CN117159534A - Application of bilobalide in preparing medicine for treating arthritis - Google Patents
Application of bilobalide in preparing medicine for treating arthritis Download PDFInfo
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- CN117159534A CN117159534A CN202311055648.4A CN202311055648A CN117159534A CN 117159534 A CN117159534 A CN 117159534A CN 202311055648 A CN202311055648 A CN 202311055648A CN 117159534 A CN117159534 A CN 117159534A
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to application technology of bilobalide, and in particular relates to application of bilobalide in preparation of a medicine for treating arthritis. The invention aims to provide a new choice for treating osteoarthritis. The technical scheme of the invention is the application of bilobalide in preparing the medicine for treating arthritis. The invention not only has new knowledge and breakthrough in the pathogenesis of OA, but also provides new opportunity and thought for searching the mechanism of bilobalide for interfering with OA, and more importantly provides new selection and thought for treating arthritis.
Description
Technical Field
The invention belongs to application technology of bilobalide, and in particular relates to application of bilobalide in preparation of a medicine for treating arthritis.
Background
Osteoarthritis (OA) is a chronic, progressive, degenerative joint disease characterized by degeneration of articular cartilage and characterized by periarticular pain and joint dysfunction. OA is currently widely recognized as a combination of mechanical and biological factors, which can lead to an imbalance in the degradation and synthesis of extracellular matrix (Extracellular matrix, ECM), and this imbalance is related to factors such as inflammatory factor mediation, biomechanical changes, chondrocyte apoptosis, and oxidative stress. The medicine for treating osteoarthritis clinically is mainly a nonsteroidal anti-inflammatory medicine, and the medicine can only relieve pain and has the defects of large side effect, medicine residue, easy recurrence after stopping medicine, incapacity of inhibiting articular cartilage degeneration and the like. Therefore, it is very necessary to develop a drug which is safe and effective, has little side effects, and has the function of preventing or treating osteoarthritis. There is increasing evidence that natural anti-inflammatory supplements may have the potential to inhibit cartilage degeneration and thus may be effective therapeutic interventions for OA.
In recent years, more and more researches focus on the anti-cartilage ECM degradation effect of up-regulating the AMPK-SIRT3 positive feedback loop in the process of protecting cartilage cells from mitochondrial damage, so that the anti-cartilage ECM degradation effect becomes an ideal target point for developing OA medicaments. Studies have shown that aging, mechanical injury and chronic inflammation lead to reduced responsiveness of AMPK/SIRT3 in articular cartilage, compromising the ability of articular tissue to repair and regenerate itself, accelerating OA progression. Interestingly, AMPK/SIRT3 signaling is important for regulating mitochondrial physiological functions, involved in chondrocyte metabolism, including ECM production and degradation, cytoskeletal rearrangement, autophagy, and apoptosis, is the core pathogenesis of osteoarthritis. Taken together, these reports underscores the typical role of AMPK/SIRT3 in maintaining mitochondrial homeostasis and chondrocyte protective responses against ECM degradation.
The ginkgo leaf is a traditional Chinese medicine, and has the effects of tonifying qi, astringing lung, resolving dampness, activating blood circulation, relieving pain and promoting blood circulation. Ginkgo leaf extractGinkgo biloba L, extract) is one of the widely used botanicals in the world. Modern pharmacological studies have shown that,the folium Ginkgo extract has effects of reducing blood lipid, resisting arteriosclerosis, and improving heart and brain circulation. Bilobalide (BB) is the only sesquiterpene compound extracted from ginkgo leaves and is receiving attention due to its anti-inflammatory, antioxidant and neuroprotective properties. However, the therapeutic effect of bilobalide on OA has not been fully elucidated.
Disclosure of Invention
The invention aims to provide a new choice for treating osteoarthritis.
The technical scheme of the invention is the application of bilobalide in preparing the medicine for treating arthritis.
In another aspect, the invention also provides the use of bilobalide in the manufacture of a medicament for delaying the progression of arthritis or reducing the severity of arthritis.
Application of bilobalide in preparing medicine for improving joint microenvironment of arthritis patient is provided.
Further, the arthritis is osteoarthritis.
In particular, the osteoarthritis is tibial or femoral arthritis.
