The Anti-Inflammatory Mechanism Study Of Huanglian Jiedu Decoction By The Regulation Of Lipid Metabolism In LPS Induced Zebrafish And High-Fat-Feeding APP/PS1 Mice

Huanglian jiedu decoction(HLJDD)was the classical traditional Chinese medicine prescription with heat-dissipating and detoxifying effects.The prescription was composed of Rhizoma coptidis,Radix scutellariae,Cortex phellodendri and Fructus gardenia at a ratio of 3:2:2:3.It had the effect of purging heat and detoxification.HLJDD had clear clinical pharmacodynamic studies in the treatment of inflammation.It could improve the redness,swelling,heat and pain caused by the inflammatory response.HLJDD could also regulate the central nervous inflammation.However,the current studies mostly focused on the evaluation of anti-inflammatory activity of HLJDD.The biological characteristics and mechanism of its "heat-clearing and detoxification" needed to be further clarified.The inflammatory response was closely related to the disorder of lipid metabolism.The proportion of phospholipids,sphingolipids and glycerolipids in cell membrane was an important characteristic of inflammatory response.The significant role of polyunsaturated fatty acids and their metabolites(lipid peroxides)in the process of promoting and resolving inflammation has been widely recognized.The disorder of lipid metabolism could accelerate the process of inflammatory degeneration.The inflammatory reaction would aggravate the imbalance of lipid metabolism and lead to the disorder of lipid metabolism.HLJDD could significantly increase the activity of lipid-metabolizing enzymes and improve the lipid levels,thus to regulate the lipid homeostasis effectively.The regulation of lipid metabolism might become an important target of HLJDD in the treatment of inflammation.In this paper,from LPS induced the systemic inflammatory response in zebrafish to the central nervous system inflammation of high-fat-feeding APP/PS1 mice,the development of inflammation could be improved by HLJDD based on the regulation of lipid metabolism.1 Study on the mechanism of HLJDD and its main active components to improve LPS-induced zebrafish inflammation.Fluorescent labeling of transgenic neutrophil zebrafish was adopted.LPS was injected into the yolk sac to establish the zebrafish inflammation model.The number of neutrophils in the yolk sac was used as the pharmacodynamic evaluation index.The results of pharmacodynamic evaluation showed that,compared with control group,after LPS stimulation,the number of neutrophils in zebrafish increased significantly.The anti-inflammatory activities of HLJDD and its three main components were evident.HLJDD had the strongest efficacy among four drugs.The expressions of 6 major proteins and 5 inflammatory factors in the TLR4/MyD88 pathway of zebrafish were detected by quantitative RT-PCR technique.All of the 11 indexes presented a significant difference in LPS group compared with control group.After HLJDD treatment,compared with the LPS group,the indexes exhibited to reverse back to the normal level in different degrees.The results of correlation analysis between kinase proteins and inflammatory cytokines suggested that HLJDD mainly regulated the upstream MyD88 connector molecule to improve the expression of inflammatory factors.Berberine was strongly correlated with NF-κB and ERK pathway.Baicalin and geniposide were strongly correlated with MAPK pathway.Based on UPLC-Q Exactive-MS/MS technology,non-targeted and targeted lipid omics analysis were carried out.The obtained data were analyzed by principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).The potential lipid biomarkers of VIP>1 and P<0.05 were selected to explore the mechanism of HLJDD based on lipid metabolism regulation to improve the inflammation induced by LPS in zebrafish.The results showed that most of the lipid markers were PCs,PEs,SMs,Cers and TGs.Then we focused on theses lipids to perform the targeted lipidomics.A total of 25 pathological potential lipid markers significantly changed and all of them were strongly correlated with each other,which supported the underlying explanation for the pathogenesis of inflammation induced by LPS.The lipids perturbations mostly ameliorated by HLJDD were mainly based on the glycerophospholipid metabolic pathway according to the KEGG database analysis.Finally,the network visualization of "potential lipid biomarkers-TLR4/MyD88 signaling pathway" for the anti-inflammatory effect of HLJDD has been constructed.The corresponding lipoproteins such as PLA2,SGMS and SMDP were observed as important intermediates between lipid homeostasis and the TLR4/MyD88 signaling pathway.Maintaining the homeostasis balance of lipid metabolism was one of the important mechanisms of HLJDD anti-inflammatory effects.2 Study on the mechanism of HLJDD to improve the central