Application of Akt pathway activator in treatment of cartilage injury and systemic reaction caused by joint bleeding

Abstract

The invention provides an application of an Akt pathway activator in treatment of cartilage injury and systemic reaction caused by joint hemorrhage. Knee joint hemorrhage modeling is carried out on mice and pigs; it is found that the Akt pathway activator sc79 and vitamin E have remarkable curative effects in treatment of cartilage injury, systemic reaction, liver injury and peripheral blood inflammation caused by joint bleeding, and a new target and a new treatment means are provided for prevention and treatment of joint bleeding.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an Akt pathway activator in treating cartilage damage and systemic reactions caused by joint bleeding. Background Art

[0002] Hemophilic arthropathy (HA) is characterized by repeated episodes of hemarthrosis. Hemarthrosis or bleeding into the joint cavity may also play a role in the pathophysiology of the development of post-traumatic osteoarthritis (PTOA) after joint injury. Hemarthrosis mainly affects large joints such as the knee, elbow, or ankle and can lead to abnormal joint remodeling. Cartilage is gradually damaged by iron deposition, lysosomal enzymes, and proinflammatory cytokines produced by the inflamed synovium, eventually leading to the formation of subarticular bone cysts. Repeated hemarthrosis leads to synovial hyperplasia and angiogenesis, and further bleeding occurs in the fragile and thickened synovium. Hemarthrosis stretches the joint capsule and ligaments, leading to joint instability, decreased joint movement due to pain, and weakness of the muscles around the joint, which can lead to joint instability. As symptoms progress, the joint becomes severely damaged due to further loss of cartilage and subchondral bone sclerosis.

[0003] There are some symptomatic treatments available, such as transfusion of replacement coagulation factors, which is the preferred method for preventing recurrent joint bleeding; aspirating blood from the joints in acute hemarthrosis can quickly eliminate inflammation; intra-articular corticosteroid therapy may be useful for selected patients; there are also conservative intra-articular treatments such as injection of hyaluronic acid or platelet-rich plasma. However, these can only delay the occurrence and progression of cartilage damage, and there is no treatment specifically targeting changes in chondrocytes and tissue pathways. Therefore, it is necessary to explore and develop safer and more reliable drugs or therapies for the treatment of joint bleeding that target cartilage tissue.

[0004] Many diseases have systemic effects. Some systemic biomarkers can reflect the destructive changes of cartilage and bone in patients with joint diseases in clinical or scientific research. In osteoarthritis (OA) and PTOA, there have been studies on biomarkers (in urine, blood and synovial fluid) related to the degeneration of cartilage, bone and synovial tissue. However, its systemic biological effects have not been reported.

[0005] The Journal of Xi'an Jiaotong University (Medical Edition) published "The mechanism of PI3K / AKT signaling pathway regulating chondrocyte autophagy and damage in bone and joint diseases", which disclosed that "PI3K / AKT signaling pathway has the effects of promoting chondrocyte proliferation, anti-apoptosis and reducing the production of inflammatory factors". However, on the one hand, although the PI3K / AKT signaling pathway has been proven to have the effects of promoting chondrocyte proliferation and anti-apoptosis, the regulatory role of the PI3K / AKT signaling pathway in chondrocyte damage and autophagy is still in the initial exploration stage. On the other hand, cartilage damage can be caused by a variety of reasons, and the treatment methods for damage caused by different factors are also different. In this document, it is mainly caused by bone and joint diseases, but other reasons, such as cartilage damage caused by joint bleeding, are not mentioned in the document.

[0006] Therefore, it is necessary to provide an Akt pathway activator for use in the treatment of cartilage damage and systemic reactions caused by joint bleeding, so as to solve the above problems existing in the prior art. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides an application of an Akt pathway activator in the treatment of cartilage damage and systemic reactions caused by joint bleeding. By modeling knee joint bleeding in mice and pigs, it was found that the Akt pathway activator sc79 and vitamin E have significant therapeutic effects in the treatment of cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding, which also provides a new target and treatment method for the prevention and treatment of joint bleeding.

[0008] In one aspect, the present invention provides a use of an Akt pathway activator for preparing an agent for preventing or treating joint bleeding, wherein the joint bleeding includes joint bleeding associated with traumatic joint injury, hemophilic arthritis, and joint surgery.

[0009] In some embodiments, it is found that the cartilage tissue treated with blood produces obvious damage. At this time, transcriptome sequencing and single-cell qPCR detection found that among the 8 common differentially expressed genes, the Akt pathway was most significantly downregulated. Therefore, in the present invention, by modeling knee joint bleeding in mice and pigs, it was found that Akt pathway activators have significant therapeutic effects in the treatment of joint bleeding.

[0010] Furthermore, the Akt pathway activator can also be used to prepare an agent for preventing or treating cartilage damage, systemic reactions and liver complications caused by joint bleeding.

[0011] In some embodiments, a model of mice with knee joint bleeding was performed, and the experimental results showed that after activating the Akt pathway, the original extracellular matrix degradation and cartilage thickness attenuation were significantly slowed down, the OARSI score was significantly reduced, the expression of cartilage matrix proteins ACAN and COLII was significantly increased, the expression of pro-inflammatory factors IL4, IL6, and IL13 in the peripheral blood was significantly reduced, and the expression of inflammatory factors IL1b, IL6, and TNFa in the liver was significantly reduced. At the same time, the serum liver function indicators AST and ALT also decreased significantly, which were close to normal indicators. It can be seen that activating the Akt pathway can reduce cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding.

