Application of oxidized berberine in preparation of Hamp inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of oxidized berberine in preparation of a Hamp inhibitor, and the affinity of the oxidized berberine and FPN protein is obviously higher than that of the existing inhibitor furfurthiamine; the LPS-induced Hamp level rise can be remarkably inhibited, the level of iron output protein FPN can be remarkably up-regulated, and the level of iron storage protein Ferritin in cells is increased; the LPS-induced BV2 cell iron disorder and inflammatory response can be obviously relieved; meanwhile, the activity of inhibiting the interaction of Hamp-FPN and the activity of resisting inflammation are achieved, so that the Hamp level is more effectively inhibited, and FPN expression is up-regulated; the inflammation-like characteristics of BV2 cell rounding, vacuole and the like caused by the LPS effect can be obviously improved; the pharmaceutical composition can effectively inhibit generation and release of LPS-induced inflammatory factors IL6 and IL1beta, and the effect of the pharmaceutical composition is obviously better than that of aspirin.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of berberine oxide in the preparation of Hamp inhibitors. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Hepcidin, encoded by the Hamp protein, is a key hormone responsible for regulating systemic iron homeostasis. It directly binds to and degrades FPN, the only iron export protein in cells, inhibiting iron absorption by intestinal epithelial cells and iron release by iron storage cells, thereby regulating the body's iron metabolism. As a key gene regulating the body's iron homeostasis, abnormal expression of Hamp can cause multi-system diseases, and inhibiting Hamp expression may be a promising therapeutic approach.

[0004] The Hamp inhibitors reported so far mainly include: specific targeted biological agents (DS28120313, NOX-H94, PRS-080, etc.), Hamp-FPN interaction antagonists (fursultiamine, thioxetine, pyrithione zinc) and Hamp upstream pathway inhibitors (LDN-193189, AG490, curcumin, SLN124, etc.), but they all have certain limitations. For example, targeted biological agents are still under research and improvement, have not yet been commercialized, and are not easy to obtain; the three Hamp-FPN interaction antagonists, fursultiamine is a vitamin B 1 The active derivatives are easily metabolized and inactivated in the body; pyrithione zinc has certain cytotoxicity and can only be used externally; and upstream pathway inhibitors, including BMP signaling pathway inhibitors and STAT3 pathway inhibitors, are not specific to Hamp. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide an application of berberine oxide in the preparation of Hamp inhibitors.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] The present invention provides application of berberine oxide in preparing Hamp inhibitor.

[0008] In some embodiments, berberine oxide is provided in the preparation of LPS-induced BV 2 Application of inhibitors in cellular iron disorders and inflammatory responses.

[0009] In some embodiments, provided is the use of berberine oxide in preparing a Hamp-FPN interaction inhibitor.

[0010] Preferably, the Hamp-FPN interaction inhibitor is used to inhibit Hamp expression and upregulate FPN expression.

[0011] In some embodiments, berberine oxide is provided for the preparation of a method for treating BV caused by LPS. 2 Application of cellular inflammatory-like signature drugs.

[0012] Preferably, the inflammatory-like features include but are not limited to BV 2 The cells become rounded or vacuolated.

[0013] In some embodiments, there is provided a use of berberine oxide in the preparation of an inhibitor of LPS-induced COX-2 up-regulation.

[0014] In some embodiments, there is provided a use of oxidized berberine in the preparation of an inhibitor of the production and release of LPS-induced inflammatory factors IL6 and IL1β.

[0015] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0016] Oxyberberin has a significantly stronger affinity for FPN protein than the existing inhibitor fursultiamine;

[0017] Berberine oxide can significantly inhibit the LPS-induced increase in Hamp levels, significantly upregulate the level of iron export protein FPN, and increase the level of intracellular iron storage protein Ferritin.

[0018] Berberine oxide can significantly alleviate LPS-induced BV 2 Cellular iron disturbances and inflammatory responses.

[0019] Oxyberberine has both the activity of inhibiting Hamp-FPN interaction and anti-inflammatory activity, thereby more effectively inhibiting Hamp levels and upregulating FPN expression.

[0020] Berberine oxide can significantly improve BV caused by LPS 2 Inflammatory features such as cell rounding and vacuolation.

