RNA hydrogel for tissue regeneration and method for producing same

Abstract

The present invention relates to an RNA hydrogel for tissue regeneration, and more specifically, to an RNA hydrogel for tissue regeneration, which can provide enhanced cell proliferation effect and anti-inflammatory effect, regenerate tissue through wound healing, can be efficiently delivered to cells, does not contain additional chemical components other than nucleic acids, thereby ensuring safety, can deliver a large amount of substances in vivo without toxicity, and can be used for tissue regeneration. The defect that the existing nucleic acid medicine is easy to decompose due to various enzymes in plasma is overcome, and meanwhile, the RNA hydrogel of adenosine is generated in quantity through cell metabolism.

Description

Technical Field

[0001] This invention relates to an RNA hydrogel for tissue regeneration. More specifically, the RNA hydrogel provides enhanced cell proliferation and anti-inflammatory effects, enabling tissue regeneration through wound healing. It can be effectively delivered into cells and does not contain any additional chemical components other than nucleic acids, thus ensuring safety and enabling high-dose delivery without in vivo toxicity. At the same time, the RNA hydrogel overcomes the disadvantage of existing nucleic acid drugs being easily degraded by various enzymes in the blood and can generate a large amount of adenosine through cell metabolism. Background Technology

[0002] Adenosine is a purine nucleoside formed by linking adenine (a nucleobase) with ribose (a pentose sugar), and it has been used to treat a variety of diseases.

[0003] As an example of a disease that can be treated using the adenosine, tissue regeneration achieved through wound healing can be cited as described in the following patent documents.

[0004] Patent Documents

[0005] Patent No. 10-2298371 (granted on August 31, 2021): "A composition for improving skin condition containing PDRN derived from the callus tissue of *Lysimachia christinae* as an active ingredient and a method thereof for preparation thereof."

[0006] However, existing therapeutic agents such as PDRN based on adenosine properties remain controversial in terms of safety and lack stability, making it difficult to fully exert their therapeutic effects. Summary of the Invention

[0007] Technical issues

[0008] This invention is proposed to solve the above-mentioned problems.

[0009] The purpose of this invention is to provide an RNA hydrogel that can provide enhanced cell proliferation and anti-inflammatory effects, and achieve tissue regeneration through wound healing.

[0010] Furthermore, the present invention aims to provide an RNA hydrogel that can be effectively delivered into cells without containing any additional chemical components other than nucleic acids, thereby ensuring safety and enabling high-dose delivery without in vivo toxicity; at the same time, the RNA hydrogel can overcome the defect of existing nucleic acid drugs being easily degraded by the action of various enzymes in the blood.

[0011] Furthermore, the present invention aims to provide an RNA hydrogel that can generate a large amount of adenosine through cellular metabolism and can be effectively delivered to immune cells, thereby inducing an anti-inflammatory response.

[0012] means for solving problems

[0013] To achieve the above objectives, the present invention is implemented through embodiments having the following configuration.

[0014] According to an embodiment of the present invention, the method for preparing the RNA hydrogel for tissue regeneration includes: a circular DNA generation step of reacting single-stranded linear DNA with primer DNA to generate circular DNA; and a gel formation step of mixing the circular DNA with RNA polymerase and performing rolling circle transcription (RCT) to form the RNA hydrogel, wherein at least 80% of the total base sequence of the single-stranded linear DNA is composed of thymine.

[0015] According to another embodiment of the present invention, in the method for preparing RNA hydrogel for tissue regeneration, the gel formation step is characterized by: mixing circular DNA, a mixture of ribonucleotide solutions, ATP, ribonuclease, and RNA polymerase in a reaction buffer, and reacting at 30–40°C for 2.7–3.3 hours to complete the gel formation step.

[0016] According to another embodiment of the present invention, in the method for preparing RNA hydrogel for tissue regeneration, the RNA hydrogel is absorbed in vivo and can generate adenosine, thereby reducing the level of pro-inflammatory cytokines and increasing the level of anti-inflammatory cytokines.

[0017] According to another embodiment of the present invention, in the method for preparing RNA hydrogel for tissue regeneration, the RNA hydrogel can promote cell proliferation and achieve tissue regeneration through wound healing.

