CN111265584A - Application of two-sided acupuncture extract in the preparation of drugs for preventing and treating inflammation in spinal fusion surgery - Google Patents

Abstract

The invention discloses the application of an extract of double-sided needles in the preparation of medicines for preventing and treating inflammation in spinal fusion surgery; the invention finds that double-sided needles can inhibit the high expression of inflammatory pathways caused by BMP-2, thereby reducing the production of inflammatory substances in cells and increasing the Tight junction protein expression. It solves the inflammatory response around spinal fusion surgery caused by BMP‑2 from the perspective of cellular mechanism, overcomes the side effects in existing drug research, and provides a basis for optimizing the clinical application of BMP‑2.

Description

Application of two-sided acupuncture extract in the preparation of drugs for preventing and treating inflammation in spinal fusion surgery

technical field

The invention belongs to the technical field of traditional Chinese medicine, and relates to the application of an extract of two-sided acupuncture in the preparation of a medicine for preventing and treating inflammation in spinal fusion surgery; in particular, it relates to an extract of two-sided acupuncture in the preparation of a medicine for preventing and treating inflammation around spinal fusion surgery caused by BMP-2 applications in .

Background technique

Bone morphogenetic protein-2 (BMP-2), as a potent osteoinductive cytokine, has been widely used in clinical practice to promote osteogenesis. In 2002, the FDA approved recombinant human bone morphogenetic protein-2 (rhBMP-2) for anterior lumbar fusion. This technology brings a leap forward in spinal fusion surgery, with a 100% spinal fusion rate significantly higher than other traditional osteogenic materials. However, follow-up showed that BMP-2 can lead to inflammation-related complications, including severe edema in the surgical field, increased postoperative drainage, and even swelling of the soft tissue anterior to the cervical spine, compressing the airway. Therefore, finding safe and effective preventive measures has become an urgent clinical problem and a major clinical difficulty.

Currently, anti-inflammatory drugs used to treat BMP-2-related side effects include methylprednisolone, corticosteroids, osteoinductive growth factor NEL-like protein 1 (Nell-1), and rapamycin. However, some poor complications limit their clinical application. For example, high-dose methylprednisolone may cause pulmonary side effects in patients with cervical spine injuries. (See Chinese Patent Application No. 201710115327.7, which discloses "an experimental method for treating spinal cord injury drugs") In addition, there are also studies on the use of biological materials to slow release BMP-2 to prevent it from causing inflammation, but this method has high costs and reduces Increasing the concentration of BMP-2 (increasing the recovery period) cannot fundamentally solve the problem. (See Chinese Patent Application No. 201810087592.3, which discloses "a double-layer composite slow-release microsphere loaded with drugs for promoting bone formation protein and eliminating arthritis symptoms and its preparation method") Therefore, it is necessary to find better clinical BMP-2 adjunctive drugs.

Two-sided acupuncture is a natural Chinese herbal medicine in Chinese folk medicine. It has been used for the treatment of various diseases such as rheumatism, stomach pain, and joint pain since ancient times. Especially in toothpaste, the ingredients of L. acupuncture are widely used for oral anti-inflammatory. It has a history of more than 30 years, and its side effects have not been reported so far, and its safety is relatively good. Recent studies have demonstrated the analgesic effects of L. japonicus extract in inflammatory pain models by targeting ERK and NF-κB signaling pathways. However, there is currently no report on the use of ampulpine in the prevention and treatment of BMP-2-induced peri-inflammation in spinal fusion surgery. Simply resisting the inflammatory response without increasing the permeability between cells, unable to control the exudation of inflammatory substances, will still cause postoperative inflammatory response. In the present invention, double-sided acupuncture can not only be anti-inflammatory, but also increase the connection between vascular endothelial cells, effectively and fundamentally resist the inflammatory response around spinal fusion surgery caused by BMP-2, which is also the working principle of the prior art unattainable.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an application of the extract of Radix Auricularia in the preparation of a drug for preventing and treating inflammation in spinal fusion surgery; especially the application of the extract from Dimeniflora in the preparation of a drug for preventing and treating inflammatory reaction around spinal fusion surgery caused by BMP-2. The present invention proposes for the first time that the extract of L. acupuncture is used to prevent and treat the inflammatory reaction around spinal fusion surgery caused by BMP-2, overcomes the side effects existing in the existing drug research, and provides a basis for optimizing the clinical application of BMP2.