[0021] Berberine oxide can effectively inhibit the production and release of LPS-induced inflammatory factors IL6 and IL1β, and its effect is significantly better than aspirin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1: is a diagram of the construction and optimization of the "HEK293T-FPN-GFP+Hamp protein+small molecule" screening system in the embodiment of the present invention, wherein the effect of PonA concentration (A) and whether serum is added (B) on the cell fluorescence intensity; (C) the effect of 1% DMSO on the cell fluorescence intensity; (D) the inhibition rate curve of different concentrations of Hamp protein on FPN-GFP cell fluorescence; (E) the effect of different concentrations of Hamp protein on FPN-GFP cell fluorescence observed under a fluorescence microscope; (n=6, data are mean ± standard error, *, *** respectively indicate P<0.05 and P<0.001 compared with the 5μm PonA induction group; ##,### Compared with the fluorescence intensity after PonA induction, P<0.01 and P<0.001, respectively).

[0024] Figure 2 This is a graph of the preliminary screening results of 1440 natural products based on the "HEK293T-FPN-GFP+Hamp protein+small molecule" system in an embodiment of the present invention. Each point in the graph represents a compound, and the blue dotted line indicates an inhibition rate of 60%.

[0025] Figure 3 In the embodiment of the present invention, Western Blot experiments verified the effects of 24 candidate compounds with an inhibition rate greater than 60% on the protein levels of FPN and Ferritin, the direct downstream targets of Hamp (Fur, fursultiamine, positive control compound);

[0026] Figure 4 : is the activity verification diagram of the candidate natural products in the embodiment of the present invention, wherein A is the MST experiment to detect the binding of three candidate natural products and the positive control Fur with FPN-GFP protein (n=3); the IC values ​​of gradient concentrations of oxidized berberine (B) and costus lactone (C) on the fluorescence intensity of cell FPN-GFP 50 Curve (n=6); D is a graph showing the effect of different concentrations of oxidized berberine (OBB) on the FPN protein level under the action of Hamp detected by Western Blot experiment.

[0027] Figure 5 1 is a test diagram of the embodiment of the present invention, in which berberine oxide has a significant improvement effect on LPS-induced abnormal expression of Hamp-FPN axis and cellular iron deposition, wherein A is the CCK8 experiment to detect the gradient concentration of berberine oxide on BV 2 Effect of different doses of oxidized berberine and positive control compounds furanthionium (Fur) and aspirin (Asp) on cell viability. 2 Cell supernatant; C is a qRT-PCR experiment to detect the effects of different doses of oxidized berberine on LPS-induced BV 2Effect of different doses of oxidized berberine on the level of Hamp in cells; D is a Western Blot experiment to detect the effect of different doses of oxidized berberine on the level of Hamp in cells treated with LPS 2 Effects of FPN and Ferritin expression levels in cells. (n=3, data are mean ± standard error, **, *** indicate P<0.01 and P<0.001 compared with the blank group; ### indicates P < 0.001 compared with the LPS group; ΔΔ,ΔΔΔ indicates P < 0.01 and P < 0.001 compared with the OOB-2.5 μm group; §§,§§§ indicates P < 0.01 and P < 0.001 compared with the OOB-10 μm group).

[0028] Figure 6 : This is a graph showing the effect of berberine oxide on LPS-induced inflammatory response in the present invention, wherein A is the BV of different doses of berberine oxide on LPS treatment. 2 A is a Western Blot experiment to detect the effects of different doses of oxidized berberine on BV treated with LPS 2 Effect of different doses of oxidized berberine on the expression levels of inflammation-related proteins COX-2 and HMGB1 in cells; qRT-PCR experiments were performed to detect the effects of different doses of oxidized berberine on BV treated with LPS 2 Effects of different doses of oxidized berberine on the mRNA levels of inflammatory factors IL6 (C) and IL1β (D) in cells; E is an ELISA experiment to detect the effects of different doses of oxidized berberine on BV 2 Effect diagram of inflammatory factor IL1β level in cell culture supernatant. (n=3, data shown are mean ± standard error; *, *** indicate P<0.05 and P<0.001 compared with the blank group; #,##,### indicates P < 0.05, P < 0.01, and P < 0.001 compared with the LPS group; Δ,ΔΔ,ΔΔΔ indicates P < 0.05, P < 0.01, and P < 0.001 compared with the OOB-5 μm group; §,§§,§§§ Denotes P < 0.05, P < 0.01 and P < 0.001 compared with the OOB-10 μm group).