Wherein the joint microenvironment is a bone joint microenvironment.
Further, the bone joint microenvironment is a femur microenvironment or a tibia microenvironment.
The invention also provides application of bilobalide in preparing a substance for activating an AMPK-SIRT3 cell pathway.
Furthermore, the invention also provides application of bilobalide in preparing a substance for inhibiting the expression of the proinflammatory factor.
Specifically, the pro-inflammatory factor is at least one of IL-1, TNF- α, IL-6 or IL-15.
On the other hand, the invention also provides application of bilobalide in preparing a substance for inhibiting oxidative stress injury.
Specifically, the substance for inhibiting oxidative stress injury is a substance for inhibiting MDA, CAT and/or GSH.
The invention has the beneficial effects that: the invention not only has new knowledge and breakthrough in the pathogenesis of OA, but also provides new opportunity and thought for searching the mechanism of bilobalide for interfering with OA, and more importantly provides new selection and thought for treating arthritis. In particular, the method has the following advantages: (1) The bilobalide can improve the disease progress and severity of osteoarthritis, and the administration of bilobalide can restore gloss on cartilage surface, and the extracellular matrix of cartilage has uniform color, and the cartilage of tibia and femur is intact, which shows that bilobalide can have potential therapeutic effect on osteoarthritis. (2) The bilobalide has an improving effect on joint tibia and femur microenvironment of osteoarthritis, and changes of POA rabbit joint gaps and subchondral bones are analyzed through X-ray and Micro-CT imaging, so that the bilobalide can improve biomechanical property and microstructure change of the subchondral bones of the rabbit osteoarthritis and inhibit abnormal bone reconstruction of the subchondral bones. (3) Analysis of the expression of AMPK, SIRT3 and p-AMPK in cartilage tissue of papain-induced osteoarthritis rabbits by immunohistochemistry and western blot, the levels of IL-1, IL-6, TNF- α and IL-15 in blood were determined, and the levels of the three key biomarkers MDA, CAT and GSH of oxidative stress were determined, indicating that bilobalide inhibits oxidative stress-mediated inflammation through AMPK/SIRT3 signaling pathways.
Drawings
Figure 1, bilobalide of the invention inhibits the progression and severity of osteoarthritis in rabbits. As shown in fig. 1A, the experimental results show that the damage degree of the tibia and the femur of the rabbits in the POA group after the BB intervention is 8 weeks, the cartilage of the rabbits in the poa+white bone lactone group is complete, the cartilage surfaces of the femur and the cartilage are restored to gloss, and the cartilage is intact as a whole. As shown in FIG. 1B, the experimental results showed that the morphological observation score of the rabbit tibia and femur cartilage of the POA group was extremely increased. The morphological scores of the rabbit tibia and femur cartilage of the POA+white bone lactone group were significantly reduced.
Fig. 2, cartilage pathological section observation. Fig. 2A shows that the rabbit cartilage surface chondrocytes are orderly arranged after the bilobalide is dry, the surface is smooth, the whole cartilage structure is complete, and the number of chondrocytes is slightly reduced after HE staining evaluation is performed on the rabbit right hindlimb tibia of each group. Fig. 2B shows that the rabbit hind limb tibia is subjected to safranine O fast green staining, and the result shows that the rabbit cartilage surface is smooth after bilobalide is dry, safranine O staining is uniform, and the staining intensity is obviously improved compared with that of POA group.
Fig. 3, rabbit knee X-ray imaging and scoring. As shown in fig. 3A, the knee joint tibia and femur joint space and osteophyte formation were taken by X-rays for each group of rabbits. The result shows that the POA+white bone lactone group joint is gradually smooth, the joint gap is gradually restored to the normal width, and no bone fragments are formed. As shown in fig. 3B, the experimental results showed that the poa+white bone lactone group X-ray score was extremely significantly reduced compared to the rabbits of the POA group. The bilobalide has an improving effect on the joint tibia and femur microenvironment of papain-induced rabbit osteoarthritis.
