Application of spirulina platensis in preparation of medicine for treating or preventing rheumatoid arthritis

Abstract

The invention discloses application of spirulina platensis in preparation of a medicine for treating or preventing rheumatoid arthritis. The invention discovers and verifies that spirulina platensis can effectively inhibit osteoclast activation and bone resorption, and can induce macrophage M2 type polarization while inhibiting macrophage M1 type polarization. In addition, the spirulina platensis can resist oxidative stress injury through an NRF2 / KEAP1 signal channel. Furthermore, in-vivo experiments of rats prove that the spirulina platensis can obviously relieve the development of RA of the rats.

Description

Technical Field

[0001] The present invention relates to a second use of Spirulina platensis, and particularly to the use of Spirulina platensis in the preparation of a medicament for treating or preventing rheumatoid arthritis. Background Art

[0002] Spirulina platensis is one of the excellent varieties of Spirulina, and is mainly distributed in regions such as Hainan, Guangdong, and Yunnan in China. Spirulina platensis mainly contains extracts of Spirulina, is rich in polysaccharides and some active substances, can improve human immunity, especially regulate the human immune system and enhance human resistance. In addition, it can also improve physical fatigue, regulate human blood lipids, reduce the cholesterol content in the blood, help people better lower blood pressure, and prevent some cardiovascular and cerebrovascular diseases. It can also improve the stomach and intestines, help treat gastric and intestinal ulcers, contains rich anti-tumor substances, helps people resist tumors, and provides various substances required by the human body.

[0003] Spirulina is one of the microalgae for large-scale production. The Spirulina commonly used for large-scale production includes Spirulina platensis, Spirulina maxima, and Spirulina subsalsa.

[0004] Spirulina platensis belongs to a kind of Spirulina, and is a micro cyanobacterium rich in nutrients, rich in proteins, polysaccharides, unsaturated fatty acids, carotenoids (β-carotene and zeaxanthin), chlorophyll, etc. In particular, the content of β-carotene is 15 times that of β-carotene in carrots. Due to its rich nutrition and high nutritional value, there are many studies on Spirulina platensis as a health food, which is used to supplement and improve body immunity, promote gastrointestinal peristalsis, and supplement the conventional elements and trace elements of the body.

[0005] Rheumatoid arthritis (RA) is a progressive systemic autoimmune disease that causes inflammation and damage to joints and other organs. The inflammation causes joint pain, stiffness, and swelling, resulting in loss of joint function due to bone and cartilage damage. RA usually leads to progressive disability. RA patients also have an increased likelihood of developing other systemic complications such as osteoporosis, anemia, cardiovascular diseases, and diseases affecting the lungs and skin. This disease shortens the average life expectancy of affected patients by 3 - 7 years. Although the disability rate caused by RA has decreased, about 30% of patients do not reach the lowest acceptable symptom state.

[0006] Currently, newly diagnosed RA patients usually take methotrexate; however, only 33% of patients show a robust response.

[0007] There is an urgent need to develop drugs that are effective in treating rheumatoid arthritis. At present, there is no report on the role of Spirulina platensis in the treatment of rheumatoid arthritis. Summary of the Invention

[0008] The object of the present invention is to provide a medical use of Spirulina platensis, which can be effectively used as a drug for treating or preventing rheumatoid arthritis.

[0009] To achieve the above object, the present invention provides the use of Spirulina platensis in the preparation of a drug for treating or preventing rheumatoid arthritis.

[0010] Optionally, the effect of the drug increases with the increase in the concentration of Spirulina platensis.

[0011] Optionally, in the drug, the concentration of Spirulina platensis is 6.25*10 5 cells / mL to 4*10 7 cells / mL.

[0012] Optionally, the drug is administered parenterally, by inhalation, topically or systemically.

[0013] Optionally, the drug is administered orally.