[0012] In some embodiments, a pig model with knee joint bleeding was also performed. The experimental results showed that after activating the Akt pathway, the original serum liver function indicators AST and ALT were significantly reduced, with no significant difference from normal indicators. This further shows that after activating the Akt pathway, the increase in serum liver function indicators AST and ALT caused by joint bleeding can be significantly reduced, thereby alleviating and treating liver damage.

[0013] Furthermore, the Akt pathway activator includes a substance that upregulates the expression of phosphorylated Akt protein.

[0014] Furthermore, the substance that can upregulate the expression of phosphorylated Akt protein includes any one or more of the CRISPR / Cas9 gene editing system targeting the Akt upstream pathway and its key proteins, RNA knockdown and overexpression and its delivery system, and small molecules.

[0015] Furthermore, the small molecule includes SC79 and vitamin E.

[0016] In some embodiments, the inventors found that in the specific case of cartilage first contact with blood, the CRISPR / Cas9 gene editing system targeting the Akt upstream pathway and its key proteins, RNA knockdown and overexpression and its delivery system, as well as small molecules sc79 and vitamin E can all upregulate the expression of phosphorylated Akt protein, and experiments have found that sc79 and vitamin E have extremely significant effects on activating the Akt pathway in mouse chondrocytes. Therefore, sc79 and vitamin E are further studied to activate the Akt pathway. Among them, the mechanism of sc79 activating the Akt pathway is: sc79 can prevent the membrane translocation of Akt protein and promote its phosphorylation by upstream kinases in the cytosol; the mechanism of vitamin E activating the Akt pathway is: blood downregulates chondrocyte phosphorylated Akt (p-Akt) by downregulating reactive oxygen species (ROS), so the use of ROS scavenger vitamin E can restore the expression of p-Akt. Therefore, in the specific case of cartilage contact with blood, VitE is also an activator of the Akt pathway.

[0017] Furthermore, the substance prevents or treats cartilage damage, systemic reactions and liver complications caused by joint bleeding by reducing cartilage matrix degradation; the cartilage matrix includes COLII and ACAN.

[0018] In some embodiments, in the specific case of cartilage's first contact with blood, it was found that sc79 can significantly reduce the degradation of cartilage matrix proteins ACAN and COLII caused by joint bleeding by activating the Akt pathway in mouse chondrocytes, and the effect is significant. This also shows that sc79 can be used as a potential therapeutic target for inhibiting cartilage matrix degradation and improving matrix synthesis caused by joint bleeding.

[0019] On the other hand, the present invention provides a use of an Akt pathway activator for preparing an agent for inhibiting apoptosis of cartilage tissue and cells or inhibiting the expression of extracellular matrix degrading enzymes, wherein the Akt pathway activator comprises a substance that upregulates the expression of phosphorylated Akt protein.

[0020] Furthermore, the "inhibition of apoptosis" includes inhibiting the expression of Cleaved caspase-3, increasing the expression of Bcl-2 protein and promoting cell proliferation; the "extracellular matrix degrading enzyme" includes MMP-3 and MMP-13.

[0021] In some embodiments, after the mouse chondrocytes were exposed to blood, on the one hand, the apoptosis marker protein cleaved The positive area of ​​caspase-3 increased significantly (the number of fluorescent staining increased significantly and the color became darker), while in the Blood+sc group, compared with the Blood group, the positive area of ​​C-caspase-3 decreased significantly (the number of fluorescent staining decreased significantly and the color became lighter), which indicates that after sc79 activates the Akt pathway, it can significantly inhibit the expression of apoptosis marker protein C-caspase-3, thereby inhibiting chondrocyte apoptosis caused by joint bleeding; on the other hand, the experiment found that in chondrocytes treated with blood, the mRNA content of the pro-survival protein Bcl-2 decreased significantly, while the mRNA expression of the pro-apoptotic protein Bax increased significantly, while in the Blood+sc group, compared with the Blood group, the mRNA content of the pro-survival protein Bcl-2 increased significantly, while the mRNA expression of the pro-apoptotic protein Bax also decreased significantly, which indicates that after sc79 activates the Akt pathway, it can significantly increase the mRNA expression of the pro-survival protein Bcl-2, while inhibiting the mRNA expression of the pro-apoptotic protein Bax, thereby inhibiting chondrocyte apoptosis caused by joint bleeding.

[0022] In another aspect, the present invention provides a use of an Akt pathway activator for preparing an agent for preventing or treating peripheral blood inflammation caused by joint bleeding, wherein the Akt pathway activator prevents or treats peripheral blood inflammation caused by joint bleeding by inhibiting the expression of inflammatory factors in peripheral blood.

[0023] Furthermore, the pro-inflammatory factors in the peripheral blood include IL4, IL6, and IL13.

[0024] In some embodiments, the experimental results show that in the Blood group of mice with joint bleeding, the expression of pro-inflammatory factors IL4, IL6, and IL13 in the peripheral blood were significantly increased; while in the Blood+sc79 group, compared with the Blood group, the expression of IL4, IL6, and IL13 was significantly decreased, indicating that after sc79 activates the Akt pathway, it can significantly reduce the expression of pro-inflammatory factors IL4, IL6, and IL13 in the peripheral blood caused by joint bleeding, thereby alleviating or treating peripheral blood inflammation.

[0025] Beneficial effects achieved by the present invention:

[0026] 1. The present invention provides new targets and means for preventing and treating joint bleeding.

[0027] 2. The present invention provides a new target for the prevention or treatment of cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding.

