Application of rhodanine in preparation of medicine for treating knee osteoarthritis

Abstract

The invention discloses application of rhodanine in preparation of a medicine for treating knee osteoarthritis, and relates to the technical field of biological medicine. The invention further discloses a medicine for treating knee osteoarthritis. The medicine comprises the rhodinine. The invention finds that the rhodanine can significantly reduce cartilage cell apoptosis and inflammation, and has a great application value in preparation of the medicine for treating knee osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of ellipticine in preparing a medicine for treating knee osteoarthritis. Background Art

[0002] Knee osteoarthritis (KOA) is a prevalent joint disease that severely impairs the quality of life of millions of people worldwide, especially as they age. Although various treatment options are available, their efficacy remains limited and adverse effects are common, emphasizing the need for new treatment strategies. Therefore, the search for new effective drugs is urgent. Summary of the invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide an application of ellipticine in the preparation of a drug for treating knee osteoarthritis. The present invention finds that ellipticine can significantly reduce chondrocyte apoptosis and inflammation, and has great application value in the preparation of a drug for treating knee osteoarthritis.

[0004] The technical solution of the present invention to solve the above technical problem is as follows: providing an application of ellipticine in the preparation of a drug for treating knee osteoarthritis.

[0005] Furthermore, ellipticine can inhibit chondrocyte apoptosis.

[0006] Furthermore, ellipticine can inhibit the expression of inflammatory cytokines IL-1β, IL-6 and TNF-α.

[0007] Furthermore, ellipticine could inhibit the expression of matrix-degrading enzymes ADAMTS4, MMP3, and MMP13.

[0008] Furthermore, the effective concentration of ellipticine is 0.5-5 μM.

[0009] Furthermore, the effective concentration of ellipticine was 2 μM.

[0010] The invention also provides a medicine for treating knee osteoarthritis, comprising ellipticine.

[0011] The present invention has the following beneficial effects:

[0012] 1. The present invention identified the important active ingredient ellipticine for the treatment of knee osteoarthritis from the traditional Chinese medicine formula of Naru Sanwei Pills through network pharmacology, and verified its function in the treatment of knee osteoarthritis and its molecular mechanism through in vitro experiments.

[0013] 2. The present invention found that ellipticine can significantly reduce oligochondrocyte apoptosis and inflammation.

[0014] 3. The present invention also clarifies the new molecular mechanism of the protective effect of ellipticine on knee osteoarthritis. Ellipticine can inhibit the expression of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and matrix degrading enzymes (ADAMTS4, MMP3 and MMP13), and has the ability to inhibit the inflammatory cascade and protect the integrity of cartilage. In addition, ellipticine can also regulate key signal transduction pathways including FOXO and TGF-β / Hippo by regulating the expression of SOD2 and SMAD2 proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the effect of different concentrations of IL-1β on the apoptosis and expression of inflammatory factors in normal human chondrocytes (C28 / I2);

[0016] Figure 2 The results of cell viability test after treatment with different concentrations of ellipticine for 24h and 48h;

[0017] Figure 3 This is a test result of the therapeutic effect of ellipticine on the cell model of knee osteoarthritis;

[0018] Figure 4 The ELISA test results of inflammatory factors IL-1β, IL-6 and TNF-α in each treatment group;

[0019] Figure 5 The expression detection results of ADAMTS4, MMP3, MMP13, COX-2 and Cleaved-Caspase3 proteins in each treatment group are shown in the figure;

[0020] Figure 6 The results of immunofluorescence detection of MMP3 protein in each treatment group are shown in Figure 2;

[0021] Figure 7 The results of key protein expression analysis in each treatment group are shown in Figure 2;

[0022] Figure 8 This is the result diagram of the effect of ellipticine on SOD2 protein and FOXO signaling pathway;

[0023] Fig. 9 This is a graph showing the effect of ellipticine on SMAD2 protein and inflammatory factors;

[0024] Fig.10 This is a graph showing the effect of ellipticine on the expression of proteins related to the Hippo and TGF-β signaling pathways. DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0026] Test Example 1

[0027] Through the TCMSP database of Chinese medicine systems pharmacology, the active ingredients in Naru Sanwei Pills (composed of Terminalia chebula, Piper longum and Radix Aconiti Kusnezoffii) that met the standards of oral bioavailability (OB)>30% and drug similarity (DL)>0.18 were screened, and the gene information related to these ingredients was obtained. At the same time, genes related to knee osteoarthritis (KOA) were collected using GeneCard and OMIM databases. Subsequently, a drug-ingredient-disease-target network diagram was constructed using Cytoscape 3.9.1 software, in which ellipticine (ELPC) was identified as a potential key active ingredient, which has a significant contribution to the effect of Naru Sanwei Pills in treating KOA.

