Application of total glucosides of paeony in preparation of medicine for preventing and / or treating gout

Abstract

The invention relates to the technical field of medicines, and discloses application of total glucosides of paeony in preparation of medicines for preventing and / or treating gout. According to the application, the total glucosides of paeony can inhibit inflammation through AMPK, so that the effect of preventing and / or treating gout is realized. The total glucosides of paeony can reduce adverse reactions such as gastrointestinal reactions and anaphylactic reactions in gout attack prevention treatment and increase treatment compliance of patients, the occurrence rate of abnormal liver functions caused by the total glucosides of paeony is relatively low, and more treatment possibilities are provided for reducing uric acid and preventing gout attack of patients with hepatic and renal insufficiency; the total glucosides of paeony have a certain uric acid reducing effect, and can reduce proliferation of synovial cells and infiltration of inflammatory cells, protect joint structures and improve prognosis of gout patients by adjusting immunity and anti-inflammatory effects.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and more specifically, to an application of total glucosides of white paeony in the preparation of a drug for preventing and / or treating gout. Background Art

[0002] The onset of gout is associated with hyperuricemia. Currently, the treatment of gout and hyperuricemia focuses on controlling the patient's blood uric acid level during the interictal period and the inflammatory response during the acute phase. Commonly used uric acid-lowering drugs are divided into two categories: promoting uric acid excretion and inhibiting uric acid production. The former is represented by benzbromarone, and drugs that inhibit uric acid production include xanthine oxidase (XOD) inhibitors allopurinol and febuxostat. However, the adverse reactions of these drugs, such as urinary stones, hypersensitivity reactions, and liver and kidney damage, are increasingly receiving attention. In the process of gout, monosodium urate (MSU) needs to be recognized by membrane-bound receptors (Toll-like receptors) and intracellular receptors (NOD receptors) to stimulate IL-1β secretion by activating their own signaling pathways. Among NOD-like receptors, the NLRP3 inflammasome can recognize and bind to MSU, playing a key role in the middle stage of gout. In addition, research on the pathogenesis of gout has found that adenosine triphosphate (ATP) is not only an energy carrier, but also acts as a stimulating signal on the purine receptor P2X ligand-gated ion channel 7 (P2X7R), promoting the secretion of cytokines such as IL-1β, IL-6, and TNF-a. The main inducing factors of acute gouty arthritis include overwork, cold, drinking, overeating, etc.; there are dramatic fluctuations in ATP in the physiological changes of the body caused by these inducing factors. ATP changes may be the second pathogenic signal that causes gouty arthritis in addition to MSU. ATP can stimulate the P2X7R signaling pathway to cause K + Outflow, activate NLRP3 inflammasome, cooperate with MSU crystal to stimulate abnormal increase in secretion of cytokines such as IL-1β, IL-6, TNF-a, and cause the onset of gouty arthritis. MSU alone is not enough to stimulate NLRP3 inflammasome to induce sufficient IL-1β secretion to cause gout. Only when ATP increases, ATP stimulates the P2X7R signaling pathway and cooperates with MSU to stimulate NLRP3 inflammasome activation, can it induce sufficient IL-1β secretion and cause gout. Therefore, in the treatment and prevention of gout, in addition to lowering uric acid, it is also necessary to inhibit the activation of the P2X7R signaling pathway.

[0003] my country has rich resources of traditional Chinese medicine, diverse chemical composition structures and numerous targets, so it is possible to screen highly effective and low-toxic xanthine oxidase inhibitors from traditional Chinese medicine. Modern phytochemical studies have shown that monoterpene glycosides, the main bioactive components in white peony extracts, have the effect of xanthine oxidase inhibitors. Animal experiments have found that total glucosides of paeonia (TGP) can reduce uric acid levels and inhibit the activity of xanthine oxidase and adenosine deaminase in mouse liver. Therefore, total glucosides of paeonia have the potential to treat gout and hyperuricemia.

