CN116999541A - Antioxidant nano-enzyme with gelatinase inhibition effect, and preparation method and application thereof - Google Patents

Antioxidant nano-enzyme with gelatinase inhibition effect, and preparation method and application thereof Download PDF

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CN116999541A
CN116999541A CN202310475695.8A CN202310475695A CN116999541A CN 116999541 A CN116999541 A CN 116999541A CN 202310475695 A CN202310475695 A CN 202310475695A CN 116999541 A CN116999541 A CN 116999541A
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sazyme
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白倩
姜伟
刘影
李治松
张瑜
谢滢滢
付红光
冯瑞霞
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Second Affiliated Hospital of Zhengzhou Unviersity
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Abstract

The application belongs to the field of biological pharmacy, relates to a novel medicine with a gelatinase inhibition effect, and in particular relates to an antioxidant nano-enzyme with the gelatinase inhibition effect, and a preparation method and application thereof. The novel brain targeting nano-drug copper monoatomic nano-enzyme (Rvg-Cu SAzyme) can effectively eliminate the accumulation of active oxygen, inhibit the activation of ROS and MMP-2/9 in the trigeminal ganglion of temporomandibular joint inflammation, and relieve the hypersensitivity of oral and facial pain. Compared with natural enzymes, the nano-enzyme has lower development cost, stronger stability and excellent recycling property, can catalyze some non-naturally occurring biological processes, and has been applied to the fields of disease diagnosis and treatment, biosensing, environmental management, antibiosis, antifouling and the like.

Description

Antioxidant nano-enzyme with gelatinase inhibition effect, and preparation method and application thereof
Technical Field
The application belongs to the field of biological pharmacy, relates to a novel medicine with a gelatinase inhibition effect, and in particular relates to an antioxidant nano-enzyme with the gelatinase inhibition effect, and a preparation method and application thereof.
Background
Temporomandibular arthritis (TMJ) is a common chronic degenerative disease, often manifested as persistent dull pain, and patients are often accompanied by temporomandibular joint pain and limited and asymmetric mandibular movement. According to data, the influence of temporomandibular joint disorder diseases on more than 10-15% of adults is shown, patients have obvious pathological changes such as osteoarthritis, and are not accompanied by psychological diseases such as anxiety, depression and the like, so that the life of the patients is greatly influenced, and how to effectively relieve temporomandibular arthritic pain becomes a key for treatment. After temporomandibular arthritis occurs, a variety of cytokines and pain-related factors are involved in this inflammatory response and hyperalgesia process, including Reactive Oxygen Species (ROS) and Matrix Metalloproteinases (MMPs). MMPs are a series of zinc-dependent endopeptidases that degrade all major protein components of the extracellular matrix, playing a vital role in inflammation-related tissue remodeling and repair. MMP-2 and MMP-9 belong to the gelatinases, MMP-2 being expressed structurally in many tissues, but MMPP-9 is highly induced, both of which are involved in the induction and sensitization of pain, and are also the most commonly expressed matrix metalloproteinases in the nervous system.
The trigeminal nerve is widely distributed on the head and neck, has sensory and motor functions, and the trigeminal ganglion participates in the reaction of oral and facial inflammation and nerve injury, and the specific mechanism is tentatively unknown.
Superoxide dismutase (SOD) is used as a core component of an intracellular antioxidant system, and has important application value in biomedicine. However, the natural SOD enzyme has unstable structure, is difficult to prepare and preserve in large quantity, and has very limited application range. The nano-enzyme is a novel artificial mimic enzyme with inherent multi-enzyme activity. The nano-enzyme is expected to overcome the limitation of the natural enzyme due to the characteristics of high stability, easy preparation, easy storage, low cost, easy regulation and the like, and can replace the natural enzyme in various application fields. The nano enzymes have broad application prospects in the aspect of oxidative stress related diseases by combining the structural stability, the preparation simplicity and the high enzyme catalysis efficiency of nano materials, and the research on the aspects of relieving pain is still an important challenge. The project monatomic copper nano enzyme takes a metal organic framework as a carrier, and utilizes copper acetylacetonate as Cu and N sources to construct the Cu-N of the metal organic framework loaded Cu monatomic nano enzyme 4 The composition has excellent CAT and SOD-like activities, and simultaneously has the effect of inhibiting the activation of ROS and MMP-2/9 in the trigeminal ganglion for treating the pain of temporomandibular arthritis, which is a difficulty in treating temporomandibular arthritis by using the copper nanoenzyme.