The purpose of this invention is to realize through the following technical solutions:

The present invention relates to the use of an extract of two-sided acupuncture in preparing a medicine for preventing and treating inflammatory reaction around spinal fusion surgery.

Further, the inflammatory response is an inflammatory response around spinal fusion surgery caused by BMP-2.

Further, the extract of L. japonicus is extracted from the roots or branches and leaves of the dicotyledon medicinal plant L. japonicus.

Further, the active ingredient of the L. japonicus extract is clinically and pharmaceutically acceptable L. japonica chlorinate.

Further, the effective dose of the cyanine dichloride is 10-100 mg/kg/d.

Based on the following mechanism, the present invention proposes the application of the extracts of L. annua in the preparation of medicines for preventing and treating inflammatory reactions around spinal fusion surgery caused by BMP-2:

First of all, syringamine has an antioxidant effect that scavenges superoxide produced in the blood and cells.

Second, syringamine has analgesic effect and can increase the pain threshold.

Finally, syringamine inhibits inflammatory factors and pathways and enhances connections between vascular cells, thereby alleviating the hyperpermeability of blood vessels (reduced inflammatory exudation) caused by inflammation against surrounding tissue Inflammatory response, which plays a crucial role in preventing and treating BMP-2-induced inflammatory response around spinal fusion surgery.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention can very effectively prevent and treat the inflammatory reaction around spinal fusion surgery caused by BMP-2 by adding an active ingredient—the extract of two-sided needles in the medicine, including significantly reducing the inflammatory response of human umbilical vein endothelial cells caused by BMP-2. High expression and high permeability of inflammatory factors/pathways; at the same time, L. japonicus extract has no effect on the viability of the cells within the effective concentration; in addition, L. japonicus extract has analgesic and antioxidant effects.

(2) The extract of L. japonica is a natural ingredient present in the roots or leaves of the dicotyledonous medicinal plant L. japonicus. From the point of view of clinical application, it has the same effect as other anti-inflammatory reactions around spinal fusion surgery caused by BMP-2. Safer than drugs.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Figure 1 is a schematic diagram of BMP-2 promoting the inflammatory expression of HUVECs; (A-C) quantitative reverse transcription polymerase chain (qPCR) reaction technology was used to detect human umbilical vein endothelial cells interleukin-1α, interleukin-1β and tumor necrosis Gene expression of factor-α, (n=3-5); (D-F) ELISA method to detect the protein expression of IL-1α, IL-1β and TNF-α in cell culture supernatant, (n=3-5); Data are expressed as mean ± standard error of the mean, *P<0.001, **P<0.01, *P<0.05; BMP-2, bone morphogenetic protein-2; IL, interleukin; TNF, tumor necrosis factor ; GAPDH, reducing glyceraldehyde-phosphate dehydrogenase;

Figure 2 is a schematic diagram of BMP-2 promoting the inflammatory pathway of HUVECs; (A-C) protein levels of p-IκB and p-p65 (n=3); (D) Immunofluorescence localization of NF-κB p65 in human umbilical vein endothelial cells and relative expression; (E-F) Calculate the average fluorescence intensity and nuclear entry of p65 in each microscopic field;

Figure 3 is a schematic diagram showing that BMP-2 increases the permeability of HUVECs; (A-B) VE-cad, p-VE-cad and occludin protein levels in HUVECs were detected (n=3); (C) VE-cad in human umbilical vein Immunofluorescence localization and relative expression in endothelial cells; (D) calculation of the mean fluorescence intensity of VE-cad in each microscopic field; (E) Dextran permeability fluorescence intensity of HUVECs monolayer determined by Transans-well assay and FITC-Dextran assay ;

Figure 4 shows the effect of different concentrations of L. japonicus extract on cell viability;

Figure 5 is a schematic diagram of the reduction of BMP-2 high inflammatory expression caused by the extract of L. japonicus; wherein, (A) ELISA method detected that treatment of L. japonicus reduced the protein expression of IL-1α in the cell culture supernatant, (B) IL-1β protein expression decreased, (C) TNF-α protein expression decreased (n=3-5);