[0014] Advantages of the technical solution of the present invention:

[0015] The present invention provides a new use of Spirulina platensis in the preparation of a drug for treating or preventing rheumatoid arthritis. The present invention discovers and confirms that Spirulina platensis can effectively inhibit osteoclast activation and bone resorption, and can inhibit M1 polarization of macrophages while inducing M2 polarization of macrophages. Moreover, Spirulina platensis can counteract oxidative stress damage through the NRF2 / KEAP1 signaling pathway. Further, in vivo experiments on rats prove that Spirulina platensis can significantly alleviate the development of RA in rats. Brief Description of the Drawings

[0016] Figure 1 It is a schematic diagram of the results of the antioxidant stress effect of Spirulina platensis in Example 1.

[0017] Figure 2 It is a schematic diagram of the results of Spirulina inhibiting M1 polarization of macrophages and inducing M2 polarization of macrophages in Example 2.

[0018] Figure 3 It is a schematic diagram of the results of Spirulina platensis inhibiting osteoclast differentiation in Example 3.

[0019] Figure 4 、 Figure 5 It is a schematic diagram of the results of Spirulina platensis treating a mouse rheumatoid arthritis model in Example 4.

[0020] Figure 6 This is a schematic diagram of the results of the in vivo biosafety assessment of Spirulina in Example 5.

[0021] Figure 7 Schematic diagram of the results of the study on the mechanism of action of Spirulina in Example 6. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. 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 creative work are within the scope of protection of the present invention.

[0023] Rheumatoid arthritis (RA) is a systemic, inflammatory, autoimmune disease that mainly affects joints throughout the body. It is mainly manifested as chronic, symmetrical, progressive polyarthritis, synovial inflammation, lymphocyte infiltration, erosion of articular cartilage and bone, etc., which eventually leads to bone destruction. Bone destruction is the core factor causing many clinical problems such as joint deformity and loss of function in RA patients, and runs through the entire development process of RA.

[0024] Studies have shown that the mechanism of RA bone destruction is related to the proliferation and differentiation of osteoclasts, the regulation of osteoclasts by intracellular signaling pathways, the effects of cytokines on bone, the expression of bone metabolism-related genes, and other factors. Osteoclasts are key cells that mediate local bone destruction and joint damage in joints. In the absence of osteoclasts, the inflammatory response itself does not directly mediate joint destruction. In other words, excessive activation of osteoclasts plays a key role in the process of RA bone destruction. RANKL / OPG is a key regulatory factor in osteoclastogenesis. Many other cytokines, such as TNF-α, IL-1, IL-6, IL-17, as well as intracellular signaling molecules and transcription factors, are also involved in the regulation of osteoclastogenesis and activation.

[0025] In addition, imbalance in macrophage polarization is closely related to the occurrence and development of RA, among which M1 macrophages play a major role in promoting inflammation and bone destruction in the RA cytokine network environment. M2 macrophages secrete interleukin IL-10, inhibit the production of proinflammatory factors, and limit the inflammatory response. The abnormal immune microenvironment of RA patients promotes metabolic reprogramming of macrophages, which in turn affects the polarization state of macrophages, destroys the M1 / M2 dynamic balance, and leads to prolonged tissue inflammation. By clearing proinflammatory M1 macrophages, inducing anti-inflammatory M2 macrophages, and achieving macrophage polarization, it is expected to alleviate RA synovial inflammation.

[0026] In addition, oxidative stress plays an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of reactive oxygen species (ROS) and ROS-generating molecules, including superoxide, peroxide, hydroxyl radicals, and reactive nitrogen species such as peroxynitrite, are present in the synovial fluid and peripheral blood of RA patients. The mitogen-activated protein kinase (MAPK) signaling pathway is closely related to cell proliferation and apoptosis and can be stimulated by oxidative stress, hormones, or cytokine activation. Oxidative stress can activate MAPK by affecting fibroblast proliferation. The main source of ROS in the body of RA patients is the large amount of superoxide anion secreted by activated macrophages. The excessive generation of ROS can affect the activity of antioxidant enzymes, reducing their ability to scavenge free radicals and prevent the generation of new free radicals. Free radicals are involved in the inflammatory lesions of the RA joint synovium and bone destruction processes, where they can damage chondrocytes, degrade collagen, and proteoglycans, thus directly or indirectly participating in synovial and bone damage. Especially during the active stage of RA, the natural enzymes superoxide dismutase (SOD) and glutathione (GSH) in body tissues are significantly reduced, resulting in a decrease in the antioxidant capacity of body tissues, an excessive increase in ROS levels, and the inability of body tissues to effectively scavenge lipid peroxidation metabolites in a timely manner. These substances can directly or indirectly participate in the destruction of the joint synovium and bone. The oxidative stress state of RA patients can induce an in vivo inflammatory response, generating many inflammatory factors such as TNF-α and IL-6, which have an important impact on the onset of RA.