[0028] 3. The present invention discloses that small molecules can be used to intervene in the target pathway Akt pathway, which is easy to use and has less toxic side effects on normal cells.

[0029] 4. The present invention provides an Akt pathway activator, a new use of sc79 or vitamin E in the treatment of joint bleeding and cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A is the result of transcriptome sequencing of cartilage tissue after blood treatment.

[0031] Figure 1 B is the result of single-cell qPCR detection of cartilage tissue after blood treatment.

[0032] Figure 1 C is a statistical chart of gene changes detected by transcriptome sequencing and single-cell qPCR in the blood treatment group.

[0033] Figure 1 D is the KEGG analysis diagram of 8 differentially expressed genes.

[0034] Figure 2 A is the result of WB detection of Akt phosphorylation levels in different groups.

[0035] Figure 2 B is the result of relative activity of chondrocytes in CCK8 experiment.

[0036] Figure 2 C is the immunofluorescence staining result of the cartilage matrix-related proteins ACAN and COLII after using sc79.

[0037] Figure 2 D is the immunofluorescence quantitative results of cartilage matrix-related proteins ACAN and COLII.

[0038] Figure 3 A is the result of detecting ROS levels in different groups using flow cytometry.

[0039] Figure 3 B is the result of detecting the activation level of Akt pathway in different groups using WB method.

[0040] Figure 3 C is the result of relative viability of chondrocytes in CCK8 experiment.

[0041] Figure 3 D is the immunofluorescence staining image of chondrocyte marker proteins ACAN and COLII after the use of vitamin E.

[0042] Figure 3 E is the expression result of chondrocyte marker proteins ACAN and COLII.

[0043] Figure 4 A is the result of SO staining of cartilage tissue after blood treatment.

[0044] Figure 4 B is the diagram of OARSI score to evaluate the degree of cartilage degradation in mice of different groups.

[0045] Figure 4 C is the immunofluorescence staining results of chondrocyte marker proteins ACAN and COLII levels.

[0046] Figure 4 D is the expression results of chondrocyte marker proteins COLII and ACAN in different groups after using sc79.

[0047] Figure 4 E is the expression results of pro-inflammatory factors IL4, IL6, and IL13 in the peripheral blood of different groups.

[0048] Figure 4 F is the expression results of inflammatory factors IL1b, IL6, and TNFa in the liver of different groups.

[0049] Figure 4 G is the result of HE staining and immunohistochemistry (CD4+T cells) of the liver after blood treatment.

[0050] Figure 4 H is the result graph of serum liver function indicators including AST and ALT in different groups.

[0051] Figure 5A This figure shows the therapeutic effect of Akt pathway activator sc79 on pig liver damage induced by joint bleeding.

[0052] Figure 6 A is the immunofluorescence image of the apoptosis marker protein cleaved caspase-3 (left) and the quantitative result of immunofluorescence cleaved caspase-3 protein (right).

[0053] Figure 6 B is the mRNA expression diagram of the pro-survival protein Bcl-2 and the pro-apoptotic protein Bax. DETAILED DESCRIPTION

[0054] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be understood by people with general skills in the field to which the present invention belongs. The reagents or instruments used without indicating the manufacturer are conventional products that can be purchased through regular channels.

[0055] Example 1: Discovery and screening process of Akt pathway activators

[0056] Joint bleeding mainly affects large joints such as the knee, elbow or ankle, and can cause abnormal joint remodeling. Cartilage is gradually damaged by iron deposition, lysosomal enzymes and proinflammatory cytokines produced by the inflamed synovium, eventually leading to the formation of subarticular bone cysts. Repeated joint effusion leads to synovial hyperplasia and angiogenesis, and further bleeding occurs in the fragile and thickened synovium. Joint bleeding stretches the joint capsule and ligaments, leading to joint instability, and pain leads to reduced joint activity, resulting in muscle weakness around the joint, which leads to joint instability. As the symptoms progress further, the joints will be severely damaged due to further loss of cartilage and subchondral bone sclerosis. In order to develop a safer and more reliable drug or therapy for the treatment of cartilage damage caused by joint bleeding, transcriptome sequencing and single-cell qPCR detection were performed on the cartilage tissue after blood treatment in this embodiment. The detection method is as follows: mouse cartilage explants are used as transcriptome sequencing objects, and cells digested from mouse cartilage explants are used as single-cell qPCR detection objects. The steps of single-cell qPCR were as follows: cells or tissues were washed twice in PBS, and then Trizol reagent (9109, TaKaRa, Japan) was added for RNA extraction. Reverse transcription was performed using a commercial kit (R323, Vazyme, China) according to the manufacturer's instructions, and qPCR reactions were performed using a TB Green PCR kit (Q711, Vazyme, China) according to the manufacturer's instructions. The steps of transcriptome sequencing were as follows: cartilage samples from newborn mice were collected in a culture dish and blood was processed. Liver specimens were obtained from the joint bleeding mouse model. RNAseq and library construction were completed by the Beijing Genomics Institute of BGI. Total RNA was extracted from tissues using Trizol (Invitrogen, USA) according to the instructions. About 60 mg of tissue was taken and ground into powder with liquid nitrogen in a 2 mL test tube to extract RNA; 25 pL to 100 pL of depc-treated water was added to dissolve the RNA. Total RNA was identified and quantified using Nano Drop and Agilent 2100 Bioanalyzer (Thermo Fisher Scientific, USA). Next, the purified mRNA was fragmented with fragmentation buffer at an appropriate temperature. Reverse transcription with primers was used to generate cDNA, and then the second-strand cDNA was synthesized. The resulting cDNA fragments were amplified by PCR, and the products were purified using Ampure XP Beads. The final library was amplified into DNA nanoballs (DNB, China) with phi29, with more than 300 copies per molecule. The DNBs were loaded into patterned nanoarrays and single-end 50 base reads were generated on the BGIseq500 platform (BGI-Shenzhen, China).