FIG. 4, micro-CT scan and three-dimensional reconstruction of bone microstructure. As shown in fig. 4A, the experimental results showed that the rabbit tibial subchondral bone of poa+white bone lactone group was slightly damaged and that trabecular bone loss was improved relative to OA group. As shown in fig. 4B, which is a three-dimensional reconstruction representative image, the experimental result shows that BB can improve biomechanical properties and microstructure changes of the subchondral bone of rabbit osteoarthritis, and inhibit abnormal bone reconstruction of the subchondral bone.
FIG. 5, AMPK and SIRT3 positive cell ratios in rabbit cartilage. As shown in fig. 5A, the experimental results indicate that bilobalide can activate the papain-induced cartilage AMPK-SIRT3 cell pathway of the rabbit with osteoarthritis. As shown in fig. 5A, the experimental results showed that the numbers of AMPK and SIRT3 positive cells in rabbit cartilage of poa+white bone lactone group were extremely significantly increased.
FIG. 6, changes in the pro-inflammatory factors IL-1, IL-6, TNF- α and IL-15. The expression of IL-1, IL-6, TNF-alpha and IL-15 in serum of POA+white bone lactone group rabbits is extremely obviously reduced. BB is shown to be effective in inhibiting inflammatory responses in rabbit organisms and to exert anti-inflammatory effects by inhibiting the expression of pro-inflammatory factors IL-1, TNF- α, IL-6 and IL-15.
Fig. 7, bilobalide inhibits oxidative stress injury caused by osteoarthritis. The CAT content in serum of the POA+white bone lactone group rabbits is obviously increased, the GSH content is obviously increased, and the MDA content is obviously reduced. The bilobalide can obviously inhibit lipid peroxidation in a papain-induced rabbit osteoarthritis model, increase water of antioxidant enzymes CAT and GSH, inhibit MDA content, inhibit oxidative damage of organisms and play a role in protecting cartilage.
Description of the embodiments
Bilobalide is the only sesquiterpene compound extracted from ginkgo leaves and is receiving attention due to its anti-inflammatory, antioxidant and neuroprotective properties. Thus, applicants consider whether bilobalide can be used to treat osteoarthritis. To verify the foregoing, the inventors constructed an osteoarthritis model using New Zealand white rabbits, and then discovered that osteoarthritis could indeed be improved by administering bilobalide.
Further, in order to clarify the mechanism of bilobalide to improve osteoarthritis. The inventors have appreciated from the literature that AMPK is a serine/threonine protein kinase, a highly conserved "energy sensor", that reacts to intracellular AMP, ADP and ATP changes. In addition, AMPK is also involved in metabolism, caloric circulation, local extracellular signaling and regulation of hormone release; activation of AMPK relieves endoplasmic reticulum stress, promotes DNA damage repair and autophagosome formation, and thereby protects cells from stress-induced phenotypic changes and apoptosis. Sirtuins are a highly conserved third class of deacetylases, most Sirtuin family members having nad+ -dependent protein deacetylase activity. SIRT3 is an NAD+ -dependent protein deacetylase downstream of the AMPK signal that enhances the activity of AMPK by LKB1 deacetylation. SIRT3 is associated with cellular metabolism, growth, autophagy, and apoptosis.
Based on the above knowledge, the inventors have conducted a related experiment and found that bilobalide is effective for inhibiting inflammation mediated by oxidative stress through AMPK/SIRT3 signal pathway, thereby improving osteoarthritis.
The main reagents used in the examples below are shown in Table 1.
TABLE 1 sources of major reagents
| Malondialdehyde (MDA) content detection kit (micro method) | Subfamily factorBiovotec A/S |
| Catalase (CAT) activity assay kit (micro-method) | Subfamily Biotech Co.Ltd |
| Reduced Glutathione (GSH) detection kit (micro method) | Subfamily Biotech Co.Ltd |
| Interleukin-1 (IL-1) test kit | Nanjing built biosciences Inc |
| Interleukin-6 (IL-6) test kit | Nanjing built biosciences Inc |
| Interleukin-15 (IL-15) test kit | Nanjing built biosciences Inc |
| Tumor necrosis factor-alpha (TNF-alpha) test kit | Nanjing built biosciences Inc |
| Safranin O staining kit | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| AMPK, SIRT3 antibodies | Affinity Biosciences, china |
| Papain | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| 4% paraformaldehyde | BEIJING SOLARBIO TECHNOLOGY Co.,Ltd. |
| Bilobalide (purity is more than or equal to 98%) | Chengdu Mandsite Biotechnology Co.Ltd |
| Horseradish peroxidase labeled goat anti-rabbit IgG (H+L) | SHANGHAI BEYOTIME BIOTECHNOLOGY Co.,Ltd. |
(1) Rabbits were randomly divided into a control group (control group, saline treatment, n=6) and a papain group (intra-articular injection of 4% papain-induced osteoarthritis model, n=12). Papain groups were divided into two subgroups (6 per group): model group (POA group), BB administration group (poa+bb group).
