Application Of Intestinal Epithelial Tlr4 Knockout Mice To Explore The Molecular Mechanism Of Anti-tumor Effects Of Glycyrrhiza Polysaccharide Intervention On Intestinal Mucosal γδT Cell Differentiation

Objective:The value of polysaccharides has long been concerned with the immune enhancement and tumor treatment.Glycyrrhiza Uralensis Polysaccharide（GCP）is one of the main medicinal components of the traditional Chinese medicine Glycyrrhiza glabra,the an-tumor effects of GCP have been confirmed within both clinical trials and experimental research.A stable method for the extraction and content determination of GCP has been established by our research group,and found that the purified GCP isolated in our laboratory has good anti-tumor activity.GCP is a macromolecules polysaccharide with extremely low levels of oral bioavailability.The previous research of group has demonstrated that oral licorice polysaccharide exerts anti-tumor effects by regulating intestinal flora,however,the specific mechanism of how GCP interferes with immune cell of intestinal mucosal to exert anti-tumor effect needs to be further discussed.γδT cells are a group of CD3⁺intrinsic immune cells expressing TCRγδand it is an important component of intestinal epithelial lymphocytes,mainly located in the basolateral aspect of small intestinal epithelial cells.γδT cells are not limited by MHC（major histocompatibility complex）.They have killing activity against a variety of tumor cells from different sources and have immunomodulatory function.They play an irreplaceable role in tumor prevention and treatment.γδT cells are a group of highly heterogeneous cells with many subtypes and changeable phenotypes,IFN-γ⁺γδT cells play an important role in tumor immune surveillance and killing.Toll-like receptor 4（TLR4）is a pattern recognition receptor which expressed on the surface of a variety of epithelial and immune cells.It could recognize lipopolysaccharide,a major component of Gram-negative bacteria,or endothelium molecules released by necrotic cells to activate immune cells.TLR4 signaling is one of the essential pathways of intestinal epithelial cells to sensitize intestinal microecological changes and promote the expression of corresponding cytokines,which in turn affects a variety of immune cells in the intestinal mucosa,such as intestinal epithelial cell-derived cytokines IL-12 and IL-18 can promote the differentiation ofγδT cells to the IFN-γ⁺subtype.In conclusion,this study firstly explored the tumor-suppressive effect of GCP on thedifferentiation of intestinal mucosalγδT cells,and intestinal epithelial Tlr4-specific knockout mice was further used to explore the molecular mechanism of GCP affected intestinalmucosalγδT differentiation.It provides an experimental basis for the anti-tumor research of licorice polysaccharide,and also enriches the connotation of the tumor treatment principle of"Fuzheng Pei Ben"in traditional Chinese medicine.Methods:1.GCP affectsγδT differentiation of intestinal mucosa and exerts anti-tumor effectsFirstly,the two groups of tumors bearing mice（SPF-PBS and SPF-GCP）were intervened by gavage with GCP and PBS（Phosphate Buffered Saline）for 21 days respectively.Obsevered the tumor emergence time,tumor volume and tumor body mass ratio,the immunohistochemical staining of tumor bearing mice were evaluated the effect of GCP on the proliferation of tumor cells and CD3⁺,γδT,CD27/γδT infiltration in tumour tissues,obsevered the changes of quality of life,spleen volume and index,detection of CD3⁺T andγδT cells in spleen by immunohistochemistry and immunofluorescence.The ratio of CD3⁺/CD4⁺/CD8ˉT,CD3⁺/CD8⁺/CD4ˉand CD3⁺/γδT in intestinal mucosa was detected by flow cytometry,and the expression of CD27,NKG2D and IFN-γactivated byγδT.It is clear that GCP exerts anti-tumor effect by affectingγδT differentiation of intestinal mucosa.2.Molecular mechanism of GCP affected the differentiation of intestinal mucosalγδT cells2.1 GCP exerted antitumor effect by affecting intestinal epithelial TLR4/My D88-independent signaling pathwayGCP was used to interfere SPF tumor-bearing mice,and scraped the intestinal mucosa of the mice.Real-time quantitative PCR（RT-q PCR）technology was used to detect the m RNA levels of key node proteins on TLR4/My D88-independent signaling pathways（TLR4,TRAM,TRIF,TBK-1,STAT-1,IRF1,IRF2）;Western