[0057] Specific results such as Figure 1As shown in A to D (in the omics analysis, genes that simultaneously meet P < 0.05 and the difference fold > 2 are defined as differentially expressed genes. The KEGG analysis results are completed using the BGI analysis system (biosys.bgi.com)), where: Figure 1 A is the result of transcriptome sequencing of cartilage tissue after blood treatment, Blood is the blood treatment group, Control is the control group (cartilage tissue not treated with blood), Figure 1 As can be seen in A, 461 genes were upregulated and 887 were downregulated after blood treatment; Figure 1 B is the result of single-cell qPCR detection of cartilage tissue after blood treatment. Figure 1 As can be seen in B, the cells after blood treatment can be divided into two main populations; Figure 1 C is the statistical diagram of gene changes detected by transcriptome sequencing and single-cell qPCR in the blood treatment group. Figure 1 As can be seen in C, in the cartilage tissue after blood treatment, the single-cell qPCR test results showed 895 significantly down-regulated genes, and the transcriptome sequencing results showed 31 significantly down-regulated genes, of which 8 were differentially expressed genes obtained by both methods; Figure 1 D is the KEGG analysis diagram of 8 differentially expressed genes. Figure 1 As can be seen in D, in the cartilage tissue after blood treatment, after KEGG analysis of 8 differentially expressed genes, 5 pathways were obtained, including P13K-Akt signaling pathway, ECM-receptor interaction, Protein digestion and absorption, Focaladhesion and Human papillomavirus infection, among which PI3K-Akt pathway was the most significantly downregulated pathway.

[0058] The experimental results showed that the cartilage tissue treated with blood produced obvious damage. At this time, 8 common differential genes were found through transcriptome sequencing and single-cell qPCR detection. By performing KEGG analysis on the 8 differential genes, 5 pathways were obtained, among which the Akt pathway was found to be downregulated most significantly. Therefore, in the present invention, knee joint bleeding models were performed on mice and pigs to further explore the role of Akt pathway activators in the treatment of cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding.

[0059] Example 2: Comparative test of the effects of sc79 and vitamin E on activating the Akt pathway in mouse chondrocytes

[0060] In order to further explore the role of Akt pathway activators in the treatment of cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding, in this example, small molecules sc79 and vitamin E that activate the Akt pathway in mouse chondrocytes through different mechanisms were compared and their effects were observed. Since the activation of the Akt pathway is indicated by the increase in the content of phosphorylated Akt detected by WB (protein immunoblotting, western blot), both small molecules can activate the Akt pathway and increase the content of phosphorylated Akt. Therefore, the specific observation index is the change in the content of phosphorylated Akt. The specific steps are as follows:

[0061] 1. Activate the Akt pathway in mouse chondrocytes by using the small molecule sc79. The specific mechanism is: sc79 can prevent the membrane translocation of Akt protein and promote its phosphorylation by upstream kinases in the cytosol. The specific operation is as follows:

[0062] (1) Mouse chondrocytes were used as test cells. 5 μg / ml sc79 (S7863, Selleck, USA) was added to 5% or 10% blood and treated with the transwell system. After 3 days of culture, the medium was replaced with normal culture medium and samples were collected after 3 days.

[0063] (2) Cells were collected and the levels of phosphorylated Akt pathway proteins were detected by Western blot. Immunofluorescence and CCK8 experiments were also performed to detect the levels of cartilage matrix-related proteins ACAN and COLII.

[0064] Specific results such as Figure 2 A~ Figure 2 As shown in D, Figure 2 A is the result of WB detection of Akt phosphorylation levels in different groups. Figure 2 B is the result of the relative activity of chondrocytes in the CCK8 experiment. Figure 2 C is the immunofluorescence staining result of cartilage matrix-related proteins ACAN and COLII after using sc79 (Merge is the fluorescence staining result of ACAN and COLII superimposed together). Figure 2 D is the immunofluorescence quantitative results of cartilage matrix-related proteins ACAN and COLII.

[0065] from Figure 2 As can be seen in A, after adding sc79, the expression of p-Akt was restored, and the color and depth were close to the control group level. Compared with the Blood group, the expression of p-Akt was significantly increased; Figure 2 As can be seen in B, the relative cell viability of normal mouse chondrocytes decreased significantly after blood treatment, while the relative cell viability increased significantly after the addition of sc79, which was significantly different from that of the Blood group. Figure 2 As shown in C, compared with the control group, in the Blood group, the positive areas of cartilage matrix proteins ACAN and COLII were significantly reduced (the number of fluorescent staining was significantly reduced and the color became lighter), indicating that ACAN and COLII were significantly degraded after blood treatment; while in the Blood+sc79 group, compared with the Blood group, the positive areas of cartilage matrix proteins ACAN and COLII were significantly increased (the number of fluorescent staining was significantly increased and the color became darker), indicating that after sc79 activated the Akt pathway, it could significantly reduce the degradation of cartilage matrix proteins ACAN and COLII caused by joint bleeding; Figure 2 As can be seen in D, after normal mouse chondrocytes were treated with blood, the expression of COLII and ACAN decreased significantly, which was significantly different from the control group. After adding sc79, the expression of COLII and ACAN increased significantly, especially the expression of COLII, which was extremely significantly different from the Blood group.

