| Aim and Significance:Based on previous work, this research focuses on the Radix Paeoniae Rubra, follows the strategy which is’originated from clinics, verified from clinics and returning to clinics’, uses evidence-based pharmacy and clinical prospective research and associates with molecular biology, network pharmacology and metabolomics to comprehensively explore the mechanism, understand the action and scientific connotation and offer the further guideline of rational application of Radix Paeoniae Rubra in clinics. So this study exhibits the feature of traditional Chinese medicine and innovative ideology with essential academic and scientific significance.Methods:1. With the formula about using Radix Paeoniae Rubra in high dose as subject and the method of evidence-based pharmacy, meta-analysis was applied to analyze the random control trial about treatment of cholestatic hepatitis and severe jaundice with formula about using Radix Paeoniae Rubra in high dose for the explicit understanding about effectiveness, serum indicator and adverse reaction.2. HPLC and UPLC-MS/MS were applied to investigate the main constituent information and chemical feature of Radix Paeoniae Rubra water extraction, and also for the identification of main compounds.3. Based on clear therapeutic effect, the mechanism of Radix Paeoniae Rubra treating cholestatic hepatitis was explored with pharmacological method and molecular biological technique on the ANIT-induced cholestatic hepatitis rat model from aspects of oxidative stress, inflammation and transport protein.4. Metabolomics and network pharmacology were applied to find out the metabolic bio marker regulation and the feature of its signaling network.5. Following the strategy of prospective clinical research, metabolomics was used to distinguish the characteristic metabolic biomarkers in serum of health people and the patients with cholestatic hepatitis before and after treatment with Radix Paeoniae Rubra. And the endogenous biomarkers reflecting prognostic of treatment of cholestatic hepatitis with Radix Paeoniae Rubra.Results:1 The evidence-based pharmacy study about high dose of Radix Paeoniae Rubra-related formula in treating cholestatic hepatitis.(1) Our research demonstrated that the cure rate of high dose of Radix Paeoniae Rubra in both single use and combination was higher than western medicine in treating cholestatic hepatitis with different courses. And the ALT, AST, TBIL and DBIL in serum of patients treated by high dose of Radix Paeoniae Rubra were lower than those in western medicine-treated patients (P<0.05) without significant difference in occurrence of adverse reactions (P>0.05). (2) Besides, high dose of Radix Paeoniae Rubra-related formula had a higher effective rate in treating severe jaundice in combination, single use or different doses than western medicine. And the TBIL, ALT and AST in serum of patients treated by high dose of Radix Paeoniae Rubra were lower than those in western medicine-treated patients (P<0.05), without significant difference in DBIL, PT, ALB, y-GT and occurrence of adverse reactions (P>0.05).2 The research of substantial basis of treating cholestatic hepatitis with high dose of Radix Paeoniae Rubra.(1) The method detecting peoniflorin and albiflorin in medicinal slices and water extraction of Radix Paeoniae Rubra were established and the changing discipline of main active components was confirmed by established fingerprint. (2) And 10 compounds were identified which turned out to be peoniflorin, albiflorin, oxypeoniflorin, gallic acid, cortex moutan glycoside E,6’-O-galloylpaeoniflroin, galloyl albiflorin, cortex moutan glycoside C, benzoylpaeoniflorin and paeonol. This result verified the chemical information of water extraction of Radix Paeoniae Rubra.3 Investigation of mechanism and effect of high dose of Paeoniae Radix Rubra on cholestatic hepatitis.