| Objective:1.The connotation of "Dampness-heat theory",the causes and pathogenic characteristics of dampness-heat,and the relationship between the occurrence of dampness-heat and visceral manifestation were systematically studied so as to form a relatively complete theoretical system of dampness-heat.The pathogenesis and intervention methods ofcolorectal cancer with dampness-heat ftagnation syndrome(SRYJ)were analyzed based on the theory of"Dampness-heat".2.To identify the characteristic biological indicators that colorectal cancer(CRC)patients differentiated fromother TCM syndrome typesthrough clinical research.3.Animal experiments were conducted to further demonstrate the effectiveness of Huangqintang decoction(HQT)combined with Radix Actinidiae chinensis(TLG)under the guidance of "Dampness-heat theory" in preventing and treating CRC with SRYJ,and preliminarily reveal its mechanism.Methods:1.By analyzing ancient and modern literatures,the causes of"dampness","heat","dampness-heat" and "dampness-heat hidden pathogen",as well as the pathogenic characteristics,and the connotation and value of "dampness-heat"theory were summarized;Meanwhile,the pathogenesis and therapeutical principle and methods of CRC with SRYJ were excavated to illuminated the feasibility of HQT and TLG combination on CRC treatment.2.Representative clinical CRC patients with different traditional Chinese medicine syndromes were selected and their KPS scores were performed.Automatic biochemical analyzer was used to detect serum ALB and biochemical index,flow cytometry was applied to analyze peripheral blood T lymphocyte subtypes.ELISA was used to detect serum levels of inflammatory factors such as IL-2,IL-6,IL-10 and IL-17 as well as tumor markers such as CEA,CA125 and CA199.16S rRNA was used to analyze the structural changes of intestinal flora.3.The mice model of CRC with SRYJ was established by intraperitoneal injection of AOM combined with 2.5%DSS solution drinking,and at the first 2 weeks supplemented by humid and hot environment intervention.Celecxib,HQT,TLG,and their combination(HQT+TLG)were administered at the same time.The physical signs,the number of death,and the symptoms of "Dampness-heat" sydrome were observed daily.At the end of the experiment,the colorectal tissue was dissected,and the tumor formation was observed and counted.HE staining and Masson staining were used to observe the histopathological changes of colon.Expression levels of TNF-α,IL-6 and IL-10 in colorectal tissue were detected byELISA,and the expression of TNF-α were observed by immunofluorescence.TUNEL method was used to observe theapoptosis of tumorcells,and immunohistochemistry was used to observe the expression of Ki-67 in tumor tissues.The mRNA expression levels of Claudin-1,Occludin,ZO-1 and IL-6 and IL-17 were detected by RT-PCR,and expression of Occludin,ZO-1,NF-κB and STAT3 protein levels were detected by Western blot.The composition of intestinal flora was analyzed by 16S rRNA.Results:1.The theory of "Dampness-heat" is generated from the theories of"dampness","heat","dampness-heat" and "insidious pathogenesis".The development of pathogenic factors of dampness and heat determine the etiologes of exogenous and endogenous dampness-heat,insidious pathogenesis,and the occurrence of the interaction between dampness and heat interaction.The most representative clinical syndrome type of CRC is SRYJ,and its main pathogenesis is lacking of vital Qi,excessive pathogenic factors,body deficiency,insidious of dampness and heat,and intestinal lesions.And the combination of HQT and TLG with functions of invigoratingthe spleen to dispeldampness and clearing away heat to remove pathogenic factors is an effective clinical prescription for the treatment of CRC with SRYJ syndrome.2.Clinical case analysis showed that KPS score of SRYJ type CRC patients was higher than that of Spleen and kidney Yin deficiency(PSYX)type.The serum ALB levels of CRC patients with SRYJsyndromewas significantly higher than that of other syndrome types.The direct bilirubin(Dbil)and serum creatinine(Scr)levels were higher in SRYJ and deficiency of Qi and blood(QXKX)colorectal cancer patients.The results of flow cytometry showed that the proportion of CD3-T cells in SRYJ CRC patients was relatively high,the proportion of CD4/CD8 was significantly higher than that of QXKX and stagnation ofliver Qi(GQYJ)CRC patients,and the serum CEA and CA199 levels were lower than other types of CRC.The levels of IL-6 and IL-12 in SRYJ and PSYX patients were significantly higher than those in GQYJ,the levels of IL-17 and IL-10 were higher in SRYJ and pixu spleen deficient and dampness resistance(PXSZ)patients,and the serum levels of IL-2 were lower in SRYJ patients.The results of bacterial community analysis showed that the intestinal flora diversity of SRYJ patients was lower.Ovatus,Stercorer,Citroniae and Gnavus had specific distribution,and Copri was the dominant flora of SRYJ CRC patients.3.Animal experiments results showed that AOM combined with drinking 2.5%DSS water solution and hygrothermal environment could successfully induce CRC model with symptoms of SRYJ,such as lethargy,thin body and loose stools.The tumorigenesis rate of survival mice in each group was 100%.HQT+TLG could significantly reduce the mortality of model mice,improve intestinal mucosal inflammatory cell infiltration and reduce high-grade intraepithelial neoplasia in model mice.HQT+TLG can reduce the levels of IL-6 and TNF-α in the colorectal tissues of model mice,and increase the total number of CD3-T cells,the proportion of CD3-CD4-T cells and the ratio of CD4/CD8.HQT+TLG can reduce mRNA expression of IL-6 and IL-17 in colorectal tissue,andcan significantly reduce the mRNA expressionof Claudin-1.HQT+TLG also could significantly change the composition of intestinal flora and increase the abundance of Firmicutes and Patescibacteria.Conclusions:1.The traditional Chinese medicine theory of "Dampness-heat"can effectively guide the diagnosis and treatment of disease with dampness-heatsyndrome;Invigoratingthe spleen function to dispel dampness and clearing away heat to remove pathogenic factors is an important method for the treatment of disease with dampness-heatsyndrome,and HQT combined with TLG is an effective combination with aforementioned functions.2.KPS score combined with serum ALB and inflammatory factor levels can be used as clinical indicators to distinguish different syndromes of CRC patients,and increased serum ALB and IL-1 7 levels may be the characteristic biological indicators ofCRC patients with SRYJ.Copri may be its characteristic flora.3.HQT and TLG combination has a good effect on improving the symptoms of damp-heat accumulation syndrome and inhibiting the progress of CRC induced by AOM-DSS and hygrothermal environmenttreatment.This function may be related to improving the composition of intestinal flora,regulating the proportion of T cell subsets in colorectal lymphoid tissue,down-regulating the expression of Claudin-1,and inhibitingactivation of IL-6/STAT3 signaling pathway to improve inflammatory response and abnormal hyperplasia of intestinal epithelium. |
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