| BackgroundGastric cancer is one of the most common malignancies threatening human health, with high stealthiness, high incidence and high mortality. Gastric precancerous lesions (GPL) are histopathologically referred to as intestine metaplasia and epithelial dysplasia. GPL frequently occurs secondary to chronic atrophic gastritis (CAG), and is closely related to gastric cancer. According to Correa hypothesis, intestinal-type gastric cancer generally goes through a gradual cascade of mucosal lesions. GPL is a bridge between chronic atrophic gastritis and gastric cancer, and also a central phase of malignant transformation. To date, there is no special drug for CAG-GPL in western medicine. Clinical reports indicated that the representative drug Vitacoenzyme could, to some extent, slow the progression of CAG and prevent malignant transformation of epithelial cells. However, Vitacoenzyme treatment lasted a long time and showed unsatisfactory therapeutic effect, which was a great limitation for its clinical use. By using Treatment Based on Syndrome Differentiation (TBSD) and special formula, Chinese Medicine treatment can not noly block the progression of CAG-GPL, but shows few side effects, multi-target effects and good safety.Jianpi Huayu Jiedu formula (Jiedu Fang, JDF), which functions to fortify the Spleen (Pi), dissipate blood stasis, and detoxify, showed a good clinical efficacy in treating chronic atrophic gastritis and GPL. Previous clinical study indicated that JDF treatment is beneficial in non-progressive GPL. In recent years, research of relationship between stem cells and malignant tumors has always been the focus. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a crucial potential biomarker of gastric stem cells or cancer stem cells. Generally, Lgr5+cells are allocated at the base of mature pyloric glands, and responsible for the long-term renewal of entire gastric mucosal epithelial cells and gastric glands. However, Lgr5+cells may differentiate abnormally and over-proliferate under integrated factors, such as Helicobacter pylori infection, inflammation, immunosuppression and chemical factors, etc. In addition, Lgr5 is an important target gene of Wnt signaling pathway. Activation of Wnt signaling pathway would lead to dysregulation of Lgr5 expression which mediate the proliferation, differentiation and immigration of cancer stem cells. Consequently, Wnt/Lgr5 signaling pathway plays a crucial role in the initiation and progression of gastric cancer. To date, rare studies focus on Wnt/Lgr5 signal pathway in elucidating the underlying mechanisms of mucosal malignant transformation and Chinese medicine intervention.Aim①To model GPL rat, and observe the effect of JDF on general conditions and macroscopic characters of GPL rat. Moreover, we would explore the effect of JDF on gastric intestinal metaplasia and epithelial dysplasia, and on ultrastructure of gastric mucosal epithelial cells in GPL rats.②We would observe the spatial distribution and number variation of Lgr5+ cells in gastric glands, and then investigate the biological role of Lgr5+ cells in GPL. In addition, we would explore how JDF affect Lgr5+ cells aberrant immigration.③We would detect the expressions of Wnt/Lgr5 pathway targets inculding Wntl、Wnt3α、β-catenin、MMP-7 and C-myc in the gastric mucosal tissues, and then observe the regulatory effects of JDF on these Wnt/Lgr5 pathway targets.MethodA total of 77 male SD rats aged 6-8 weeks,120-140 g, were randomly divided into control group (n=10), model group (n=15), vitacoenzyme group (n=13,0.2 g·kg-1·d-1), JDF 15gg·kg-1·d-1 dose group (n=13, A-JDF), JDF 7.5g·kg-1·d-1 dose group (n=13,B-JDF) and JDF 3.75g·kg-1·d-1 dose group (n=13, C-JDF). Rats in control group were fed routinely. To model GPL rat, the rats in the other groups were free to MNNG solution (200 u g·mL-1) every day, and subjected to hunger-satiety shift every other day, with the administration of Xiaochengqi Decotion (2 mL per rat) on the hunger day by gastrogavage. All procedures proceeded for 19 continuous weeks. At the 9th week,2 rats randomly selected from the model group were found to suffer the GIM lesion in the gastric mucosal tissue. Rats in the treated groups were given corresponding drugs for 10 weeks. At the end of 19th week, all the rats were sacrificed, the mucosal tissue from gastric anturm and body of stomach were collected. Alcian blue-periodic acid Schiff (AB-PAS) staining was performed to observe GIM lesion in gastric mucosal tissues (the scope and degree of gastric metaplasia lesion). Hematoxylin-eosin (HE) staining was performed observe GED lesion in gastric mucosal tissues (dysplastic alteration of gastric epithelial cells, disorder of glandular structure and cell abnormal differentiation). Transmission electron microscope was used to observe the ultrastructure of gastric epithelial cells (shape, size, chromatin distribution, intranuclear voids, ratio of nucleus to cytoplasm, cytoplasmic vacuolation, mitochondrial structure and microtubularvesicle structure in chief and parietal cells). Real-time quantitative RT-qPCR was performed to detect C-myc mRNA expression. EnVision immunohistochemistry was performed to detect Lgr5 protein expression. Spatial distribution and number variation of Lgr5+ cells in gastric glands were observed under light microscope. Protein expression of MMP-7, Wntl, Wnt3a, and β-catenin of gastric mucosal tissues in various groups were detected using EnVision immunohistochemistry.Results①HE staining showed that model rats suffer from GED lesion characterized by incomplete and crowded tubular glandular, and also with a back-to-back tubular structure. Dysplastic glands were lined by atypical cells with overlapping, hyperchromatic and cigar shaped nuclei that showed loss of polarity, increased karyokinesis. AB-PAS staining revealed that model rats suffer from diffuse GIM lesion manifested by regions with blue dye. Data suggested that incidence of severe GIM is significantly decreased in JDF groups when compared with that in model group (P<0.01). Compared with model group, incidence of moderate-severe GED lesion was significantly decreased in C-JDF group (P<0.05), but no significant decrease was found in A-and B-JDF groups (P>0.05).