[0028] Test Example 2

[0029] In order to construct an effective cell model of knee osteoarthritis and evaluate the potential therapeutic effect of ellipticine, we first investigated the effects of different concentrations of IL-1β on the apoptosis and expression of inflammatory factors in normal human chondrocytes (C28 / I2). Figure 1 As shown, A is the apoptosis rate of each group, and B, C and D are the changes in IL-1β, IL-6 and TNF-α levels. The results showed that IL-1β treatment significantly increased the apoptosis rate and the expression levels of IL-6 and TNF-α, and all were concentration-dependent. In particular, when the IL-1β concentration was 20 ng / mL, the apoptosis rate reached 35.66%, which met the IC30 standard, and was accompanied by a significant upregulation of inflammatory factors. Therefore, this concentration was selected as the induction condition for the knee osteoarthritis cell model.

[0030] Subsequently, in order to determine the optimal concentration and time of ellipticine on C28 / I2 cells, the CCK-8 method was used to evaluate the cell viability after 24h and 48h treatment with different concentrations of ellipticine. Figure 2 As shown in the figure, A is the cell proliferation inhibition rate after 24 hours of treatment, and B is the cell proliferation inhibition rate after 48 hours of treatment. The results show that ellipticine significantly inhibited cell activity and showed concentration dependence. It is worth noting that at a concentration of 2μM, the cell inhibition rate after 48 hours of treatment is close to IC30 and is more stable than 24 hours of treatment, so this condition was selected for subsequent experiments.

[0031] Finally, in order to verify the therapeutic effect of ellipticine on IL-1β-induced knee osteoarthritis cell model injury, the model was constructed using 20 ng / mL IL-1β and treated with 2 μM ellipticine for 48 h. Flow cytometry and CCK-8 were used to detect the cell survival rate and apoptosis rate. Figure 3 As shown, A is the cell survival rate and B is the cell apoptosis rate. The results showed that ellipticine significantly reduced the cell apoptosis rate in the model group and restored cell activity, indicating that ellipticine can effectively antagonize IL-1β-induced C28 / I2 cell damage and show activity in treating knee osteoarthritis.

[0032] Test Example 3

[0033] (1) We further explored the potential mechanism of ellipticine in treating IL-1β-induced C28 / I2 knee osteoarthritis cell model damage. Specifically, we constructed an OA cell model (using 20 ng / mL IL-1β to treat C28 / I2 cells), and then treated the model group cells with a culture medium containing 2 μM ellipticine. After 48 hours, ELISA, Western Blot (WB) and immunofluorescence techniques were used to detect the expression changes of inflammatory-related factors in each group of cells. The results are as follows: Figure 4-6 shown.

[0034] Figure 4 In the figure, A, B and C represent the changes in IL-1β, IL-6 and TNF-α levels, respectively. ELISA results show that compared with the Control group, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the supernatant of the Model group were significantly increased, indicating that IL-1β successfully induced an inflammatory response; after the addition of ellipticine, the levels of these inflammatory factors were significantly reduced, indicating that ellipticine has an anti-inflammatory effect.

[0035] Figure 5 In the figure, A is the WB analysis result, and B is the comparison of protein expression. The results show that compared with the Control group, the expression levels of ADAMTS4, MMP3, MMP13, COX-2 and Cleaved-Caspase 3 proteins in the Model group cells were significantly increased, indicating that the OA model was successfully constructed and accompanied by extracellular matrix degradation and activation of apoptosis pathways; while ellipticine treatment significantly reduced the expression levels of these proteins, indicating that ellipticine can inhibit inflammation-mediated matrix degradation and cell apoptosis.

[0036] Figure 6Immunofluorescence detection visually demonstrated the expression of MMP3 protein in cells. Compared with the Control group, cells in the Model group showed enhanced fluorescence intensity, indicating increased expression of MMP3 protein; while the fluorescence intensity of cells in the ellipticine-treated group was significantly reduced, indicating that MMP3 protein expression was inhibited, further supporting the role of ellipticine in inhibiting the pathological process of OA.

[0037] In summary, the results showed that ellipticine could effectively inhibit the expression of inflammatory factors and the activation of related proteins in the IL-1β-induced C28 / I2 knee osteoarthritis cell model, thereby promoting the recovery of cell damage, providing a scientific basis for ellipticine as a drug for the treatment of OA.

[0038] (2) The key proteins of ellipticine in the treatment of OA cell model were screened by proteomic sequencing, and the expression of key proteins was analyzed by RT-qPCR and western blotting. The results are as follows: Figure 7 As shown in the figure, AF represents the gene expression levels of PTGS1, CASP3, RBX1, NDUFS3, RPL3, SOD2 and SMAD2 detected by RT-qPCR, and GI represents the protein expression levels of SOD2 and SMAD2 detected by WB.

