Application of targeting CD36 in treatment of rheumatoid arthritis

Abstract

The invention discloses application of targeting CD36 in treatment of rheumatoid arthritis, and relates to the technical field of biological materials, and the technical key points are as follows: the CD36 is an effective target spot for treating the rheumatoid arthritis, the content of the CD36 in the body of an RA patient is reduced, and RA-FLS proliferation and release of inflammatory factors can be inhibited; a new molecular mechanism that CD36 participates in RA-FLS activation by regulating fatty acid metabolism is found, CD36 is a new target for relieving RA joint inflammation and injury, and a new treatment direction is provided for clinical treatment of RA.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to the application of CD36 targeting in the treatment of rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease based on chronic synovitis that can lead to the destruction of bone and cartilage within the joints. Its pathogenesis is not fully understood, but studies show that immune cell infiltration and altered synovial cell phenotypes are major factors contributing to synovial inflammation. Although an increasing number of biologics and small molecule inhibitors have significantly improved the prognosis of RA, a considerable proportion of patients still experience persistent synovitis and progressive joint damage.

[0003] Fibroblast-like synoviocytes (FLS) are the main cells in the synovium of patients with rheumatoid arthritis (RA), exhibiting a "pro-inflammatory" or "invasive / tissue-damaging" phenotype, leading to persistent joint inflammation and bone damage. Therefore, inhibiting RA-FLS activation can effectively control synovial hyperplasia and inflammation, prevent cartilage erosion, and ultimately reduce or delay the progression of joint damage. Fatty acid metabolism is a core process of cellular energy supply and lipid homeostasis, mainly including fatty acid uptake, oxidation (FAO), and synthesis. Abnormal fatty acid metabolism participates in regulating inflammatory responses and immune cell function, but its specific mechanism in RA-FLS activation remains unclear.

[0004] Therefore, the present invention aims to provide an application of CD36 targeting in the treatment of rheumatoid arthritis to solve the above-mentioned problems. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide an application of CD36 targeting in the treatment of rheumatoid arthritis. A new molecular mechanism by which CD36 participates in the activation of RA-FLS by regulating fatty acid metabolism has been discovered. CD36 is a new target for alleviating joint inflammation and damage in RA, providing a new therapeutic direction for the clinical treatment of RA.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides an application of CD36 targeting in the treatment of rheumatoid arthritis (RA). CD36 is an effective target for treating RA, providing a theoretical basis for developing CD36-targeted RA drugs. Reducing the CD36 level in RA patients can inhibit RA-FLS proliferation and the release of inflammatory factors.

[0008] By targeting and knocking down CD36 in RA-FLS, the level of CD36 can be reduced, which can induce mitochondrial damage and participate in intracellular fatty acid metabolism reprogramming, inhibiting RA-FLS proliferation, migration, invasion and release of inflammatory factors.

[0009] By targeting and knocking down CD36 in RA-FLS, the level of CD36 can be reduced, thereby inhibiting the EMT process, reducing the migration and invasion capabilities of RA-FLS, alleviating synovial inflammation, and reducing bone damage and erosion.

[0010] Compared with existing technologies, the beneficial effects of this solution are:

[0011] This invention discovers a novel molecular mechanism by which CD36 participates in RA-FLS activation by regulating fatty acid metabolism. CD36 is a new target for alleviating RA joint inflammation and damage. Inhibiting the feedback loop of CD36-fatty acid metabolism reprogramming-inhibiting RA-FLS activation provides a promising potential complementary treatment for RA. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the increased CD36 level expression in RA synovial tissue in an embodiment of the present invention;

[0013] A shows representative HE staining images of CD36 in RA and TC synovial tissues, n=6; B and C show...

[0014] Representative immunohistochemical staining images and quantitative analysis of CD36 in synovial tissues of RA and TC (n=3); D, E. Western blot detection of CD36 protein expression in synovial tissues of RA (n=6) and TC (n=6); F, G. Detection of CD36 protein expression in primary FLS of RA (n=6) and TC (n=6) using Western blot; H. Detection results of free fatty acid levels in synovial fluid of RA patients.