Disclosure of Invention
In order to effectively relieve temporomandibular arthritic pain, the application provides an antioxidant nano-enzyme with a gelatinase inhibition effect, and a preparation method and application thereof.
The technical scheme of the application is realized as follows:
a preparation method of antioxidant nano-enzyme with gelatinase inhibition effect comprises the following steps:
(1) Preparing a methanol solution of 2-methylimidazole as solution A, in which Zn (NO 3 ) 2 And Cu (acac) 2 Adding the solution B into the solution A, performing high-pressure heating reaction in a Teflon-lined stainless steel pressure cooker of 100 mL, centrifugally separating the reaction solution, washing the reaction solution with DMF three times, washing the reaction solution with methanol twice, and finally drying the reaction solution in vacuum overnight to obtain Cu SAzyme; in the preparation process, the solution B needs ultrasonic stirring for 15min to form transparent solutionThe solution is then stirred vigorously at room temperature for 1 hour;
(2) Dissolving DSPE-PEG-COOH in chloroform-methanol mixed solvent, rotary evaporating, drying, adding Cu SAzyme aqueous solution, performing hydration reaction on lipid membrane formed by rotary evaporation, and performing ultrasonic treatment to obtain nano enzyme Rvg-Cu SAzyme.
The concentration of 2-methylimidazole in the solution A in the step (1) is 10-100 mg/mL, and Zn (NO) in the solution B 3 ) 2 Is 10-100 mg/mL, cu (acac) 2 The concentration of (C) is 10-50 mg/mL.
The volume ratio of the solution A to the solution B is 1:2-4.
The high-pressure heating reaction is carried out to obtain the temperature of 90-120 ℃ and the time of 2-4 h; the temperature of vacuum drying is 60-90 ℃.
The volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 2:1.
The concentration of the Cu SAzyme aqueous solution is 1-5 mg/mL, and the volume is 2 mL; the ultrasonic treatment time is 5-15 min.
The antioxidant nano-enzyme prepared by the method.
The application of the antioxidant nano-enzyme in preparing antioxidant medicines.
The application of the antioxidant nano-enzyme in preparing medicaments for inhibiting the activation of ROS and MMP2/9 in the trigeminal ganglion of temporomandibular arthritis.
The antioxidant nano-enzyme has the function of preparing medicines for relieving temporomandibular arthritic pain.
The application has the following beneficial effects:
1. the novel brain-targeted liposome packaging monoatomic nano-enzyme Rvg-Cu SAzyme not only provides a novel method for in-situ synthesis synergistic nano-therapy, but also discloses an application mechanism in resisting oxidative stress induced temporomandibular arthritic pain.
2. The application provides a novel nano-drug with a function of relieving temporomandibular arthritis pain, which is characterized in that pain hypersensitivity occurs on the 1 st day after CFA induced temporomandibular arthritis operation, the peak value is reached on the 3 rd day after operation, and the pain hypersensitivity is maintained to the 10 th day after operation. Systemic nano-administration eliminates the accumulation of active oxygen, inhibits the activation of MMP-2 and MMP-9 in TMJ mice, and reduces pain hypersensitivity. The use of nano-scale within a suitable time window not only delays the progression of joint inflammation, but also produces an effective analgesic effect. In summary, the present study used brain targeting peptide modified copper monoatomic nanoezymes (Rvg-Cu SAzyme),
3. the Rvg-Cu SAzyme has the particle size smaller than 200 nm, is beneficial to the nano enzyme assembly to smoothly reach the brain through the blood brain barrier, plays the role of antioxidant enzyme, and adjusts the oxidative stress level.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of an animal experiment.
