Medicine for treating lupus nephritis and application thereof

Abstract

The invention particularly discloses a medicine for treating lupus nephritis and application thereof, and relates to the technical field of biological medicine. According to the application of the extracellular vesicles and the tripterygium wilfordii in preparation of the medicine for treating lupus nephritis, the mesenchymal stem cell apoptosis vesicles and the tripterygium wilfordii are combined for treatment, the immunoregulation, anti-inflammation and other characteristics of the tripterygium wilfordii and the mesenchymal stem cell apoptosis vesicles are combined, the dosage of the tripterygium wilfordii can be reduced, the toxic and side effects of the tripterygium wilfordii can be greatly reduced, and the curative effect of the tripterygium wilfordii is improved. , such as reproductive system toxicity, gastrointestinal reactions, and liver and kidney function impairment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a drug for treating lupus nephritis and an application thereof. Background Art

[0002] Mesenchymal stem cell apoptotic vesicles are vesicles released during apoptosis and possess multiple biological functions and therapeutic potential. Derived from the apoptotic process, these vesicles contain proteins, DNA, RNA, lipids, and metabolites, and play an important role in regulating cell proliferation, migration, differentiation, and immune regulation. Mesenchymal stem cell apoptotic vesicles can interact with receptor cells, activating intracellular signaling pathways and influencing cell fate. Studies have shown that mesenchymal stem cell apoptotic vesicles play a significant role in tissue regeneration.

[0003] Mesenchymal stem cells have achieved good results in treating autoimmune diseases such as lupus nephritis. However, mesenchymal stem cells become undetectable within a few days of infusion into the human body, yet their immunomodulatory effects can persist for several months. Furthermore, studies have shown that mesenchymal stem cell apoptotic vesicles, like mesenchymal stem cells, possess the immunomodulatory and immune tolerance-inducing properties. In fact, mesenchymal stem cell apoptotic vesicles possess distinct advantages in intercellular communication. Apoptotic vesicles can release a variety of mesenchymal stem cell-derived components, and these effector molecules can exert a variety of biological effects directly on cells or after being internalized by receptor cells.

[0004] Tripterygium wilfordii has immunosuppressive, anti-inflammatory and inflammatory factor-regulating effects. It can be used as an auxiliary treatment for systemic lupus erythematosus, especially for mild cases or combined treatment.

[0005] Systemic lupus erythematosus (SLE) is an autoimmune disease that primarily affects young women and can affect multiple organs. Pathologically, the kidneys are almost 100% affected, with 50-80% presenting as nephritis, also known as lupus nephritis. Lupus nephritis presents with a protracted course and frequent recurrences, posing challenges to clinical treatment. Consequently, lupus nephritis is the leading cause of death in SLE patients.

[0006] The combination of immunosuppressants and steroids is currently the mainstay of clinical treatment for lupus nephritis, achieving favorable outcomes in most patients. However, some patients with severe or refractory disease remain ineffective against these conventional medications, resulting in a poor prognosis. Furthermore, long-term use of steroids and immunosuppressants can be associated with serious side effects, such as metabolic disorders, osteoporosis, and immune suppression, which can increase the risk of infection.

[0007] Mesenchymal stem cells have low immunogenicity and strong immunomodulatory capabilities. They have shown certain potential and effectiveness in the treatment of lupus nephritis. Several clinical studies have shown that mesenchymal stem cell transplantation can significantly improve the clinical symptoms and disease activity of patients with lupus nephritis. For example, in some studies, patients' disease activity scores were significantly reduced and proteinuria levels decreased. Although mesenchymal stem cell therapy has shown potential in the treatment of lupus nephritis, it also has some shortcomings and deficiencies: the effects of mesenchymal stem cells from different sources vary in clinical trials, and some studies have failed to show significant improvement; in some cases, they may still cause infection; in some animal models, mesenchymal stem cells have been found to promote tumor growth; and although mesenchymal stem cells have low immunogenicity, they may still trigger an immune response in some cases.

[0008] Therefore, exploring different products derived from mesenchymal stem cells and establishing combined drug treatment plans to avoid the above adverse effects and better treat lupus nephritis are of great significance and have good application prospects.

[0009] After reviewing domestic literature and patents, no reports were found on the treatment of lupus nephritis with apoptotic vesicles of mesenchymal stem cells combined with tripterygium wilfordii polyglycosides. Summary of the Invention

[0010] (1) Technical problems solved

[0011] In view of this, one of the main purposes of the present invention is to provide the use of extracellular vesicles and tripterygium wilfordii extract in the preparation of drugs for treating lupus nephritis. Mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii polyglycosides were used to treat lupus nephritis. The results showed that after treatment with mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii polyglycosides, the enlargement of the lymph nodes and spleen of mice was significantly alleviated; the titer of serum autoantibodies - anti-double-stranded DNA antibodies, creatinine and urine protein were significantly reduced; and the renal pathology was significantly alleviated. In short, mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii polyglycosides can effectively treat lupus nephritis, and its effect is significantly better than treatment with mesenchymal stem cell apoptotic vesicles alone or tripterygium wilfordii polyglycosides alone.

