BuPiHeWei Decoction Ameliorate 5-Fluorouracil-Induced Intestinal Microecological Disorder In Rats By Regulating The Th17/Treg Signaling Pathway

Objective:To observe the protective effect of BuPiHeWei Decoction on 5-Fu-induced intestinal microecological disorders in rats and to explore the mechanism based on Thl7/Treg-TLR-4/NF-κB signaling pathway.Methods:The 35 SD rats were randomly divided into four groups:blank control group,5-Fu che-motherapy-only group,5-Fu+Bupihewei Decoction group and 5-Fu+Bacillus Lichenifor-mis Capsule group,8 rats in the blank control group and 9 rats in the other groups.The rats in the blank control group were intraperitoneally injected with an equal volume of saline.Rats in 5-Fu group,5-Fu+Bupihewei Decoction group and 5-Fu+Bacillus Licheniformis Capsule group were injected with 5-Fu injection at 30mg/Kg for 5 consecutive days(dl-d5).The rats in blank control group and the 5-Fu group were administered by gastric perfUsion with 2ml saline on dl-d5,once a day.The rats in 5-Fu+Bupihewei Decoction group were administered by gastric perfusion with 10.5g/kg Bupihewei Decoction on d1-d5,once a day.The rats in 5-Fu+Bacillus Licheniformis Capsule group were administered by gastric perfusion with 0.2g/kg Bacillus Licheniformis Capsule physiological saline on dl-d5,once a day.Before chemotherapy(d0),d3,and after chemotherapy(d6),the defecation of the rats was observed and the body mass of the rats was measured every morning.In day6,0.5g feces were collected and the rats were sacrificed,laparotomized to obtain small intestine tissue.The retained specimens were stored in a-80℃ refrigerator and tested as follows:① Detected of fecal microbiota changes using 16S rDNA high-throughput sequencing.② Observed of pathological changes of intestinal mucosa by HE.③The expression of RORγt,Foxp3 and IL-17、IL-21、IL-10 were detected by Western blot and qPCR.④The expression of TLR-4 and NF-κB were detected by Western blot and qPCR.⑤ The levels of TNF-α,IL-1β,IL-6 and TGF-β were detected by Elisa.Results:1、The body weight of rats on d3 and d6 was significantly decreased in the chemotherapy-only group than that of the blank control group(P<0.01),while the diarrhea score was sig-nificantly increased(P<0.01).HE staining showed that the height of villi was shortened in the 5-Fu group,with shedding and enhanced edema.16S rDNA high-throughput sequencing showed that in the 5-Fu chemotherapy-only group the content of Firmicutes and Bacteroidetes were significantly reduced and the proportion of Proteobacteria was significantly increased in phylum level.In addition,the content of Escherichia-Shigella,Ruminococcaceae_NK4A214 and Ruminococcaceae-UCG-005 were significantly increased and the proportion of Prevotellaceae-Alloprevotella was reduced in genus level.2、Compared to the 5-Fu group,the body weight on d3 and d6 was significantly increased(P<0.01),while the diarrhea score was significantly decreased(P<0.01,P<0.05)in the Bupihewei Decoction group and the Bacillus Licheniformis Capsule group.Compared with the Bacillus Licheniformis Capsule group,the difference of the diarrhea score on the 6th day between the Bupihewei Decoction group and the 5-Fu group group was more obvious.In addition,the height of villi was increased,with attenuated shedding and edema in the Bupihewei Decoction group than that in the 5-Fu group and Bacillus Licheniformis Capsule group.Moreover,compared with the 5-Fu group,the proportion of Proteobacteria,Escherichia-Shigella were significantly reduced in the Bupihewei Decoction group(P<0.05).Compared with the Bacillus Licheniformis Capsule group,the proportion of Proteobacteria and Escherichia-Shigella between the Bupihewei Decoction group and the 5-Fu group group was more obvious.3、Compared with the blank control group,the expression of RORγt,Foxp3 in protein and RORγt,Foxp3,IL-17,IL-21,IL-10 in mRNA were significantly increased in the 5-Fu gro-up(P<0.01).However,compared with the 5-Fu group,the expression of RORγt,Foxp3 in protein RORγt,Foxp3,IL-17,IL-21,IL-10 in mRNA were significantly decreased(P<0.05)in the Bupihewei Decoction group,and the expression of RORγt,IL-17 and IL-10 mRNA were significantly lower than that in the Bacillus Licheniformis Capsule group(P<0.05).4、Compared with the blank control group,the expression of the inflammatory factors(including TNF-α,IL-1β and IL-6,TGF-p)were significantly increased in the 5-Fu group(P<0.01).However,compared with the 5-Fu group,the expression of the inflammatory factors(including TNF-α,IL-1β and IL-6,TGF-β)were significantly decreased(P<0.05)in the Bupihewei Decoction group and the Bacillus Licheniformis Capsule group,and compared with the Bacillus Licheniformis Capsule group,the content of TNF-α and IL-6 between the Bupihewei Decoction group and the 5-Fu group group was more obvious.5、Compared with the blank control group,the expression of TLR-4 and NF-κB in protein and mRNA were significantly increased in the 5-Fu group(P<0.01).While,compared with the 5-Fu group,the levels of TLR-4 and NF-κB in protein and mRNA were siguificantly decreased in the Bupihewei Decoction group(P<0.01,P<0.05),and the expression of TLR-4 and NF-κB in protein were significantly decreased than that in the Bacillus Licheniformis Capsule group(P<0.01,P<0.05).Conclusions:1、In our experiment,the multiple and low-doses intraperitoneal injection of 5-Fu could successfully simulate intestinal micro-ecological disorders in rats,such as the change of Intestinal flora and Intestinal nucosal inflammation.2、BPHW Decoction could significantly regulate Intestinal microecological disorder in rats,which manifested in regulate the structure of the flora and reduce the intestinal pathological damage.3、The effects of BPHW Decoction to regulate Intestinal microecological disorder for 5-Fu were based on the Thl7/Treg-TLR-4/NF-κB signaling pathway.