| Primary hepatocellular carcinoma(HCC)has the characteristics of high incidence and concealed onset.In China,the incidence and mortality of HCC remain high,and the number of deaths is on the rise.HCC is a major health problem in China,which seriously reduces the quality of life of patients,affects the lives and health of residents,and also causes huge social and economic burdens to them and their families.In the early stage of HCC,surgical resection is the best treatment.However,due to the poor medical environment and patient’s awareness of disease prevention,it is often found in the middle and late stages.At this time,the curative effect of surgical resection is limited,and other treatments such as TACE may also have the risk of recurrence and metastasis.Precancerous lesions of the liver are widely studied in recent years.In short,they refer to a series of changes that occur before the carcinogenesis of liver tissue.Their occurrence and development are regulated by many factors,including environmental factors,gene mutations,signaling pathways and the cytokines regulated by them,which can affect the occurrence of HCC.It is a multi-factor,multi-step and multi-way process of disease.At this stage,hepatic oval cells are the core link.When stimulated by special pathological factors,they can be activated and transformed into hepatocellular carcinoma cells.Since reversal is possible at this stage,it is considered to be an ideal target for the prevention and treatment of HCC.However,due to the limited research,its regulatory mechanism has not been fully clarified.Wnt pathway is a signal pathway that has been studied more,and its mechanism has been clearer.At present,classical Wnt pathway is the most thorough and meticulous one.It has been proved that the classical Wnt pathway is closely related to the occurrence and development of primary hepatocellular carcinoma.Its abnormal activation can run through the whole process of HCC from occurrence to development and metastasis,so it is also an important research direction in the prevention and treatment of HCC.Many studies have shown that traditional Chinese medicine can treat primary liver cancer by regulating the classical Wnt pathway,but there are relatively few studies on precancerous lesions related to traditional Chinese medicine.Liangxue Jiedu Formula is an empirical compound based on the principle of invigorating Qi and invigorating spleen and cooling blood and detoxifying.It has been proved to be effective in the adjuvant treatment of severe hepatitis and hepatocellular carcinoma for many years.Studies have shown that it can regulate the proliferation and differentiation of hepatic oval cells in the rat model of precancerous lesion(hepatic oval cell proliferation model),but its specific mechanism has not been clarified.Therefore,this study hopes to study the blocking effect of Liangxue Jiedu Recipe on precancerous lesions of liver by establishing rat model of precancerous lesions of liver,using RNA scope double staining,HE staining and immunohistochemical staining,and further study its possible mechanism,that is,the regulating effect of classical Wnt pathway,in order to provide scientific basis for the prevention and treatment of precancerous lesions of liver by traditional Chinese medicine,and for the followup.Further study of therapeutic targets in related pathways will provide some basis and support.The first part Blocking effect of Liangxue Jiedu Recipe on precancerous lesions of liver in ratsObjective And Significance To establish a rat model of precancerous lesions of liver,observe the morphology and structure of rat liver cells in different groups under microscope,compare their changes,and conduct HE staining and immunohistochemical staining to study the blocking effect and influence of Liangxue Jiedu Recipe on precancerous lesions of liver in rats.Research methods1 30 rats were randomly divided into three groups,10 in each group.Group A:model group of precancerous lesions of rat liver(model group);Group B:normal control group;Group C:Liangxue Jiedu recipe group.Among them,two groups A and C constructed rat models of precancerous lesions of liver:gastric administration of 0.15g/L 2-acetaminofluorene(2-AAF)for one week+2/3 hepatectomy was used to establish the model;group C was used to intervene:rats were gavaged with Liangxue Jiedu recipe.Modeling was completed after 5 weeks.2 HE staining technique was used to observe and compare the morphological and structural characteristics of hepatocytes in different groups of rats.3 Immunohistochemical(IHC)staining technique was used to stain paraffin sections of hepatocytes of rats in each group with OV6 and CD34.The staining conditions of different groups were observed,the distribution and staining degree of different groups were compared,and the expression differences were analyzed semi-quantitatively.Results1 HE staining:At the end of the 5th weekend,the hepatic lobules in group A(model group)disappeared,the proliferated bile ducts were observed in the portal area,the aggregated hepatic oval cells and inflammatory cells were observed nearby,and some hepatic oval cells grew into the hepatic parenchyma around the portal area,and there were different degrees of damage near the hepatic plate in group B(normal pair).There was no obvious pathological change in the control group compared with the normal liver tissue;the liver tissue structure in group C(intervention group)was relatively intact,and the structure of hepatic lobules was more intact than that in group A(model group).The pathological changes of hepatic oval cells were mild,and a small number of variant cells could be seen.2 Immunohistochemical staining:In group A(model group),significant brown-yellow staining was observed near the portal area and in hepatic lobules,indicating that OV6 and CD34 were highly expressed;in group B(normal control group)and group C(intervention group),a small amount of brown-yellow staining was observed,indicating that OV6 and CD34 were low expressed,and there was no significant difference between groups.Conclusions1 The precancerous lesions of rat liver can be observed to be different from the normal liver tissues.Although the degree of precancerous lesions is not as obvious as that of hepatocellular carcinoma,a large number of hepatic oval cells can be observed to proliferate,which can be regarded as a special change at this stage.2 Through the intervention of matrine and traditional Chinese medicines such as Liangxue Jiedu recipe,we can see that