Blot technology detected the expression of key proteins in the TLR4/My D88-independent pathway（TLR4,TRAM,TRIF,TBK-1,STAT-1,p-STAT-1,IRF1,IRF2）;ELISA was used to detect the concentrations of intestinal mucosal cytokines TNF-α,IL-2,IL-7,IL-12,and IL-21.The effect of GCP on the TLR4/My D88-independent pathway of intestinal mucosa was clarified,thereby promoting the release of related cytokines.2.2 Using intestinal epithelial Tlr4-specific knockout mice to explore the molecular mechanism of GCP affected intestinal mucosalγδT differentiationCRISPR/Cas9 technology was applied to construct intestinal epithelial Tlr4-specific knockout mice(Tlr4^f/fcre T),GCP and PBS gavage,respetictaly.The ratio of CD3+/γδT in intestinal mucosa,the expression of CD27,NKG2D and IFN-γactivated byγδT was detected by flow cytometry to investigate the effect of GCP on intestinal mucosalγδT differentiation in Tlr4^f/fcre T tumor-bearing mice.The tumor emergence time,tumor volume,tumor body mass ratio,and the immunohistochemical staining of tumor bearing mice were calculated to evaluate the effect of GCP on the proliferation of tumor cells.The number of CD3⁺and CD27/γδT infiltration into tumor tissues,the changes of quality of life,spleen volume and index,the number of CD3⁺T cells in spleen was detected by immunohistochemistry,and the number of spleenγδT cells was detected by immunofluorescence to clarify the molecular mechanism of GCP affecting the tumor-inhibitory effects of intestinal mucosalγδT cell differentiation.Results1.GCP affectsγδT differentiation of intestinal mucosa and exerts anti-tumor effectsThe experimental results showed that compared with the SPF tumor-bearing mice in the PBS group,the tumor emergence time of SPF tumor bearing mice in GCP group was delayed,the tumor body mass ratio decreased significantly,the expression of tumor proliferation marker Ki67 decreased significantly,and the tumor tissue infiltrated CD3⁺and CD27/γδT increased significantly.The quality of life was significantly improved,the volume and index of spleen were significantly increased,and the number of CD3⁺T cells andγδT increased significantly.Compared with SPF tumor bearing mice in PBS group,the ratio of CD3⁺/γδT cells in the intestinal mucosa of SPF tumor bearing mice in GCP group was significantly increased,and the expressions of CD27,NKG2D and IFN-γwere significantly increased in the activated phenotype ofγδT.2.Molecular mechanism of GCP affected the differentiation of intestinal mucosalγδT cells2.1 GCP exerted antitumor effect by affecting intestinal epithelial TLR4/My D88-independent signaling pathwayThe results showed that,compared with PBS-SPF tumor-bearing mice,the key nodes TLR4,TRAM,TRIF,TBK-1,STAT-1,IRF1,IRF2 of intestinal epithelial TLR4/My D88independent pathway of SPF tumor bearing mice in GCP group were significantly increased,and the protein levels of the above key node proteins were also increased,and the level of p-STAT-1 was significantly increased.The local cytokines TNF-α,IL-2,IL-7,IL-12 and IL-21 in the intestinal mucosa were significantly increased by ELISA.2.2 Using intestinal epithelial Tlr4-specific knockout mice to explore the molecular mechanism of GCP affected intestinal mucosalγδT differentiationThe experiment results showed that there was no significant difference in the proportion ofγδT cells in gavaged with GCP Tlr4^f/f cre T tumor bearing mice and gavaged with PBS Tlr4^f/fcre T tumor bearing mice,and was also no significant difference in the expression of CD27,NKG2D and IFN-γactivated byγδT.It was further found that there was no significant difference in tumor emergence time,tumor volume and tumor body mass ratio between the two groups,there was no significant difference in the expression of tumor proliferation marker Ki67and the tumor tissue infiltrated CD3⁺and CD27/γδT,no significant in quality of life,the volume and index of spleen,and the number of CD3⁺T cells andγδT in spleen also had no significant.Conclusion1.We demonstrated that GCP exerted anti-tumor effects through regulated intestinal mucosalγδT differentiation2.It is clarified that GCP affects the release of intestinal mucosal cytokines through the intestinal epithelial TLR4/My D88-independent signaling pathway,and interferes the differentiation ofγδT cells to exert the molecular mechanism of anti-tumor.