The Regulation Effects Of Ciji-Hua’ai-Baosheng Ⅱ Formula On Gastrointestinal Microecology With Transplanted H₂₂ Hepatocellular Carcinoma In Mice Receiving Chemotherapy

Background:Ciji-Hua’ai-Baosheng Ⅱ Formula(CHB)is improved by Ciji-Hua’aiBaosheng formula.After many years of clinical research,it has been proved that the formula can reduce the gastrointestinal discomfort symptoms of cancer patients after chemotherapy.It can also improve appetite,enhance immunity,and increase their quality of life.The dynamic balance of human intestinal microecology regulates the state of the whole gastrointestinal tract.This prescription may not only alleviate the gastrointestinal discomfort,but also affect the gastrointestinal microecology.However,it is not clear whether the formula has regulatory effect on gastrointestinal microecology and the mechanism of this prescription in cancer patients after chemotherapy.Making a profound study of its mechanism of action can provide a scientific basis for clinical medication guidance for patients with cancer after chemotherapy.Aim:To explore the effect of CHB on the intestinal flora,intestinal barrier and some correlation factors in signal pathways with transplanted H22 hepatocellular carcinoma in mice receiving chemotherapy,to further clarify the effect of CHB on the gastrointestinal microecology of mice after chemotherapy.Methods:A total of 96 Kunming mice of SPF grade(specific pathogen free)were injected subcutaneously with H22 hepatoma cells in the right anterior armpit(the suspension of H22 cells was adjusted to 2×107/mL).One week later,the transplanted tumor appeared in mice.The model of transplanted H22 hepatocellular carcinoma in mice receiving chemotherapy was made by a single abdominal injection of high does 5-fluorouracil(5-FU),the concentration of 5-FU is 200mg/kg,which is equivalent to the concentration of 0.2mL/10g with mouse.After the model was established successfully,36 mice were randomly selected one day before the drug was used.0.2g of feces of mice was taken under sterile condition.After the mice were euthanized,a section of ileum tissue(>500mg)from the same part of the mice was taken for detecting the flora.The remaining 60 mice were divided into six groups according to their weight and gender(10 mice in each group):normal saline control group(NS-Contr),5-fluorouracil control group(5-FU-Contr),Yangzheng-Xiaoji capsule group(YZXJC),CHB-high dose group(CHB-H,65g/kg),CHB-middle dose group(CHB-M,32.5g/kg)and CHB-low dose group(CHB-L,16.25g/kg).On the 7th day of treatment,0.2g feces of mice in each group were taken under sterile condition and then the feces were stored in-80℃ refrigerator for future detection.After 14 days of treatment,mice were fasted for 12 hours,then they were euthanized for collecting the experimental samples.0.2g feces of mice in each group were taken under sterile condition,0.2g feces of each group of mice were taken under aseptic condition,the eyeball serum was removed to get blood,tumor,liver,spleen,thymus and were taken for future detection.The morphological changes of the tissues of stomach,jejunum,ileum and colon were stained by HE and photographed under light microscope.Ultra high performance liquid chromatography(UHPLC)and a high resolution electrospray ionization mass(HR-ESI-MS)were used to analyze the bioactive components in the water solution of CHB.The contents of Tumor Necrosis Factor-α(TNF-α),Interleukin-22(IL-22),Interleukin-6(IL-6),Interleukin-1β(IL-1(3),Interleukin-8(IL-8),Interleukin-10(IL-10),Transforming Growth Factor-β(TGF-β)in serum and Secretory Immunoglobulin A(SIgA)in jejunum of mice were determined by Enzyme linked immunosorbent assay(ELISA).The protein and mRNA expressions of Zonula occuluden-1(ZO-1),Occludin,Claudin-1,Mucin-2(MUC-2),Nuclear Factor kappa B(NF-κB)and I-kappa-B-alpha(IκB-α)in colon tissues of mice were detected by Western blotting,Immunohistochemistry,and Realtime fluorescence quantitative polymerase chain reaction.The species composition of bacteria in mice feces and ileum were detected by 16S rDNA metagenomic sequencing.Results:The following 31 major components(including 3 unknown components)were identified in CHB decoction by UHPLC-MS:Naringin,hesperidin,nobiletin,tangeretin,5,7,8,4’-tetramethoxyflavone,3,5,6,7,8,3’,4’-heptamethoxyflavone,phenylalanine,salvianic aid A,protocatechualdehyde,caffeic acid,lithospermic acid,rosmarinic acid,salvianolic acid B,tanshinone IIA,cryptotanshinone,dihydrotanshinone I,tangshenoside I,tangshenoside Ⅱ,kukoamine B,thalictrine,spinosin,nicotinic acid,pingpeimine B,pingpeimine A,pingpeimine C,peimisine,peimine,and peiminine.After CHB treatment,compared with NS contr group,the mice in high,medium and low dose CHB groups had significantly increased weight,improved appetite,increased immune organ function,and improved general living conditions.The contents of IL-10,TGF-β,SIgA,TNF-α,IL-1β,IL-6,IL-8 and IL-22 in the serum of mice of all the CHB treated groups were higher than those of 5-FU group and negative control group,while the protein and mRNA expressions of ZO-1,occludin,MUC-2,NF-κB,IκB-α and gene decreased.In the analysis of fecal flora of mice in the middle period of treatment,compared with NS-Contr group,the number of Actinobacter,AF12,Adlercreutzia,Clostridium and Coriobacterae increased significantly in the CHB-H group,and the number of Paraprevotella increased significantly in the CHB-M group.In the analysis of fecal flora of mice in the later period of treatment,compared with NS-Contr group,the number of Deferribacteres,Lactobacillus increased significantly in the CHB-H group,while the number of Musipirillum,Odoribacter,RF32 and YS2 decreased significantly.The number of Lactobacillus increased significantly in the CHB-Mgroup,while the number of Rikenellaceae and YS2 decreased.In the analysis of the bacterial population in the mice ileum,compared with NS-Contr group,the number of Lactobacillus,Ruminococcus,Bacteroides and S24-7 increased significantly in the CHB-H group,while the number of Musipirillum and Prevotella decreased significantly.The number of Rikenellaceae and YS2 decreased significantly in the CHB-M group.Conclusion:CHB could not only inhibit tumor growth,but also relieve the gastrointestinal side effects of 5-FU chemotherapy in mice with liver cancer transplanted tumor.The mechanism may be to reduce NF-κB signal pathway,promote the expression of the pro-inflammatory cytokine and inhibit the expression of inflammatory suppressing cytokine,as well as the change of related flora,so as to reduce the intestinal mucosal damage,enhance the intestinal barrier function,alleviate the gastrointestinal inflammation,finally regulate microecological balance of gastrointestinal tract in mice after chemotherapy.