| Objective To study the regulation of water metabolism by Veronicastrum axillare decoction on SD rats gatric injury model through the analysis of the amount of gastric juice, gastric acidity, and pepsin activity; to study the protective effects of serum containing Ⅴ. axillare on ethanol injured human gastric epithelial cell (GES-1) by measuring the survival rate of GES-1; to explore the anti-ulcer mechanism of Ⅴ. axillare based on water trans-membrane regulation by detecting the expression levels of AQP1, AQP3, AQP4 and the key factors of NF-κB signaling pathway of GES-1 and the gastric mucosal of rats.Methods (1) 36 SD rats (SPF) were randomly divided into 6 groups:the normal group (0.9% saline), the model group (0.9% saline), the Ranitidine group (0.027g/(kg-d), approximately 3 times human dose of Ranitidine), the low dose V. axillare group (0.7g/(kg.d)), the medium dose Ⅴ. axillare group (1.4g/(kg.d)) and the high dose Ⅴ. axillare group (2.8g/(kg.d)),6 in each group. Rats in each group were gavaged once daily for 14 consecutive days. One hour after the last gavage, rats in the normal group were administered with 5mL/kg 0.9% normal saline, while the others were administered with 5mL/kg ethanol. The rats were killed one hour later. Gastric juice volume, gastric acidity, and activity of pepsin were determined respectively. mRNA and protein expression levels of AQP1, AQP3 and AQP4 were measured by Real Time PCR and Enzyme-linked immunosorbent assay (ELISA). (2) 20 male SD rats were divided into five groups and prepared by 14 consecutive daily intragastric administration of 0.9% saline, Ranitidine (0.027g/(kg.d)), and Ⅴ. axillare decoction (0.7g/(kg.d),1.4g/(kg.d),2.8g/(kg.d) respectively for low/mid/high dose groups). At the end of the preparation period the rats were terminated and the serum from each group was collected. GES-1 cells were cultured with DMEM containing 10% fetal calf serum for 24h, then equally divided into 10 groups, the normal group, the model group, the Ranitidine group, the low dose Ⅴ. axillare group, the medium dose Ⅴ. axillare group, the high dose Ⅴ. axillare group, the low dose Ⅴ. axillare+PMA group (PMA:PKC activator), the medium dose Ⅴ. axillare+ PMA group, the high dose Ⅴ. axillare+ PMA group, and the PMA group. GES-1 cells were cultured in DMEM with 10% serum containing different concentration of V. axillare decoction for 24h. PMA was added at 100ng/mL when needed. Cell injury was induced by 5% ethanol. The cell viabilities were measured by MTT assays. The mRNA expressions for AQP1, AQP3, AQP4, NF-κB, TNF-α, TNFR-1, IKKβ and IκBα were measured by RT-PCR. The protein expressions of AQP1, AQP3 and AQP4 were measured by Western blot, while those of NF-κB, TNF-α, TNFR-1, IKKβ and IκBα were detected by ELISA.Results (1) The first part of the experimental results show that:Gastric juice volume and gastric acid output of the model group were the highest, and the pepsin activity of the model group was the strongest. Gastric juice volume and pepsin activity of the different dose V. axillare groups decreased significantly compared with the model group in a dose-dependent manner(P<0.01), and those of the high dose V. axillare group was the most effective among all the groups (P<0.05 or P<0.01). Compared with the model group, the expression of AQP3 and AQP4 in gastric mucosa tissue of each dose V. axillare group decreased significantly (P<0.05 or P<0.01), while those of AQP1 increased significantly (P>0.05 or P<0.01). (2) The second part of the experimental results show that:Compared with the normal group, cells were significantly less active after being damage by 5% ethanol, and the protein and mRNA expression of NF-κB, TNF-α, IκBα and IKKβ of the model group and PMA group were significantly increased (P<0.05 or P<0.01); Compared with the model group and PMA group, cell viabilities were significantly increased on the medium and high dose V. axillare groups (P<0.01), and the protein and mRNA expression of NF-κB, TNF-α, IκBα and IKKβ were significantly down-regulated in serums containing different doses of V. axillare decoction (P<0.05 or P<0.01); Meanwhile, the expression of TNFR-1 was also regulated to some degree (P<0.05 or P<0.01). (3) The third part of the experimental results show that:The expression of AQP1, AQP3 and AQP4 in GES-1 treated with different dose of V. axillare in vitro were consistent with the animal experiment results. Each V. axillare dose could down-regulate the expressions of AQP1 and up-regulate the expressions of AQP3 and AQP4 in GES-1 cells respectively (P<0.05 or P<0.01). Meanwhile, the expression of AQP1, AQP3 and AQP4 can be indirectly influenced by the NF-κB signaling pathway. Compared with PMA group, Ⅴ. axillare effectively reversed the effects of PMA on the expression of AQP1, AQP3 and AQP4 (P<0.05 or P<0.01).Conclusion (1) The water extract of Ⅴ. axillare has significant protective effects on gastric mucosa of rats and GES-1 in vitro. Ⅴ. axillare decoction reduced gastric ulcer induced by ethanol, and the serum containing Ⅴ. axillare protected GES-1 cells against ethanol injury in a dose-dependent manner. (2) The anti-ulcer mechanism of Ⅴ. axillare decoction was closely related to the regulation of water metabolism balance, i.e. regulation of gastric juice secretion, total gastric acid excretion, and pepsin activity, the high dose Ⅴ. axillare group was the most effective; (3) Ⅴ. axillare influenced the expression of AQP1, AQP3, AQP4, and the key factors-(NF-κB, TNF-a, IκBα, IKKβ,TNFR-1) of NF-κB signaling pathway, so as to regulate the water metabolism balance of GES-1 cells and achieve the anti-ulcer function, the high dose Ⅴ. axillare group was the most effective for regulating NF-κB signaling pathway and AQPs. |
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