[0018] According to another embodiment of the present invention, in the method for preparing RNA hydrogel for tissue regeneration, the linear DNA is composed of SEQ ID NO. 1 and the primer is composed of SEQ ID NO. 2.

[0019] According to another embodiment of the present invention, in the RNA hydrogel for tissue regeneration of the present invention, at least 80% of the bases constituting the RNA hydrogel are adenine; and, after the RNA hydrogel is absorbed in vivo, it can generate adenosine, reduce the level of pro-inflammatory cytokines, and increase the level of anti-inflammatory cytokines.

[0020] The effects of the invention

[0021] The present invention achieves the following effects through the above embodiments.

[0022] This invention can provide enhanced cell proliferation and anti-inflammatory effects, thereby achieving tissue regeneration through wound healing.

[0023] Furthermore, this invention can be effectively delivered into cells and does not contain any additional chemical components other than nucleic acids, thereby ensuring safety. It enables high-dose delivery without in vivo toxicity and overcomes the disadvantage of existing nucleic acid drugs being easily degraded by various enzymes in the blood.

[0024] Furthermore, the present invention can generate a large amount of adenosine through cell metabolism and effectively deliver it to immune cells, thereby inducing an anti-inflammatory response. Attached Figure Description

[0025] Figure 1 The diagram is for illustrative purposes only and illustrates a method for preparing an RNA hydrogel according to an embodiment of the present invention and its working principle.

[0026] Figure 2 A digital image of an RNA hydrogel according to an embodiment of the present invention.

[0027] Figure 3 This is a field emission scanning electron microscope (FE-SEM) image of an RNA hydrogel according to an embodiment of the present invention.

[0028] Figure 4 The digital images illustrate how RNA hydrogels according to an embodiment of the present invention can be prepared in various different forms.

[0029] Figure 5 The figure shows the result of nuclease treatment on an RNA hydrogel according to an embodiment of the present invention.

[0030] Figure 6 A graph showing the rheological properties of the RNA hydrogel as a function of RCT reaction time.

[0031] Figure 7 and Figure 8 This is a digital image used to confirm the efficiency of RNA hydrogel formation under different base sequence conditions.

[0032] Figure 9 This is a graph used to confirm the cellular uptake efficiency of an RNA hydrogel according to an embodiment of the present invention.

[0033] Figure 10 A graph used to confirm the adenosine production and anti-inflammatory effects of an RNA hydrogel according to an embodiment of the present invention.

[0034] Figure 11The figure illustrates the results of an in vitro wound healing experiment using human skin fibroblasts (HDF) with RNA hydrogel according to an embodiment of the present invention.

[0035] Figure 12 In the middle, Figure (A) shows the... Figure 11 The quantitative analysis results of the distance between 15 wound areas are shown in Figure (B), which shows the results of evaluating wound closure efficiency by comparing the initial wound distance with the wound distance after 48 hours.

[0036] Figure 13 A graph used to confirm the wound healing effect of RNA hydrogel according to an embodiment of the present invention.

[0037] Figure 14 This is a diagram used to confirm the tissue regeneration effect of the RNA hydrogel according to an embodiment of the present invention. Detailed Implementation

[0038] The RNA hydrogel for tissue regeneration and its preparation method according to the present invention will be described below with reference to the accompanying drawings. Unless otherwise defined, all terms used in this specification have the same ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains; if there is any conflict between the meaning of a term used in this specification and its ordinary meaning, the definition made in this specification shall prevail. Furthermore, to avoid unnecessarily obscuring the essence of the invention, well-known functions and structures will not be described in detail. In this specification, when a part is referred to as "comprising" a constituent element, it does not exclude the inclusion of other constituent elements unless otherwise expressly stated to the contrary.

[0039] Reference Figure 1 A method for preparing an RNA hydrogel for tissue regeneration according to an embodiment of the present invention will be described, the method comprising: a circular DNA generation step of reacting single-stranded linear DNA with primer DNA to generate circular DNA; and a gel formation step of mixing the circular DNA with RNA polymerase and performing rolling circle transcription (RCT) to form the RNA hydrogel.