(2) Rabbits from the papain group were injected with 0.1 mL of 4% papain in the joint cavities of the right knee at 1 d, 4 d and 7 d, respectively. After each intra-articular injection, the animals were allowed to move freely in the cages.
(3) Two weeks after the joint cavity is injected with papain to establish a rabbit osteoarthritis model, the rabbits in the POA+BB group are infused with 80 per day mg per kg BB, and continuously intervene for 8 weeks. The lavage volume of each rabbit BB was 3 mL.
(4) 8 weeks after dosing, the rabbits of each group were euthanized and knee joint and blood samples were taken. The collected blood was centrifuged at 2500 r/min for 20 min at room temperature, and the supernatant was collected and stored at-80 ℃.
(1) And collecting knee joint samples of all joints of each group of rabbits, shooting knee joint tibia and femur surface images by using a Nikon D5300 AF-SD Nickel 18-55 mm single phase inverter, and observing the surface morphology change of joint cartilage.
(2) Changes in articular cartilage surface morphology were observed and scored on a scale of 0-4 according to the Pelletier scoring criteria: 0 = smooth cartilage surface, normal color; 1 = rough cartilage surface, yellowish in color; 2 = cartilage surface damage or extension to the middle layer; 3 = cartilage surface ulcers and extends to deeper layers; 4 = cartilage loss and subchondral bone exposure.
The results are shown in FIG. 1: the cartilage surface of the control group is smooth and glossy, and the cartilage surface is ruddy, and no damage or destruction is seen. The POA group rabbits had severe articular surface ulcers, damaged femoral cartilage surfaces, severe femoral rim wear (arrow), and bare subchondral bone (box). The tibial surface appeared dark red and had lost gloss, the distal cartilage of the tibia eroded (indicated by the arrow), the tibial margin frayed severely, and the subchondral bone exposed (indicated by the box). The cartilage of the rabbit joint in the POA+BB group is complete, the cartilage surfaces of the femur and the cartilage are slightly rough (shown by arrows), but the cartilage surfaces of the tibia restore gloss, and the cartilage is complete as a whole.
Compared with rabbits in a control group, the morphological observation scores of the tibia and the femur of the rabbits in the POA group are greatly increasedP<0.01). Compared with the rabbits in the POA group, the morphological scores of the tibia and the femur cartilage of the rabbits in the POA+BB group are greatly reducedP<0.01）。
(1) Rats and rabbits were resected for tibia and femur, and EDTA was decalcified for 4 weeks (EDTA was changed weekly). 4% formaldehyde fixation 24 h, tap water rinse 1 h.
(2) The following different liquids are filled into the glass cylinders, and transferred into the different glass cylinders according to the following sequence, and the alcohol with concentration of 70%, 80%, 95% and 100% is dehydrated step by step, so that the xylene is transparent.
(3) Dehydrated bone tissue was placed in paraffin, embedded 1 h, sliced every other day, baked at 60 ℃, dewaxed with xylene, and dehydrated stepwise with 100%, 95%, 85%, and 75% alcohol by volume, respectively.
(4) Conventional HE staining: hematoxylin staining for 10 min, water washing, 1% hydrochloric acid alcohol 5 s, running water reverse blue for 10 min, eosin for 10 min, water washing and optical resin sealing.
(5) Toluidine blue staining: the toluidine blue dye liquor with the mass fraction of 2 percent is dyed for 30 minutes, washed and dried by tap water, dehydrated step by 70 percent, 80 percent, 95 percent and 100 percent of alcohol, transparent dimethylbenzene and sealed by optical resin.
