Effect Of Zuojin Pill And Anti-zuojin Pill On Histone Modification And Related ECM In Rat With Cold Syndrome And Heat Syndrome During The Progression Of Colotectal Cancer

ObjectiveColorectal Cancer（CRC）is the third most common cancer in the world,which has caused serious clinical problems due to its high incidence,mortality and recurrence..Colorectal cancer is caused by complex interactions of non-modifiable（eg,genetic and epigenetic alterations）and modifiable（eg,environmental,diet and lifestyle）risk factors.Epigenetic mechanisms,such as DNA methylation,histone modification and noncoding RNA,have emerged as molecular transducers of environmental stimuli to control gene expression.The extracellular matrix is an important part of the tumor microenvironment,which is considered to be an important regulatory factor in various aspects of tumor initation,development,transformation,invasion,and metastasis.In the process of tumor development accompany with extracellular matrix remodeling,and It is regulated by a myriad of factors that converge on the transcription of genes encoding extracellular matrix protein,a process the epigenetic machinery plays a pivotal role Histone modification as an important part of the epigenetics,which can perform instantly response to surrounding environment due to their special structural characteristics,so histone modifications may participate in extracellular matrix remodeling.Our studied showed that occurrence and development patterns of tumors were different under the influence of cold and heat in different internal environments.Zuojin pill and anti-zuojin pill can play an anti-tumor role by affecting the activity of methyltransferase and blocking the expression of DNA methylation,but their specific mechanism of action in CRC is not thorough.Therefore,this study established a rat model of colorectal cancer with different syndromes of cold and heat,studied the differences in histone modification and extracellular matrix pathological characteristics in the occurrence and development of colorectal cancer in the two different syndromes,and explored the effect of intervention Zuojin Pill and Anti-zuojin Pill on CRC.MethodsTo establish an animal model of heat syndrome and cold syndrome,heat syndrome group were treated with 20%ethanol solution combined with capsaicin 5 weeks.Cold syndrome group was treated with cold distilled water（10mg/kg）and soaked in ice water（10℃）.The body surface characteristics and biochemical indexes of rats with cold syndrome and heat syndrome were evaluated.DMH-induced colorectal cancer model was used on the basis of cold syndrome and heat syndrome from the 6th week.DMH-induced colorectal cancer model is the research group the slow carcinogenesis model explored in the previous research is to simulate as much as possible that the tumor is produced under the long-term interaction between the constitution and the environment.Groups of Cold syndrome and heat syndrome respectively provided with decoction of Zuojin Pill and Fanzuojin Pill via intragastric gavage untill the end of different phases of experiments.The rat’s physical characteristics,such as ear condition,paws condition,tongue condition and excrement character,body weight and food intakes were assessed at different time points.Biochemical induces were assyed by microplate reader,including succinate dehydrogenase（SDH）activity,lactate dehydrogenase（LDH）activity,the Na⁺-K⁺-A TPase and Ca²⁺-Mg²⁺-ATPase activity in TPase in the supernatant of colon tissue homogenate.Colorectal cancer models were evaluated by colonic morphology,histopathology,tumor type,carcinogenesis rate and other indicators.The colon tissues at 27,29,32,35,and 38 weeks were taken.Global levels of H3K9ac,H3K18ac,H3K27ac,H4K16ac and histones H3K4me3,H3K9me3,H3K27me3,H4K20me in colon mucosa were assessed by western blot;Enzyme-linked immunoassay（Elisa）was used to detect the expression levels of histone deacetylases（HDACs）HDAC1,HDAC2,HDAC3,HDAC8 and methylases EZH2,G9a,LSD1,KTM5C in the supernatant of colon tissue homogenate.Serial issue sections were obtained from each sample with the microtome and then were stained with hematoxylin and eosin（HE）,picrosirius red,Masson’s trichrome（MT）,and Weigert’s