FIG. 2 is a graph showing changes in ROS in TG and SP5C when TMJ mice develop pain hypersensitivity and joint inflammation is evident; wherein A, B, C, D: the TMJ mice were prompted for 3 days post-operative oral facial mechanical pain and facial pain scores for 3 days post-operative pain hypersensitivity. E: TMJ mice have rugged temporomandibular joint cartilage surface, reduced chondrocyte numbers, and inflammatory cell infiltration. F. H. increased ROS (8-OhdG as a marker of oxygen radical) expression in TMJ mouse TG. G. E: ROS expression was increased in TMJ mouse SP 5C.
FIG. 3 is a QT-PCR map; (A, B), west blotting (E, F), immunofluorescence (I, J, K), QT-PCR (C, D, N, P), west blotting (G, H), immunofluorescence (I, L, M), west blotting (O, R).
FIG. 4 is a graph showing the effect of nano-enzyme administration on TMJ alleviation without significant toxic or side effects; wherein the graph B, C: nanoenzymes (8 mg/kg) did not affect liver and kidney function in both groups of mice, and did not show in vivo toxicity; D. no abnormal change in body weight was found in both groups of mice during the test period; diagram F-I: nanoenzyme administration can reduce hyperalgesia of TMJ mice; J-O: the nano-enzyme can delay the progress of TMJ mice temporomandibular joint inflammation.
FIG. 5 is a graph showing that nanoenzyme administration can reduce MMP2/9 and ROS expression in the TG region; QT-PCR (A-C, G-I), west blotting (D-F, J-L), immunofluorescence (M, N, Q, R) after nano-dosing; FIG. O, S is a graph showing that nanoenzymes reduce ROS expression in TG; figure P, T suggests that nanoenzymes inhibited astrocyte activation in TG.
FIG. 6 is a graph showing the effect of nanoenzyme administration on MMP-2/9 and ROS in the SP5C region; after nano administration, QT-PCR (A-C, G-I), west blotting (D-F, J-L) and immunofluorescence (M, N, Q, R) indicate that the expression of MMP2/9 gene, which is a key protein for pain in SP5C region of TMJ mice, is obviously reduced, and the protein expression is not obviously changed. Figure O, S is that nanoenzymes reduced ROS expression in SP 5C. FIG. P, T is a graph showing the inhibition of astrocyte activation by nanoenzymes in SP 5C.
FIG. 7 is a transmission electron microscope image of a monoatomic nanoenzyme.
Detailed Description
The technical solutions of the present application will be clearly and completely described in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without any inventive effort, are intended to be within the scope of the application.
Example 1
The preparation method of the antioxidant nano-enzyme with the gelatinase inhibition effect comprises the following steps:
(1) 2-methylimidazole (1314 mg) was dissolved in 15 ml of methanol with stirring as solution a; zn (NO) 3 ) 2 (1190 mg) and Cu (acac) 2 (846, mg) dissolved in 30, mL methanol and stirred ultrasonically for 15min to form a transparent solution, followed by stirring vigorously at room temperature for 1 hour as solution B; adding the firing solution B into the solution A; and then transferred to a 100 mL teflonThe liner was placed in a stainless steel autoclave and heated at 120℃for 4 hours. The product obtained was isolated by centrifugation, followed by three washes with DMF, two washes with methanol and finally dried overnight under vacuum at 70℃to give Cu SAzyme.
(2) DSPE-PEG-COOH was dissolved in chloroform/methanol (v/v=2:1) and then dried under a rotary evaporator. Thereafter, the dried lipid film was hydrated with Cu SAzyme solution (1 mg/mL,2 mL), followed by ultrasonic treatment with a probe for 5 minutes, to obtain Rvg-Cu SAzyme, and further confirming the synthesis of the monoatomic nanoenzyme by a transmission electron microscope as shown in FIG. 7, and it was found from FIG. 7 that the synthesized nanoenzyme exhibited a dodecahedral structure.