[0027] In summary, oxidative stress and inflammatory responses play crucial roles in the occurrence and development of RA, and act jointly on macrophages and osteoclasts, polarizing macrophages and inducing osteoclast activation. After activation, these two types of cells secrete a large amount of pro-inflammatory factors and release ROS, exacerbating local inflammation, so they are mutually causal. On the other hand, immune cells involved in the pathogenesis of RA directly upregulate the expression of receptor activator of NF-κB ligand (RANKL), or indirectly increase RANKL expression through the secretion of pro-inflammatory cytokines, promoting osteoclast formation, differentiation, and activation. This is manifested as osteoporosis in the early stage of RA and bone destruction in the late stage. Therefore, inhibiting osteoclast differentiation helps in the treatment of early RA, preventing disability and reducing mortality in RA patients. However, the research and development of current drugs against oxidative stress and anti-inflammatory responses are still in the initial stage and have not been widely used in clinical practice. There are no reports on drugs that can simultaneously resist oxidative stress and anti-inflammatory responses and inhibit osteoclastic resorption.

[0028] The present invention discovers that Spirulina platensis can effectively inhibit osteoclast activation and bone resorption, and can inhibit M1 polarization of macrophages while inducing M2 polarization of macrophages. Moreover, Spirulina platensis can counteract oxidative stress damage through the NRF2 / KEAP1 signaling pathway. Further, in vivo experiments on rats prove that Spirulina platensis can significantly alleviate the development of RA in rats. The following is a detailed description in combination with examples.

[0029] Example 1 Antioxidant stress effect of Spirulina platensis

[0030] Lipopolysaccharide (LPS) was used as an oxidative stress inducer, and the cell line was macrophage Raw264.7. Well-grown Raw264.7 cells were seeded in a confocal dish at a density of 1×10 4 cells per well, with 3 replicates in each group. After culturing with DMEM complete medium (DMEM containing 10% FBS and 100 U / mL double antibiotics) for 24 hours, 100 ng / mL of lipopolysaccharide and different concentration gradients of Spirulina platensis (the concentrations from low to high were 2.5*10 6 cells / mL, 5*10 6 cells / mL, 1*10 7 cells / mL) were co-incubated with Raw264.7 cells at 37°C for 24 hours. Among them, the blank group was without Spirulina platensis and lipopolysaccharide, and the control group was only added with lipopolysaccharide without Spirulina platensis.

[0031] The intracellular ROS content of Raw264.7 cells was fluorescently labeled using a reactive oxygen species detection kit. As Figure 1 shown, in the control group without Spirulina platensis, the amount of ROS was the largest. As the concentration of Spirulina platensis increased, the amount of ROS gradually decreased. When the concentration of Spirulina platensis was about 1*10 7 cells / mL, ROS was basically invisible. It can be proved that Spirulina platensis can inhibit or scavenge the generation of ROS during the polarization of macrophage Raw264.7.

[0032] Example 2 Inhibition of M1 polarization of macrophages and induction of M2 polarization of macrophages by Spirulina platensis

[0033] Lipopolysaccharide LPS was used as an inducer of macrophage inflammation, and the cell line was macrophage Raw264.7. After co-incubating different concentrations of Spirulina platensis with Raw264.7 cells in a 37°C constant temperature incubator (5% CO2) for 24 hours, the expression of related genes TNFα, iNOS, IL-1β, IL-6 and IL-12 in Raw246.7 cells was detected by polymerase chain reaction (PCR).

[0034] The experimental groups were as follows: PBS negative control group (A), LPS positive control group (B), LPS + Spirulina platensis group (C, the concentration of Spirulina platensis was 2.5*10 6 cells / mL), LPS + Spirulina platensis group (D, the concentration of Spirulina platensis was 5*10 6 cells / mL), LPS + Spirulina platensis group (E, the concentration of Spirulina platensis was 1*10 7 cells / mL).

