阅读说明：本技术 萝卜硫素在制备抑制和/或治疗放射性肌纤维化的药物中的应用 (Application of sulforaphane in preparation of drugs for inhibiting and/or treating radioactive muscle fibrosis ) 是由 姜新 辛颖 杨格 王斌 赵钦 魏金龙 王欢欢 于 2021-09-30 设计创作，主要内容包括：本发明提供了萝卜硫素在制备抑制和/或治疗放射性肌纤维化的药物中的应用,涉及生物医药技术领域。本发明所述萝卜硫素(SFN)作为Nrf2激动剂,可通过干预氧化应激、炎症、细胞周期、增殖、凋亡及血管生成和转移等相关的信号传导通路发挥抑制肿瘤的效果,且萝卜硫素可保护多种组织和器官免于纤维化。本发明所述SFN可通过调控Nrf2介导的氧化应激和TGF-β1/Smad信号通路来抑制纤维化基因的表达,减少细胞外基质(ECM)的过度积累,从而显著改善放射引起的肌纤维化,对未来在临床上应用具有一定的意义。(The invention provides an application of sulforaphane in preparation of a medicine for inhibiting and/or treating radioactive muscle fibrosis, and relates to the technical field of biological medicines. The Sulforaphane (SFN) as an Nrf2 agonist can play a role in inhibiting tumors by interfering in relevant signal transduction pathways such as oxidative stress, inflammation, cell cycle, proliferation, apoptosis, angiogenesis and metastasis, and the like, and the sulforaphane can protect various tissues and organs from fibrosis. The SFN provided by the invention can inhibit the expression of fibrosis genes by regulating Nrf 2-mediated oxidative stress and TGF-beta 1/Smad signal pathways, and reduce the excessive accumulation of extracellular matrix (ECM), so that the muscle fibrosis caused by radiation is obviously improved, and the SFN has a certain significance for clinical application in the future.)

1. Application of sulforaphane in preparation of drugs for inhibiting and/or treating radioactive muscle fibrosis.

2. Application of sulforaphane in preparation of a preparation for up-regulating expression level of endogenous antioxidant substances.

3. The use of claim 2, wherein the endogenous antioxidant substances comprise expression proteins of downstream antioxidant genes CAT and HO-1 of Nrf2/ARE signaling pathway.

4. Application of sulforaphane in preparation of reagents for inhibiting TGF-beta 1/Smad signaling pathways.

5. A medicament for inhibiting and/or treating radioactive muscle fibrosis is characterized in that the effective component of the medicament comprises sulforaphane.

6. The medicament of claim 5, further comprising a pharmaceutically acceptable excipient.

7. The medicament of claim 5 or 6, wherein the dosage form of the medicament comprises an injection.

8. The pharmaceutical composition of claim 7, wherein the injection dose of the pharmaceutical composition is 0.5mg/kg of sulforaphane.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of sulforaphane in preparation of a medicine for inhibiting and/or treating radioactive muscle fibrosis.

Background

Radiotherapy kills tumor cells by Ionizing Radiation (IR) and causes different degrees of damage to normal tissues in the target area. However, the toxicity of long-term radiotherapy on normal tissues is still unavoidable, wherein the radioactive myofibrosis (RIMF) is one of the common serious long-term complications caused by radiotherapy, which not only has serious adverse effects on the appearance and the quality of life of patients, but also can cause interruption and delay of radiotherapy and even influence the treatment effect and the overall life cycle of patients. At present, an anti-fibrosis drug for preventing and reversing radioactive muscle fibrosis is not clinically approved, and a treatment strategy is only symptomatic support treatment, such as steroid hormone ointment of mometasone furoate, betamethasone and the like and emulsifiable paste of biyafen and the like, but the effect is not ideal, the anti-fibrosis drug only plays a certain role in prevention and treatment, and the requirements of clinical patients are difficult to meet.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of sulforaphane in preparation of a drug for inhibiting and/or treating radioactive muscle fibrosis, wherein the sulforaphane can mobilize and activate the endogenous antioxidant defense system of the body, and up-regulate a plurality of endogenous antioxidant substances to effectively prevent and treat the radioactive muscle fibrosis.

