Study On The Mechanism Of Gexia Zhuyu Decoction In Preventing And Treating Metabolic Associated Fatty Liver Disease By Inhibiting S1P-related Inflammatory Reaction

【Purposes】In this study,cell experiments and animal experiments were conducted to explore the preventive and therapeutic effect and related mechanism of Gexia Zhuyu Decoction on Metabolic Associated Fatty Liver Disease(MAFLD)from the point of view of inhibiting S1P-related inflammatory reaction.【Methods】Animal experiment:After one week of adaptive feeding,36 SD rats were randomly divided into 6 groups:normal group(Con),model group(Model),low dose group of Gexia Zhuyu Decoction(GXZYT-L),middle dose group of Gexia Zhuyu Decoction(GXZYT-M),high dose group of Gexia Zhuyu Decoction(GXZYT-H)and aspirin group(AS).Among them,the normal group was fed ordinary feed,and the other groups were fed a high-fat diet with free drinking water.The rats in each group were given intragastric administration of drugs from the day of modeling.The low,middle and high dose groups of Gexia Zhuyu Decoction were gavaged according to the crude drugs of 7.2//kg/d,14.4//kg/d and28.8g//kg/d respectively,the aspirin group was perfused according to the dose of 9mg/kg/d,and the normal group and model group were given the same amount of normal saline.The liver index was calculated by measuring the body weight and liver weight of rats and the pathological changes of the liver were observed by liver morphology,HE and oil red O,including steatosis,lipid deposition and inflammatory cell infiltration.Serum liver function(ALT,AST and GGT),bilirubin(TBIL and DBIL),blood lipids(TC,TG,LDL-C,HDL-C),liver lipids(TC,TG),serum and liver inflammatory cytokines(IL-1α,IL-1β,TNF-α,IL-6)were detected by biochemical kit,and hemorheology related indexes were detected by automatic rheometer.The platelet aggregation rate and adhesion rate were measured by platelet aggregation meter and adhesion meter ball,the levels of plasma PAF and CD62 P and S1 P in serum and liver were detected by biochemical kit,and the protein expressions of SPHK1,S1PR1,S1PR2 and S1PR3 in liver tissue were detected by Westemblot.Cell experiment:The concentration of FFA for establishing MAFLD cell model was screened by CCK-8 kit,and the success of MAFLD cell model was determined by oil red O staining and TC and TG detection.After successful establishment of the model,normal group(Control),model group(Model),low dose group of Gexia Zhuyu Decoction(GXZYT-L),middle dose group of Gexia Zhuyu Decoction(GXZYT-M),high dose group of Gexia Zhuyu Decoction(GXZYT-H)and aspirin group(AS)were set up.The lipid deposition in MAFLD cell model was observed by oil red O staining,TC and TG detection.QPCR was used to detect the m RNA expression of NF-kappa B / p65,TNF-α,IL-6 and SGPL1 in MAFLD cell model.【Result】Animal experiment:(1)Compared with the normal group,the body weight,liver wet weight and liver index of the model group were significantly higher than those of the model group(P<0.05).The body weight,liver wet weight and liver index of rats in the Gexia Zhuyu decoction and aspirin group decreased significantly(P <0.05).(2)The morphological observation showed that the liver of the model group was enlarged,and dark yellow,the capsule was tense and the edge was round and obtuse.Compared with the model group,the liver was significantly improved in Gexia Zhuyu decoction and aspirin group.The results of HE staining showed that in the model group,hepatocytes were arranged disorderly,and there were a large number of fat vacuoles,accompanied by balloon degeneration,severe damage of hepatic lobules,infiltration of inflammatory cells and necrotic lesions.Gexia Zhuyu decoction and aspirin can significantly reduce fat vacuoles and balloon-like changes,and relieve inflammatory cell infiltration.The results of oil red O staining showed that there were a large number of red lipid droplets and inflammatory cell infiltration in the model group,and Gexia Zhuyu decoction and aspirin could significantly reduce the liver lipid droplets and reduce the infiltration of inflammatory cells.(3)Compared with the normal group,the levels of serum liver function(ALT,AST and GGT),bilirubin(TBIL and DBIL),blood lipids(TC,TG,LDL-C,HDL-C),liver lipids(TC,TG),serum and liver inflammatory cytokines(IL-1α,IL-1β,TNF-α,IL-6)increased significantly in the model group.Compared with the model group,Gexia Zhuyu decoction and aspirin could significantly reduce the above-mentioned indexes(P <0.05).(4)Compared with the normal group,the hemorheological indexes of the model group were significantly higher(P < 0.05),including whole blood viscosity(high,middle and low shear),hematocrit,erythrocyte sedimentation rate,plasma viscosity and fibrinogen.Compared with the model group,Gexia Zhuyu decoction and aspirin could reduce the related indexes of hemorheology(P <0.05).(5)Compared with the normal group,the levels of plasma PAF and CD62 P in the model group were significantly higher than those in the model group,and the levels of plasma PAF and CD62 P in Gexia Zhuyu decoction and aspirin group were significantly lower than those in the model group.(6)Compared with the normal group,the level of S1 P in serum and liver in the model group increased significantly(P < 0.05),and compared with the model group,the levels of S1 P in serum and liver in Gexia Zhuyu decoction and aspirin group decreased significantly(P <0.05).(7)Compared with the normal group,the expressions of SPHK1,S1PR1,S1PR2 and S1PR3 in the liver tissue of the model group were significantly increased,and compared with the model group,the expressions of SPHK1,S1PR1,S1PR2 and S1PR3 in the liver tissue of rats in Gexia Zhuyu decoction and aspirin group were significantly decreased.Cell experiment:(1)After L02 cells were treated with 1m M FFA for 24 hours,oil red O showed a large number of red lipid droplets in hepatocytes;compared with the normal group,the contents of TC and TG in the model group increased,indicating that the MAFLD cell model was established successfully.(2)Compared with the normal group,the contents of TC and TG in the model group were significantly higher than those in the model group,and the levels of TC and TG in MAFLD cell model in the Gexia Zhuyu decoction and aspirin group were significantly lower than those in the model group.The results of oil red O staining showed that a large number of red lipid droplets appeared in the model group,which surrounded the cells and even connected them into slices.The lipid droplets were significantly decreased in the low,middle and high-dose serum and aspirin-containing serum group.(3)Compared with the normal group,the expressions of NF-κB/p65,TNF-α and IL-6m RNA in the model group were significantly increased,and compared with the model group,the expressions of NF-κB/p65,TNF-α and IL-6 m RNA in MAFLD cell model were significantly decreased in Gexia Zhuyu decoction group and aspirin group.(4)Compared with the normal group,the expression of SGPL1 m RNA in the model group was significantly increased,and the expression of SGPL1 m RNA in MAFLD cell model was significantly decreased in the Gexia Zhuyu decoction and aspirin group compared with the model group.【Conclusion】The prevention and treatment of MAFLD with Gexia Zhuyu decoction may inhibit S1P-related inflammatory reaction by reducing the synthesis of S1 P,increasing the degradation of S1 P and hindering the activation of S1 P specific receptor.