Study On Mechanism Of Optimization Of Fermentation Process And Protective Effect Of Puerarin Hovenia Dulcis Seed On Alcoholic Liver Injury

Objective: Puerariae lobatae radix and Hovenia dulcis thunb are commonly used as anti-alcohol medicinal materials.Both of them contain a large number of flavonoids with anti-alcohol effect.In order to improve the content of flavonoids and enhance the anti-alcohol effect.After screening and determining the fermentation strains and extracting solvents,the optimal fermentation process of Puerariae Lobatae Radix-Hovenia dulcis Thunb was obtained by orthogonal test using multi-index comprehensive weighted scoring method,and the flavonoids before and after fermentation were analyzed.A mouse model of acute alcoholic liver injury was established to evaluate the difference in efficacy of Puerariae Lobatae Radix-Hovenia dulcis Thunb.drug pair before and after fermentation.A macrophage M1 polarization model was established to study the protective mechanism of Puerariae Lobatae Radix-Hovenia dulcis Thunb.drug pair before and after fermentation on alcoholic liver injury.Methods: 1.Select the best combination of drugs,the best extraction solvent,and the best fermentation strain based on the effectiveness of anti alcohol and anti intoxication.2.Through a single factor experiment of initial p H value,inoculation amount,inoculation ratio,and fermentation time,determine the factors and levels of the orthogonal experiment,and use the multiple index comprehensive weighted scoring method to conduct orthogonal design experiments to optimize the optimal fermentation process.3.The content of total flavonoids before and after drug fermentation was detected,and the components of puerarin,daidzein,daidzein,myricetin,and dihydromyricetin were analyzed.4.By establishing a mouse model of acute alcoholic liver injury,the levels of serum transaminase and inflammatory factors in mice and superoxide dismutase in liver were detected by enzyme linked immunosorbent assay(ELISA);HE staining,immunohistochemistry,and Western Blot methods were used to observe the pathological morphological changes in liver tissue,and to detect the regulatory effects of drugs on the expression of TLR4,My D88,ADH1,and ALDH2.5.A model of macrophage M1 polarization was established and cell polarization was detected by flow cytometry;Immunohistochemical method was used to detect the expression of polarization related proteins in liver tissue;The expression of TLR4,STAT1,SOCS3,IL-10,i NOS,and Arg1 in liver tissue was detected by Western Blot technique;To explore the protective mechanism of drugs regulating immune cell polarization against alcoholic liver injury.Results: 1.The fermentation drug pair is pueraria lobata and Hovenia dulcis.The extraction solvent is ethanol extraction,and the fermentation strain is lactobacillus plantarum+lactobacillus paracasei.2.After fermentation,the activity of AST and ALT in serum and the content of inflammatory factors IL-6 and TNF-α were decreased,and the liver cell injury caused by alcohol was inhibited.It can also increase the secretion of the main antioxidant enzyme SOD,increase the metabolic rate of reactive oxygen species in the body,and reduce the content of lipid peroxidation products.3.HE staining of liver tissue showed that compared with before fermentation,the drug pair after fermentation could maintain normal liver morphology and reduce hepatocyte necrosis.4.It was verified by Western blotting of liver tissue that Puerariae Lobatae Radix-Hovenia acerba after fermentation could up-regulate the expression of ADH1 and ALDH2 protein by down-regulating the expression of TLR4 and My D88,so as to improve ethanol metabolism and reduce inflammation in vivo.5.It was verified by Western blotting in Raw264.7 cells that ethanol could up-regulate TLR4,STAT1 and SOCS3-related polarized proteins to make macrophages tend to M1 polarization.The fermented Puerariae Lobatae Radix-Hovenia dulcis Thunb.drug pair could down-regulate the expression of TLR4,STAT1 and SOCS3,and up-regulate the expression of IL-10,i NOS and Arg1 to make macrophages tend to M2 polarization,so as to achieve the effect of preventing and treating alcoholic liver injury.Conclusion: 1.The optimal fermentation process was feasible and stable.The total flavonoid content of Puerariae Lobatae Radix-Hovenia dulcis Thunb drug pair was increased from 2.752 mg / g to 3.695 mg / g,an increase of 33 %.The content of puerarin,daidzin,daidzein and myricetin was analyzed,and the total content was increased by 30 %,which provided a new route for the extraction process of Puerariae Lobatae Radix-Hovenia dulcis Thunb drug pair.2.Compared with before fermentation,fermentation can improve the effect of Puerariae Lobatae Radix-Hovenia dulcis Thunb on enhancing ethanol metabolic activity in vivo,reduce the oxidative damage of ethanol metabolic intermediates,and protect the structural integrity of liver cells.3.Compared with before fermentation,Puerariae Lobatae Radix-Hovenia dulcis Thunb after fermentation could improve the expression of ADH1 and ALDH2 protein and improve the ability of ethanol metabolism.It can regulate the expression of TLR4,STAT1,SOCS3,IL-10,i NOS,Arg1 protein,promote the polarization of macrophages to M2 type,reduce M1 type cells,and protect the liver.