nervous system inflammation in high-fat-feeding APP/PS1 miceQuantitative RT-PCR was used to detect the mRNA level of mice brain inflammatory cytokines TNF-α and IL-1β,respectively.The results showed that,compared with the control group,the expression of the TNF-α and IL-1β in the high-fat-feeding AD group increased significantly.After drug intervention,low and high doses of HLJDD could dose-dependently reduce the mRNTA levels of TNF-α and IL-1β.The high-dose HLJDD group exhibited the significant difference(P<0.001).UPLC-AB Sciex Triple Quad 6500+ was used to establish the method for the detection of neurotransmitters in the mice brain samples.The concentration changes of neurotransmitters in high-fat-feeding AD mice were determined after HLJDD treatment.The results of methodology verification showed the UPLC-MS/MS method was suitable for the determination of endogenous neurotransmitters in large quantities of biological samples.Compared with the control group,14 indexes showed significant changes in the high-fat-feeding AD group(P<0.05).After HLJDD treatment,most of these indexes had a tendency to reverse back.Among of them,5 indexes showed significant differences(P<0.05).Lysine and arginine showed dose-dependent improvement after HLJDD administration.Through the pathway enrichment analysis,HLJDD could improve the metabolism of amino acids.There was a tendency to improve the excitatory amino acid,inhibitory amino acid and cholinergic system.Some of them had significant differences.Based on the results of pharmacodynamic evaluation,the mechanism of HLJDD in improving central nervous system inflammation was further explored on the lipid metabolism.Based on the changes of lipid on the cell membrane,the semi-quantitative targeted lipids were studied by UPLC-QQQ-MS/MS method focusing on phospholipids and sphingolipids.The results showed that compared with the control group,26 lipids showed significant changes in the high-fat-feeding AD group(P<0.05).14 lipids had a tendency to reverse back after HLJDD administration.PC(20:5/22:6),PE(O-18:1/22:6)and PE(16:0/20:4)showed significant differences after HLJDD administration.COX-2,5-LOX.12-LOX and CYP4A12a enzymes in the high-fat-feeding AD mice were quantitatively determined to elucidate the changes of specific enzymes in the central nervous system,and explored the regulatory mechanism of HLJDD treatment.Western blot results showed that,compared with the control group,the expression of 5-LOX enzyme was significantly decreased in the high-fat-feeding AD group.After HLJDD treatment,the concentration of COX-2,5-LOX and 12-LOX showed a dose-dependent tendency of improvement.Among of them,high dose HLJDD could significantly decrease the concentration of COX-2 and 5-LOX enzymes.To further elucidate the mechanism of HLJDD based on the regulation of lipid metabolism to improve the neuro inflammation.4 PUFAs and 19 oxidized lipids were determined by UPLC-AB Sciex Triple Quad 6500+ technique to detect the concentrations of PUFAs and oxidized lipids in high-fat-feeding AD mice after HLJDD administration.The results of PUFAs and oxidized lipids methodology verification showed the UPLC-MS/MS method has been verified to be accurate,sensitive and reliable.It was suitable for the determination of endogenous lipid metabolism in large quantities of biological samples.In the high-fat-feeding AD group,the concentrations of PGs and TXs were significantly increased,while the concentrations of EETs and DiHETs were decreased remarkably.Low and high doses of HLJDD treatments showed a certain tendency to reverse back,and most of them showed significant changes in the high dose HLJDD group.Among of them,EETs showed a dose-dependent change afted HLJDD treatment.To sum up,HLJDD and its main components could significantly improve the inflammation induced by LPS in zebrafish.The regulation of lipids and associated lipoproteins was one of the important ways for HLJDD to exert its anti-inflammatory effect.HLJDD could also improve the central nervous system inflammation of high-fat-feeding APP/PS1 mice from multiple levels by regulating the expression of specific enzymes in the polyunsaturated fatty acid pathways and their metabolites.This paper basically clarified the mechanism that HLJDD could improve the inflammatory microenvironment disorder through the regulation of lipid metabolism(lipids,specific enzymes of polyunsaturated fatty acid pathway,lipid peroxides).Based on clinical practice and the theory of traditional Chinese medicine,this study provided the theoretical and experimental basis for further enriching and improving the pathogenesis of antipyretic and detoxifying traditional Chinese medicine on inflammation and central nervous system inflammation,hoping that traditional Chinese medicine could be further and better applied in clinical multi-discipline.