Example 2
The preparation method of the antioxidant nano-enzyme with the gelatinase inhibition effect comprises the following steps:
(1) 2-methylimidazole (10 mg) was dissolved in 15 mL methanol with stirring as solution a; zn (NO) 3 ) 2 (10 mg) and Cu (acac) 2 (50 mg) dissolved in 30 mL methanol and stirred ultrasonically for 15min to form a transparent solution, followed by stirring vigorously at room temperature for 1 hour as solution B; adding the firing solution B into the solution A; then transferred to a 100 mL teflon lined stainless steel autoclave and heated at 100 ℃ for 4 hours. The product obtained was isolated by centrifugation, followed by three washes with DMF, two washes with methanol and finally dried overnight under vacuum at 100deg.C to give Cu SAzyme.
(2) DSPE-PEG-COOH was dissolved in chloroform/methanol (v/v=2:1) and then dried under a rotary evaporator. Thereafter, the dried lipid membrane was hydrated with Cu SAzyme solution (5 mg/mL,5 mL), followed by probe sonication for 1 minute, yielding Rvg-Cu SAzyme.
Example 3
The preparation method of the antioxidant nano-enzyme with the gelatinase inhibition effect comprises the following steps:
(1) 2-methylimidazole (1514 mg) was dissolved in 15 mL methanol with stirring as solution a; zn (NO) 3 ) 2 (1200 mg) and Cu (acac) 2 (1000 mg) ultrasonic stirring dissolved in 30 mL methanolStirring for 15min to form a transparent solution, and then stirring for 1 hour at room temperature under high force to obtain a solution B; adding the firing solution B into the solution A; then transferred to a 100 mL teflon lined stainless steel autoclave and heated at 90 ℃ for 4 hours. The product obtained was isolated by centrifugation, followed by three washes with DMF, two washes with methanol and finally dried overnight under vacuum at 80℃to give Cu SAzyme.
(2) DSPE-PEG-COOH was dissolved in chloroform/methanol (v/v=2:1) and then dried under a rotary evaporator. Thereafter, the dried lipid membrane was hydrated with Cu SAzyme solution (10 mg/mL,10 mL), followed by probe sonication for 10 minutes, yielding Rvg-Cu SAzyme.
Example 4
The preparation method of the antioxidant nano-enzyme with the gelatinase inhibition effect comprises the following steps:
(1) 2-methylimidazole (657 mg) was dissolved in 15 mL methanol with stirring as solution a; zn (NO) 3 ） 2 (10-1200-mg) and Cu (acac) 2 (500 mg) dissolved in 30 mL methanol and stirred ultrasonically for 15min to form a transparent solution, followed by stirring vigorously at room temperature for 1 hour as solution B; adding the firing solution B into the solution A; then transferred to a 100 mL teflon lined stainless steel autoclave and heated at 115 ℃ for 4 hours. The product obtained was isolated by centrifugation, followed by three washes with DMF, two washes with methanol and finally dried overnight under vacuum at 90℃to give Cu SAzyme.
(2) DSPE-PEG-COOH was dissolved in chloroform/methanol (v/v=2:1) and then dried under a rotary evaporator. Thereafter, the dried lipid membrane was hydrated with Cu SAzyme solution (6 mg/mL,8 mL), followed by probe sonication for 6 minutes, yielding Rvg-Cu SAzyme.
Comparative example
The content of comparative example 1 is substantially the same as that of example 1 except that: rvg is not added in the step (2), and finally the monoatomic nano-enzyme is prepared.
Examples of the effects
1. Experimental animal
Animal experiments were approved by the ethical committee of the second affiliated hospital of Zhengzhou university (ethical number: 2021056) and met the guidelines of the national institutes of health. C57BL male mice were purchased from Henan province animal experiment center for 6-8 weeks. The cages were normalized to 5 mice per cage and food and water were freely obtained at the appropriate temperature (22.+ -. 2 ℃) and 12 hours of light and shade cycle (light cycle: 8:00 am-8:00 pm). All animals were acclimatized at least 3 days before any procedure began.
2. Temporomandibular joint pain model
Under isoflurane anesthesia, CFA (10 ul, 1mg/ml, sigma) or physiological saline (10 ul) was injected into TMJ. As previously described, we injected CFA and normal saline into the superior joint space of the bilateral temporomandibular joint of the mice.
