Protective Mechanism Of HNYSPJ60 On Acetic Acid-induced Chronic Gastric Ulcer In Rats

Gastric ulcer is the most common disorder of upper digestive tract,with a high morbidity and recurrence rate.The pathophysiology of gastric ulcer has not been fully elucidated.It is believed that it occurs,mainly due to an imbalance between aggressive(HC1 and pepsin)and defensive(mucus secretion and bicarbonate)factor of the gastric mucosa resulting in ulcerative lesions.The indiscriminate use of NSAIDs,poor eating habits,smoking,alcohol,stress and infection with H.pylori have also been implicated in the etiology of gastric ulcer.HNYSPJ60 was isolated from the dry bark of Albizia chinensis(Osbeck)Merr.Previous study showed that HNYSPJ60 significantly improved gastric ulcer in some experimental models of gastric ulcer.The present study was designed to investigate the effect of HNYSPJ60 on chronic gastric ulcer induced by acetic acid in rats.After the 12-day treatments,the effects of HNYSPJ60 were evaluated using the changes of general behavior,gastric tissue morphology and the ulcer area.As shown in results,treatments with HNYSPJ60(200 mg·kg=1,i.g.)for 12 days effectively relieved mucosal damage induced by acetic acid and reduced gastric ulcer area by 38.4%(P<0.05).Gastric mucosal inflammation may play key roles in gastric ulcer.In acetic acid-induced gastric ulcers,persistent infiltration of neutrophils and macrophages into scarred mucosa,and the release of pro-inflammatory cytokines(TNF-??IL-6?IL-1?)to the site of inflammation worsen the ulcerative process.Stress-activated MAP kinase(MAPKs),comprised of JNK and p38,play prominent roles in the inflammation.Compared with model group,treatments with HNYSPJ60(200 mg·kg-1,i.g.)for 3 days reduced the activity of MPO by 37.3%(P<0.01).Meanwhile the contents of TNF-a and IL-6 respectively decreased by 43.8%(P<0.01)and 32.7%(P<0.01)compared with model group.Otherwise,HNYSPJ60(200 mg·kg-1,i.g.)could obviously suppress the phosphorylation of p38,but the phosphorylation of JNK and expression of p38 and JNK remained almost unaltered.Gastric ulcer healing encompasses cell proliferation,epithelial regeneration,gland reconstruction,formation of granulation tissue,neovascularization,interaction between various cells and the matrix and tissue.Cell proliferation is essential for ulcer healing,because it supplies epithelial cells crucial for the re-epithelialization of mucosal surface and the reconstruction of gastric gland.PCNA,a key factor of DNA polymerase ?,was shown to act as a cell proliferation maker.Western blot analysis detected higher levels of PCNA in the model group compared with control group at 12 d after operation.The PCNA levels were significantly higher in the HNYSPJ60(200 mg·kg-1,i.g.)treated group compared with the model group.Ulcer healing is controlled by the cytokines and growth factor.Epidermal growth factor(EGF)activates epithelial cell migration and proliferation and accelerates wound/ulcer healing in vivo and in vitro by binding to their specific receptors(EGFR)on the cell surface.Ras/Raf/ERK1/2 pathway is one of the signaling pathways downstream of activated EGFR,which play an essential role in cell proliferation.Western blot analysis showed that p-ERK1/2 levels were not significantly different in the model group at 12 d after operation.The p-ERK1/2 levels were significantly higher in the HNYSPJ60(200mg·kg-I,i.g.)treated group compared with the model group.At 7 d after operation,the animals were sacrificed and stomachs were rapidly removed and processed in order to investigate the upstream protein.Western blot analysis showed that p-MEK1/2,p-c-Raf and EGFR levels were not significantly different in the model group at 7 d after operation.But the operation decreased the expression of EGF.Compared with the model group,the HNYSPJ60(200 mg·kg-1,i.g.)treated rats showed significantly increase in the levels of p-MEK1/2,EGF and EGFR at 12 d after operation.However,phosphorylation of c-Raf and expression of MEK1/2 and c-Raf remained almost unaltered.Immunohistochemical investigation showed weak reactivity for EGF and EGFR in the bottom of the gastric glands of rats at 7 d compared to control group.The expression of EGF and EGFR was enhanced in both the mucosal proliferative zone and the bottom of the gastric glands of the HNYSPJ60(200 mg kg-1,i.g.)treated rats in comparison with that in the model rats.In summary,HNYSPJ60(200 mg·kg-1,i.g.)exhibits a protective effect on acetic acid-induced chronic gastric ulcer,which might be relevant to anti-inflammation through inhibition of the phosphorylated p38 and the proliferation through activation of the EGF-R-ERK transduction pathway.