[0040] The circular DNA generation step is a step of generating circular DNA by reacting single-stranded linear DNA with primer DNA. The linear DNA contains base sequences at both ends that can bind complementary to the primer DNA, and its middle portion contains a base sequence composed of a large amount of thymine. The linear DNA contains base sequences that enable the final prepared RNA hydrogel to exert tissue regeneration effects. Among various RNA-based therapeutic formulations, hydrogels exhibit superior effects in achieving tissue regeneration through wound healing; therefore, the linear DNA contains base sequences capable of forming hydrogels (e.g., base sequences capable of forming cross-linkable quadruplexes and having a fast polymerization rate). Preferably, at least 80% of the total base sequence of the linear DNA is composed of thymine; more preferably, the linear DNA is composed of the sequence shown in SEQ ID NO:1.

[0041] The gel formation step involves mixing the circular DNA with RNA polymerase and performing rolling circle transcription (RCT) to form an RNA hydrogel. For example, the gel formation step can be completed by mixing the circular DNA, a mixture of ribonucleotide solutions, ATP, ribonuclease, and RNA polymerase in a reaction buffer and reacting at 30–40°C for 2.7–3.3 hours.

[0042] In the RNA hydrogel prepared by the above method, at least 80% of the bases constituting the RNA hydrogel are adenine. After absorption in vivo, the RNA hydrogel can generate adenosine, reducing the level of pro-inflammatory cytokines and increasing the level of anti-inflammatory cytokines, and achieving tissue regeneration by promoting cell proliferation and wound healing. The RNA hydrogel can be effectively delivered into cells without the need for toxic cell delivery carriers and can prevent easy degradation by enzymes in vivo, thereby generating a large amount of adenosine, inducing an anti-inflammatory response, and effectively achieving tissue regeneration through cell proliferation and wound healing. Furthermore, DNA-based therapeutic agents need to cross the cytoplasm and nuclear membrane, while RNA-based therapeutic agents only need to cross the cytoplasm to regulate the activity of intracellular proteins. Therefore, compared with DNA hydrogels, RNA hydrogels can provide superior therapeutic effects. Furthermore, in RNA-based therapeutic agents used to produce tissue regeneration effects, various formulations such as particles and hydrogels can be considered. However, in tissue regeneration applications, a system that can continuously adhere to and release adenosine at the wound site (in vitro) is required. When treating wound sites requiring tissue regeneration with nanoparticles in solution form, effective delivery is difficult to achieve. RNA hydrogels prepared in hydrogel form can be used like ointments, thus they can be directly applied to the wound site as therapeutic agents or used to promote skin regeneration at surgical sites. Therefore, preparing RNA-based therapeutic agents as RNA hydrogels can further enhance their tissue regeneration effects.

[0043] Another embodiment of the present invention relates to a composition for tissue regeneration comprising the said RNA hydrogel.

[0044] Another embodiment of the present invention relates to a method for preparing a tissue regeneration composition comprising the RNA hydrogel.

[0045] The present invention will be further described in detail below through embodiments, but these embodiments are only used to provide a more specific description of the present invention and do not constitute a limitation on the scope of the present invention.

[0046] <Example 1> Preparation of RNA hydrogel

[0047] 1. Prepare the linear DNA (SEQ ID NO. 1) and primer DNA (SEQ ID NO. 2) shown in Table 1 below.

[0048] 2. To generate circular DNA, 10 μM of linear DNA and 10 μM of primer DNA were mixed at the final concentration and incubated at 95°C for 2 minutes. The temperature was then slowly lowered to 25°C over 1 hour to form a mixture. Subsequently, ligation buffer (30 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, and 1 mM ATP) and T4 DNA ligase (0.06 U μL) were added to the mixture. -1 The reaction proceeds overnight at room temperature, thereby generating circular DNA (cirDNA).

[0049] 3. To generate an RNA hydrogel via rolling circle transcription (RCT), the above-mentioned circular DNA (1 μM), ribonucleotide solution mixture (2 mM), ATP (12 mM), and ribonuclease inhibitor (0.8 U μL) were added. -1 T7 RNA polymerase (10 U / μL) -1 The mixture was prepared with 2X reaction buffer (80mM Tris-HCl, 4mM nonylamine, 12mM MgCl2 and 2mM DTT) and reacted at 37°C for 3 hours to generate an RNA hydrogel (A-rich RNA hydrogel).