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

the invention provides application of sulforaphane in preparation of a medicament for inhibiting and/or treating radioactive muscle fibrosis.

The invention also provides application of the sulforaphane in preparation of a preparation for up-regulating the expression quantity of endogenous antioxidant substances.

Preferably, the endogenous antioxidant substances comprise expression proteins of downstream antioxidant genes CAT and HO-1 of a Nrf2/ARE signal path.

The invention also provides application of the sulforaphane in preparing a reagent for inhibiting TGF-beta 1/Smad signal pathways.

The invention also provides a medicament for inhibiting and/or treating the radioactive muscle fibrosis, and the effective component of the medicament comprises sulforaphane.

Preferably, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicament comprises an injection.

Preferably, the injection dosage of the medicine is 0.5mg/kg calculated by sulforaphane.

Has the advantages that: the invention provides an application of sulforaphane in preparation of a medicament for inhibiting and/or treating radioactive muscle fibrosis, wherein the Sulforaphane (SFN) serving as an Nrf2 agonist can play a role in inhibiting tumors by interfering in relevant signal transduction pathways such as oxidative stress, inflammation, cell cycle, proliferation, apoptosis, angiogenesis and metastasis, and the sulforaphane can protect various tissues and organs from fibrosis. The invention prevents the radioactive muscle fibrosis caused by radiation by using the safe SFN without side effect through the antioxidation effect of the SFN, and in the specific embodiment, the invention constructs a mouse radioactive muscle fibrosis and SFN treatment model, and explores the effectiveness of the SFN in preventing the radioactive muscle fibrosis in vivo. The result shows that the SFN can inhibit the expression of fibrosis genes by regulating Nrf 2-mediated oxidative stress and TGF-beta 1/Smad signal pathways, reduce excessive accumulation of ECM, thereby obviously improving the muscle fibrosis caused by radiation, and having certain significance for clinical application in the future.

Drawings

FIG. 1 is a graph of SFN-induced myofibrosis alleviating results, wherein A is a graph of H & E staining results (20 μm), B is a graph of Masson staining results (20 μm), and C is a statistical result of the area occupied by collagen fibers;

FIG. 2 is a graph showing the result of expression of ECM component inhibition by SFN (20 μm);

FIG. 3 is a graph showing the results of oxidative stress of skeletal muscle weakened by SFN, wherein A is a graph showing ELLISA results of ROS expression levels, and B and C are graphs showing Westernblot results of 3-NT;

FIG. 4 is a graph showing the results of strong inhibition of TGF- β 1 expression by SFN in RIMF mice;

FIG. 5 is a graph of the results of significant reduction of phosphorylated Smad2/3 protein expression in muscle tissue of mice in the IR/SFN group for SFN (20 μm);

fig. 6 is a graph (20 μm) of SFN activation Nrf2/ARE signal path results.

Detailed Description

The invention provides application of sulforaphane in preparation of a medicament for inhibiting and/or treating radioactive muscle fibrosis.

The Sulforaphen (SFN) is an Isothiocyanate (ITC) compound extracted from daily-eaten cruciferous vegetables, such as broccoli, cauliflower, kale and cabbage, and has a structure shown in a formula I:

in the embodiment of the invention, the radioactive muscle fibrosis animal model is used for verifying that after SFN is given, muscle fibers are arranged regularly, no central nuclear muscle fiber is seen, a small amount of fibrous connective tissue is generated in the interstitium, and inflammatory cells are dissipated; and the generation of collagen fibers is reduced, the gap between muscle fibers is reduced, the structure is regular, the proportion of the collagen fibers in the total area of the observed visual field is obviously reduced, and the SFN is prompted to obviously reduce the fibrosis degree of the muscle tissue of the model. Meanwhile, the SFN can also remarkably reduce the expression of collagen-I (NCBI ID:12842) and CTGF (NCBI ID:14219) in mouse muscle tissues, weaken the oxidative stress reaction of skeletal muscles, inhibit a TGF-beta 1/Smad signal pathway and activate an Nrf2/ARE signal pathway, thereby showing the remarkable effect of preventing and treating the radioactive muscle fibrosis. In the present invention, when the sulforaphane is used to act on a human body, the significant genes or proteins are all of human origin, collagen-I human origin (NCBI ID:1277), CTGF human origin (NCBI ID:1490), CAT human origin (NCBI ID:847), HO-1 human origin (NCBI ID: 3162).