[0004] In recent years, studies have found that total glucosides of white peony are the general name of paeoniflorin, hydroxypeoniflorin, paeoniflorin, paeoniflorin lactone glycosides, and benzoylpeoniflorin. It has the effects of vasodilation, analgesia, sedation, anti-inflammatory and anti-ulcer, antipyretic and antispasmodic, and diuresis. It is a bidirectional immunomodulator and is dose-dependent. It affects the cellular immunity, humoral immunity, and inflammatory process of autoimmune diseases in multiple links. It has been widely used to treat rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome and many other diseases. In primary Sjögren's syndrome, paeoniflorin can downregulate ATP-induced inflammatory response and the expression of P2X7R in PBMCs. This study first found that paeoniflorin (PF) significantly inhibited the expression of P2X7R induced by ATP in vitro and significantly reduced the production of IL-1β and IL-6 in pSS-PBMCs. This study shows that total glucosides of white peony may downregulate the secretion of inflammatory mediators and inflammatory cytokines by inhibiting the expression of P2X7R, providing a theoretical basis for total glucosides of white peony to prevent the onset of gouty arthritis.

[0005] At present, there is no report on the application of total glucosides of white paeony in the preparation of drugs for preventing and / or treating gout. Summary of the invention

[0006] The purpose of the present invention is to explore the mechanism of action of total glucosides of white paeony in preventing and / or treating gout, and to provide the application of total glucosides of white paeony in preparing drugs for preventing and / or treating gout, so as to reduce or eliminate the adverse reactions of traditional drugs for treating gout.

[0007] In order to achieve the above objectives, this application adopts the following technical solutions:

[0008] In a first aspect, the present application provides the use of total glucosides of paeony peony in the preparation of a drug for preventing and / or treating gout.

[0009] Furthermore, the total glucosides of white peony inhibit inflammation through the AMPK pathway, thereby playing a role in preventing and / or treating gout.

[0010] In a second aspect, the present application provides a drug for preventing and / or treating gout, wherein the drug comprises total glucosides of paeony glycosides as an active ingredient.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0012] Furthermore, the pharmaceutical dosage forms include tablets, powders, granules, capsules, oral solutions, injections or sustained-release preparations.

[0013] In summary, this application has the following beneficial effects:

[0014] 1. Safety: Reduce adverse reactions such as gastrointestinal reactions and allergic reactions in the prevention of gout attacks, and increase patient compliance with treatment. Since the incidence of abnormal liver function caused by total glycosides of white paeony is relatively low, it provides more treatment possibilities for lowering uric acid and preventing and treating gout attacks in patients with liver and kidney dysfunction;

[0015] 2. Effectiveness: Total glucosides of white peony have a certain effect of lowering uric acid. By regulating immunity and anti-inflammatory effects, it can reduce the proliferation of synovial cells and the infiltration of inflammatory cells, protect the joint structure, and improve the prognosis of gout patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Changes in the swelling degree of the paws of mice after TGP intervention.

[0017] Figure 2 :Changes in the swelling index of the paw of mice after TGP intervention. (*p value indicates the difference between the control group, *** indicates P<0.001; #p value indicates the difference between the MSU crystal intervention group, # indicates P<0.05)

[0018] Figure 3 :Changes in the expression of genes and proteins related to inflammation in the soles of mice after TGP intervention. (*p value indicates the difference between the control group, * indicates P<0.05, ** indicates P<0.01; #p value indicates the difference between the MSU crystal intervention group, # indicates P<0.05)

[0019] Figure 4 : Changes of pAMPK protein expression in the soles of mice after TGP intervention.

[0020] Figure 5 :AMPKα1 flox / flox AMPKα2 flox / flox Mice and AMPKα1 flox / flox AMPKα2 flox / flox Lyz2 Cre+ / - Mouse gene identification. ((A) Lyz2 gene; (B) AMPKα1; (C) AMPKα2;)

[0021] Figure 6 : AMPK after TGP intervention fl / fl Mice and AMPK fl / flThe degree of paw swelling in Lyz2-Cre mice.

