Use of phlorizin in the treatment and / or prevention of lupus nephritis

Abstract

The application of phlorizin in the medicine for treating and / or preventing lupus nephritis, the molecular formula of phlorizin is C 21 H 24 O 10 , the application promotes the differentiation of regulatory T cells (Treg) by applying phlorizin to up-regulate the PI3K / Akt signal pathway, thereby reducing the symptoms of lupus nephritis, and provides an effective treatment method with less side effects. In the application, the traditional Chinese medicine monomer phlorizin can effectively promote the differentiation of Treg cells, and compared with compound traditional Chinese medicine, the use of single component has more advantages in drug efficacy control and efficacy evaluation. In addition, phlorizin is widely sourced, low in cost and non-toxic, and is very suitable for long-term treatment of lupus nephritis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural medicine, and particularly relates to application of phlorizin in medicine for treating and / or preventing lupus nephritis. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs, characterized by abnormal immune activation, almost every organ system is involved, especially the impact on the kidney is particularly significant. The incidence of lupus nephritis can be as high as 60%, and is often associated with poor prognosis of SLE. In addition, lupus nephritis significantly increases the incidence and mortality of SLE. In recent years, glucocorticoids, cyclophosphamide, mycophenolate mofetil (MMF) and calcineurin inhibitors have been clinically used for the treatment of lupus nephritis. However, the complete remission rate of lupus nephritis after six months of standard treatment is only 20%-30%, which is far lower than expected, and there are also problems of large side effects and strong drug resistance. Therefore, it is urgent to find more effective drugs to treat lupus nephritis.

[0003] The intrarenal pathogenesis of lupus nephritis involves the combination of antibodies with multiple autoantigens in the kidney, as well as the deposition of circulating immune complexes, which is derived from the destruction of self-tolerance. CD4+CD25+Foxp3+ regulatory T cells (Treg) as a unique subset of CD4+ T cells play a key role in inhibiting the abnormal activation of self-reactive lymphocytes and maintaining immune system tolerance. The number of Treg cells in the peripheral blood of patients with lupus nephritis is significantly reduced, and the Treg cell count is negatively correlated with the clinical activity of the disease. In NZB / W F1 mice, Treg cell depletion accelerates the development of lupus nephritis, while Treg cell transfer slows down the disease progression. Therefore, increasing the level of Treg cells provides a promising method for the treatment of lupus nephritis.

[0004] Traditional Chinese medicine monomers are an important source of clinical drugs, and their safety and effectiveness are widely recognized. Studies have shown that some traditional Chinese medicine monomers have the characteristics of regulating immunity, antioxidant, anti-inflammatory, etc., and have certain protective effect on kidney damage. However, the molecular targets and mechanisms of many traditional Chinese medicine monomers in the treatment of kidney disease are still unclear, which is also the difficulty of developing drugs for treating kidney disease based on traditional Chinese medicine monomers. Therefore, screening and verifying the effect of traditional Chinese medicine monomers, and finding the key target of their direct action, have important significance for applying traditional Chinese medicine monomers to clinical treatment of lupus nephritis, improving kidney function and relieving kidney disease progression. In-depth study of the therapeutic effect of traditional Chinese medicine monomers on lupus nephritis and revealing their specific action pathways and molecular mechanisms in the treatment of lupus nephritis can further provide scientific basis for the development of drugs for treating lupus nephritis based on traditional Chinese medicine monomers.

[0005] Phlorizin (PHZ) is a dihydrochalcone compound belonging to the flavonoid family, mainly found in the inedible parts of apple trees, such as seeds, bark, and unripe fruits. Numerous studies have shown that phlorizin exhibits anti-diabetic properties by inhibiting sodium-glucose co-transporter 1 and 2, down-regulating absorption in the kidney and intestine, or increasing insulin sensitivity. In addition, studies have demonstrated that phlorizin exerts antioxidant effects by regulating molecules related to oxidative stress, such as peroxisome proliferator-activated receptor gamma, nuclear factor erythroid 2-related factor 2, and NADPH oxidase. Phlorizin also exhibits anti-inflammatory properties by inhibiting LPS-induced inflammation through the NF-κB or MAPK signaling pathways. Furthermore, phlorizin has been found to reduce inflammation and synovial hyperplasia in rheumatoid arthritis by regulating the mTOR pathway. However, the role of phlorizin in lupus nephritis remains unknown.

