Application of perfluorooctane sulfonic acid in construction of early-stage and middle-stage osteoarthritis model

Abstract

The invention relates to the technical field of model construction, in particular to application of perfluorooctane sulfonic acid in construction of an early and middle-stage osteoarthritis model. The invention relates to an application of perfluorooctane sulfonic acid in construction of an early-and-middle-stage osteoarthritis model. According to the application, the perfluorooctane sulfonic acid solution is placed in a knee joint cavity of an animal once a week for continuous 8 weeks. When the animal is a mouse, the concentration of the perfluorooctane sulfonic acid in the solution is 500 [mu] mol / L, and the usage amount of the perfluorooctane sulfonic acid solution each time is 10 [mu] L. A perfluorooctane sulfonate solution is injected into a knee joint cavity of a mouse to successfully construct the mouse early-and-middle-stage osteoarthritis model, including partial degradation of cartilage matrix, surface cartilage injury, inflammatory response of synovial tissue and the like. The model can be used for better researching the pathogenic mechanism, inflammation development process and joint pathological change of the early and middle-stage knee osteoarthritis and researching the treatment strategy for the early and middle-stage knee osteoarthritis.

Description

Technical Field

[0001] The invention relates to the technical field of model construction, and in particular to the application of perfluorooctane sulfonic acid in constructing an early and middle stage osteoarthritis model. Background Art

[0002] The main pathological manifestations of osteoarthritis are cartilage destruction, osteophyte formation, subchondral bone sclerosis, synovial thickening, and structural changes in the ligaments and surrounding muscles around the joints. Since its etiology and etiology are still unclear, it is difficult to recover due to the lack of effective clinical diagnosis and treatment, so it is necessary to establish an osteoarthritis model.

[0003] Animal experimental research is an important way to explore the pathogenesis and treatment mechanism of knee osteoarthritis. However, different research methods have their own advantages and disadvantages in the model making. Existing osteoarthritis modeling methods include invasive induction methods, such as the Hulth method, anterior cruciate ligament transection or meniscectomy. The model surgery is traumatic, with the risk of postoperative infection, and often forms cartilage terminal pathological changes, that is, late osteoarthritis. Non-invasive induction methods, such as joint immobilization, have a narrow range of model applications. Hereditary osteoarthritis phenotypic modeling methods, such as knocking out Col11a1, Col2a1 genes, or Del1, type II, type IX collagen gene mutations, or cartilage matrix gene defects. However, the success rate of transgenic animals is low, the theory and technology are not yet perfect, and the transgene is only expressed in specific cells. Since cartilage tissue is a non-regenerative tissue, patients with late-stage osteoarthritis generally undergo surgical treatment, such as total knee replacement, which increases the economic burden on families and society. There is currently a lack of targeted treatment strategies for patients with early and middle-stage osteoarthritis. They can only take symptomatic treatments such as anti-inflammatory and analgesic treatments, which cannot effectively cure early and middle-stage osteoarthritis.

[0004] Therefore, the development of new non-invasive early and middle-stage osteoarthritis models will help scholars better study the pathological changes of early and middle-stage osteoarthritis and develop corresponding treatment strategies. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of perfluorooctane sulfonic acid in constructing an early and middle stage osteoarthritis model. The present invention successfully constructs an early and middle stage osteoarthritis model in mice by injecting perfluorooctane sulfonic acid (PFOS for short) into the knee joint cavity of mice, including partial degradation of cartilage matrix, surface cartilage damage and inflammatory response of synovial tissue. This model can better study the pathogenic mechanism, inflammatory development process, and pathological changes of joints in early and middle stage knee osteoarthritis, as well as explore treatment strategies for early and middle stage osteoarthritis. Based on this, the present invention proposes a new method for constructing an early and middle stage knee osteoarthritis animal model, a method for constructing an early and middle stage osteoarthritis model based on the injection of perfluorooctane sulfonic acid into the mouse joint cavity.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides the use of perfluorooctane sulfonic acid in constructing an early and middle stage osteoarthritis model.

[0008] Preferably, the application comprises the following steps:

[0009] Perfluorooctane sulfonate solution was placed in the knee joint cavity of the animals once a week for 8 consecutive weeks.