[0022] Figure 7 : AMPK after TGP intervention fl / fl Mice and AMPK fl / fl Lyz2-Cre mouse paw swelling index. (All p values ​​represent the difference between groups compared with the previous grouping, *** indicates P<0.001; # indicates P<0.05; $ indicates P<0.05)

[0023] Figure 8 : AMPK after TGP and MSU intervention fl / fl Mice and AMPK fl / fl Lyz2-Cre serum IL-1β and IL-6 expression changes. (All p values ​​represent the difference between groups compared with the previous grouping, * indicates P < 0.05, ** indicates P < 0.01; # indicates P < 0.05; $ indicates P < 0.05)

[0024] Fig. 9 :Changes in IL-1β and IL-6 secretion in the supernatant of RAW264.7 cells after TGP intervention. (*p value indicates difference from the Control group, ** indicates P<0.01, *** indicates P<0.001; #p value indicates difference from the MSU crystal intervention group, # indicates P<0.05, ## indicates P<0.01)

[0025] Fig.10 :Changes in IL-1β, IL-6 mRNA and NLRP3 protein expression in RAW264.7 cells after TGP intervention. (*p value indicates difference from the Control group, ** indicates P<0.01, *** indicates P<0.001; #p value indicates difference from the MSU crystal intervention group, # indicates P<0.05, ## indicates P<0.01)

[0026] Fig.11 : Changes of pAMPK protein expression in RAW264.7 after TGP intervention.

[0027] Fig.12 :After Compound C intervention in RAW264.7, pAMPK expression and supernatant IL-1β, IL-1β and IL-6 mRNA expression, and NLRP3 protein level changed. (*p value indicates difference from the Control group, *** indicates P<0.001; #p value indicates difference from the MSU crystal intervention group, ### indicates P<0.001; $p value indicates difference from the MSU crystal intervention group, $ indicates P<0.05) DETAILED DESCRIPTION

[0028] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0029] It is worth noting that the materials used in the examples are commercially available unless otherwise specified, and the methods used in the examples are existing methods unless otherwise specified.

[0030] Example 1: Animal Experiment

[0031] 1. TGP intragastric administration relieves inflammation in mice with gouty arthritis

[0032] 1. TGP intragastric administration improves inflammation in gout mice: MSU crystals were injected into the soles of the mice 2 weeks after TGP intragastric administration, and the swelling index of the soles of the mice was measured 8 hours later. The intervention of TGP significantly reduced the swelling index of the soles of the mice ( Figure 1 and Figure 2 ).

[0033] The mouse serum was taken for ELISA to detect the level of IL-1β. It can be seen that TGP pretreatment can reduce the levels of IL-1β and IL-6 in mouse serum ( Figure 3 ). The mRNA of the mouse paw tissue was extracted for qPCR detection of the expression of inflammatory cytokines, and the protein of the paw tissue was extracted for WB detection of the expression level of NLRP3 protein. It can be seen that TGP treatment significantly downregulated the expression of IL-1β, IL-6 and the protein level of NLRP3 ( Figure 3 ).

[0034] 2. TGP acts through the AMPK pathway

[0035] 1. TGP gavage up-regulates pAMPK expression in the soles of mice: In vivo experimental results suggest that TGP treatment increases the expression of pAMPK in the soles of mice ( Figure 4 ).

[0036] 2. Identification of AMPKα1 and AMPKα2 targeted knockout mice in myeloid cells: To better explore the mechanism of TGP, AMPKα1 flox / flox Mouse, AMPKα2 flox / flox With Lyz2 Cre+ / - AMPKα1 flox / flox AMPKα2 flox / flox Lyz2 Cre+ / - Mouse and AMPKα1 flox / flox AMPKα2 flox / flox Mouse; DNA was extracted from mouse tails and amplified by PCR and then analyzed by agarose gel electrophoresis. Figure 5As shown; the bright band near 700kd is Lyz2 Cre+ / - The wild type has no band in this region; the bright band near 500kd is AMPKα1 flox / flox The band around 300 kd is the wild type; as shown in the figure, the bright band around 500 kd is AMPKα2 flox / flox , the band around 300 kd is the wild type; for the convenience of subsequent description, AMPKα1 flox / flox AMPKα2 flox / flox AMPK in mice fl / fl Mouse, AMPKα1 flox / flox AMPKα2 flox / flox Lyz2 Cre+ / - AMPK in mice fl / fl Lyz2-Cre mice.

