Study On The Effect Of Guiqi Baizhu Prescription On The Proliferation Of Gastric Cancer Cells And T Cell Activity Based On PD-L1 And HER2 Combined Targets

Objective:Previous studies have confirmed that Guiqi Baizhu Prescription(GQBZP)can effectively improve the adverse reactions and life quality of patients with gastric cancer(GC),However,the material basis and molecular mechanism of GQBZP are still unclear.In this study,chemical informatics-molecular docking-molecular dynamics combined with protein detection and cell biology method were used to reveal substance basis of efficacy that GQBZP could promote the immune activity by Jianpi Fuzheng,targeting PD-L1(Programmed cell death ligand 1)to regulate immune cell function,and at the same time,it can also inhibit tumor proliferation by Huayu Quxie,targeting HER2(Human epidermal growth factor receptor 2)to inhibit tumor cell proliferation.In addition,whose effect characteristics of multi-component“multi-points significant effect,synergistic synergy”were investigated.Methods:1.Clinical treatment targeting combined with database were used to screen the target of GQBZP in the treatment of GC.The structure library of small molecular components of GQBZP was constructed by chemical informatics method.HER2 and PD-L1,which promoted the proliferation of tumor cells and inhibited lymphocyte activity respectively,were selected as the target proteins.The crystal structure and active sites of HER2/PD-L1 were determined.The molecular docking technology was used to screen the representative target components which could effectively bind to the target protein,and then molecular dynamics simulation,binding free energy calculation and microscale thermophoresis(MST)were carried out to verify whose binding affinity with the target proteins.Enzyme activity test was used to detect the effect of small molecules of traditional Chinese medicine(TCM)on HER2.2.The anti-tumor effect of small molecules of TCM targeting HER2 in different GC cell lines was detected.Small molecules of TCM targeting HER2 were used to interfere GC MKN-45cells stimulated by EGF(Epidermal growth factor),whose effects on the ability of proliferation and clone formation of GC cells,and the expression of PI3K-AKT(Phosphatidylinositol 3kinase,PI3K/Protein kinase B,AKT)pathway related proteins and genes were detected.Small molecules of traditional Chinese medicine targeting PD-L1 were used to interfere human peripheral blood T lymphocytes stimulated by soluble PD-L1,whose effects on T-cell proliferation and expression of cell factor(IFN-?,Interferon-gamma/IL-2,Interleukin-2),changes in the number of PD-1⁺(Programmed cell death protein 1)T cells and expression of T-cell function related proteins and genes were detected.3.The co-culture system of human GC MKN-45 cells and human peripheral blood T lymphocytes was constructed,which was interfered by the small molecules of TCM formononetin targeting PD-L1,quercetin targeting HER2,and combined quercetin with formononetin.The influences for small molecules intervention of TCM on T cell proliferation,expression of cell factor(IFN-?/IL-2)and T cell apoptosis were detected.The intervention effects for small molecules of TCM on T cell function inhibition in co-culture system were observed.At the same time,the influences of small molecules of TCM on the proliferation and apoptosis of GC cells were detected,and whose inhibitory effect on the proliferation of GC cells was investigated.Results:1.HER2 and PD-L1 were selected as the effective targets to study the material basis and mechanism of GQBZP in the treatment of GC.2.385 small molecules of TCM in GQBZP docking with HER2 protein scored less than-4,189 small molecules of TCM docking with PD-L1 protein scored less than-4.The small molecules with higher scores were selected to conduct MST,daidzein in Astragalus membranaceus and quercetin in Astragalus membranaceus/Glycyrrhiza uralensis had higher binding affinity with HER2,K_d were 3.7?M/490 n M,respectively;isorhamnetin/formononetin in Astragalus membranaceus/Glycyrrhiza uralensis had higher binding affinity with PD-L1,K_d were 667 n M/355 n M,respectively.50ns molecular dynamics simulation results showed that the binding mode of quercetin with HER2 was more stable,the binding free energy was-26.55 Kcal/mol;and the binding mode of formononetin with PD-L1 was more stable,the binding free energy was-19.41 Kcal/mol.The results of enzyme activity test showed that quercetin could significantly inhibit the activity of HER2 kinase with IC₅₀ of 570.7 n M,daidzein had no obvious inhibitory effect on HER2.3.By interfering GC cells stimulated by EGF and T lymphocytes stimulated by PD-L1 alone,it was found quercetin could inhibit the proliferation and clone formation of GC cells induced by EGF(P<0.05),and inhibit the expression of HER2,PI3K,AKT proteins and genes as well(P<0.05).Quercetin could inhibit GC cell proliferation by blocking PI3K/AKT signaling pathway;formononetin could reduce the expression of PD-1 in T lymphocytes to a certain extent,increase the expression of cell factor(IFN-?/IL-2)in cell supernatant and the expression of PI3K,AKT,p-zap70(Zeta chain of T cell receptor associated protein kinase 70)proteins(P<0.05).It was through blocking PD-1/PD-L1 signaling pathway and activating downstream PI3K/AKT signaling pathway that PD-L1 can relieve the inhibition of T lymphocyte function.4.Small molecules of TCM targeting HER2 and PD-L1 interfered with the co-ulture system of GC cells and T lymphocytes together.The results showed that the intervention of formononetin,quercetin,and combined formononetin with quercetin could relieve the inhibition of T lymphocyte proliferation,reduce T lymphocyte apoptosis,increase the expression of cell factor(IFN-?/IL-2)and reduce the expression of PD-1 in T lymphocytes(P<0.05),inhibit the proliferation of GC cells(P<0.05),promote the apoptosis of GC cells,and reduce the expression of targeting protein(HER2/PD-L1)in GC cells.The combined intervention of small molecules of TCM had better effect,indicating that the combined intervention could improve the activity of T lymphocytes in the co-culture system and inhibit the proliferation of GC MKN-45 cells.Conclusion:1.From the perspective of big data,the results of molecular docking preliminarily show that GQBZP has not only the small molecules which can combine with HER2,the target protein for promoting tumor cell proliferation,but also the small molecules which can combine with PD-L1,the target protein for inhibiting lymphocyte activity.Through the“multi-points significant effect,synergistic synergy”,which block HER2 inhibiting tumor cell proliferation while intervening PD-L1 pathway to promote immune cell activity.2.Cell biology research revealed that the material basis of GQBZP“multi-points significant effect,synergistic synergy”can promote immune activity and inhibit tumor,and the mechanism of Jianpi Huayu method in the treatment of GC.3.This study revealed that the scientific connotation of GQBZP in treating GC by Jianpi Huayu,providing more specific chemical and biological information support for the effectiveness of Guiqi Baizhu Prescription,providing basis for the development and transformation of GQBZP,and at the same time,it can provide methodological reference for the modernization of TCM compound material research.