[0015] Figure 2This is a schematic diagram illustrating CD36 knockdown and SSO intervention inducing mitochondrial damage and cellular fatty acid metabolism reprogramming in RA-FLS cells, as described in embodiment A. In A, a lentivirus interfering with CD36 (sh-CD36) is transfected into RA-FLS cells. The negative control group consists of uninfected RA-FLS cells (sh-NC). The SSO group is stimulated with 20 μM of the CD36 inhibitor SSO for 24 hours. In AC, Western spectroscopy is used to analyze the RA-FLS cells. blot and qRT-PCR analyses were used to assess the CD36 knockdown efficiency; D and E are representative immunofluorescence images of CD36; F is a GO enrichment bar chart in the transcriptome analysis after CD36 knockdown, showing the biological process of enrichment; G is a transmission electron microscope image of mitochondrial morphology in RA-FLS with CD36 knockdown and inhibition, with red arrows indicating normal mitochondrial morphology, no mitochondrial swelling, and clear mitochondrial cristae; green arrows indicate mitochondrial damage, mitochondrial swelling, vacuolization, and cristae breakage, as well as the formation of autophagosomes; H is a representative image of lipid droplets stained with Bodipy 493 / 503 in RA-FLS; I and J are representative images and quantitative analysis of lipid droplets stained with Oil Red O in RA-FLS; KM is a Western blot analysis of the expression of fatty acid oxidation (FAO)-related proteins (carnitine palmitoyltransferase 1A, CPT1A) and de novo fatty acid synthesis (DNL)-related proteins (fatty acid synthase, FASN).

[0016] Figure 3 This is a schematic diagram illustrating how CD36 knockdown and SSO intervention inhibit the release of inflammatory factors and MMPs in RA-FLS in an embodiment of the present invention; A shows the expression of IL-1β, IL-6, TNF-α, MMP1, and MMP9 mRNA by qRT-PCR after transfecting RA-FLS and stimulating RA-FLS with 20 μM CD36 inhibitor SSO for 24 hours; BE shows the expression levels of MMP3, pro-IL-1β, and NLRP3 proteins detected by Western blot; F and G show the supernatant levels of IL-1β and IL-6 in different groups (n=3) detected by ELISA.

[0017] Figure 4 This diagram illustrates how CD36 knockdown and SSO intervention inhibit RA-FLS proliferation and promote RA-FLS apoptosis in this embodiment of the invention; A represents the cell cycle GSEA analysis in the transcriptome sequencing results after CD36 knockdown; BD represents the expression levels of cell cycle proteins CDK4 and Cyclin D1 detected by Western blot; E represents the cell proliferation capacity of different groups detected by CCK8 assay; FI represents the expression levels of apoptosis-related proteins Bcl-2 and Bax detected by Western blot.

[0018] Figure 5 This is a schematic diagram illustrating how CD36 knockdown and SSO intervention inhibit RA-FLS migration and invasion in embodiments of the present invention; A and B show the effect of CD36 on RA-FLS migration ability detected by scratch healing assay; CE shows the effect of CD36 on RA-FLS migration and invasion ability detected by Transwell assay; FI shows the protein expression levels of E-cadherin, N-cadherin, and Vimentin in RA-FLS after CD36 knockdown and SSO intervention detected by Western blot.

[0019] Figure 6 In this embodiment of the invention, CD36 knockdown and SSO intervention in RA-FLS can activate AMPK and inhibit PI3K-AKT-mTOR pathway activation; A and B are differential gene volcano plots and differential gene expression heatmaps in the transcriptome sequencing results after CD36 knockdown; C is the KEGG enrichment analysis of the identified genes by transcriptome sequencing; the size and color of the dots represent the number of genes and the p-value range, respectively; DF is the evaluation of PRKAA2 expression and phosphorylated AMPKα2 protein expression level by Western blot and qRT-PCR analysis; GJ is the detection of PI3K-AKT-mTOR pathway protein expression level by Western blot.

[0020] Figure 7 This is a schematic diagram illustrating how inhibiting CD36 can alleviate joint inflammation and bone erosion in CIA rats in this embodiment of the invention; A is the animal experiment timeline, including CIA rat modeling and treatment; B is a representative image of the rat paw; C and D are the effects of the CD36 inhibitor SSO on the degree of rat paw pad swelling and arthritis index; E and F are histological manifestations. Representative images of rat ankle joint hematoxylin-eosin (HE) staining and safranin O / fast green staining; G is a MicroCT image of the rat ankle joint; H and I are the results of Western blot detection of CD36 expression in synovial tissue of different groups; J and N are the results of MicroCT bone analysis parameters of rat ankle joint (BV / TV: Bone Volume / Total Volume; BS / BV: Bone Surface / Bone Volume; BS / TV: Bone Surface / Total Volume; Tb.N: Trabecular Number; BMD: Bone Mineral Density).