[0037] 3. AMPK gene knockout reverses the effect of TGP: TGP gavage and MSU crystal intervention in mice, such as Figure 6 and Figure 7 As shown in Figure 2, TGP pretreatment can significantly alleviate the MSU crystal-induced swelling of the mouse paw, while AMPK fl / fl Lyz2-Cre mice, compared with AMPK fl / fl The swelling of the paws in the TGP-treated group was significantly increased (p<0.05). fl / fl The levels of IL-1β (p<0.05) and IL-6 (p<0.05) in the serum of Lyz2-Cre mice were significantly higher than those of AMPK fl / fl This suggests that after AMPK knockout, the ability of TGP to relieve inflammation is inhibited ( Figure 8 ).

[0038] Example 2: Cell experiment

[0039] 1. TGP pretreatment of RAW264.7 cells and MSU crystal intervention reduced the inflammatory level of cells

[0040] 1. TGP reduces the level of MSU-induced inflammation in RAW264.7 cells: RAW264.7 cells were pretreated with TGP, and MSU crystals were added for intervention 1 hour later. Cell supernatants were collected 24 hours later, and ELISA was used to detect the protein levels of IL-1β and IL-6 in the supernatant. Total cell mRNA was extracted to detect the mRNA expression levels of inflammatory cytokines IL-1β and IL-6, and total cell protein was extracted to detect the expression of NLRP3 protein. Fig. 9 and Fig.10As shown in the results, TGP intervention can downregulate the increase in IL-1β and IL-6 levels in cell supernatants induced by MSU crystals, reduce the mRNA expression of intracellular inflammatory cytokines IL-1β and IL-6, and downregulate the expression level of NLRP3 protein. The above results suggest that TGP pretreatment alleviates the cellular inflammatory response induced by MSU crystals.

[0041] 2. TGP acts through the AMPK pathway

[0042] 1. TGP intervention in RAW264.7 activates AMPK: To further explore the mechanism of TGP's anti-inflammatory effect in MSU crystal-induced inflammation, RAW264.7 was pretreated with TGP, and total cell protein was extracted after MSU crystal intervention to detect changes in pAMPK expression. The results showed that pAMPK expression increased in a dose-dependent manner ( Fig.11 ).

[0043] 2. Inhibition of AMPK activation, the effect of TGP was reversed: RNAW264.7 was pretreated with AMPK inhibitor Compound C, and then given TGP pretreatment and MSU crystal stimulation. The experimental results indicate that Compound C effectively reduces the phosphorylation level of AMPK. When cells were pretreated with Compound C, it can be seen that the IL-1β level in the supernatant was significantly increased compared with the TGP intervention group, the mRNA levels of IL-1β, IL-6 and iNOS were increased, the expression of pAMPK was downregulated, and the expression of NLRP3 protein was increased, indicating that after the activity of AMPK was inhibited, the anti-inflammatory effect of TGP was reversed ( Fig.12 ).

[0044] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Application of total glucosides of white paeony in the preparation of drugs for preventing and / or treating gout.

2. The use according to claim 1, characterized in that: The total glucosides of white peony inhibit inflammation through the AMPK pathway, thereby playing a role in preventing and / or treating gout.

3. A drug for preventing and / or treating gout, characterized in that: Includes total glucosides of paeony ash as an active ingredient.

4. The drug according to claim 3, characterized in that It also includes pharmaceutically acceptable carriers or excipients.

5. The drug according to claim 3, characterized in that The pharmaceutical dosage forms include tablets, powders, granules, capsules, oral liquids, injections or sustained-release preparations.

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