Application of Klotho in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention belongs to the field of medicines for treating rheumatoid arthritis, discloses application of Klotho in preparation of medicines for treating rheumatoid arthritis, and particularly relates to application of Klotho in improvement of the inflammatory state of rheumatoid arthritis by regulating a PI3K-AKT signal channel. Klotho can effectively improve clinical inflammation indexes of RA (rheumatoid arthritis). The Klotho can improve the inflammatory state of the RA by adjusting a PI3K-AKT signal channel. Animal experiments prove that Klotho can be applied to treatment of rheumatoid arthritis diseases.

Description

Technical Field

[0001] The present invention belongs to the field of rheumatoid arthritis therapeutic drugs, and particularly relates to the application of Klotho in the preparation of rheumatoid arthritis therapeutic drugs. Background Art

[0002] Rheumatoid arthritis (RA) is one of the most common autoimmune diseases. It is a systemic, polyarticular, chronic, progressive inflammatory musculoskeletal disease of the synovial joints that may be influenced by genetic, epigenetic, and environmental factors. It is characterized by inflammatory changes in the synovial tissue of the joints, cartilage, and bone, and less commonly, in extra-articular locations. Initially, only a few joints are affected, such as the proximal interphalangeal and metacarpophalangeal joints; however, in later stages, many joints are affected, and extra-articular areas can also be affected, including the lungs, heart, blood vessels, skin, and eyes, which can be life-threatening.

[0003] αKlotho (herein referred to as Klotho) was discovered accidentally by Kuro-o et al. in 1997 and is considered a longevity gene, associated with antioxidant, anti-inflammatory, anti-apoptotic, and anti-aging effects. The NHANES data analyzed the relationship between serum klotho levels and all-cause mortality and cardiovascular mortality in RA patients. A significant U-shaped association was observed between serum klotho levels and all-cause mortality, indicating that lower serum klotho was significantly associated with higher all-cause mortality in RA patients. The results suggest that klotho has a protective effect on RA patients. However, the mechanism of action of klotho on RA has not yet been clarified. Summary of the Invention

[0004] To overcome the deficiencies of the prior art, the present invention provides the use of Klotho in the preparation of a drug for treating rheumatoid arthritis, and verifies the effect of Klotho on inflammation-related signaling pathways using a collagen-induced arthritis (CIA) model.

[0005] The first purpose of the present invention is to protect the use of Klotho in preparing drugs for treating rheumatoid arthritis.

[0006] Furthermore, the use of Klotho in the preparation of a drug for treating rheumatoid arthritis is specifically used to improve the inflammatory state of rheumatoid arthritis by regulating the PI3K-AKT signaling pathway.

[0007] Furthermore, the drug for treating rheumatoid arthritis is a drug for human use.

[0008] Another object of the present invention is to protect a drug for treating rheumatoid arthritis, the ingredient of the drug comprising Klotho.

[0009] Furthermore, the drug for treating rheumatoid arthritis includes Klotho and excipients, wherein the excipients are one or more of purified water, surfactants, chelating agents, antioxidants, stabilizers, and osmotic pressure regulators.

[0010] Furthermore, the Klotho content in the drug for treating rheumatoid arthritis is 50-80%.

[0011] The present invention offers the following advantages over existing technologies: Klotho can effectively improve clinical inflammatory markers in RA. Klotho improves RA inflammation by regulating the PI3K-AKT signaling pathway. Animal studies have confirmed that Klotho can be used to treat rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0013] Figure 1 HE staining and result scores of ankle joint sections in each group;

[0014] Figure 2 HE staining and result scores of knee joint sections in each group;

[0015] Figure 3 is the Western blot image of PI3K and AKT in spleen;

[0016] Figure 4 It is a statistical chart of the ratio of PI3K and AKT to β-acrtin. DETAILED DESCRIPTION

[0017] The present invention is described in detail below by specific examples, but the scope of protection of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0018] Example 1

[0019] Animal Experiment: 8-10-week-old male WT and HE mice were randomly divided into a wild-type control group (WT NC), a heterozygous control group (HE NC), a wild-type CIA group (WT CIA), and a heterozygous CIA group (HE CIA). Chicken type II collagen and complete Freund's adjuvant (CFA) were added to two sterile glass syringes at equal volumes. The syringes were connected via a three-way valve and repeatedly injected at low temperature until completely emulsified. CIA mice were injected subcutaneously at the base of the tail with a volume of 0.1 ml. On day 21 after immunization, a second injection of chicken type II collagen emulsified with incomplete Freund's adjuvant was performed using the same method and conditions approximately 2 cm from the site of primary immunization. On day 21 after the second immunization, 40 μg of LPS was injected intraperitoneally into CIA mice using PBS buffer.