[0010] Preferably, when the animal is a mouse, the concentration of perfluorooctane sulfonic acid in the perfluorooctane sulfonic acid solution is 500 μmol / L, and the amount of the perfluorooctane sulfonic acid solution used each time is 10 μL.

[0011] Preferably, the perfluorooctane sulfonic acid solution further contains Tween 80, and the mass percentage of Tween 80 in the perfluorooctane sulfonic acid solution is 2%.

[0012] The present invention also provides the use of perfluorooctane sulfonic acid in preparing a reagent for promoting the expression of inflammation in human knee joint synovial cells.

[0013] Preferably, the perfluorooctane sulfonic acid promotes the secretion of inflammatory factors in synovial cells.

[0014] Preferably, the inflammatory factors include one or more of IL-1β, IL-6, TNF-α, CXCL2, CXCL3 and CXCL8.

[0015] The invention also provides the use of perfluorooctane sulfonic acid in preparing a reagent for promoting secretion of degradation enzymes in human knee joint chondrocytes.

[0016] Preferably, the human knee chondrocyte degrading enzyme includes one or more of MMP9 protease, MMP13 protease and ADAMTS5 protease.

[0017] The present invention also provides the use of perfluorooctane sulfonic acid in preparing a reagent for inducing mild to moderate synovial inflammation and / or cartilage damage of the knee joint.

[0018] Beneficial effects of the present invention:

[0019] The purpose of the present invention is to provide a simple injection-induced knee osteoarthritis model, to better construct a knee osteoarthritis model caused by environmental organic compound damage that is similar to the clinical pathogenesis, to study the pathogenic mechanism, inflammatory development process, and pathological changes in the joints of knee osteoarthritis caused by environmental organic compound damage, and to propose a better treatment plan.

[0020] The present invention successfully established an early and middle stage osteoarthritis model in mice by injecting PFOS into the knee joint cavity of mice, including partial degradation of cartilage matrix, damage to surface cartilage, and inflammatory response of synovial tissue, etc. This model can better study the pathogenic mechanism, inflammatory development process, and pathological changes of joints in early and middle stage knee osteoarthritis, as well as explore treatment strategies for early and middle stage osteoarthritis.

[0021] The invention is rapid, minimally invasive, has a high success rate, strong repeatability, low cost, simple operation, and little stress on animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 The mRNA expression level of inflammatory factors in human synovial cells after PFOS treatment of the present invention;

[0024] Figure 2 The expression level of matrix degrading enzyme mRNA in human chondrocytes after PFOS treatment of the present invention;

[0025] Figure 3 The results of safranin fast green staining and hematoxylin-eosin staining of the knee joints of mice of the present invention and the Mankin and Synovitis scoring results;

[0026] Figure 4 The protein expression of iNOS and MMP13 in the synovial tissue of the knee joint of the mice of the present invention;

[0027] Figure 5 The protein expression of iNOS and MMP13 in the knee cartilage tissue of the mice of the present invention;

[0028] Figure 6 This is an evaluation of the toxic and side effects of PFOS in the present invention. DETAILED DESCRIPTION

[0029] The present invention provides the use of perfluorooctane sulfonic acid in constructing an early and middle stage osteoarthritis model. In the present invention, the use preferably comprises the following steps: placing a perfluorooctane sulfonic acid solution in the knee joint cavity of an animal once a week for 8 consecutive weeks.

[0030] In the present invention, when the animal is preferably a mouse, the concentration of perfluorooctane sulfonic acid in the perfluorooctane sulfonic acid solution is preferably 500 μmol / L, and the amount of the perfluorooctane sulfonic acid solution used each time is preferably 10 μL.

[0031] In the present invention, the perfluorooctane sulfonic acid solution preferably further contains Tween 80, and the mass percentage of Tween 80 in the perfluorooctane sulfonic acid solution is preferably 2%. In the present invention, the Tween 80 functions as a solubility aid.

[0032] The present invention does not specifically limit the method of placing the perfluorooctane sulfonic acid solution in the animal knee joint cavity, and those skilled in the art can use conventional methods, such as injection. The present invention preferably punctures the knee joint cavity from the lateral source of the patellar ligament to inject the perfluorooctane sulfonic acid solution into the animal knee joint cavity.

