Application of phlorizin in medicine for treating and / or preventing lupus nephritis

Abstract

According to the application of the phlorizin in the medicine for treating and / or preventing the lupus nephritis, the molecular formula of the phlorizin is C21H24O10, the phlorizin is applied to up-regulate a PI3K / Akt signal channel and promote differentiation of regulatory T cells (Treg), so that the symptoms of the lupus nephritis are relieved, and an effective treatment means with small side effects is provided. The traditional Chinese medicine monomer phlorizin can effectively promote Treg cell differentiation, and compared with a compound traditional Chinese medicine, the use of a single component has more advantages in efficacy control and curative effect evaluation. In addition, phlorizin is wide in source, low in cost, free of toxic and side effects and very suitable for long-term treatment of lupus nephritis.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicine, and specifically relates to an application of phlorizin in a drug for treating and / or preventing lupus nephritis. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple organs and is characterized by abnormal immune activation that affects almost every organ system, especially the kidneys. The incidence of lupus nephritis can be as high as 60% and is often associated with a poor prognosis in SLE. In addition, lupus nephritis significantly increases the morbidity and mortality associated with SLE. In recent years, drugs such as glucocorticoids, cyclophosphamide, mycophenolate mofetil / mycophenolic acid, and calcineurin inhibitors have been clinically used to treat 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, there is an urgent need to find more effective drugs to treat lupus nephritis.

[0003] The intrarenal pathogenesis of lupus nephritis involves antibody binding to multiple autoantigens in the kidney and the deposition of circulating immune complexes, which stems from the destruction of self-tolerance. CD4+CD25+Foxp3+ regulatory T cells (Tregs), as a unique subset of CD4+T cells, play a key role in inhibiting the abnormal activation of autoreactive lymphocytes and maintaining immune system tolerance. Treg cells are significantly reduced in the peripheral blood of patients with lupus nephritis, and Treg cell counts are negatively correlated with clinical disease activity. In NZB / W F1 mice, Treg cell depletion accelerates the development of lupus nephritis, while Treg cell transfer slows the disease progression. Therefore, increasing Treg cell levels provides a promising approach for the treatment of lupus nephritis.

[0004] As an important source of clinical drugs, the safety and effectiveness of Chinese medicine monomers are widely recognized. Studies have shown that some Chinese medicine monomers have the characteristics of regulating immunity, anti-oxidation, and anti-inflammatory, and have a certain protective effect on kidney damage. However, the molecular targets and mechanisms of action of many Chinese medicine monomers in the treatment of kidney disease are still unclear, which is also the difficulty in developing drugs for the treatment of kidney disease based on Chinese medicine monomers. Therefore, screening and verifying the effects of Chinese medicine monomers and finding their direct key targets are of great significance for applying Chinese medicine monomers to the clinical treatment of lupus nephritis, improving renal function and alleviating the progression of kidney disease. In-depth research on the therapeutic effects of Chinese medicine monomers on lupus nephritis and revealing their specific pathways and molecular mechanisms in the treatment of lupus nephritis can further provide a scientific basis for the development of drugs for the treatment of lupus nephritis based on 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, root bark, and immature fruits. Several studies have shown that phlorizin exhibits antidiabetic properties by inhibiting sodium-glucose cotransporters 1 and 2 and downregulating renal and intestinal absorption or improving insulin sensitivity. In addition, studies have also demonstrated that phlorizin exerts antioxidant effects by regulating molecules associated with oxidative stress, such as peroxisome proliferator-activated receptor γ, nuclear factor erythroid 2-related factor 2, and NADPH oxidase. Phlorizin also exhibits anti-inflammatory properties by inhibiting LPS-induced inflammation through the inhibition of NF-κB or MAPK signaling pathways. In addition, 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 the treatment of lupus nephritis. Treg cells reduce pathological damage by inhibiting overactive immune responses and preventing autoimmune attacks. They also reduce inflammatory responses and promote the repair of renal tissue by secreting anti-inflammatory cytokines such as IL-10 and TGF-β. At the same time, Treg cells play a key role in maintaining immune tolerance, reducing relapse rates, and maintaining renal function by regulating immune responses and reducing renal damage. Therefore, therapeutic strategies targeting Treg cells are expected to become an effective treatment for lupus nephritis. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide a new drug for treating and / or preventing lupus nephritis by using phlorizin, which promotes the differentiation and function of Treg cells by upregulating the PI3K / Akt signaling pathway, thereby inhibiting the inflammatory response and improving kidney function. This is achieved specifically through the following technical solutions:

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

[0009]

[0010] Furthermore, phlorizin is used as the sole active ingredient in the preparation of drugs for treating and / or preventing lupus nephritis.

[0011] Furthermore, phlorizin promotes the differentiation and function of Treg cells by upregulating the PI3K / Akt signaling pathway, thereby inhibiting inflammatory response and improving kidney function.

