The Treatment Effect Of HCQ In 5-fluorouracil-induced Intestinal Mucositis And Its Molecular Mechanism

ObjectiveChemotherapeutic drugs,especially 5-fluorouracil(5-FU),frequently give rise to intestinal mucositis,featuring severe diarrhea and loss of crypts in histopathology,which is a treatment-and life-threaten cause in clinic.However,there is no effective therapy on it.Its mechanism may be related to recognition and activation of immune cells and pro-inflammatory cytokines release.Hydroxychloroquine(HCQ),widely used in the treatment of autoimmune diseases,such as rheumatoid arthritis(RA)and systemic lupus erythematosus(SLE),is an immunosuppressive drug that can also inhibit inflammation.Therefore,we here investigated the effect of HCQ on 5-FU-induced intestinal mucositis in mice.MethodsIntestinal mucositis was induced in sex-matched C57BL/6J mice by once daily administration of 5-FU(50 mg/kg)for 5 days in vivo and the effect of HCQ(60mg/kg/day,once daily for 6 days(days 0-5),starting at 1 day before the first 5-FU injection)on body weight change of mice,diarrhea score,histology,length of small intestine,TNEL staining,inflammatory cytokine and the signal pathway were then investigated.Double-strand DNA(dsDNA)concentrations in the culture supernatant of HCT-116 cells treated with indicated concentrations at 72h or with 5-FU(1μM)at indicated times in vitro.Bone marrow-derived macrophages or dendritic cells were treated with HCQ at indicated concentration for 1h before transfected in vitro with CT-DNA at the indicated concentration for 6h,and TLR9 expression in cells and IL-1βlevel in supernatant were analyzed.ResultsIn vivo,consecutive administration of 5-FU to mice caused severe intestinal mucositis,which was histologically characterized by the destruction of villi and loss of crypts,accompanied by body weight loss,diarrhea and shorten of small intestine.Daily HCQ administration one day before 5-FU reduced the severity of intestinal mucositis,body weight loss,diarrhea,TUNEL-positive cells,pro-inflammatory cytokine IL-1βsecretion in serum,and also strongly downregulated the expression of TLR9 and NF-κB in small intestine.In vitro,dsDNA in the cell culture supernatant of HCT-116 cells treated with 5-FU released in a dose-and time-dependent manner.HCQ efficiently inhibited TLR9 expression of dsDNA stimulated BMDMs and BMDCs,and also reduced IL-1β secretion in in cell culture supernatant.ConclusionsHCQ alleviated 5-FU-induced intestinal mucositis in mice resulting from suppression of pro-inflammatory cytokines generation via inhibition of dsDNA-activated TLR9-NF-κB signal pathway.Thus,HCQ may be a useful option for preventing intestinal mucositis during 5-FU chemotherapy.