Research On The Mechanism Of Qiyu Sanlong Decoction On NSCLC Via PD-1/PD-L1 And PTEN/PI3K/Akt Pathway

1 Objective Under the guidance of theory in Traditional Chinese Medicine(TCM)“dialectical unity between Whole and Part”,we explored the TCM etiology and pathogenesis of Lung cancer,in order to reveal its essence and treatment.From PD-1/PD-L1 and PTEN/PI3K/Akt signaling pathway,we established model of subcutaneous allografts in C57BL/6 mice using LLC.Through testing related molecular expression changes in pathway after drug intervention,we observed the effect of Qiyu Sanlong Decoction(QYSL),to explore its molecular mechanism of the treatment in lung cancer,which will provide scientific basis for TCM in adjuvant therapy for lung cancer.2 Methods2.1 Theoretical Research Collecting and filtrating literature about TCM etiology and pathogenesis of Lung cancer,to concluse its development and reveal the essence and treatment.At the same time,through the literature research,we expounded the structure of PD-1/PD-L1,PTEN/PI3K/Akt and its correlation with lung cancer,to provide theoretical basis for further experimental study on TCM in adjuvant therapy for lung cancer.2.2 Experimental Research2.2.1 Establish Model We established model of subcutaneous allografts in C57BL/6 mice using LLC.Regularly cultured cells,selected the logarithmic phase cells,and digestted them with pancreatic enzyme,centrifugated them in 1800rpm/min×5min,then diluted and adjusted its cell concentration in 1×107/m L with DMEM medium free serum.0.2 m L cell suspension was injected in left axillary region of C57BL/6 mice,to construct subcutaneous transplantation tumor model.2.2.2 Animal Group and Dosing Method Testing into tumorigenesis in mice after 10 d of cells-inoculation,according to the criteria which touching 3-5mm3 hard objects in subcutaneous.The tumor-burdened mice were randomly divided into model group(M),chemotherapy group(C),high dosage,middle dosage,low dosage group of QYSL(QH,QM,QL)and comibination group(CQH),each group contained8 mice.On the 11 th day,the QH,QM and QL group were respectively given intragastric administration of 80.48,40.24,20.12g/kg·d,the C group were intraperitoneal injected with 0.4ml cisplatin,the CQH group were administrated with high dosage of QYSL and cisplatin,the M group were administrated with the same amount of normal saline,once a day for ten days.2.2.3 Detection Index2.2.3.1 Whether has effects of Fu-zheng of QYSL The survival state of mice that contained mental state,activity condition,diet state,emaciation degree,hair changes was observed after the delivery everday.The pathological morphological changes of lung tissues by HE.The PD-1,PD-L1 m RNA and protein expressions in spleen tissues and tumor tissues were detected by RT-q PCR assay and Western blot assay respectively.ELISA method measured the expression of Th1,Th2 in the spleen of mice.2.2.3.2 Whether has effects of Xiao-ji of QYSL Tumor volume was detected and then drawn the tumor growth curve.Tumours were weighed and then calculated tumor inhibition rates.The ultrastructural of tumor cells were observed under the transmission electron microscope.RT-q PCR and Western blot respectively detected the related molecular in gene transcription level and protein expression of the PTEN/PI3K/Akt signaling pathway.3 Results3.1 Theoretical Research The TCM etiology and pathogenesis of lung cancer was the deficiency of vital qi,phlegm,blood stasis,and cancerous toxin accumulating,then formed tangible lung tumor for a long time.Activation of PD-1/PD-L1 pathway in lung cancer could cause turmor cells to produce the immune escape.Modern medical research concluded that vital qi is closely related to immune.PI3K/Akt/m TOR was one of the most common maladjusted pathway in lung cancer.mi RNA21,as one of the oncogene,which target gene PTEN could negatively regulate it.QYSL had the effects of supplementing qi and nourishing yin,detoxificating toxin and resisting cancer,so it could mediate the above two signal pathway to adjuvantly treat lung cancer using its effects of Fu-zheng and Xiao-ji.3.2 Experimental Research3.2.1 QYSL have effect of Fu-zheng3.2.2.1 Effects of QYSL on the survival condition and lung pathological morphological changes in mice Survival condition : The M group had general mental state,activity condition and diet state.The C group had the worst survival condition and emaciation state.Good survival condition is belong to every dose of QYSL.The CQH group was worse than QYSL,but better