②Transmission electron microscope analysis showed that both chief and parietal cells show up as nuclei atypia, increased caryoplasm ratio and chromatic abnormality in gastric epithelium of GPL rats. Morphological changes including karyopyknosis, dilated intranuclear space, nuclear fragmentation and cytoplasmic vacuolation could be observed in some of the cells. Furthermore, endoplasmic reticulum number in chief cells and mitochondrion number in parietal cells were decreased. Mitochondrial structure was fractured, normal structure of microtubularvesicle in parietal cells was destructed, or some microtubularvesicles were obviously dilated. Conversely, pathological changes of chief and parietal cells were improved in JDF treated rats, which were manifested by partially normalized caryoplasm ratio, reduced vacuolation and restored number of endoplasmic reticulum.③We found that very few scattered Lgr5+cells were found at the bottom of the normal gastric gland units, at the crypt of gastric mucosa. On the contrary, Lgr5+ cells were densely located at gastric epithelium in GPL rats, so Lgr5+ cells might immigrate from the crypt to the gastric epithelium. In treated groups, Lgr5+ cells were almost distributed at the gastric epithelium, and number of Lgr5+ cells was clearly reduced. The above mentioned changes in C-JDF group were the most prominent. Our data also demonstrated that Lgr5 protein expression is increased notably in GPL rats as compared with control rats (P<0.01). C-JDF could suppress Lgr5 protein over-expression in GPL rats (F<0.05).④Level of C-myc mRNA expression in gastric epithelial cells of GPL rats in model group was significantly higher than that in control group (P<0.01). Compared with model group, levels of C-myc mRNA expression in all JDF groups were significantly decreased (P<0.01). Compared with vitacoenzyme, level of C-myc mRNA expression in C-JDF group was significantly decreased (P<0.01). Compared with A-and B-JDF groups, level of C-myc mRNA expression in C-JDF group was significantly decreased (P<0.05).⑤Level of Wntl protein expression in gastric epithelium of GPL rats in model group was significantly higher that of control group (P<0.01). Compared with model group, Wntl protein level in all JDF groups was significantly decreased (P<0.01 or P<0.05). In addition, Wntl protein level in C-JDF group was significantly decreased as compared to that of vitacoenzyme group (P<0.05), however, no significant decrease was found in A-and B-JDF groups (P>0.05).⑥Level of Wnt3a protein expression in gastric epithelium of GPL rats in model group was significantly higher that of control group (P<0.01). Compared with model group, Wnt3a protein level in all treated groups was decreased slightly with no significant difference (P<00.05).⑦Level of β-catenin protein expression in gastric epithelium of GPL rats in model group was significantly higher that of control group (P<0.01). Compared with model group, β-catenin protein level in all treated groups was significantly decreased (P<0.01 or P<0.05). Compared with vitacoenzyme group, β-catenin protein level in all JDF groups was significantly decreased (P<0.05).⑧Level of MMP-7 protein expression in gastric epithelium of GPL rats in model group was significantly higher that of control group (P<0.01). Compared with model group, MMP-7 protein level in C-JDF group was significantly decreased (P<0.01), but no significant difference was shown in the vitacoenzyme, A-and B-JDF groups (P>0.05). Compared with vitacoenzyme group, β-catenin protein level in C-JDF group was significantly decreased (P<0.05), however, no significant decrease was found in A-and B-JDF groups (P>0.05).Conclusion①In this study, we use combined methods of MNNG solution free-drinking, hunger-satiety shift and administration of Xiaochengqi Decotion to model GPL rat successfully.②JDF could, to some degree, relieve GIM and GED lesions in GPL rats. Moreover, JDF could ameliorate aberrant ultrastucture of chief-and parietal cells in gastric epithelium of GPL rats.③Lgr5+cells might not locate at the crypt of gastric gland units, but immigrate form the the crypt to gastric epithelium in gastric tissue with GPL. Lgr5+cells divorce from normal microenvironment could cause abnormal differentiation and over proliferation of gastric epithelial cells in GPL, which might be involved in malignant transformation of gastric mucosa. JDF could inhibit Lgr5+ cell over-proliferation.④Dysfunction of Wnt/β-catenin signaling pathway might be closely related to Lgr5+ cell aberrant immigration. JDF could treat or reverse GPL, and this might be due to its capacity of suppressing abnormal activation of Wnt/Lgr5 pathway and the relative targets. |
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