Figure 6 is a schematic diagram of the high expression of inflammatory pathway induced by BMP-2 inhibited by the extract of L. japonicus; (A-C) determination of p-IκB and p-p65 protein levels (n=3); (D) NF-κBp65 in human umbilical vein endothelial cells Immunofluorescence localization and relative expression in ; (E-F) Calculate the mean fluorescence intensity and nuclear entry of p65 in each microscopic field;

Fig. 7 is a schematic diagram of the high permeability of cells caused by the reduction of BMP-2 by the extracts of A. japonicus; (A-D) protein levels of VE-cad, p-VE-cad and occludin (n=3); (E) calculation of each microscopic field The mean fluorescence intensity of VE-cad; (F) Dextran permeability fluorescence intensity of HUVECs monolayer determined by Transans-well assay and FITC-Dextran assay; (G) Immunofluorescence localization of VE-cad in human umbilical vein endothelial cells and relative expression;

FIG. 8 is a conceptual diagram of the reduction of BMP-2-induced cellular inflammation and high permeability by two-sided acupuncture.

Detailed ways

The present invention will be described in detail below with reference to the embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several adjustments and improvements can be made without departing from the inventive concept. These all belong to the protection scope of the present invention.

Example 1

The present embodiment relates to the specific implementation process and efficacy verification of the two-sided needle extract for preventing and treating inflammation around spinal fusion surgery caused by BMP-2:

1. Cell Culture

Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection. In a high glucose endothelial cell culture medium (Dulbecco's Modified Eagle Medium, Sigma, 211-500) containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin, 37°C, 5% _CO in a wet cell incubator nourish. When the whole cell fusion rate reached about 80%, drug treatment was carried out.

2. Medication

Modeling: rhBMP-2 with a concentration gradient of 0, 10 and 20 ng/ml acted on human umbilical vein endothelial cells for 8 hours.

Two-sided needle extract intervention + modeling: 4 hours before the 20ng/ml rhBMP-2 intervention, 3 doses of the two-sided needle extract (extractable or commercially available, this example) were given respectively. The extract of Zhongliangzhi was purchased from Xi'an Qingzhi Biotechnology Co., Ltd.) to interfere with human umbilical vein endothelial cells.

After the BMP-2 intervention, the cells were collected for mRNA and protein expression determination, and the supernatant was used for cytokine quantification. Human umbilical vein endothelial cells without any intervention were used as control group.

3. Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted with Trizol reagent (Invitgen, Carlsbad CA) and quantified by nanotiter. RNA was then reverse transcribed using the PrimeScript RT kit (Takara Bio). Add samples according to the manufacturer's instructions, mix gently, and place them in a PCR machine for reverse transcription. The process was 37°C for 15 minutes (reverse transcription reaction), 85°C for 5 seconds (reverse transcriptase inactivation), and 4°C as the termination temperature. After the reaction, it was stored at -20°C until use. Using this cDNA as a template, qPCR was performed with TB GreenTM PreMixEx TaqTM II (Tli RNAseH Plus) (Takara; RR820A). The specific primers used in the experiments were from PrimerBank (Table 1). The qPCR process was a three-step reaction (denaturation at 95°C for 15s, annealing at 60°C for 30s, and extension at 72°C for 24s, repeated 40 times). The experiment was performed on the ABI7900 real-time PCR system (Applied Biosystems), and all data were selected from each gene amplification the linear stage. The relative ratio of the relevant mRNA to GAPDH expression was calculated and normalized to the control group.

Table 1 qPCR primers

4. Enzyme-linked immunosorbent assay (ELISA)

Seed 5*10 ⁵ HUVECs on 96-well plate, and start drug treatment until the cells are confluent. Collect the culture supernatant of each group of cells (control group, BMP-2 treatment groups with different concentrations (10ng/ml, 20ng/ml), BMP-2+Zhang-acupuncture treatment groups with different concentrations (1, 10, 20μg/ml)) , use the kit according to the instructions of Leidse Bio, including IL-1α, IL-β and TNF-α.