[0020] Hematoxylin and eosin (HE) staining: (1) Dewaxing: Dewax the joint tissue in a gradient manner. Xylene 10 min, 2 times; anhydrous ethanol 5 min, 2 times; 95% ethanol 5 min, 1 time; 90% ethanol 5 min, 1 time; 80% ethanol 5 min, 1 time. (2) Staining: Hematoxylin staining for 2 min; distilled water washing for 1 min; eosin staining for 1 min. (3) Sealing: After staining, the slices were dehydrated in a gradient manner. The order was 80% ethanol 2 s, 1 time; 90% ethanol 2 s, 1 time; 95% ethanol 2 s, 1 time; anhydrous ethanol 1 min, 3 times; neutral gum was added and coverslips were used to seal the slices. (4) Observation and photography: After sealing, the slices were sliced ​​and the histological changes of the synovium of each group of rats were observed and photographed. According to the inflammation scoring standard, 0 points are normal; 1 point is mild infiltration of inflammatory cells; 2 points is moderate infiltration of inflammatory cells; 3 points is severe infiltration of inflammatory cells.

[0021] Western blot detection of protein expression: (1) Lysis: Take spleen tissues from each group of mice, add these tissues to tissue protein lysis buffer, and mince them. Discard the supernatant and add RIPA:PMSF = 100:1. Place it on ice and let it stand. Then use an ultrasonic disruptor to fully lyse the tissue. After standing for 30 minutes, centrifuge at 12000r for 30 minutes, and take the supernatant for storage. (2) Protein quantification: Follow the instructions for use of BCA reagent. Use an enzyme-linked microplate reader to measure the absorbance of the sample, and calculate the corresponding data based on the absorbance of the standard well and the known concentration. Draw a standard curve and deduce the concentration of the sample to be tested. (3) Sample preparation: Mix the sample of known concentration with protein loading buffer in proportion, place it in a metal bath, and heat it at 100℃ for 10 minutes to prepare a protein electrophoresis sample. After cooling the sample to room temperature, store it in a -20℃ refrigerator for use. (4) Gel preparation: Prepare a 5% upper layer of concentrated gel and a 10% lower layer of separation gel. (5) Loading. (6) Electrophoresis: In the experiment, first perform electrophoresis at 80V, 360mA at a constant current for 30 minutes. Then adjust the voltage to 120V, 360mA and continue running at a constant current. (7) Cut PVDF membranes of equal size according to the size of the target protein gel and place these membranes in anhydrous ethanol for activation. (8) Place the transfer tank in an ice water tank. Set the current to 360mA and the voltage to 300V, and select the appropriate transfer time according to the molecular weight under the condition of constant voltage. (9) Blocking: Wash with TBST solution for 5 minutes, then add 5% BSA blocking solution and block at room temperature for 2 hours. (10) Primary antibody incubation and washing. (11) Secondary antibody incubation and washing. (12) Development. (13) Result analysis.

[0022] The experimental results showed that in addition, HE staining was performed on the tissue sections of the knee and ankle joints of each group. The results showed that there was no inflammatory cell infiltration and proliferation in the synovial membrane of the knee and ankle joints of the control group WT NC and HE NC, the cells were arranged in an orderly manner, and the surface of the articular cartilage and bone tissue was smooth. However, the synovial membrane of the knee and ankle joints of CIA had obvious proliferation, the cells were arranged in a disorderly and sparse manner, the inflammatory cells were severely infiltrated, new blood vessels were formed, and the bone tissue was eroded and damaged. Among them, the inflammatory infiltration of the synovial membrane of the knee and ankle joints of HE CIA was more severe than that of WT CIA, and the inflammation score was higher. The above results suggest that in CIA, the loss of Klotho protein promotes bone erosion, cartilage destruction and joint inflammation.

[0023] Western blot analysis was used to analyze the effect of Klotho on the PI3K / AKT signaling pathway in CIA. The results showed that PI3K and AKT expression in the spleens of HE NCs was higher than that in WT NCs. After CIA induction, PI3K and AKT expression was significantly elevated, with HE CIA expressing significantly higher levels of PI3K and AKT than WT CIA.

[0024] The results showed that Klotho exerted anti-inflammatory effects through PI3K / AKT, improved CIA inflammation and bone destruction, and alleviated the disease progression.

[0025] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. Application of Klotho in the preparation of drugs for the treatment of rheumatoid arthritis.

2. The use of Klotho in preparing a drug for treating rheumatoid arthritis according to claim 1, characterized in that Specifically, Klotho is used to improve the inflammatory state of rheumatoid arthritis by regulating the PI3K-AKT signaling pathway.

3. A drug for treating rheumatoid arthritis, characterized in that: Ingredients include Klotho.

4. The drug for treating rheumatoid arthritis according to claim 3, characterized in that The ingredients include Klotho and excipients, wherein the excipients are one or more of purified water, surfactants, chelating agents, antioxidants, stabilizers, and osmotic pressure regulators.

5. The drug for treating rheumatoid arthritis according to claim 4, characterized in that The Klotho content in the medicine for treating rheumatoid arthritis is 50-80 wt%, and the remainder is auxiliary materials.

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