Application of CircSS18 in drugs for treating osteoarthritis and drug preparation method

Abstract

The present invention discloses the use of CircSS18 in a drug for the treatment of osteoarthritis and a method for its preparation. Local intra-articular injection of a nano-controlled-release hydrogel composite overexpressing circSS18 has significant potential clinical value in the targeted treatment of osteoarthritis: 1. Compared with current clinical local intra-articular injections of sodium hyaluronate and platelet-rich plasma (PRP), this method has a clear target and mechanism of action, making the treatment more targeted and effective, and a curative approach. 2. Compared with current clinical oral symptomatic treatments such as NSAIDs and glucosamine, this method can fundamentally inhibit the progression of osteoarthritis. 3. Compared with surgical procedures such as arthroscopy and joint replacement, this method is less invasive, less expensive, more feasible, and more practical, making it more acceptable to patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to use of circSS18 in drugs for treating osteoarthritis and a method for preparing the drug. Background Art

[0002] Osteoarthritis (OA) is one of the most common degenerative diseases. The number of people suffering from OA is rapidly increasing. The aging population and the lack of effective early treatments have led to an increasingly heavy social and economic burden from OA. Therefore, exploring effective interventions to delay the onset and progression of OA is a key breakthrough in OA treatment.

[0003] The current treatments for OA are mainly early rehabilitation physical therapy and drug therapy. The drugs mainly include symptomatic anti-inflammatory and analgesic drugs (NSAIDs) and glucosamine, as well as intra-articular injections of sodium hyaluronate and platelet-rich plasma (PRP) for treatment. Since the pathogenesis and treatment targets of OA have not been fully clarified, the above drugs are all symptomatic treatments with uncertain efficacy, and cannot effectively inhibit the progression of OA in the early stages. Arthroscopic debridement is performed in the middle and late stages of the disease. Although arthroscopic debridement is less invasive, its efficacy is limited, and it is difficult to cure OA fundamentally. In the late stages of the disease, artificial joint replacement is often required. Artificial joint replacement surgery is highly invasive and expensive, and the artificial joint may require a secondary revision surgery after a certain service life, which greatly increases the patient's trauma and socioeconomic burden. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention proposes the application of CircSS18 in the treatment of osteoarthritis.

[0005] Use of CircSS18 in drugs for treating osteoarthritis, and use of a circSS18 overexpressing plasmid in the preparation of drugs for treating osteoarthritis.

[0006] Preferably, the nano-controlled release hydrogel is combined with a plasmid overexpressing circSS18 in the preparation of a drug for treating osteoarthritis.

[0007] Preferably, a nano controlled-release hydrogel containing a composite overexpression of circSS18 plasmid is injected into the knee joint cavity.

[0008] The beneficial effects of the present invention are:

[0009] The present invention utilizes nano-controlled release hydrogel compounded with overexpressed circSS18 for local intra-articular injection, which has great potential clinical value in the targeted treatment of OA:

[0010] 1. Compared with the current clinical local joint cavity injection of sodium hyaluronate and platelet-rich plasma (PRP) treatment, the present invention has a clear target and mechanism of action, and the treatment is more targeted and effective, and is a curative means;

[0011] 2. Compared with the current oral medications for symptomatic treatment such as NSAIDs and glucosamine, the present invention can fundamentally inhibit the progression of OA;

[0012] 3. Compared with arthroscopy, joint replacement and other surgeries, the present invention has less trauma, lower cost, higher feasibility and operability, and is easily accepted by patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 Schematic diagram of significantly differentially expressed circular RNAs detected by microarray in chondrocytes of human adipose-derived stem cells (hADSCs) at 3 and 21 days of chondrogenic differentiation;

[0015] Figure 2 Schematic diagram for validating the top ten differentially expressed circular RNAs in the IL-1β osteoarthritis cell model;

[0016] Figure 3 Schematic diagram for validating the top ten differentially expressed circular RNAs in the TNF-α osteoarthritis cell model;

[0017] Figure 4 Schematic diagram to verify the high expression of circSS18 in the early and middle stages of hADSC chondrogenic differentiation;

[0018] Figure 5 Schematic diagram of the expression of TNF-α in 9 pairs of normal and osteoarthritis cartilage tissues;

