| Background and purpose: The incidence and mortality of chronic hepatitis B-related liver cancer are increasing year by year.Although there are many hypotheses about the pathogenesis of liver cancer,the exact mechanism has not yet been clarified as a key scientific problem that hinders the improvement of the level of prevention and treatment.Over the years,the strategies for the prevention and treatment of liver cancer have mainly focused on anti-virus and liver cancer cells themselves,ignoring the influence of host factors in the human body.With the introduction of the concept of liver cancer microenvironment,it has gradually attracted the attention of researchers as a key factor affecting the occurrence and development of liver cancer.The abnormal liver regeneration microenvironment formed by liver inflammation and fibrosis of chronic liver disease promotes the occurrence and development of liver cancer.Therefore,research to reduce the occurrence of liver cancer and inhibit its progression and metastasis by improving the liver regeneration microenvironment has great clinical reference value and far-reaching scientific significance.This article combines two parts of clinical experiment and in vitro experiment to explore the effect and mechanism of Diwuyanggan Capsule in reducing the risk of HBeAg-negative chronic hepatitis B patients by improving the liver regeneration microenvironment.The first part is to observe the effect of Diwuyanggan Capsule on the incidence and risk of liver cancer in patients with HBeAg-negative chronic hepatitis B.The second part reveals the related mechanism of TGF-?1 activation of hepatic stellate cells LX2 and the imbalance of EMT/MET under co-culture conditions,analyzes its effect on the proliferation,apoptosis and invasion of hepatocellular carcinoma cells HCCLM3,and discusses the passage of Diwuyanggan capsule The mechanism of improving the liver regeneration microenvironment,regulating the imbalance of EMT/MET and the disorder of TGF-?/Smad signal pathway to prevent the occurrence and development of liver cancer.Method:1.This experiment passed the previously completed randomized controlled clinical trial for HBeAg-negative chronic hepatitis B(Chinese Clinical Trial Registry Registration Number: Chi CTR-TRC-12002962),which used Diwuyanggan Capsules alone or Entecavir alone or a combination of both Patients who have been treated for 48 weeks will undergo a prospective follow-up observation for up to 108 months(entecavir will not be stopped during the follow-up period).The blood routine,liver and kidney function,coagulation function,hepatitis B two pairs of semi-quantitative,and serum HBV will be tested every 6 months.DNA levels and alpha-fetoprotein(AFP).Main efficacy evaluation index: Observe the incidence of liver cancer during the end-point event during the follow-up period.Secondary evaluation indicators: observe the biochemical and virological responses during follow-up.Among them,the cumulative incidence of liver cancer was calculated using the Kaplan-Meier method and compared with the log-rank method.2.In vitro experiment by establishing a co-culture model of human hepatic stellate cell LX-2 activated by TGF-?1 and human hepatocarcinoma cell line HCCLM3 to simulate the liver regeneration microenvironment of liver cancer.The experiment was divided into LX2 control group(LX2-NS),co-culture model group(Co-culture model),co-culture DWYG group(Co-culture DWYG),and the drug-containing serum of Diwu Yanggan Capsule was given for intervention.8.Cell biology methods such as flow cytometry and Transwell invasion experiment were used to observe the effects of activated HSCs on the proliferation,apoptosis and invasion of liver cancer cells under co-culture conditions.Co-culture was studied by Western Blotting,q RT-PCR and other molecular biological methods The mechanism of the imbalance between activated HSCs and EMT/MET under conditions,analyze the expression patterns of Smad2,Smad3,T?RI,Smad7 and other m RNA and protein in the TGF-?/Smad signaling pathway during this process,and explore how Diwu Yanggan Capsules can improve liver regeneration.The environment,regulation of EMT/MET imbalance and TGF-?