5. Western blot

Cellular proteins were extracted and Western blotting was performed using established methods. Proteins were extracted from HUVEC cells with RIPA buffer (R0010; Solarbio, Beijing, China) containing phenylmethanesulfonyl fluoride (PMSF, Solarbio, Beijing, China). The protein concentration was determined with BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Protein samples (10 μg each) were separated by 10% SDS PAGE and transferred to nitrocellulose membranes. 5% skim milk was dissolved in Tris buffer containing Tween-20 to block non-specific antibody binding, followed by primary antibody application (VE-cad, P-VE-cad, Occuldin, p65) overnight at 4°C. , p-p65, I-κB, p-IκB and GAPDH (Abcam)). Then, peroxidase-labeled secondary antibodies (goat anti-rabbit antibody and goat anti-mouse antibody, ZSGB-BIO, Beijing, China) were added for 1 h at 1:2500 at room temperature. Protein bands were then visualized and analyzed using the Syngene Imaging Department (Cambridge, UK) and ImageJ (National Institutes of Health, Bethesda, MD, USA). Relative proportions of relevant protein and GAPDH expression were calculated and normalized to the control group.

6. Immunofluorescence (IF)

Immunofluorescence staining of HUVECs. First, prepare the cell sheet, wash with 1×PBS for 3×5min, and dry the water around the cell sheet. Blocked in PBS containing 0.3% BSA (Solarbio, China) for 1 h at room temperature, washed with 1×PBS for 3×5 min, incubated with primary antibody overnight, incubated with secondary antibody for 1 h at room temperature, and washed with 1×TBST for 3×5 min. DAPI (Boster, China) nuclei staining was then performed before covering the slides with coverslips. The relative expression levels and expression positions of p65 and VE-cad were detected. Nuclei were localized with DAPI. Fluorescence microscopy images were quantified using ImageJ. For the measurement of the relative expression levels of p65 and occludin, the total expression was obtained from 5 random fields, and then divided by the number of DAPI to obtain the average expression, which was then normalized to the control group. To compare differences in p65 expression in the nucleus, the percentage of cells containing p65 in the nucleus was calculated and normalized to the control group.

7. Cell Viability Assay Kit (CCK-8)

According to the manufacturer's instructions (Nanjing KeyGen Biotech), the CCK-8 method was used to detect the activity of HUVECs under the action of the extract of L. japonica. Human umbilical vein endothelial cells (2500 cells/well) were seeded in 48-well plates, with 6 replicates per group, and cultured in 100 μl of various concentrations of amphibiana extract (1, 10, 20, 50, 100 μg/ml) or serum-free cultured in medium for 24h. CCK8 solution (10 mL) was added to each well and incubated at 37°C for 2 h. Then, the absorbance at 450 nm of the supernatant was measured with a microplate reader (Victor X; PerkinElmer) (n=6/group).

8. Cell Permeability Analysis

In vitro permeability assays were performed using Transwell plates. HUVECs were seeded in the upper chamber with 200 μL of extracellular matrix, and the lower chamber was implanted with 500 μL of extracellular matrix. The various combinations of interventions described above were performed when the plates reached 100% monolayer cell coverage. Then, cells were incubated with FITC-dextran (1 mg/ml) for 1 h, and a microplate reader (Biotech, highland park, USA) was used to detect the fluorescence expression of the medium under excitation light of 494 nm and 521 nm. Finally, the values of each group were normalized to the control group.

9. Statistical analysis

All experiments were performed three times or more. GraphPad Prism 7.0 statistical software was used to analyze the data for mean ± standard deviation, and one-way ANOVA was used for significance test, followed by post hoc LSD test. P<0.05 was considered significant.

Figure 1 is a schematic diagram of BMP-2 promoting the inflammatory expression of HUVECs; cells were treated with Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum until the cell fusion rate reached about 80%. Cells were then treated with BMP-2 (0, 10 and 20ng/ml) for 24h. It can be seen from Figure 1 that BMP-2 can promote the expression of IL-1α, IL-1β and TNF-α at the gene and protein level.