[0033] The present invention also provides the use of perfluorooctane sulfonic acid in the preparation of a reagent for promoting the expression of inflammation in human knee joint synovial cells. In the present invention, the perfluorooctane sulfonic acid preferably promotes the secretion of inflammatory factors in synovial cells. In the present invention, the inflammatory factors preferably include one or more of IL-1β, IL-6, TNF-α, CXCL2, CXCL3 and CXCL8.

[0034] The present invention also provides the use of perfluorooctane sulfonic acid in the preparation of a reagent for promoting secretion of a degrading enzyme in human knee chondrocytes. In the present invention, the degrading enzyme in human knee chondrocytes preferably includes one or more of MMP9 protease, MMP13 protease and ADAMTS5 protease.

[0035] The present invention also provides the use of perfluorooctane sulfonic acid in preparing a reagent for inducing mild to moderate synovial inflammation and / or cartilage damage of the knee joint.

[0036] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] PFOS promotes inflammatory expression in human knee synovial cells

[0039] 1. Test materials:

[0040] Human synoviocytes were extracted by two-step enzymatic digestion.

[0041] 2. Test methods:

[0042] After surgical resection of the synovium, the synovium was immediately placed in whole culture for mechanical and enzymatic digestion to obtain a single-cell suspension. Most of the fat tissue was removed with ophthalmic scissors, and the synovial tissue was minced under sterile conditions. The synovial tissue was digested with 0.25% trypsin containing EDTA (Invitrogen) for 30 min and 300 U / mL type I collagenase (Worthington Biochemical) at 37°C for 30–45 min. All digestion solutions were filtered through a filter with a pore size of 70 μm (Fisher Scientific). The filtrate was centrifuged at 300 g for 5 min, the supernatant was removed, and the cells were resuspended in DMEM complete medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin. The cells were seeded in 6-well plates. When the cells grew to P3, a PFOS solution with a concentration of 200 μmol / L was added, and an equal amount of DMSO solution was added to the control group. After treatment for 3 h, fluorescent quantitative PCR was performed to evaluate the expression of inflammatory factors.

[0043] The specific steps of fluorescence quantitative PCR detection are as follows:

[0044] (1) Total RNA extraction

[0045] After PFOS treatment, the 6-well plate was collected, the culture medium was discarded, 1 mL PBS was added to each well for washing once, PBS was discarded, 1 mL Trizol was added to each well, and it was allowed to stand for 5 min. After repeated mixing with a 1 mL pipette tip, it was transferred to a 1.5 mL RNase-free EP tube. 200 μL chloroform (trichloromethane) was added to each centrifuge tube and shaken up and down for 15 min. – 30s, let stand for 5min, and centrifuge at 12000rpm for 15min at 4℃. After centrifugation, take out the centrifuge tube, aspirate 400μL of the upper transparent aqueous phase into a new 1.5mL RNase-free EP tube, add 500μL of isopropanol to each centrifuge tube, shake up and down for 15s, let stand for 10min, and centrifuge at 12000rpm for 10min at 4℃. After centrifugation, take out the centrifuge tube, discard the supernatant, add 1mL of pre-cooled 75% anhydrous ethanol to each centrifuge tube, and centrifuge at 7500rpm for 5min at 4℃. After centrifugation, put the centrifuge tube in an ice box, discard the supernatant, centrifuge again at 7500rpm for 1min at 4℃, discard the residual supernatant, and dry for 10min. Add appropriate amount (20 – Dissolve RNA in 40 μL RNase-free ultrapure water and mix well before measuring RNA concentration.

[0046] (2) Reverse transcription to synthesize cDNA

[0047] The sample volume was calculated with 1 μg of total RNA as the template, and the reverse transcription reaction system was prepared according to the reverse transcriptase instructions, with a total volume of 20 μL. The reverse transcription conditions were: 5 min at 25°C, 30 min at 42°C, 5 min at 85°C, and maintained at 4°C.