/Smad signaling pathway disorder to prevent the occurrence and development of liver cancer.Result:(1)Comparison of enrollment baseline data and follow-up time: 170 patients who met the inclusion criteria were randomly divided into three groups,of which group A was the Diwu Yanggan capsule treatment group(56cases),and the Diwu Yanggan capsule was taken orally;B;Group is the Diwu Yanggan Capsule combined with Entecavir treatment group(57 cases),simultaneously using Diwu Yanggan Capsule and Entecavir;Group C is the Entecavir control group(57 cases),oral entecavir,108 M for patients whose treatment period reaches 48 weeks Follow up.At the time of enrollment,the age,gender,blood routine(including neutrophil NEU,lymphocyte LYM,neutrophil to lymphocyte ratio NLR and platelet count PLT),PT international normalized ratio INR,liver function included(ALT,AST,GGT,ALB,TP,TBIL)and serum HBV-DNA levels were not significantly different(P>0.05).The follow-up period was from January 1,2012 to January 11,2021,with a total follow-up of 108 months,with an average follow-up time of 84.03 months and a median follow-up time of 94.00 months.The average follow-up time and median follow-up time of Diwu Yanggan Capsule group were 90.75 months and 99.00 months,respectively.The average follow-up time and median follow-up time of Diwu Yanggan Capsule combined with Entecavir group were 78.82 months and 92.50 months,respectively.Months,the average follow-up time and median follow-up time of the entecavir group were 82.78 months and 91.00 months,respectively.A total of 151 patients were followed up,and the total loss to follow-up rate was 11.18%(19/170).Among them,the Diwu Yanggan Capsule group completed follow-up of 48 cases,the Diwu Yanggan Capsule combined with Entecavir group completed follow-up of 50 cases,and the Entecavir group completed follow-up of 53 cases.The loss to follow-up rate of the three groups was 14.29%(8/56)and 12.28%(7/57),7.02%(4/57),there was no significant difference in the loss to follow-up rate between the groups(P>0.05).(2)Comparison of Diwu Yanggan Capsule's biochemical response to HBeAg-negative chronic hepatitis B patients: The blood biochemical results of the three groups of patients with different intervention programs were analyzed,and it was found that there was no significant difference in the levels of ALT and AST among the three groups.(P>0.05).Other biochemical indicators such as ALP,TP,ALB,GELO,TBIL,DBIL,IBIL,etc.were not significantly different among the three groups(P>0.05).The blood biochemical indexes of the three groups of follow-up patients were compared with those at the time of entry.It was found that the liver function ALT and AST of the three groups of follow-up patients were lower than before(ALT:23.50±15.40 vs 34.70±17.51,25.96±27.45 vs 35.87±18.07,28.18± 20.30 vs40.25±21.20;AST: 22.08±5.87 vs 28.72±11.24,23.11±11.17 vs 31.91±11.61,27.57±16.85 vs 33.72±14.35),the difference is statistically significant(P<0.01),while ALP,TP,ALB There were no significant differences in biochemical indicators such as,GELO,TBIL,DBIL,IBIL and the previous entry(P>0.05).(3)Comparison of the virological response of Diwu Yanggan Capsules to HBeAg-negative chronic hepatitis B patients: The levels of HBV DNA in each group of patients with different treatment regimens were significantly lower than when they were enrolled(P<0.01).There was no significant difference in HBV DNA levels among the three groups during the follow-up(P>0.05).The levels of HBs Ag in each group were compared with those at the time of entry,and the levels of HBs Ag in the three groups were significantly lower than before(P<0.05).The follow-up results showed that the HBs Ag drop >1500IU/ml in the three groups was 34.21%(13/38),35.71%(10/28),28.57%(8/28),and there was no significant difference between the three groups.(P>0.05).(4)The effect of Diwu Yanggan Capsule on the cumulative incidence of liver cancer in patients with HBeAg-negative chronic hepatitis B: Follow-up to 108 months,the Kaplan-Meier(product limit method)method was used to draw the incidence and survival curve of liver cancer,and the Diwu Yanggan capsule group and Diwu Yanggan capsule were calculated.The cumulative incidence of liver cancer events in the Wuyanggan capsule combined with entecavir group and entecavir group were 0%(0/48),2%(1/50),11.32%(6/53),and then the logrank test was used to log Rank(Mantel-Cox)conducted analysis and test,and the results showed that the cumulative incidence of liver cancer in the three groups was