| Background:Primary liver cancer is a highly malignant and deadly disease that poses a significant threat to global health.The immune ecosystem of liver cancer is very complex,involving multiple pathways and mechanisms,and exhibiting continuous,dynamic,and reversible characteristics.Although immunotherapy for liver cancer has become a research hotspot,its efficacy is still limited,mainly due to the suppressive immune microenvironment of liver cancer.Activation of the TGF-β pathway induces the formation of a tumor-permissive extracellular matrix,preventing the infiltration of CD8+T cells into the tumor parenchyma and reducing the efficacy of immunotherapy.However,targeting TGF-β,programmed cell death protein 1(PD-1),and programmed cell death ligand 1(PD-L1)simultaneously can promote T cell infiltration into the tumor center and stimulate a strong anti-tumor immune response.Therefore,finding new treatment strategies and drugs,especially using traditional Chinese medicine(TCM)to treat liver cancer,is of great significance.Biejiajian pill is a commonly used Chinese patent medicine in clinical practice with certain anti-tumor effects.Exploring the regulatory effects of the modified Biejiajian pill and its separated prescriptions on the TGF-β/Smad3 pathway in the immune microenvironment of liver cancer,as well as its impact on the PD-1/PD-L1-mediated immune escape mechanism,is expected to provide new theoretical support and scientific basis for TCM in the treatment of liver cancer and open up new avenues for immunotherapy of liver cancer.This study has a positive significance for promoting the further development of the field of liver cancer treatment and improving the treatment efficacy for patients.Objective:(1)Using scientometric analysis and visualization analysis to comprehensively analyze the 10-year scientific achievements of TCM in the treatment of liver cancer from 2010 to 2020,and explore its emerging research trends,in order to provide reference and guidance for future research.(2)Through factor analysis,explore the distribution characteristics of TCM syndrome and syndrome elements in primary liver cancer,and to supplement and enrich the understanding of TCM in primary liver cancer.(3)Based on the TGF-β/Smad3 pathway,explore the regulatory effect of the modified Biejiajian pill and its separated prescriptions(Ruanjiansanjie formula and Fuzheng formula)on the PD-1/PD-L1-mediated liver cell immune escape mechanism,aiming to provide new theoretical support and scientific basis for TCM immunotherapy.Methods:(1)The first part of the study collected relevant literature on TCM treatment for liver cancer from January 1st,2010 to December 31st,2020 by searching the China National Knowledge Infrastructure database,and managed and visualized the data using Excel 2019 and Citespace software.(2)The second part of the study,a cross-sectional survey was conducted to collect the four diagnostic methods of patients with primary liver cancer and establish an information database.Factor analysis was used to explore and analyze this information to objectively evaluate the distribution of disease location and pathogenic factors in patients with primary liver cancer.(3)The third and fourth parts of the study established an in vitro culture model of H22 liver cancer cells and conducted experiments using the modified Biejiajian pill and its separated prescriptions.The viability of H22 cells was detected using the CCK8 assay,and the invasion and migration abilities of H22 cells were evaluated using the Transwell assay.The expression of epithelial-mesenchymal transition(EMT)molecular markers(E-cadherin,N-cadherin,Vimentin)in H22 cells was detected using immunofluorescence,and the expression levels of EMT molecular markers,p-Smad3,and TGF-β in H22 cells were examined using Western blot.(4)A subcutaneous transplantation tumor model of H22 liver cancer was established in animal experiments of the fifth and sixth parts of study,and the modified Biejiajian pill and its separated prescriptions were used to intervene in H22 tumor-bearing mice.The change in tumor volume was dynamically observed,and flow cytometry was used to detect the PD-1+ratio in CD8+T cells.The expression levels of EMT molecular markers,p-Smad3,TGF-β,PD-1,and PD-L1 in tumor tissue were detected using Western blot,and the expression of p-Smad3,TGFβ,PD-1,and PD-L1 was observed using immunohistochemistry.Results:(1)The first part of the study included 2,751 relevant articles,indicating that a large-scale research output has been generated in the field of TCM treatment for liver cancer between 2010 and 2020,with an overall upward trend despite fluctuations.Shanghai University of Chinese Medicine School of Basic Medicine has published the most articles,but there is less collaboration between different research teams.Researcher Wu Xiaoxiong has been the most active and promising author in this field in recent years,publishing the most articles with the strongest impact.Currently,cutting-edge research directions include TCM external application for prevention and treatment of liver cancer,TCM combined with radiofrequency ablation for the treatment of liver cancer,liver cancer syndrome differentiation and the standardization of TCM scales,exploration of the mechanism of TCM formulas or monomers in the treatment of liver cancer,and quality control evaluation of TCM formulas and monomers.Clinical research remains a hot topic for future research,and multidisciplinary cross-fusion research combined with artificial intelligence and data mining technology will become a new trend in the field of TCM treatment for liver cancer.