[0066] It can be seen from this that in the specific case of cartilage's first contact with blood, sc79 can significantly reduce the degradation of cartilage matrix proteins ACAN and COLII caused by joint bleeding by activating the Akt pathway in mouse chondrocytes, and the effect is significant. This also shows that sc79 can be used as a potential therapeutic target for inhibiting cartilage matrix degradation and improving matrix synthesis caused by joint bleeding.

[0067] 2. Activate the Akt pathway in mouse chondrocytes by small molecule vitamin E (VitE). The specific mechanism is: VitE can remove reactive oxygen species (ROS), thereby activating the expression of the p-Akt pathway. The specific operation is as follows:

[0068] (1) Mouse chondrocytes were seeded in a 6-well plate. When the cell confluence reached about 70%, 5% mouse whole blood, 100ul / well mouse whole blood containing 75μM VitE, and an equal volume of PBS were added as a control. The ROS content was determined (using the DCFH-DA fluorescent probe, according to the instructions (Biyuntian, S0033S), and the cell fluorescence value of the FITC channel measured at the end reflected its ROS content).

[0069] (2) For WB experiments, cells were incubated with primary antibodies against p-Akt or Tubulin and corresponding secondary antibodies, and then imaged using a chemiluminescence imaging system (Shanghai Qinxiang).

[0070] (3) For qPCR experiments, the RNA of the cells is first extracted and then reverse transcribed into cDNA. The Ct value of the specific cDNA amplification process is measured using SYBR dye.

[0071] (4) For CCK8 experiments, the assay was performed according to the manufacturer's instructions (Tongren Chemical, CK04), and the absorbance values ​​were normalized to relative cell viability.

[0072] Specific results such as Figure 3 As shown in A to E, the Blank group is the blank group: normal mouse chondrocytes, the Control group is the control group: an equal volume of PBS is added to normal mouse chondrocytes, the Blood group is the positive control group: 5% mouse whole blood is added to normal mouse chondrocytes, and the Blood+VitE group is the experimental group: an equal volume of mouse whole blood containing 75 μM VitE is added to normal mouse chondrocytes, wherein, Figure 3 A is the result of detecting ROS levels in different groups using flow cytometry. Figure 3 As can be seen in A, compared with the positive control group, after adding VitE, the ROS content in mouse chondrocytes decreased significantly, approaching the ROS content level of the blank group and the control group, indicating that ROS was cleared by VitE; Figure 3 B is the result of using WB method to detect the activation level of Akt pathway in different groups. Figure 3 As can be seen in B, after adding VitE, the expression of p-Akt was restored, and the color and depth were close to the control group level. The Tubulin group is the internal reference group, which is a reference and represents the control variable, indicating that the total amount of cells involved in the experiment is the same. It also shows that this experiment is effective and the results are reliable. Figure 3 C is the relative activity result of chondrocytes in CCK8 experiment. Figure 3 As can be seen in C, the relative cell viability of normal mouse chondrocytes decreased significantly after being treated with blood, while the relative cell viability increased significantly after adding VitE, which was significantly different from the Blood group; Figure 3 D is the immunofluorescence staining of chondrocyte marker proteins COLII and ACAN after using vitamin E. Figure 3 E is the expression result of chondrocyte marker proteins COLII and ACAN. Figure 3 As can be seen in D and E, the expression of COLII and ACAN proteins in normal mouse chondrocytes significantly decreased after blood treatment, which was significantly different from the control group. After adding VitE, the expression of COLII and ACAN proteins increased significantly, especially the expression of COLII protein, which was significantly different from the Blood group.

[0073] The experimental results show that blood downregulates chondrocyte phosphorylated Akt (p-Akt) by downregulating reactive oxygen species (ROS). In this example, the use of ROS scavenger VitE can restore the expression of p-Akt. Therefore, in the specific case of cartilage contact with blood, VitE is also an activator of the Akt pathway.

[0074] And from Figure 2 D and Figure 3 It can be seen from the expression of the contents of E chondrocyte marker proteins COLII and ACAN that in cartilage damage caused by initial joint bleeding, compared with the use of vitamin E as an Akt pathway activator, the recovery of the contents of chondrocyte marker proteins COLII and ACAN was significantly improved after the addition of sc79.

[0075] Therefore, in the present invention, in the specific case of cartilage contacting blood for the first time, it was found that both sc79 and vitamin E can activate the Akt pathway in mouse chondrocytes, and when sc79 is preferred, it can significantly reduce the degradation of cartilage matrix proteins ACAN and COLII caused by joint bleeding, and the effect is more significant. It also shows that sc79 can be used as a potential therapeutic target for inhibiting cartilage matrix degradation and improving matrix synthesis caused by joint bleeding.

[0076] Example 3: Joint bleeding mouse modeling and drug administration and testing of the therapeutic effect of Akt pathway activator sc79 on joint bleeding-induced mouse cartilage matrix degradation, peripheral blood inflammatory response and liver damage

[0077] To further explore the role of Akt pathway activator sc79 in treating cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding, in this example, joint bleeding models were established and administrated to mice, and the effect of Akt pathway activator sc79 was observed. The specific steps are as follows:

[0078] 1. Mouse model of knee joint bleeding

[0079] The knee joint bleeding mouse model was established by injecting blood (8ul / leg) into the knee joint cavity of mice (C57BL / 6, male, 10 weeks old) 4 times, once a week. The C57BL / 6 mice were killed 1 week after the last injection.