[0035] As Figure 2 shown, with the increase in the concentration of Spirulina platensis, the expressions of genes TNFα, iNOS, IL-1β, IL-6 and IL-12 related to macrophage M1 polarization all gradually decreased. When the concentration of Spirulina platensis was 1*10 7 cells / mL, it could significantly inhibit the expressions of genes TNFα, iNOS, IL-1β, IL-6 and IL-12 related to macrophage M1 polarization. It can be proved that Spirulina platensis can effectively inhibit the polarization of M1 pro-inflammatory macrophages and has an anti-inflammatory effect. As mentioned above, macrophage polarization is an important part in the occurrence and development of RA. Therefore, Spirulina platensis is expected to counteract the development of RA.

[0036] Example 3 Inhibition of osteoclast differentiation by Spirulina platensis

[0037] Male wild-type mice at 4 - 6 weeks old were sacrificed by cervical dislocation. After disinfection, the long bones of both lower limbs were separated using sterile instruments in a laminar flow hood. A 1 mL syringe was used to aspirate the prepared α-MEM complete medium (containing 10% fetal bovine serum (FBS), 100 U / mL penicillin-streptomycin double antibody and 30 ng / mL recombinant M-CSF) to flush out all the bone marrow into a 10 cm dish, and a 1 mL pipette was used to pipette and mix to obtain homogeneous bone marrow cells. After 3 - 5 days of differentiation culture, the primary bone marrow cells were differentiated into primary bone marrow monocytes (BMMs). When the cells grew confluent, the primary bone marrow monocytes in good growth state were seeded into 96-well plates or 6-well plates at a density of 8×10 3 cells per well, with 3 replicates in each group; after culturing with α-MEM complete medium (α-MEM containing 10% FBS, 100 U / mL double antibody and 30 ng / mL M-CSF) for 24 hours, the medium was replaced with osteoclast differentiation induction medium (α-MEM containing 10% FBS, 100 U / mL double antibody, 30 ng / mL M-CSF, 50 ng / mL RANKL) and recorded as day 0 of osteoclast differentiation. At the same time, different concentration gradients (from low to high in concentration were 2.5*10 6 cells / mL, 5*10 6cells / mL, 1*10 7 cells / mL) of *Spirulina platensis* to stimulate the cells. Change the medium every other day, and terminate the differentiation on the 5th day of osteoclast differentiation for subsequent Trap staining ( Figure 3 a) of Figure 3 phalloidin staining, protein extraction and immunoblotting ( Figure 3 b) of

[0038] RNA extraction and qPCR ( Figure 3 c) of Figure 3 the experiment. The internal reference protein was β-actin.

[0038] Figure 3 As shown in the experimental results in Figure 3 Figure 3 Figure 3

[0039] Example 4 In Vivo Experiment: Treatment of a Mouse Model of Rheumatoid Arthritis with *Spirulina platensis*

[0040] Experimental Method

[0041] Take 24 DBA mice and give the first immunization injection at 8 weeks of age (inject PBS in the control group), subcutaneously inject 100 μL of bovine type II collagen + Freund's incomplete adjuvant (prepared at 1:1), and give a booster injection 3 weeks later. Divide them into 4 groups with 6 mice in each group: Group A is the control group (Sham group); Group B is the model group (RA model group); Group C is the model + *Spirulina platensis* group; Group D is the model + methotrexate group. Perform tail vein drug treatment three days after the two immunizations. Inject PBS subcutaneously in Group B, inject *Spirulina platensis* (4*10 7 cells / mL) in Group C, and inject methotrexate (1 mg / mL) in Group D. The injection dose is 100 μL. Treat once every three days and perform limb swelling score and clinical score every two days.

