| Objective:1.To explore the pathogenesis of acute-on-chronic liver failure(ACLF)constructed under the guidance of the theory of "toxic turbidity" : the new theory of immune staging is of practical significance for the study of ACLF outcome and treatment.2.Elucidating the role of exosome mi R-223-3p/NLRP3/IL-17 pathway in immunomodulation in ACLF rats.3.Clarify the effect target of detoxification and stasis II.formula regulating the mi R-223-3p/NLRP3/IL-17 pathway of exosomes in ACLF rats.Method:1.Sort out the treatment ideas and experiences of liver failure in ancient and modern literature,and explore the intrinsic correlation between ACLF’s "toxic and turbidity-causing disease" theory and immune regulatory network.2.Observation of exosome mi R-223-3p expression in ACLF rat plasma and its correlation with biochemical indexes and the intervention effect of detoxification and stasis II: The ACLF model of rats was constructed by using CCL4 combined with D-Gal N/LPS,which was divided into normal group,cirrhosis group,model group and detoxification and stasis II.group,and after successful molding,Masson staining,HE staining was observed to observe the degree of fibrosis and necrosis of liver tissue,and the survival rate of each group was observed.Plasma extraction,differential centrifugation to isolate exosomes,TEM,NTA,WB analysis,identification of exosomes,PCR detection of exosomes mi R-223-3p expression,flow cytometry to detect the proportion of serum Th17,WB detection of serum IL-17 protein content,ELISA detection of serum NLRP3,IL-17,IL-1β,IL-18,TNFα,IL-6,AST,ALT content,automatic biochemical analyzer to detect serum TBi L.3.Observation of exosome mi R-223-3p/NLRP3/IL-17 in ACLF rat liver tissue expression law and detoxification of stasis II.regulatory effect:experimental molding,grouping method 2,liver tissue extraction after successful intervention,differential centrifugation to isolate exosomes,TEM,NTA,WB analysis,identification of exosomes,PCR quantitative analysis of exosome mi R-223-3p,PCR detection of liver tissue NLRP3,IL-17 expression,WB detection of NLRP3,WB detection of NLRP3,Protein content of IL-17,IL-1 β,IL-18,TNF α,IL-6,ELISA detected the content of liver tissue homogenate NLRP3,IL-1β,IL-18,IL-17,IL-6,TNF-α;TUNEL method detected the degree of apoptosis of hepatocytes.4.Construct a LPS-induced Kupffer immune-inflammatory damage cell model.The cells were divided into six groups:(1)blank control group,(2)LPS group,(3)mi R-223-3p miomon group,(4)mi R-223-3p miomon control group,(5)mi R-223-3p mimic + JDHY group,(6)miromic control group +JDHY group,each group added interventions,PCR detected the expression of cell mi R-223-3p,NLRP3,IL-17,WB detected the protein content of cell NLRP3,IL-17,ELISA detects the content of cells IL-1β,IL-18,IL-6,TNF-α.Results:1.As the cause of liver failure,the "poison" of Traditional Chinese medicine is said in ancient and modern times,and ancient doctors refer to "poison" as the evil of external epidemic and the state of various evils.Modern physicians represented by "poison turbidity" have expanded the concept of "poison",believing that "poison" is divided into inside and outside,both or cause disease alone,or both,combined with the modern concept of immunology,the new strategy of immunization staging treatment of Chinese medicine ACLF has been constructed,and the essence of "turbidity" is mainly a series of immune response products produced by various imbalanced immune substances and their interactions in the process of the body’s immune response,which partially overlaps with the concept of "poison",which is equivalent to the precursor part of "internal poison",and the