3. Drug treatment
Half an hour after CFA injection, mice were randomized into treatment and control groups. Mice in the treatment group were injected with nanoenzyme or mmp-2/9 inhibitor, minocycline, by systemic administration via the tail vein. The dose of nanoenzyme was 5mg/kg (iv., 0.1 ml) dissolved in PBS at a concentration of 1 mg/ml; the minocycline dose was 3mg/kg (iv., 0.1 ml) dissolved in PBS at a concentration of 0.6 mg/ml. Control mice were treated with the same volume of PBS.
4. Mechanical pain behavioural test
Pain behavioural tests mice were assessed for oral facial mechanical hyperalgesia. All experiments were performed by one person blinded to the treatment group. The threshold for head withdrawal was determined by applying a stimulus through von-Frey filaments and measuring the mechanical hyperalgesia of the orofacial area. Mice were placed in a 10 cm long, constraining plexiglas cylinder with their head and forepaws extended, but the restraint prevented them from turning around. After 5 minutes of acclimation, the filaments were applied to the area of skin innervated by the trigeminal nerve branches. Each filament was applied 5 times to the trigeminally innervated skin area for a period of 1-2 seconds, 10 seconds apart, starting with the lowest filament force (0.08 g) and continuing in ascending order. A positive response is defined as a sharp withdrawal of the head after stimulation. The head retraction threshold is calculated as the force that produces a positive response in the five stimuli.
5. Mouse ghost analysis
The Mouse Ghost Scale (MGS) is a facial expression-based nociceptive coding system used for spontaneous pain testing in mice. After the mice were acclimatized in the plexiglas jar for 5min, 30min video was recorded with a high resolution digital video camera at different time points before and after CFA or normal saline injection in the temporomandibular joint. A clear facial image was selected every 3 minutes. For each photo we observe the following three facial expressions: (1) orbital compression, i.e. narrowing of the orbital area, eyelid tightening or eye compression (representing wrinkles around the eye) (2) nasal bridge doming, circular extension of visible skin on the nasal bridge (3) ear position: characterized by the ear pulling away from the baseline position and back, or by the ear tip pulling back to form a vertical ridge. These facial expressions are scored according to the values 0 (no present), 1 (medium visible) and 2 (obvious). Individual and composite scores were calculated from the average of 10 images per mouse at each time point.
6. Body weight measurement
Sufficient water and food are given to the mice, ventilation is good, and temperature and humidity are proper. Starting from two days before the molding of the mice, the body weight was weighed and recorded at 9-10 points daily, and the change in body weight of the mice was observed for the duration of 3 days after the operation.
7. Serum biochemical index monitoring
The mice were deeply anesthetized three days later with PBS solution or Nano solution (in PBS,5 mg/kg), and were collected by needle stick orbit blood collection, placed in a 1.5ml centrifuge tube, and allowed to stand overnight in a 4 degree refrigerator. The next day at 3000 rpm in the centrifuge, for 10 minutes, the supernatant was extracted. According to the biochemical kit step (Lei Du, shenzhen), the corresponding index of serum is detected.
8. Temporomandibular joint HE staining
Mice were sacrificed the third day after administration of CFA or normal saline, tail vein or PBS under isoflurane anesthesia in the temporomandibular joint, and intact temporomandibular joint tissue, including synovium, articular disc, cartilage and mandibular condyle, were isolated, fixed with 4% paraformaldehyde, and then demineralized with 10% edta. After decalcification, temporomandibular joint tissue was dehydrated in gradient, paraffin-embedded, and all paraffin-embedded specimens were serially sectioned 4mm thick, and hematoxylin-eosin stained.