[0080] 2. Drug administration

[0081] Based on the knee joint bleeding mouse model, 25 μg / ml sc79 (S7863, Selleck, USA) was added to the blood, and the surgical procedure was the same as the knee joint bleeding mouse model. C57BL / 6 mice were killed 1 week after the last injection.

[0082] 3. Joint sample processing and testing

[0083] (1) Detection of cartilage degradation in mice:

[0084] ① Joint sampling: Cut off the entire joint of the mouse and use ophthalmic scissors to remove the muscle tissue as cleanly as possible.

[0085] ② Fixation: Soak the joint in 4% paraformaldehyde in the dark for more than 24 hours, rinse with running water overnight to completely rinse off the fixative;

[0086] ③ Decalcification: Place the joint in decalcification solution for 3 weeks, changing the solution 2-3 times during this period. The femur and tibia become soft at this time, and the degree of decalcification can be judged by cutting with scissors. Rinse with running water overnight to completely rinse off the decalcification solution;

[0087] ④ Prepare paraffin sections and perform subsequent SO staining and immunofluorescence staining such as ACAN and COLII.

[0088] ⑤Evaluate the degree of cartilage degradation in mice through OARSI scoring, etc.

[0089] 4. Peripheral blood sample processing and testing

[0090] (1) Peripheral blood ELISA test

[0091] Peripheral blood sampling: Blood was collected from the eyeballs of mice and stored in disposable blood collection tubes; serum was collected from peripheral blood according to the instructions of the kits with catalog numbers ml064310 (IL-4) and ml063159 (IL-6) of ELISA, or according to the instructions of the kit with catalog number D721112 (IL-13) of Bioengineering.

[0092] The systemic inflammatory response of mice was evaluated by peripheral blood inflammatory factor analysis, and the liver function of mice was evaluated by peripheral blood biochemical analysis.

[0093] 5. Liver sample processing and testing

[0094] (1) Detection of inflammation levels in liver samples

[0095] ① Liver sampling: Collect mouse liver and use ophthalmic scissors to cut off the connective tissue as much as possible.

[0096] ② Fixation: Soak the liver in 4% paraformaldehyde and keep it away from light for more than 24 hours, then rinse it with running water overnight to completely rinse off the fixative;

[0097] ③ Make paraffin sections and perform subsequent HE staining and immunohistochemical staining.

[0098] (2) qPCR detection of liver samples

[0099] ① Extraction of mRNA: The liver was washed twice in PBS, and then Trizol reagent (9109, TaKaRa, Japan) was added for RNA extraction.

[0100] ② Reverse transcription: A commercial kit (R323, Vazyme, China) was used according to the manufacturer's instructions.

[0101] ③qPCR reaction: qPCR reaction was performed using TB Green PCR kit (Q711, Vazyme, China) according to the manufacturer's instructions.

[0102] The specific experimental results are as follows Figure 4 As shown in A~H. Figure 4 A is the result of SO staining of cartilage tissue after blood treatment; Figure 4 B is a graph showing the degree of cartilage degradation of mice in different groups evaluated by OARSI scoring; Figure 4 C is the immunofluorescence staining result of chondrocyte marker proteins ACAN and COLII; Figure 4 D is the expression results of chondrocyte marker proteins COLII and ACAN in different groups; Figure 4 E is the expression results of pro-inflammatory factors IL4, IL6, and IL13 in peripheral blood of different groups; Figure 4 F is the expression results of inflammatory factors IL1b, IL6, and TNFa in the liver of different groups; Figure 4 G is the result of HE staining and immunohistochemistry (CD4+T cells) of the liver after blood treatment; Figure 4 H is the result graph of serum liver function indexes including AST and ALT in different groups. In the above figure, Control refers to mice that were raised normally and not treated in any way, which is the control group; Blood refers to the experimental group mice that were injected with blood (8ul / leg) 4 times in the knee joint cavity, once a week, and were killed 1 week after the last injection; Blood+sc79 refers to the experimental group mice that were injected with 25μg / ml sc79 in the blood on the basis of Blood.

[0103] from Figure 4 As can be seen from A, compared with the control group, the extracellular matrix (ECM) and cartilage thickness in the Blood group were significantly degraded; while in the Blood+sc79 group, the ECM degradation and cartilage thickness attenuation were significantly slowed down, which indicates that sc79 can significantly alleviate joint damage in mice caused by joint bleeding.

[0104] from Figure 4 As can be seen from B, compared with the control group, the OARSI score (which assesses the severity of arthritis, the higher the score, the more severe the arthritis) of the Blood group reached 3, indicating that the cartilage knee joints of the mice in the Blood group were significantly damaged, which was significantly different from that of the control group; while in the Blood+sc79 group, the OARSI score was about 1.2, which was significantly different from that of the Blood group, indicating that sc79 can significantly alleviate the joint damage of mice caused by joint bleeding.

[0105] from Figure 4 As can be seen in C, compared with the control group, in the Blood group, the positive areas of cartilage matrix proteins ACAN and COLII were significantly reduced (the number of fluorescent stainings was significantly reduced and the color became lighter), indicating that ACAN and COLII were significantly degraded after blood injection; while in the Blood+sc79 group, compared with the Blood group, the positive areas of cartilage matrix proteins ACAN and COLII were significantly increased (the number of fluorescent stainings was significantly increased and the color became darker), which indicates that sc79 can inhibit the degradation of cartilage matrix proteins ACAN and COLII caused by joint bleeding, and activating the Akt pathway can reduce the cartilage matrix damage caused by joint bleeding.

