Based On Macrophage Polarization To Explore The Cytological Basis Of Phlegm Syndrome Of Colorectal Cancer In Mice With High Fat Diet

ObjectiveA high-fat diet can increase the risk of colorectal cancer?CRC?by affecting macrophage polarization.In this study,a mice model of CRC with phlegm syndrome?PS-CRC?was constructed by high-fat diet combined with AOM/DSS.Then Erchen decoction,a classical prescription for resolving phlegm,was used as the therapeutic treatment for the disease.The relationship between macrophage polarization and PS-CRC and therapeutic effect of Erchen decoction on CRC were clarified,and the effect of Erchen decoction on macrophage polarization was further verified in a macrophage cell culture model.The main purposes of this study were to explore the cellular and molecular mechanisms of the formation of PS-CRC,study the immune basis for the prevention and treatment of CRC by traditional Chinese medicine?TCM?,,and search the TCM drugs for CRC.Method1.In vivo experiment:110 5-week-old SPF ICR male mice were randomly divided into5 groups with two kinds of diets:control group and CRC group in normal diet,phlegm syndrome group,phlegm syndrome CRC group and phlegm resolving CRC group in high-fat diet,with 20 mice in each.For each diet part,five more mice were fed for the detection of phlegm syndrome model use in the fifth week.After 4 weeks of feeding,the signs,behaviors and blood fat of the mice and were used for assessment of the phlegm syndrome.After phlegm syndrome modeling,the phlegm resolving CRC group was administered by gavage with Erchen decoction(8.7g·kg^-1·D^-1)until the end of the experiment,and the other groups were infused with saline at the same dose.From the 8th week,the azoxymethane?AOM,0.15ml/g?was injected into CRC group and phlegm syndrome CRC group,combined with dextran sodium sulfate?DSS,2%free drinking for 1 week?in the 9th,14th and 19th week to induce CRC,the remaining groups were injected with saline and fed with drinking water at the corresponding time.The body weights of mice were measured weekly and the behavioral observation was made every week.After 26 weeks,all mice were euthanized for pathological and molecular biological study.The total cholesterol?TC?,triglyceride?TG?and blood glucose?GLU?levels in serum were tested and the occurrences of tumor were recorded.Hematoxylin eosin?HE?staining was used to observe the intestinal pathological changes of mice;immunohistochemistry was used to detect the expression of macrophage marker CD68,M1 macrophage marker iNOS,M2 macrophage marker CD206,etc.in colorectal tissues of mice in each group.2.In vitro experiment:Erchen decoction was prepared,and CCK-8 method was used to screen the concentration which had no obvious inhibitory effect on cell proliferation,mitochondrial respiratory function was analyzed by Seahorse XF96 cell energy metabolism analyzer to identify the effect of Erchen decoction on macrophage polarization.For RAW264.7?M?phase?,M1 model was induced by LPS?100ng/ml?+IFN-??20 ng/ml?.M2macrophage model was induced by IL-4?20ng/ml?.Cell morphology was observed under microscope after 24 h,Cells were collected and the expression of surface protein markers of M??F4/80+CD11b?,M1?CD11c?,M2?CD206?macrophage was detected by flow cytometry after adding antibodies.The Phenotypic signature gene expression of M1?TNF-?,iNOS?and M2?Arg-1,MMR?was analyzed by q-PCR to determine whether the models were successfully established.After polarization,the control group?M??,model group?M1,M2?,model intervention group?M1+EC,M2+EC?,and the Erchen decoction group intervened in M??EC?would be set up.Duplicated the model group and intervention group with the above-mentioned methods.Erchen decoction?2mg/ml?was added to the three intervention groups?M1+EC,M2+EC,EC?,cells were collected after 24hrs of treatment.The expression of surface markers and phenotype specific genes were detected for each group to identify the effect of Erchen decoction on the macrophage polarization.Result1.In vivo experiments:?1?Model evaluation:?1?Phlegm syndrome model:compared with the control group,the body weight of the model group,the TG and TC at the 5th week,and GLU,TG and TC at the 26th week were significantly higher?P<0.05?;after phlegm resolving,the body weight,GLU and TC in the phlegm resolving CRC group were significantly decreased compared with the phlegm syndrome CRC group?P<0.05?.?2?CRC model:During CRC modeling,the mice in CRC group and the phlegm syndrome CRC group had hematochezia and diarrhea;Comparece of tumor of the two groups were 66.7%and 92.9%respectively,which were significantly higher than that of the control group.Combined with the results of pathological analysis,CRC modeling was successful.?2?Immunohistochemical test.Compared with the control group,the expression of CD68 in the CRC group,PS-CRC group and phlegm resolving CRC group was significantly increased?P<0.05?.After the treatment of Erchen decoction,the expression of CD68 in the phlegm resolving CRC group was significantly decreased?P<0.05?.Compared with the control group,the expression of iNOS of the CRC group,phlegm syndrome group and phlegm syndrome CRC group increased significantly?P<0.05?.After Erchen decoction was used to eliminate the phlegm,the expression of iNOS in the phlegm resolving CRC group was significantly lower than that in the CRC group,phlegm syndrome group and phlegm syndrome CRC group?P<0.05?;Compared with the control group,the expression of CD206was significantly increased in the phlegm syndrome CRC group and the phlegm resolving CRC group?P<0.05?.After Erchen decoction,the expression of CD206 was decreased in the phlegm-resolving CRC group compared with the Phlegm-Syndrome CRC group,but the proportion of CD206 in total macrophages increased significantly.2.In vitro experiments:?1?Effects of Erchen decoction on the energy metabolism of macrophage RAW264.7,When at a concentration below 2 mg/ml,Erchen decoction can increase the basal and maximal respiration of macrophage RAW264.7,promote mitochondrial oxidative phosphorylation,and enhance the reserve respiratory capacity.?2?mRNA expression of M1 and M2 marker gene.Compared with the M1 model group,expression of TNF-?and iNOS gene in the M1+EC group decreased significantly?P<0.05?.Compared with the M2 model group,expression of Arg-1 in M2+EC group increased significantly?P<0.01?,and MMR showed an upward trend?P>0.05?;Arg-1 and MMR mRNA expression in the EC group with Erchen decoction intervention alone were significantly increased compared with the control group?P<0.05?.?3?Expression of marker proteins on the cell surface of M1 and M2:Compared with the M1 model group,the expression of marker protein CD11c on the cell surface in the M1+EC group decreased significantly?P<0.05?;compared with the M2 model group,the expression of CD206 in the M2+EC group showed an upward trend?P>0.05?;the expression of cell surface marker protein CD206 in EC group with Erchen decoction intervention alone was12.39 times higher than that in control group,with significant statistical difference?P<0.01?.Conclusion1.The increase of inflammatory M1 macrophages in intestine may play a role in the cytological pathophysiology of phlegm syndrome in CRC;2.Erchen decoction can promote macrophages to M2 phenotype;3.Phlegm-resolving method was suggested to regulate macrophage polarization to affect the incidence of CRC.