Based On Hippo Signaling Pathway To Explore The Colorectal Cancer Liver Metastasis Spleen Deficiency Syndrome And Chinese Medicine Intervention Mechanism

Colorectal cancer(CRC)is one of the most common malignant tumors of the digestive tract in China.The liver is the most common metastatic site in CRC,and colorectal cancer liver metastases are also the leading cause of death in these patients.Colorectal cancer is called "gut mushroom","anal hemorrhoids","intestinal effusion","gut slippery" and so on.Traditional Chinese medicine refers to the transfer of CRC as"transmission".At present,there are many reports on the pathogenesis of CRLM,which are mainly related to the dissipation of positive qi by cancer,and the interaction between positive and deficient.Zhengxu includes spleen deficiency,qi deficiency,blood deficiency,yin deficiency,and yang deficiency,and standard reality includes qi stagnation and blood stasis,phlegm dampness condensation,and heat toxicity.The correlation between Zhengxu and true is the pathological basis of CRC,and sputum poisoning is the basic way of CRLM.Some scholars have reported that CRC TCM syndromes have a certain relationship with the occurrence of liver metastasis,but what is the key pathogenesis of liver metastasis in CRC?At present,there is no special research in this area.Hippo signaling pathway is related to many tumors.Some scholars have found that the recurrence,metastasis and drug resistance of colon cancer are closely related to the core-oncogene Yes-associated protein(YAP)of Hippo signaling pathway.Recent studies have found that G protein-coupled receptors Signal pathways(hereinafter referred to as GPCRs)can regulate Hippo signaling pathways.The regulatory mechanism is:lysophosphatidic acid(hereinafter referred to as LPA),sphingosine 1-phosphate(hereinafter referred to as SIP).LPA and SIP inhibit LATS1/2 kinase activity and activate YAP by activating G12/13 or Gq/11 coupled receptors.In contrast,epinephrine(AD or EPI)and glucagon(GC)inhibit YAP activation.Finding potential drugs that interfere with the Hippo pathway through GPCRs is of great value.Hippo signaling pathway plays an important role in the occurrence and development of colorectal cancer liver metastasis,but its mechanism has not been fully elucidated.Its correlation with CRLM TCM syndromes has not been reported in China.Therefore,the purpose of this experiment is to carry out a prospective study of 128 patients with CRLM(taking CRC patients as a control)and find out what are the key pathogenesis of liver metastasis in CRC.Based on this,an animal model of CRLM disease and syndrome was established to study the effects of Chinese medicine The CRLM disease syndrome combined with the effects of the animal model Hippo signaling pathway to explore the target of intervention of traditional Chinese medicine is expected to open a new path for further exploration of the modern scientific connotation of the syndrome and the bottleneck problem of traditional Chinese medicine to overcome the high metastasis rate of the disease.Part Ⅰ Study on TCM Syndrome and Related Factors of Patients with Liver Metastasis of Colorectal CancerObjective To analyze the TCM syndromes and clinically relevant factors of patients with colorectal cancer and liver metastases from colorectal cancer,and to explore the relevant factors and objective indicators of TCM pathogenesis of liver metastases from colorectal cancer.Methods The prospective epidemiological survey was used to analyze and analyze the clinical epidemiological data and tumor marker levels of patientsResults Colorectal cancer patients had deficiency syndromes:72 cases of spleen deficiency,55 cases of qi deficiency,21 cases of blood deficiency,5 cases of yang deficiency,7 cases of yin deficiency,evidence:47 cases of phlegm dampness,3 cases of qi stagnation,14 cases of blood stasis,and heat poisoning.8 cases of colorectal cancer liver metastasis deficiency syndrome:89 cases of spleen deficiency,62 cases of qi deficiency,24 cases of blood deficiency,3 cases of yang deficiency,6 cases of yin deficiency,evidence:37 cases of phlegm dampness,6 cases of qi stagnation,and blood stasis 20 Cases,6 cases of fever,the liver metastasis of colorectal cancer was statistically significant(P<0.05),and related to pathological