| Purpose: The preliminary work of this project is based on a solid foundation.Wang Wenyan,the famous Chinese doctor in the country,has achieved very good results in the clinical practice of traditional Chinese medicine for liver fibrosis for more than 20 years.In the long-term clinical practice,he has analyzed and summarized the pathogenesis of liver fibrosis,mainly including liver depression and spleen deficiency,qi stagnation and blood stasis,and the treatment principle is mainly to dredge the liver and strengthen the spleen,promote blood circulation and soften the hardness,Summarize the clinical effective experience and develop the hospital preparation Ruangan decoction for the prevention and treatment of liver fibrosis and early cirrhosis.Ruangan decoction has the effect of soothing the liver and strengthening the spleen,activating the blood and softening the firmness.It is mainly used to treat the syndrome types of liver stagnation and spleen deficiency,qi stagnation and blood stasis in bloating(liver fibrosis and cirrhosis).It is composed of bupleurum chinense,atractylodes macrocephala,turmeric,turtle shell,salvia miltiorrhiza,zedoary rhizome,cassia,corydalis,alisma orientalis,eupatorium,forsythia,and safflower seed.The main symptoms are hypochondriac pain,abdominal distension,lack of appetite,numbness,loose stools,fatigue and fatigue,or lump under the hypochondriac,purple or dull tongue,or tooth marks,ecchymosis,and thin or weak pulse can be felt.However,its biological mechanism of anti-liver fibrosis needs further study.In this study,combined with network pharmacology and animal experimental research,we explored the mechanism of Ruangan decoction on liver fibrosis.Objective: 1.Use the method of network pharmacology to study the core drugs of the empirical formula Ruangan decoction,and explore the action link,target and biological signal transduction pathway of Ruangan decoction in the treatment of nonalcoholic fatty liver fibrosis,This provides research ideas for the study of Ruangan decoction in improving and even reversing nonalcoholic fatty liver fibrosis from the perspective of basic experiments and is supported by animal experiments.2.The rat model of nonalcoholic fatty liver fibrosis was established through in vivo experiments,and the therapeutic effect and possible mechanism of Ruangan decoction on the rat model of nonalcoholic fatty liver fibrosis were further explored at the level of living animals according to the previous basis and the analysis of biological information.Material and method: 1.Using TCMSP database and consulting relevant literature,we screened the effective biological components and action targets of the empirical formula Ruangan decoction.Collect the relevant targets of liver fibrosis disease through Gene Cards website and OMIM website and conduct protein interaction and topological analysis to obtain the core target,find out the main components corresponding to the core target,further analyze the GO function enrichment and KEGG pathway enrichment,and further analyze and explore the biological signal pathway and action link of Ruangan decoction in the treatment of nonalcoholic fatty liver fibrosis.2.Except the blank control group,the rats in other groups were randomly divided into the model group,the traditional Chinese medicine Ruangan decoction group and the western medicine colchicine group,with 10 rats in each group.The model group was given 40% CCl4 by gavage according to 2mg/kg,and 2ml/kg olive oil by gavage,while the blank group was only given olive oil.40% CCl4(2mg/Kg)and olive oil(2ml/Kg)were given every four days.Interventional treatment drugs were given after the fifth week of modeling.The positive drug group was given 0.04mg/ml colchicine 1ml;The Chinese medicine group was given Ruangan decoction,which was prepared into a liquid with a concentration of 0.5 g/ml for 2 ml/d.The rest groups were given physiological saline by gavage.At the 8th week of modeling,pathological staining showed the formation of liver fibrosis in the model group.After 10 weeks,samples were taken for tests of pathology and expression level of serum IL-6 and TNF-α,AFP and ALT protein indicators,and detection of NF κ B protein,P65 mRNA,I κB-α,and the protein and mRNA expression levels of upstream and downstream molecules of TNF-α/ NF-κB pathway such as TNF-α and Caspas3.Results:1.Through the bioinformatics method,244 active ingredients of the empirical prescription of liver