[0050] [Table 1]

[0051]

[0052] <Example 2> Characterization of RNA Hydrogel

[0053] 1. The RNA hydrogel prepared in Example 1 was photographed with a digital camera, and the RNA hydrogel stained with GelRed was also photographed after being irradiated with ultraviolet light. The results are shown in the figure. Figure 2 The RNA hydrogel described above was imaged using field emission scanning electron microscopy (FE-SEM), and the results are shown below. Figure 3 RNA hydrogels prepared using a silicone mold of a specific shape were photographed with a digital camera, and the results are shown below. Figure 4 The RNA hydrogel was treated with different types of nucleases (DNase I, RNase I, RNase III) under the same conditions, and then photographed with a digital camera. The absorbance at 260 nm and the RNA concentration were measured. The results are shown below. Figure 5 .

[0054] 2. From Figure 2 It can be seen that RNA hydrogels have been successfully prepared; from Figure 3 It can be seen that RNA hydrogels are membrane-like structures with a large number of particles embedded within a monolithic structure; from Figure 4 It is known that RNA hydrogels with various morphologies can be easily prepared; from Figure 5 It is known that the RNA hydrogel is only degraded and its absorbance and concentration increase when treated with RNase I, a nuclease specific to single-stranded RNA. Therefore, it can be concluded that the RNA hydrogel is composed of single-stranded RNA.

[0055] <Example 3> Evaluation of RNA hydrogel formation efficiency

[0056] 1. Characterization of RNA hydrogels based on RCT reaction time

[0057] (1) Except for changing the RCT reaction time, all other conditions were the same as in Example 1. RNA hydrogels were prepared, and the rheological changes of each RNA hydrogel with increasing shear strain were analyzed. The results are shown in... Figure 6 .

[0058] (2) From Figure 6 It can be seen that the RNA hydrogel prepared by the RCT reaction time of 3 hours in Example 1 has a higher storage modulus than the RNA hydrogel prepared by the RCT reaction time of 1 hour or 6 hours, and the gel properties can be maintained for a longer time. This indicates that before the RCT reaction time reaches 3 hours, the RNA production rate is much faster than the hydrolysis rate, while after 3 hours, the RNA production efficiency begins to decline.

[0059] 2. Verify the formation of RNA hydrogels under different base sequences.

[0060] (1) Except for the use of the following linear DNA, the other conditions were the same as in Example 1. RNA structures were prepared and photographed with a digital camera. The results are shown in... Figure 7 Using SEQ ID NO. 3 (5'-ATA GTG AGT CGT ATT ATT TTT TTT TTTAAC GGA CCA ACG CAC ACG CAG GAG GAG GGA CGG GGA AAA GCG GGC CGA GGG AGC CGGATC CCT-3'), approximately 20% of its total nucleotide sequence is thymine; using SEQ ID NO. 4 (5'-ATA GTG AGT CGTATT ATT TTT TTT TTT TTT TTT TTT TTT TTT TTT AAC GGA CCA ACG CAC ACG CAG GAGGAG GGA CGG GGA AAA ATC CCT-3'), approximately 40% of its total nucleotide sequence is thymine; using SEQ ID NO. 5 (5'-ATA GTG AGT CGT ATT ATT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT AAA CGG ACC The sequence AAC GCA CAC GCA ATC CCT-3' is approximately 60% thymine; SEQ ID NO. 6 (5'-ATA GTG AGT CGT ATT ATT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT ATC CCT-3') is approximately 80% thymine.