[0012] (2) Technical solution

[0013] In order to achieve the above object, the present invention provides the use of extracellular vesicles and tripterygium wilfordii in the preparation of a medicament for preventing and / or treating lupus nephritis.

[0014] In one embodiment, the extracellular vesicles are secreted by stem cells.

[0015] In one embodiment, the stem cells include one or a combination of embryonic stem cells, adult stem cells, perinatal stem cells, or induced pluripotent stem cells.

[0016] In one embodiment, the stem cells are adult stem cells.

[0017] In one embodiment, the adult stem cells include mesenchymal stem cells and / or neural stem cells.

[0018] In one embodiment, the adult stem cells are mesenchymal stem cells.

[0019] In one embodiment, the stem cells include one or a combination of proliferating stem cells, differentiating stem cells, senescent stem cells, or apoptotic stem cells.

[0020] In one embodiment, the stem cell is an apoptotic stem cell.

[0021] In one embodiment, the extracellular vesicles are derived from apoptotic mesenchymal stem cells.

[0022] In one embodiment, the Tripterygium wilfordii extract comprises a Tripterygium wilfordii extract.

[0023] In one embodiment, the Tripterygium wilfordii extract is an organic solvent extract.

[0024] In one embodiment, the organic solvent extract is tripterygium wilfordii polyglycosides.

[0025] In one embodiment, the amount of the extracellular vesicles is 1×10 6 -1×10 7 / times and / or the dosage of tripterygium wilfordii polyglycosides is 10-20 mg / kg.

[0026] In one embodiment, the amount of the extracellular vesicles is 1×10 7 / Second-rate.

[0027] In one embodiment, the dosage of the tripterygium wilfordii polyglycosides is 20 mg / kg.

[0028] In one embodiment, the amount of the extracellular vesicles is 1×10 6 / Second-rate.

[0029] In one embodiment, the dosage of the tripterygium wilfordii polyglycosides is 10 mg / kg.

[0030] In one embodiment, the amount of the extracellular vesicles is 1×10 6 / time and the dosage of tripterygium wilfordii polyglycosides is 10 mg / kg.

[0031] In another aspect, the present invention further provides a pharmaceutical composition comprising:

[0032] (1) a therapeutically effective amount of the extracellular vesicles and Tripterygium wilfordii extract described above;

[0033] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0034] In another aspect, the present invention further provides a pharmaceutical preparation, which comprises the above-mentioned pharmaceutical composition.

[0035] In another aspect, the present invention further provides a product comprising the above-mentioned pharmaceutical preparation.

[0036] Beneficial effects

[0037] The present invention provides the use of extracellular vesicles and a tripterygium wilfordii extract in the preparation of a medicament for treating lupus nephritis. Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The isolation and production of mesenchymal stem cell apoptotic vesicles are simple, and the quality measurement is easy to standardize.

[0039] 2. The treatment of mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii extract not only combines the immunomodulatory and anti-inflammatory characteristics of tripterygium wilfordii extract and mesenchymal stem cell apoptotic vesicles, but also reduces the dosage of tripterygium wilfordii extract, greatly reducing its toxic side effects, such as reproductive system toxicity, gastrointestinal reactions, and liver and kidney function damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0041] Figure 1 Isolation and identification of MSC apoptotic vesicles; A. Morphology of apoptotic MSCs under a microscope; B. Extraction of vesicles from apoptotic MSCs; C. Detection of vesicle markers of apoptotic MSCs by western blot; D. Vesicle size of apoptotic MSCs.

[0042] Figure 2 The effect of treating lupus nephritis in mice; A. Anti-double-stranded DNA antibodies, creatinine and urea nitrogen in mouse serum; B. Spleen weight, lymph node weight and urine protein in mice.

[0043] Figure 3 This is a diagram showing the detection of inflammatory infiltration in the kidneys of mice with lupus nephritis after treatment. DETAILED DESCRIPTION

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0045] Currently, the mainstay of clinical treatment for lupus nephritis remains a combination of immunosuppressants and steroids, which achieves good results in most patients. However, some patients with severe or refractory disease do not respond well to these conventional medications and have a poor prognosis. Furthermore, long-term use of steroids and immunosuppressants can cause serious side effects, such as metabolic disorders, osteoporosis, and immune suppression, which can increase the risk of infection.

