| Colorectal cancer(CRC)is a common malignant tumor of digestive tract,and it is also one of the malignant tumors with the highest morbidity and mortality in the world.CRC is a systemic disease with heterogeneous characteristics.Customizing personalized diagnosis and treatment programs based on the clinicopathologicaLand molecular characteristics of different patients in the process of diagnosis and treatment of CRC can improve the prognosis of CRC patients.Traditional Chinese medicine(TCM)“Treatment based on syndrome differentiation”plays an important role in the diagnosis and treatment of CRC.“syndrome differentiation”can summarize the nature of pathological changes in different bodies at a certain stage.Making personalized treatment plan according to the results of“syndrome differentiation”is helpful to improve the prognosis of CRC patients.However,it is still difficult to clarify the correlation between TCM syndrome classification and colorectal carcinogenesis paradigm.TCM theory holds that tumor is a complex and systematic disease formed by long-term interaction between the body and internal-external environments,while modern tumor biology holds that tumor is the result of the interaction between tumor cells and their surrounding microenvironment under the continuous pressure of internal and external environment,so it seems that there is a natural relationship between them.The TCM syndrome classification of CRC actually summarizes the characteristics of different stages of tumor occurrence and development at macro level,while TCM syndrome classification can show the characteristics of the interaction between tumor cells and their surrounding microenvironment at micro level.Extracellular matrix(ECM)is the main component of tumor microenvironment,ECM remodeling plays a key role in the occurrence and development of CRC,and the molecular characteristics of ECM malignant remodeling can be used as an important feature to predict different stages of tumor.In TCM theory,cold syndrome and heat syndrome are a pair of guiding principles that reflect the nature of diseases.Cold-heat syndrome differentiation plays an important role in guiding the diagnosis and treatment of diseases in TCM.In the previous study,we found that there were significant differences in colonic tissue length and colorectal carcinogenesis paradigm in rats between cold syndrome and heat syndrome,however,the reason for this difference has not been clarified.ECM remodeling is an important factor leading to changes in the length of colon tissue.Therefore,this study aims to investigate the mechanism behind the differences in the occurrence and development of CRC between cold syndrome and heat syndrome from the point of view of the characteristics of ECM malignant remodeling.Objective:The mechanism under the different paradigm of CRC between the cold syndrome and heat syndrome was explored from the perspective of the malignant remodeling characteristics of ECM,which provids a scientific basis for the TCM diagnosis and treatment of CRC based on syndrome differentiation.Methods:1.Establishment and evaluation of cold-syndrome and heat-syndrome CRC rat modelsThe rats were divided into control group,model group,heat-syndrome model group(heat model group)and cold-syndrome model group(cold model group).Control group and model group were given pure water intragastric administration once a day.The heat model group was intragastric with capsaicin/ethanol solution every day,and the cold model group was intragastric with 0℃ ice water and then immediately placed in a 2-6℃ refrigerator for 2 h.Colorectal cancer was induced by continuous injection with DMH for 12 weeks.The rats in each group were scored based on the criteria of cold syndrome and heat syndrome,and the content of energy metabolism enzyme,anal temperature,body weight,diet and water were measured to evaluate whether the cold syndrome or heat syndrome model was established successfully.The colorectal carcinogenesis paradigm between cold syndrome and heat syndrome were evaluated by using several methods such as observing the colonic morphology,calculating the number and size of tumors,evaluating the pathological stages of colon with H&E staining according to the criteria of histology of tumor,and methyblue staining was used to calculate the abnormal crypt foci to evaluate the risk of precancerous lesions.2.Differential analysis of characteristics of ECM components between cold-syndrome and heat-syndrome CRC rat modelsMovat staining method was used to evaluate the remodeling of colon ECM in CRC rats with cold syndrome and heat syndrome.The total collagen density of the colon of rats was determined by sirius red staining and polarized light microscopy.The contents,area of collagen I and collagen III,and the ratio of collagen I/collagen III were detected by immunohistochemistry and qRT-PCR to evaluate the collagen deposition in the colon of CRC rats with cold syndrom and heat syndrom.3.Differential analysis of ECM biomechanical characteristics between cold-syndrome and heat-syndrome CRC rat modelsTo evaluate the difference of colonic collagen cross-linking degree in CRC rats with different syndromes of cold and heat.The cross-linking morphological parameters of collagen fibers in the colon of rats were analyzed by using the sirius red staining method and the CT-FIRE software.The contents of LOX and LOXL2 were