| ObjectiveSpleen deficiency internal environment is closely related to the occurrence and progression of hepatocellular carcinoma(HCC),and spleen deficiency syndrome is one of the important syndromes in TCM diagnosis and treatment of HCC.However,the role of spleen deficiency internal environment in the pathogenesis of HCC remains unclear.Therefore,this study first summarizes the role and efficacy of spleen-strengthening therapy in HCC.Furthermore,this study elaborated the influence of spleen deficiency internal environment on cancer stem cells(CSCs)expression of HCC,and explored the effect and mechanism of Oatps and PTEN/AKT pathway expression in spleen deficiency internal environment of CSCS expression of HCC.MethodsThe first part discusses the effect,efficacy and safety evaluation of TCM spleenstrengthening therapy in the treatment of HCC based on clinical data.The first part is divided into two steps: The first step is to select the TCM medication data of patients diagnosed with HCC or postoperative hepatocellular carcinoma in the outpatient department of Traditional Chinese Medicine of the First Affiliated Hospital of Sun Yat-sen University in recent 10 years.Python and Excel software were used to conduct statistical analysis on the drug use of HCC patients mentioned above by frequency statistics and clustering classification,so as to clarify the proportion of spleen-strengthening drugs in the treatment of HCC.The second step: Use computer to search CNKI,WANFANG Date,VIP,Cochrane Library,Clinical controlled trial Database,The Dutch Excerpta Medica Database(EMBASE),Pubmed database collection the randomized controlled study on the combination of TCM strengthening spleen and invigorating qi therapy with conventional Western medicine therapy in the treatment of HCC from the establishment of the database to June 2021.According to the inclusion and exclusion criteria,14 literatures that met the criteria were screened out from the above literatures.Cochrane bias risk assessment tool was used for literature evaluation.The short-term efficacy,KPS score,survival rate,immune function and adverse reactions of 14 literatures screened by Revman 5.3 were systematically analyzed.The second part confirmed the effect of spleen deficiency environment on CSCs expression of DEN drug-induced hepatocellular carcinoma mice through in vitro and in vivo experiments.C57BL/6 mice were randomly divided into four groups: blank group,spleen deficiency group,DEN drug-induced hepatocellular carcinoma(HCC)group and spleen deficiency with DEN drug-induced hepatocellular carcinoma(SD-HCC)group.Reserpine was used to establish spleen deficiency mice model,and diethylnitrosamine(DEN)and carbon tetrachloride were used to establish liver cancer mice model.The changes of diet and body weight in each group were observed.After the experiment,liver tissue was stained with HE for pathological analysis.RT-PCR and Western Blot were used to detect the m RNA and protein expression of AFP and CD133 in lung,liver and kidney tissues.Primary tumor cells were extracted from liver tumors of HCC group and spleen SD-HCC group,and the effects of spleen deficiency internal environment on CSCs expression of DEN drug-induced HCC mice were evaluated by suspension pellet-forming experiment,migration experiment and cloning experiment.The third part explore the effect and mechanism of Oatps expression and PTEN/AKT signaling pathway changes in CSCs expression of DEN drug-induced HCC mice induced by spleen deficiency environment in animal experiments.RT-PCR and Western Blot were used to detect Oatp2a1,Oatp1b2 and Oatp4a1 m RNA and protein expressions in lung,liver and kidney tissues of mice in each group to observe the transfer of "turbid dampness" to each viscera in spleen deficiency environment.Meanwhile,RT-PCR and Western Blot were used to detect PTEN and AKT m RNA and protein expressions in lung liver and kidney tissues to explore the possible mechanism of spleen deficiency environment on the CSCs expression of DEN drug-induced HCC mice.ResultsThe first part of drug analysis showed that the prescriptions of 1263 HCC patients,including 248 drugs.Among them,44 drugs were used more than 5% in frequency,and Hedyotis diffussa was the most frequently used drug for Antipyretic,detoxifying and dispersing knot,which was used in about 79% prescriptions.The second frequently used drugs were Radix pseudostellariae(69%),processed glycyrrhiza(58%)and Poria cocos(58%),all of that are spleen-invigorating and qi replenishing drugs.Among the 44 core drugs,there were 17 drugs function of strengthen spleen,benefit qi and reduce phlegm,with a total prescription frequency of 5916 times.The results indicated that the drugs for spleen-invigorating and qi replenishing,regulating qi and stomach accounted for a high proportion and frequency in the core drugs of TCM in the comprehensive treatment of HCC.The second part of first step showed that 14 randomized controlled trials were included in this systematic