[0039] Depend on Figure 7 It can be seen that at the gene expression level, compared with the Control group, the gene expressions of PTGS1, CASP3, NDUFS3, RPL3, SOD2 and SMAD2 in the Model group were significantly upregulated, and the expression of SOD2 protein was also significantly increased, indicating that the inflammatory response induced by IL-1β led to significant changes in these genes and proteins. However, compared with the Model group, the gene expressions of PTGS1, CASP3, SOD2 and SMAD2 in the ellipticine-treated group were significantly downregulated, and the expression of SOD2 and SMAD2 proteins was also significantly reduced, indicating that ellipticine can effectively inhibit the abnormal expression of genes and proteins caused by IL-1β. It is particularly noteworthy that ellipticine has a significant reversal effect on the expression of PTGS1 (related to the synthesis of inflammatory mediators prostaglandins), CASP3 (apoptosis execution protein) and SOD2 (superoxide dismutase, related to oxidative stress), further supporting the therapeutic value of ellipticine in alleviating the inflammatory response of chondrocytes. In summary, this study verified the proteomic screening results by RT-qPCR and western blot analysis, and revealed the specific molecular mechanism of ellipticine in the treatment of IL-1β-induced C28 / I2 knee osteoarthritis cell model injury, providing strong evidence for its further development as a therapeutic drug.

[0040] Test Example 4

[0041] This study first constructed a SOD2 overexpression (OE) and interference (si-SOD2) cell system and verified its effectiveness through experiments. The mechanism of ellipticine in the treatment of OA was further explored. Figure 8 As shown in the figure, A is the cell apoptosis rate of each group, B is the cell proliferation rate of each group, CE are the changes in IL-1β, IL-6 and TNF-α levels of each group, and FG are the detection results of p-FOXO1A protein expression levels of each group.

[0042] The results showed that ellipticine effectively reversed the apoptosis and inflammatory response of OA cell models by inhibiting the expression of SOD2, and this process may be achieved through the FOXO signaling pathway. These findings not only reveal the potential mechanism of ellipticine in the treatment of OA, but also provide important clues for the future development of OA treatment strategies targeting SOD2.

[0043] Test Example 5

[0044] (1) This study first constructed a SMAD2 overexpression (OE) and interference (si-SMAD2) cell system and verified its effectiveness through experiments. We further evaluated how ellipticine affects the functional state of the OA cell model by regulating SMAD2. Fig. 9 As shown, A is the cell apoptosis rate of each group, B is the cell proliferation rate of each group, and CE are the changes in IL-1β, IL-6 and TNF-α levels in each group, respectively.

[0045] The experimental results showed that compared with the Model group, the cell apoptosis rate in the ellipticine-treated group and the si-SMAD2 group was significantly reduced, while the cell proliferation rate was significantly increased. In contrast, the OE-SMAD2 group showed a higher cell apoptosis rate and a lower cell proliferation rate. It is worth noting that when ellipticine was added, the changes in the OE-SMAD2 group were effectively reversed, indicating that ellipticine can counteract the abnormal cell function caused by SMAD2 overexpression. At the same time, the results of ELISA test showed that ellipticine can significantly inhibit the expression of inflammatory-related factors such as IL-1β, IL-6 and TNFα promoted by SMAD2 overexpression, further confirming its positive role in anti-inflammatory.

[0046] (2) In order to further explore the potential mechanism of ellipticine regulating SMAD2, the expression levels of Hippo signaling pathway-related proteins (TEAD1 and p-YAP) and TGF-β signaling pathway-related proteins (TGF-β and p-SMAD2) in each group of cells were detected. Fig.10 shown.

[0047] The results showed that ellipticine significantly affected the expression of key proteins in the Hippo signaling pathway (through TEAD1 and p-YAP) and the TGF-β signaling pathway (through TGF-β and p-SMAD2). This suggests that ellipticine exerts its biological effects by simultaneously regulating the Hippo and TGF-β signaling pathways.

[0048] In summary, the results of this experiment showed that ellipticine effectively reversed the apoptosis and inflammatory response of OA cell models by inhibiting the expression of SMAD2, and this process may involve a complex regulatory network of Hippo and TGF-β signaling pathways. These findings not only provide a theoretical basis for the potential application of ellipticine in the treatment of OA, but also point out the direction for further exploration of its molecular mechanism.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of ellipticine in the preparation of a drug for treating knee osteoarthritis.

2. The use according to claim 1, characterized in that The ellipticine can inhibit chondrocyte apoptosis.

3. The use according to claim 1, characterized in that The ellipticine can inhibit the expression of inflammatory cytokines IL-1β, IL-6 and TNF-α.

4. The use according to claim 1, characterized in that The ellipticine can inhibit the expression of matrix degradation enzymes ADAMTS4, MMP3 and MMP13.

5. The use according to claim 1, characterized in that The effective concentration of ellipticine is 0.5-5 μM.

6. A drug for treating knee osteoarthritis, characterized in that: The invention comprises the ellipticine as claimed in claim 1.

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