detected by immunohistochemistry and qRT-PCR.4.Differential analysis of the molecular characteristics of pre-metastatic niche formation related to ECM between cold-syndrome and heat-syndrome CRC rat modelsqRT-PCR,immunohistochemistry and western blot was used to detect the genes and protein expression of LAMC2,Integrin β1,FAK and p-FAK/FAK in rat colon tissue.The interaction intensity of LAMC2 protein and Integrin β1 in rat colon tissue was determined by Duolink in-situ experiment.The contents of pro-inflammatory cytokines IL-1β,TNF-αand IL-6 in serum of rats were determined by Enzyme-linked immunoassay(ELISA).5.Differential analysis of the molecular characteristics of EMT related to ECM between cold-syndrome and heat-syndrome CRC rat modelsqRT-PCR,immunohistochemistry and Western blot were used to detect the gene and protein levels of Snail,Fibronectin,E-cadherin and N-cadherin in rat colon tissues.6.Differential analysis of malignant progression degree between clinical deficiency cold syndrome and excessive heat syndrome of CRCThe cancer tissues and distal normal tissues of 30 CRC patients were collected.The malignant progression of colorectal cancer in clinical deficiency cold syndrome and excessive heat syndrome was evaluated according to the diagnostic criteria of Traditional Chinese Medicine and western medicine as well as pathological staging standards and chi-square test.7.The molecular basis of the difference in CRC malignant progression between clinical deficiency cold syndrome and excessive heat syndrome was explored based on the characteristics of ECM malignant remodeling(1)Movat staining was used to evaluate the ECM remodeling of CRC between clinical deficiency cold syndrome and excessive heat syndrome.Analyze the total collagen density in clinical samples by using sirius staining combined with the polarized light microscope,further using immunohistochemical and qRT-PCR technique to detect the content and distribution area of type Ⅰ collagen and type Ⅲ collagen,and the ratio of type Ⅰcollagen and type Ⅲ collagen.(2)The cross-linking morphological parameters(including width,length,stiffness and Angle)of collagen fibers in clinical samples were detected by using the sirius staining method and software CT-FIRE in order to analyze the cross linking of collagen fibers.The gene and protein levels of LOX and LOXL2 were detected by qRT-PCR and immunohistochemistry method.(3)qRT-PCR and immunohistochemistry were performed to detect the gene and protein levels of LAMC2,Integrin β1,FAK and p-FAK/FAK in clinical samples.Duolink in-situ experiment was used to detect the interaction signal of LAMC2 and Integrin β1 protein in clinical samples.(4)qRT-PCR and immunohistochemistry were used to detect the gene and protein levels of Snail,Fibronectin,E-cadherin and N-cadherin in clinical samples.Results:1.Establishment and evaluation of cold-syndrome and heat-syndrome CRC rat modelsThe results of signs score,anal temperature measurement,body weight,diet-water quantity and energy metabolism enzyme measurement showed that the established rats with cold and heat syndrome were consistent with the description characteristics of existing literature on cold and heat syndrome animal model.The results showed that the average size and number of tumors in the cold model group were higher than those in the heat model group.H&E staining showed that at week 17,the rates of colonic atypical hyperplasia in both hot and cold mode groups were 100%,which could be classified as precancerous lesions.At the last stage of the experiment,the malignant degree of colorectal cancer in cold-model rats was the highest,and lymphatic metastasis appeared in cold model group.The results of abnormal crypt foci showed that the number of abnormal crypt foci in the cold model group was significantly higher than that in the heat model group during precancerous lesions,indicating that the cold model group had a higher risk of precancerous lesions.The results showed that colonic length of rats in cold mode group was the shortest,and colonic tissue fibrosis of rats in cold mode group was the most obvious.2.Differential analysis of characteristics of ECM components between cold-syndrome and heat-syndrome CRC rat modelsThe results of movat staining showed that the ECM remodeling characteristics of the colon tissues of cancerous rats were significantly different from those of normal tissues.The results of total collagen density measurement showed that the content of total collagen in cold model group was the highest,followed by that in heat model group.The results of immunohistochemistry and qRT-PCR showed that the colonic collagen deposition was relatively high in the cold model group at precancerous stage.3.Differential analysis of ECM biomechanical characteristics between cold-syndrome and heat-syndrome CRC rat modelsThe results of collagen fiber morphology parameters showed that the colonic fiber morphology was longer and wider in cold model group from the precancerous stage on.In addition,the protein and gene expressions of LOX and LOXL2 were higher in cold model group,suggesting that the cross-linking degree of colonic collagen fiber was higher in cold model group than in heat model group.4.Differential analysis of the molecular characteristics of pre-metastatic niche formation related to ECM between cold-syndrome and heat-syndrome CRC rat modelsThe