(2)The second part of the study included 64 patients with primary liver cancer,of whom 52 were male and 12 were female,with a mean age of 58±8.596 years and a mean duration of illness of 33.37 ± 36.5 months.53 patients were infected with hepatitis B virus,3 were infected with hepatitis C virus,1 had dual infection,and 7 were not infected.Patients often presented with a mixture of deficiency and excess syndromes,with the most common syndromes being liver-kidney yin deficiency,liver depression and spleen deficiency,and qi stagnation and blood stasis.The main affected organs were the liver,the semi-exterior and semi-interior,the gallbladder,the stomach,the kidney,the heart and mind,and the spleen.The main pathological syndromes were yin deficiency,heat,fluid deficiency,dryness,blood heat,qi deficiency,qi stagnation,qi descent,dampness,summer-heat,yang deficiency,phlegm,yang excess,blood deficiency,essence depletion,blood stasis,and blood activation.(3)Results of the third part of the in vitro experiments showed that the inhibitory rates of RJSJ-H,FZ-H,and BJJW-H groups were 57.22%,61.80%,and 59.39%,respectively,as determined by the CCK8 assay after 12 hours of treatment,and these experimental conditions were used for subsequent experiments.Transwell results showed that the inhibitory effects on H22 cell migration and invasion were BJJW-H>FZ-H>RJSJ-H,and the differences were statistically significant compared to the Control group(P<0.01).Immunofluorescence and Western Blot results suggested that compared with the Control group,the BJJW-H group and its separated prescriptions group could increase the expression of the epithelial-related molecule E-cadherin protein while decreasing the expression of the stroma-related molecules Vimentin and N-cadherin protein.(4)In the fourth part of the study,it was found that the B JJW-H group and its separated prescriptions group downregulated the protein expression of p-Smad3 and TGF-β(P<0.05,P<0.01).Further rescue Western blot experiments showed that BJJW-H could increase the expression of E-cadherin and decrease the expression of Vimentin,with statistical differences compared to the Control group(P<0.05).There was a trend of decreased expression of Ncadherin,but there was no statistical difference.When BJJW-H was added with TGF-β1,the key proteins of EMT showed the opposite trend.(5)The results of the in vivo experiment in the fifth part of the study showed that the average tumor volume and weight of the TGF-β inhibitor group(LY3200882),BJJW-H group,FZ-H group,and RJSJ-H group on the 21st day were significantly smaller than those of the Model group(P<0.05,P<0.01),but there was no significant change in the average tumor volume and weight of the BJJW-H+TGF-β1 group.The tumor inhibition rates of LY3200882,BJJW-H,RJSJ-H,and FZ-H were 72.56%,64.08%,51.44%,and 33.28%,respectively,while the tumor inhibition rate of the BJJW-H+TGF-β1 group was only 15.12%.Western blot results showed that compared with the Model group,LY3200882,BJJW-H,RJSJ-H,FZ-H,and BJJWH+TGF-β1 activator group increased the expression of E-cadherin,while reducing the expression of N-cadherin and Vimentin,with significant differences(P<0.01).(6)The flow cytometry results of the sixth part of the study showed that the expression rate of CD8+T PD-1+was highest in the Model group(59.73 ± 0.07%).Compared with the Model group,the expression rates of PD-1 in CD8+T cells were lower in the LY3200882,BJJWH,RJSJ-H,FZ-H,and BJJW-H+TGF-β1 activation groups,with statistical differences(P<0.01).The CD8+T cell PD-1 expression rate was reduced the most in the LY3200882 group(8.35 ± 1.17%),followed by the BJJW-H group(10.70 ± 0.36%);When BJJW-H was combined with TGF-β1 activator,the CD8+T cell PD-1 expression rate increased(47.19 ± 0.46%),with statistical differences compared with BJJW-H(P<0.01).Western blot and immunohistochemical results showed that compared with the Model group,the protein expression levels of PD-1 and PD-L1 were down-regulated in all groups,and the protein expression levels of p-Smad3 and TGF-β were inhibited.The LY3200882 inhibitor group showed the most down-regulation of PD-1,PD-L1,p-Smad3,and TGF-β proteins,followed by the BJJW-H group.When BJJW-H was combined with TGF-β1 activator,the expression of PD1,PD-L1,p-Smad3,and TGF-β proteins increased with statistical differences compared with BJJW-H(P<0.05).Conclusion:(1)The field of TCM in the treatment of liver cancer is showing an increasing trend.The interdisciplinary fusion of research combining artificial intelligence and data mining technology will become a new trend in the field of TCM in the treatment of liver cancer.(2)64 patients with primary liver cancer present with a mixture of deficiency and excess syndromes.The location-based patterns of the syndromes involve the liver,semi-exterior and semi-interior,gallbladder,stomach,kidneys,mind,and spleen.The major pathological patterns include yin deficiency,heat,fluid deficiency,dryness,blood heat,qi deficiency,qi stagnation,qi descent,dampness,summer-heat,yang deficiency,phlegm,yang excess,blood deficiency,essence depletion,blood stasis,and blood activation.(3)The modified Biejiajian pill and its separated prescriptions(Ruanjiansanjie formula and Fuzheng formula)have differential therapeutic effects on liver cancer,with the modified Biejiajian pill showing the best efficacy.(4)The modified Biejiajian pill and its separated prescriptions may exert anti-tumor effects in the prevention and treatment of liver cancer by suppressing the EMT process via downregulating the TGF-β/Smad3 signaling pathway,decreasing the protein expression of PD1 and PD-L1,reducing the PD-1 expression rate of CD8+T cells,improving the exhausted state of CD8+T cells,reshaping the liver cancer immune microenvironment. |
PDF Full Text Request