[0061] (2) Except for the use of the following linear DNA, the other conditions were the same as in Example 1. RNA structures were prepared, and the GelRed-stained RNA structures were irradiated with ultraviolet light and then photographed with a digital camera. The results are shown in... Figure 8Using SEQ ID NO. 7 (5'-ATA GTG AGT CGT ATT AGG GGG GGG GGG GGG GGG GGG TGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG ATC CCT-3'), the total base sequence is mostly guanine; using SEQ ID NO. 8 (5'-ATA GTG AGT CGT ATT AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA ACA AAA AAA AAA AAA AAA AAA AAA ATC CCT-3'), the total base sequence is mostly adenine; using SEQ ID NO. 9 (5'-ATA GTG AGT CGT ATT ACC CCC CCCCCC C ... CCC CCC CCC CCC CCC CCT CCCCCC ATC CCT-3'), whose total base sequence is mostly cytosine. Figure 8 In the diagram, A represents the RNA hydrogel prepared in Example 1, C represents the RNA structure prepared using the linear DNA of SEQ ID NO. 7, U represents the RNA structure prepared using the linear DNA of SEQ ID NO. 8, and G represents the RNA structure prepared using the linear DNA of sequence variation 9.

[0062] (3) From Figure 7 It is known that hydrogels only form when using linear DNA with approximately 80% of its total base sequence being thymine; from Figure 8 It is known that hydrogels only form in RNA structures where the majority of the total base sequence is adenine. Therefore, to prepare RNA hydrogels, at least 80% of the total base sequence of RNA must be adenine. Thus, the two necessary conditions for RNA hydrogel formation are: a base sequence capable of forming cross-linkable quadruplexes, and a relatively fast polymerization rate.

[0063] <Example 4> Evaluation of cellular uptake efficiency of RNA hydrogel

[0064] 1. To evaluate the cell uptake efficiency of the RNA hydrogel prepared in Example 1, Raw 264.7 cells were treated with the RNA hydrogel (A-rich RNA hydrogel) prepared in Example 1, stained with 10X GelGreen. The cells were then washed with DPBS, and the cell membranes were stained with WGA and the nuclei with Hoechst 33342. Images were subsequently obtained using a confocal laser scanning microscope (CLSM), and the cell flow cytometry results obtained using a nucleocounter are shown below. Figure 9 .

[0065] 2. From Figure 9 As shown in A, GelGreen fluorescence is visible in the cytosol, indicating that the RNA component containing a large amount of adenine has been internalized and distributed in the cytosol. Furthermore, from the results of cell flow cytometry analysis... Figure 9 As indicated by B, the RNA component containing a large amount of adenine has been successfully delivered to the cell.

[0066] Example 5: Confirmation of anti-inflammatory effects related to wound healing

[0067] 1. The circular DNA (1 μM), dNTP mixture (2 mM), reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT) prepared in Example 1 and Phi29 DNA polymerase (1 U / μL) were mixed and reacted at 30°C for 4 hours. Then, primer-2 (50 pM) of sequence listing SEQ ID NO:10 (5'-ACGCAGTATTATGGACTG-3') and primer-3 (50 pM) of sequence listing SEQ ID NO:11 (5'-TGGTACGTTAGGAACATC-3') were added, and the reaction was continued at 30°C for 16 hours to obtain DNA hydrogel (D. Gel).

[0068] 2. To evaluate the adenosine production and anti-inflammatory effects related to wound healing of the RNA hydrogel (R. Gel) prepared in Example 1, the same concentrations of the RNA hydrogel (R. Gel) prepared in Example 1, the DNA hydrogel (D. Gel) prepared in step 1 of Example 5, and ATP were used to treat LPS-treated Raw 264.7 cells. After 24 hours of reaction, the adenosine concentration was measured using an adenosine assay kit, and the concentrations of IL-6, TNF-α, IL-10, TIMP-1, and arginase-1 were measured using a mouse IL-6, TNF-α, IL-10, TIMP-1, and arginase-1 ELISA kit. The results are shown in... Figure 10.

[0069] 3. From Figure 10 As shown in A, the adenosine concentration was significantly higher in RNA hydrogels than in DNA hydrogels; changes in cytokine release associated with M1 and M2 were observed. Figure 10 As shown in B to F, when RNA hydrogels were treated, the release of pro-inflammatory cytokines TNF-α and IL-6 decreased significantly, while the release of factors related to anti-inflammatory effects, such as IL-10, TIMP-1, and arginase-1, increased, similar to the positive control ATP.

[0070] <Example 6> In vitro wound healing experiment of RNA hydrogel

[0071] 1. Conduct in vitro wound healing experiments.

