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CN110680905A - Application of TSLP in preparation of medicine for treating lumbar disc herniation - Google Patents

️Tue Jan 14 2020

Application of TSLP in preparation of medicine for treating lumbar disc herniation Download PDF

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Publication number

CN110680905A

CN110680905A CN201911016907.6A CN201911016907A CN110680905A CN 110680905 A CN110680905 A CN 110680905A CN 201911016907 A CN201911016907 A CN 201911016907A CN 110680905 A CN110680905 A CN 110680905A Authority

China

Prior art keywords

tslp

expression

tgf

beta

jak

Prior art date

2019-10-24

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Pending

Application number

CN201911016907.6A

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Chinese (zh)

Inventor

祝勇

杨学军

达逸峰

黄智�

王文磊

李莉

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Second Affiliated Hospital Of Inner Mongolia Medical University

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Second Affiliated Hospital Of Inner Mongolia Medical University

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2019-10-24

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2019-10-24

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2020-01-14

2019-10-24 Application filed by Second Affiliated Hospital Of Inner Mongolia Medical University filed Critical Second Affiliated Hospital Of Inner Mongolia Medical University

2019-10-24 Priority to CN201911016907.6A priority Critical patent/CN110680905A/en

2020-01-14 Publication of CN110680905A publication Critical patent/CN110680905A/en

Status Pending legal-status Critical Current

Links

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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

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Abstract

The invention discloses application of TSLP in preparation of a medicament for treating lumbar disc herniation. TSLP can promote infiltration of macrophages into disc tissues by inducing the expression of monocyte chemotactic factor-1 (MCP-1) to be increased, thereby promoting the resorption process of the herniated lumbar disc tissues.

Description

Application of TSLP in preparation of medicine for treating lumbar disc herniation

Technical Field

The invention relates to the field of medical research, in particular to application of TSLP in preparation of a medicament for treating lumbar disc herniation.

Background

Lumbar disc herniation refers to rupture of nucleus pulposus from the intervertebral disc caused by degenerative change of the lumbar disc or fiber rupture caused by trauma, and presses lumbar nerves, so that the lumbar disc herniation causes the symptom of the radiation pain, and is called as lumbar disc herniation. Currently, only a few patients with prolapse type intervertebral disc and prolapse of lumbar intervertebral disc, whose conservative treatment symptoms are not relieved, receive surgical treatment. Studies by Shujohn et al have shown that the herniated disc tissue can heal itself through a resorption process, but the underlying mechanism is not yet clear. The Wangzhi and Yuan Hu propose that macrophage infiltration participates in the process of reabsorption, so that the protruded intervertebral disc tissue can be obviously reduced. This may be associated with macrophage-mediated release of several factors, including Matrix Metalloproteinases (MMPs) and Vascular Endothelial Growth Factor (VEGF).

Thymic Stromal Lymphopoietin (TSLP) is an IL-7-like cytokine found in the supernatant of mouse thymus stromal cells by Friend et al in 1994, and is secreted mainly by non-hematopoietic cells, such as fibroblasts, epithelial cells, stromal cells, and the like. TSLP mRNA is mainly expressed in epithelial cells, stromal cells and mast cells of tissues such as thymus, lung, skin, small intestine, etc., and is closely related to asthma and airway allergic inflammatory reaction. Moret et al found that TSLP plays an important role in rheumatoid arthritis; koyama et al demonstrated increased expression of TSLP in vitro experimental studies of synovial fibroblasts from patients with rheumatoid arthritis and osteoarthritis, suggesting that TSLP is also expressed in bone tissue and involved in inflammatory responses. Ohba et al have demonstrated that TSLP is also expressed in mouse and human disc tissues, and that tumor necrosis factor-alpha (TNF-alpha) can up-regulate the expression of TSLP in vitro experiments by nuclear factor-kappa B (NF-kappa B), while TSLP can cause macrophage infiltration into disc tissues by promoting the expression of monocyte chemotactic factor-1 (MCP-1) and increased expression of TSLP is also found in disc tissues of patients with disc herniation, and TSLP is considered to be an important factor in mediating resorption. However, it is still unknown whether the body up-regulates the expression of TSLP and how to regulate TSLP after herniated disc.

Disclosure of Invention

The invention aims to provide application of TSLP in preparation of a medicament for treating lumbar disc herniation.

In a previous study, the applicant found that when the inflammatory environment of lumbar disc herniation is simulated by stimulating mouse disc tissues with the inflammatory factor TNF-alpha, an increase in TSLP and MCP-1 in the culture supernatant of the experimental group compared to the control group was observed by ELISA. The applicant thus assumes that: high expression of TSLP can facilitate the resorption process.