[0021] Figure 8 This is a diagram illustrating the mechanism by which CD36 knockdown inhibits RA-FLS activation in an embodiment of the present invention;

[0022] Figure 1-8The dashed box in the middle represents the magnified area; the results are expressed as mean ± SEM; all are representative results of three independent experiments; including β-actin as an internal reference gene; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The invention will now be described in detail with reference to the embodiments.

[0025] Example:

[0026] 1.1 CD36 is highly expressed in synovial tissue and primary FLS of RA patients.

[0027] Synovial tissue was collected from patients with TC and RA to verify the expression of CD36 in RA. HE staining was used to observe pathological changes in the synovial tissue. The results showed inflammatory cell infiltration, synovial thickening, and extensive angiogenesis in the RA group, with some areas exhibiting fibrosis. In contrast, synovial tissue from TC patients showed no significant cell thickening, loose tissue, and no significant inflammatory cell infiltration or angiogenesis in the interstitium. Figure 1 A). IHC showed that CD36 expression levels in RA synovium were higher than those in TC synovium. Figure 1 B, C). Furthermore, Western blot results showed that the expression level of CD36 in the synovial tissue of RA was significantly increased compared to TC patients. Figure 1 D, E). To further clarify the expression of CD36 in RA-FLS, primary FLS were isolated and cultured from RA and TC synovial tissues. Western blotting showed that CD36 expression in RA-FLS was higher than that in TC FLS. Figure 1 F, G). Elevated levels of free fatty acids in the synovial fluid of RA patients were also detected. Figure 1 H). The above results suggest that RA patients have high expression of CD36 in synovial tissue and RA-FLS, which may be related to the increased level of free fatty acids in synovial fluid and may be involved in the pathological progression of RA synovial tissue.

[0028] 1.2 CD36 knockdown induces RA-FLS mitochondrial damage and fatty acid metabolism reprogramming

[0029] Following CD36 knockdown transfection of RA-FLS cells using lentiviral vectors, transfection efficiency was analyzed by Western blot and RT-qPCR, and transcriptome sequencing was performed to further investigate the specific mechanism by which CD36 affects RA-FLS function. Compared with the control group, CD36 mRNA expression, CD36 protein level, and CD36 fluorescence intensity were decreased in the CD36 knockdown group and the CD36 inhibitor (Sulfosuccinimidyl oleate sodium, SSO) intervention group. Figure 2 AE). Transcriptome sequencing results showed that CD36 knockdown can affect biological functions such as cell proliferation and migration, apoptosis, inflammatory response, intercellular signal transduction, and lipid metabolism regulation. One of the functions of CD36 is to participate in intracellular fatty acid metabolism (AE). Figure 2 F). Mitochondria are the core organelles of fatty acid metabolism. Transmission electron microscopy was used to observe mitochondrial damage in cells and to explore the mechanism by which CD36 regulates fatty acid metabolism in RA-FLS. It was found that CD36 knockdown and SSO intervention promoted mitochondrial vacuolization, swelling, and cristae breakage in RA-FLS, and a large amount of autophagy was also observed in the cytoplasm. Figure 2 G).

[0030] The relationship between mitochondrial damage and fatty acid metabolism was investigated. Bodipy 493 / 503 and Oil Red O staining were used to observe lipid droplet formation and metabolism. Western blot analysis was performed to detect fatty acid metabolism-related proteins to clarify intracellular fatty acid metabolism. Bodipy 493 / 503 and ORO staining results showed that CD36 knockdown and SSO intervention reduced lipid accumulation in RA-FLS (mitochondrial-associated fatty acid septa). Figure 2 Western blot results showed that, compared with the control group, knockdown of CD36 and SSO intervention in RA-FLS increased the expression of fatty acid β-oxidation protein CPT1A and fatty acid synthesis protein FASN (Figure KM), suggesting that CD36 reduction in RA-FLS promoted fatty acid oxidation. Although the endogenous lipid synthesis pathway increased, the number of intracellular lipid droplets decreased, indicating that fatty acid oxidation in RA-FLS may be stronger than that of the endogenous lipid synthesis pathway. In summary, the above results indicate that knockdown and inhibition of CD36 can induce mitochondrial damage and fatty acid metabolism reprogramming in RA-FLS.