[0006] Treg differentiation is crucial in treating lupus nephritis. Treg cells prevent autoimmune attacks by suppressing overactive immune responses, thereby reducing pathological damage. They also reduce inflammatory responses by secreting anti-inflammatory cytokines such as IL-10 and TGF-β, promoting the repair of kidney tissue. At the same time, Treg cells play a key role in maintaining immune tolerance, reducing relapse rates, and maintaining kidney function by modulating immune responses and reducing kidney damage. Therefore, therapeutic strategies targeting Treg cells are expected to be effective treatments for lupus nephritis. SUMMARY

[0007] To address the problems in the prior art, the application is designed to provide a new application of phlorizin in treating and / or preventing lupus nephritis. This application upregulates the PI3K / Akt signaling pathway, promotes the differentiation and function of Treg cells, thereby inhibiting inflammatory responses and improving kidney function. The technical solutions are as follows:

[0008] The first aspect of the application provides the use of phlorizin in the preparation of a drug for treating and / or preventing lupus nephritis. The molecular formula of the phlorizin is C 21 H 24 O 10 , and the structural formula is:

[0009]

[0010] Further, phlorizin is used as the only active ingredient in the preparation of a drug for treating and / or preventing lupus nephritis.

[0011] Further, phlorizin upregulates the PI3K / Akt signaling pathway, promotes the differentiation and function of Treg cells, and achieves the effects of inhibiting inflammatory responses and improving kidney function.

[0012] Further, promoting the differentiation of Treg cells specifically refers to enhancing downstream FOXP3 protein and FOXP3-mediated Treg cell differentiation.

[0013] Further, phlorizin achieves the effect of improving lupus nephritis by promoting the differentiation of primary naive CD4+ T cells into Tregs.

[0014] The second aspect of the present application provides a drug for treating and / or preventing lupus nephritis, which contains the effective component phlorizin.

[0015] The present application explores the pathological changes caused by the decrease of Treg cell differentiation in the kidney tissue of lupus nephritis, studies the molecular mechanism of phlorizin promoting Treg differentiation, and provides a new idea for the application of traditional Chinese medicine monomers in the treatment of lupus nephritis. This helps to better understand the mechanism of the disease and provides theoretical support for the development of new drugs.

[0016] Compared with the prior art, the traditional Chinese medicine monomer phlorizin in the present application can effectively promote the differentiation of Treg cells. Compared with compound traditional Chinese medicine, the use of single component has more advantages in efficacy control and efficacy evaluation. In addition, phlorizin is widely sourced, low in cost and has no toxic side effects, and is very suitable for long-term treatment of lupus nephritis. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure for the mechanism of phlorizin in the present application;

[0018] Figure 2 Figure for the improvement of kidney function and kidney pathological changes of lupus nephritis mice by phlorizin in Example 1;

[0019] Figure 3 Figure for the influence of phlorizin on the differentiation of Treg cells in Example 2;

[0020] Figure 4 Figure for the mechanism of phlorizin promoting the differentiation of Treg cells in lupus nephritis mice in Example 3;

[0021] Figure 5 Figure for the promotion of phlorizin in the differentiation of primary naive CD4+ T cells in Example 4. DETAILED DESCRIPTION

[0022] The present application is further described below in conjunction with the drawings and specific examples in the specification, so as to better understand the technical solutions.

[0023] The present application first evaluates the therapeutic effect of Phlorizin on lupus nephritis in animal experiments using MRL / lpr mice. Combined with in vitro cell experiments, experimental data reveal that Phlorizin slows down the progression of lupus nephritis by up-regulating the PI3K / Akt pathway, enhancing downstream FOXP3 protein and FOXP3-mediated Treg cell differentiation, and the mechanism diagram is shown in Figure 1 .

