| Objective: 1、By means of systematic evaluation and Meta analysis,we were scientifically evaluated the methodological quality and relevant data of the clinical randomized controlled trial of “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment ” in the treatment of hepatocellular carcinoma.2、The pharmacological prediction and the analysis of its mechanism for treating the liver cancer of Diwu Yanggan capsule,which shape for the rule of “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”,based on network pharmacology that provide a basis for subsequent cell experiments.3、To study the effect and mechanism of Diwu Yanggan capsule on liver cancer cells in co-culture condition through bone marrow stem cells and its microenvironment.To explore the therapeutic mechanism of “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”(Diwu Yanggan capsule)to regulate the imbalance of “ marrow stem cells differentiate into liver cells”/ “ marrow stem cells fail to differentiate into liver cells””to prevent the occurrence and development of HCC.Methods: 1、By searching the China journal full-text database(CNKI),Wanfang database,VIP Database for Chinese Technical Periodicals,China Biology Medicine disc(CMB),Chinese clinical trial registry database(http://www.chictr.org/cn/)and MEDLINE,EMBASE,the Cochrane Central Register of Controlled Trials(Central),the Cochrane Hepato-Biliary Group Controlled Trials Register to collect the paper of Clinical randomized controlled trial of traditional Chinese medicine,which represent the rule of “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”,in the treatment of primary liver cancer.On this basis,the quality of these clinical literatures was evaluated and the data were extracted strictly according to the method of evidence-based medicine.The clinical efficacy and related laboratory indexes were analyzed by using software Rev Man 5.3 to heterogeneity test,Meta analysis and sensitivity analysis.The adverse reactions were analyzed in a descriptive way,and a funnel graph was used to analyze the bias of publication.All of this is to provide evidence-based medical evidence for the treatment of liver cancer by “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”.2、The mechanism of Diwu Yanggan capsule in treating cancer was predicted and analyzed by the method of network pharmacology.The experimental steps are as follows.Firstly,five kinds of traditional Chinese medicine components of Diwuyanggan capsule: Radix Rehmanniae Praeparata,Capillary artemisia,Schisandra chinensis,liquorice,Curcumin as the central words,were queried in TCMSP database,and their corresponding target proteins were screened according to OB ≥ 30% or DL ≥ 0.18.Secondly,three databases,Onco DB.HCC,Liverome and TCMSP,were used to search the genes associated with liver cancer.Thirdly,the Uniprot database was used to search the ID number of liver cancer related genes to see if they were in accordance with human genes and to sort them out at the same time.Finally,the final screening compounds were mapped with human hepatoma related genes,and the composition target network map of Diwu Yanggan capsule was constructed by Cytoscape software.At the same time,these eligible target proteins were input into the String database to construct the protein-protein interaction network.The enrichment of KEGG Pathway was analyzed by Clue One software,and the mechanism of Diwu Yanggan capsule in treating liver cancer was obtained.3、Based on the network pharmacology and the previous research results of the research group,the cell experiment part will focus on the Wnt/β-catenin pathway.The cell experiment will be conducted in two parts.The first experiment: The IC50 of Diwuyanggan capsule on liver cancer cell line HCCL-M3 was determined by MTT method.The low drug group was designed as 20 μg/ml,the middle group was 40 μg/ml,and the high group was 80 μg/ml.Three groups of drugs were used to treat liver cancer cells respectively.24 h,48h and 72 h cells were collected respectively.The proliferation and apoptosis of HCCL-M3 cells were detected by MTT assay and flow cytometry.Detection of m RNA expression of Wnt Pathway-related markers by RT-PCR.The second experiment: Using the 10% Diwu Yanggan Drug Serum on Co-culture system for 21 days.Using CCK8 and Transwell tumor invasion to observe the effect of Drug Serum on Hepatocellular carcinoma cells in Co-culture system with 24 h.Observation of apoptosis in hepatocellular carcinoma cells by flow cytometry on day 14 and day 21.Western Blot and RT-PCR were used to detect the expression of Wnt pathway markers.Result: 1 、 Clinical