[0019] Figure 6 Schematic diagram showing that the circSS18 overexpression plasmid was successfully constructed by PCR experiment;

[0020] Figure 7 Schematic diagram of the expression of extracellular matrix (ECM) synthesis indicator COL2A1 and decomposition indicator MMP13 detected by transfection of circSS18 overexpression plasmid;

[0021] Figure 8 Schematic diagram for detecting the cartilage protective effect of circSS18 in mice. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of 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. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0023] With the advancement of sequencing and bioinformatics analysis technologies, studies have revealed that circular RNA (circRNA) structures are stable, highly conserved, and highly tissue- and stage-specific. Furthermore, circRNAs possess diverse biological functions, playing important roles in sponging miRNAs, binding or translating proteins, and regulating the expression of parental genes. A growing number of research institutions are dedicated to designing and delivering novel engineered circRNAs to overcome the limitations of linear mRNA technology and enhance the therapeutic potential of RNA in a variety of diseases, including cancer, autoimmune diseases, and genetic disorders. Consequently, circRNAs have become one of the most promising targets in the diagnosis and treatment of numerous diseases.

[0024] Differentially expressed circSS18 was screened out during the chondrogenic differentiation of human adipose-derived stem cells (hADSCs). Preliminary in vitro and in vivo experimental studies found that EIF4A3 may promote the nuclear export of circSS18, and HuR competitively binds to IGF2BP2 with circSS18, thereby inhibiting the activity of the cGAS-STING signaling pathway, ultimately promoting the chondrogenic differentiation of hADSCs and inhibiting PHC degeneration. The application of nano-controlled release hydrogels to deliver overexpressed circSS18 has become one of the therapeutic strategies for delaying the progression of OA in the early stage.

[0025] The use of circSS18 in drugs for treating osteoarthritis, including the use of a circSS18 overexpressing plasmid in the preparation of a drug for treating osteoarthritis; the use of a nano-controlled release hydrogel composited with a circSS18 overexpressing plasmid in the preparation of a drug for treating osteoarthritis, and the specific preparation process of the nano-controlled release hydrogel composited with a circSS18 overexpressing plasmid: pcDNA3.1(+)-S-hsa_circ_0002164 plasmid primer sequences: F: 5'-TGCAGCCAAACCAAGGTATC-3', R: 5'-GTGGTGCTGGTAAAAGAGAC-3', dissolving quaternized xylan (QAX), cellulose nanocrystal (CNC) colloidal solution and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) in water, and adding ammonium persulfate (APS) and N,N-methylenebisacrylamide (MBA). The PVA solution and the pcDNA3.1(+)-S-hsa_circ_0002164 plasmid were added dropwise to the homogenous mixture while stirring. After adding tetramethylethylenediamine (TMEDA), the mixture was polymerized at -20°C for 12 hours and then thawed at room temperature for 1 hour. After repeating the freeze / thaw cycle twice, a hydrogel was obtained. The hydrogel was then soaked in ultrapure water for three days to remove impurities, completing the preparation.

[0026] The application method is to inject nano-controlled release hydrogel containing overexpressed circSS18 plasmid into the knee joint cavity.

[0027] Instructions attached Figure 1-5 The chip screened and verified the expression pattern of circSS18. The chip screened and verified that circSS18 was highly expressed in the early and middle stages of chondrogenic differentiation and normal cartilage tissue, suggesting that circSS18 can promote cartilage differentiation and play a role in protecting cartilage. Figure 1 The middle part is the cartilage microarray of human adipose-derived stem cells (hADSC) that were differentiated into chondrogenic microspheres for 3 days and 21 days, and the significantly differentially expressed circular RNA was detected; the attached Figure 2 The top ten circular RNAs with significantly differential expression in the chip were verified in the osteoarthritis cell model constructed by IL-1β, indicating that circSS18 was significantly underexpressed in these osteoarthritis cells, suggesting that circSS18 can inhibit the progression of osteoarthritis. Figure 3 The top ten circular RNAs with significant differential expression in the chip were verified in the osteoarthritis cell model constructed by TNF-α, indicating that circSS18 was also significantly low-expressed in these osteoarthritis cells, again suggesting that circSS18 can inhibit the progression of osteoarthritis. Figure 4 The results showed that circSS18 was highly expressed in the early and middle stages of hADSC chondrogenic differentiation, suggesting that circSS18 could promote chondrogenic differentiation. Figure 5 The results of a study in 9 pairs of normal and osteoarthritis cartilage tissues showed that circSS18 was highly expressed in normal cartilage tissue, but the opposite was true in osteoarthritis cartilage tissue, suggesting that circSS18 may play a role in protecting cartilage.

