The Clinical Efficacy Of YQCL On Hcc After Interventional Operation And The Mechanism Of Inducing Tumor Cell Apoptosisand

ObjectiveTo study the clinical efficacy and prognosis of YQCL in the treatment of patients with hepatocellular carcinoma after interventional surgery,and to further explore the molecular mechanism of YQCL in inhibiting the progression of liver cancer through animal experiments,so as to provide real world research basis and experimental basis for the clinical application of YQCL.MethodThe first part:Literature research.The progress of Traditional Chinese Medicine（TCM）in the treatment of hepatocellular carcinoma is summarized,and the origin and modern research on the prescription of YQCL are discussed.The second part:1.Retrospective analysis was made on the clinical efficacy and the correlation with the endpoint event of the YQCL in the treatment of HCC patients who accepted interventional therapy.Patients with HCC diagnosed by in-patient diagnosis and out-patient diagnosis of chronic diseases in the Renmin Hospital of Wuhan University and the East Branch of Renmin Hospital of Wuhan University from Jan.2018 to Jun.2019 were collected,and those who met the inclusion criteria and exclusion criteria were selected.Record the baseline data of the patients,including gender,age,stage,whether there is hepatitis,liver cancer stage,Child-pugh classification,surgical intervention,presence or absence of other diseases,and time of use of YQCL,etc..The medical records were collected to record the TCM symptoms and KPS three months before and after medication,as well as the results of tumor markers,serum enzyme indexes and liver imaging results.OS was taken as the outcome of the study,and the way of death medical records or total survival time of telephone follow-up were consulted.The patients were divided into observation group（YQCL was used for three months or more as the observation group,combined with symptomatic treatment）and control group（symptomatic treatment）.2.Compare two groups of patients with baseline data,the progress of the tumor before and after treatment for 3 months,Chinese medicine symptom integral,KPS,the levels of AFP and PIVKA,serum enzyme levels（ALT,AST,γ-GGT ALP,CK and LDH）,such as record progression-free survival（PFS）and OS.3.Epidata3.1 software was used for double data entry and consistency test.SPSS23.0 software was used for statistical analysis of the data.Measurement data in line with normal distribution were expressed as x±s,measurement data in non-normal distribution as M(Q₂₅-Q₇₅),and counting data as percentage.The independent or paired sample T test or Mann-Whitney U test was used for comparison between metrology data sets,and the chi-square was used for analysis for comparison of counting data.The incidence of different outcomes in each group was calculated,the cumulative incidence of endpoint events in the two groups was calculated by Kaplan-Meier method,and the differences among each group were compared by log-rank test.Cox proportional Hazard model was used to analyze the association between each related factor and the occurrence of liver cancer related adverse events.P<0.05 was considered statistically significant.The third part:The possible molecular mechanism of inhibiting the progression of hepatocellular carcinoma in H₂₂tumor-bearing rats by YQCL.1.Sixty SPF male&female BALB/C rats were randomly divided into blank group,model group,low-dose group（YQCLL group）,medium-dose group（YQCLM group）,high-dose group（YQCLH group）,and Huaier granule group（HE group）,with 10 rats in each group.In addition to the blank group,the remaining rats constructed the H₂₂liver cancer mouse model.Blank group,model group rats daily physiological saline lavage,YQCLL group was given YQCL low dose lavage（5 g/kg/d）,YQCLM group was given YQCLmedium dose（10 g/kg/d）,YQCLH group was given YQCL high dose（20 g/kg/d）to fill the stomach,HE group to huaier particle（1.6 g/kg/d）to fill the stomach,measure and record each weight,abdominal diameter,the change of food intake in rats.All rats were fed for10 days.2.10 days later,rats were sacrificed and orbital vein blood was collected.Blood analysis（WBC,Hb,PLT）,liver and kidney function（ALT,AST,BUN,Cr）were detected in each group.Serum interleukin-6（IL-6）levels were detected by ELISA.The spleen,thymus and liver were taken and the thymus index,spleen index and liver index of each group were calculated.3.The ascites of rats in each tumor-bearing group was taken,and the apoptosis of H₂₂cells in the ascites of rats in each group was observed by Hochest staining,and the apoptosis rate of H₂₂cells was detected by flow cytometry.4.Westernblot was used to detect the expression of JAK2/STAT3 pathway related proteins（JAK2,P-JAK2,STAT3,p-STAT3）in H₂₂cells of rats in each group.The expression of apoptosis-related factors（BAX,BCL-2,Caspase-3）was detected,and the relative optical density was calculated.5.All data were analyzed by SPSS23.0 software and expressed as mean±standard deviation（x±s）.For data comparison between multiple groups,antecedent homogeneity test of variance was used;one-way ANOVA was used for homogeneity of variance;rank sum test was used for heterogeneity of variance.T-test or multiple-comparison T-test was used for intra-group comparisons.P<0.05 was considered statistically significant.ResultsThe second part of the clinical study results:1.112 cases were evaluated in the enrolled group,including 58 cases in the observation group and 54 cases in the control group.Three months after treatment,all indicators of the two groups were improved,and the difference was statistically significant compared with that before treatment（P<0.05）.Three months after treatment,the observation group had higher stabilization rate of liver cancer solid tumor than the control group,and could significantly improve symptoms such as stress pain,abdominal distension,lack of sodium and food,limb languor and fatigue,etc.KPS was significantly increased,and the single score and total score of TCM symptoms were significantly decreased,and the difference was statistically significant compared with the control group.AFP and PIVKA were significantly reduced in the observation group,and the difference was statistically significant compared with the control group.In the comparison of serum enzymes,the levels of ALT,AST and LDH in the observation group were significantly reduced after treatment,and the difference was statistically significant compared with the control group.2.In terms of survival time comparison,the results of PFS were similar between the two groups,with no statistically significant difference between the observation group（4.72±0.204）months and the control group（4.47±0.193）months.The OS in the observation group was longer than the control group,（13.97±0.638）months vs.