[0106] from Figure 4 As can be seen from D, compared with the control group, in the Blood group, the expressions of cartilage matrix proteins ACAN and COLII decreased significantly, which was significantly different from the control group; while in the Blood+sc79 group, compared with the Blood group, the expressions of COLII and ACAN proteins increased significantly, especially the expression of ACAN protein, which was significantly different from the Blood group. This further indicates that after sc79 activates the Akt pathway, it can significantly reduce the degradation of cartilage matrix proteins ACAN and COLII caused by joint bleeding.

[0107] from Figure 4 As can be seen from E, compared with the control group, in the Blood group, the expression of proinflammatory factors IL4, IL6, and IL13 in the peripheral blood were significantly increased; while in the Blood+sc79 group, compared with the Blood group, the expression of IL4, IL6, and IL13 was significantly decreased, which indicates that after the joint cavity injection of sc79 activates the Akt pathway, it can significantly reduce the expression of proinflammatory factors IL4, IL6, and IL13 in the peripheral blood caused by joint bleeding, thereby alleviating or treating peripheral blood inflammation.

[0108] from Figure 4 As can be seen from F, compared with the control group, in the Blood group, the expressions of inflammatory factors IL1b, IL6, and TNFa in the liver were significantly increased, which was significantly different from the control group; while in the Blood+sc79 group, compared with the Blood group, the expressions of IL1b, IL6, and TNFa were significantly decreased, which indicates that after intra-articular injection of sc79 activates the Akt pathway, the expression of inflammatory factors IL1b, IL6, and TNFa in the liver caused by joint bleeding can be significantly reduced, thereby alleviating and treating liver inflammation.

[0109] from Figure 4As can be seen in G, compared with the control group, in the Blood group, the infiltration of immune cells (such as CD4+T cells) was significantly enhanced, and the color around the cells was significantly darker; while in the Blood+sc79 group, compared with the Blood group, the infiltration of immune cells (such as CD4+T cells) was significantly reduced, and the color around the cells was significantly lighter, close to that of the control group; this indicates that after sc79 activates the Akt pathway, it can significantly reduce the infiltration of hepatic inflammatory cells caused by joint bleeding.

[0110] from Figure 4 As can be seen from H, compared with the control group, in the Blood group, the serum liver function indicators AST and ALT increased significantly; while in the Blood+sc79 group, compared with the Blood group, the serum liver function indicators AST and ALT decreased significantly, and there was no significant difference compared with the control group. This indicates that after sc79 activates the Akt pathway, it can significantly reduce the increase of serum liver function indicators AST and ALT caused by joint bleeding, thereby alleviating and treating liver damage.

[0111] It can be seen from this that sc79 can inhibit cartilage damage, systemic reactions, liver damage and peripheral blood inflammation caused by joint bleeding, and activating the Akt pathway can reduce systemic peripheral blood inflammatory reactions and liver damage caused by joint bleeding.

[0112] Example 4: Modeling and drug administration of pigs with knee joint bleeding and testing of the therapeutic effect of Akt pathway activator sc79 on pig liver damage and peripheral blood inflammation induced by joint bleeding

[0113] 1. Pig model of knee joint bleeding

[0114] The knee joint bleeding pig model was established by injecting blood (2 ml / leg) into the knee joint cavity of Yorkshire pigs (male, 6 months old) 8 times, twice a week. The Yorkshire pigs were killed 1 week after the last injection.

[0115] 2. Drug administration

[0116] Based on the pig model of knee joint bleeding, 25 μg / ml sc79 (S7863, Selleck, USA) was added to the blood, and the surgical procedure was the same as that of the pig model of knee joint bleeding. Yorkshire pigs were killed one week after the last injection.

[0117] 3. Peripheral blood sample processing and testing

[0118] Peripheral blood sampling: Blood was collected from the jugular vein of the pigs and stored in disposable blood collection tubes; liver function was evaluated by peripheral blood biochemical analysis.

[0119] The specific experimental results are as follows Figure 5A As shown, Figure 5AThis is a graph showing the therapeutic effect of Akt pathway activator sc79 on liver damage induced by joint bleeding in pigs. Among them, Control refers to pigs that were raised normally and not treated in any way, which is the control group; Blood refers to pigs in the experimental group that were injected with blood (2 ml / leg) 8 times, twice a week, into the knee joint cavity and were killed 1 week after the last injection; Blood+sc79 refers to pigs in the experimental group that were injected with 25 μg / ml of sc79 in addition to Blood.

[0120] from Figure 5A It can be seen that: 1) Compared with the control group, in the Blood group, serum liver function indicators AST, ALT, and GGT all increased significantly, while TP, ALP, and A / G all decreased significantly, which was significantly different from the control group; in the Blood+sc79 group, compared with the Blood group, serum liver function indicators AST, ALT, and GGT decreased significantly, while TP, ALP, and A / G increased significantly, and the index content was close to the control group, with no significant difference. This shows that after sc79 activates the Akt pathway, it can significantly reduce the increase of serum liver function indicators AST, ALT, and GGT caused by joint bleeding, and increase the decrease of serum liver function indicators TP, ALP, and A / G caused by joint bleeding, thereby alleviating and treating liver damage. This further shows that sc79 can be used as a potential therapeutic target for liver function damage caused by joint bleeding.