[0024] Example 1: Phlorizin improves kidney function and pathological changes of lupus nephritis mice

[0025] 1) Animal grouping and intervention: 7-8 week old female MRL / lpr mice were provided by Shanghai SLRC Experimental Animal Co., Ltd. The mice were randomly divided into three groups: (1) control group, (2) Phlorizin treatment group, (3) dexamethasone (DXSM) treatment group. From 12 weeks of age, Phlorizin group mice were orally administered Phlorizin at a dose of 40 mg / kg / day; DXSM group was orally administered dexamethasone at a dose of 2.5 mg / kg / day, for 10 weeks. At 22 weeks of age, the mice were anesthetized, 1 mL of blood was collected by cardiac puncture to determine IgG levels, and then the spleen and kidney were removed.

[0026] 2) Kidney histology: After the mice were anesthetized, the kidneys were removed, quickly fixed in 4% formalin, and embedded in paraffin according to the regular procedure. 5 μm thick sections were mounted on glass slides and stained with hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) to study morphological changes and kidney inflammation.

[0027] 3) Urine analysis: Urine samples were manually collected at 21 weeks of age in mice and centrifuged at 3500 rpm for 10 minutes at 4°C. After mixing the urine samples of each mouse, they were stored at -80°C until analysis. According to the manufacturer's instructions, commercial kits were used to determine the total protein and albumin concentrations in the urine.

[0028] Experimental results:

[0029] To study the effect of Phlorizin on lupus nephritis, we used female MRL / lpr mice, which begin to develop severe LN symptoms at about 16 weeks of age. From 12 to 22 weeks of age, female MRL / MpJ and MRL / lpr mice received saline, Phlorizin, or daily treatment of Phlorizin. Subsequently, we evaluated the kidney structure and function of MRL / lpr mice. Histologically, PAS and H&E staining of kidney sections showed that MRL / lpr mice had inflammatory cell infiltration Figure 2A, B). It was found that Phloretin could reduce kidney injury in MRL / lpr mice, which was reflected in the detailed grading of PAS-stained sections, which evaluated crescent formation, glomerular deposits, intraglomerular proliferation, and immune cell infiltration. In addition, to evaluate the effect of Phloretin on urine organs, urine samples were collected and albumin and total protein levels were measured. As expected, after Phloretin treatment, the total protein in the urine of MRL / lpr mice was significantly reduced, and the level of microalbumin was also reduced Figure 2 C, D), which indicated that Phloretin treatment had a significant improvement on kidney function. Considering that immune complex deposition is a key pathological mechanism in LN, we also evaluated the IgG level in serum. The results showed that Figure 2 E indicated that the serum IgG level was significantly reduced in MRL / lpr mice after Phloretin treatment. At the same time, we also evaluated the transcription level of inflammatory factors in the kidney, including IL-10, IL-6, IL-17, TNF-a, CXCL15 and IFN-g. Real-time PCR results showed that Phloretin significantly reduced kidney inflammation by down-regulating pro-inflammatory factors (IL-6, IL-17, TNF-a, CXCL15, IFN-g) and up-regulating anti-inflammatory factor IL-10 Figure 2 F-K). Overall, these findings confirmed that Phloretin could protect the kidney from injury.

[0030] Example 2: Phloretin promotes Treg differentiation in lupus nephritis mice

[0031] Experimental method:

[0032] To analyze Treg cell differentiation, a single-cell suspension of the spleen was prepared, and the cells were labeled using antibodies provided by Thermo Fisher Scientific, including PE-cy7 anti-mouse CD4, APC-cy7 anti-mouse CD25, BV510 anti-mouse CXCR5, and BV650 anti-mouse PD-1. After permeabilizing the cells with fixation buffer, intracellular staining of Foxp3 was performed using Foxp3 buffer, and finally, AF647 anti-mouse Foxp3 antibody was labeled, and flow cytometry was performed.

[0033] Experimental results:

[0034] This example further explores how Phloretin affects Treg cell differentiation. Flow cytometry analysis showed that the proportion of Treg cells was higher in Phloretin-treated MRL / lpr mice compared to MRL / MpJ mice Figure 3 ).