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

[0013] Furthermore, phlorizin improves lupus nephritis by promoting the differentiation of primary naive CD4+T cells into Tregs.

[0014] The second aspect of the present invention provides a drug for treating and / or preventing lupus nephritis, the drug containing the effective ingredient phlorizin.

[0015] The present invention explores the pathological changes caused by the reduced differentiation of Treg cells in the kidney tissue of lupus nephritis, studies the molecular mechanism of phlorizin in promoting Treg differentiation, and provides new ideas for the application of Chinese medicine monomers in the treatment of lupus nephritis. This helps to deeply understand the mechanism of the disease and provide theoretical support for the development of new drugs.

[0016] Compared with the prior art, the Chinese medicine monomer phlorizin of the present invention can effectively promote Treg cell differentiation. Compared with compound Chinese medicine, the use of a single ingredient has more advantages in drug efficacy control and efficacy evaluation. In addition, phlorizin has a wide source, low cost and no toxic side effects, and is very suitable for long-term treatment of lupus nephritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the mechanism of action of phlorizin of the present invention;

[0018] Figure 2 This is a diagram showing the improvement of renal function and renal pathological changes in mice with lupus nephritis by phlorizin in Example 1;

[0019] Figure 3 This is a diagram showing the effect of phlorizin on the differentiation of Treg cells in Example 2;

[0020] Figure 4 This is a diagram showing the mechanism of action of phlorizin in promoting Treg differentiation in mice with lupus nephritis in Example 3;

[0021] Figure 5 This is a diagram showing the effect of phloridzin on promoting differentiation of primary naive CD4+T cells in Example 4. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments to provide a better understanding of the present technical solution.

[0023] This invention is the first to use MRL / lpr mice in animal experiments to evaluate the therapeutic effect of phlorizin on lupus nephritis. Combined with in vitro cell experiments, experimental data revealed that phlorizin up-regulated the PI3K / Akt pathway, enhanced downstream FOXP3 protein and FOXP3-mediated Treg cell differentiation, thereby slowing down the progression of lupus nephritis. Its mechanism of action is shown in the figure below. Figure 1 shown.

[0024] Example 1: Phlorizin improves renal function and renal pathological changes in mice with lupus nephritis Experimental method steps:

[0025] 1) Animal grouping and intervention: Female MRL / lpr mice aged 7-8 weeks were provided by SLRC Laboratory Animal Co., Ltd., Shanghai, China. The mice were randomly divided into three groups: (1) control group, (2) phlorizin treatment group, and (3) dexamethasone (DXSM) treatment group. Starting from 12 weeks of age, mice in the phlorizin group were orally treated with phlorizin at a dose of 40 mg / kg / day; mice in the DXSM group were orally treated with dexamethasone at a dose of 2.5 mg / kg / day for 10 weeks. At 22 weeks, the mice were anesthetized, and 1 mL of blood was collected by cardiac puncture to measure IgG levels, and then the spleen and kidneys were removed.

[0026] 2) Renal histology: The kidneys were removed from mice after anesthesia, quickly fixed in 4% formalin, and embedded in paraffin according to routine procedures. 5 μm thick sections were mounted on slides and stained with hematoxylin-eosin (H&E) and periodic acid-Schiff (PAS) to study morphological changes and renal inflammation.

[0027] 3) Urine analysis: Urine samples were manually collected from mice at 21 weeks of age and centrifuged at 3500 rpm for 10 minutes at 4°C. Urine samples from each mouse were pooled and stored at -80°C until analysis. Total protein and albumin concentrations in urine were determined using commercial kits according to the manufacturer's guidelines.

[0028] Experimental results:

[0029] To investigate the role of phlorizin in lupus nephritis, we used female MRL / lpr mice, which begin to develop severe LN symptoms at approximately 16 weeks of age. Female MRL / MpJ and MRL / lpr mice were treated with saline, phlorizin, or daily phlorizin from 12 to 22 weeks of age. Subsequently, we evaluated the renal structure and function of MRL / lpr mice. Histologically, PAS and H&E staining of kidney sections showed the presence of inflammatory cell infiltration in MRL / lpr mice ( Figure 2A, B). The study found that phlorizin reduced renal damage in MRL / lpr mice, as demonstrated by detailed grading of PAS-stained sections, evaluating crescent formation, glomerular deposition, intraglomerular proliferation, and immune cell infiltration. In addition, to evaluate the effects of phlorizin on urinary organogenesis, urine samples were collected and albumin and total protein levels were measured. As expected, after phlorizin treatment, total urine protein was significantly reduced in MRL / lpr mice, and microalbumin levels were also reduced ( Figure 2 C, D), indicating that phlorizin treatment significantly improved renal 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 showed that serum IgG levels were significantly reduced after phlorizin treatment in MRL / lpr mice. At the same time, we also evaluated the transcription levels of inflammatory factors in the kidney, including IL-10, IL-6, IL-17, TNF-α, CXCL15, and IFN-γ. Real-time PCR results showed that phlorizin significantly reduced renal inflammation ( Figure 2 FK). Overall, these findings confirm that phlorizin can protect the kidneys from damage.