trials based on Meta-analysis: a total of 18 literatures were included,with a total of 2213 patients with primary liver cancer.In clinical effective rate,according to the different treatment methods of the control group,it was divided into interventional chemotherapy group,radiotherapy group and cryoablation group.The results showed that the therapeutic effect of the experimental group was better than that of the control group,RR=1.07(95%CI:1.03 to 1.12).The results of TCM symptoms and signs,quality of life score of KPS,the AFP and AST showed that there was a significant effect between the two groups,and the curative effect of the experimental group was significantly better than that of the control group,RR=1.71(95%CI:1.51 to 1.93),RR=1.13(95%CI:1.07 to 1.20),RR=-0.32(95%CI:-0.58 to-0.05),RR=-0.51(95%CI:-0.76 to-0.26).In aspect of NK cell,being standard for 3 months,divided into 2 groups subgroup analysis concluded that under the treatment for 3 months,due to the heterogeneity among group P<0.00001,I2=98%,selected high quality literature sensitivity analysis found that the experimental group the number of NK cells significantly after treatment higher than the control group,RR=0.17(95%CI:0.03 to 0.30).The number of NK cells in the experimental group was significantly higher than that in the control group after treatment for less than 3 months,RR=1.63(95%CI:1.04 to 2.22).In terms of adverse reactions,the incidence of adverse reactions and the extent of adverse reactions in experimental group were less than those in the control group.2、The results of network pharmacological analysis showed that 56 target proteins related to human cancer were screened according to the criteria of OB ≥ 30% or DL ≥ 0.18.The composition-target network diagram was obtained by Cytoscape software.It was found that the total effective compound molecules of Diwu Yanggan capsule were 98,and the total target was 56.String database,the network of protein-protein interaction is constructed.The interaction between protein and protein of Diwu Yanggan capsule for the prevention and treatment of cancer was obtained,and 415 interactions were obtained in the figure,with an average nodal degree of 15.7.Through the enrichment analysis of KEGG Pathway,106 pathways related to the prevention and treatment of cancer by Diwu Yanggan capsule were obtained,including P53,PPAR,TNFN,WNT,VEGF,which provided the basis for the subsequent cell experiment.3、Cell experiment: The first experiment(the effect and its mechanism of Diwu Yanggan capsule on hepatoma cells)results:(1)MTT results show that the inhibition rate of hepatoma cells in the low concentration group of 24 hours was(0.137 ±0.010),that in the middle concentration group was(0.232 ±0.006),and that in the high concentration group was(0.305 ±0.003).The inhibition rate of the low,medium and high drug group was significantly higher than that of the control group.The statistical results showed that the difference was significant(P < 0.05).At the same time,the inhibition rate of drug cells increased gradually in the low,middle and high groups,and the difference was significant(P < 0.05).The inhibition rate of hepatoma cells in the low concentration drug group was(0.291 ±0.012),in the middle concentration group was(0.399 ±0.017),and in the high concentration drug group was(0.600 ±0.007),which was significantly higher than that in the control group(P < 0.05).At the same time,the inhibition rate of liver cancer cells in high concentration drug group was significantly higher than that in middle concentration drug group and low concentration drug group,and that in middle concentration drug group was significantly higher than that in low concentration drug group(P < 0.05).It has statistical significance.After 72 hours,the cell inhibition rate of the low concentration group was(0.319 ±0.028),the middle concentration group was(0.578 ±0.003),and the high concentration group was(0.713 ±0.007).The three groups were significantly higher than the control group,the difference was significant(P < 0.05).At the same time,the difference among the three groups was significant(P < 0.05).In addition,the cell inhibition rates of the three groups at 24 h,48 h and 72 h were compared between the same groups.The results showed that the cell inhibition rate of the middle and high concentration drug groups increased gradually with the passage of time,and the difference was significant(P < 0.05).The results of flow cytometry showed that the early apoptosis was(7.76 ±0.56)in the low concentration group,(7.88 ±0.81)in the middle concentration group,and(10.38 ±1.13)in