[0121] Example 5: Test on the effect of Akt pathway activator sc79 on reducing chondrocyte apoptosis caused by joint bleeding

[0122] To further explore the role of Akt pathway activator sc79 in the treatment of chondrocyte apoptosis caused by joint bleeding, in this example, the following operations were performed: mouse chondrocytes were seeded in the lower chamber of a 24-well transwell plate, and when the degree of confluence reached about 70%, 5% mouse whole blood, an equal volume of mouse whole blood containing 5μg / ml sc79, or an equal volume of PBS were added to the upper chamber of the transwell. Among them, the group with an equal volume of PBS was the control group (Control), the group with 5% mouse whole blood was the experimental group (Blood), and the group with an equal volume of mouse whole blood containing 5μg / ml sc79 was the experimental group (Blood+sc79).

[0123] Three days later, the cells were fixed with paraformaldehyde for immunofluorescence experiments, and the cells were incubated with the primary antibody corresponding to C-caspase-3, the corresponding fluorescent secondary antibody and DAPI, and then photographed with a fluorescence microscope (Olympus, Japan).

[0124] At the same time, the cells were lysed with trizol for qPCR experiments. For qPCR experiments, the RNA of the cells was first extracted and then reverse transcribed into cDNA, and the Ct value of the specific cDNA amplification process was determined using SYBR dye.

[0125] The specific experimental results are as follows Figure 6 A and Figure 6 As shown in B. Figure 6 A is the immunofluorescence image of the apoptosis marker protein cleaved caspase-3 (left) and the quantitative result of immunofluorescence cleaved caspase-3 protein (right). Figure 6 B is the mRNA expression diagram of the pro-survival protein Bcl-2 and the pro-apoptotic protein Bax.

[0126] from Figure 6 As shown in A, compared with the control group, in the Blood group, after blood treatment, the positive area of ​​the apoptosis marker protein cleavedcaspase-3 (C-caspase-3) increased significantly (the number of fluorescent staining increased significantly and the color became darker) (left), and the content of C-caspase-3 increased significantly (right); while in the Blood+sc group, compared with the Blood group, the positive area of ​​C-caspase-3 decreased significantly (the number of fluorescent staining decreased significantly and the color became lighter) (left), and the content of C-caspase-3 decreased significantly (right), and the content was close to that of the control group, indicating that after sc79 activated the Akt pathway, it could significantly inhibit the expression of the apoptosis marker protein C-caspase-3, thereby significantly inhibiting the apoptosis of chondrocytes caused by joint bleeding (DAPI is the result of cell nucleus staining, Merge is the result of two fluorescence superpositions of cell nucleus staining and apoptosis marker protein C-caspase-3 staining, which serves as a comparative reference for the staining results of apoptosis marker protein C-caspase-3).

[0127] from Figure 6 As shown in B, compared with the control group, in the Blood group, the mRNA content of the pro-survival protein Bcl-2 decreased significantly, while the mRNA expression of the pro-apoptotic protein Bax increased significantly, both of which were significantly different from the control group; in the Blood+sc group, compared with the Blood group, the mRNA content of the pro-survival protein Bcl-2 increased significantly, while the mRNA expression of the pro-apoptotic protein Bax also decreased significantly, indicating that after sc79 activated the Akt pathway, it could significantly increase the mRNA expression of the pro-survival protein Bcl-2, while inhibiting the mRNA expression of the pro-apoptotic protein Bax, thereby inhibiting chondrocyte apoptosis caused by joint bleeding.

[0128] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. Use of an Akt pathway activator for preparing a reagent for preventing or treating joint bleeding, It is characterized in that The joint bleeding includes joint bleeding associated with traumatic joint injury, hemophilic arthritis, and joint surgery.

2. The use according to claim 1, It is characterized in that The invention can also be used for preparing agents for preventing or treating cartilage damage, systemic reactions and liver complications caused by joint bleeding.

3. The use according to claim 2, It is characterized in that The Akt pathway activator includes a substance that upregulates the expression of phosphorylated Akt protein.

4. The use according to claim 3, It is characterized in that The substance that can upregulate the expression of phosphorylated Akt protein includes any one or more of the CRISPR / Cas9 gene editing system targeting the Akt upstream pathway and its key proteins, RNA knockdown and overexpression and its delivery system, and small molecules.

5. The use according to claim 4, It is characterized in that The small molecules include SC79 and / or vitamin E.

6. The use according to claim 5, It is characterized in that The substance prevents or treats cartilage damage, systemic reactions and liver complications caused by joint bleeding by reducing cartilage matrix degradation; the cartilage matrix includes COLII and ACAN.

7. Use of an Akt pathway activator for preparing an agent for inhibiting apoptosis of cartilage tissue and cells or inhibiting the expression of extracellular matrix degrading enzymes, It is characterized in that The Akt pathway activator includes a substance that upregulates the expression of phosphorylated Akt protein.

8. The use according to claim 7, It is characterized in that The “inhibition of apoptosis” includes inhibiting the expression of Cleaved caspase-3, increasing the expression of Bcl-2 protein and promoting cell proliferation; the “extracellular matrix degrading enzyme” includes MMP-3 and MMP-13.

9. Use of an Akt pathway activator for preparing a reagent for preventing or treating peripheral blood inflammation caused by joint bleeding, It is characterized in that The Akt pathway activator prevents or treats peripheral blood inflammation caused by joint bleeding by inhibiting the expression of inflammatory factors in peripheral blood.

10. The use according to claim 9, It is characterized in that The pro-inflammatory factors in the peripheral blood include IL4, IL6, and IL13.

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