evaluation,with a total of 1402 subjects,including 700 in the intervention group(spleen-strengthening therapy + conventional Western medicine treatment)and 702 in the control group(conventional Western medicine treatment alone).The results of literature quality evaluation showed that all 14 studies had moderate risk of bias.Meta-analysis showed that spleen-strengthening therapy combined with conventional Western therapy had better short-term efficacy in primary liver cancer than conventional Western therapy alone,the difference has statistically significant [OR= 2.61,95%CI(2.03,3.37),P < 0.01].KPS score of quality of life of spleen-strengthening therapy combined with conventional Western therapy group was higher than that of conventional treatment group,the difference has statistically significant [OR=9.61,95%CI(4.98,14.24),P < 0.01].The half-year survival rate was similar between the two groups,no statistical significance was observed [OR=1.24,95%CI(0.62,2.47),P < 0.54].In terms of immune function: The values of CD3+,CD4+,CD4+/CD8+ in the treatment of HCC in the group of conventional Western medicine therapy combined with Spleen-strengthening Chinese medicine were higher than those in the group of conventional Western medicine therapy alone,and CD8+were lower than those in the group of conventional Western medicine therapy alone,the differences were statistically significant,all the difference has statistically significant[ CD3+: OR=6.63,95%CI(4.67,8.59),P < 0.01;CD4+: OR=7.14,95%CI(5.61,8.68),P <0.01;CD4+/CD8+: OR=0.5,95%CI(0.39,0.61),P < 0.00001;CD8+: OR=-3.35,95%CI(-5.56,-1.14),P = 0.003].In terms of safety,conventional Western medicine combined with spleen-strengthening Chinese medicine group had therapeutic advantages in terms of leukopenia and incidence of nausea and vomiting,which were all lower than those in the simple conventional Western medicine group [OR=0.51,95%CI(0.32,0.82),P < 0.005;OR=0.33,95% CI(0.12,0.92),P = 0.03],but there was no significant difference in the incidence of thrombocytopenia and liver function damage [OR=0.63,95%CI(0.38,1.05),P= 0.08;OR=0.31,95%CI(0.08,1.23),P = 0.1].In conclusion,spleen-strengthening therapy combined with conventional western medicine therapy can improve the short-term efficacy of HCC patients,improve KPS score,enhance immune function and reduce the side effects of western medicine treatment.In the second part of animal experiments,it was found that the body weight of mice decreased significantly during the modeling of spleen deficiency,and the weight difference before and after modeling was statistically significant(SD-HCC group: P=0.0001 < 0.05).After the completion of the spleen deficiency model,the body weight of the two groups of mice recovered in different degrees,and the body weight of the HCC group(normal saline injection)recovered significantly more than that of the SD-HCC group(reserpine injection).After the completion of HCC model,there was no significant difference in body weight before and after modeling in HCC group,while the body weight after modeling in the spleen deficiency with SD-HCC group decreased significantly,and the difference was statistically significant(P=0.007 < 0.05).Comparison of food intake between the two groups during the whole modeling period: compared with the HCC group,the food intake of the SD-HCC group was reduced,and the difference has statistically significant(P=0.0014 < 0.05).The pathological results of liver tissue after modeling showed that the liver volume of the SD-HCC group was significantly higher than that of HCC group,and the pathological grade of liver cancer was higher.The above studies showed that DEN drug induced HCC model was successful,the diet decreased,body weight decreased,and tumor volume increased of SD-HCC mice.The results of in vitro cloning experiment showed that the proliferation and cloning of SD-HCC cells increased significantly after spleen deficiency mice serum stimulation,and the proliferation area of HCC cells increased significantly compared with that of HCC cells(P=0.005 < 0.05).The results of cell migration experiment showed that at 24 h,the area between scratch was slightly decreased in HCC group,but significantly decreased in SDHCC group,the difference has statistical significance(P=0.001 < 0.05).After 48 hours,the area between scratch was significantly reduced in SD-HCC group,which was statistically significant compared with HCC group(P=0.001 < 0.05).The results of cell suspension pellet-forming experiment showed that the average number of stem cell pellet-forming in SD-HCC group was significantly higher than that in HCC group,and the difference has statistically significant(P=0.001 < 0.05).Cell experiments confirmed that the spleen deficiency environment could increase the ability of stem self-renewal on hepatocellular carcinoma cells.AFP m RNA expression in liver tissues of HCC group was significantly higher than that of blank group(P=0.007<0.05).AFP m RNA expression in liver tissue of HCC group