fibrosis,1225 targets of the empirical prescription of liver fibrosis,1075 targets of liver fibrosis diseases,and 211 targets of the empirical prescription of Ruangan Granule for the treatment of liver fibrosis were selected.Through the analysis of protein-protein interaction network and topological characteristics,it was concluded that CASP3,MAPK1,IL-2,IL-6,TNF,ESR1,AKT1,VEGFA,EGFR and other targets are potential therapeutic core targets of Ruangan Granule.GO enrichment analysis showed that the biological processes involved mainly involved ERK1 and ERK2 cascade,gene expression,cell proliferation,MAP kinase activity,RNA polymerase II promoter transcription,angiogenesis and positive regulation of smooth muscle cell proliferation,negative regulation of apoptosis process,angiogenesis and protein phosphorylation.KEGG enrichment analysis showed that the empirical formula of liver fibrosis treated liver fibrosis through TNF pathway,Fox O pathway,HIF-1 pathway,VEGF pathway,Erb B pathway and other signal pathways.2.Animal experiment(1)The HF model has been successfully replicated in this study,which can be used for clinical efficacy evaluation and new drug research.Effect on liver pathology of rats with liver fibrosis: After 10 weeks,the rats were killed,and immunohistochemical staining(HE(hematoxylin-eosin)staining)was completed.The model group showed that the hepatocytes had bullous fatty degeneration,forming a large number of collagen fibers.Hepatitis changes and pseudolobular tissue structure can be seen in local lesions.The pathological sections of the liver in the Chinese medicine group and the colchicine group showed that the fibrous septum was significantly reduced,the infiltration of focal necrotic cells,lymphocytes and neutrophils was significantly reduced,and no pseudolobular structure was found.(2)Detection of liver ALT,IL-6,TNF-α and AFP of each group by ELISA method.The experimental results showed that ALT,IL-6,TNF-α,AFP was significantly higher than that in the blank group(P<0.05 or P<0.001).Give Ruangan decoction or colchicine intervention to detect serum expressions of ALT,TNF-α,IL-6 and AFP in the Ruangan decoction group was significantly lower than that in the model group(P<0.01),indicating that Ruangan decoction could significantly reduce the serum transaminase,IL-6,TNF-α and AFP.There was no statistical difference between the Ruangan decoction group and the colchicine group.(3)Western blot and RT-PCR were used to detect the expression of key proteins and mRNA in the pathway TNF-α/ NF-κB.The experimental results showed that NF-κB protein increased significantly in the liver of rats in the model group,significantly higher than that of normal rats(P<0.001).The expression of rat liver NFκ B protein in Ruangan decoction group was significantly lower than that of model group(P<0.05).The expression of NF κ B protein in liver of rats in colchicine group also decreased significantly compared with the model group(P<0.05).The expression of P65 mRNA in the liver of rats in the model group was significantly higher than that in the normal group(P<0.001).The expression of P65 mRNA in Ruangan decoction group was significantly lower than that in model group(P<0.001).The expression of P65 mRNA protein in the liver of rats in the colchicine group was also significantly lower than that in the model group(P<0.001).The expression level of rat liver IκB-α protein in the model group was significantly higher than that in the normal group(P<0.001).The protein expression of rat liver IκB-α in Ruangan decoction group was significantly lower than that of the model group(P<0.01).Compared with the model group,the protein expression of rat liver IκB-α of the colchicine group also decreased,but the difference was not statistically significant(P>0.05).The expression of IκB α mRNA increased significantly in the model group,significantly higher than that of normal rats(P<0.001).Rat liver IκBα mRNA expression in Ruangan decoction group was significantly lower than that of model group(P<0.001).Rat liver IκBα mRNA expression in the colchicine group also decreased significantly compared with model group(P<0.001).