the structure and morphology of liver have a great change compared with the model group,and the hepatic oval cells are significantly reduced,suggesting that traditional Chinese medicines may have the effect of blocking precancerous lesions of liver,and its mechanism may be through regulating the proliferation and differentiation of hepatic oval cells.The second part Regulating effect of Yiqi Liangxue Jiedu Recipe on classical Wnt pathwayObjective and Significance1 To study the localization and expression of Wnt1 and beta-catenin,the key components of the classical Wnt pathway,in each group,and to compare their differences and analyze the influence of Liangxue Jiedu Recipe on the classical Wnt pathway in rat hepatocellular precancerous lesions.2 To study the expression of Wntl and beta-catenin,the key components of the classical Wnt pathway,in different groups,and to compare their differences and analyze the effects of Liangxue Jiedu Recipe on the classical Wnt pathway in rat hepatocellular precancerous lesions.Methods1 Six wax blocks were randomly selected from each group.The expression and localization of Wntl and beta-catenin in each group were detected by RNA scope 2.5 visible double staining,and the differences among the groups were analyzed and compared.2 Immunohistochemical staining was used to detect the localization and expression of Wntl and beta-catenin proteins in the liver tissues of rats in each group,and the differences among the groups were analyzed and compared.Results1 Wntl(red)and beta-catenin(green)were detected in group A(model group),which were mainly expressed near the portal area of liver tissue.The contents of Wnt1(red)and betacatenin(green)in group B(normal control group)and group C(intervention group)were less or not detected.2 Wntl and beta-catenin protein were strongly positive in group A(model group).They were brown-yellow stained near the portal tract and dispersed in the membrane and cytoplasm of oval cells or hepatocytes.In group C(intervention group),the staining degree of the two proteins decreased,and the yellow area decreased compared with group A.In group B(normal control group),a small amount of Wntl and beta-catenin proteins were observed in the cell membrane and cytoplasm of hepatocytes.Conclusions1 The high expression of Wnt pathway key molecules Wnt1 and beta-catenin in liver tissue of rats with precancerous liver lesions indicates that the classical Wnt pathway may be abnormally activated in precancerous liver lesions,and its expression decreases after the intervention of Liangxue Jiedu Formula,suggesting that Liangxue Jiedu Formula may block the progression of precancerous liver lesions by regulating Wnt pathway.2 The high expression of Wnt pathway key molecules Wntl and beta-catenin in liver tissue of rats with precancerous liver lesions indicates that the classical Wnt pathway may be abnormally activated in precancerous liver lesions,and its expression decreases after the intervention of Liangxue Jiedu Formula,suggesting that Liangxue Jiedu Formula may block the progression of precancerous liver lesions through the regulation of Wnt pathway.The third part Expression of Wnt1 and beta-catenin protein in human hepatocellular carcinomaObjective and Significance1 To detect the expression of Wnt1 and beta-catenin proteins in human hepatocellular carcinoma and its adjacent liver cirrhosis tissues,compare the differences of protein localization and quantification between the two groups.2 Explore the relationship between Wnt1 and beta-catenin proteins and pathological parameters between the two groups.Research methods1 HE staining was used to observe the structural characteristics and pathological changes of hepatocellular carcinoma and cirrhotic tissues adjacent to the cancer.2 To observe the expression of Wnt1 and beta-catenin protein in hepatocellular carcinoma group and cirrhosis group by immunohistochemical staining and compare their differences.Results1 Microscopically,the hepatocellular carcinoma showed a thin beam or pseudoglandular duct type.The cancer cells were polygonal with abundant cytoplasm,eosinophilic,round nucleus,obvious nucleolus and mild atypia.Most of the patients had intrahepatic tumors that did not invade the capsule.A few of the patients had invasion under the capsule,and the surrounding liver tissues showed cirrhosis.2 Immunohistochemistry:Wntl was mainly localized in the cytoplasm of hepatocellular carcinoma cells,followed by the cell membrane,which was stained yellow-brown and positive.Most of the corresponding adjacent tissues were negative,and a few of the cytoplasms or membranes were stained with light yellow granules.Quantitative analysis showed that the integrated optical density of Wnt1 in hepatocellular carcinoma group was significantly higher than that in adjacent tissues.In cirrhosis group,there was significant difference between the two groups(t=13.64,P<0.001).In hepatocellular carcinoma,betacatenin was localized in the cell membrane and cytoplasm of cancer cells,but also distributed in the nucleus,stained yellow-brown or brown,which was positive expression.In the corresponding adjacent tissues,beta-catenin was positive expression,which was distributed in the cell membrane of hepatocellular cells.It resembles a grid.Quantitative analysis of integral optical density showed that beta-catenin in hepatocellular carcinoma group was significantly higher than that in para-cancerous cirrhosis group(t=7.31,P<0.001).3 Comparing the relationship between Wntl and beta-catenin and clinical parameters of patients,we found that beta-catenin was related to the degree of differentiation of hepatocellular carcinoma(t=3.45,P=0.02)(see tables 9 and figures 12 to 14,tables 10 and figures 15 to 17),and there was a negative correlation between them(Spearman correlation coefficient was0.764,P<0.001)(see table 11).Conclusions1 The expression of 1 Wntl and beta-catenin in hepatocellular carcinoma and its adjacent tissues is significantly different.Both of them are stronger in hepatocellular carcinoma than in adjacent tissues,suggesting that the classical Wnt pathway plays an important role in the occurrence and development of hepatocellular carcinoma.2 The expression of beta-catenin was negatively correlated with different pathological grades of hepatocellular carcinoma.The lower the degree of differentiation,the higher the expression of beta-catenin,suggesting that beta-catenin may be related to the prognosis of patients. |
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