Investigation Of Paeonol-Geniposide On Acute Alcoholic Liver Injury Based On Uniform Design Method

Objective:To explore the optimal dosage and compatibility effect of Paeonol and Geniposide composition on acute alcoholic liver injury in mice,and to explore the role of p38MAPK/JNK/NF-κB signaling pathway in Pae-Gen alleviating acute alcoholic liver injury in mice.Methods:In this study,Lieber-De Carli alcohol liquid diet was used to induce the mouse model of acute alcoholic liver injury.The experimental grouping scheme was designed by uniform design method,and the combined dosage of two drugs was designed by two factors and six levels uniform design method.A total of 64 C57BL/6male mice were randomly divided into eight groups:blank group,model group,and Uniform Design 1–6 groups.The rats were modeled for a total of 16 days,with the first 5 days as the transition period of liquid feed.On the 6th to 15th days,the model group was fed with full alcohol liquid feed and given the corresponding drug intervention by gavage,while the control group was given the corresponding normal saline by gavage.The rats in the model group were given an acute alcohol gavage at 7 am on the 16th day,while those in the control group were given corresponding dextrin by gavage and the samples were taken at 4 pm.The activity status and diet of the mice were observed during the modeling period.After the modeling,the serum and liver samples were collected and stored for further test.The pathological changes in the liver tissue of the mice were observed by HE staining,and the contents of TC and TG in the liver tissue were detected.With liver tissue TC and TC content as screening indicators,the regression equation was obtained by quadratic polynomial regression analysis through DPS data processing system,and the combined dose of Pae and Gen was determined to be the"optimal formula"when the liver tissue TC and TG content was the lowest.The validation test was conducted using the same modeling method as the screening test with the single components Pae and Gen in the formulation and in the formulation as the observation objects and silybin as the reference.According to the results of the screening test,48 C57BL/6 male mice were divided into Control,Model,Pae+Gen,Pae,Gen and silybin groups.After modeling,mouse serum and liver were taken and stored for further test.The pathological changes in the liver tissue of the mice were observed by HE staining.The kit was used to detect liver injury indicators TC,TG,ALT and AST levels in the mice,and to detect oxidative stress-related indicators in the liver tissue:reduced GSH,CAT,SOD and MDA.The levels of serum inflammatory factors IL-1β,IL-6,and TNF-αwere detected by ELISA.The expression levels of p38MAPK,JNK,and NF-κB P65-related proteins in the liver were detected by Western-blot,IHC method,and immunofluorescence.Results:In that proportion dose screening experiment,compared with the blank control group,there was significant steatosis in the liver tissue of the model group.The contents of TC and TG in mouse liver tissue were dependent variables Y₁,Y₂,Gen and Pae as independent variables x₁and x₂,respectively.The regression equation was obtained after quadraticpolynomialregressionanalysis:Y₁=0.151 8-0.000 918x₂+0.000 001 48x₁²+0.000 002 07x₂²+0.000 000 38x₁x₂;Y₂=0.663 2-0.001 14x₂+0.000 024 3x₁²+0.000 002 9x₂²-0.000 005 655x₁x₂,（adjusted r₁²=0.874,P<0.001;r₂²=0.65,P<0.01）.It was concluded that when x₁=20 mg·Kg^-1,and x₂=220mg·Kg^-1,the expected efficacy of the two drugs was the best.In the verification test,compared with the blank group,the liver tissue of the model group had significant fatty degeneration,and the liver specific gravity was increased.The serum ALT and AST levels and liver tissue TC and TG levels were significantly increased.The levels of GSH,CAT and SOD related to oxidative stress were decreased,while the level of MDA was increased.The levels of serum inflammatory factors IL-1β,IL-6,and TNF-αwere significantly increased.The expressions of NF-κB P65,p-p38 MAPK and p-JNK proteins in liver tissue were up-regulated.After the intervention with Pae and Gen,the liver injury index,liver lipid level,oxidation products and inflammatory factors were significantly reduced,the antioxidant enzyme activity was significantly increased,and the protein expression levels of p-p38MAPK,p-JNK and NF-κB P65 were significantly down-regulated.The combination group of Pae and Gen had better effects on alcoholic liver injury than the single drug group.Conclusion:The optimal dosage of Pae-Gen could be effectively optimized by uniform design and pharmacodynamic analysis.The combination of the two drugs could reduce the alcoholic liver injury by reducing the lipid level in mouse liver tissue,oxidative stress injury and inflammatory response,and its effect might be related to the targeting of p38MAPK/JNK/NF-κB channel.