Research On Mechanism Of Regulation Of CD163/sCD163 Expression In Alf Rats By Jiedu Huayu Granule Though IκB/NF-κB Signaling Pathway Based On Damp-turbid Theory In Febrile Diseases Doctrine

Objective:1 Starting from the literature related to damp turbidity of epidemic febrile diseases,this paper discusses the historical origin and connotation of the pathogenic theory of"toxin evil-toxin turbidity"of liver failure,so as to provide theoretical support for the consistency of"theory,method,formula and medicine"of Jiedu Huayu Granule（JDHY）applied to acute liver failure（ALF）,promote the inheritance and innovation of epidemic febrile diseases theory,and provide solid theoretical support for the clinical application and development of TCM infectious diseases in difficult and severe cases such as liver failure.2 It is planned to explore though in vivo and in vitro experiments IκB/NF-κB signal pathway affects the expression of CD163/sCD163,and to make clear that Jiedu Huayu Granule affects IκB/NF-κB to elucidate the mechanism of CD163/sCD163 expression in the treatment of ALF and provide the curative mechanism for early prevention and treatment of ALF with traditional Chinese medicine.Methods:1 The literature research ResearchThis paper systematically arranged the literature discussion on acute yellow in the past dynasties of traditional Chinese medicine,took the theory of epidemic febrile diseases as the main guidance together with four books of the Theory of Warm heat,Differentiation of Damp Heat,Differentiation of Epodemic Febrile Diseases and Longitude and Latitude of Warm Heat as the theoretical framework,and made"acute yellow","liver failure","dampness turbidity","Damp heat"and"Jiedu Huayu Granule"as the key words in CNKI,VIP,Wanfang and other databases to collect and sort out relevant literature.The literature method,comprehensive analysis method and comparison method were applied to sort out and summarize relevant historical records and modern related works.2 The Experimental Research（1）Animal experiments:Animal experiment:ALF rat model was established by one-time intraperitoneal injection of D-galactosamine combined with LPS.Control group,ALF group,ALF+Bay group and ALF+JDHY group were set up.Peripheral serum and liver tissue of rats in each group were taken for index detection.（1）Detection of IKKβ,p-IKKβ,IκBα,p-IκBα,NF-κB,p-NF-κB,Bax,Bcl-2,CD163 and PCNA in liver tissue by Western blot;（2）Detection of IL-10,IL-6,IL-13,IL-1β,TNFα,IFNγ,and CD163m RNA levelin rat liver tissue by q PCR;（3）Detection of IL-10,IL-6,IL-13 IL-1βm,TNF-α,IFNγ,and sCD163in peripheral serum by ELISA;（4）Detection of CD⁴⁺T cells and CD⁸⁺T cells in peripheral serumby flow cytometry,and the ratio of CD⁴⁺/CD⁸⁺was calculated;（5）Detection of hepatocyte apoptosis in liver tissue by TUNEL;（6）Detection of liver histopathology by HE staining;（7）Detection of AST,ALT,ALP,TBIL,PTA and PLT in serum by biochemistry.（2）Cell experiment:The liver Kupffer cell injury model was constructed by LPS stimulation and activation.The drμg containing serum medium of Jiedu Huayu Granule was used for intervention.Control group,model group and JDHY group were set up.The cells and supernatant were collected for index detection.（1）Detection of p-nf-κB in nucleus,NF-κB,p-IκB,IκB in cytoplasm,CD163 and PCNA expression in Kupffer cells by Western blot;（2）Detection of inflammatory factor TNFα,IL-6,and anti-inflammatory factor IL-10 in cell supernatant by ELISA;（3）Detection of CD163 in Kupffer cells and the apoptosis rate of Kupffer cells by flow cytometry;（4）Detection of apoptosis of Kupffer cells by TUNEL.Results:1 The literature ResearchThis paper systematically arranges the etiology,pathogenesis and syndrome differentiation and treatment of acute yellow disease in the literature of traditional Chinese medicine,and traces the origin and development of traditional Chinese medicine’s understanding of the disease;And clarify the hierarchical interaction of multiple pathogenic factors such as"poison,dampness,heat,blood stasis and deficiency"in the pathogenesis and progress of acute liver failure layer by layer,put forward the understanding of its etiology and pathogenesis from the perspective of traditional Chinese medicine infectious diseases,and form a systematic understanding of acute liver failure with"damp and turbid theory"as the core,It enriches and develops the connotation of new pathogenic theory of"toxic evil-toxic turbidity"of liver failure,and provides a new theoretical evidence of febrile diseases for Jiedu Huayu Granule and the prescription and syndrome of acute liver failure.2 The Experimental research（1）The results of animal experiments:（1）Western blot showed that compared with the control group,the levels of CD163,PCNA,and Bcl-2 were decreased（P<0.05）,the expressions of p-IKKβ,p-IκBα,p-NF-κB and Bax in the liver tissue of the other three groups significantly increased（P<0.05）,but the expression of IKKβ,IκBαand p-NF-κB did not change significantly（P>0.05）,and the ratio of p-IKKβ/IKKβ,p-IκBα/IκBα,p-NF-κB/NF-κB increased significantly（P<0.05）;Compared with ALF group,the level of CD163,PCNA,and Bcl-2increased（P<0.05）,the expressions of p-IKKβ,p-IκBα,p-NF-κB and Bax in ALF+JDHY group and ALF+Bay Group decreased significantly（P<0.05）,but the expression of IKKβ,IκBαand p-NF-κB did not change significantly（P>0.05）,and the ratio of p-IKKβ/IKKβ,p-IκBα/IκBα,p-NF-κB/NF-κB decreased significantly（P<0.05）.