Inhibiting TLR4 Alleviates Methotrexate Induced Intestinal Toxicity

Background: Methotrexate(MTX),a kind of folic acid anti-tumor drug,has being extensively used in autoimmune diseases for some years.However,methotrexate can have serious side effects,including intestinal toxicity that can lead to the interruption of chemotherapy.Clinically,it is a common practice for patients to take leucovorin(LV)to cope with side effects during the methotrexate treatment.Our previous studies suggest that leucovorin could alleviate methotrexate-induced intestinal toxicity via gut microbiota.Toll-like receptors(TLRs)identify gut microbiota and its metabolites,as molecular sensors of gut microbiota in intestinal inflammation.To further clarify the pathological changes of leucovorin alleviating methotrexate intestinal toxicity may beneficial to the development of new therapeutic targets to methotrexate-induced intestinal toxicity.Objective: To investigate leucovorin ameliorated methotrexateinduced intestinal toxicity via gut microbiota modulation of TLR4,and the effect of inhibiting TLR4 on intestinal toxicity induced by methotrexate.Methods: Experiment I: Male Balb/c mice were reared in the same cage for 2 weeks to assimilate the gut microbiota,divided into four groups:control(CON)group,methotrexate(MTX)group,methotrexate combined with calcium leucovorin(MTX+LV)group and calcium leucovorin(LV)group,6 mice in each group.According to the clinical dose of methotrexate and the previous dose exploration,dissolve methotrexate in sodium bicarbonate for administration at a dose of 50 mg/kg.And dissolve leucovorin in drinking water for administration at a dose of 10 mg/kg.The MTX group was administered intragastrical with methotrexate once three days and used the drinking water;the LV group was administered leucovorin dissolved in drinking water at 10 mg/kg for 48 hours after intragastric administration of sodium bicarbonate for 12 h.The body weight of the mice was recorded daily,fecal samples,serum samples and intestinal tissue samples were collected.HE staining was used to observe the changes of intestinal tissue pathology;ELISA was used to detect protein in intestinal tissue of TNF-α、IL-1β and IL-10;Metagenome was used to detect differences of gut microbiota;Metabolomics was used to detect concentration of LPS in serum;Western Blot and RT-PCR were used to detect the protein and m RNA expression TLR4、My D88 and NF-κB in intestinal tissue.Experiment II: Male Balb/c mice were reared in the same cage for 2weeks to assimilate the gut microbiota,divided into four groups: control(CON)group,methotrexate(MTX)group,methotrexate combined with calcium leucovorin(MTX+LV)group and methotrexate combined with TAK-242(MTX+ TAK-242)group,10 mice in each group.According to the clinical dose of TAK-242 and the previous dose exploration,administration at a dose of 3 mg/kg.The methotrexate group was administered intragastrical with methotrexate once three days,and used the drinking water;the LV group was administered leucovorin dissolved in drinking water at 10 mg / kg for 48 hours after intragastric administration of sodium bicarbonate for 12h;TAK-242 was injected at 3 mg/kg after methotrexate administration for 24 h.When the mice apparently died,the experiment was terminated.The survival status of the mice was recorded daily,intestinal tissue samples were collected.HE staining was used to observe the changes of intestinal tissue pathology.Results:(1)Leucovorin ameliorated methotrexate-induced weight loss and intestinal damage: compared with MTX group,the body weight of mice in the MTX+LV group was significantly increased(P<0.05),and intestinal damage was alleviated(P<0.01).(2)Leucovorin ameliorated methotrexate-induced intestinal inflammation: the results of ELISA showed that compared with the MTX group,expression of TNF-α(P<0.05)and IL-1β(P<0.05)protein were reduced in the MTX+LV group,and the content of IL-10(P<0.01)increased.(3)Leucovorin modulated the methotrexate-induced intestinal microbiota and metabolite composition: gram-negative bacteria in MTX group increased significantly,and the results of metabolomics showed that compared with the MTX+LV group,the concentration of LPS(Lipopolysaccharide)(P<0.01)in the MTX group increased.(4)Leucovorin modulated TLR4/NF-κB pathway: the results of Western Blot and RT-PCR showed that compared with MTX group,the protein expression of TLR4(P<0.001)and NF-κB(P<0.05)decreased in MTX+LV group,the m RNA expression of NF-κB(P>0.05)decreased.(5)Inhibiting TLR4 alleviated methotrexate-induced intestinal toxicity: compared with MTX group,the body weight of mice in the MTX+TAK-242 group increased significantly,and their intestinal damages(P<0.01)were alleviated.Conclusion: Leucovorin alleviate methotrexate induced intestinal toxicity via modulation of the gut microbiota-LPS-TLRs;TLR4 inhibitor TAK-242 can alleviate the intestinal toxicity of methotrexate.