The source of the SFN in the present invention is not particularly limited, and conventional commercial products in the field can be used, and can also be extracted or synthesized by itself, and in the examples, the SFN is preferably obtained from a leaf organism company.

The invention also provides application of the sulforaphane in preparation of a preparation for up-regulating the expression quantity of endogenous antioxidant substances.

The endogenous antioxidant substances preferably comprise expression proteins of downstream antioxidant genes CAT (NCBI ID:12359) and HO-1(NCBI ID:15368) of Nrf2/ARE signal channels.

The invention also provides application of the sulforaphane in preparing a reagent for inhibiting TGF-beta 1/Smad signal pathways.

TGF-beta 1/Smad is a key fibrosis-promoting signal pathway, and Western blot results in the embodiment of the invention show that the expression of TGF-beta 1 in muscle tissues of mice in an irradiation group (IR group) is remarkably increased compared with that in a model blank control group (CON group). The expression of TGF- β 1 was significantly reduced in muscle tissue of mice in the irradiation/SFN group (IR/SFN group) compared to the IR group, suggesting that SFN strongly inhibits TGF- β 1 expression in RIMF mice. The immunohistochemistry result shows that the SFN significantly reduces the expression of phosphorylated Smad2/3 protein in the muscle tissue of the IR/SFN group mice, while the expression level of the total Smad2/3 protein is not obviously changed, and in conclusion, the SFN inhibits the expression of a fibrotic gene by a TGF-beta 1/Smad signaling pathway.

The invention also provides a medicament for inhibiting and/or treating the radioactive muscle fibrosis, and the effective component of the medicament comprises sulforaphane.

The medicine of the invention preferably also comprises pharmaceutically acceptable auxiliary materials. The dosage form of the medicine provided by the invention preferably comprises an injection, and the injection dosage of the medicine is 0.5mg/kg calculated by sulforaphane.

The following examples are provided to illustrate the application of sulforaphane in the preparation of drugs for inhibiting and/or treating radioactive muscle fibrosis, but they should not be construed as limiting the scope of the present invention.

Example 1

Animals were grouped:

constructing an animal model: the animal model of the radioactive muscle fibrosis is established by a single irradiation of the left thigh skeletal muscle of the mouse using an X-RAD 320X-ray irradiator.

(1) Blank control group (CON): performing non-ray irradiation treatment, injecting corresponding dose of normal saline subcutaneously for 5 days continuously every week, continuously injecting for 4 weeks, and observing to 8 weeks;

(2) SFN group (SFN): non-radiation treatment, receiving subcutaneous injection of SFN (0.5mg/kg) in synchrony with IR/SFN group, injecting continuously for 4 weeks, observing to 8 weeks;

(3) illumination set (IR): the left thigh skeletal muscle of the mouse was irradiated with a single 40Gy dose of SFN formulated solvent (normal saline) on the day of irradiation for 5 consecutive days per week for 4 consecutive weeks, observed to week 8;

(4) irradiation/SFN group (IR/SFN): mice left thigh skeletal muscle received a single 40Gy radiation exposure and given SFN (0.5mg/kg) subcutaneous injection on the day of exposure for 5 consecutive days per week for 4 weeks, observed to week 8;

mice were sacrificed at week 8 after the start of the experiment and observed for muscle damage.