classification,in which ulcerative adenocarcinoma was more prone to liver metastasis(P<0.05),Related to the degree of tumor invasion,invasion of subserosal or paraperitoneal tissues without peritoneal coverage is more prone to liver metastasis(P<0.01),and related to the number of lymph node metastases(P<0.01),It has statistical significance with serum CEA,CA242,CA19-9,CA50,CA724,CA125,CA153,and plasma D-dimer(P<0.05).The diagnostic efficacy of ROC curve analysis:combined detection>serum CEA>serum CA242>Serum CA199>D-dimer of plasma>CA125 of serum>CA724 of serum>CA50 of serum>Serum CA125.The positive rates of diagnosis of serum CEA,CA199,CA125,and plasma D-dimer in patients with spleen deficiency in colorectal cancer and colorectal cancer with liver metastasis were higher than those in patients with non-spleen deficiency(P<0.05).Conclusions TCM syndrome differentiation,age,tumor pathological classification,degree of invasion,number of intestinal lymph node metastases,serum CEA,CA153,CA242,CA199,CA50,CA724,CA125,plasma D-dimer and colorectal liver metastasis Highly correlated.The combined detection of several tumor indicators can increase the diagnosis rate.The first study found that serum CEA,CA199,CA125,and plasma D-dimers can be used as quantitative indicators of spleen deficiency syndrome in colorectal cancer and colorectal cancer.The monitoring,diagnosis and traditional Chinese and western medicine treatment of liver metastases have clinical guiding significance.Part Ⅱ Establishment of a mouse model of colorectal cancer with liver metastasis and spleen deficiencyObjective To compare three animal models of liver metastasis of colorectal cancer in mice,and to construct mouse models of spleen deficiency and disease syndrome of liver metastasis of colorectal cancer.Methods 1)The relationship between cancer cell concentration and liver metastasis rate/mouse survival rate was explored using a spleen retention model.The screened cell concentration was used to evaluate the advantages and disadvantages of the splenic retention model,the total splenectomy model and the splenic hemisection model and their correlation with CEA,CA199,and AFP.2)Forty BALB/c mice were randomly divided into a sham operation group as group A,a spleen deficiency group as group B,a colon cancer liver metastasis group as group C and a colon cancer liver metastasis group as spleen deficiency group.In group A and group B,mice were injected with the lower pole of the spleen into the cell suspension without tumor cells,and the injected hemi-spleen was excised after the abdomen was closed.The lower pole was resected and the abdomen was closed.On the third day after surgery,groups A and C were given saline for 7 days,and groups B and D were given rhubarb decoction for 7 days.The mice spontaneously moved,and liver HE staining confirmed the success of the modelingResults 1)On day 10 of colonic adenocarcinoma(CT26)inoculation in mice,the liver metastasis rate was 100%.When 0.15 mL of CT26 cells were injected(0.5 × 107/mL to 1.5× 107/mL),the mouse survival rate was high(P<0.05).The splenic retention model,the complete splenectomy model and the splenic hemisection model were all successfully modeled.The hepatic metastasis rate was the highest on day 7 of the splenic hemisection model(P<0.05),and the life expectancy of the splenic hemisection model was the longest(P<0.05).The serum CEA and CA199 of the three models were higher than those of the control group(P<0.05).The serum AFP of the three models was lower than that of the control group(P<0.05).2)Open field experiment results:There was no significant difference between the total distance and the average speed in the 10 minutes before the modeling of the four groups(P>0.05),and there was no significant difference in the total distance and the average speed of the group A before and after the modeling(P>0.05).After modeling,the total distance of 10 minutes and the average speed of group B,C,and D were lower than those of group A,and the difference was statistically significant(P<0.05).Conclusions 1)The splenic hemisection model is a better model for colorectal cancer liver metastasis in mice.2)The spleen hemisection model combined with rhubarb gavage can establish a disease-syndrome-combined animal model of liver metastasis of colorectal