| AObjective:1.Randomized controlled trial(RCT)was used to observe the curative efficacy on“tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment ”treat the third stage prevention program for liver cancer and to provide evidence-based medical evidence for clinical application.2.This study was based on 16 Sr DNA technology to study the effect of the third stage prevention program for hepatocellular carcinoma(HCC)on intestinal flora diversity in patients with advanced HCC,and to explore the therapeutic mechanism of the third stage prevention program for HCC based on“tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment ”by regulating intestinal microecology to improve the microenvironment of liver regeneration.Methods:1.A total of 111 patients diagnosed as advanced hepatocellular carcinoma from May 2017 to January 2019 in the outpatient department of liver disease and ward of Hubei Traditional Chinese Medicine Hospital were randomized divided into Western medicine control group and “tonifying thekidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” combined treat group and “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment”single treat group.After3 months and 6 months of treatment,the patients survival rates of 3 months and 6 months、solid tumor efficacy、TCM symptom score and quality of life score of the three groups were compared,and clinical biochemical indexes(ALT、AST、TBil、ALB、WBC、PLT、NLR、PT、 PTA)were observed.2.The fecal specimens of 40 patients(including 10 normal subjects)were further collected.Illumina Mi Seq was used as the sequencing platform.The number of OTUs,alpha and beta diversity,richness index(CHAO,ACE)and diversity index(Simpson,Shannon)of intestinal flora in 16 Sr DNA V3-V4 region were analyzed by using software such as Mothur,Graphpad,R language and Metastats.Comprehensive analysis and evaluation were made on the differences among different phylum,genus and intestinal flora groups.Results:1.Survival rate comparison of the three groups: At the 3 months post-treatment time point,the patient survival rates of 3 months in the“TTK”combined treat group were 85.71%;the patient survival rates of 3months in the“TTK”single treat group were 68.57%;the patient survival rates of 3 months in the “WMC” group were 47.05%;and the differences between groups were statistically significant(P<0.05).At the 6 months post-treatment time point,the patient survival rates of 6 months in the “TTK”combined treat group were 68.57%;the patient survival rates of 6 months in the“TTK”single treat group were 54.28%;the patient survival rates of 6 months in the“WMC”group were 32.35%;and the differences between groups were statistically significant(P<0.05).2.Comparison of curative effect of solid tumors in three groups: After 12 weeks of treatment,the short-term curative effect was evaluated by RECIST1.1 criterion,and no patient got CR;the proportion of patients with SD was17.65%(6/34),34.29%(12/35),28.57%(10/35);the proportion of patients with PR was 8.82%(3/34),14.28%(5/35),11.43%(4/35);the proportion of patients with PD was 73.53%(25/34),51.43%(18/35),60%(21/35);the curative effect of solid tumors in the “WMC” group,the“TTK”combined treat group and the“TTK”single treat group were 26.47%,48.57% and 40%.The curative effect of SD and solid tumors(including the“TTK”combined treat group and the“TTK”single treat group)was significantly different from that in the control group(P<0.05).3.Comparison of TCM symptom scores before and after treatment in the three groups:After 12 weeks of treatment,TCM symptom scores in the three groups decreased significantly,especially in the “TTK”combined treat group which was significantly different from that in the“TTK”single treat group and the“WMC” group(22.23±3.58vs25.3±4.34vs27.13±5.80)(P<0.05).The third stage prevention program for liver cancer can significantly improve the clinical symptoms of patients.Its main manifestations are improving abdominal distension,chest pain,fatigue,weakness of body,etc.4.Comparison of clinical biochemical indicators in three groups: A fter 12 weeks of treatment,the ALB level in the“TTK” combined treat group was significantly higher than that in the “TTK”single treatment group and the “WMC”group(38.86g/L ± 6.29g/LVS37.31g/L ± 6.01g/L VS33.86g/L ± 6.73g/L)(P<0.05);PLT level was significantly higher t han that in the“TTK” single treat group and the “WMC”group(191.29± 57.60VS141.88 ± 58.20VS151.86±98.30(P<0.05);TBil in the “TT K” combined treat group was significantly lower than that in the“TTK”single treat group and the“WMC”group(P<0.05);ALT was significa ntly lower than that in the “TTK” single treat group and the“WMC”group(P<0.05).There were significant differences(32.07±20.68VS41.00±25.8VS64.44±56.33)(P<0.05);AST decreased significantly,compared with the “TTK” single treat group and the“WMC”group(58.9±49.42VS85.75±99.79VS115.56 ±145.95)(P<0.05);NLR decreased significantly,com pared with the “TTK”single treat group and the“WMC”group(3.45 ±1.94 VS5.24±5.93VS4.02 ± 4.38)(P<0.05);5.Survival scale scores before and after treatment in the three groups:The scores of survival scale before and after treatment in the three groups were compared: there was no significant difference among the three groups in body pain(BP),mental health(MH),energy(VT),emotional function(RE),but there was significant difference among the three groups in body health(HT),physiological function(RP),social function(SF),physiological function(PF),general health status(GH).There was a significant difference between the groups(P < 0.05)indicated