[0048] (3) Real-time quantitative fluorescence PCR

[0049] The reaction system is as follows:

[0050] Table 1 Reaction system

[0051] Reagents Volume (μL) SYBRGREEN 10 ForwardPrimer 4 ReversePrimer 4 cDNA 2

[0052] Set the loop conditions as follows:

[0053] Table 2 Cycle conditions

[0054] 95℃ 2min 95℃ 15s 58℃ 15s 72℃ 1min 40 cycles

[0055] (4) Primer information

[0056] Table 3 Primer information

[0057]

[0058]

[0059] 3. Test results:

[0060] Figure 1 The results showed that after 3 hours of PFOS treatment, the secretion of inflammatory factors such as IL-1β, IL-6, TNF-α, CXCL2, CXCL3, and CXCL8 in synovial cells increased significantly (P<0.05), indicating that PFOS can promote the inflammatory expression of synovial cells in human knee joints.

[0061] Example 2

[0062] PFOS promotes the secretion of degradative enzymes in human knee chondrocytes

[0063] 1. Test materials:

[0064] Human chondrocytes were extracted by two-step enzymatic digestion.

[0065] 2. Test methods:

[0066] Cartilage was obtained after knee replacement surgery. The cartilage tissue was cut into small pieces under sterile conditions and washed in pre-cooled PBS. The cartilage tissue was digested with 0.25% trypsin containing EDTA (Invitrogen) for 30 minutes and digested with 600-900U / mL type I collagenase (Worthington Biochemical) at 37°C for 2-3 hours. All digestion solutions were filtered through a filter with a pore size of 70 μm (Fisher Scientific). The filtrate was centrifuged at 300g for 5 minutes, the supernatant was removed, and the cells were resuspended in DMEM complete medium containing 10% fetal bovine serum, 100μg / mL streptomycin, and 100U / mL penicillin. The cells were seeded in 6-well plates. When the cells grew to P3, a 200μmol / L PFOS solution was added. The same amount of DMSO solution was added to the control group. After treatment for 3 hours, fluorescent quantitative PCR was performed to evaluate the expression of matrix-degrading proteases.

[0067] The fluorescent quantitative PCR detection steps are the same as those in Example 1, and the primer sequences of the chondrocyte matrix degradation protease-related genes are as follows:

[0068] Table 4 Primer information

[0069]

[0070] 3. Test results:

[0071] Figure 2 The results showed that PFOS treatment for 3 h significantly increased the expression of chondrocyte catabolic proteases, such as MMP9, MMP13, and ADAMTS5 (P<0.05), indicating that PFOS can promote the secretion of catabolic proteases related to matrix degradation.

[0072] Example 3

[0073] PFOS induces early and middle stage synovial inflammation and cartilage damage in the knee joints of mice

[0074] 1. Test materials:

[0075] 10-week-old male C57BL / 6 mice.

[0076] 2. Test methods:

[0077] The 10-week-old mice were divided into two groups: the PFOS group, i.e. the experimental group (PFOS injection OA model construction group) and the control group. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and the body weight was weighed and recorded. Sterile towels were laid on the operating table, the mice were fixed, the right knee joint was fully exposed, the hair around the right knee joint was shaved, and the right knee joint was disinfected 3 times with 75% alcohol. The area above the patellar ligament was found, and a white structure was observed through the skin in front of the mouse knee joint, which is the patellar ligament. Under microscope observation, the corresponding reagents were extracted with a 33G microinjection needle (Hamilton), and the dose was 10 μL. The concentration of PFOS in the experimental group was 500 μmol / L, and the mass percentage of Tween 80 was 2%. The experimental group mice were injected with PFOS+PBS+2% Tween80 (8W, 1 time / W) and the control group was injected with PBS+2% Tween80 (8W, 1 time / W). The mass percentage of Tween 80 in the reagent in the control group was 2%; the pH value of PBS was 7.2. Puncture into the knee joint cavity from the lateral edge of the patellar ligament and inject the reagent. After the injection, the mice were placed in a cage and the vital signs were observed until the anesthesia was awakened. Until the 8th week, the right knee joints of the mice were collected for paraffin-embedded sections and stained.