（2）q PCR showed that compared with the control group,the expression levels of IL-10,IL-13 and CD163 m RNA in ALF group,ALF+JDHY group and ALF+Bay group were significantly lower（P<0.05）,IL-6 and IL-1β,TNFα,IFNγincreased significantly（P<0.05）;Compared with ALF group,the expression levels of IL-10,IL-13 and CD163 m RNA in ALF+JDHY group and ALF+Bay group were significantly higher（P<0.05）,IL-6 and IL-1β,TNFα,IFNγdecreased significantly（P<0.05）.（3）ELISA showed that compared with the control group,the levels of IL-10 and IL-13 in serum of ALF group were significantly lower（P<0.05）,and the levels of IL-6,IL-1β,TNFα,IFNγincreased significantly（P<0.05）;Compared with the ALF group,the levels of IL-10 and IL-13 in serum of ALF+JDHY group and ALF+Bay group were significantly increased（P<0.05）,and the levels of IL-6,IL-1β,TNFα,IFNγdecreased significantly（P<0.05）;compared with the control group,the expression level of sCD163 in the serum of the other three groups decreased significantly（P<0.05）;compared with the ALF group,the expression of sCD163 in serum of ALF+JDHY group and ALF+Bay group significantly increased（P<0.05）.（4）FCM showed that compared with control group,the proportion of CD⁴⁺T cells decreased,the proportion of CD⁸⁺T cells increased significantly,the ratio of CD⁴⁺/CD⁸⁺decreased and the ratio of CD⁴⁺/CD⁸⁺increased significantly in ALF group（P<0.05）;compared with the ALF group,the proportion of CD⁴⁺T cells in peripheral blood increased significantly in both ALF+JDHY group and ALF+Bay group the proportion of CD⁸⁺T cells changed not obviousy,and the ratio of CD⁴⁺/CD⁸⁺also increased（P<0.05）.（5）TUNEL test found that the number of apoptosis cells in the liver tissue of the liver failure model group was significantly higher than that in the control group（P<0.05）,and the number of apoptotic cells in ALF+JDHY group and ALF+Bay group were significantly lower than those in ALF group（P<0.05）.（6）HE staining showed that the structure of hepatocytes in control group was normal,hepatocytes were closely arranged,the nucleus was round,the lobule structure was normal,and hepatocytes and sinusoidal cells were arranged radially around the central vein;In ALF group,there were massive necrosis of hepatocytes,destruction of hepatic lobule structure,infiltration of inflammatory cells around lobules and portal vein,and blood stasis in hepatic sinuses;Compared with ALF group,hepatocyte necrosis,inflammatory cell infiltration and lobular structure were significantly reduced in ALF+JDHY group and ALF+Bay group.（7）Biochemical test showed that the levels of AST,ALT,ALP and TBIL in serum in ALF model group were higher than those in control group,while the levels of PTA and PLT were lower than those in control group（P<0.05）;The levels of AST,ALT,ALP and TBIL in ALF+JDHY group and ALF+Bay group were significantly lower than those in the model group,and the levels of PTA and PLT were higher than those in the model group（P<0.05）.In all the test results,there was no significant difference between the ALF+Bay group and the ALF+JDHY groups（P>0.05）.（2）The results of animal experiment:（1）Western Blot detection showed that p-NF-κB in cytoplasm protein and NF-κB,p-IκB,IκB in cytoplasmprotein in the model group were higher than that in the normal group and that in the JDHY group were lower;the expression of PCNA in model group decreased and that in JDHY group increased（P<0.05）.（2）The inflammatory factors as TNFα,IL-6 in the cell supernatant of the model group detected by ELISA were higher in the model group but lower in the JDHY group while the anti-inflammatory factor IL-10 in the model group was lower in the model group and higher in the JDHY group（P<0.05）.（3）FCM showed that compared with control group,the expression of CD163 in model group decreased significantly and that in jdhy group increased significantly.Compared with model group,the expression of CD163 in jdhy group increased significantly（P<0.05）;compared with the control group,the apoptosis rate of Kupffer cells increased significantly in model group and JDHY group（P<0.05）,but decreased significantly in JDHY group than in model group（P<0.05）..（4）TUNEL showed that compared with control group,Kupffer cell apoptosis increased in model group and decreased in JDHY group（P<0.05）.Conclusions:Based on the theory of"damp-turbid"of febrile diseases in ALF from the perspective of infectious diseases of traditional Chinese medicine,this paper holds that epidemic toxin lurks into the body,attacks the spleen,stomach,liver and gallbladder and other viscera,and transforms into evil such as dampness,heat and blood stasis deficiency.The toxin and other evil promote each other,become turbid with each passing day,and turbid with dampness;The damp-turbid theory of febrile diseases further interprets the connotation of the theory of"toxin evil toxin and turbidity",and provides a new theoretical evidence of febrile diseases for Jiedu Huayu Granule in the treatment of ALF.Experimental studies show that in the pathological state of ALF,“NF-κB signaling pathway（activation）-proinflammatory factors IL-6,IL-1β,TNFα,IFNγ（up-regulation）-CD163/sCD163 expression（inhibition）”cascade aggravates the progression/deterioration of ALF;Jiedu Huayu Granule can reversely regulate this cascade reaction,clear away heat and dampness,remove turbidity,detoxify and expel blood stasis,play an anti-inflammatory and immune regulating role,significantly reduce hepatocyte apoptosis,promote hepatocyte regeneration,and effectively antagonize liver failure..