[0031] 1.3 CD36 knockdown inhibits the release of RA-FLS inflammatory factors

[0032] To observe the effects of CD36 knockdown and CD36 inhibition on the release of inflammatory factors in RA-FLS, CD36 was knocked down and SSO intervention was performed in RA-FLS. RT-qPCR was used to detect the mRNA expression of inflammatory factors, MMP1, and MMP9. Western blot analysis was used to analyze the protein expression levels of MMP3, IL-1β, and NLRP3, and ELISA was used to detect the levels of IL-1β and IL-6 in the cell supernatant. Compared with the control group, the mRNA expression of IL-1β, IL-6, TNF-α, and MMP1 and MMP9 was significantly reduced in RA-FLS after CD36 knockdown and SSO intervention. Figure 3 A) The expression levels of MMP3, pro-IL-1β, and NLRP3 proteins were downregulated. Figure 3 BE), the levels of IL-1β and IL-6 in cell supernatant decreased ( Figure 3 These results indicate that CD36 knockdown and SSO intervention can inhibit the release of RA-FLS inflammatory factors and MMPs. CD36 may be an important molecule involved in regulating RA synovial inflammation.

[0033] 1.4 CD36 knockdown inhibits RA-FLS proliferation and promotes RA-FLS apoptosis

[0034] To observe the effects of CD36 knockdown and CD36 inhibition on RA-FLS proliferation and apoptosis, GSEA transcriptome sequencing results showed that CD36 knockdown led to cell cycle arrest (…). Figure 4 A) Western blot was used to detect the expression of cell cycle proteins CDK4 and Cyclin D1 in each group. It was found that CD36 knockdown and SSO intervention in RA-FLS reduced the protein expression levels of CDK4 and Cyclin D1. Figure 4 BD). CCK8 proliferation assay showed decreased cell proliferation capacity ( Figure 4 E) suggests that RA-FLS cell cycle arrest occurs in the G1 phase, preventing cell transition to the S phase and reducing cell proliferation. Knockdown of CD36 and SSO intervention in RA-FLS resulted in increased expression of the pro-apoptotic protein Bax and decreased expression of the anti-apoptotic protein Bcl-2, with a significantly increased Bax / Bcl-2 ratio. Figure 4 (FI). The results showed that RA-FLS knockdown and CD36 inhibition could suppress cell proliferation and promote apoptosis. CD36 may be an important molecule involved in regulating RA synovial hyperplasia.

[0035] 1.5 knockdown of CD36 reduces RA-FLS in vitro migration and invasion.

[0036] Further investigation was conducted into the effects of CD36 on RA-FLS migration and invasion. Scratch assays showed that CD36 knockdown and SSO intervention significantly reduced the migration ability of RA-FLS cells. Figure 5 A, B), which is consistent with the results of the transwell migration experiment. Figure 5 C, D). Subsequent studies investigated the effects of CD36 knockout and SSO intervention on the invasive ability of RA-FLS. CD36 knockout and SSO intervention significantly reduced the invasive ability of RA-FLS. Figure 5 C, E). In RA, FLS undergoes epithelial-mesenchymal transition (EMT) to enhance migration and invasion, and E-cadherin, N-cadherin, and Vimentin also play important roles in EMT. Western blot analysis of the protein expression levels of E-cadherin, N-cadherin, and Vimentin showed that in RA-FLS, CD36 knockdown and SSO intervention significantly inhibited N-cadherin protein expression, while the protein expression levels of E-cadherin and Vimentin did not change significantly. Figure 5 The above results indicate that CD36 knockdown and SSO intervention may reduce the migration and invasion capabilities of RA-FLS by inhibiting the EMT process.

[0037] 1.6 Knockdown of CD36 activates AMPK and inhibits PI3K-AKT-mTOR pathway activation

[0038] Following CD36 knockdown transfection of RA-FLS using a lentiviral vector, transcriptome sequencing and differentially expressed genes were performed to further explore the specific pathways by which CD36 affects RA-FLS function. Volcano plots revealed 232 differentially expressed genes, including 139 upregulated genes and 93 downregulated genes. After CD36 knockdown, the level of the PRKAA2 gene, which is closely related to energy metabolism, was significantly upregulated. Figure 6 A, B), KEGG results showed that CD36 knockdown can participate in the pathogenesis of RA through multiple signaling pathways, including AMPK, PI3K / AKT / mTOR, and TGF-β signaling pathways. Figure 6 C). RA-FLS with CD36 knockdown was validated by Western blotting and RT-qPCR to further verify transcriptome-related results. It was found that CD36 knockdown and SSO intervention increased the expression levels of total PRKAA2 protein and phosphorylated AMPKα2, and increased PRKAA2 mRNA levels. CD36 knockdown and SSO intervention activated the AMPK pathway. Figure 6DF). Western blot results showed that, compared with the control group, the expression of PI3K, AKT and mTOR proteins in RA-FLS after CD36 knockdown and SSO intervention was inhibited to some extent. Figure 6 (GJ) Knockdown of CD36 and SSO intervention can inhibit the activation of the PI3K / AKT / mTOR pathway. These results suggest that CD36 may participate in the pathogenesis of RA by dually regulating the AMPK signaling pathway and the PI3K / AKT-mTOR signaling pathway.

