| Breast cancer,as one of the current leading killer,has horribly threaten to women’s health.In recent years,the number of women with breast cancer and its incidence has been increasing in our country.Paclitaxel(PTX)is a commonly used chemical reagent for breast cancer,while these patients have been suffered from its side effects,such as intestinal reaction,neurotoxic effects and myelotoxicity.Intestines is an important organ for metabolism and immunity.The interaction of intestinal mucosa barrier and gut microbiota plays an important role in maintaining the human health.Meanwhile,gut microbiota could influence the tumorigenesis and treatment by means of impacting immunoreaction of host.ESGL is the extraction extracted from the broken spore of Ganoderma lucidum,which possess the activities of immunoregulation and anti-tumor.However research of the enhancement and detoxication effect of ESGL with PTX was seldom reported.This study aims to explore the mechanism of enhancement and detoxication effect of ESGL with PTX on breast cancer,supplying theory basis for adjuvant agent in routine chemotherapy.Objective:The study aims to investigate the effect of anti-breast cancer enhancement of ESGL with PTX and the repairment ability of ESGL on intestinal mucosal barrier under the injury by PTX.Method:1.Anti-breast cancer enhancement of ESGL combined with PTX(1)The cell activity of breast cancer 4T1 cell influenced by PTX combined with ESGL was determined by MTT assay(2)Female BALB/c mice were randomly divided 6 groups,Normal group,Model group,PTX group,ESGL+P groups(100,200,400 mg/kg).The 4T1-breast cancer model was established by injecting subcutaneously(s.c.)into the right forleg of subjected BALB/c mice.On the 2th day after modeling,PTX group was intraperitoneally injected(i.p.)with PTX at a dose of 12.5 mg/kg twice a week.The ESGL+P group was administrated orally with ESGL(100,200,400 mg/kg,p.o.)once a day,in addition to the twice-a-week intraperitoneal injection of PTX(12.5mg/kg).Normal group and Model group received equal volume of saline.In the following 21 days,tumor growth was observed.(3)During the 21-day experiment,body weight change and tumor growth were observed and recorded.On the 22th day,all mice were sacrificed and their blood,thymus,tumor and small intestine were harvested for the following assays.(4)The ratio of T lymphocytes and their major subsets(Th,Tc),and the expression of costimulating factor CD28 and immune checkpoints proteins(PD-1,CTLA-4,Tim-3)in peripheral blood,thymus and tumor microenvironment(tumor infiltrating lymphocyte,TIL)were analyzed by flowcytometry analysis(FCM).2.The repairment effect of ESGL on intestinal mucosal barrier under the injury by PTX(1)Parts of the small intestine was fixed in 4%formaldehyde(PH=7.2)and embedded in paraffin.Some sections were subjected to Haematoxylin and Eosin(HE)staining to analyze the structure of small intestine tissues,some were subjected to TdTmediated dUTP Nick-End Labeling assay(TUNEL)to analyze the apoptosis level and the others were subjected to immunohistochemistry(IHC)to analyze the expression of proliferating cell nuclear antigen(PCNA).(2)The expression of junction proteins(ZO-1,E-cadherin,β-catenin,Occludin)in small intestinal tissues were detected by Western Boltting(WB)and those for the corresponding genes were detected by Real-time Quantitative Polymerase Chain Reaction(RT-qPCR).Lipopolysaccharide(LPS)content in serum was detected by Enzyme-Linked Immunosorbent Assay(ELISA).3.The influence of ESGL with PTX on gut microbiota on tumor-bearing miceGut microbiota in cecum was detected by 16S rRNA gene sequencing.Results:1.Anti-breast cancer enhancement of ESGL combined with PTX(1)Results of MTT assay indicated that the 48h-and 72h-IC50 of PTX on 4T1 were 243.2ng/mL and 151.9ng/mL,respectively.There is no significant difference in cell viability between monotherapy and combination,indicating that ESGL would not strength the cytotoxicity of PTX on 4T1 cells.(2)Tumor volume and weight droped obviously in mice of PTX group when compared with those of Model group,and those of ESGL+P groups showed more decrease,suggesting that ESGL could enhance the anti-tumor activity of PTX.