（12.17±0.561）months,and the difference was statistically significant.3.COX regression analysis results:Univariate regression analysis showed that the survival time was related to the use of YQCL,gender,tumor size,tumor number,Child-pugh grading,hepatitis and liver cancer stage,and the difference was statistically significant;The survival time was not related to the age of the patients,the combination of diseases and the choice of intervention methods.The same trend was shown in the multi-factor regression analysis.The use of YQCL,liver cancer stage,tumor size and tumor number were all key factors affecting the OS of HCC patients.Subgroup stratification analysis results showed that in the age group of 55～64 years old,for liver cancer stage I,tumors greater than 5cm,multiple tumors,etc.,the risk of death of patients can be significantly reduced by YQCL.In the age group of18～54,the survival benefit was related to hepatitis and stage.In the age group over 65 years old,the survival benefit was related to gender,liver cancer stage and tumor size.The third part of the experiment study results:1.Comparison of body weight,abdominal diameter and food intake of rats in each group showed statistically significant differences between the tumor-bearing group and the blank group.The weight and food intake of rats in the YQCLH and HE groups increased,and the abdominal diameter decreased,with statistically significant differences compared with the model group.There was no statistically significant difference between YQCLH group and HE group.2.In terms of the comparison of thymus index,spleen index and liver index,the difference between each tumor-bearing group and the blank group was statistically significant.The thymus index and spleen index were higher in YQCLH group and HE group,while the liver index was lower,and the difference was statistically significant compared with the model group.There was no statistically significant difference between YQCLH group and HE group.3.In the comparison of blood analysis,WBC and Hb values of rats in each tumor-bearing group decreased,and the difference was statistically significant compared with the blank group.There was no statistically significant difference between the tumor-bearing groups.There was no significant difference in PLT between the tumor-bearing group and the blank group.The WBC values of YQCLH group and HE group were slightly higher,and the difference was statistically significant compared with the model group.There was no statistically significant difference between YQCLH group and HE group.In the comparison of liver and kidney functions,the difference between the tumor-bearing group and the blank group was statistically significant.ALT,AST,BUN and Cr in YQCLH group and HE group were lower than those in the model group,and the differences were statistically significant.There was no statistically significant difference between YQCLH group and HE group.4.In the comparison of serum IL-6,the difference between each tumor-bearing group and the blank group was statistically significant.The il-6 values of YQCLH group and HE group were lower than those of the model group,and the difference was statistically significant.There was no statistically significant difference between YQCLH group and HE group.5.The Hochest results showed that the number of blue fluorescent cells in the model group,YQCLL group,YQCLM group,YQCLH group and HE group were not consistent.In the model group,a small number of blue fluorescent cells were observed.More blue fluorescent cells were observed in YQCLL group,YQCLM group,YQCLH groupand HE group,among which,a large number of blue fluorescent cells were observed in YQCLH group and HE group,and typical apoptotic bodies were also observed,with chromatin concentration and nuclear fragmentation into round bodies of different sizes.H₂₂cells gradually appeared apoptosis when the dose of gas supplementing and tumor removing prescription increased,and the higher the drug concentration was,the more obvious the apoptotic characteristics were,and the more rare the cells with normal morphology were.The results were better than that of HE group.The apoptosis rate of H₂₂cells was detected by flow cytometry.The apoptosis rate of H₂₂cells in YQCL groups and HE group was significantly increased compared with model group.The apoptosis rate of YQCLL group,YQCLM group and YQCLH group increased gradually according to the increasing drug concentration,and the difference was statistically significant compared with the model group.The apoptosis rate of YQCLH group was the highest,and the difference was statistically significant compared with that of HE group.6.Intervention effect of YQCL party in JAK2/STAT3 signaling pathway:Westernblot results showed that the expression levels of JAK2,p-Jak2,STAT3and p-Stat3 proteins in YQCL groups and HE group were decreased.Among them,histone expression in YQCLM group,YQCLH group and HE group decreased significantly,and the difference was statistically significant compared with the model group.The expression levels of JAK2 and p-STAT3 in the YQCLH group decreased,and the difference was statistically significant compared with that in the HE group.7.Westernblot results showed that the expressions of BAX and Caspase-3in the model group were decreased,while those in the YQCLL group,YQCLM group,YQCLH group and HE group were significantly increased.The differences between the YQCLM group,YQCLH group and the model group were statistically significant.There was no significant difference between YQCLH group and HE group.The expression of BCL-2 increased in the model group,but decreased to different degrees in the YQCL groups and HE group.The difference was statistically significant when compared with the model group.The difference between YQCLH group and HE group was statistically significant.ConclusionYQCL can significantly improve the clinical symptoms of HCC patients after interventional therapy,improve the quality of life of the patients,delay the progression of liver cancer,reduce the risk of death,and prolong the overall survival.The antitumor mechanism of YQCL may be through induced by inhibiting the serum IL-6 level,inhibiting the activation of the JKA2/STAT3signaling pathway,regulating the expression of the apoptotic factors BAX,Bcl-2 and Caspase-3,and thus inducing the apoptosis of tumor cells.