| Aim To observe the effect of "Qi Huang Decoction" on the tight junction of intestinal mucosa of rat model of Jianpi Tong,and to explore its mechanism.Methods1.Modeling and Grouping method The rat model was established by gastrectomy.A total of 90 Wistar male rats were numbered from low to high according to body weight.Then they were randomly divided into 6 groups,15 in each group.They were normal group,sham operation group(abdominal incision only,unresected stomach),nutrition group and high,medium and low dose of Qihuang decoction.2.Intervention methods of each group Normal diet and drinking water were used in normal group and sham operation group after operation.The nutrition group and the traditional Chinese medicine Qihuang Decoction group were fasting after operation.In the nutrition group,the enteral nutrition solution 120KJ/kg.d was injected daily through the silicone tube after modeling,and the Chinese herbal medicine Qi Huang Decoction group was injected with enteral nutrition liquid plus Chinese herbal decoction.The high,middle and low doses of Qihuang Decoction group were 20,10,5g/kg.d,respectively.A total of 7 d.3.Observation index and detection method3.1 Jianpi Tongli traditional Chinese medicine(Qihuang Decoction)through effect on intestinal epithelial tight junction structure: by establishing the rat model of gastric surgery trauma,given intestinal dripping of Qihuang decoction,observe the morphology and structure of tight junctions under the transmission electron microscope and measuring the width(um).3.2 Jianpi Tongli traditional Chinese medicine regulates the expression of Occludin,claudin and ZO-1m RNA: the expression level of m RNA was detected by real-time fluorescence quantitative RT-PCR,and the transcription of Occludin,claudin and ZO-1 genes were observed.3.3 Medicinal herbs through the tight junction molecules Occludin,claudin,ZO-1distribution and location adjustment: under the fluorescence microscope,distribution by immunofluorescence detection of Occludin,claudin and ZO-1 in intestinal epithelial cells,the existence of three co localization and co localization of strength.In order to evaluate whether the three can be distributed together at the near cell membrane of the cell edge,the intensity of distribution,and the combination of each other together.4.Statistical method The experimental data of all the measurement data were expressed by mean± standard deviation(`x±s).Two groups of data were compared by single factor(AVOVA)variance analysis,and processed by software SPSS 22.P<0.05 thought the difference was statistically significant.Results1.The morphological structure of tight junction of intestinal mucosa1.1 The morphology and structure of the electron microscope are seen:The tight junction between the groups can be displayed in the electron microscope,tight junctions located in epithelial cells near the free surface at the top side,a narrow ribbon(welding line or ridge);tight junction fusion point continuous visible on the surface of cell membrane or anastomosis,the continuity and integrity of each group is different,on the whole,the normal group is closely connected to its continuity and integrity than operation group.Compared with the nutrition group,the surface integrity and continuity of the cell membrane on the surface of the Chinese medicine group were relatively good.1.2 Transmission electron microscope to measure intestinal mucosal epithelial tight junction width,normal group,sham operation group,nutrition group and decoction of low,medium and high dose group is tight junction to the average width were0.7668± 0.1276 um,0.5265 ± 0.1020 um,0.1944 ± 0.0075 um,0.2930 ± 0.0122 um,0.3114 ± 0.0470 um,0.3710 ± 0.263 um.Compared with the normal group,the tight junction of intestinal epithelial cells was destroyed in the sham operation group,the nutrition group and the traditional Chinese medicine group,and the width of the tight junction was narrowed(P<0.01).Compared with the nutrition group,the tight junction width of intestinal mucosa in rats was well repaired and protected after one week of treatment with three dose groups(P<0.01).But compared with the sham operation group,the width of the intestinal mucosa of the rats in each dose group had not recovered to the sham operation group(P<0.01).2.The tight junction protein ZO-1,Claudin-1 and Occludin m RNA expression2.1 Compared with the normal group,sham operation group,nutrition group and Chinese medicine group Claudin-1 m RNA expression was significantly increased(P<0.01);compared with the sham group,the expression of Claudin-1 m RNA nutrition group and Chinese medicine group increased significantly(P<0.01);compared with the nutrition group,decoction of low,medium and high dose group Claudin-1 the expression of m RNA increased gradually(P<0.01).2.2 Compared with the normal group,the expression of the sham operation group,nutrition group and Chinese medicine group Occludin m RNA and ZO-1 m RNA decreased significantly(P<0.01);compared with the nutrition group,decoction of low,middle and high dose groups of Occludin m RNA and ZO-1 m RNA expression gradually increased(P<0.01);compared with the sham group,the expression of Occludin m RNA and traditional Chinese medicine ZO-1 m RNA has increased,but not to the level of sham operation group group(P<0.01).3.Tight junction molecules Occludin,Claudin-1,ZO-1 distribution and localization and co localization strength.3.1 Laser confocal fluorescence microscopy was used to observe the distribution and co localization of three protein molecules in intestinal epithelial cells.Under the fluorescence microscope,we can directly see that occludin and Claudin-1 protein are expressed on the surface of the epithelial cell membrane,and the ZO-1 protein molecules are expressed in the cytoplasm.3.2 The co localization strength was determined by counting the number of positive cells that were located in each visual field under the fluorescence microscope.Normal group,sham operation group,nutrition group and decoction of low,medium and high dose group of Claudin-1 and the co localization of ZO-1 positive cells were 155 ± 8、132 ± 10、68 ± 9、80 ± 5、90 ± 7、111± 8,Occludin and ZO-1 were co localization of positive cell number150 ± 9、129 ± 7、70 ± 10、83 ± 6、93 ± 7、114± 8,and compared with the normal group,nutrition group and Chinese Medicine Group Co localization of protein decreased(P<0.01).Compared with the nutrition group,after the use of traditional Chinese medicine,the co localization strength between the three kinds of traditional Chinese medicine histone was enhanced(P<0.01).Compared with the sham operation group,the co localization intensity of the three doses of the traditional Chinese medicine histone was not recovered to the sham operation group(P<0.01).Conclusions1.The intestinal epithelial cells of Jianpi Tongli Decoction on gastric resection caused by surgical trauma to the tight junction between the damage has a protective effect,can make the damaged tight junction integrity and continuity and get better recovery,Qihuang decoctio can cause intestinal epithelial cells damaged by the tight junction between the increase of the width.Qihuang decoction can promote the expression of Occludin,Claudin-1 and ZO-1 molecule m RNA.2.Qihuang decoction can promote the expression of Occludin,Claudin-1 and ZO-1molecule m RNA.3.The distribution of Occludin and Claudin-1 on the surface of the cell membrane,the distribution of ZO-1 in the cytoplasm,and the co localization between Claudin-1and Occludin and ZO-1.Qihuang decoction can strengthen the distribution of Occludin,Claudin-1 and ZO-1 molecules in the proximal intestinal epithelial cells and combine with each other. |
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