was better than positive group, and with no depilation. The mental status, activity of prevention group was slightly better than the treatment group. In the process of the experiment, 3 mouse died in prevention group, 2 mouse died in model group, positive group, treatment group, respectively.2 The general observation of the mouse′s esophagus in different groupCut the esophagus longitudinally, at the end of 14 week, esophageal mucosa had no abnormality seen in normal group mouse. The esophageal mucosa of model group mouse was hyperemia, or gloomy, the local was uplifted, gray spots and plaque could be seen. The esophageal mucosa of prevention group mouse was red, without the formation of grey evection, spots and plaque. At the end of the experiment, naked eye lesions was not found in normal group. Papilloma, ulcer was appeared in model group mouse′s esophagus mucosa. Papilloma and white plaque was seen in the esophagus mucosa of positive group, prevention group and treatment group.3 The esophageal histopathological change of the mouse in different group observed by light microscopeAt the end of 14, 24 week, there was no paraplasm on normal group esophageal epithelial lamina. In model group heterotypic cells occupied two-thirds of under full-thickness at the end of 14 week, cancer cell appeared, with enlarged, hyperchromatic nuclear and pathological fission at the end of 24 week. In the positive group, heterotypic cells occupied between 1/3 and 2/3 of the epithelial full-thickness mostly at the end of 24 week. In the prevention group, heterotypic cells were not exceed 1/3 of the epithelial full-thickness at the end of 14 week, heterotypic cells occupied between 1/3 and 2/3 of the epithelial full-thickness at the end of 24 week. In the treatment group, heterotypic cells occupied more than 2/3 of the epithelial full-thickness mosly, and only a little occupied between 1/3 and 2/3 of the epithelial full-thickness at the end of 24 week.4 The esophageal cells ultrastructure observed by transmission electron microscope in different groupIn the model group, the intercellular space was widen, the number of desmosomes was reduced, the cytoplasmic became edema, chromatin was variated compared with normal group; In the positive group, prevention group and treatment group, the intercellular space was tighter slightly, the number of desmosomes was more compared with model group. In the prevention group and treatment group, the intercellular space was widen, the number of desmosomes was reduced compared with positive group. In the prevention group, the intercellular space was tighter, the number of desmosomes was more than the treatment group.5 The expression of β-catenin,Wnt1 and c-myc in different group detected by IHC-SPIn the normal group, the expression of β-catenin protein was little, β-catenin put color on cell membrane. In the model group, β-catenin accumulated on the cytoplasm and nucleus, the cytoplasm and nucleus was buffy. The expression level of Wnt1 protein was low in the normal group, Wnt1 protein accumulated on the cytoplasm in the model group, the cytoplasm appeared buffy. The expression level of c-myc protein was low in the normal group, c-myc protein mainly expressed on nucleus, a little was expressed on cytoplasm in the model group, the nucleus and cytoplasm appeared buffy. The average expression level of β-catenin and c-myc protein in the model group was obviously higher than normal group(P<0.05). The expression level of the positive group, treatment group and prevention group was much lower than the model group(P<0.05). There was no significant difference between the prevention group and the positive group(P>0.05). The treatment group was higher than positive group(P<0.05). The prevention group was much lower than the treatment group(P<0.05). The expression level of Wnt1 in the model group was much higher than normal group(P<0.05). The expression level of the positive group, treatment group and prevention group was much lower than the model group(P<0.05). The prevention group and treatment group were higher than the positive group(P<0.05). There was no significant difference between the prevention group and the treatment group(P>0.05).6 The expression of β-catenin,Wnt1 and c-mycm RNA in different groupThe total number of RNA in different group was equal and Olig(d T)15 anchor primers was used for reverse transcription.β-catenin, Wnt1, c-myc primers were used to proceed PCR amplification on reverse transcription product, then 192 bp, 623 bp, 263 bp specific amplification band was appeared respectively. The amplification band in model group was the brightest and was the darkest in normal group. In the positive group, prevention group and treatment group the amplification band was darker compared with model group. The ratio of goal gene amplified fragment integral absorbance and GAPDH amplified fragment intergral absorbance was used to denotate the expression level of β-catenin, Wnt1, c-mycm RNA. Statistics analysis showed: The ratio of both β-cateninm RNA/GAPDHm RNA and c-my cm RNA/ GAPDHm RNA in model group was much higher than innormal group(P<0.05). The positive group, treatment group and prevention group were much lower than the model group(P<0.05). There was no significant difference between the prevention group and positive group(P>0.05). The treatment group was higher than the positive group(P<0.05). The prevention group was much lower than the treatment group(P<0.05). The ratio of Wnt1 m RNA/GAPDHm RNA in model group was much higher than in normal group(P<0.05). The positive group, treatment group and prevention group were much lower than the model group(P<0.05). The ratio in prevention group and treatment group was higher than the positive group(P<0.05). There is no significant difference between the prevention group and the treatment group(P>0.05).Conclusions:1 4NQO is an effective induced cancer agent for the establishment of esophageal precancerous lesions animal model.2 As the aggravating of esophageal pathology damagement degree, the expression of Wnt pathway related protein β-catenin, Wnt1 and c–myc is elevated.3 YIGE formula can reduce the expression of β-catenin, Wnt1 and c-myc, and can block the occurrence of esophageal cancer. Preventive medicine has obvious advantages.4 The mechanism of YIGE formula block the occurrence of esophageal cancer may was that it reduced the expression of Wnt pathway related protein β-catenin, Wnt1 and c-myc. |
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