The Protective Effect Of Amifostine On High Dose Methotraxate-induced Small Intestinal Mucositis

ObjectiveConstructing HDMTX-induced small intestinal mucositis damage model in mice, to evaluate the effects of amifostine in preventing intestinal mucosal damage caused by high dose methotrexate (HDMTX) in mice model, and to provide the laboratory basis of prevent and protective of intestinal mucosal for the oncology patients in the clinic.Methods1This study was designed to construct animal model of HDMTX induced small intestinal mucosa damage by administration of intraperitoneal injection.2Trial animal group and administration:mice were divided into5experimental groups randomly:Group A (normal control group)(n=18) intraperitoneal injection of saline; Group B(HDMTX group)(n=18):intraperitoneal injection of a single dose of MTX (260mg/kg); Group C(CF group)(n=18):intraperitoneal injection of MTX(260mg/kg), then muscle injection of CF (2.6mg/kg), every6hours per time, three times; Group D (n=18):hypodermic injection of amifostine (200mg/kg) and intraperitoneal injection of MTX (260mg/kg); Group E (n=18):hypodermic injection of amifostine (200mg/kg) and intraperitoneal injection of MTX(260mg/kg), then muscle injection of CF (2.6mg/kg), every6hours per time, three times.3Histological analysis:after3days,5days, the jejunum segment was removed for morphological analyses, intestinal mucosal damage, mucosal structural changes, villus height and crypt depth.4Mortality rate:observing the mice daily, record death and survival, and statistical analysis of its survival.5WBC level was measured in d0, dl, d3, d5following HDMTX injection.6Bax and bcl-2mRNA expression levels were determined by RT-PCR, enterocyte proliferation and enterocyte apoptosis were determined in d3, d5following MTX injection.Results1The weight of mice decreased significantly in group B compared with group A, the weight of amifostine and CF group is better than other groups, the difference was statistically significant in d3, d5, d7(P<0.05).2Diarrhea rate in different groups, the difference was statistically significant (P<0.05). The total diarrhea rate in normal control group, HDMTX group, CF group, Amifostine group, amifostine and CF group is0%,100%,77.78%,66.67%,50%respectively, besides of normal control group, the rate of diarrhea in amifostine and CF group is lower than other groups.3Mucosal atrophy was observed in each group. Amifostine-CF group mice demonstrated greater villus height and crypt depth than other groups.4Mortality rate in different groups, the difference was statistically significant (P<0.05). The total mortality rate in normal control group, HDMTX group, CF group, Amifostine group, amifostine and CF group is0%,66.67%,44.44%,50%,16.67%. respectively Besides of normal control group, the rate of mortality in amifostine and CF group is lower than other groups.5WBC levels were decreased significantly in group B compared with group A. Amifostine-CF group mice demonstrated higher WBC level than other groups. 6A greater index of proliferation was measured in the jejunum compared to MTX animals, and was significantly increased in group E compared with group B. A significant decrease in enterocyte apoptosis in the jejunum of MTX-amifostine-CF mice (vs. MTX, MTX-amifostine, MTX-CF) was accompanied by decreased bax and increased bcl-2mRNA expression.Conclusions1This experiment successfully constructed HDMTX induced small intestinal mucosa damage model, and found that administration of intraperitoneal injection is the easiest way to establish the small intestinal mucosa damage model in mice.2Treatment with CF, amifostine, amifostine and CF prevents mucosal injury and improves intestinal recovery following MTX injury in mice, but the effect of amifostine and CF is better than other groups. Application amifostine preventively can reduce the degree of weight lose, the incidence of diarrhea and mortality, and has certain protective effect in bone marrow suppresion.3Amifostine raises the expression of bcl-2, downs the expression of bax, restrains the intestine epithelial cell apoptosis, reduces the damage degree of small intestinal mucosa inflammation, which provide the theory basis of preventing chemotherapy induced mucositis by cell protectants amifostine for clinical medicine.