[0035] Example 3: Mechanism of action of Phloretin in promoting Treg differentiation in lupus nephritis mice

[0036] Experimental method:

[0037] CD4+T cells extracted from MRL / lpr mice after drug or control treatment were lysed using RIPA buffer containing protease and phosphatase inhibitors. CD4+T cells treated with Phloretin / Dexamethasone or untreated were also lysed in the same manner. Lysates (20 pg) were separated by SDS-PAGE and immunoblot analysis was performed using the following antibodies: phosphorylated AKT (Thr308), total AKT, phosphorylated PI3K (Tyr467 / 199), total PI3K, FOXOl, Foxp3, and b-actin.

[0038] Results of the experiment:

[0039] The results of this example show that phloretin administration promotes the differentiation of Treg cells in vivo. KEGG pathway analysis showed that the PI3K / Akt signaling pathway plays a key role in mediating the effect of phloretin on lupus nephritis. To verify this, CD4+T cells were purified from in vivo samples and the expression levels of the relevant proteins were evaluated. As shown in Figure 4 p-Akt and p-PI3K levels were significantly increased, while the overall protein levels of PI3K and Akt did not change much after phloretin treatment. These results suggest that phloretin can promote the differentiation of Treg cells by enhancing the activation of the PI3K / Akt pathway in CD4+T cells. FoXOl, as a key transcription factor regulated by the PI3K / Akt pathway, plays an important role in the development of Treg cells. Therefore, the level of FoxOl in vivo was detected in this example, and the results showed that phloretin significantly increased the expression of FoxOl in CD4+T cells. FoxOl is known to upregulate the expression of Foxp3, thereby promoting the development of Treg cells. Therefore, the protein expression of Foxp3 in CD4+T cells was evaluated, and it was found that the protein level of Foxp3 increased after phloretin treatment. The data suggest that phloretin treatment promotes the differentiation of Treg cells by regulating the PI3K-Akt-FoxOl pathway.

[0040] Example 4: Phloretin promotes the differentiation of naive CD4+T cells into Treg cells in vitro

[0041] Experimental methods:

[0042] Mouse CD4+ T Cell Isolation Kit to obtain naive CD4+ T cells from the spleen of MRL / lpr mice by negative selection. FACS analysis confirmed cell purity over 98%. After isolation, naive CD4+ T cells were placed in RPMI-1640 medium, added with 10% heat-treated fetal bovine serum, and activated with 1 pg / mL anti-mouse CD3 and 0.5 pg / mL anti-mouse CD28. The addition of 5 ng / mL TGF-b induced Treg cell polarization, and FACS analysis was performed after five days of culture.

[0043] Experimental results:

[0044] To further investigate the direct effect of PHZ on Treg cell development, this example evaluated the proportion of Treg cells with and without PHZ treatment. The results of the study showed that PHZ significantly enhanced the differentiation of Treg cells Figure 5 A-B). Consistently, this example also evaluated the levels of Akt, p-Akt, PI3K, p-PI3K, FoxOl and Foxp3. As shown in Figure 5 C-D, PHZ treatment led to over-activation of the PI3K / Akt pathway, manifested by an increase in p-Akt and p-PI3K. In addition, the levels of FoxOl and Foxp3 were also significantly upregulated after PHZ treatment. These findings highlight that PHZ effectively activated the PI3K / Akt / FoxOl pathway, thereby improving the differentiation of Treg cells.

Claims

1. The use of phlorizin in the treatment and / or prevention of lupus nephritis, wherein the molecular formula of phlorizin is C 21 H 24 O 10 The structural formula is: ； Phlorizin is the sole active ingredient in the preparation of drugs for the treatment and / or prevention of lupus nephritis.

2. The application as described in claim 1, characterized in that, Phlorizin promotes the differentiation and function of Treg cells by upregulating the PI3K / Akt signaling pathway, thereby inhibiting inflammatory responses and improving kidney function.

3. The application as described in claim 2, characterized in that, The promotion of Treg cell differentiation specifically involves enhancing downstream FOXP3 protein and FOXP3-mediated Treg cell differentiation.

4. The application as described in claim 1, characterized in that, The phlorizin improves lupus nephritis by promoting the differentiation of primary naive CD4+ T cells into Treg cells.

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