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

[0031] Experimental methods:

[0032] To analyze Treg cell differentiation, spleen single cell suspensions were prepared and cells were labeled with 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 permeabilization of cells with fixation buffer, intracellular staining of Foxp3 was performed with Foxp3 buffer, and finally labeled with AF647 anti-mouse Foxp3 antibody for flow cytometry.

[0033] Experimental results:

[0034] This example further explores how phlorizin affects the differentiation of Treg cells. Flow cytometry analysis showed that the proportion of Treg cells in MRL / lpr mice treated with phlorizin was higher than that in MRL / MpJ mice ( Figure 3 ).

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

[0036] Experimental methods:

[0037] After drug or control treatment, CD4+T cells extracted from MRL / lpr mice were lysed using RIPA buffer containing protease and phosphatase inhibitors. CD4+T cells treated with phlorizin / dexamethasone or untreated were also lysed in the same manner. Lysates (20 μg) were separated by SDS-PAGE and analyzed by immunoblotting using the following antibodies: phosphorylated AKT (Thr308), total AKT, phosphorylated PI3K (Tyr467 / 199), total PI3K, FOXO1, Foxp3, and β-actin.

[0038] Experimental results:

[0039] The results of this study showed that phlorizin administration promoted the differentiation of Treg cells in vivo. KEGG pathway analysis showed that the PI3K / Akt signaling pathway played a key role in mediating the effects of phlorizin on lupus nephritis. To verify this, CD4+T cells were purified from in vivo samples and the expression levels of related proteins were evaluated. Figure 4 As shown, the levels of phosphorylated Akt (p-Akt) and phosphorylated PI3K (p-PI3K) increased significantly, while the overall protein levels of PI3K and Akt did not change much after phlorizin treatment. These results suggest that phlorizin may promote the differentiation of Treg cells by enhancing the activation of the PI3K / Akt pathway in CD4+T cells. As a key transcription factor regulated by the PI3K / Akt pathway, FoXO1 plays an important role in the development of Treg cells. Therefore, this example detects the level of FoxO1 in vivo, and the results show that phlorizin significantly increases the expression of FoxO1 in CD4+T cells. FoxO1 is known to upregulate the expression of Foxp3, thereby promoting the development of Treg cells. Therefore, the present application evaluated the protein expression of Foxp3 in CD4+T cells and found that the protein level of Foxp3 increased after phlorizin treatment. The data show that phlorizin treatment promotes the differentiation of Treg cells by regulating the PI3K-Akt-FoxO1 pathway.

[0040] Example 4: Phlorizin promotes the differentiation of primary naive CD4+ T cells into Treg

[0041] Experimental methods:

[0042] Using Mouse CD4+T Cell Isolation Kit obtains naive CD4+T cells from the spleen of MRL / lpr mice by negative selection. FACS analysis confirms that the cell purity is over 98%. After isolation, naive CD4+T cells are placed in RPMI-1640 medium supplemented with 10% heat-treated fetal bovine serum and activated with 1μg / mL anti-mouse CD3 and 0.5μg / mL anti-mouse CD28. 5ng / mL TGF-β is added to induce Treg cell polarization, and FACS analysis is 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 showed that PHZ significantly enhanced the differentiation of Treg cells ( Figure 5 AB). Consistently, the present example also evaluated the levels of Akt, p-Akt, PI3K, p-PI3K, FoxO1, and Foxp3. Figure 5 As shown in Figures CD, PHZ treatment led to overactivation of the PI3K / Akt pathway, as evidenced by an increase in p-Akt and p-PI3K. In addition, the levels of FoxO1 and Foxp3 were also significantly upregulated after PHZ treatment. These findings highlight that PHZ effectively activates the PI3K / Akt / FoxO1 pathway, thereby improving the differentiation of Treg cells.

Claims

1. The use of phlorizin in treating and / or preventing lupus nephritis, wherein the molecular formula of the phlorizin is C 21 H 24 O 10 , the structural formula is:

2. The use according to claim 1, characterized in that The phlorizin is used as the sole active ingredient in the preparation of drugs for treating and / or preventing lupus nephritis.

3. The use according to claim 1, characterized in that The phlorizin promotes the differentiation and function of Treg cells by upregulating the PI3K / Akt signaling pathway, thereby inhibiting inflammatory response and improving kidney function.

4. The use according to claim 3, characterized in that The promotion of Treg cell differentiation specifically includes enhancing downstream FOXP3 protein and FOXP3-mediated Treg cell differentiation.

5. The use according to claim 1, characterized in that The phlorizin improves lupus nephritis by promoting the differentiation of primary naive CD4+T cells into Tregs.

6. A drug for treating and / or preventing lupus nephritis, characterized in that: This medicine contains the active ingredient phlorizin.

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