pathogenic damage is not as good as "poison" "Severe,but sickly,tricky to treat." Detoxification and stasis II.is a representative of the immune breakthrough stage and hyperactivity phase in the ACLF immune staging treatment strategy.2.Plasma exosome mi R-223-3p decreased in the LC group and ac LF group,and the ac LF decline was more obvious(P<0.05),the JDHY group plasma exosome mi R-223-3p was significantly higher than that of ac LF group(P<0.05);NLRP3 and IL-17 were increased in the LC group and ACLF group,and the ACLF increase was more significant(P<0.05),JDHY group NLRP3,IL-1β,IL-18,IL-17,IL-6 and TNF-α were significantly lower than those in aclf group(P<0.05);TBi L,AST,ALT were increased in LC group and ACLF group,and ACLF was more significantly elevated(P<0.05),TBi L,AST and ALT in JDHY group decreased significantly compared with ACLF group(P<0.05),rat survival rate was increased(P<0.05),plasma exosome mi R-223-3p expression and TBi L,AST,The content of ALT was negatively correlated and positively correlated with survival.3.The hepatic tissue exosome mi R-223-3p decreased in the LC group and the ACLF group,and the ac LF decreased more significantly(P<0.05),and the hepatic tissue exosome mi R-223-3p in the JDHY group was significantly higher than that in the ACLF group(P<0.05);NLRP3,IL-1β,IL-18,Th17,IL-17,IL-6,TNF-α,and hepatocellular apoptosis rate were increased in the LC group and the ACLF group.Compared with< the ACLF group,NLRP3,IL-1β,IL-18,Th17,IL-17,IL-6,TNF-α,and hepatocyte apoptosis decreased significantly(P<0.05)in the JHDY group and the ACLF group.4.Cell experimental results:(1)compared with the normal group,the mi R-223-3p of the LPS group was reduced(P<0.05),NLRP3,IL-1β,IL-18,IL-17,IL-6,TNF-α were all elevated(P<0.05);(2)Compared with the LPS group and the control group,the mi R-223-3p of the mi R-223-3p of the milime group(P<0.05),NLRP3,IL-1β,IL-18,IL-17,IL-6,IL-6,TNF-α decrease(P<0.05);(3)Compared with the LPS group,the expression upregulation of mi R-223-3p in the JDHY group and the mi R-223-3p group of the SIMULATION group + JDHY group(P<0.05),NLRP3,IL-1β,IL-18,IL-17,IL-6,TNF-α level decreased(P<0.05);(4)compared with the simple mimic group,Mi R-223-3p levels in the mimetic group + JDHY group increased(P<0.05),NLRP3,IL-1 β,IL-18,IL-17,IL-6,TNF-α levels decreased(P<0.05);(5)Mi R-223-3p was significantly higher(P<0.05),NLRP3,IL-1β,IL-18,IL-17,IL-6,IL-6,TNF-α decreased(P<0.05).Conclusion:1.The combination of ACLF TCM "poisoning and turbidity" theory and modern immunology helps to interpret the new immune staging strategy of ACLF in TCM,and plays a certain role in enlightening the clinical and basic research of ACLF.Detoxification and stasis II.is a representative of the immune breakthrough stage and hyperactivity stage in the treatment of ACLF immune staging.2.The activation of exosome mi R-223-3p can downregulate the expression of NLRP3,reduce IL-1β,IL-18,inhibit Th17 cell activity,reduce IL-17,IL-6,TNF-α levels,antagonize hepatocyte apoptosis,reduce ACLF hepatocellular inflammatory necrosis,and protect liver function.The exosome mi R-223-3p/NLRP3/IL-17 pathway participates in the disease process of ACLF by regulating the Th17 cell immune network.3.Detoxification and stasis II.formula is inhibited by exosome mi R-223-3p/NLRP3/IL-17 pathway.The activity of the Th17/IL-17 inflammatory axis,inhibiting excessive hyperactivity of T cell immune response,reducing immune damage,antagonizing hepatocyte apoptosis,reducing hepatic inflammatory necrosis,reversing ACLF,improving survival rate,has the effect of re-establishing ACLF immune balance. |
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