(6) Safranin O-fast green staining: adding freshly prepared Weibert staining solution for 5 min, and washing with water. Acid differentiation also differentiated 15 s. Infection was performed in the fast green staining solution for 5 min and sections were washed with lactic acid solution 15, s. And (5) infecting in the safranin staining solution for 5 min. Dehydrating with 95% ethanol 3 s and anhydrous ethanol 3 s for 1 min. The xylene is transparent and the optical resin is sealed.
(7) Histopathological features were scored semi-quantitatively according to the international society of osteoarthritis research (Osteoarthritis Research Society Internationa, OARSI) grading system.
The results are shown in FIG. 2. In rabbits of the control group, cells of the cartilage shallow layer, the transition layer, the radioactive layer and the calcified layer are arranged in a column shape from top to bottom, the cells are orderly arranged, and the cartilage surface is smooth without damage. The rabbit cartilage of the POA group is severely damaged, the articular cartilage surface is rough, the staining intensity of the matrix is weakened, obvious cracks are visible, superficial chondrocytes are lost, cells are hypertrophic and cavitation are carried out, and chondrocyte cells are arranged in clusters. The cartilage cells on the surface of the rabbit cartilage of the POA+BB group are orderly arranged, the surface is restored to be smooth, the whole cartilage structure is complete, and the number of the cartilage cells is slightly reduced.
Pathological changes in rabbit cartilage ECM were observed in each group by safranin O fast green staining of rabbit hind limb tibia. Safranin O is a polyanionic cationic dye that binds to anionic groups in ECM polysaccharides of cartilage tissue, and eosinophilic bone and acid dye bind fast green to green or blue, in contrast to cartilage that appears red, to distinguish cartilage tissue from bone tissue. The rabbit cartilage structure of the control group has clear and complete layers, the chondrocytes are orderly arranged, and the ECM is uniformly dyed. The rabbit tibia cartilage of the POA group is uneven in ECM staining and low in intensity, proteoglycan in the ECM is seriously lost, articular cartilage is seriously damaged, obvious cracks to the middle and lower layers appear, the cartilage surface is uneven, and the surface layer chondrocyte is seriously lost. The rabbit cartilage surface of the POA+BB group is smooth, the safranin O is uniformly dyed, and the dyeing intensity is obviously improved compared with that of the POA group.
(1) Drug intervention the knee joints of rabbits and mice were evaluated orthotopic and lateral radiographically.
(2) Each group of knee X-rays was scored on a scale of 0-4, 0 = no osteophyte formation, 4 = severe osteophyte formation.
The results are shown in FIG. 3: the formation of the knee joint tibia and femur joint space and osteophytes of each group of rabbits was photographed by X-rays. The joint surface of the rabbit of the control group is smooth and neat and compact shadow, the outline is smooth and clear, and the joint gap is normally in low-density shadow. The shadow area of the soft tissue of the POA rabbit joint is enlarged, the joint surface is eroded, the joint gap is reduced (shown by an arrow), and the osteophyte exists. The joints of the POA+BB group are gradually smooth, the joint gap is gradually restored to the normal width, and no bone fragments are formed. Compared with rabbits in a control group, the X-ray scores of the POA group are extremely obviously increasedP<0.01 Obvious increase of POA+BB groupP<0.05). Compared with rabbits in POA group, the X-ray score in POA+BB group is extremely obviously reducedP<0.01). The BB has an improving effect on the joint tibia and femur microenvironment of papain-induced rabbit osteoarthritis.
(1) To observe the changes in bone microstructure and parameters by BB, micro-CT scans were performed on rabbit knee samples of the control group, POA group and POA+BB group, which were fixed.
(2) The scanning parameters are as follows: the tube current 200 uA, voltage 85 KV, scan the entire object, scan resolution 10.13 μm, exposure time 384 ms, scan angle 180 degrees. And (3) calibrating the scanning phantom under the same condition, and obtaining an original image after scanning.
(3) The reconstruction of the selected region is performed on the original image using three-dimensional reconstruction software NRecon (version V1.7.4.2). Before reconstruction, the reconstructed image is previewed.
