Pueraria Flos Alleviates Alcoholic Liver Injury Via Regulation of PPAR α and MAOA: Deciphering the Effective Forms and Potential Mechanism Based on a Bioinformatics-Integrated Metabolic Prole Strategy and Experimental Validation

12 Background: Pueraria Flos, a representative medicinal and edible antidote for alcoholism, has 13 rich clinical experience and remarkable curative effect in the treatment of alcoholic liver disease 14 (ALD). However, its effective forms and hepatoprotective mechanism are remained unknown. 15 Methods: A strategy based on UPLC-QTOF-MS combined with mass defect filtering technique 16 was established for comprehensively identifying prototypes and metabolites absorbed and 17 excreted into rat plasma, urine, bile and feces after oral administration . Then, the absorbed 18 constituents with a relative high level were subjected to the network pharmacology, functional 19 enrichment analysis and molecular docking to clarify the potential mechanism in the treatment 20 of ALD . Furthermore, the therapeutic effect of PF on ALD and predicted mechanisms was 21 further evaluated using a rat model of alcohol-induced liver injury and Western blot analysis. 22 Results: 25 absorbed prototype constituents and 82 metabolites were identified or tentatively 23 characterized with glucuronidation, sulfation, methylation, hydroxylation and reduction as their 24 major metabolic pathways. The constructed absorbed constituent-target-pathway-disease 25 network and docking analysis revealed that 4 metabolic components Te-7XG, genistein-7G-4'S, 26 tectoridin-4'S and Te-7XG-4'S, 2 targets MAOA and PPARA, and 6 pathways related to lipid 27 regulation and amino acid metabolism may play crucial roles in the PF mediated protection 28 against ALD. An in vivo validation in rat further demonstrated that PF alleviated liver injury 29 via activating and suppressing the PPARA and MAOA expression, respectively. 30 Conclusions: The present results not only increase the understanding on the effective form and 31 molecular mechanism of PF mediated protection aga inst ALD, but also promote better 32 application of PF as supplement food and herbal medicine for the treatment of ALD.


Introduction
With the development of social economy, the alcoholic drinkers have been increasing in 41 recent years. Alcohol abuse and alcoholism have emerged as a common public health problem 42 all round the world. As an important organ responsible for alcohol metabolism, the liver is the 43 main target organ for alcohol toxicity. Accordingly, the incidence rate of alcoholic liver disease 44 (ALD) caused by long-term heavy drinking is increasing year by year. Therefore, exploring the  were explored. Furthermore, the predicted key targets of PF against ALD were validated in an 100 alcohol-induced liver injury rat model, which would promote better application of PF that is a 101 medical resource for developing a supplement food or an herbal medicine for the treatment of 102 ALD ( Figure 1).  163 The condition of chromatographic separation and mass detection was almost the same as 164 those reported in literature [11]. The only difference is the change of elution gradient, which 165 was listed as follows: 0-1 min, 5%-8% B; 1-8 min, 8%-13% B; 8-9.5 min, 13%-15% B; 9.  190 The absorbed constituent-target-pathway-disease network was constructed by utilizing the 191 network visualization software Cytoscape 3.2.1, which supplies a method for data integration, 192 analysis and visualization for complicated network analysis. In the network plot, a "node" 193 signifies an ingredient, target or pathway, an "edge" represents interaction among different 194 targets. The "degree" of a node was in agreement with the number of its connected edges. meaningful, which mean that there was a binding between constituents and targets. the protein was transferred using a PVDF and then blocked in 5% milk with TBST for 2 hours.

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The membranes were immersed in respective primary antibody of PPARα (1:1000 dilution),   235 All values were expressed as mean ± standard deviation (SD). Differences between 236 different groups were analyzed with one-way analysis of variance (ANOVA) using GraphPad Prism 8.0.1. The value p < 0.05 was considered as statistically significant. 240 In order to identify the absorbed constituents and in vivo metabolites in rats, a total of 43 In total, 25 prototype components and 82 metabolites, including 93 flavonoids, 13 252 saponins and 1 phenolic acid were detected in vivo. In addition, the peak area of each absorbed 253 constituent was also recorded automatically using the MetaboLynx TM system. After 254 summarizing the peak area of all constituents, the percentage that calculated by the ratio of their 255 peak area to total peak area in each biological sample was described as relative content. The 256 detail data of identified results were listed in Table 1. These compounds could be generally 257 divided into two categories, namely flavonoids−related and saponins−related metabolites. was 16 Da (O) higher than that of aglycone, which was identified as 1-hydroxyl-soyasapogenol 304 B as C-1 was the active site according to related research [18].

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P22~P25 and M77 could be grouped due to possessing the same aglycone sophoradiol.     1 In order to understand important effective components, the relative content of each 2 metabolite was calculated by area normalization and expressed as the percentage of its peak 3 area to the total peak areas in each kind of bio-samples (Table 1). 13 candidate components that 4 were detected in rat plasma and/or bile samples with relative content more than 3% were 5 screened for the further network pharmacology analysis. In all, 104 and 5338 related genes were 6 obtained as the targets of compounds and disease after removing duplicates, respectively. Their

Experimental validations of the pharmacological effects and the molecular mechanisms
76 of PF against ALD 77 We further verified the pharmacological effects and the prediction mechanisms of PF 78 against ALD based on the rat model. As shown in Figure 6A, ALT and AST levels were

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In this paper, the metabolic profile of PF in vivo was systematically described based on   Table 1