9. Western immunoblotting
Mice were sacrificed under isoflurane anesthesia at temporomandibular joint with CFA or normal saline, tail vein administration or the third day after PBS, TG tissues were taken and homogenized in RIPA lysate. Protein concentration was determined by BCA protein assay, and the loaded solution was prepared for electrophoresis and transfer of protein to PVDF membrane. Blocking for 1 hour at room temperature, incubation of the antibody overnight at 4℃with primary antibodies including GAPDH Polyclonal antibody (1:5000; proteintech), MMP-2 Polyclonal antibody (1:1000; proteintech), MMP-9 (1:1000; N-termjnal) Rabbit Polyclonal antibody), EMMPRIN (B-5) (1:200; SANTACRUZ BIOTECHNOLOGY). Secondary antibody incubation, washing and exposure. The secondary antibodies included HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1:5000; proteintech), and Western banding intensity was analyzed using an exposure machine and related software imageJ.
10. Real-time fluorescence quantitative PCR (QT-PCR)
After isoflurane anesthetizing the mice, TG and SP5C were rapidly removed in 1.5ml enzyme-free tubes, placed in liquid nitrogen for quick freezing, and then placed in a-80 ℃ refrigerator for storage. RNA in the tissue was extracted using an RNA extraction kit (Ai Kerui), and PCR was performed on a Bio-Rad CFX96 real-time PCR system according to the instructions. Wherein the primer sequence is upstream 5'-TGATAACCTGGATGCCGTCG-3' and downstream 5'-CCAGCCAGTCTGATTTGATGC-3' of MMP 2; MMP9 upstream 5'-GCTGGCAGAGGCATACTTGTAC-3', downstream 5'-GGTGTTCGAATGGCCTTTAGTG-3'; GAPDH upstream 5'-CCTCGTCCCGTAGACAAAATG-3', downstream 5'-TGAGGTCAATGAAGGGGTCGT-3'.
11. Immunofluorescence
For immunofluorescent labeling, paraffin sections were incubated overnight at 4deg.C with primary antibodies, including MMP-2 Polyclonal antibody (1:100; proteintech), anti-MMP-9 Mouse mAb (1:500; servicebio), anti-DNA/RNA Damage antibody [15A3] (1:200; abcam). Sections were incubated with secondary antibody for one hour at room temperature. The secondary antibodies included CoraLite594-conjugatedGoat Anti-Mouse IgG (H+L) (1:200; proteintech), coraLite488-conjugated Goat Anti-Rabbit IgG (H+L) (1:200; proteintech) slides were observed on a fluorescence microscope and immunofluorescence intensity was determined using the ImageJ program.
12. Statistical analysis
Statistical analysis was performed using GraphPad Prism 7 software, and the results of the analysis are shown. Data from the 3 independent classification groups were compared using one-way or two-way analysis of variance (ANOV a). The comparison between the two groups used a unpaired t test. P values less than 0.05 were considered significant in all statistical tests. Data are expressed as mean ± SEM.
TMJ mice develop orofacial pain hypersensitivity and increased MMP2/9 and ROS expression in the trigeminal ganglion and increased ROS expression in the nucleus of the trigeminal nerve fascicle:
as compared to the saline-injected group in joint cavities in fig. 1, CFA-induced mice became apparent hyperalgesia on the first day post-surgery for temporomandibular arthritic pain and maintained at least on day 10 post-surgery (fig. 1C). The scoring of facial pain expression also suggested that TMJ mice had significant pain changes within 3 days post surgery (fig. 2B, C, D), pain changes with a trend substantially consistent with the trend of mechanical pain in the oral face of the mice (fig. 2A), and that the joints showed significant inflammatory lesions (fig. 2E) and significant increases in ROS expression in TG (fig. 2F, H) and SP5C (fig. 2G, E), increased expression of the pain-critical protein MMP-2/9 in TG (fig. 3A, B, E, F, I, J, K), significant downregulation of the MMP-2/9 expression gene in SP5C (fig. 3C, D), no significant changes in protein (fig. 3I, G, H, L, M), and no significant changes in EMMPRIN expression in TG and SP5C (fig. 3N-R).
