Study On The Mechanism Of Yangyin Fuzheng Jiedu Prescription In Treating HBV-related Liver Cancer Based On NK Cell Co-inhibitory Molecule

Objectives:First,a retrospective cohort study is used to establish a simple visualization of the Nomogram model based on the number of NK cells to predict the long-term survival of patients with hepatocellular carcinoma and to provide a simple and quick decision-making tool for the clinic.Second,we observe the effect of Yangyin Fuzheng Jiedu prescription on the phenotype and function of NK cell exhaustion in patients with HBV-HCC through a prospective cohort study.Methods:1 A retrospective cohort study included 121 patients diagnosed with HCC from January 2012 to December 2014.The Cox multivariate regression was used to screen out the independent risk factors affecting the 5-year overall survival of patients with HCC,establish a simple visual Nomogram model to predict overall survival and use the decision tree to show the 5-year overall survival rate under different risk factors.2 A prospective cohort study included 133 patients diagnosed as HBV-HCC from June 2018 to December 2019,divided into integrated Chinese and Western medicine group(Yangyin Fuzheng Jiedu prescription on the basis of western medicine treatment group for 24 weeks)and Western medicine treatment group.We isolated and extracted peripheral blood mononuclear cells(PBMC)from patients with HBV-HCC.Polychromatic flow cytometry was used to detect the expression levels of NK cell surface co-stimulatory and co-inhibitory molecules and the apoptotic function of each subset of NK cells,cytokine secretion function(IL-12/15/18 in vitro stimulation),the killing ability,transcription factor level(T-bet,Eomes),and proliferation ability.To observe the effect of Yangyin Fuzheng Jiedu prescription on the phenotype and function of NK cells in patients with HBV-HCC and on the prognosis of patients.Results:1 A retrospective cohort study found that absolute NK cell counts(≤124.5 cells/ul),tumor size(>5 cm),number of tumors(multiple),and level of AFP(>400 ng/ml)were independent risk factors affecting the 5-year overall survival of patients with HCC.A Nomogram model based on the number of NK cells was established to predict the 5-year overall survival rate of patients with HCC.This model C index is 0.73.2 The prospective cohort study found that:(1)The proportion of peripheral blood total NK cells in HBV-HCC patients was significantly reduced compared with the normal,CHB and HBV-LC patients.Further,in the proportion of each subset of NK cells,the proportion of CD56dim NK cell subsets was reduced,and the proportion of subsets of CD56-NK cells was significantly increased.(2)The expression of activated receptors NKG2D in NK cells in patients with HBV-HCC was reduced,and the expression of inhibitory receptors TIGIT,TIM-3,2B4,PD-1,BTLA and CD39 was increased.(3)NK cell exhaustion characteristics were found in both TIGIT+NK cells and TIM-3+NK cells in patients with HBV-HCC.Compared with TIGIT-NK cells,the characteristics of high expression exhaustion in TIGIT+NK cells were as follows:the proportion of activated receptor CD226 was decreased,the proportion of inhibitory receptors 2B4,PD-1,TIM-3 and LAG-3 was increased;the proportion of cytokine secretion(IFN-γ,TNF-α)was decreased;the ability of cell killing and proliferation were decreased;and the level of apoptosis was increased.Compared with TIM-3-NK cells,TIM-3+NK cells also expressed the following exhaustion characteristics:2B4,TIGIT,BTLA,CD39,and LAG-3 all showed increased molecular expression ratio;cytokine secretion function,killing ability and proliferation ability were significantly decreased.(4)The co-expression of TIGIT and TIM-3 co-mediated NK cell exhaustion in patients with HBV-HCC.Compared with TIGIT+TIM-3-NK cells,TIGIT-TIM-3+NK cells,TIGIT-TIM-3-NK cells in patients with HBV-HCC,the expression of cytokines ratio(IFN-γ,TNF-α)decreased,the ability of cell killing and proliferation were decreased;and the characteristic of exhaustion of high expression T-betloEomeshi NK cells.(5)The 48 week PFS of HBV-HCC patients in the integrated Chinese and Western medicine group after matching with the 1:1 propensity score were significantly better than those in the Western medicine treatment group(p=0.009).The PFS of patients with HBV-HCC in the integrated Chinese and Western medicine group were still significantly better than those in the Western medicine treatment group,especially in BCLC stage C-D,Child B+C,tumor multiple and AFP>400 ng/ml.(6)The proportion of total NK cells was slightly increased in HBV-HCC patients treated with Yangyin Fuzheng Jiedu prescription(p=0.0463),CD56dimNK cell subsets increased and CD56-NK cell subsets decreased.Meanwhile,Yangyin Fuzheng Jiedu prescription could reduce the expression levels of TIM-3 and TIGIT+TIM-3+in total NK cells in patients with HBV-HCC.And in the patients with high expression levels of TIM-3hi and TIGIT+TIM-3+hi,the PFS of the integrated Chinese and Western medicine group were significantly better than those of the Western medicine treatment group.Conclusion:1 A visual Nomogram was used to establish a predictive model based on the number of NK cells(tumor size,number of tumors,level of AFP and number of NK cells)to predict the 5-year overall survival rate of patients with HCC,and to provide a convenient and quick decision-making tool for the clinic.2 Yangyin Fuzheng Jiedu prescription can prolong the PFS of HBV-HCC patients,especially when the tumor is advanced,the liver function grade is worse,and the tumor load is heavier.Compared with the western medicine treatment group,the effect of 48 weeks in the integrated Chinese and western medicine group was more obvious.The expression level of NK cell exhaustion phenotype in HBV-HCC patients can be regulated by Yangyin Fuzheng Jiedu prescription.3 TIGIT,TIM-3,and TIGIT+TIM-3+ co-expression mediated NK cell exhaustion in patients with HBV-HCC.Meanwhile,Yangyin Fuzheng Jiedu prescription significantly prolonged the PFS of patients with high TIM-3hi and TIGIT+TIM-3+hi expression.