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Objective By simulating acute hypoxic conditions, an experimental model of intestinal stress injury in plateau mice was established to explore the pathogenic mechanism of acute gastrointestinal diseases in plateau, and to lay foundation for preventive and therapeutic measures.MethodsThirty-six SPF-grade adult male BALB/c mice were randomly divided into four groups: normoxic 24 h, normoxic 72 h, hypoxic 24 h, and hypoxic 72 h, based on body weight using a randomized numerical table method, with nine mice in each group. Mice in the normoxic group were kept in a conventional barrier environment, while those in the hypoxic group were placed in a hypoxic chamber within the barrier environment with oxygen concentration set at 10% to simulate plateau conditions. They were subjected to stress for 24 h and 72 h, respectively, in order to establish a model of intestinal injury induced by acute hypoxia. After modeling, the mice were weighed, anesthetized with 1% pentobarbital sodium, and then euthanized by cervical dislocation. Duodenal and colonic tissues were collected. Histopathological morphology of intestinal tissues was observed after HE staining. Western blotting and immunohistochemistry were used to detect the expression levels of tight junction-related proteins in intestinal tissues. Real-time fluorescence quantitative PCR was performed to measure the expression levels of inflammatory cytokines and chemokines. TUNEL staining was used to assess apoptotic activity of intestinal epithelial cells, thus evaluating intestinal injury-related phenotypes in this model.Results Compared with the normoxic groups, mice in the 24 h and 72 h hypoxia groups showed weight loss, shortened duodenal villi, abnormal crypt structure, and decreased villus/crypt ratio. The colonic mucosa was infiltrated with inflammatory cells and irregular crypt structure. Expression levels of Occludin and zonula occludens-1 (ZO-1) were significantly decreased in duodenal and colonic tissues of mice in the 24 h and 72 h hypoxia groups (P<0.05). The expression of pro-apoptotic protein Bax was significantly up-regulated while expression of anti-apoptotic protein Bcl-2 was significantly down-regulated in duodenal tissues (P<0.05). Apoptotic activity of intestinal epithelial cells was significantly enhanced (P<0.05). In addition, interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) mRNA levels were significantly increased in duodenal tissues after 24 and 72 h of hypoxic stress(P<0.05). After 24 h of hypoxic stress, there was no significant change in the expression levels of inflammatory cytokines in colon tissues (P>0.05), but after 72 h, the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, MCP-1, and anti-inflammatory factor IL-10 mRNAs significantly increased in colon tissues of mice (P<0.05).Conclusion The usage of a hypoxia chamber to simulate an acute hypoxic environment in plateau can lead to abnormal intestinal tissue structure, intestinal barrier dysfunction, and induce intestinal epithelial cell apoptosis, triggering an intestinal inflammatory response in stress mice. These findings indicate the successful construction of a mouse model for an acute hypoxic stress-induced intestinal injury.