(4) In order to reduce the image artifacts to achieve the optimal reconstruction effect, the reconstruction parameters are adjusted and set to the following values: smoothing=5 (smoothness), beam-harding=8 (light intensity), and Ring artifacts=25% (Ring tail), among other parameters. And after the parameters are set, starting image reconstruction.
The results are shown in FIG. 4. In the coronal plane and the transverse plane of the knee joint of the rabbit of the control group, the optical density and the contrast of the line piece have no obvious difference, and the surface of the tibia has no obvious abnormal such as abrasion, defect and the like. The bone density, cortical thickness and bone surface of the three-dimensional reconstructed control group were also unchanged and defective. The POA group observed loss of tibial trabeculae (shown by arrows) on the coronal plane of Micro-CT scan, and the cross section observed abrasion and destruction of subchondral bone at the central position of tibial plateau, with the trabeculae discontinuous. After three-dimensional reconstruction, obvious cracks (shown by a box) appear on the tibia of rabbits in the POA group, the tibia surface is not smooth, subchondral bone erosion appears, and abnormal bone reconstruction occurs on subchondral bones. The rabbit tibia of the POA+BB group had slight damage to subchondral bone, and bone trabecular loss was improved relative to that of the POA group (indicated by arrows), and after three-dimensional reconstruction, obvious bone defects and damage were found in the rabbit tibia (indicated by boxes). The BB can improve the biomechanical property and microstructure change of the cartilage of the rabbit osteoarthritis and inhibit abnormal bone reconstruction of the cartilage.
The experimental results above illustrate: the bilobalide not only protects joint health by inhibiting inflammation, resisting oxidation and matrix metabolism, but also can improve subchondral bone injury to protect joint and inhibit osteoarthritis. Based on these findings, bilobalide is considered to have an ameliorating effect on osteoarthritis, and is a potential therapeutic agent for osteoarthritis.
Key catabolic enzymes, inflammatory factors and pathway proteins in the various groups of rabbit articular cartilage were semi-quantified by immunohistochemical staining. Protein is extracted from tissues to be used as an antigen, a specific antibody is obtained by immunizing animals, the antigen in the tissues is detected and combined by the antibody, and then the secondary antibody with a color reagent label is used for detection, so that qualitative and positioning research on the protein in the tissues can be realized. The specific operation method is as follows:
(1) Paraffin sections with a thickness of 3-5 μm were placed in an oven (72 ℃) to bake the sections 2 h.
(2) Slice dewaxing hydration procedure: the slices were sequentially placed in xylene I and II for 12 min each, absolute ethanol I and II for 3 min each, 95% ethanol for 5 min,85% ethanol for 5 min, and distilled water for 3 min.
(3) The tissue repair adopts a high-temperature high-pressure method: the citric acid antigen retrieval buffer 1000 mL with ph=6.0 was added to the pressure cooker, and the slices were inserted into a plastic rack and placed into the pressure cooker. The pressure cooker 1600 and W are preheated to boiling, the pressure valve 1300 and W are expanded, and the repair is carried out for 2 minutes.
(4) Slicing after antigen retrieval, preventing the slices from being soaked in distilled water for 2 min. Placed in endogenous peroxidase blocker 3% H 2 O 2 And 4 min. Washing with tap water for 2 min and PBS for 2 min.
(5) Taking out the slice, wiping the surrounding liquid, dripping 10% BSA blocking solution to uniformly cover the tissue, and blocking at 37 ℃ for 40 min. 60 μl of primary antibody was added dropwise and incubated overnight in a wet box at 4deg.C in a refrigerator.
(6) The sections were washed 3 times with PBS for 3 min each. After the tissue is sufficiently washed, the liquid around the tissue is wiped off, and then horseradish enzyme-labeled goat anti-mouse IgG polymer is dripped, and the tissue is incubated for 50 minutes at room temperature.
(7) The sections were washed 3 times with PBS for 3 min each. The DAB color development liquid prepared at present is dripped (50 mu L of DAB concentrated liquid is dripped into 1 mLDAB substrate liquid), and the color development is carried out for 5-20 min at room temperature.