(4)Detection key molecules expression of upstream and downstream of the pathway TNF-α/ NF-κB by Western Blot and RT-PCR.The experimental results showed that the protein level of TNF-α of rats in the model group was significantly higher than that of normal rats(P<0.001).While in Ruangan decoction group was significantly lower than that of the model group(P<0.001).In the colchicine group,the protein expression of TNF-α also decreased significantly compared with the model group,(P<0.05).The expression of TNF-α mRNA was significantly higher than that of normal rats(P<0.001).While in the Ruangan decoction group TNF-α mRNA expression was significantly lower than that of model group(P<0.01).In the colchicine group,TNF-α mRNA expression also decreased significantly compared with model group(P<0.01).Caspas 3 protein in the liver of rats in the model group was significantly higher than that in the normal group(P<0.001).While in the Ruangan decoction group,the protein expression of Caspas 3 was significantly lower than that of the model group(P<0.001).The protein expression of caspas 3 in the liver of rats in the colchicine group was also significantly lower than that in the model group(P<0.05).The expression of Caspas 3 mRNA in the liver of rats in the model group was significantly higher than that in the normal group(P<0.001),and the expression of Caspas 3 mRNA in the Ruangan decoction group was significantly lower than that of in the model group(P<0.001).The expression of caspas 3 mRNA in the liver of rats in the colchicine group was also significantly lower than that in the model group(P<0.001).(5)Immunohistochemical staining method and digital pathological image analysis based on artificial intelligence learning were used to detect the expression levels of rat liver NFκB、I κB1 and Caspas3 positive cells in each group.The results showed that the proportion of NFκ Bpositive cells in liver tissue of rats in the model group increased significantly(P<0.001).The cell proportion of expression of NFκB in the Ruangan decoction group was significantly lower than that of the model group(P<0.001),and there was no statistical difference compared with the blank control group(P>0.05).The expression of NF-κB in the Colchicine group also decreased significantly compared with the model group(P<0.001),and there was no statistical difference compared with the blank control group(P>0.05).The cell proportion of expression of IκB1in liver tissue of rats in the model group was significantly higher than that of normal rats(P<0.05).The cell proportion of expression of IκB1 in liver tissue of rats in Ruangan decoction group was significantly lower than that of the model group,but there was no statistical significance(P>0.05).The expression of IκB1 in liver tissue of rats in colchicine group showed an increasing trend compared with the model group(P<0.05).The proportion of cells expressing Caspas3 in the liver tissue of rats in the model group increased,which was higher than that of rats in the normal group,but there was no statistical difference(P>0.05).The proportion of cells expressing Caspas3 in the liver tissue of rats in the Ruangan decoction group was significantly lower than that in the model group(P<0.001).The expression of Caspas3 in the liver tissue of rats in the colchicine group showed an increasing trend compared with the model group(P<0.05).Conclusion:1.Ruangan decoction which have the effects of soothing the liver,strengthening the spleen,promoting blood circulation.Indicating qi stagnation and blood stasis,as well as liver stagnation and spleen deficiency type bloating,it is consistent with the etiology,pathogenesis,and treatment principles determined by the platform.2.Caspas3,IL-6,TNF-α,etc.may be the core targets of the empirical formula Ruangan decoction in the treatment of liver fibrosis.The treatment of liver fibrosis with Ruangan decoction may be mainly through the TNF-α/NF-κB signal pathway and other pathways.3.The whole animal experiment showed that Ruangan decoction could significantly reduce the CCl4-induced liver fibrosis rats by regulating the expressions of NF-κB、IκB1 protein and mRNA to regulate the development of liver fibrosis.4.Ruangan decoction could reduce the expression levels of TNF-α/NF-κB and inflammatory factors,apoptotic factor protein and mRNA in the upstream and downstream of signal pathway to a certain extent,inhibiting the highly activated state of key molecules in the pathway,inhibiting inflammatory reaction,and inhibiting TNF-α/ NF-κB so as to mightly inhibit the activation of HSC and anti-apoptosis,and then delayed the process of liver fibrosis,thus showing the effect of improving liver fibrosis.It is a potential anti-fibrosis drug. |
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