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Objective Given that psychosocial stress can contribute to a series of diseases,such as inflammatory bowel disease and irritable bowel syndrome,we aimed to establish an experimental chronic restraint mouse intestinal stress injury model as a basis for exploring the pathogenic mechanism of chronic restraint stress-induced gastrointestinal diseases,and for developing preventive and curative measures.Methods Eighteen male SPF-grade BALB/c mice were acclimatized for 7 days and then divided into a control group and a chronic restraint stress group according to body weight,using a randomized numerical table method.The mice were subjected to restraint stress for 3 hours per day for 14 days to establish an intestinal injury model.The model was evaluated by observing body weight,pathological changes in intestinal histomorphology,expression of tight junction proteins,apoptosis of intestinal epithelial cells,and mRNA expression levels of inflammatory cytokines.Results After 14 days of chronic restraint stress,model mice showed weight loss,shortened duodenal villus height,abnormal crypt structure,a decreased villus/crypt ratio,colonic mucosal inflammatory cell infiltration,and irregular crypt structure.Protein immunoblotting,immunohistochemistry,and immunofluorescence staining showed that the expression levels of the duodenal and colonic tight junction proteins Occludin and Claudin-1 were significantly decreased in mice after chronic restraint stress(P＜0.05),while expression levels of the apoptotic protein cleaved-caspase-3 in intestinal epithelial cells were significantly increased(P＜0.05).Regarding the mRNA expression levels of intestinal inflammatory factors and chemokines,chronic restraint stress for 14 days significantly increased the gene expression levels of interleukin(IL)-1β,IL-6,monocyte chemoattractant protein-1(MCP-1),tumor necrosis factor-α,and IL-10 in the duodenum of mice(P＜0.05),and significantly increased the gene expression levels ofIL-1β,IL-6,and MCP-1 in the colon(P＜0.001).Conclusions The use of a behavioral restriction device to restrain mice continuously for 14 days led to abnormal intestinal tissue structure,intestinal barrier dysfunction,and intestinal epithelial cell apoptosis,and triggered an intestinal inflammatory response in the stressed mice,indicating successful establishment of a mouse model of intestinal injury by chronic restraint stress.
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Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website.The primers were linked to pCAS9/gRNA1 vector.Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method.After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained.The western-blot results showed that the cell lines did not express MATP protein.Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.
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Objective To establish an animal model by placing one end of PICC in the hepatic portal vein of a beagle dog and leaving the other end out of its body.Methods Six Beagle dogs were given respiration anesthesia through orotracheal intubation.An incision was made through the right rectus abdominalis to locate the superior mesenteric vein (SMA) and the main hepatic portal vein.The left branch of SMA was separated and cut to put PICC into the main hepatic portal vein before being ligated and fixed.The other end of PICC was elicited through the right abdominal wall and passed beneath the skin to the back neck and fastened in case of movement.Results The anesthetic effect was good and all the operations were successful.The mean operation time was about an hour and the mean blood loss was about 15 ml.The incision healed 5-7 d after operation.Conclusion The establishment of the model can improve the effects of liver-targeting drugs,which can cut down the dosage,lower the cost of treatment and experiment and reduce the adverse effect of medicines.Through PICC,we can directly draw blood from the hepatic portal vein to measure the blood concentration before the first pass elimination.Then according to the concentration,we can calculate the absorption rate in the gastrointestinal tract,which can facilitate related experimental studies.
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Objective To analyze the effect of Noggin silencing on the BMP and Wnt signaling pathways in hair follicle development.Methods The expression of BMP-2, BMP-4, BMPR-IA, BMP-6, BMP-7, LEF-1 andβ-catenin in Noggin silencing MC3T3-E1 stable cell line was detected by RT-PCR and western blot.Results RT-PCR results showed that the expressions of five genes in BMP signaling pathway were all significantly influenced by Noggin silencing, the ex-pressions of BMP-2 (P<0.001), BMP-4 (P<0.01), BMP-6 (P<0.001) and BMP-7 (P<0.001) were all increased and the expression of BMPR-IA (P<0.01) was decreased.While the expressions of the two genes LEF-1 (P<0.001) and β-catenin ( P<0.001) in Wnt signaling pathway were significantly decreased.Western blot results showed that the ex-pressions of these proteins in the two signaling pathways were also affected.The expressions of BMP-2 (P<0.05), BMP-4 (P<0.05), BMP-6 (P<0.05) and BMP-7 (P<0.05) were all increased, while the expressions of BMPR-IA (P<0.05), LEF-1 (P<0.01) andβ-catenin (P<0.001) were decreased.Conclusions There may be a negative feedback regulation of Noggin on the BMP signaling pathway in vitro, but a positive feedback regulation on the Wnt signaling pathway in vitro.It provides certain evidence for studies on the effect of Noggin gene on BMP and Wnt signaling pathways in vivo. There may be an interaction between hair follicle development-related signaling pathways, which still needs further experi-ments to prove.