[0029] Figure 7 In the present invention, the MST experiment detected the signal changes after gradient concentrations of oxidized berberine and the positive control fursultiamine bound to the FPN protein (n=3). DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] The present invention provides a natural product of traditional Chinese medicine, berberine oxide, which can specifically target the Hamp-FPN axis. Its affinity and inhibitory activity with FPN protein are significantly stronger than those of the existing inhibitor fursultiamine. The specific screening test is as follows:

[0032] High-throughput screening experiments

[0033] According to the literature, a high-throughput screening system of "HEK293T-FPN-GFP+Hamp protein+small molecule" was constructed. After a series of experimental optimizations, 100 ng / ml Hamp protein+10 μM small molecule was used as the final screening condition ( Figure 1 ). After adding Hamp protein and small molecules, the culture plate was returned to the cell culture incubator for 24 hours, and then the fluorescence intensity of each well was detected. Each 96-well plate was set with a positive control well (PonA) and a negative control well (PonA+Hamp). The inhibition rate of small molecule compounds on FPN-GFP fluorescence (%FPN-GFP) = (fluorescence intensity of test well - fluorescence intensity of negative control well) / (fluorescence intensity of positive control well - fluorescence intensity of negative control well) * 100%.

[0034] Based on the constructed high-throughput screening system, a preliminary evaluation of the inhibitory rate of 1440 natural products on Hamp-FPN interaction was performed. In the first round of screening, 70 compounds had an inhibition rate of greater than 50% on Hamp-FPN, 42 compounds had an inhibition rate of greater than 60%, and 23 compounds had an inhibition rate of greater than 70% ( Figure 2 ). Compounds with inhibition rates greater than 60% were retested for two rounds, and the final results showed that 24 compounds had inhibition rates greater than 60% on Hamp-FPN.

[0035] Western Blot Experiment Screening

[0036] In order to prevent the influence of compound autofluorescence and other operational factors on fluorescence intensity, thereby causing false positive results, the expression levels of FPN-GFP protein and downstream ferritin affected by FPN in cells treated with "Hamp protein + small molecule compound" were detected by Western Blot experiment. The results showed that ( Figure 3), among the above 24 natural products, 43C, 37B, 228A, and 65D had no significant effect on the protein level of FPN-GFP; 37C, 279A, and 273B had no significant effect on the protein level of Ferritin, and it was speculated that they may not have a significant improvement effect on intracellular iron deposition caused by reduced FPN expression. Therefore, the above 7 compounds were excluded in subsequent studies. In addition, 156D (androstenone) is a stimulant and a controlled substance, so no subsequent research was conducted. Finally, 16 natural products were included in the subsequent study.

[0037] Evaluation of drugability of candidate natural products

[0038] Evaluating the drugability of small molecule compounds in the early screening stage can help select candidate drugs that are well absorbed in the body and easy to drug, which helps to reduce time and economic costs in the subsequent development process. The drugability of the 16 candidate compounds that have been verified by the initial screening and WB experiments was evaluated (the results are shown in Table 1), of which 9 compounds have high gastrointestinal absorption rate (GI-A) and can pass through the blood-brain barrier (BBB-P), and are considered to have good drugability. Further investigation of the biological functions and research progress of each compound revealed that Staurosporine is a broad-spectrum ATP competitive kinase inhibitor that can bind to many kinases with high affinity, has low selectivity and poor specificity; Cyclopamine is a selective Smoothened antagonist (IC 50 =46nM), and is also an antagonist of the Hedgehog pathway with poor specificity. After excluding the above two compounds, 295B-Oxyberberine (OBB), 2811D-Micheliolide (MCL) and 1510H-Germacrone (GER) were finally listed as the compounds of focus according to the inhibition rate.

[0039] Table 1 Druggability-related parameters of 16 candidate natural products

[0040]

[0041]

[0042] Activity evaluation of key candidate natural products

[0043] First, the MST experiment was used to test whether the above three candidate compounds were direct inhibitors of Hamp-FPN. The results showed that oxyberberine and costusroot lactone could directly bind to FPN, and their affinity for FPN was stronger than that of the positive control fursultiamine ( Figure 4 A), while the affinity of germacron to FPN is weak, so it was not studied further.

[0044] Based on the “HEK293T-FPN-GFP+Hamp protein+small molecule” system, gradient concentrations of oxyberberine and costus lactone were added and the fluorescence value of each well was detected after 24 hours of action, and the IC 50 Curve, get the IC of berberine oxide 50 The value is 0.2422±0.065μm( Figure 4 B), IC of costus lactone 50 The value is 1.074±0.101μm( Figure 4 C).

[0045] Western Blot experiments were further used to detect the effect of berberine oxide (0.1μm, 0.2μm, 1μm, 2μm) on the FPN protein level under the action of Hamp.