[0078] 3. Test results:

[0079] Figure 3 The results of A and B showed that the articular cartilage structure, chondrocytes and cartilage tissue staining were normal in the control group; the surface cartilage was damaged, the cells were moderately damaged, the cartilage tissue staining was moderately reduced, the content of proteoglycan in the cartilage matrix was reduced, and the Mankin pathological score was significantly increased (P < 0.05) ( Figure 3 (middle D). Figure 3 The results of hematoxylin-eosin staining in C showed that the mice in the PFOS model group showed mild synovitis, with obvious thickening of the synovium, an increase in the number of synovial cells, and a significant increase in the Synovitis synovitis score (P < 0.05) ( Figure 3 Furthermore, immunofluorescence staining of iNOS and MMP13 was performed, and the results showed that PFOS injection caused a significant increase in the expression of iNOS and MMP13 in the synovium and cartilage tissue of the mouse knee joint ( Figure 4 and 5 ), indicating that PFOS injection can cause mild to moderate synovial inflammation and cartilage damage in the knee joints of mice, which is consistent with the phenotype of early and middle-stage osteoarthritis. PFOS knee joint injection may become an effective new method for modeling early and middle-stage osteoarthritis.

[0080] Example 4

[0081] PFOS toxicity and side effects assessment

[0082] 1. Test materials:

[0083] 10-week-old male C57BL / 6 mice.

[0084] 2. Test methods:

[0085] The 10-week-old mice were divided into two groups: the PFOS group, i.e. the experimental group (PFOS injection OA model construction group) and the control group. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and the body weight was weighed and recorded. Sterile towels were laid on the operating table, the mice were fixed, the right knee joint was fully exposed, the hair around the right knee joint was shaved, and the right knee joint was disinfected 3 times with 75% alcohol. The area above the patellar ligament was found, and a white structure was observed through the skin in front of the mouse knee joint, which is the patellar ligament. Under microscope observation, the corresponding reagent was extracted with a 33G microinjection needle (Hamilton), and the dose was 10 μL. The concentration of PFOS in the reagent in the experimental group was 500 μmol, and the mass percentage of Tween 80 was 2%. The experimental group mice were injected with PFOS + PBS + 2% Tween80 (8W, 1 time / W) and the control group was injected with PBS + 2% Tween80 (8W, 1 time / W). The mass percentage of Tween 80 in the reagent in the control group was 2%; the pH value of PBS was 7.2. Puncture into the knee joint cavity from the lateral edge of the patellar ligament and inject the reagent. After the injection, the mice were placed in a mouse cage and the vital signs were observed until the anesthesia awakened. Until the 8th week, the periocular blood of the mice was collected for blood routine and biochemical index detection, and the paraffin-embedded sections of the heart, liver, spleen, lung and kidney tissues were collected for staining.

[0086] 3. Test results:

[0087] like Figure 6 Further analysis of the toxicity of PFOS showed that hematoxylin-eosin staining of the heart, liver, spleen, lungs and kidneys showed no obvious inflammatory infiltration or damage, and the main biochemical (liver function and kidney function, etc.) and blood routine results were no different among the groups.

[0088] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of perfluorooctane sulfonate in constructing early and middle stage osteoarthritis models.

2. The use according to claim 1, characterized in that: The application comprises the following steps: Perfluorooctane sulfonate solution was placed in the knee joint cavity of the animals once a week for 8 consecutive weeks.

3. The use according to claim 2, characterized in that: When the animal is a mouse, the concentration of perfluorooctane sulfonic acid in the perfluorooctane sulfonic acid solution is 500 μmol / L, and the amount of the perfluorooctane sulfonic acid solution used each time is 10 μL.

4. The use according to claim 3, characterized in that: The perfluorooctane sulfonic acid solution also contains Tween 80, and the mass percentage of Tween 80 in the perfluorooctane sulfonic acid solution is 2%.

5. Application of perfluorooctane sulfonic acid in the preparation of reagents that promote inflammatory expression in human knee synovial cells.

6. The use according to claim 5, characterized in that: The perfluorooctane sulfonic acid promotes the secretion of inflammatory factors in synovial cells.

7. The use according to claim 6, characterized in that: The inflammatory factors include one or more of IL-1β, IL-6, TNF-α, CXCL2, CXCL3 and CXCL8.

8. Application of perfluorooctane sulfonic acid in the preparation of reagents for promoting the secretion of degradative enzymes in human knee chondrocytes.

9. The use according to claim 8, characterized in that: The human knee joint chondrocyte degrading enzyme includes one or more of MMP9 protease, MMP13 protease and ADAMTS5 protease.

10. Use of perfluorooctane sulfonic acid in the preparation of reagents for inducing mild to moderate synovial inflammation and / or cartilage damage in the knee joint.

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