| Objective Veronicastrum axillare is a resource-rich medicinal plant in Zhejiang province.It often used for external treatment of bruises,mumps,furunculosis and so on,oral treatment of pleural effusion,nephritis,cirrhosis ascites and gastric ulcer and many other inflammation-related diseases.The previous researches indicated that the water extract of V.axillare significantly reduced the gastric mucosal damage of SD rats induced by ethanol,and V.axillare-loaded rat serum has significant protective effects on human gastric epithelial cells（GES-1）cultured in vitro.The water extract of V.axillare and V.axillare-loaded serum significantly decreased the ethanol-induced high expression of the key factors of NF-κB signaling pathway,AQP3 and AQP4,while increased the expression of AQP1.The results indicated that the effect of V.axillare on treatment of ethanol-induced damage was related to anti-inflammatory and regulation of water metabolism.In order to clarify whether V.axillare has a direct protective effect on GES-1 cells in vitro and its efficacy components and mechanisms,the polysaccharides of V.axillare was firstly extracted and separated,and then the ethanol demaged GES-1cell was used to study the protective effect of the water extract of V.axillare and polysaccharides from V.axillare,and the effects of the water extract of V.axillare on the apoptosis and expression levels of the key factors of NF-κB signaling pathway,AQP1,AQP3 and AQP4.The goal was to investigate the anti-ethanol damage components and mechanism of V.axillare and provide experimental basis for clinical application and further utilization of V.axillare.Methods（1）Using the method of water extraction and ethanol precipitation,crude polysaccharide was obtained from V.axillare.Savag deproteinzation and DEAE-cellulose 52 ion exchange chromatography（IEC）were used to separate V.axillare polysaccharide components.（2）GES-1 cells were cultured in vitro,and the survival rates of GES-1 cells treated with water extract of V.axillare,Ranitidine（positive control）,polysaccharide of V.axillare and its components separated by IEC were detected by Sulforhodamine B（SRB）assay,and the optimal dosage was screened.（3）A model of GES-1 cells damaged by 5%ethanol was established,and the effects of water extract and polysaccharide components of V.axillare on the survival rate of GES-1 cells was detected by SRB assay and flow cytomety.（4）The m RNA and prtotein expression of TNFR-1,NF-κB,TNF-α,IκBα,IKKβ,AQP1,AQP3 and AQP4 of 5%ethanol-injured cells were measured by q RT-PCR,Western blot and ELASA in order to investigate the regulatory effect of V.axillare on NF-k B signaling pathway and AQPs in GES-1 cells.Results（1）The yield of polysaccharides in V.axillare was 3.10%after water extraction,alcohol precipitation and Savag deproteination.Six polysaccharide compoments of V.axillare were obtained by IEC separation,and the yield were1.45%,0.20%,0.15%,0.45%,0.30%and 0.15%,respectively.（2）The toxicity of each component to GES-1 cells was detected by SRB method.The results showed that the TC0 of water extraction,undeproteinized polysaccharide,deproteinized polysaccharide,and polysaccharide components of IEC to GES-1 cells converted to dosage of V.axillare were 160μg/m L,856μg/m L,10268μg/m L,19465μg/m L,3078μg/m L,6242μg/m L,2983μg/m L,1264μg/m L and 622μg/m L,respectively.The toxicity of each polysaccharide component to GES-1 cells was component 6>component 5>component 4>component 2>component 3>component 1.（3）The viability of GES-1 cells injured by 5%ethanol was significantly lower than those of normal group（P<0.01）,while the apoptosis rate was significantly higher than those of normal group（P<0.01）.After preculture with low,medium and high concentrations of V.axillare water extracts for 48 h,the viability of ethanol-injured GES-1 cells was significantly increased in a dose-dependent manner,and the apoptosis rate was significantly decreased（P<0.01）.Meanwhile,the polysaccharide of V.axillare and six components separated by IEC significantly improve the viability of ethanol-injured GES-1 cells in a dose-dependent manner,and the protective effects of components 4,6 and 5 to GES-1 cells was higher than those of components 1,2 and 3.（4）Compared with normal group,the m RNA and protein expression levels of TNFR-1,NF-κB,TNF-α,IκBα,IKKβ,AQP3 and AQP4 in model group were increased significantly（P<0.05,P<0.01）,while the expression of AQP1 was significantly down-regulated（P<0.05,P<0.01）.Compared with model group,the low,medium and high dose of V.axillare water extracts showed a significant down-regulation to protein and m RNA expression of TNFR-1,NF-κB,TNF-αand IκBαIKKβ,AQP3 and AQP4（P<0.05,P<0.01）,and an up-regulation to those of AQP1（P<0.05,P<0.01）.Conclusion（1）Six polysaccharide components of V.axillare were obtained by water extraction,alcohol precipitation,Savag deproteinized and DEAE-cellulose 52 ion exchange chromatography.The total yield of polysaccharides was 2.80%,in which the yield of component 1 was the highest.It was 1.45%.（2）Under the same dosage of V.axillare,the toxicity of V.axillare polysaccharide and its components on GES-1cells was significantly lower than that of V.axillare water extracts.（3）V.axillare water extracts and polysaccharide both had protective effects on GES-1 cells,and V.axillare water extracts significantly reduced the apoptosis rate of ethanol-injured GES-1 cells.（4）V.axillare water extracts modulated TNF-α-mediated NF-κB signaling pathway and the expression of AQPs.Therefore,it protects GES-1 cells against ethanol damage by anti-inflammation and regulation of water metabolism. |
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