[0046] The results showed that oxidized berberine increased the FPN-GFP protein level in a dose-dependent manner. At 2 μM, FPN-GFP was restored to the initial level induced by PonA, which was stronger than the positive control Fur( Figure 4 D).

[0047] Berberine oxide can significantly alleviate LPS-induced BV 2 Cellular iron disturbance and inflammatory response

[0048] LPS-induced BV was constructed 2 The microglial inflammation model was used to explore the effect of berberine oxide on Hamp upregulation caused by inflammation and its downstream links. First, the CCK8 experiment was used to detect the effect of berberine oxide at a gradient concentration of 0-80 μM on BV 2 The results showed that when the concentration of berberine oxide was less than 10 μM, it had no significant effect on BV 2 There was no significant effect on cell viability ( Figure 5 A). After the action of LPS and berberine oxide, it can be seen with the naked eye that LPS treatment caused the cell culture supernatant to turn yellow significantly, and berberine oxide improved this phenomenon in a dose-dependent manner. 2 The color of the cell supernatant is closest to ( Figure 5 B).

[0049] At the molecular level, LPS treatment induces BV 2 The expression of Hamp in cells was upregulated, FPN expression was downregulated, and iron content increased. Berberine oxide significantly inhibited the increase of Hamp level induced by LPS in a dose-dependent manner ( Figure 5 C), and significantly upregulated the level of iron export protein FPN and increased the level of intracellular iron storage protein Ferritin ( Figure 5D). Fur, an inhibitor of Hamp-FPN interaction, had no significant effect on the level of Hamp, but by inhibiting the degradation of FPN by Hamp, it significantly upregulated FPN expression, promoted iron excretion, and reduced intracellular iron levels. Other studies have shown that aspirin (Asp), a classic antipyretic, analgesic and anti-inflammatory drug, can inhibit LPS-induced Hamp upregulation by inhibiting the IL6-JAK2 / STAT3 pathway. The experimental results also showed that 50 μm Asp significantly reduced Hamp levels ( Figure 5 C), but only slightly increased the FPN level (P = 0.2665), and had no significant effect on the intracellular ferritin level ( Figure 5 D).

[0050] Compared with the two positive compounds Fur and Asp, berberine oxide showed a better inhibitory effect on the expression of Hamp at a lower dose (P<0.001 and <0.01), and more effectively upregulated the expression of FPN (P<0.01 and <0.001). The above results suggest that berberine oxide may have both the activity of inhibiting Hamp-FPN interaction and anti-inflammatory activities, thereby more effectively inhibiting Hamp levels and upregulating FPN expression.

[0051] Different concentrations of berberine oxide significantly improved BV induced by LPS 2 Inflammatory features such as cell rounding and vacuolation ( Figure 6 A). COX-2 is a rate-limiting enzyme that catalyzes the synthesis of prostaglandins from arachidonic acid in the body. It is not only a marker of inflammation, but also considered a marker of lipid peroxidation and ferroptosis. It stimulates the synthesis of pain mediators (IL6, SP, CGRP, etc.) in primary sensory neurons, leading to hyperalgesia and promoting the occurrence and development of NP. The experimental results showed that berberine oxide significantly inhibited the upregulation of COX-2 induced by LPS, and was significantly better than aspirin at a lower dose (P<0.001, Figure 6 The results of the detection of inflammatory factors in cells and culture supernatant showed that oxidized berberine could effectively inhibit the production and release of LPS-induced inflammatory factors IL6 and IL1β, and the effect was significantly better than that of aspirin ( Figure 6 CE). Fur, an inhibitor of Hamp-FPN interaction, had no significant effect on the level of IL6, but significantly reduced the level of IL1β in the supernatant ( Figure 6 in, CE).

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of berberine oxide in the preparation of Hamp inhibitors.

2. Application of berberine oxide in the preparation of inhibitors of LPS-induced iron disorder and inflammatory response in BV2 cells.

3. Application of berberine oxide in the preparation of Hamp-FPN interaction inhibitors.

4. The use according to claim 3, characterized in that: The Hamp-FPN interaction inhibitor is used to inhibit Hamp expression and upregulate FPN expression.

5. Application of berberine oxide in the preparation of drugs for treating BV2 cell inflammatory-like characteristics caused by LPS.

6. The use according to claim 5, characterized in that: The inflammation-like features include, but are not limited to, rounding or vacuolation of BV2 cells.

7. Application of berberine oxide in the preparation of LPS-induced COX-2 up-regulation inhibitors.

8. Application of berberine oxide in the preparation of inhibitors of the production and release of LPS-induced inflammatory factors IL6 and IL1β.

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