Resorcin-Fuschin（WRF）.Sirius red staining combined with linear cross-polarized light microscope to observe the cross-linked morphology of collagen;in addition,the protein levels of LOXL2,COLI,COL Ⅲ collagen and elastin were detected by Western blot.Quantitative Real-time PCR（qRT-PCR）was used to detect the expression levels of type Ⅰ collagen,type Ⅲ collagen,elastin and LOXL2 mRNA in rat colon tissue;IHC was used to detect the ratio of colon COLI/COLⅢ and distribution analysis,LOXL2 expression.Take 27,29,32,35,and 38 weeks of colon tissue sections and use IHC to detect the distribution of colon tissue MMP1,MMP2,MMP9,TIMP1;then use qRT-PCR to detect the expression of colon tissue MMP1,MMP2,MMP9,TIMP1.Results（1）From the appearance,general condition,physical sign scores of Zuojin Pill and Anti-Zuojin Pill,colon tissue morphology and pathological results of rats with cold and heat syndromes,the modelling of colorectal cancer with cold and heat syndromes was successful.The activities of SDH,LDH,Na⁺-K⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase in cold syndrome rats were significantly inhibited.The SDH,LDH,Na+-K--ATPase,and Ca²⁺-Mg²⁺-ATPase of rats with heat syndrome all increased to varying degrees.The SDH,LDH,Na+K+-ATPase and Ca²⁺Mg²⁺-ATPase activities of Zuojin Pill group and Anti-Zuojin Pill group were improved compared with cold and heat syndrome groups.Compared with the heat syndrome group,the cold syndrome group has a higher tumor incidence and metastasis rate.In the early stage of colorectal cancer,the incidence and polymorphism of tumors in Zuojin Pill and Anti-Zuojin Pill were lower than those of cold and hot syndrome groups,but they had no significant effect on the occurrence of tumors in the late stage.The colorectal cancer of the cold module occurred earlier than the heat syndrome group and the results of HE staining indicated that the pathological condition was more serious.To the middle stage of the tumor,the colorectal cancer of the cold syndrome groupoccurred more slowly,and the colorectal cancer of the heat syndrome group occurred more quickly.A large number of tumors appeared in both cold and heat syndrome group in the advanced stage of the tumor,but the pathological condition of cold syndrome group was more serious.（2）The changes of histone modification detected by WB method in colorectal cancer tissues at different periods showed that H3K9ac,H3K27ac,H3K4me3,H3K9me3 and H4K20me expressions were increased in the cold syndrome group compared with the heat syndrome group in the early stage of colorectal cancer.On the contrary,the expression of H4K16ac and H3K27me3 in cold syndrome group was lower than that in heat syndrome group.There was no significant difference in histone expression in middle and late stage of colorectal cancer with cold and heat syndrome group.Zuojin pill and Anti-zuojin Pills can inhibit the expression of H3K9ac,H3K27ac,H3K4me3,H3K9me3 and H4K20me in the early stage,but have no obvious effect on the expression in the middle and late stage.（3）Enzyme-linked immunoassay（ELISA）was used to detect the expression of histone modification related enzymes in colorectal cancer tissues in different periods.The expression of methylase EZH2,G9a,SUV420H2 and deacetylated HDAC1,HDAC2,HDAC3,HDAC8 and in colon tissue supernatant of colorectal cancer rats in the early stage of colorectal cancer was increased.Conversely,LDS1 expression was reduced.Zuojin pills and Anti-zuojin Pills can inhibit the expression of EZH2,G9a,SUV420H2 and deacetylated HDAC1,HDAC2,HDAC3 and HDAC8 in the early stage,and increase the expression of LSD1,but the effect is not obvious in the middle and late stage.