different(P <0.05).Among them,the cumulative incidence of liver cancer in the Diwu Yanggan capsule group(0%)was significantly lower than that of the entecavir positive control group(11.32%)(P <0.05).The cumulative incidence of liver cancer(2%)in the Diwu Yanggan Capsule combined with Entecavir group was lower than that of the Entecavir positive control group(11.32%)(P <0.05).The incidence of liver cancer in the Diwu Yanggan capsule group(0%)was lower than the incidence of liver cancer in the Diwu Yanggan capsule combined with Entecavir(2%),but the difference was not statistically significant(P >0.05).It is suggested that compared with entecavir antiviral therapy alone,Diwuyanggan capsule alone or combined entecavir can significantly reduce the incidence of liver cancer in patients with HBeAg-negative chronic hepatitis B.(5)TGF-?1 induces the activation of LX2 and the morphological observation of LX2 in the LX2/HCCLM3 co-culture system: LX2 activated precursors are irregularly shaped as polygonal stars,with underdeveloped cell growth and tight intercellular connections;through TGF-?1(10ng/ml)after induction and activation,the cell volume increases,the cell processes are stretched,the connections between the cells are loose,and the cell proliferation speed is accelerated;after co-cultivation with human liver cancer cell HCCLM3,the pseudopodia of LX2 cells are more stretched and grow in a stretched bipolar shape.Proliferation increased significantly.(6)CCK-8 method to detect the optimal concentration of DWYG drug-containing serum: CCK-8 method was used to detect the effect of different concentrations of DWYG drug-containing serum on the proliferation of HCCLM3 in the LX2/HCCLM3 co-culture system.The experimental results showed that compared with the control group,with the increase of serum concentrations of 5%,10%,15%,20%,etc.,the A450 values measured by the cells of each group gradually decreased,and the proliferation activity of HCCLM3 cells decreased significantly.(P<0.01),the cell proliferation inhibition rate gradually increased(P<0.01),and 10% was selected as the best experimental serum drug concentration.(7)The effect of DWYG medicated serum on the proliferation of HCCLM3 in the LX2/HCCLM3 co-culture system:(1)The effect of DWYG medicated serum on the proliferation of HCCLM3 in the LX2/HCCLM3 co-culture system: Compared with the A450 value of the M3 control group(0.69±0.03),the co-culture model group(0.75±0.01)increased the proliferation of HCCLM3 cells(P<0.01).Compared with the A450 value of the M3 control group(0.69±0.03),the proliferation activity of HCCLM3 cells in the co-cultured DWYG group(0.65±0.01)decreased(P>0.05).Compared with the A450 value of the co-culture model group(0.75±0.01),the proliferation activity of HCCLM3 cells in the co-culture DWYG group(0.65±0.01)was significantly reduced(P<0.01).It can be seen that the proliferation rate of HCCLM3 in the co-culture model group(111.45%)was significantly higher than that of the M3 control group(100%),while the proliferation rate of HCCLM3 cells in the co-culture DWYG group(90.77%)was significantly lower than that of the co-culture model group(111.45%).It is suggested that the LX2/HCCLM3 co-culture system may promote the proliferation of HCCLM3 liver cancer cells,and the DWYG medicated serum may inhibit the proliferation of HCCLM3 in the LX2/HCCLM3 co-culture system.(2)LX2 proliferation in the LX2/HCCLM3 co-culture system: Compared with the LX2 control group(0.62±0.02),the co-culture model group(0.71±0.02)LX2 cell proliferation activity increased significantly(P<0.01).Compared with the LX2 control group(0.62±0.02),the co-cultured DWYG group(0.63±0.01)had no significant difference in LX2 cell viability(P>0.05).Compared with the co-culture model group(0.71±0.02),the co-culture DWYG group(0.63±0.01)decreased the proliferation ability of LX2 cells(P<0.01).Compared with the LX2 control group(100%),the co-culture model group has a higher cell proliferation rate(120.35%).Compared with the co-culture model group(120.35%),the proliferation rate(90.77%)of LX2 cells in the co-culture DWYG group decreased significantly.It is suggested that the LX2/HCCLM3 co-culture system may promote the proliferation of human hepatic stellate cells LX2,and the DWYG medicated serum can inhibit the proliferation of LX2 in the LX2/HCCLM3 co-culture system.