than the C group obviously.Lung pathological morphological changes:Tumor metastases could beseen in the lung of M,C,and QL group.While there were no tumor metastases in QH,QM and CQH group.3.2.1.2 Effects of QYSL on expression of PD-1 and PD-L1 m RNA Compared with M group,QH group could reduce PD-1,PD-L1 m RNA(P<0.01).There was no statistical significance in reducing PD-L1 m RNA of C group(P>0.05).CQH group had no statistical significance of the PD-1m RNA and PD-L1 m RNA expression(P>0.05).3.2.1.3 Effects of QYSL on expression of PD-1 and PD-L1 protein Compared with M group,both QYSL and cisplatin could decrease PD-1and PD-L1 protein expression levels(P<0.01).Compared with C group,CQH group had no interaction effect on the expression of PD-1 protein(P>0.05),but had interaction effect on PD-L1 protein(P<0.05).3.2.1.4 Effects of QYSL on content of Th1 and Th2 cytokine in spleen Th1 : Compared with M group,IL-2 and IFN-γ content levels were significantly increased of QH group(P<0.01).Compared with C group,IL-2content level was obviously increased of every dose of QYSL group,but CQH group was not obvious(P=0.56).IFN-γ content level were increased both every QYSL group and CQH group(P<0.01).Th2:Compared with M group,QH group could obviously decrease the IL-4 and IL-10 levels(P<0.01).CQH group slightly reduced IL-4 level(P<0.05).Compared with C group,both every QYSL group and CQH group could decrease content of IL-4 and IL-10.3.2.2 QYSL have effect of Xiao-ji3.2.2.1 Effects of QYSL on macrostructure and ultrastructure of tumor Macrostructure of tumor : Cisplatin could obviously reduce the tumor volume and lower the tumor quality(P<0.01).The inhibition effects on the tumor volume and quality of QYSL were not obvious(P>0.05).The joint interaction also has no statistical significance(P>0.05).The tumor inhibitionrate between CQH group and C group was equal.Ultrastructure of tumor : M group has the typical malignant tumor characteristic.Every QYSL group showed apoptosis characteristic,especially in QH group which appear apoptotic body.Typical crescent apoptosis could be seen in C group.The nuclear chromatin gathered,organelles structure almost entirely disappeared,and there were cytoplasm edema and serious necrosis in CQH group.Apoptosis factors: Compared with M group,QH and C group could reduce Bcl-2 m RNA level(P<0.01).Compared with C group,Bcl-2 m RNA expression could be reduced in QH and CQH group(P < 0.01).But no difference between CQH and QH group(P>0.05).Compared with M group,QH and C group could significantly increase Bax m RNA level(P<0.01).Compared with C group,Bax m RNA had increasing trend but without statistical significance in CQH group(P>0.05).3.2.2.2 Effects of QYSL on mi RNA21 m RNA in tumor Compared with M group,there had decreasing trend of mi RNA21 m RNA in every QYSL group,especially statistically significant in QH group(P<0.01).C group also could reduce mi RNA21 m RNA(P<0.05).But compared with C group,there was no obvious difference in joint use(P>0.05).3.2.2.3 Effects of QYSL on PTEN m RNA and protein in tumor Compared with M group,PTEN protein and PTEN m RNA relative expression were highest in QH group(P<0.01).C group also could increase the expression of PTEN protein and PTEN m RNA(P<0.01).Compared CQH group with C group,its interaction effect was also statistically significant(P < 0.05).3.2.2.4 Effects of QYSL on related molecular of PI3K/Akt/m TOR pathway in tumor PI3 K p85α,PI3 K p110α:QH,C,CQH group could reduce PI3 K p85α,PI3K p110α protein expression(P<0.01).The interaction effect between QYSL and cisplatin is statistical significance(P<0.01).Akt、p-Akt:Compared with M group,the expression of Akt and p-Akt were lower in QH group and C group(P<0.05).The interaction effect between QYSL and cisplatin is also statistical significance(P<0.01).m TOR、p-m TOR:Compared with M group,the expression of m TOR and p-m TOR were lower in QH group and C group(P<0.05).There was no statistical significance on m TOR expression between QYSL and cisplatin(P>0.05),but was statistically significant on p-m TOR(P<0.01).4 Conclusion QYSL,Based on the “Fu-zheng and Xiao-ji”therapy created,had effects of improving survival status and immunologic function of tumor-bearing mice.Also,QYSL could inhibit growth of transplantation tumor,which the mechanism may be related to PD-1/PD-L1 and PTEN/PI3K/Akt pathway by down-regulating expression of PD-1,PD-L1,and mi RNA21,and up-regulating expression of PTEN,then negatively regulate the PI3K/Akt/m TOR signal pathway.