that the“TTK”combined treat group and the“TTK”single treat group could effectively improve the quality of life,and the“TTK”combined treat group was better than“TTK”single treat group.6.Safety testing: After treatment,the serum safety of the three groups was tested to be non-toxic and side effects.No adverse reactions and side effects were found during the treatment of the three groups.7.OTU: A total of 1363877 valid sequences were obtained from 40 stool samples.The effective length of the sequences was 420-460 bp,and the coverage of sequencing was good.At 97% similarity level,1299 types of OTUs were obtained respectively.Specaccum species accumulation curve and abundance grade curve of intestinal flora in all groups constructed by the experiment tended to be flat with the increase of sequencing depth,and the amount of sequencing data was sufficient.8.Using OTU,Ace,Chao,Shannon and Simpson index to analyze the alpha diversity of intestinal flora,we found that compared with the normalgroup,the intestinal flora richness index(Chao and ACE)and diversity index(Simpson and Shannon)of hepatocellular carcinoma patients decreased significantly(P<0.05);compared with the “WMG” group,the intestinal flora richness index(Chao and ACE)of “TTK” combined treat group and “TTK”single treat group increased significantly(P<0.05).The richness index(Chao and ACE)and diversity index(Simpson and Shannon)of intestinal flora increased significantly(P<0.05).9.After sequencing,the intestinal flora of each group showed differences in Firmicutes,Thermi,Synergistetes,Planctomycetes,TM7,Tenericutes,Gemmationadetes and Bacteroidetes(P<0.01);Metastats analysis was used to compare the differences of intestinal flora among groups.There were differences in phylum,class,order,family and genus between the two groups before and after treatment.In this study,the number of beneficial bacteria in intestinal flora of patients with hepatocellular carcinoma was significantly reduced,such as: Lachnospriaceae,Ruminococcaceae,Blautia,Clostridiales,etc.The increasing abundance of bacteria was mostly common opportunistic pathogenic bacteria in clinic,such as Streptococcus,Enterobacteria and Klebsiella,Megasphaera,Veillonella,Haemophilus and so on.After treatment,the abundance of beneficial bacteria Blautia,Clostridium and Bifidobacterium in each group increased significantly(P < 0.05);while the abundance of harmful bacteria Phascolartobacterium,Klebsiella,Faecalibacterium in each group decreased significantly(P<0.05)10.Principal component analysis(PCA)was used to investigate the beta diversity of intestinal flora in patients.Compared with the normal group,the bacterial flora of the “WMC” was obviously separated,indicating that there was significant difference in the structure of bacterial flora between the two groups,suggesting that the improvement of intestinal microecology was not obvious in the “WMC” group;“TTK”combined treat group and“TTK” singletreat group both tended to be normal group,and “TTK” combined treat group was closer to normal group,indicating that the flora structure of“TTK”combined treat group was similar to that of normal group,and there was a certain relationship between them,which indicated that “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment”treat the third stage prevention program for liver cancer could significantly improve the intestinal flora of patients.11.Based on species abundance matrix and sample grouping data,a partial least squares discriminant analysis(PLSDA)model was constructed to analyze the relationship between intestinal flora differences among groups.The results showed that there was no intersection between the normal group and the“WMC”group,indicating that there was no correlation between the community structure diversity of the normal group and the“WMC”group,suggesting that the improvement of intestinal microecology was not obvious in the “WMC”group;there was intersection between the “TTK”combined treat group and “TTK” single treat group and the normal group,indicating that there was a certain correlation among the three groups,which indicated that“tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment”treat the third stage prevention program for liver cancer could significantly improve the intestinal flora of patients.At the same time,“TTK”combined treat group was closest to the normal group,indicating that there was a strong correlation between the diversity of community structure between the two groups.Conclusion: “Tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” treat for the third stage prevention program for liver cancer can significantly increasing the patients survival rates of 3 months and 6 months and improving liver function,one of the mechanism may be its play a role in regulating liver cancer cells and theirmicroenvironment.Diversity of OUT,alpha and beta intestinal flora,richness index(CHAO,ACE)and diversity index(Simpson,Shannon)as well as differences among different flora,genus and intestinal flora groups were analyzed by 16 sr DNA sequencing in patients with hepatocellular carcinoma.The results showed that “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” treat the third stage prevention program for liver cancer could inhibit the occurrence and development of hepatocellular carcinoma by increasing the beneficial bacteria of intestinal flora in patients and decreasing the harmful bacteriaof intestinal flora in patients,thereby regulating the intestinal microecology and improving the microenvironment of liver regeneration. |
PDF Full Text Request