（4）The abnormal accumulation of collagen and elastin in colonic tissues of the cold and hot syndrome group was observed by masson and Sirius red staining and Weigert’s resorcinol fuchsin staining,and the content of collagen fibers and elastic fibers was higher in the cold and heat syndrome group than in the cold and heat syndrome groups.Collagen quantitative analysis showed that compared with the normal group,the length,width,angle and verticality of collagen in the cold syndrome groupand the hot syndrome group were increased,and the verticality of collagen in the cold syndrome group was higher than that in the hot syndrome group.The results showed that the expression of COL1,COL3,Elastin and LOXL2 was increased in cold syndrome group and heat syndrome group compared with normal group,and the expression of COL1,Elastin and LOXL2 in cold syndrome group was higher than that in heat syndrome group.The above changes were more obvious in the early stage of colorectal cancer,and there was little difference in the expression of each group in the late stage of colorectal cancer.The protein expression levels of MMP1,MMP2,MMP9 and TIMP1 increased in cold and hot modules.The expression of cold syndrome group was high.（5）The expression level of H3K4me3 in the promoter region of extracellular matrix component genes in colon tissues was investigated by Chip-qPCR.The results showed that the expression levels of activated histone modified H3K4me3 associated with activated genes were significantly increased in COLI,COLIII,Elstin,LOXL2,MMP1,MMP2,MMP9 and TIMP1 promoter regions in colon tissue at week 27 compared with the normal group.The expression levels of histone H3K4me3 in COLI,LOXL2,MMP2,MMP9 and TIMP1 promoter regions increased in cold syndrome group at week 27 compared with heat syndrome group.At week 27,Zuojin Pill and Anti-zuojin Pill could reduce the expression of histone H3K4me3 in COLI,COLIII,Elstin,LOXL2,MMP1,MMP2,MMP9 and TIMP1 promoter regions to varying degrees.Conclusion（1）The results of appearance,general condition,sign score,antisyndrome effect of Zuojin Pill and Anti-zuojin Pill as well as colonic tissue morphology and pathology showed that the model of colon cancer with cold and hot syndrome was successfully established.It is feasible to use the above method to prepare the model of colon cancer with cold and heat syndrome.It can provide a good animal model for studying the relationship between the occurrence and development of TCM syndromes,colorectal cancer,epigenetics and tumor microenvironment.（2）In the early stages of tumorigenesis,the expression of histones in colorectal cancer rats with different syndromes of cold and heat is significantly different,which has potential clinical application value for the diagnosis and treatment of colorectal cancer.The changes in histone methylase and histone acetylase levels may be one of the reasons for the changes in histone methylation and acetylation levels in colorectal cancer.（3）The differences of colonic tissue stiffness,tumor incidence and polymorphism in colorectal cancer with different syndromes of cold and heat may be related to different tumor microenvironment.The extracellular matrix（ECM）of colonic tissues in cold and heat syndrome group rats was profoundly reshaped,including angle,length,width and straightness,which had a great influence on the progression of colorectal cancer.Overexpression of LOXL2,MMP1,MMP2,MMP9 and TIMP1,accompanied by increased collagen and elastin deposition,may be one of the mechanisms leading to ECM remodeling.Understanding the role of extracellular matrix and extracellular matrix related remodeling enzymes in the pathogenesis of colon cancer is helpful to understand the molecular mechanism of colorectal cancer progression and provide new ideas for the diagnosis and treatment of CRC.（4）Zuojin pill and Anti-Zuojin pill had obvious effect on DMH-induced colorectal cancer in the early stage,but had little effect in the late stage of colorectal cancer.The antitumor effects of Zuojin pill and Anti-Zuojin pill may be related to regulating the expression of histone and histone modification enzyme,inhibiting the deposition of extracellular matrix and regulating the expression of matrix metalloproteinase.The efficacy of Zuojin pill and anti-Zuojin pill is closely related to dosage and compatibility ratio.（5）In the early stage of colorectal cancer,histone H3K4me3 may be involved in the development of colorectal cancer by regulating the expression of extracellular matrix and extracellular matrix related regulatory enzyme promoter region genes.The inhibition of Zuojin pill and anti-Zuojin pill on genes in extracellular matrix and extracellular matrix related regulatory enzyme promoter regions may be partly attributed to the inhibition of their expression by histone modification.