(8) Counterstaining the nuclei: hematoxylin dye solution is used for dyeing 40 and s, and water is used for washing for 1 min. Differentiation of 1% hydrochloric acid-alcohol solution 3 s, tap water rinsing, hematoxylin bluing 10 s, and running water rinsing.
(9) And (3) removing the water sealing piece: the slices were sequentially placed in 85% and 95% ethanol for 5 min each, absolute ethanol I for 5 min.
(10) And (3) transparency: the slices are sequentially placed in xylene I and II for 2 min each, the slices are taken out of the xylene and are dried slightly, and the neutral resin is sealed.
(12) Image acquisition and analysis: image-Pro Plus software was used to analyze the optical density of the pictures.
The results are shown in FIG. 5. Compared with rabbits in a control group, the ratio of AMPK and SIRT3 positive cells in cartilage of the rabbits in the POA group is increased, but the change is not obvious [ (]P>0.05). Compared with rabbits in the POA group, the number of AMPK and SIRT3 positive cells in the cartilage of the rabbits in the POA+BB group is greatly increasedP<0.01). Indicating that BB can activate the papain-induced cartilage AMPK-SIRT3 cell pathway of the osteoarthritis rabbit.
The content of inflammatory factors IL-1 beta, TNF-alpha, IL-6 and IL-15 in rabbit serum is measured by ELISA method, and the intervention effect of BB on systemic inflammatory reaction of organism is observed. The specific operation method is as follows:
(1) The method is characterized in that the method is strictly operated according to the specification of a kit manufacturer, the kit is placed at 4 ℃ for 30 min in advance for room temperature balance, 50 mu L of corresponding reagent is sequentially added into a standard hole, a sample hole and a blank hole, then a sample is added into the bottom of the hole of the ELISA plate, the hole wall is not touched as much as possible, and the mixture is gently shaken and mixed uniformly.
(2) 100 mu L of enzyme-labeled reagent is added to each well except for the blank wells, and the wells are incubated at 37 ℃ for 60 min. After washing the washing solution for 5 times, 50. Mu.L of each of the color developers A and B was sequentially added, and the stop solution was stopped.
(3) OD values were measured for each well using a multifunctional microplate reader 450 nm wavelength with a blank Kong Diaoling. And (3) building a Logistic four-parameter regression model by using ELISAcal software, calculating a regression equation of a standard curve by taking the concentration of a standard substance as an abscissa and the OD value as an ordinate, and further calculating the concentration of a sample.
As shown in FIG. 6, the pro-inflammatory factors IL-1, IL-6, TNF-alpha and IL-15 in the serum of rabbits in the POA group were significantly increased as compared with the control groupP<0.01). Compared with the rabbits in the POA group, the expression of IL-1, IL-6, TNF-alpha and IL-15 in the serum of the rabbits in the POA+BB group is extremely obviously reducedP<0.01). BB is shown to be effective in inhibiting inflammatory responses in rabbit organisms and to exert anti-inflammatory effects by inhibiting the expression of pro-inflammatory factors IL-1, TNF- α, IL-6 and IL-15.
Oxygen radicals act on unsaturated fatty acids of lipids to produce peroxidized lipids, which gradually decompose into a complex series of compounds including Malondialdehyde (MDA). The level of lipid peroxidation can be detected by detecting the level of MDA. The operation was performed according to the manufacturer's instructions of the kit (model KTB1050, subfamily biotechnology limited) as follows: the kit is placed at 4 ℃ for 30 min in advance for room temperature balance, and the enzyme label instrument is preheated for more than 30 min in advance. A blank tube was set, 300. Mu.L of Reaction Mix and 100. Mu.L of deionized water were added, and 300. Mu.L of Reaction Mix and 100. Mu.L of sample were added to the measurement tube and thoroughly mixed. Incubation in a 95℃water bath for 30 min, cooling in an ice bath, and centrifuging at 10 min at 10 r/min. 200 μl of supernatant was added to a 96-well plate and absorbance at 532 nm and 600 nm was measured.