[0046] Mesenchymal stem cells can significantly improve clinical symptoms and disease activity in patients with lupus nephritis. For example, studies have shown that disease activity scores and proteinuria levels in patients with lupus nephritis are significantly reduced. However, mesenchymal stem cells have some drawbacks and limitations: clinical effects vary between batches; they may cause infection; they may promote tumor growth; and they may trigger an immune response.

[0047] Mesenchymal stem cell apoptotic vesicles contain mesenchymal stem cell-derived proteins, DNA, RNA, lipids, and metabolites, and share the immunomodulatory and immune tolerance-inducing properties of mesenchymal stem cells. Apoptotic vesicles can release a variety of mesenchymal stem cell-derived components, which can directly enter cells or be internalized by recipient cells to exert a variety of biological effects. The preparation method of mesenchymal stem cell apoptotic vesicles can be standardized, eliminating variability between batches and facilitating quality control. They can be prepared in large quantities, are easy to store and transport, and are inexpensive, making them more readily applicable for clinical treatment.

[0048] Tripterygium wilfordii extract is suitable for patients with mild to moderately active systemic lupus erythematosus, especially those with joint pain, skin erythema, or mild kidney involvement. It can be used as an adjunct to glucocorticoids or immunosuppressants to reduce drug dependence.

[0049] Mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii extract have immunosuppressive, anti-inflammatory and immunomodulatory effects. The combined use of the two can reduce the dosage of mesenchymal stem cell apoptotic vesicles and tripterygium wilfordii extract, thereby reducing the toxic side effects of tripterygium wilfordii extract and the potential side effects of mesenchymal stem cell apoptotic vesicles.

[0050] Terms and Definitions

[0051] As used herein, the term "pharmaceutical composition" refers to a composition comprising a drug formulated together with one or more pharmaceutically acceptable carriers.

[0052] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated using methods known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration to a mammal. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonades, perfumed waters, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic solutions, capsules, creams, lozenges, tinctures, pastes, pills and soft or hard gelatin capsules.

[0053] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0054] In the composition, pharmaceutically acceptable carriers include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch or talc, or liquid carriers, such as water, fatty oils or liquid paraffin. Other examples of the carrier include culture media, such as DM EM or RPMI; and low temperature storage media, comprising components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates and ion concentrations that balance the intracellular state at low temperatures; and mixtures of organic solvents and water.

[0055] The pharmaceutical composition disclosed in the present invention can be in the form of granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, or metered sprays.

[0056] The pharmaceutical compositions of the present invention can be administered using any known method.The term "administering" or "administering" a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those skilled in the art.

[0057] For example, the compound or agent can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter).

[0058] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and effective dosage levels include the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors well known in the medical art.

[0059] As used herein, the term "treating" a condition or patient means taking steps to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially improving or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition, or disorder, and avoiding harmful or unpleasant symptoms. Treating also refers to accomplishing one or more of the following: (a) lessening the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and / or (e) limiting the recurrence of symptoms in patients who were previously free of symptoms of the condition.

[0060] As used herein, the terms "prevention," "preventing," and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms (eg, diabetes or cancer).

[0061] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0062] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0063] Example 1

[0064] Preparation of apoptotic vesicles from mesenchymal stem cells:

[0065] 1. Mesenchymal stem cell culture and apoptosis induction: Third-generation umbilical cord blood-derived mesenchymal stem cells stored in liquid nitrogen were cultured in DF-12 medium containing 10% fetal bovine serum for 48 hours. The cultured cells were then irradiated with ultraviolet light at 200 mJ / cm² for 5 hours to induce apoptosis.

[0066] 2. Isolation and identification of apoptotic vesicles of mesenchymal stem cells: Collect cells after ultraviolet irradiation and centrifuge at 800g for 10 minutes at room temperature, then at 2,000g for 10 minutes, and finally at 16,000g for 30 minutes. Collect the precipitate after centrifugation, which is the apoptotic vesicles of mesenchymal stem cells (vesicles secreted during the culture process require ultracentrifugation and other methods to obtain and can basically be excluded).

[0067] Western blot was used to identify proteins in apoptotic vesicles, such as cleaved caspase3;

[0068] The diameter of apoptotic vesicles was detected by ZetaView nanoparticle analyzer.

[0069] The results are as follows Figure 1 As shown in Figure 2, apoptotic mesenchymal stem cells (MSCs) undergo morphological changes ( Figure 1 A); Isolate apoptotic MSC vesicles according to the method reported in the literature (Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell recept or signaling, Bioact Mater, 2023, 25: 472-484) Figure 1 B); Western blot was used to detect and compare the surface markers of MSC and MSC apoptotic vesicles. The results showed that MSC apoptotic vesicles had cleaved caspase 3, while MSC did not express it ( Figure 1 C); The results of the nanoparticle analyzer showed that the particle size of the extracted apoptotic MSC vesicles was between 50-250nm, which was consistent with the characteristics of vesicles ( Figure 1 D).