was significantly increased than that of spleen deficiency group(P=0.036<0.05).AFP m RNA expression in liver,lung and kidney tissues of SD-HCC group was increased than that of HCC group(liver P=0.001<0.05,lung P=0.001<0.05,kidney P=0.009<0.05).AFP m RNA expression in spleen deficiency group was significantly increased than that of blank group(P=0.001<0.05).Compared with HCC group,CD133 m RNA expression in liver and lung tissues of SD-HCC group was significantly increased(liver P=0.025<0.05,lung P=0.03<0.05),but decreased in kidney tissue(P=0.017<0.05).Compared with the blank group,the expression of CD133 m RNA in liver tissue of spleen deficiency group was significantly increased(P=0.029<0.05),but it was decreased in kidney tissue(P=0.013<0.05).CD133 protein was mainly expressed in the cell membrane and cytoplasm of liver tissue,and positive expression of CD133 was found in the liver tissues of the four groups of mice.The expression of CD133 in liver tissues of the SD-HCC group was significantly increased compared with the other three groups.The result indicate the increased expression of AFP and CD133 in liver promoting the self-renewal and multidirectional differentiation of HCC stem cells in spleen deficiency environment.Lung and kidney are involved in the occurrence and development of HCC in spleen deficiency environment.CD133 expression trend in lung is consistent with that in liver,while CD133 expression trend in kidney is opposite to that in liver.The results of the third part of Oatps expression show: compared with HCC group,the expression of Oatp2a1 m RNA in lung,liver and kidney tissues in SD-HCC group was significantly increased(lung P=0.027<0.05,liver P=0.007<0.05,kidney P=0.037<0.05).Compared with blank group,Oatp2a1 m RNA expression in lung tissues of spleen deficiency group was increased(P=0.01<0.05).Compared with blank group,Oatp4a1 m RNA expression in liver and lung tissues of spleen deficiency group was significantly increased(liver P=0.049<0.05,lung P=0.0002<0.05),but significantly decreased in kidney tissue(P=0.017<0.05).Compared with HCC group,Oatp4a1 m RNA expression in liver and lung tissues of SD-HCC group was significantly increased(liver P=0.043<0.05,lung P=0.002<0.05).Compared with blank group,Oatp1b2 m RNA expression in lung,liver and kidney tissues of spleen deficiency group was significantly decreased(lung P=0.028<0.05,liver P=0.025<0.05,kidney P=0.039<0.05).Compared with HCC group,Oatp1b2 m RNA expression in kidney tissue of SD-HCC group was significantly decreased(P=0.012<0.05).The above results showed that high expression of Oatp4a1 and Oatp2a1 in liver may be associated with the accumulation of toxic drugs in liver of SD-HCC mice,while the expression of CD133 in liver was increased.The expression of Oatp4a1 and Oatp2a1 were increased in lung tissues,while CD133 expression in lung was increased.The expression of Oatp1b2 expression was decreased in kidney tissues,while CD133 expression was decreased.Oatps expression may be the internal cause of CD133 expression change induced by turbid dampness transport disorder under spleen deficiency environment.PTEN/AKT pathway expression: compared with blank group,the expression of PTEN m RNA in liver tissue of spleen deficiency group was significantly decreased(P=0.047<0.05).Compared with HCC group,the expression of PTEN m RNA in liver tissue of SD-HCC group was significantly decreased(P=0.047<0.05).Compared with blank group,PTEN m RNA expression in spleen deficiency group was significantly increased(P=0.0002<0.05).Compared with HCC group,PTEN m RNA expression in lung tissues of SD-HCC group was significantly increased(P=0.0008<0.05).The expression of AKT m RNA in SD-HCC group was higher than that in HCC group,but there was no significant statistical difference.These results suggest that the decreased expression of PTEN and the increased expression of CD133 in liver may be one of the internal mechanisms to accelerate the occurrence and development of HCC in spleen deficiency environment.However,the increased expression of PTEN in lung tissue and the unchanged expression of PTEN in kidney tissue may be one of the anti-tumor and self-protection mechanisms of lung and kidney under the dampness metabolism disorder in spleen deficiency environment.ConclusionSpleen-strengthening therapy combined with conventional western medicine therapy in the treatment of HCC can improve the short-term efficacy,improve the immune function of patients,and reduce the side effects of western medicine treatment better than the simple conventional western medicine treatment group.In spleen deficiency environment,the occurrence and development of liver cancer can be affected by increasing the CSCs expression of DEN drug-induced HCC mice,and the mechanism may be related to the oatps-mediated "turbid and dampness" substance transport disturbance in spleen deficiency environment,resulting in the decreased expression of PTEN. |
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