The invention researches the effect of TSLP expression on reabsorption of lumbar disc tissues and provides the application of TSLP in preparing a medicament for treating lumbar disc herniation.

Further, high expression of TSLP can promote resorption of the herniated lumbar disc tissue.

Compared with the prior art, the invention has the beneficial effects that: the invention provides that the high expression of TSLP can promote the reabsorption of the tissues of the herniated lumbar disc. The application of the compound in preparing the medicine for treating the lumbar disc herniation provides a new molecular theoretical basis for researching the pathophysiological process of the lumbar disc herniation in the future, and provides a new target for seeking the best anti-inflammatory time and conservatively treating the lumbar disc herniation by utilizing an organism autogenous repair mechanism in the future.

Drawings

FIG. 1 is a drawing of a rat nucleus pulposus cell culture at 2X magnification and at 4X magnification.

FIG. 2 is a graph showing the identification of nucleus pulposus cells by immunofluorescence assay, wherein A is the Collagen II stain (200-fold), B is the nuclear stain (200-fold), C is the Collagen II stain (400-fold), and D is the nuclear stain (400-fold).

FIG. 3 is a graph showing the phosphorylation levels of NF-. kappa.B in tissues detected by immunohistochemistry, showing positive nuclear staining in the brown-yellow or brown-brown color, and the ABC method x40, in which A is a control group, B is a TNF-. alpha.group, C is a TGF-. beta.group, and D is a TGF-. beta.inhibitor group (HTS group).

FIG. 4 shows the expression of p-NF-. kappa.B protein detected by immunoblotting (Western-blot) after culturing mouse intervertebral disc tissues for 0, 3, 6, 12 and 24 hours using 10ng/ml TNF-. alpha.10 ng/ml TGF-. beta.and 1uM HTS (TGF-. beta.pathway inhibitor).

FIG. 5 is a graph showing the detection of the expression of each gene by qRT-PCR technique under the action of IL-6 and JAK/STAT inhibitors (BP-1-102). A is the expression of JAK1, STAT3 and TSLP mRNA under the stimulation of IL-6(10 ng/ml); b is the expression of STAT3, TSLP mRNA under the action of JAK/STAT inhibitor (BP-1-102,500 nM). (represents P <0.05, represents P <0.01, represents P <0.001)

FIG. 6 is a graph showing the expression of TSLP protein detected by Western-blot technique under the action of IL-6 and JAK/STAT inhibitor (BP-1-102).

FIG. 7 is a graph showing the expression of TGF-. beta.in cell culture supernatants 72h after the action of IL-6, TGF-. beta./SMAD pathway inhibitor, JAK/STAT pathway inhibitor. (represents P <0.05)

Detailed Description

The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:

first, main material and reagent

DMEM/F12, trypsin, fetal bovine serum (Gibico, USA); trizol (Ambion, USA); reverse transcription kit (term, USA) qPCR kit (KAPA, switzerland); collagenase type II (biofvexx, sweden); mouse anti-Smad 2, rabbit anti-Smad 7, mouse anti-p-

STAT

3, rabbit anti-TSLP, HRP labeled secondary antibody (Shanghai Biotechnology engineering Co., Ltd., Shanghai, China); flasks, plates (NEST, Thermo, USA).

Second, separation and culture of rat nucleus pulposus cells

4-week-old female SD rats (provided by the animal testing center of Sigan university of transportation) were sacrificed by intraperitoneal overdose with anesthetic and soaked in 75% ethanol for 10 min. Separating the spine along the back on an aseptic experiment table, stripping the attached tissues around the spine, washing with aseptic PBS for 3 times, separating the intervertebral disc under a microscope (keeping the upper and lower end plates), cutting open the fibrous ring, carefully collecting the jelly-like nucleus pulposus tissue to a 1.5ml centrifuge tube, cleaning the collected nucleus pulposus tissue with PBS liquid containing antibiotics and antibiotics for 3 times, centrifuging at 1500rpm for 5min, and discarding the supernatant; adding 0.25% pancreatin, digesting at 37 deg.C for 30min, shaking once every 15min, stopping digestion with DMEM containing serum, centrifuging at 1000rpm for 5min, and removing supernatant; using DMEM/F12 (20% FPS, double antibody) culture medium containing 0.2% type II collagenase for heavy suspension, standing and digesting for 4h at 37 ℃, shaking once every 1h, filtering by using a 70um cell filter screen after the flocculent tissue disappears, centrifuging at 900rpm of filtrate for 5min, and discarding the supernatant; DMEM/F12 (20% FPS, double antibody) was resuspended, inoculated into a 25cm2 air-permeable flask, and placed in a CO2 incubator at 37 ℃ with saturated humidity and 5% volume fraction. The first fluid change was performed after 5 days, and the fluid change was performed every 2 days thereafter. Cell attachment and growth were observed under a microscope as shown in FIG. 1. When the rate of the rat nucleus pulposus cell adnexal growth fusion reaches 80%, subculturing the cells. The cells were washed twice with PBS, the supernatant was discarded, and the cells were digested with 0.25% trypsin. Observing under microscope, digesting for 1-2min to see that cells are separated from each other and become round, i.e. digestion is completed. Rapidly removing pancreatin, adding complete culture medium, blowing to make cells, preparing single cell suspension, subculturing according to the proportion of 1:4, carrying out amplification culture at 37 ℃ under the condition of 5% CO2 saturated humidity, and taking the nucleus pulposus cells of the second and third generations of rats for subsequent tests.