[0042]

[0043]

[0044] 1) Take pictures of the four limbs, front view and side view, to evaluate the diameter, length, width, and thickness of the four limbs;2) Limb score, with a total score of 0 - 4 points. Among them, normal is counted as 0 points; mild redness of the ankle joint or tarsal joint is counted as 1 point; slight redness and swelling from the ankle to the tarsus is counted as 2 points; moderate redness and swelling from the ankle joint to the metatarsal joint is counted as 3 points; severe redness and swelling of the ankle, foot, and fingers is counted as 4 points;

[0045] 3) Clinical score, with a total score of 0 - 4 points. Among them, normal is counted as 0 points; only one finger has erythema and edema is counted as 0.5 points; erythema and mild edema of the paw pad, ankle joint, or the second to fifth fingers is counted as 1 point; erythema and moderate edema of two joints (paw pad, ankle joint, the second to fifth fingers) is counted as 2 points; erythema and severe edema of the entire sole is counted as 3 points; swelling and deformation resulting in loss of limb mobility is counted as 4 points.

[0046] 60 - day endpoint assessment: 1) Gait; 2) Posterior knee diameter, length, width, thickness; 3) Pain; 4) Heat tolerance.

[0047] The experimental results are as Figure 4 shown: Figure 4 Figure a shows that at the 60 - day endpoint, the photographed results of the mouse limbs show that spirulina has an obvious anti - inflammatory effect, and the effect is similar to that of the RA clinical treatment drug methotrexate (MTX). Figure 4 Figure b shows the clinical score results at different time points. From the results of the mouse limb and clinical scores, it can be seen that Spirulina platensis can significantly relieve the swelling of the paw in the process of RA occurrence and development and reduce the clinical score. Figure 4 Figure c is the X - ray photograph of the mouse limbs, indicating that Spirulina platensis can inhibit the bone destruction of the ankle joint in the process of RA development. Figure 4 Figure d is the result of the ankle thickness at different time points, indicating that Spirulina platensis can significantly relieve the swelling of the paw in the process of RA occurrence and development. Figure 4 Figure e is the pain sensation score. The higher the threshold, the lower the pain sensitivity. The results show that Spirulina platensis may relieve the pain sensitivity caused by RA by relieving inflammation. Figure 4 Figure f is the erosion index score. The lower the score, the smaller the bone destruction. The results show that Spirulina platensis can relieve the bone destruction of the talus and increase the bone mass of the talus. Figure 4 The experimental results prove that after treatment with Spirulina platensis, the clinical score of the rats increases and the average palm thickness decreases significantly, indicating that Spirulina platensis treatment can significantly improve rheumatoid arthritis in mice and relieve the pain of mice.

[0048] Figure 5 Figure a is a schematic diagram of the mouse walking gait, indicating that Spirulina platensis can significantly improve the mouse walking gait. Figure 5 Figure b is the quantitative analysis of the maximum contact area of the mouse limbs, indicating that Spirulina platensis can significantly improve the mouse walking gait and increase the maximum contact area between the mouse limbs and the ground. Figure 5c is a quantitative analysis of the footprint area of ​​mice walking with their limbs, indicating that Spirulina platensis can significantly improve the walking gait of mice and increase the footprint area of ​​mice walking.

[0049] Depend on Figure 4 , Figure 5 Experimental analysis has shown that Spirulina platensis can significantly improve RA in mice, and can further improve the walking ability and walking quality of mice.

[0050] Example 5 In vivo biosafety assessment of Spirulina platensis

[0051] The mouse RA model was established as above and treated with Spirulina platensis (same dosage as in Example 4) and methotrexate (same dosage as in Example 4). At the 60-day endpoint, the mice were killed after anesthesia, and the heart, liver, spleen, lung and kidney of each group of mice were sliced ​​and stained to evaluate the biosafety of Spirulina platensis in mice.

[0052] like Figure 6 As shown in the figure, the H&E staining results of different organ tissues of RA mice treated with Spirulina indicated that Spirulina platensis had no toxicity to important organs such as heart, liver, spleen, lung, and kidney. The experimental groups were as follows: Group A was the sham operation group; Group B was the RA model group; Group C was the Spirulina platensis treatment group; and Group D was the methotrexate treatment group.