1. SFN mitigates radiation-induced muscle fibrosis: h & E staining was performed in the reference (PMID:32032580), and the results showed that muscular fiber atrophy occurred in the muscle tissue of the IR group mice, cell nuclei appeared in the center of the muscular fibers, and massive fibrous connective tissue proliferation occurred in the interstitium, with a small amount of inflammatory cell infiltration. Compared with the IR mice, the muscle fibers of the IR/SFN group are aligned, the central nuclear muscle fibers are not seen, a small amount of fibrous connective tissue is generated in the interstitium, and inflammatory cells are dissipated (A in figure 1). The Masson staining (PMID: 31481954) observed that the IR group slightly atrophied red muscle fibers, the number of blue collagen fibers increased significantly, the muscle fiber gaps widened, and the proportion of collagen fibers in the total area of the observed field of view increased, indicating a significant increase in fibrosis, compared to the CON group; in contrast, the IR/SFN group had reduced collagen fibril formation, reduced muscle fiber space, and a regular structure, with a significantly reduced proportion of collagen fibers in the total area of the observed field of vision, suggesting a significantly reduced degree of muscle fibrosis (B and C in FIG. 1).

2. SFN inhibits expression of ECM components: the results of Western Blot (reference PMID:32032580, PMID:28596000) showed that the levels of collagen-I were dramatically increased in the IR group compared to the CON group, while the expression of collagen-I was much lower in the IR/SFN group than in the IR group, and the results of IHC (reference PMID:32032580) were consistent with the results of Western Blot, indicating that SFN administration was able to down-regulate the expression of collagen-I in mouse muscle tissue. CTGF is effective in stimulating the division and proliferation of fibroblasts, promoting collagen synthesis and connective tissue proliferation, and causing muscle fibrosis. The significantly increased expression of CTGF in the IR group compared to the CON group, whereas CTGF expression was much lower in the IR/SFN group than in the IR group, indicating a significant inhibition of ECM expression by SFN (fig. 2).

3. SFN attenuates oxidative stress of skeletal muscle: elisa results showed that ROS expression levels were significantly increased in IR compared to CON group, and significantly decreased after SFN exposure (a in fig. 3). Meanwhile, the results of Westernblot showed that the oxidative stress injury index 3-nitrotyrosine (3-NT) was significantly increased in the muscle tissue of the IR group mice as compared to the CON group. The expression of 3-NT in the IR/SFN group was significantly reduced compared to the IR group (B and C in FIG. 3), indicating that radiation can cause oxidative muscle damage and increase the expression of 3-NT, while SFN can inhibit the production of ROS and damage caused by oxidative stress, thus exerting an antioxidant effect.

4. SFN inhibits the classical TGF-. beta.1/Smad signalling pathway: TGF-. beta.1/Smad is a key profibrotic signaling pathway. Western blot results showed that the expression of TGF-. beta.1 was significantly increased in muscle tissue of mice in the IR group compared to the CON group. The expression of TGF-. beta.1 was significantly reduced in muscle tissue of the mice in the IR/SFN group compared to the IR group, suggesting that SFN strongly inhibited TGF-. beta.1 expression in RIMF mice (A and B in FIG. 4). The immunohistochemistry results found that SFN significantly reduced phosphorylated Smad2/3 protein expression in muscle tissue of IR/SFN group mice, while total Smad2/3 protein expression level was not significantly changed (a and B in fig. 5). Together, the above results indicate that SFN inhibits the TGF-beta 1/Smad signaling pathway from inhibiting the expression of fibrotic genes.

5. SFN activates Nrf2/ARE signal path: western blotting results show that in muscle tissues of mice in an SFN group, the expression of Nrf2 is obviously increased, and the expression of Nrf2 in muscle tissues of mice in an IR group is obviously reduced compared with that of the SFN group; the expression of Nrf2 in muscle tissue of mice in the IR/SFN group was significantly higher than in CON and IR groups (a and B in fig. 6). In addition, compared with the CON group, the protein expression of antioxidant genes CAT and HO-1 downstream of Nrf2 in muscle tissues of mice in the SFN group is obviously increased; compared with the CON group, the IR group has no significant difference in the expression of the genes; whereas the protein expression was significantly increased for CAT and HO-1 in the IR/SFN groups compared to the IR group (A in FIG. 6, C, D and E in FIG. 6). The above results indicate that SFN is able to activate Nrf2/ARE signaling pathway in RIMF mice.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.