Thirdly, identifying nucleus pulposus cells by immunofluorescence labeling

Cultured rat nucleus pulposus cells in logarithmic growth phase are digested by 0.25% trypsin, collected in a 1.5ml EP tube, and after acidification treatment of a sterile slide, the cells are dropped on the slide. The cells were cultured in a 5% CO2 incubator at 37 ℃. When the cell wall attachment fusion rate reaches 80%, the slide is taken out, and the supernatant is discarded. PBS was used for 3 dips for 3min each. The slides were washed 3 times with PBS again after 15 minutes, 3min each time, using 4% paraformaldehyde drop wise. Excess paraformaldehyde is washed away. 0.5% Triton X-100 (in PBS) was allowed to permeate for 20min at room temperature. The slides were washed 3 times with PBS for 3min each time. And (3) sucking the redundant PBS by using absorbent paper, dripping the prepared normal goat serum on the slide, and sealing the slide at room temperature after the completion. After 30min, the blocking solution was again sucked off with absorbent paper, and without rinsing, a sufficient amount of rabbit anti-mouse type II collagen antibody and polyprotein antibody (1: 100) was again added dropwise to each slide and placed in a wet box and incubated overnight at 4 ℃. The next day, the slides were rinsed 3 times with PBS, 3min each time. The excess PBS on the slide was blotted dry with absorbent paper, and diluted fluorescent (Cy3) -labeled goat anti-rabbit IgG (1: 100) was dropped onto the slide, and all subsequent processing steps were transferred to the dark room from the addition of the fluorescent secondary antibody. The slide added with the secondary antibody is placed in a wet box and incubated for 1h at the temperature of 20-37 ℃. After being taken out, the glass slide is soaked and washed by PBS for 3 times, each time for 3min, and DAPI is dripped to stain the core of the specimen. After incubation in the dark for 5min, the slides were rinsed with PBS for 6 times, 5min each time, and excess DAPI was washed off; after completion, excess liquid on the slide was blotted with absorbent paper. Mounting the slide with mounting solution containing an anti-fluorescence quencher, and then observing the prepared slide under a fluorescence microscope and collecting an image, as shown in FIG. 2.

Fourthly, extracting the rat intervertebral disc tissue protein by freezing and grinding

Fifthly, real-time fluorescent quantitative PCR (qRT-PCR)

Adjusting cell number to 2.5X 10⁵Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing for 12h to 70%, adding IL-6(10ng/ml), TGF-beta pathway inhibitor (SB431542,150ng/ml), JAK/STAT pathway inhibitor (BP-1-102,500ng/ml), and alpha-MEM at 37 deg.C for control group, and 5% CO₂After culturing for 72h, mRNA is extracted by using a Trizol method, and the expression of JAK1, JAK2, STAT3, SMAD2/3/7 and TSLP mRNA is detected by using a qRT-PCR technology. The primers were synthesized by Beijing Optimalaceae New Biotechnology Co., Ltd (see Table 1 for primer sequences). The specific conditions are as follows: 95 ℃, 3min, 1 cycle; 40 cycles of 95 deg.C, 3s and 60 deg.C, 20s, using non-reagentDissociation curve determination by the anisotropic amplification method.