[0053] With the occurrence and development of RA, KEAP1 expression increases, causing NRF2 to be ubiquitinated and degraded. As the most important transcription factor in the cellular oxidative stress pathway, NRF2 can enter the nucleus to upregulate the expression of various antioxidant proteases such as SOD and GPX, remove excess free radicals in the body, and maintain cell homeostasis. Therefore, the degradation of NRF2 will lead to a reduction in the corresponding antioxidant enzymes, and the oxidative stress burden of the cells will increase. It can be seen that the endogenous pathway that regulates the level of oxidative stress in macrophages and plays an important antioxidant role is the NRF2 / KEAP1 signaling pathway. This reminds us that if the NRF2 / KEAP1 signaling pathway can be targeted and the protein level expression of KEAP1 can be reduced, then the ubiquitination level of NRF2 will inevitably decrease, its degradation will be inhibited, and protein expression will increase, which can become an important measure to alleviate the pathological mechanism of RA. The following is a detailed description in conjunction with Example 6.

[0054] Example 6 Study on the mechanism of action of Spirulina platensis

[0055] The cell line used was macrophage Raw264.7. Raw264.7 cells with good growth status were cultured at 3×10 5 The cells were inoculated on a 6-well plate at a density of 100 μg / mL. After culturing for 24 hours in DMEM complete medium (DMEM containing 10% FBS and 100 U / mL double antibody), different concentration gradients (from low to high, 6.25*10 5cells / mL, 1.25*10 6 cells / mL, 2.5*10 6 cells / mL, 5*10 6 cells / mL, 1*10 7 cells / mL) of Spirulina platensis were co-incubated with Raw264.7 cells at 37 °C for 24 hours, followed by subsequent protein extraction and immunoblotting ( Figure 7 a), and cell immunofluorescence assay (the concentrations of Spirulina platensis from low to high were 2.5*10 6 cells / mL, 5*10 6 cells / mL, 1*10 7 cells / mL, Figure 7 b), and the internal reference protein was β-actin.

[0056] As Figure 7 shown in a), as the concentration of Spirulina platensis increased, the expression of KEAP1 protein decreased, the expression of NRF2 protein increased, and the corresponding antioxidant proteins such as SOD2 and HO-1 also increased with the increase in the concentration of Spirulina platensis. However, GPX1 and GPX4 showed a trend of increasing first and then decreasing, which may be due to the different expression times of different antioxidant enzymes. Moreover, with the increase in the concentration of Spirulina platensis, LC3 autophagosomes increased and co-localized with KEAP1. It was speculated that this co-localization would enable autophagosomes to phagocytose KEAP1 and digest KEAP1 protein after binding to lysosomes ( Figure 7 b). As KEAP1 was digested, its function of assisting in ubiquitination of NRF2 and degrading it was affected, resulting in an increase in the expression of NRF2 and its entry into the nucleus, activating the antioxidant pathway and increasing the expression of antioxidant proteins SOD2, HO-1, GPX1, and GPX4. This proved that the protective effect of Spirulina platensis on macrophages was achieved by regulating the NRF2 / KEAP1 pathway and activating the antioxidant stress process.

[0057] In summary, the present invention discovered a new medical use of Spirulina platensis for health food. Through experiments, it was proved that Spirulina platensis has the effects of antioxidant stress, inhibiting M1 polarization of macrophages and inducing M2 polarization of macrophages, and inhibiting osteoclast differentiation, and can effectively treat or prevent rheumatoid arthritis. And through animal model experiments, it was proved that Spirulina platensis has an effective treatment and biological safety for murine rheumatoid arthritis. Further, the present invention also explored the theoretical basis for the ability of Spirulina platensis to treat or prevent rheumatoid arthritis through mechanism research experiments.

[0058] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and alternatives to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. Use of Spirulina platensis in the preparation of a medicament for treating or preventing rheumatoid arthritis.

2. The use according to claim 1, characterized in that, The effect of the medicament increases with the increase in the concentration of Spirulina platensis.

3. The use according to claim 2, characterized in that, In the said drug, the concentration of Spirulina platensis is 6.25*10 5 cells / mL to 4*10 7 cells / mL.

4. The use according to claim 1, characterized in that, The medicament is administered parenterally, by inhalation, topically or systemically.

5. The use according to claim 1, wherein, The medicament is administered orally.

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