CAT is a common antioxidant enzyme that catalyzes the breakdown of hydrogen peroxide (H2O 2) into water and oxygen, H2O2 is highly damaging to cells and excessive accumulation leads to oxidation of cellular targets (proteins, lipids or DNA) leading to cell death. The operation was performed according to the manufacturer's instructions of the kit (model KTB1040, subfamily biotechnology limited) as follows: the kit is placed at 4 ℃ for 30 min in advance for room temperature balance, and the enzyme label instrument is preheated for more than 30 min in advance. Blank wells, standard wells, assay wells and positive control groups were set in 96-well plates, each reagent was added sequentially according to instructions, mixed well, incubated at room temperature for 5 min, and absorbance was measured 540 and nm.
GSH is the most important antioxidant sulfhydryl compound in cells, plays an important role in cell antioxidant, protein sulfhydryl protection and amino acid transmembrane transport, and the measurement of GSH content can reflect the redox state of cells. The operation was performed according to the manufacturer's instructions of the kit (model KTB1600, subfamily biotechnology limited) as follows: the kit is placed at 4 ℃ for 30 min in advance for room temperature balance, and the enzyme label instrument is preheated for more than 30 min in advance. Blank holes, standard holes and measuring holes are arranged in a 96-well plate, all reagents are added in sequence according to the instruction, the mixture is fully and uniformly mixed, the mixture is incubated for 2 minutes at room temperature, and the absorbance is measured 412 and nm.
As shown in FIG. 7, the MDA content in the serum of the rabbits in the POA group is extremely significantly increased compared with that of the rabbits in the control groupP<0.01). Compared with rabbits in the POA group, the MDA content in the serum of the rabbits in the Cel group and the POA+BB group is extremely obviously reducedP<0.01). Compared with rabbits in a control group, CAT and GSH levels in serum of the rabbits in the POA group are extremely obviously reducedP<0.01). Compared with rabbits in POA group, the CAT content in serum of the rabbits in POA+BB group is extremely obviously increasedP<0.01 GSH content is obviously increasedP<0.05). The results show that BB can obviously inhibit lipid peroxidation in a papain-induced rabbit osteoarthritis model, increase the level of antioxidant enzymes CAT and GSH, inhibit oxidative damage of organisms and play a role in protecting cartilage.
Claims (10)
1. Application of bilobalide in preparing medicine for treating arthritis is provided.
2. Application of bilobalide in preparing medicine for delaying arthritis progress or reducing arthritis severity is provided.
3. Application of bilobalide in preparing medicine for improving joint microenvironment of arthritis patient is provided.
4. A use according to any one of claims 1 to 3, characterized in that: the arthritis is osteoarthritis.
5. A use according to claim 3, characterized in that: the joint microenvironment is a bone joint microenvironment.
6. Use of bilobalide in the preparation of a substance that activates the AMPK-SIRT3 cell pathway.
7. Use of bilobalide in the preparation of a substance that inhibits the expression of a pro-inflammatory factor.
8. The use according to claim 7, characterized in that: the pro-inflammatory factor is at least one of IL-1, TNF-alpha, IL-6 or IL-15.
9. Application of bilobalide in preparing substances for inhibiting oxidative stress injury is provided.
10. The use according to claim 9, characterized in that: the substances for inhibiting oxidative stress injury are substances for inhibiting MDA, CAT and/or GSH.
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| CN113786400A (en) * | 2021-10-29 | 2021-12-14 | 山西中医药大学 | Application of bilobalide in preparing medicine for treating or preventing Th17 cell related diseases |
| CN114272237A (en) * | 2022-01-21 | 2022-04-05 | 苏州大学附属第一医院 | Ginkgo biloba extract for treating osteoarthritis |
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| CN113786400A (en) * | 2021-10-29 | 2021-12-14 | 山西中医药大学 | Application of bilobalide in preparing medicine for treating or preventing Th17 cell related diseases |
| CN114272237A (en) * | 2022-01-21 | 2022-04-05 | 苏州大学附属第一医院 | Ginkgo biloba extract for treating osteoarthritis |
Non-Patent Citations (1)
| Title |
|---|
| TIANWEN MA等: "Natural product, bilobalide, improves joint health in rabbits with osteoarthritis by anti-matrix degradation and antioxidant activities", 《FRONTIERS IN VETERINARY SCIENCE》, vol. 9, 20 October 2022 (2022-10-20), pages 1 - 13 * |
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