[0072] After seeding 100,000 SVEC4 cells into 4-well cell culture plates, they were cultured overnight in a CO2 incubator. Wounds were then created by scraping the bottom of the culture plates with 20–200 μL pipette tips. Subsequently, DNA hydrogel (D. Gel, 20 μL) prepared in Step 1 of Example 5 and RNA hydrogel (R. Gel, 20 μL) prepared in Example 1 were added directly to the cell culture plates. After 24 hours, the plates were washed with PBS, and fresh cell culture medium was added. The wound spacing was observed. At 24 and 48 hours, the wound distance between cells was observed using an optical microscope alongside untreated cells and is shown below. Figure 11 In addition, regarding Figure 11 The wound spacing in the images was quantitatively labeled, and the wound closure efficiency was calculated. The results are shown in... Figure 12 The above method for calculating wound closure efficiency is to compare the initial wound distance with the wound distance after 48 hours, and calculate it according to formula 1 – {(distance after 48 hours) / (initial distance)}.

[0073] 2. From Figure 11 and Figure 12 It can be seen that, compared with untreated or treated DNA hydrogels, the treatment of RNA hydrogels resulted in rapid cell proliferation, a rapid reduction in the distance between wounds, and a significant wound closure effect.

[0074] <Example 7> In vivo wound healing experiment using RNA hydrogels

[0075] 1. Using a surgical bio-punch, a full-thickness skin lesion with a diameter of 8 mm was created on the back of 4-week-old male Balb / c mice, and the RNA hydrogel prepared in Example 1 and the DNA hydrogel prepared in step 1 of Example 5 were injected into the mouse wound site (100 μL).

[0076] 2. After injection, images were taken with a digital camera after a certain period of time, and the wound length was measured based on the images. The results are shown below. Figure 13 In addition, 12 days after injection, skin tissue was harvested from the wound site, dehydrated with 4% paraformaldehyde and ethanol, embedded in paraffin, and sectioned to a thickness of 5 μm. The resulting sections were stained with H&E and analyzed using an optical microscope to obtain images. The results are shown in [Figure number missing]. Figure 14 .

[0077] 3. From Figure 13 It can be seen that the wound healing efficiency is higher when using RNA hydrogels than when using DNA hydrogels; from Figure 14 It can be seen that the tissue regeneration effect is better when using RNA hydrogels than when using DNA hydrogels.

[0078] The applicant has described the preferred embodiments of the present invention above. However, these embodiments are only one way to implement the technical concept of the present invention. Any changes or modifications to the embodiments under the premise of implementing the technical concept of the present invention should be interpreted as falling within the scope of the present invention.

Claims

1. A method for preparing an RNA hydrogel for tissue regeneration, characterized in that, include: The circular DNA generation step involves reacting single-stranded linear DNA with primer DNA to generate circular DNA. as well as The circular DNA is mixed with RNA polymerase and subjected to rolling circle transcription to form an RNA hydrogel. In the single-stranded linear DNA, at least 80% of the full-length base sequence is thymine.

2. The method for preparing RNA hydrogel for tissue regeneration as described in claim 1, characterized in that, The gel formation step is performed by mixing circular DNA, a ribonucleotide solution mixture, ATP, ribonuclease, and RNA polymerase in a reaction buffer and reacting at 30–40°C for 2.7–3.3 hours.

3. The method for preparing RNA hydrogel for tissue regeneration as described in claim 2, characterized in that, The RNA hydrogel is absorbed in vivo and generates adenosine, which reduces the level of pro-inflammatory cytokines while increasing the level of anti-inflammatory cytokines.

4. The method for preparing RNA hydrogel for tissue regeneration as described in claim 3, characterized in that, The RNA hydrogel can promote cell proliferation and achieve tissue regeneration through wound healing.

5. The method for preparing RNA hydrogel for tissue regeneration as described in claim 4, characterized in that, The linear DNA consists of the sequence shown in SEQ ID NO. 1, and the primers consist of the sequence shown in SEQ ID NO.

2.

6. An RNA hydrogel for tissue regeneration, characterized in that, In the RNA hydrogel, adenine accounts for more than 80% of the bases. The RNA hydrogel is absorbed in vivo and generates adenosine, which reduces the level of pro-inflammatory cytokines while increasing the level of anti-inflammatory cytokines.

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