(8)The effect of DWYG medicated serum on the apoptosis of HCCLM3 and LX2 cells in the LX2/HCCLM3 co-culture system:(1)The effect of DWYG medicated serum on the apoptosis of HCCLM3 cells in the LX2/HCCLM3 co-culture system: According to the results of flow cytometry,the co-culture model group(6.23±0.54)compared with the M3 control group(7.52±0.17).The mortality rate dropped significantly(P<0.01).Compared with the M3 control group(7.52±0.17),the co-culture DWYG group(24.02±0.25)had a significantly higher cell apoptosis rate(P<0.01).Compared with the co-culture model group(6.23±0.54),the co-culture DWYG group(24.02±0.25)significantly increased the apoptosis rate(P<0.01).It is suggested that the LX2/HCCLM3 co-culture system inhibits the apoptosis of HCCLM3,and the DWYG medicated serum can promote the apoptosis of HCCLM3 in the LX2/HCCLM3 co-culture system.Compared with the M3 negative control group(6.23±0.54),the M3 DWYG group(24.02±0.25)had a significantly higher apoptosis rate(P<0.01).It is suggested that the LX2/HCCLM3 co-culture system inhibits the apoptosis of HCCLM3,and the DWYG medicated serum can promote the apoptosis of HCCLM3 in the LX2/HCCLM3 co-culture system.(2)The effect of DWYG medicated serum on the apoptosis of LX2 cells in the LX2/HCCLM3 co-culture system: Compared with the LX2 control group(6.37±0.32),the co-culture model group(5.15±0.29)significantly decreased the apoptosis rate(P<0.01).Compared with the LX2 control group(6.37±0.32),the co-culture DWYG group(7.36±0.37)increased cell apoptosis(P<0.01).Compared with the co-culture model group(5.15±0.29),the co-culture DWYG group(7.36±0.37)significantly increased cell apoptosis(P<0.01).It shows that the LX2/HCCLM3 co-culture system can inhibit the apoptosis of LX2 cells to a certain extent,and DWYG medicated serum can attenuate the inhibitory effect and promote the apoptosis of human hepatic stellate cells LX2 in the LX2/HCCLM3 co-culture system.(9)The effect of DWYG medicated serum on the invasion ability of HCCLM3 in the LX2/HCCLM3 co-culture system: Compared with the M3 control group(104.00±4.18),the co-culture model group(148.80±4.09)has significantly enhanced HCCLM3 invasion ability,and the difference is significant.Statistically significant(P<0.01).Compared with the M3 control group(104.00±4.18),the co-culture DWYG group(78.00±6.60)had a lower invasion ability(P<0.01).Compared with the co-culture model group(148.80±4.09),the cell invasiveness of the co-culture DWYG group(78.00±6.60)was significantly decreased(P<0.01).It is suggested that the LX2/HCCLM3 co-culture system can enhance the invasion ability of HCCLM3,while the DWYG medicated serum can reduce the high HCCLM3 invasion ability induced by the co-culture system.(10)The effect of DWYG medicated serum on the expression of EMT related m RNA expression in LX2 cells of the LX2/HCCLM3 co-culture system:RT-PCR detection technology was used to detect the expression of E-cadherin and Vimentin m RNA related to EMT.The statistical results showed that compared with the LX2 control group,the expression of E-cadherin m RNA in the co-culture model group decreased(P<0.01),and the expression of Vimentin m RNA was obvious Increase(P<0.01).Compared with the LX2 control group,the expression of E-cadherin m RNA in the co-cultured DWYG group increased(P<0.01),but there was no significant difference in the expression of Vimentin m RNA(P>0.05).Compared with the co-culture model group,the expression of E-cadherin m RNA in the co-culture DWYG group was significantly increased(P<0.01),and the expression of Vimentin m RNA was significantly decreased(P<0.01).It is suggested that the LX2/HCCLM3 co-culture system can significantly promote the expression of Vimentin m RNA in LX2 cells and inhibit the expression of E-cadherin m RNA,while the DWYG medicated serum promotes the expression of E-cadherin m RNA in LX2 cells in the LX2/HCCLM3 co-culture system,and inhibits Vimentin.m RNA expression.(11)The effect of DWYG medicated serum on the expression of EMT and TGF-?/Smad signaling pathway related m RNA expression in LX2 cells of the LX2/HCCLM3 co-culture system:The expression of Smad2,Smad3,T?RI,and Smad7 m RNA related to the TGF-?