the high concentration group,which was significantly higher than that in the control group(3.22 ±0.12)(P < 0.05),and the difference between the two groups was significant(P < 0.05).The results showed that the apoptotic rate increased gradually with the increase of drug concentration,and the difference was significant(P < 0.05).The late stage apoptosis was(9.65 ±0.76)in the middle concentration group and(11.85 ±0.79)in the high concentration group,which was significantly higher than that in the control group(4.62 ±0.18)(P < 0.05).At the same time,the late apoptotic rate of low,middle and high concentration drug groups were compared.The results showed that with the increase of drug concentration,the apoptotic rate increased gradually,and the difference was significant(P < 0.05).The total apoptosis was(12.15 ±0.86)in the low concentration group,(17.53 ±0.84)in the middle concentration group and(22.23 ±1.24)in the high concentration group.The total apoptosis in the three groups was significantly higher than that in the control group(7.84 ±0.22)(P < 0.05),and the total apoptosis in the low,middle and high concentration groups increased with the increase of the concentration.The apoptotic rate increased gradually with significant difference(P < 0.05).The early apoptosis in the low concentration drug group(10.69 ±0.46),the middle concentration group(14.79 ±0.82)and the high concentration group(12.99 ±0.60)were significantly higher than those in the control group(3.92 ±0.27)at 48 h.The difference among the four groups was significant(P < 0.05).The late apoptosis in the low concentration drug group was(5.11 ±0.30),the middle concentration drug group was(15.05 ±0.26),the high concentration drug group was(29.43 ±0.87),the three groups were significantly higher than the control group(4.81 ±0.12),the difference was significant(P < 0.05).The late apoptosis increased with the increase of drug concentration,and the difference between groups was significant(P < 0.05).The total apoptotic rate was(15.80 ±0.76)in the low concentration group,(29.84 ±1.04)in the middle concentration group and(42.42 ±0.91)in the high concentration group.The total apoptosis rate of the three groups was significantly higher than that of the control group(8.74 ±0.39)(P < 0.05),and the total apoptosis rate of the three groups increased with the increase of drug concentration.The apoptotic rate increased gradually with significant difference(P < 0.05).The early apoptosis was(8.20 ±0.43)in the low concentration group at 72 h,(13.04 ±0.36)in the middle concentration group and(11.11 ±0.99)in the high concentration drug group.All the three groups were significantly higher than the control group(4.66 ±0.33).The difference among the four groups was significant(P < 0.05).The late apoptosis in the low concentration group was(8.84 ±0.59),in the middle concentration group was(26.15 ±0.94),and in the high concentration group was(56.71 ±0.61),which was significantly higher than that in the control group(5.08 ±0.30),P <0.05.With the increase of drug concentration,the late apoptosis became more and more obvious,and the difference between the two groups was significant(P < 0.05).The total apoptotic rate of low concentration drug group was(17.05 ±0.92),that of middle concentration drug group was(39.19 ±1.30),and that of high concentration drug group was(67.82 ±1.44),which was higher than that of control group(9.75 ±0.42),the difference was significant(P < 0.05).At the same time,the total apoptosis rate of three groups increased with the increase of drug concentration.The difference was significant(P < 0.05).In addition,the total apoptosis at the three time points of 24 h,48 h and 72 h in the low,middle and high groups were compared respectively.It was found that with the prolongation of the time of drug action,the total apoptosis of hepatoma cells increased gradually in each group.The difference was significant(P < 0.05).The result of RT-PCR for Wnt signal Pathway:At 24 h,the expression of β-catenin m RNA was(0.48 ±0.08)in the middle concentration drug group and(0.44 ±0.06)in the high concentration drug group,which was significantly lower than that in the control group(P < 0.05),and was significantly lower in the middle and high concentration drug group than in the low concentration drug group(0.95 ±0.15)(P < 0.05).At 48 h,the low concentration drug group was(0.42 ± 0.08),the medium concentration drug group was(0.26±0.01),the high concentration drug group was(0.09±0.02),the three groups were significantly lower than the control group,and the difference was significant,P<0.05.At the same time,the