[0070] Example 2

[0071] Mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii polyglycosides for the treatment of lupus nephritis:

[0072] 1. Preparation of Lupus Nephritis Mouse Model:

[0073] 2. Treatment of Lupus Nephritis Mice: ① Mesenchymal Stem Cell Apoptotic Vesicles Alone Treatment Group: The apoptotic vesicles prepared in Example 1 were dissolved and counted using sterile PBS, and then 1×10 7 The mice were intraperitoneally injected with a dose of 10 mg / kg once a week for 4 consecutive weeks; ② The group treated with tripterygium wilfordii polyglycosides alone (Tripterygium wilfordii polyglycosides tablets, Shanghai Fudan Fuhua Pharmaceutical Co., Ltd., specification 10 mg / tablet) was gavaged with a 20 mg / kg dose per mouse using a gavage needle, and the treatment was continued for 4 consecutive weeks; ③ The group treated with apoptotic vesicles derived from mesenchymal stem cells combined with tripterygium wilfordii polyglycosides was treated with the apoptotic vesicles prepared in Example 1 (1×10 6 ) intraperitoneally once a week; concurrently, administer 10 mg / kg of Tripterygium wilfordii polyglycoside suspension orally daily. Combined treatment continued for 4 weeks.

[0074] 3. Evaluation of the efficacy of mesenchymal stem cell apoptotic vesicles combined with tripterygium wilfordii polyglycosides in the treatment of lupus nephritis: After 4 weeks of treatment, serum was collected and tested for autoantibodies, creatinine, and urea nitrogen; urine protein was tested; spleen and lymph node weights were tested; and kidneys were collected and stained with H&E and other methods to observe pathological changes in the mouse kidneys.

[0075] The results showed that the mesenchymal stem cell-derived apoptotic vesicles treatment group alone, the tripterygium wilfordii polyglycosides treatment group alone, and the mesenchymal stem cell-derived apoptotic vesicles combined with tripterygium wilfordii polyglycosides treatment group could significantly reduce the serum anti-double-stranded DNA antibodies, creatinine, and urea nitrogen levels of mice with lupus nephritis, but the mesenchymal stem cell-derived apoptotic vesicles combined with tripterygium wilfordii polyglycosides treatment effect was more significant ( Figure 2 A). At the same time, the mesenchymal stem cell-derived apoptotic vesicles treatment group alone, the tripterygium wilfordii polyglycosides treatment group alone, and the mesenchymal stem cell-derived apoptotic vesicles combined with tripterygium wilfordii polyglycosides treatment group all significantly reduced the weight of the spleen and cervical lymph nodes in mice with lupus nephritis ( Figure 2 B), significantly reduced the urine protein level in mice with lupus nephritis ( Figure 2 B), however, the effect of the mesenchymal stem cell-derived apoptotic vesicles combined with tripterygium wilfordii polyglycosides treatment group was the most significant ( Figure 2 B).

[0076] In terms of renal pathological changes, the mesenchymal stem cell-derived apoptotic vesicles treatment group and the tripterygium wilfordii polyglycosides treatment group had little effect on improving the infiltration of inflammatory cells in the kidneys of mice with lupus nephritis. However, the mesenchymal stem cell-derived apoptotic vesicles combined with tripterygium wilfordii polyglycosides treatment group had a significant effect on reducing the infiltration of inflammatory cells in the kidneys ( Figure 3 ).

[0077] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0078] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of extracellular vesicles and tripterygium wilfordii in the preparation of a medicament for preventing and / or treating lupus nephritis.

2. The use according to claim 1, characterized in that The extracellular vesicles are secreted by stem cells.

3. The use according to claim 2, characterized in that The stem cells include one or a combination of embryonic stem cells, adult stem cells, perinatal stem cells or induced pluripotent stem cells.

4. The use according to claim 3, characterized in that The stem cells are adult stem cells.

5. The use according to claim 4, characterized in that The adult stem cells include mesenchymal stem cells and / or neural stem cells.

6. The use according to any one of claims 2 to 5, characterized in that: The stem cells include one or a combination of proliferating stem cells, differentiating stem cells, senescent stem cells, and apoptotic stem cells.

7. The use according to claim 6, characterized in that The stem cells are apoptotic stem cells.

8. The use according to claim 7, characterized in that The amount of extracellular vesicles used is 1×10 6 -1×10 7 / times and / or the dosage of Tripterygium wilfordii extract is 10-20 mg / kg.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (1) a therapeutically effective amount of the extracellular vesicles and Tripterygium wilfordii extract according to any one of claims 1 to 8; (2) A pharmaceutically or immunologically acceptable carrier or excipient.

10. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 9.

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