Effect Of Steamed Rehmannia Root On The Alimentary Mucositis Induced By High-dose Methotrexate

Chemotherapy is one of the main methods of cancer treatment,but it can easily lead to a variety of side effects,such as alimentary mucositis(AM).AM caused by chemotherapy is often diagnosed as Qi and Yin deficiency in TCM,and the prescription often contains steamed rehmannia root(SRR).The previous research of our group had confirmed that SRR could alleviate AM caused by methotrexate(MTX)chemotherapy and the mechanism was mainly due to anti-oxidation and anti-inflammation.However,high-dose chemotherapy is commonly used in clinical practice,and the effect of SRR on AM induced by high-dose MTX(HDMTX)chemotherapy has not been reported yet.In order to further evaluate the effectiveness and scientificity of SRR in the prevention and treatment of AM induced by HDMTX chemotherapy(HDMTX-AM),we established a mouse model of HDMTX chemotherapy,and evaluated the effect of SRR on HDMTX-AM.The focus was to reveal the time-effect relationship of SRR to HDMTX chemotherapy,the active part of SRR was also explored.Section 1.Effect of SRR on the mortality of mice induced by HDMTX chemotherapyIn this study,we evaluated the effects of SRR(1.56g/kg,ig),ciprofloxacin(CPFX,0.13g/kg,ig)and the combination of SRR and CPFX on the mortality of HDMTX(300mg/kg,ip)treated mice.The results showed that the mortality rate in the model group was 27.3%,9.1%in the CPFX group,and 0.0%in the remaining groups on the eighth day after chemotherapy.At the same time,it was found that HDMTX caused a significant fatigue and decreased body weight and food intake,and some mice suffered diarrhea.SRR and CPFX could improve this condition to some extent.Section 2.Time-effect relationship on prevention and treatment of SRR on HDMTX-AMIn this study,samples were taken 6,12,24,and 48h after HDMTX chemotherapy,MTX concentration in blood,and some indexes of oxidative damage and inflammation were determined.The results showed that:(1)Intestinal villi became significantly shortened after 24h;(2)The blood concentration of MTX dropped rapidly after 12h;(3)The level of malondialdehyde(MDA)gradually increased,while the activity of superoxide dismutase(SOD)and catalase(CAT)decreased;(4)The content of nuclear transcription factor-κB(NF-κB)in the intestinal mucosa peaked at 12h and then dropped;(5)The levels of inducible nitric oxide synthase(iNOS)and myeloperoxidase(MPO)increased significantly after HDMTX chemotherapy and reached to their highest level at 24h and 48h respectively;(6)The level of serum creatinine(SCr)increased significantly 6h and 24h after chemotherapy,however,the alterations of blood urea nitrogen(BUN),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)did not reach to a significant level.Compared to the mice treated with HDMTX alone,SRR administration could significantly decrease NF-κB and SCr 6h after chemotherapy,MDA at 12h,SCr,MDA and iNOS at 24h,MPO at 48h,while significantly increase the height of intestinal villi at 24h,and CAT level at 48h,however SRR has no significant influence on the blood concentration of MTX.The results indicate that SRR could alleviate intestinal mucosal injury by anti-oxidation and anti-inflammation from the early stage of HDMTX chemotherapy.Section 3.The effect of SRR on the intestinal microbiota in mice caused by HDMTXThe effects of SRR on intestinal microbiota of HDMTX chemotherapy mice were further evaluated,the result indicates that HDMTX chemotherapy lead to dysbiosis of intestinal microbiota,manifested mainly as:(1)At the phylum level,the relative abundance of Firmicutes decreased gradually,while that of Bacteroidetes increased;Proteobacteria showed a trend of decrease at first and then increase,while Actinobacteria was the opposite;(2)At the genera level,the relative abundances of Helicobacter,Clostridium,Flexispira and Escherichia increased,while those of Ruminococcus,Lactobacillus,Prevotella and Odoribacter decreased.In addition,the level of plasma endotoxin(LPS)was found peaked 12 h after the chemotherapy.SRR could(1)significantly reduce the number of intestinal microbiota species in HDMTX treated mice;(2)affect the relative abundance of intestinal microbiota to some extent,especially significantly inhibit the increase of Clostridium 6h and 12h after chemotherapy.However,plasm LPS was not affected by SRR administration to a significant level.Section 4.Evaluating the active fractions of SRR in relieving HDMTX-AMIn this study,5 fractions of SRR were prepared by extracting with methanol and ethyl acetate,included(1)SRR after extracting with methanol(F-deMeOH);(2)SRR after extracting with ethyl acetate(F-deEtOAc);(3)ethyl acetate fraction(extracted from SRR water extract,F-EtOAc);(4)methanol fraction(extracted from the dried residue of SRR after ethyl acetate extraction,F-MeOH);(5)macromolecular fraction(the remaining part of SRR after extracting by ethyl acetate and methanol,F-MAC).Firstly,the browning degree and fingerprint spectrum of each fraction were compared.Two animal experiments were conducted to evaluate the effects of the 5 samples on HDMTX-AM with water extract of SRR as control.The results showed that SRR water extract was the best one,the antioxidative and anti-inflammatory activities of the other samples decreased to varying degrees.The results of this study showed that significant oxidative damage and inflammation occurred 6h after HDMTX chemotherapy and the condition became worsen gradually,which suggested the importance of prevention for the protection of alimentary mucosa.The prophylactic administration of SRR did not affect the elimination of MTX,could significantly reduce the oxidative damage and inflammation of intestinal mucosa,and completely avoid the death of mice caused by HDMTX chemotherapy.In addition,it was firstly found that SRR could affect intestinal microbiota,inhibit the proliferation of harmful bacteria and promote that of probiotics,which suggested that regulation of intestinal microbiota was one of the mechanisms of SRR to alleviate HDMTX-AM.Considering that the activity decreased after extracting with methanol and ethyl acetate,SRR has a characteristic of multi-component and multi-target.in alleviating HDMTX-AM.