(3)The results of FCM assay showed that ESGL combinated with PTX could promote the expression of CD28 in T cells,Th cells and Tc cells and depress the expression of PD-1 in T cells in peripheral blood,raise the ratio of Tn cells and Te cells in thymus,decreased the expression of PD-1,CTLA-4,Tim-3 in TILs.The finding illustrated the combination could strengthen the function of immune surveillance.2.The effect of repairment of ESGL on intestinal mucosal barrier under injury by PTX.(1)The results of HE staining showed that ESGL could restore the intestinal mucosal structure destroyed by PTX.After the combination treatment,the intestinal muscle layer become intact,and the villi were prolonged with close arrangement.The results of TUNEL assay indicated that ESGL could reduce the intestinal goblet cell apoptosis damaged by PTX,and the IHC assay suggested ESGL could promote the expression of PCNA and proliferation of crypt cells.(2)The results of WB and RT-qPCR analysis revealed that PTX could inhibit the expression of ZO-1,E-cadherin,β-catenin,Occludin in both proteins and genes level.But ESGL combined with PTX could up-regulated the above-mentioned proteins and genes.In addition,ELISA analysis reflected that combination could decrease the LPS level in serm compared with PTX alone.3.The influence of ESGL with PTX on gut microbiota in tumor-bearing mice(1)The results of classification and identification in OTU showed that the number of specific OTU in PTX group was significantly lower than that in other groups.(2)The results of α-diversity analysis showed that Chao1 index and ACE index in all groups were significantly lower compared with Normal group,suggesting an evident decline in the microbiota abundance of ESGL+P groups.(3)The results of β-diversity analysis based on PCoA principal coordinate analysis and ANOSIM analysis showed that Model group has the similarity in microbial community structure with Normal group,while those of PTX group and ESGL+P groups exhibited obvious difference with Normal group.(4)The results of Taxonomic analysis showed that all groups has obvious difference with each others in classification of genus and phylum.Abundance of Bacteroidetes in ESGL+P groups was higher than that in PTX group and Normal group,while abundance of Firmicutes in these groups was not different,leading an increase in ratio of Bacteroidetes and Firmicutes.LEfSe analysis indicated that there existed several specific genera in each group.In brief,relative abundances of Dorea and Ruminococcus were highest in Normal group.Coprococcus,Parabacteroides,and Prevotella were enriched in Model group.Desulfovibrio,Ochrobactrum,Odoribacter,and Turicibacter were specific in PTX group.Bacteroides,Ruminococcus,and other 5 genus were significantly enriched in ESGL+P groups.Conclution:Firstly,it was found that combination of ESGL and PTX evidently strengthened tumor suppression of PTX.By the combination,T cells and their major subsets were increased in peripheral blood and thymus of the tumor-bearing mice,and expression of CD28 protein was up-regulated in T cells of peripheral blood.Furthermore,ESGL synergized with PTX could inhibite the expression of immune checkpoint proteins in TILs.These results indicated that ESGL could promote the anti-tumor activity of PTX via enhancing the function of immune surveillance in tumor-bearing mice.Secondly,combination of ESGL and PTX could repair the structure damage in small intestine induced by PTX,reduce the apoptosis and recover the function of proliferation and increase the expression of the tight junction protein and gene in intestine tissue and decreased the secretion of LPS in serm.Theses results indicated that ESGL could recover intestinal mucosa barrier and thus eliminate the side effects caused by PTX.Finally,the combination also could change the diversity and abundance in gut microbiota,enrich the predominant bacterium,and ameliorate the microbiota disbiosys caused by PTX.In summary,the combination of ESGL and PTX could enhance the effect of anti-breast cancer and alleviate the injury in intestinal mucosa barrier induced by PTX,revealing practical application value of ESGL in adjuvant chemotherapy. |
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