The Mechanism Of Tanshinol Regulating The Killing Effect Of NK Cells On Breast Cancer Based On TGF-?₁

Background and PurposeThe balance of coagulation-anticoagulation system in tumor patients was broken,and the blood was in a state of"concentration,viscosity,coagulation and aggregation".The state of blood stasis was accompanied by the pathophysiological process of tumorigenesis and development.The content of immunosuppressive factor TGF-?1 in peripheral blood is significantly increased under hypercoagulable state,which can inhibit tumor immunity mediated by Natural Killer(NK)cells in many ways and promote the occurrence and development of tumor.In view of the hypercoagulable state of the body,salvia miltiorrhiza can improve the hypercoagulable state of peripheral blood and promote the immunity of the body.In this study,Tanshinol,a representative component of Salvia miltiorrhiza Bunge,a traditional Chinese medicine for promoting blood circulation and removing blood stasis,was used to investigate the effect of Tanshinol on the inhibition of NK cells by TGF-?1 on breast cancer.Research Contents and ResultsIn this study,NK cells(NK92MI)from patients with human non-Hodgkin's lymphoma(NHL)and human breast cancer cell line ZR-75-1,were used to investigate the killing effect of NK92MI cells on ZR-75-1 cells and the intervention of TGF-?1 in this process.The dose of Tanshinol was determined to reverse the killing effect of TGF-?1 on NK92MI cells in vitro.The effect of TGF-?1 on NKG2DL-NKG2D axis was observed by positive and negative experiments,and the correction effect of Tanshinol on this process was observed.Western Blot,RT-PCR and immunofluorescence assay were used to investigate the effect of Tanshinol on the expression of NKG2DL on the surface of human breast cancer cell line.The results showed that TGF-?1 could significantly down-regulate the expression of NKG2DL.Different doses of Tanshinol can be corrected.Western Blot,RT-PCR and immunofluorescence assay were used to investigate the effects of Tanshinol on the transcription and protein level of NKG2D and its molecular chaperone DAP 10 on the surface of NK92MI cells.Studies have shown that different doses of Tanshinol can reverse the transcription and translation levels of NKG2D/DAP10 mediated by TGF-?1.The expression and nuclear accumulation of p-smad2/3,an important transcription factor in NK92MI cells,were observed by Western Blot,immunofluorescence assay and laser confocal 3D imaging.The results showed that Tanshinol could significantly reverse the down-regulation of p-smad2/3 expression and nuclear accumulation mediated by TGF-?1,which indicated that Tanshinol could regulate the downstream cellular immune killing related signaling pathway by regulating its protein expression and nuclear translocation.The formation of NKG2D-DAP10 complex on the surface of NK cells was investigated by immunoprecipitation and immunofluorescence technique.The results showed that Tanshinol could reverse the inhibitory effect of TGF-?1 on the binding ability of NKG2D-DAP10 and restore the interaction between them.Western Blot was used to investigate the activation of PI3K-ERK1/2-PLCy 2,a signal pathway related to the release of killing mediators in NK cells.The results showed that TGF-?1 could significantly down-regulate the expression of this signaling pathway and reverse it after different doses of Tanshinol.Flow cytometry was used to detect the expression of CD107a on the surface of NK cells.Tanshinol could improve the down-regulation of CD 107a expression on the surface of NK cells by TGF-?1,and promote the degranulation effect of NK cells to play an immune killing role.Intracellular factor staining combined with flow cytometry was used to detect the degranulation related indexes of NK92MI cells.TGF-?1 could significantly inhibit the intracellular expression of Perforin,Granzyme B and IFN-?.At the same time,through RT-PCR and ELISA experiments,Tanshinol can reverse the synthesis and release of killing mediators mediated by TGF-?1,thus mediating the immune killing effect of NK cells on tumor cells.Conclusion and SignificanceWe found that TGF-?1 could destroy the NKG2DL-NKG2D signal axis,down-regulate the expression of p-smad2/3 and affect the expression and nuclear accumulation of TGF-?1.It inhibited the activation of PI3K-ERK1/2-PLCy2 pathway of degranulation of NK cells,and the expression of degranulation marker CD 107a,and then affect the release of anti-tumor cytotoxic killing medium of NK cells.Tanshinol can interfere with the negative regulation of TGF-?1 on the function of NK cells,mainly by promoting the expression of NKG2D and its molecular chaperone DAP 10 and promoting the formation of NKG2D-DAP10 complex.Tanshinol causes NK cells to activate and release various killing mediators to carry out immune attacks on tumor cells.This study confirmed that TGF-?1,an immunosuppressive factor released in large quantities in peripheral blood hypercoagulable state of tumor patients,could inhibit the anti-tumor immune function of NK cells,and Tanshinol was the representative of traditional Chinese medicine for promoting blood circulation and removing blood stasis.From the point of view of innate immunity,the immunosuppressive effect of TGF-?1 on NK cells was reversed,the immunity of the body was restored,and the occurrence and development of tumor was inhibited.