medium concentration drug group was also lower than the low concentration drug group,and the difference was significant.The expression of beta-catenin m RNA in the lower,middle and high three groups of drug groups at the time of P<0.05.72 h Respectively(0.17 ± 0.05),(0.24 ± 0.10),(0.13 ± 0.05)were significantly lower than those in the control group,and statistically significant difference was found in P<0.05.In addition,the expression of β-catenin m RNA at 24 h,48 h and 72 h in the middle and high concentration group was significantly lower than that in the 24 h group(P < 0.05),and the expression of β-catenin m RNA at 48 h was significantly lower than that at 24 h(P < 0.05).The expression of β-catenin m RNA at three time points(24 h,48 h and 72 h)in the low concentration group was compared with the same group.It was found that the expression of β-catenin m RNA decreased gradually with the prolongation of time,and the difference was significant(P < 0.05).The expression of GSK3 beta m RNA in 24 h high concentration drug group was(3.34 + 0.92),significantly higher than that in the control group,with significant difference,P<0.05.At the same time,the expression of GSK3 beta m RNA in the high concentration drug group was significantly higher than that of the low concentration drug group(1.21 ± 0.48),and the medium concentration drug group was(1.37±0.46),the difference was significant,P <0.05.In the 48 h concentration drug group(3.63±1.48)and the high concentration drug group(8.69±1.63),both groups of drugs were significantly higher than the control group GSK3,and the difference was significant,P<0.05;At the same time,the high concentration drug group was significantly higher than the medium concentration group and the low concentration drug group(1.19±0.30),and the difference was significant,P<0.05.In the 72 h low-concentration drug group GSK3,the m RNA expression was(5.25±1.39),the medium concentration drug group was(8.55±3.31),and the high-concentration drug group was(9.55 ± 3.65),significantly higher than the control group,and the difference was significant,P<0.05.In addition,the difference of 48 h and 72 h GSK3 in the low-concentration drug group was significant,P<0.05.The results of Cyclin D1 m RNA showed that the expression of Cyclin D1 m RNA in the low concentration drug group of 48 h was(0.25 ±0.06),the medium concentration drug group was(0.13±0.02),the high concentration drug group was(0.09±0.02),the three group was significantly lower than the control group,and the difference between the groups was significant,P<0.05.At the same time,the high concentration drug group and the medium concentration drug group were also lower than the low concentration drug group.The difference was also lower.Significantly,the expression of GSK3 beta in P<0.05.72 h low concentration drug group(0.08±0.01),medium concentration drug group(0.10±0.04),high concentration drug group(0.06±0.01)was significantly lower than that of the control group.The difference was significant after statistical P<0.05.In addition,the expression of Cyclin D1 m RNA decreased gradually at 24 h,48 h and 72 h in low concentration group(P < 0.05),and the expression of Cyclin D1 m RNA decreased gradually at 24 h or 48 h in high concentration group(P < 0.05).The results of second experiment(the co-culture system of human bone marrow mesenchymal stem cell and hepatoma cells)showed :The CCK8 method was used to detect the proliferation of hepatoma cells in the co-culture system.The results showed that the OD value of the serum group was(0.623 ±0.014),which was significantly lower than that of the normal control group(0.840 ±0.019)and the blank serum control group(0.812 ±0.021).The difference among the three groups was significant(P < 0.05).The cell inhibition rate calculated by the formula was(0.257 ±0.034)in the drug serum group and(0.033 ±0.003)in the blank serum group.The difference between the two groups was significant(P < 0.05).Flow cytometry was used to detect the apoptosis of hepatoma cells in co-culture system.On the 14 th day,the results showed that the early apoptotic rate was(14.89 ±2.50)and the total apoptotic rate was(22.26 ±1.54)in the drug serum group,(10.42 ±0.92)and(14.50 ±1.99)in the blank serum control group,which were significantly higher than those in the normal control group(4.79 ±0.22)and(6.54 ±0.16).The difference among the three groups was significant(P < 0.05).The early apoptosis and total apoptosis were significantly higher in the serum group than in the control group(P < 0.05),and the difference between the two groups was significant(P < 0.05).On the 21 st day,the