[0039] 1.7 Inhibition of CD36 can alleviate joint inflammation and bone erosion in CIA rats.

[0040] In vitro experiments have confirmed that CD36 knockdown and administration of the CD36 inhibitor SSO can inhibit the activation of the malignant phenotype of RA-FLS. In vivo experiments were conducted to evaluate the therapeutic effect of CD36 inhibition in animal models of arthritis. In a rat model of CIA, the CD36 inhibitor SSO was administered by gavage (…). Figure 7 A). Compared with the CIA model group, the degree of paw pad swelling in rats in the CD36 inhibitor group began to decrease on day 8 after treatment. Around 14 days after the first immunization, the arthritis index in both the CIA and SSO groups was significantly higher than that in the normal control group. Compared with the CIA model group, the arthritis index in the SSO group was significantly lower starting on day 8 after treatment. Figure 7 D). Compared with the CIA group, the SSO group showed reduced synovial inflammation and cartilage damage. Figure 7 (E, F). In summary, these findings indicate that in a rat model of RA, inhibiting CD36 can effectively alleviate synovial inflammation and reduce bone damage and erosion, providing a theoretical basis for future clinical applications.

[0041] 2. Discussion

[0042] The main pathological feature of rheumatoid arthritis (RA) is chronic synovial inflammation. Activated RA-FLS, as the main effector cells in synovial inflammation and joint injury, not only participate in the release of pro-inflammatory cytokines but also exhibit tumor-like behavior of migrating and invading articular cartilage. Energy metabolism is considered a key factor in RA-FLS activation. This application explores the role of CD36 in the pathogenesis of RA. It was found that in the synovial inflammatory microenvironment, increased free fatty acids in synovial fluid promote CD36 expression, enhancing the functional activation of RA-FLS and participating in RA pathogenesis. Knockdown of CD36 in RA-FLS and SSO intervention can induce mitochondrial damage and participate in intracellular fatty acid metabolism reprogramming, inhibiting RA-FLS proliferation, migration, invasion, and the release of inflammatory factors. Figure 8Furthermore, this application also investigated the therapeutic effects of inhibiting CD36 on synovial inflammation and bone erosion in a CIA animal model. Specifically, the current study identified CD36 as a key molecule regulating lipid metabolism and activation in RA-FLS.

[0043] In summary, this application reveals the pleiotropic regulatory role of CD36 in the pathological process of rheumatoid arthritis (RA) and elucidates the molecular mechanism by which it drives synovial inflammation and joint destruction through its involvement in fatty acid metabolism reprogramming and signaling pathway interactions. Specifically, knockdown and inhibition of CD36 in RA-FLS lead to mitochondrial dysfunction and fatty acid metabolism reprogramming, further promoting RA-FLS apoptosis and inhibiting its proliferation, migration, and invasion, as well as suppressing the secretion of pro-inflammatory cytokines. The AMPK and PI3K / AKT / mTOR signaling pathways are involved in these processes. This feedback loop of inhibiting CD36-fatty acid metabolism reprogramming-inhibiting RA-FLS activation provides a promising potential complementary therapeutic approach for RA. In conclusion, this study provides new insights into the role of CD36 in the pathogenesis of RA and supplements the theoretical support for this potential therapeutic target.

[0044] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. An application of CD36 targeting in the treatment of rheumatoid arthritis, characterized by: CD36 is an effective target for treating rheumatoid arthritis. Drugs that reduce CD36 levels can be prepared. These drugs can reduce CD36 levels in RA patients and inhibit RA-FLS proliferation and the release of inflammatory factors.

2. The application of CD36 targeting in the treatment of rheumatoid arthritis as described in claim 1, characterized in that: By targeting and knocking down CD36 in RA-FLS, the level of CD36 can be reduced, which can induce mitochondrial damage and participate in intracellular fatty acid metabolism reprogramming, inhibiting RA-FLS proliferation, migration, invasion and release of inflammatory factors.

3. The application of CD36 targeting in the treatment of rheumatoid arthritis as described in claim 1, characterized in that: By targeting and knocking down CD36 in RA-FLS, the level of CD36 can be reduced, thereby inhibiting the EMT process, reducing the migration and invasion capabilities of RA-FLS, alleviating synovial inflammation, and reducing bone damage and erosion.

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