The nano enzyme administration can reduce TMJ inflammatory pain and joint inflammation, and effectively inhibit the expression of MMP2/9 and ROS in trigeminal ganglion, and the ROS activation in the nucleus of trigeminal ganglion:
nanoenzyme (5 mg/kg) and carrier (PBS) were administered via the tail vein 30 minutes after complete Freund's adjuvant or normal saline. Behavioural testing was performed starting on day 2 before surgery and going to day 3 after surgery. The results are shown in fig. 4F-O, where the pain hypersensitivity was significantly reduced and the joint inflammation was significantly inhibited in mice given the nanoenzyme group TMJ. From FIG. 5, it can be seen that ROS (FIG. 5O, S) expression and astrocyte (FIG. 5P, T) activation in TG were inhibited after nanoenzyme administration, and that QT-PCR (FIG. 5A-C, G-I), west blotting (FIG. 5D-F, J-L), immunofluorescence (FIG. 5M, N, Q, R) suggested a significant decrease in the expression of MMP2/9, a key protein for pain in the TG region of TMJ mice. ROS (FIG. 6O, S) expression and astrocyte activation in SP5C were inhibited (FIG. 6P, T), QT-PCR (FIG. 6A-C, G-I), west blotting (FIG. 6D-F, J-L), immunofluorescence (FIG. 6M, N, Q, R) suggested that TMJ mice had significantly reduced expression of MMP2/9 gene, a key pain protein in the TG region, and no significant change in protein expression. The change in MMP-2/9 within the trigeminal ganglion is consistent with the effects of the prior nonspecific inhibitor minocycline.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the application.

Claims (10)

1. The preparation method of the antioxidant nano-enzyme with the gelatinase inhibition effect is characterized by comprising the following steps:
(1) Preparing a methanol solution of 2-methylimidazole as solution A, in which Zn (NO 3 ) 2 And Cu (acac) 2 The methanol solution of (2) is used as a solution B, then the solution B is added into the solution A for high-pressure heating reaction, and the reaction solution is centrifugally separated, cleaned and dried in vacuum to obtain Cu SAzyme;
(2) Dissolving DSPE-PEG-COOH in chloroform-methanol mixed solvent, rotary evaporating, drying, adding Cu SAzyme aqueous solution, hydration reaction, and ultrasonic processing to obtain nano enzyme Rvg-Cu SAzyme.
2. The method for preparing the antioxidant nano-enzyme with the gelatinase inhibiting effect according to claim 1, which is characterized in that: the concentration of the 2-methylimidazole in the solution A in the step (1) is 10-100 mg/mL, and the concentration of Zn (NO) in the solution B is 10-100 3 ) 2 Is 10-100 mg/mL, cu (acac) 2 The concentration of (C) is 10-50 mg/mL.
3. The method for preparing the antioxidant nano-enzyme with the gelatinase inhibiting effect according to claim 2, which is characterized in that: the volume ratio of the solution A to the solution B is 1:2-4.
4. The method for preparing the antioxidant nano-enzyme with the gelatinase inhibiting effect according to claim 2, which is characterized in that: the high-pressure heating reaction is carried out to obtain the temperature of 90-120 ℃ and the time of 2-4 h; the temperature of vacuum drying is 60-90 ℃.
5. The method for preparing the antioxidant nano-enzyme with the gelatinase inhibiting effect according to any one of claims 1 to 4, which is characterized in that: the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 2:1.
6. The method for preparing the antioxidant nano-enzyme with the gelatinase inhibiting effect according to claim 5, which is characterized in that: the concentration of the Cu SAzyme aqueous solution is 1-5 mg/mL, and the volume is 2 mL; the ultrasonic treatment time is 5-15 min.
7. An antioxidant functional nanoenzyme prepared by the method of any one of claims 1-4 and 6.
8. The use of the antioxidant function nano-enzyme according to claim 7 in the preparation of antioxidant drugs.
9. Use of the antioxidant functional nanoenzyme of claim 7 for the preparation of a medicament for inhibiting the activation of ROS and MMP2/9 in the trigeminal ganglion of temporomandibular arthritis.
10. The use of the antioxidant function nanoenzyme of claim 7 in the preparation of a medicament for alleviating temporomandibular arthritic pain.
CN202310475695.8A 2023-04-28 2023-04-28 Antioxidant nano-enzyme with gelatinase inhibition effect, and preparation method and application thereof Pending CN116999541A (en)

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