early apoptosis rate(18.07 ±0.72),the late stage apoptosis rate(7.65 ±0.61)and total apoptosis rate(25.72 ±0.95)in serum group were significantly higher than those in control group(10.34 ±0.86),late apoptosis rate(4.60 ±0.50)and total apoptosis rate(14.93 ±1.26).After statistical analysis,the difference was significant(P < 0.05).Meanwhile,the early apoptosis rate,late apoptosis rate and the total apoptosis rate were compared with the control group.It was found that the serum of Diwu Yanggan medicine group was significantly higher than that of the blank serum control group,and the difference between the two groups was significant(P<0.05).In addition,the total apoptosis rate at 21 days was significantly higher in serum group than that in 14 days,and the difference was significant(P < 0.05).Detection of apoptosis of Bone Marrow Mesenchymal Stem cells in Co-culture system by flow Cytometry.The 14 th day showed that the late apoptotic rate(4.82 ± 0.37)and the total apoptosis rate(15.30±0.83)in the serum group were significantly lower than those in the blank serum control group(8.77±0.51)and the total apoptosis rate(19.25±1.23).On the 21 st day,the results showed that the late apoptosis rate(6.37 ±0.38)and the total apoptosis rate(15.04 ±1.07)were significantly lower than those in the control group(10.64 ±0.84)and(21.45 ±1.45)(P<0.05).Transwell tumor invasion test showed that the group of Diwu Yanggan serum was(60.00 ±6.25),which was significantly lower than that of the serum control group(89.33 ±4.16)and the normal control group(96.66 ±5.03).The difference between the two groups was significant(P<0.05).The expression of β-catenin m RNA was detected by RT-PCR.On the 14 th day,the levels of β-catenin m RNA were(0.63±0.19)group of Diwu Yanggan serum and(0.67 ±0.18)in serum control group,respectively.The difference was significant(P < 0.05).On the 21 st day,the levels of(0.38 ±0.16)and(0.53 ±0.11)in serum group and control group were significantly lower than those in normal control group(P<0.05),and the difference was significant(P<0.05).The results of GSK-3β m RNA expression showed that on the 7th day,the expression was(1.90 ±0.38)and(1.61 ±0.35)in the group of Diwu Yanggan serum and the serum control group,respectively,which was significantly higher than that in the normal control group(0.92 ± 0.11),and the difference was significant(P < 0.05).On the 14 th day,the group of Diwu Yanggan serum was(3.35 ±0.66),the blank serum control group was(2.48 ±0.46),the two groups were significantly higher than the normal control group,the difference between the three groups was significant(P < 0.05),at the same time,the Diwu Yanggan serum group was also higher than the blank serum control group(P < 0.05).On the 21 st day,the Diwu Yanggan serum group was(5.45 ±0.53)and the blank serum control group was(4.85 ±0.52),which was significantly higher than that of the normal control group,and the difference between the two groups was significant(P < 0.05).The results of C-MYC m RNA expression.On the 7th day,the expression of C-MYC was(0.60 ±0.14)and(0.69 ±0.13)in the Diwu Yanggan serum group and the blank serum control group respectively.The two group were lower than the control group(0.93 ±0.18).The difference was significant(P<0.05).On the fourteenth day,the Diwu Yanggan serum group(0.45±0.12)and the blank serum control group(0.65±0.15)were lower than those of the normal control group.The difference between the three groups was significant,P<0.05.At the same time,the Diwu Yanggan serum group was also lower than the blank serum control group,P<0.05.The difference was significant,and the difference was statistically significant.On the 21 st day,the difference between the two groups was significantly lower than that in the normal control group(P<0.05),and the difference was significant(P<0.05)between the two groups(0.22±0.04)and the control group(0.37 ±0.08)(P< 0.05).The results of Cyclin D1 m RNA expression.On the 7th day,the expression of Cyclin D1 in the Diwu Yanggan serum group was(0.78 ±0.05),which was significantly lower than that in the normal control group(1.06±0.13),the difference was significant(P < 0.05).On the 14 th day,the Diwu Yanggan serum group(0.53±0.12)and the blank serum control group(0.68±0.11)were lower compared with the normal control group,and the difference was significant,P<0.05.On the 21 day,the Diwu Yanggan serum group(0.25±0.04) and the blank serum control group(0.39±0.07)were significantly lower than those in the control group,and the difference was significant,P<0.05.At the same time,the Diwu Yanggan serum group was lower than that in the