/Smad signaling pathway in LX2 cells was detected experimentally.The result analysis showed that the expression of Smad2,Smad3,and T?RI m RNA in the co-culture model group increased compared with the LX2 control group.,Smad7 m RNA expression decreased(P<0.01).Compared with the LX2 control group,the expression of T?RI m RNA in the co-cultured DWYG group decreased(P<0.01),the expression of Smad7 m RNA increased(P<0.01),and the expression of Smad2 and Smad3 m RNA decreased(P>0.05).Compared with the co-culture model group,the expression of Smad2,Smad3 and T?RI m RNA in the co-culture DWYG group decreased(P<0.05),and the expression of Smad7 m RNA increased(P<0.05).It is suggested that the LX2/HCCLM3 co-culture system can significantly promote the expression of Smad2,Smad3,T?RI m RNA in LX2 cells,and reduce the expression of Smad7 m RNA,while the DWYG medicated serum can promote the expression of Smad7 m RNA in LX2 cells in the LX2/HCCLM3 co-culture system,and inhibit Smad2.,Smad3,T?RIm RNA expression.(12)The effect of DWYG medicated serum on the expression of EMT related proteins in the LX2/HCCLM3 co-culture system:Compared with the LX2 control group,the expression of E-cadherin protein in the co-culture model group decreased(P<0.01),and the expression of Vimentin protein was significantly increased(P<0.01).Compared with the LX2 control group,the expression of E-cadherin protein in the co-cultured DWYG group was enhanced(P<0.01),and there was no significant difference in the expression of Vimentin protein(P>0.05).Compared with the co-culture model group,the expression of E-cadherin protein in the co-culture DWYG group increased(P<0.01),and the expression of Vimentin protein decreased(P<0.01).It is suggested that the LX2/HCCLM3 co-culture system can significantly promote the expression of Vimentin protein and inhibit the expression of E-cadherin protein in LX2 cells,while the DWYG medicated serum promotes the expression of E-cadherin protein in the LX2/HCCLM3co-culture system and inhibits the expression of Vimentin protein.(13)The effect of DWYG medicated serum on the expression of TGF-?/Smad signaling pathway related proteins in the LX2/HCCLM3co-culture system:The expression of Smad2,Smad3,T?RI,and Smad7 proteins related to the TGF-?/Smad signaling pathway in LX2 cells was detected experimentally.The analysis of the results showed that the expression of Smad2,Smad3,and T?RI proteins in the co-culture model group was increased compared with the LX2 control group.,Smad7 protein expression decreased(P<0.01).Compared with the LX2 control group,the expression of Smad2,Smad3 and T?RI protein in the co-cultured DWYG group decreased,while the expression of Smad7 protein increased(P<0.01).Compared with the co-culture model group,the expression of Smad2,Smad3,T?RI protein in the co-culture DWYG group decreased,and the expression of Smad7 protein increased(P<0.01).It is suggested that the LX2/HCCLM3 co-culture system can significantly promote the expression of Smad2,Smad3,T?RI protein in LX2 cells,and reduce the expression of Smad7 protein,while DWYG medicated serum can promote the expression of Smad7 protein in LX2 cells in the LX2/HCCLM3 co-culture system and inhibit Smad2,Smad3,T?RI protein expression.Conclusion:(1)According to the clinical trial data of 170 cases of Diwuyanggan Capsules in the treatment of HBeAg-negative CHB RCT,follow-up to 108 months,the biochemical response and virological response of the Diwuyanggan capsule group alone,the entecavir group and the combination group The effect is equivalent.Diwuyanggan capsules alone or combined with Entecavir can significantly reduce the incidence of liver cancer.(2)The constructed activated LX2 and HCCLM3 co-culture system simulates the liver regeneration microenvironment of liver cancer,which can promote the proliferation of LX2 and HCCLM3,inhibit the apoptosis of LX2 and HCCLM3,and enhance the invasion ability of HCCLM3 cells.The mechanism may be through the activation of TGF-?/ The Smad signaling pathway promotes EMT.DWYG medicated serum can significantly inhibit the proliferation of LX2 and HCCLM3 in the co-culture system,promote the apoptosis of LX2 and HCCLM3,and reduce the invasion ability of HCCLM3 cells.The mechanism may be through inhibiting the excessive activation of TGF-?/Smad signaling pathway and regulating EMT/MET Imbalance,improve the liver regeneration micro-environment of liver cancer,and play a role in preventing the occurrence and development of liver cancer. |
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