[0028] Figure 6-7 To detect the phenotype and possible mechanism of action of circSS18. Figure 6 The expression of circSS18 and its parent gene SS18 was detected by transfection of circSS18 overexpression plasmid. PCR test showed that the circSS18 overexpression plasmid was successfully constructed. Figure 7 The expression of ECM synthesis indicator COL2A1 and degradation indicator MMP13 was detected by transfection of circSS18 overexpression plasmid. Western blot test showed that after overexpression of circSS18, the synthesis indicator COL2A1 in the extracellular matrix was significantly upregulated, while the degradation indicator MMP13 in the extracellular matrix was significantly downregulated, which identified that circSS18 exerts cartilage protection by promoting extracellular matrix synthesis.

[0029] Attachment Figure 8 This is an osteoarthritis mouse model. The figure shows that after the osteoarthritis mouse model (DMM) was constructed, two groups were divided. One group of mice was injected into the joint cavity with a nano-controlled-release hydrogel compound overexpression plasmid control group, and the other group of mice was injected into the joint cavity with a nano-controlled-release hydrogel compound overexpression circSS18 plasmid. The joint cavity sections were then stained with HE, Safranin O Fast Green, COL2A1 and MMP13, all of which indicated that circSS18 can play a role in protecting cartilage in mice.

[0030] Animal experiments further found that local intra-articular injection of nano-controlled-release hydrogels combined with overexpressed circSS18 has great potential clinical value in the targeted treatment of OA:

[0031] 1. Compared with the current clinical local joint cavity injection of sodium hyaluronate and platelet-rich plasma (PRP) treatment, the present invention has a clear target and mechanism of action, and the treatment is more targeted and effective, and is a curative means;

[0032] 2. Compared with the current oral medications for symptomatic treatment such as NSAIDs and glucosamine, the present invention can fundamentally inhibit the progression of OA;

[0033] 3. Compared with arthroscopy, joint replacement and other surgeries, the present invention has less trauma, lower cost, higher feasibility and operability, and is easily accepted by patients.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of a plasmid overexpressing circSS18 in the preparation of a medicament for treating osteoarthritis.

2. Use of nano-controlled release hydrogel combined with a plasmid overexpressing circSS18 in the preparation of a drug for the treatment of osteoarthritis.

3. Use of the nano-controlled release hydrogel combined with a plasmid overexpressing circSS18 according to claim 2 in the preparation of a medicament for treating osteoarthritis, characterized in that: Nano-controlled release hydrogel containing circSS18 overexpressing plasmid was injected into the knee joint cavity.

4. Use of the nano-controlled release hydrogel combined with a plasmid overexpressing circSS18 according to claim 2 in the preparation of a medicament for treating osteoarthritis, characterized in that: The specific preparation process of the nano-controlled release hydrogel composite overexpression circSS18 plasmid is as follows: pcDNA3.1(+)-S-hsa_circ_0002164 plasmid primer sequence: F: 5'-TGCAGCCAAACCAAGGTATC-3', R:5'-GTGGTGCTGGTAAAAGAGAC-3', quaternized xylan, cellulose nanocrystal colloidal solution, and 2-acrylamido-2-methylpropanesulfonic acid were dissolved in water, and ammonium persulfate and N,N-methylenebisacrylamide were added; PVA solution and pcDNA3.1(+)-S-hsa_circ_0002164 plasmid were added dropwise to the above uniform mixture under stirring; tetramethylethylenediamine was added to the mixture, polymerized at -20°C for 12 hours, and then thawed at room temperature for 1 hour; a hydrogel was obtained after repeating the freeze / thaw cycle twice; the hydrogel was then soaked in ultrapure water for three days to remove impurities, completing the preparation and ready for use.

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