Sixth, the immunity mark test (Western-Blot)

Adjusting cell number to 2.5X 10⁵Cells/ml, seeded in 6-well plates at 37 ℃ with 5% CO₂Culturing for 12h to 70%, adding IL-6(10ng/ml) and TGF-beta pathway inhibitor (SB431542,150ng/ml) respectively, JAK/STAT pathway inhibitor (BP-1-102,500ng/ml) was cultured for 72 hours at 37 ℃ in 5% CO2, washed 3 times with PBS, cells were sufficiently lysed by adding cell lysate containing 1% protease inhibitor (PMSF) (whole procedure on ice), centrifuged at 12000rpm for 5 minutes at 4 ℃, separated on 10% SDS-polyacrylamide gel (SDS-PAGE) for 1 hour, wet-transferred to polyvinylidene fluoride membrane (PVDF membrane) for 1.5 hours, 5% skim milk powder was blocked at room temperature for 1 hour, primary antibody was added, incubated overnight at 4 ℃, washed 3 times with Tris-HCl buffer salt solution (TBST), incubated with HRP-labeled secondary antibody at room temperature for 1 hour, washed 3 times with TBST for 5 minutes each, and photographed by exposure.

Enzyme-linked immunosorbent assay (ELISA)

The supernatant of the 72-hour cell culture was collected, 100ul of the supernatant of IL-6(10ng/ml), TGF- β pathway inhibitor (SB431542,150ng/ml), JAK/STAT pathway inhibitor (BP-1-102,500ng/ml) was added to each well of the polystyrene plate, and the plate was kept at 4 ℃ overnight in the dark. The next day, incubation in the dark at 37 ℃ for 1h, discarding the supernatant, washing with 1% TBST for 3 times, 3min each time, adding primary antibody, incubation in the dark at 37 ℃ for 1h, washing with TBST for 3 times, adding secondary antibody, incubation in the dark at 37 ℃ for 1h, adding color-developing agent in the dark, developing at 37 ℃ for 30min, adding terminator and detecting the OD 450nm value on a spectrophotometer.

Statistical analysis

The database was built using Excel statistical software (Microsoft, USA) and statistical analysis was performed. The two groups of mean comparisons were tested with independent samples t, and differences of P <0.05 were statistically significant.

Analysis of results

One, the IL-6 mediated JAK/STAT signaling pathway promotes the resorption process of herniated disc tissue by modulating expression of TSLP.

Expression of JAK/STAT pathway-associated factors JAK1, STAT3, and TSLP mRNA following treatment of rat MSCs with IL-6, JAK/STAT pathway inhibitors (BP-1-102), respectively (see FIG. 5). IL-6 stimulation upregulates the expression of JAK1, STAT3, TSLP mRNA in rat cells. While STAT3, TSLP expression was significantly reduced in the presence of inhibition of the JAK/STAT pathway (BP-1-102). In terms of protein expression, a significant increase in TSLP protein expression was also observed with IL-6 stimulation using the immunoblot assay (Western-blot), whereas in the presence of JAK/STAT inhibitors, TSLP protein expression was significantly reduced (see FIG. 6). This suggests that the IL-6 mediated JAK/STAT signaling pathway is involved in the resorption process of herniated disc tissue by upregulating TSLP expression.

Secondly, the endogenous TGF-beta has the inhibition effect on the NF-kappa B pathway

Previous experiments of the applicant found that after rat intervertebral disc tissues are co-cultured with 10ng/ml TNF-alpha, 10ng/ml TGF-beta and 1uMHTS (TGF-beta pathway inhibitor) for 6 hours, the phosphorylation level of NF-kappa B in the tissues is detected by using an immunohistochemistry method, and the staining of cell nucleus is positive to brown or brown, and the result shows that: the TNF-alpha group had the highest number of positive cells, followed by the TGF-beta inhibitor group (HTS group), while only a few positive cells were seen in the TGF-beta group and the control group, as shown in FIG. 3. Only a small amount of phosphorylated NF-kappa B positive cells, 9.4% + -3.3%, were seen in the control group; the most phosphorylated NF-kappa B positive cells of the BTNF-alpha group are 92.4% +/-1.4%; the TGF-beta group expresses a small number of phosphorylated NK-kappa B positive cells which are 33.7% +/-8.8%; the number of positive cells in the TGF-. beta.inhibitor group (HTS group) was 74.1%. + -. 6.7% second only in the TNF-. alpha.group.

The differences in each group were statistically significant (F: 167.669, P: 0.000); any two groups of comparison differences were statistically significant (mean P ═ 0.000).

Murine intervertebral disc tissue was co-cultured with 10ng/ml TNF-. alpha.10 ng/ml TGF-. beta.1 uM HTS (TGF-. beta.pathway inhibitor) for 0, 3, 6, 12, 24h, then histones were extracted by cryomilling and the expression of phosphorylated NF-. kappa.B protein was detected using immunoblot assay (Western-blot), as shown in FIG. 4: after 3 and 6 hours of culture, compared with a control group, the expression of phosphorylated NF-kappa B protein is obviously increased in a TNF-alpha group and a TGF-beta inhibitor group (HTS group), which indicates that endogenous TGF-beta can inhibit the phosphorylation of NF-kappa B.