Figure 2 is a schematic diagram of BMP-2 promoting the inflammatory pathway of HUVECs; all photos in the figure were taken with a 40x magnification. It can be seen from Figure 2 that BMP-2 can promote the protein expression of p-IκB and p-p65 and increase the expression of p65 into the nucleus.

Figure 3 is a schematic diagram of the increase of the permeability of HUVECs by BMP-2; the photos in (C) are all taken with a magnification of 40 times, and (D) is the calculation of the average fluorescence intensity of VE-cad in each microscopic field. Human umbilical vein endothelial cells were seeded on transwell plates in complete medium to achieve 100% monolayer cell coverage. These cells were then stimulated with BMP-2 by replacing the complete medium in the upper chamber with medium containing 1 mg/ml FITC-Dextran. Cells were cultured in a cell culture incubator for 2 h, (E) Dextran-permeable fluorescence intensity of HUVECs monolayer was measured by Transans-well assay and FITC-Dextran assay. It can be seen from Figure 3 that BMP-2 can inhibit the protein expression of p-VE-cad and Occludin and reduce the permeability of cells.

Figure 4 shows the effect of different concentrations of L. japonicus extract on cell viability. It can be seen from Figure 4 that the low and medium concentrations of L. japonicus will not affect the cell viability, while the excessively high concentration of L. japonicus extract will reduce the cell viability.

Fig. 5 is a schematic diagram showing the reduction of high inflammatory expression caused by BMP-2 by the extract of L. japonicus; it can be seen from Fig. 5 that L. japonicus can inhibit the expression of IL-1α, IL-1β and TNF-α at the protein level.

Figure 6 is a schematic diagram of the high expression of inflammatory pathway caused by the inhibition of BMP-2 caused by the extracts of the two-sided needle Fluorescence intensity and nuclear entry. It can be seen from Figure 6 that the two-sided needle reduces the expression of p-IκB and p-p65 protein levels, and reduces the nuclear entry of p65.

Figure 7 is a schematic diagram of the high permeability of cells caused by the reduction of BMP-2 by L. japonicus extract; wherein, human umbilical vein endothelial cells were seeded on transwell plates in complete medium to achieve 100% monolayer cell coverage . These cells were then stimulated with BMP-2 and ZN by replacing the complete medium in the upper chamber with medium containing 1 mg/ml FITC-Dextran. Cells were cultured in a cell culture incubator for 2 h. Figure (F) Dextran-permeable fluorescence intensity of monolayer HUVECs was measured by Transans-well assay and FITC-Dextran assay. It can be seen from Figure 7 that the two-sided needle increases the expression of p-VE-cad and occludin protein levels, and reduces the cell permeability.

FIG. 8 is a conceptual diagram of the reduction of BMP-2-induced cellular inflammation and high permeability by two-sided acupuncture. It can be seen from Figure 8 that BMP-2 induces the production of inflammatory factors, activates the NF-κB pathway signal, and reduces the expression of VE-cad and Occludin. Two-sided acupuncture can inhibit the production of inflammatory factors induced by BMP-2, inhibit the activation of NF-κB pathway signaling, and reduce the expression of VE-cad/Occludin.

To sum up, in the present invention, it was found that LMZ can inhibit the high expression of the inflammatory pathway caused by BMP-2, thereby reducing the production of cellular inflammatory substances and increasing the expression of tight junction proteins. It solved the inflammatory response around spinal fusion surgery caused by BMP-2 from the level of cellular mechanism, overcome the side effects existing in existing drug research, and provided a basis for optimizing the clinical application of BMP-2.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention.

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Claims (5)

1. The use of an extract of two-sided acupuncture in the preparation of a medicine for preventing and treating inflammatory reactions around spinal fusion surgery. 2 . The use according to claim 1 , wherein the inflammatory response is an inflammatory response around spinal fusion surgery caused by BMP-2. 3 . 3 . The use according to claim 1 , wherein the extract of L. japonicus is extracted from the roots or branches and leaves of the dicotyledon medicinal plant L. japonicus. 4 . 4 . The use according to claim 1 , wherein the active ingredient of the L. japonica extract is clinically and pharmaceutically acceptable L. japonica chlorinate. 5 . 5. purposes according to claim 1, is characterized in that, the effective dosage of described syringamine chloride is 10-100mg/kg/d.

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