blank serum control group significantly,and the difference was significant,P<0.05.Detection of β-catenin protein expression by Western Blot.On the 14 th day,the expression of beta-catenin protein in the Diwu Yanggan serum group was(0.36±0.05),lower than that of the normal control group(0.76±0.13)significantly,and the difference was statistically significant,P<0.05.At the same time,the expression of Diwu Yanggan serum group was significantly lower than that in the blank serum control group(0.57±0.05),and the difference was significant,P<0.05.On the 21 day,Diwu Yanggan serum group(0.24±0.03)and the blank serum control group(0.68±0.04)were lower than the normal control group(0.82±0.02),and the difference was statistically significant between the three groups(P<0.05).At the same time,the expression of beta-catenin protein in Diwu Yanggan serum group was also lower than that in the blank serum control group,and the difference was significant,P<0.05.Expression of GSK-3β protein: on the 14 th day,the expression of GSK-3 β protein was(0.94 ±0.22)in Diwu Yanggan serum group and(0.71 ±0.12)in the blank serum control group,respectively,which was significantly higher than that in the normal control group(0.45±0.06).The difference was significant(P<0.05).On day 21,the expression of GSK-3β protein in Diwu Yanggan serum group was(1.05±0.11)and the blank serum control group was(0.76±0.06),which was higher than the normal control group(0.39±0.07),and the difference was statistically significant,P<0.05.At the same time,the expression of GSK-3βprotein in Diwu Yanggan serum group was also higher than that in the blank serum control group,which was statistically significant.The results of C-MYC protein expression showed that: on the 14 th day,the expression was(0.34 ±0.03)in Diwu Yanggan serum group and(0.57 ±0.06)in the blank serum control group,which was lower than that in the normal control group(0.73 ±0.03),the difference was significant(P<0.05).At the same time,the expression of c-MYC protein in Diwu Yanggan serum group was lower than that in blank serum control group(P < 0.05).On the 21 st day,Diwu Yanggan serum group was(0.36 ±0.12)and(0.78 ±0.14)in the blank serum control group,which was lower than that in the normal control group(0.89 ±0.15)(P < 0.05),and the expression of Diwu Yanggan serum group was also lower than that in blank serum control group.The difference was significant(P<0.05).The results of Cyclin D1 protein expression showed that: On the 7th day,the level in Diwu Yanggan serum group(0.37 ±0.07)was lower than that in normal control group(0.61±0.10),the difference was significant(P<0.05).On the 14 th day,the Diwu Yanggan serum group(0.23±0.04)and the blank serum control group(0.37±0.10)were lower than the normal control group(0.50±0.02),and the difference was significant,P<0.05.At the same time,the expression of Cyclin D1 in Diwu Yanggan serum group was also lower than that in the blank serum control group,and the difference was statistically significant,P<0.05.On day 21,the expression of Cyclin D1 in Diwu Yanggan serum group(0.21±0.04)was lower than that of the normal control group(0.74±0.03),and the difference was significant statistically.At the same time,Diwu Yanggan serum group was also lower than the blank serum control group(0.66±0.06),and the difference was P<0.05.Conclusion:(1)The results of Meta analysis showed that the method of “tonifying the kidney to regulate liver regeneration and repair by affecting stem cells and their microenvironment”combined with western medicine was more effective than western medicine alone in treating liver cancer with significant curative effect and fewer adverse reactions.(2)Through network pharmacological analysis,it is found that the mechanism of Diwu Yanggan capsule acting on liver cancer may be closely related to Wnt signaling pathway.(3)The results of two cell experiments showed that Diwu Yanggan capsule could inhibit the proliferation of hepatoma cells,not only in hepatoma cells but also in the co-culture system and the effect of reducing its invasiveness and inducing its apoptosis is remarkable.In co-culture system,it was found that Diwu Yanggan capsule could protect bone marrow stem cells from apoptosis of hepatoma cells.That is it can tonifying kidney to form bone marrow stem cell.By Western Blot and RT-PCR,it was found that the mechanism of Diwu Yanggan capsule on hepatoma cells might be to improve the microenvironment of liver regeneration by inhibiting Wnt signaling pathway to prevent the occurrence and development of liver cancer. |
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