Effect Of Hedyotis Diffusa Willd Formula Alleviates 5-FU-induced Intestinal Damage In Colorectal Cancer

Aim:To evaluate the effect of Hedyotis diffusa Willd Formula(HDWF)on alleviating 5-fluorouracil-induced intestinal mucositis in vivo,and to explore the underlying mechanisms.The aim of this study was to provide theoretical basis for the clinical application of the drug.Methods:After detached through trypsinization.CT-26 cells were resuspended in serum-free RPMI-1640.2×106 cells mixed with matrigel(1:1)were injected subcutaneously into the right flank region of each mouse.When the tumor size reached 100 to 300 mm3,the mice were randomly divided into 4 groups(n=8):control group,HDWF group,5-FU group,and 5-FU +HDWF group.2 g/kg of HDWF was intragastrically given,daily for 4 days,and 5-FU(150 mg/kg)was administered intraperitoneally only on the first day of treatment,the control group was given normal saline with the same volume of intraperitoneal injection and intragastric perfusion.The general condition of the mice(hair'color,diet,activity degree,etc.)was observed every day.The weight and volume of the mice were measured and recorded.And diarrhea score was calculated every day.On the fifth day,after the eyeball took blood,the mice were killed by cervical dislocations,and the thymus,spleen and jejunal tissues were sacrificed.The thymus and spleen were weighed.The excised jejunum tissues fixed in 4%paraformaldehyde were embedded in paraffin and sectioned at 4 ?m thickness.Then,the number of red blood cells,white blood cells and platelets in the blood of mice was detected by an automatic blood analyzer,and the thymus and spleen index of mice were calculated,haematoxylin and eosin(H&E)staining was used to observe the effect of HDWF on the morphology of jejunum,Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay was used to detect cell apoptosis.Immunohistochemical(IHC)staining was performed to determine the expression of Bcl-2,Bax,PCNA,Cyclin D1,CDK4,p21 in the jejunum tissue.Enzyme-linked immuno sorbent assay(ELISA)was used to detect the expression of inflammatory factors(TNF-?,IL-1?,IL-6,IL-10)in the mouse serum.Results:Compared with the control group and HDWF group,from the beginning of the third day,the weight of mice in 5-FU group were significantly decreased,tumor volume decreased significantly,and there were symptoms of diarrhea(P<0.05),after giving HDWF,the weight loss did not improve(P>0.05),and compared with the 5-FU group,the volume of tumor has no difference(P>0.05),however,it significantly alleviated 5-FU-induced diarrhea(P<0.05),The blood test results showed that 5-FU had no effect on the number of red blood cells(P>0.05),but it can significantly reduce the number of white blood cells and platelets(P<0.05),HDWF treated can improve mice leukopenia cases(P<0.05),by calculating the thymus and the spleen index,we can see that the 5-FU can significantly reduce the thymus and spleen index of mice(P<0.05),but the treated with HDWF did not restore the situation(P>0.05).The pathological observation to the intestinal mucosa appeared different degree of damage,such as intestinal mucosal villi shortened,intestinal epithelial cell edema,inflammatory cell infiltration,lamina propria edema,HDWF could significantly alleviated 5-FU-induced intestinal damage in experimental animals.TUNEL assay indicated that 5-FU challenge significantly increased the percentage of TUNEL-positive intestinal crypt cells in xenograft mice(P<0.05),as compared with mice in either control or HDWF alone group,which however was reduced by HDWF treatment(P<0.05).In addition,data from IHC staining showed that 5-FU significantly reduced Bcl-2,PCNA,CDK4,Cyclin D1 protein level in intestinal crypt cells,whereas the level of Bax,p21 protein were significantly increased after 5-FU challenge.Compared with the 5-FU group,in the combined group,the expression of Bcl-2,PCNA,CDK4,Cyclin D1 were increased significantly(P<0.05),the expression of Bax,p21 decreased significantly(P<0.05).Futhermore,the results of ELISA showed that 5-FU significantly reduced TNF-?,IL-1?,IL-6 protein level in the mouse serum(P<0.05).However,the level of IL-10 was significantly increased after treating 5-FU(P<0.05).Moreover,there were no obvious differences between,the control group and HDWF group of tumor volume,body weight of mice,diarrhea,blood cells,thymus and spleen index,jejunum tissue damage,intestinal cell apoptosis and related protein expression of proliferation and apoptosis and inflammation(P>0.05).Conclusions:(1)HDWF can significantly alleviate 5-FU-induced diarrhea and the symptoms of diarrhea in CT-26 tumor-bearing mice,suggesting that HDWF had a protective effect on 5-FU-induced intestinal damage.(2)HDWF reduced intestinal injury caused by 5-FU by regulating the expression of intestinal cell proliferation and apoptosis related proteins and the levels of inflammation,which may be one of the mechanisms of HDWF reduce 5-FU-induced intestinal injury.