results of qRT-PCR,IHC and Western Blot showed that the gene and protein expression levels of LAMC2,Integrin β1,FAK and p-FAK/FAK were significantly higher in cold model group than in heat model group.The results of Duolink showed that the signal of LAMC2-Integrin β1 interaction was almost not expressed in the normal group at week 17 and 24,and the intensity of LAMC2-Integrin β1 interaction was significantly higher in the colons of cold model group compared with any other groups.The levels of pro-inflammatory cytokines IL-1β and IL-6 in cold model group were significantly higher than those in heat model group from the precancerous stage on.5.Differential analysis of the molecular characteristics of EMT related to ECM between cold-syndrome and heat-syndrome CRC rat modelsThe results of qRT-PCR,western blot and immunohistochemistry showed that compared with the model group and the heat model group,the gene and protein levels of Snail,Fibronectin and N-cadherin were higher in the cold model group,while the expression levels of E-cadherin were significantly lower in the cold model group.6.Differential analysis of malignant progression degree between clinical deficiency cold syndrome and excessive heat syndrome of CRCThe probability of tumor progression to advanced stage in CRC patients with clinical deficiency cold syndrome was higher than that in patients with excessive heat syndrome.The proportion of distant metastasis was higher in patients with deficiency cold syndrome.7.The molecular basis of the difference in CRC malignant progression between clinical deficiency cold syndrome and excessive heat syndrome was explored based on the characteristics of ECM malignant remodeling(1)Differential analysis of ECM main components in deficiency-cold syndrome and excessive-heat syndrome CRC patientsThe results of Movat staining showed that ECM collagen fibers were widely distributed in clinical colon cancer tissues.The results of total collagen density measurement showed that the content of total collagen in deficiency cold syndrome group was higher than that in excessive heat syndrome group.The protein expression and area ratio of COL Ⅰ and COL Ⅰ/COL Ⅲ in the deficiency cold syndrome group were significantly higher than those in the excessive heat syndrome group,indicating that colonic collagen deposition in CRC patients with deficiency cold syndrome was more serious and earlier than that in CRC patients with excessive heat syndrome.(2)Differential analysis of biomechanics related molecular characteristics in deficiency-cold syndrome and excessive-heat syndrome CRC patientsThe results of collagen morphological parameters showed that the width and length of collagen in deficiency cold syndrome group were higher than that in excessive heat syndrome group.The results of qRT-PCR and immunohistochemistry showed that the gene and protein expression of LOX and LOXL2 in the cancer tissues of the deficiency and cold syndrome group were significantly higher than those in the excessive heat syndrome group,suggesting a high degree of collagen crosslinking in the CRC tissues of the deficiency cold syndrome group.(3)Differential analysis of molecular characteristics related to pre-metastasis niche in deficiency-cold syndrome and excessive-heat syndrome CRC patientsThe results of qRT-PCR and immunohistochemistry showed that the gene and protein expression of LAMC2,Integrin β1 and FAK in cancer tissues were higher than those in normal tissues.In cancer tissue,the gene levels of LAMC2,Integrin β1 and FAK in deficiency cold syndrome group was higher than that in excessive heat syndrome group.The Duolink results showed that in cancer tissue,LAMC2 and Integrin β1 interaction signal intensity in deficiency cold syndrome group was higher than that in excessive heat syndrome group.(4)Differential analysis of molecular characteristics related to EMT in deficiency-cold syndrome and excessive-heat syndrome CRC patientsThe results of qRT-PCR and immunohistochemistry showed that the gene and protein expressions of EMT markers Snail,Fibronectin and N-cadherin in cancer tissues were higher in deficiency cold syndrome group than in excessive heat syndrome group,while E-cadherin was significantly lower than in excessive heat syndrome.Conclusion:1.There were significant differences in colorectal carcinogenesis paradigm in CRC rats between cold syndrome and heat syndrome,and the degree of malignant tumor progression in CRC rats with cold syndrome was higher than that with heat syndrome,the underlying mechanism of which may contribute to the differential expression of characteristics of ECM remodeling including the collagen deposition and crosslinking characteristic molecules,LAMC2-Integrin β1 interaction mediated pre-metastasis niche characteristic molecules,and ECM-related EMT characteristic molecules.2.The malignant metastasis rate of CRC in clinical deficiency cold syndrome was higher than that in excessive heat syndrome,and the differential expression of characteristics of ECM remodeling including the collagen deposition and crosslinking characteristic molecules,LAMC2-Integrinβ1 interaction mediated pre-metastasis niche characteristic molecules,and ECM-related EMT characteristic molecules may be the molecular basis for the higher malignant metastasis rate of CRC in clinical deficiency cold syndrome. |
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