cancer in line with clinical syndrome types.Part Ⅲ Explore the mechanism of traditional Chinese medicine intervention in mice with colorectal cancer liver metastasis and spleen deficiency based on Hippo signaling pathway Objective To study the role of Hippo signaling pathway in a mouse model of hepatic metastasis and liver metastasis from colorectal cancer,and to initially explore the target of intervention of Jiawei Sijunzi Decoction on mouse model of colorectal cancer with liver metastasis and spleen deficiency.Methods 1)Twenty BALB/c mice were randomly divided into a sham operation group and an experimental group.In the sham operation group,the mice were opened and closed,and the experimental group was injected with colon adenocarcinoma CT26 cells into the lower pole of the spleen and resected to establish a liver metastasis model.After 7 days,the mice were sacrificed and dissected.Enzyme-linked immunosorbent assay(ELISA)to detect epinephrine(EPI),glucagon(GC),lysobisphosphatidic acids(LPA),and 1-phosphate sphingosine(LPA)in eye blood of mice sphingosine-1 phosphate(SIP)content;HE staining results confirmed the success of splenic hepatectomy for liver transplantation tumors.Immunohistochemical analysis of Yes-associated protein 1(YAP1)and phosphorylated YAP1(phospho-yes)-associated protein 1,p-YAP1)was used for localization,and the expression levels of YAP 1 and p-YAP1 in liver tissue were detected by Western blot.2)Establish 70 mice with disease and syndrome combined with liver and spleen deficiency syndrome in colorectal cancer.Animal models were randomly divided into model control group A,capecitabine(chemotherapeutic agent)group B,fingolimod hydrochloride(SIP receptor inhibition Agent)Targeted group C,Kang’ai Pingwan Chinese patent medicine group D,Modified Sijunzi Decoction low,medium,high E,F,G group.After 14 days of treatment,the mice’s eyeballs were blooded,sacrificed and dissected.The serum levels of LPA,SIP,GC,AD,and serum in mice were measured by ELISA,and the expression levels of YAP 1 and p-YAP1 in liver tissue were detected by Western blotResults 1)The liver metastasis rate was 100%7 days after the establishment of a mouse colorectal cancer hemi-spleen resection model.The plasma EPI and GC contents in the sham operation group were significantly higher than those in the experimental group(both P values<0.01).The levels of LPA and S1P in rat plasma were significantly higher than those in the sham operation group(both P values<0.01);Tissue HE staining results showed typical characteristics of poorly differentiated adenocarcinoma with mitosis.In the sham operation group,YAP1 protein was expressed in cytoplasm[nucleus YAP1(-),cytoplasm YAP1(+)],p-YAP1 protein was expressed in cytoplasm and nucleus[nucleus p-YAP1(+),cytoplasmic p-YAP1(+)];YAP1 protein was expressed in cytoplasm and nucleus in liver tumor tissue of the experimental group[nucleus p-YAP1(+),cytoplasm p-YAP1(+)],p-YAP1 protein was in nucleus and cytoplasm No expression was found[nucleus p-YAP1(-),cytoplasm p-YAP1(-)].The expression level of YAP1 in liver tumor tissues in the experimental group was significantly higher than that in the sham operation group(P<0.01),and the expression level of p-YAP1 in liver tumor tissues in the experimental group was significantly lower than that in the sham operation group(P<0.01).2)Compared with group A,the plasma levels of EPI and GC in mice of groups B,C,D,and G were higher than those in group A,and the levels of LPA and SIP were lower than those in group B.The differences were statistically significant(P<0.01)There was no statistically significant difference between group E and F and group A(P>0.05).Compared with group A,the expression of YAP 1 protein in groups B,C,and G was significantly down-regulated,and the difference was statistically significant(P<0.01).Compared with group A,the expression of YAP 1 protein in group B was significantly up-regulated,and the difference was statistically significant.Significance(P<0.01).Conclusions 1)It was found that EPI,GC and YAP1 expression were negatively correlated with p-YAP1 expression,and LPA,S1P and YAP1 were positively correlated with p-YAP1.2)Modified Sijunzi Decoction may play an antitumor effect by regulating Hippo signaling pathway,down-regulating YAP1,and up-regulating p-YAP1.