(1) The results showed that Radix Paeoniae Rubra could reduce the serum ALT, AST and ALP and the activity of y-GT(p<0.05) in rat cholestatic hepatitis induced by ANIT dose-dependently, down-regulate the content of serum TBIL, DBIL and TBA (p<0.05), relieve edema, necrosis of cells and infiltration of inflammatory cells in liver tissue and improve pathological state of liver tissue. (2) In terms of regulating anti-oxidative system, high dose of Radix Paeoniae Rubra administration could enhance the expression of Nrf2 transportation to nucleus by up-regulating Akt. Nrf2 transportation from cytoplasm to nucleus could induce the increase of GCLca and GCLm expression, thus improving expression of GSH(p<0.05) and enhancing anti-oxidative stress of liver tissue. (3) In terms of inhibiting inflammatory reaction, high dose of Radix Paeoniae Rubra could depress the expression of pro inflammatory factor IL-1β (p<0.05) induced by ANIT obviously by inhibiting NF-κB nuclear translocation and NLRP3 expression. (4) In terms of regulating transport protein, high dose of Radix Paeoniae Rubra could reverse the decrease of NTCP, BSEP and MRP2 causing by ANIT remarkably. By regulating intake and excretion of bile, Paeoniae Radix Rubra in turn alleviated the state of cholestasis in cholestatic hepatitis.4 Network research of mechanism of high dose of Paeoniae Radix Rubra on cholestatic hepatitis.(1) Metabolomics was used to identify 12 metabolite biomarkers including L(D)-arginine, carnitine, pantothenate, L-tryptophan,2-benzylacetamide, 11-deoxycortisol, glycocholic acid, glycochenodeoxycholate, taurocholate, protoporphyrin Ⅸ,4-guanidinobutanoate and L-palmitoylcarnitine, all of which were biomarkers relevant to alleviation of ANIT-induced cholestatic hepatitis by high dose of Paeoniae Radix Rubra. These metabolite biomarkers respectively interfered in metabolism of D-arginine, ornithine, taurine, hypotaurine and phenylalanine, biosynthesis of pantothenate、coenzyme A and primary bile acid, degradation of diaminocaproic acid, metabolism of arginine, proline and porphyrin and so forth multiply signal pathways. (2) Meanwhile, from network pharmacology, Akt and NF-kB were proved to be critical regulator proteins of treating cholestatic hepatitis by Radix Paeoniae Rubra, and it also revealed the mechanism of treating cholestatic hepatitis by Radix Paeoniae Rubra in the formulae was strongly related to T cell receptor signaling pathway, Toll-like receptor signaling pathway and NOD-like receptor signaling pathway.5 The metabolomics about treatment of cholestatic hepatitis with high dose of Radix Paeoniae Rubra based on clinics.(1) Self-control clinical trial found out that high dose of Radix Paeoniae Rubra could dramatically reduce the serum TBIL, DBIL, ALT and AST in patients with effective rate to be 83.3%. (2) Metabolomics revealed 7 relevant metabolite biomarkers such as all-trans-dehydroretinal, bilirubin diglucuronide, glycochenodeoxycholic acid, bilirubin glucuronide, glycocholic acid, L-valine and glycodeoxycholic acid, which verified the metabolomics results in animal.Conclusions:(1) The results verified that the efficiency of high dosage of Radix Paeoniae Rubra related prescriptions in dose of 25-150 g/60kg·d used in single or combination was higher than normal therapy of western medicine in severe obstructive jaundice and cholestatic hepatitis based on the evidence-based pharmacy. (2) The results from the fundamental research demonstrated that Radix Paeoniae Rubra exhibited therapeutic effects on cholestatic hepatitis in dose of 20-80 g/kg with paeoniflorin compounds as the main active ingredients in a dose-dependent way. Radix Paeoniae Rubra treated cholestatic hepatitis mainly via activating Akt-Nrf2-GSH signaling pathway and down-regulating NF-κB-NLRP3, as well as regulating the transprotor of BSEP、MRP2、NTCP in and out. On the other hand, Radix Paeoniae Rubra affected bile acid, metabolic pathway, T cell receptor, toll-like receptor, NOD-like receptor to further attenuate the overall symptoms. (3) Glycodeoxycholic acid and glycocholic acidwere found to be the potential biomarkers for treating cholestasis with Radix Paeoniae Rubra in dose of 300 g/60kg·d in the further prospective clinical study. This study demonstrated the scientific connotation and dosage characteristic of Radix Paeoniae Rubra in high dose treating cholestatic hepatitis, and also offered theoretical basis and direct guideline for usage of Radix Paeoniae Rubra in a high dose in clinical application. |
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