The results show that after 3 and 6 hours of culture, the expression of phosphorylated NF-kB is obviously increased in a TGF-beta inhibitor group (HTS group) and a TNF-alpha group compared with a control group, and the inhibition of the activity of TGF-beta (HTS group) and the TNF-alpha group have the effect of inducing the expression of phosphorylated NF-kB protein.

And thirdly, the influence of IL-6 on the expression of TGF-beta in nucleus pulposus cells of rats.

Rat nucleus pulposus cells were cultured by using IL-6(10ng/ml), TGF-beta pathway inhibitor (SB431542,150ng/ml) and JAK/STAT pathway inhibitor (BP-1-102,500ng/ml), respectively, and cell culture supernatants were collected at 72 hours, and the TGF-beta concentration in the supernatants was measured by enzyme-linked immunosorbent assay (ELISA), and the results showed that: under IL-6 stimulation, the TGF-beta concentration in the cell culture supernatant was increased, and the TGF-beta pathway inhibitor group and the JAK/STAT pathway inhibitor group were decreased compared with the control group (see FIG. 7).

Compared with the control group, the concentration of TGF-beta in the cell culture supernatant fluid is increased under the stimulation of IL-6; a decrease in TGF-. beta.concentration in the supernatant was observed following inhibition of the TGF-. beta.and JAK/STAT pathways.

Conclusion of the experiment

First, IL-6 regulates the expression of TSLP through the JAK/STAT signal pathway and participates in the reabsorption of herniated disc tissue.

Studies have shown that TSLP can promote infiltration of macrophages into disc tissue by inducing increased expression of monocyte chemotactic factor-1 (MCP-1), thereby promoting the resorption process of herniated disc tissue. In the experiment, the applicant observes that JAK1, STAT3 and TSLP mRNA is in a high expression state after exogenous IL-6 stimulation is added for 72 hours, and the expression of JAK/STAT pathway key factors STAT3 and TSLP mRNA is obviously reduced after JAK/STAT pathway inhibitor (BP-1-102) is added. Immunoblotting experiments also demonstrated a significant increase in TSLP protein expression following stimulation with added IL-6. This suggests that IL-6 induces an increase in expression of TSLP through the JAK/STAT signaling pathway, and is involved in the resorption process of disc herniation.

And secondly, IL-6 influences the activity of a TGF-beta signal channel through a JAK/STAT signal channel, thereby influencing the expression of TSLP and further participating in the reabsorption process of the intervertebral disc protrusion.

Applicants' studies demonstrate that under physiological conditions the TGF- β signaling pathway in disc tissue assumes a certain activation state and inhibits TSLP expression. In this experiment, the applicant observed that the mRNA expression of Smad2, a TGF-beta pathway marker, was significantly increased after IL-6 stimulation, while the expression of Smad2 was significantly decreased after JAK/STAT pathway inhibitor addition (BP-1-102), and the immunoblot assay (Western-blot) also showed a significant increase in Smad2 protein expression after IL-6 addition. At the same time, we observed an increase in TGF- β concentration in cell culture supernatants after IL-6 addition (P <0.05) and a decrease in TGF- β concentration in supernatants after JAK/STAT pathway inhibition (P <0.05) in enzyme-linked immunosorbent assays (ELISA). It follows that under IL-6 stimulation, the JAK/STAT signaling pathway is activated, which in turn leads to activation of the TGF- β pathway.

Under normal physiological conditions, TSLP expression continues to be inhibited, and this inhibition is caused by the continuous, minimal expression of TGF- β by disc tissue. When the lumbar disc herniates, this inhibition is broken, TSLP expression is elevated, which in turn elevates MCP-1 expression, which can induce macrophage infiltration into the herniated disc tissue, causing and promoting the resorption process. In the experiment, the applicant finds that the inflammatory factor IL-6 can activate JAK/STAT pathway to up-regulate TSLP expression, which provides a new thought for researching inflammation generation and reabsorption mechanism of the lumbar intervertebral disc protrusion and a new target for conservative treatment of the lumbar intervertebral disc protrusion. In addition, the IL-6/JAK/STAT family affects the TGF-. beta./Smads family, suggesting a potentially important role for IL-6 in maintaining disc local microenvironment homeostasis.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.

Sequence listing

<110> second subsidiary hospital of inner Mongolia medical university

Application of <120> TSLP in preparation of medicine for treating lumbar disc herniation

<160>12

<170>SIPOSequenceListing 1.0

<210>1

<211>24

<212>DNA/RNA