Application of TFEB agonist in preparation of medicine for treating intervertebral disc degeneration

Abstract

The invention relates to the technical field of drugs for treating intervertebral disc degeneration, in particular to application of a TFEB agonist in preparation of drugs for treating intervertebral disc degeneration. The key regulation effect of the transcription factor TFEB in intervertebral disc degeneration is provided. Researches on intervertebral disc specimens with different degeneration degrees and rat and mouse models find that the expression level of TFEB is obviously reduced along with the increase of the degeneration degree of the intervertebral disc. In addition, abnormal mechanical stresses and other stimuli result in a significant reduction in TFEB expression in nucleus pulposus cells, accompanied by disorders of lipid metabolism. Therefore, the TFEB plays a key role in maintaining the lipid metabolism homeostasis of the nucleus pulposus cells. The deletion of TFEB aggravates abnormal mechanical load and age-related disc degeneration and leads to a disorder in the biological behavior of nucleus pulposus cells. On the contrary, overexpression of TFEB can significantly relieve the degeneration phenomenon, and normal functions and lipid metabolism balance of nucleus pulposus cells are recovered.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating intervertebral disc degeneration, and in particular to the use of a TFEB agonist in preparing a drug for treating intervertebral disc degeneration. Background Art

[0002] The transcription factor TFEB belongs to the MiTF family of transcription factors and has motif structures such as glutamine-rich, helix-loop-helix and leucine zipper. Under normal conditions, TFEB exists in the cytoplasm in a phosphorylated form and is in an inactive state; when cells are stimulated by external stimuli (such as starvation, lysosomal dysfunction), TFEB is dephosphorylated and enters the nucleus, and recognizes specific DNA elements to initiate the transcription of corresponding target genes to exert its biological effects. On the one hand, TFEB can promote lipid droplet autophagy by regulating the biosynthesis and function of lysosomes, thereby regulating lipid metabolism; on the other hand, TFEB can directly regulate the expression of genes related to lipid catabolism to regulate lipid metabolism.

[0003] Curcumin is a natural compound isolated from turmeric and other ginger plants. It has multiple physiological activities such as antioxidant and anti-inflammatory, as well as good therapeutic effects on a variety of diseases. However, its bioavailability in the body is limited, which seriously limits its clinical application. The prior art screened a series of synthetic monocarbonyl analogs of curcumin and found that the curcumin analog 1,5-bis(2-methoxyphenyl)penta-1,4-diene-3-one (TA for short) has good brain tissue bioavailability, can directly bind to TFEB and promote TFEB nuclear entry. For example, Chinese patent 202110429206.6 discloses small molecules that curcumin enhances autophagy and lysosomal biosynthesis through TFEB. Small molecules can prevent the accumulation of toxic protein aggregates to treat neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease.

[0004] The prior art does not disclose the role of TFEB in regulating intervertebral disc degeneration. The technical problem to be solved by the present invention is how to provide a drug for treating intervertebral disc degeneration. Summary of the invention

[0005] In view of the existing phenomenon, the present invention provides the use of a TFEB agonist in the preparation of a drug for treating intervertebral disc degeneration, which at least solves one of the above technical problems.

[0006] The first technical solution provided by the present application is the use of a TFEB agonist in the preparation of a drug for treating intervertebral disc degeneration.

[0007] Furthermore, TFEB agonists can treat intervertebral disc degeneration by activating TFEB to regulate lipid metabolism.

[0008] Furthermore, TFEB agonists are used to improve lipid metabolism disorders in nucleus pulposus cells caused by abnormal mechanical loading.

[0009] Furthermore, TFEB agonists are used to alleviate decreased viability and / or apoptosis of nucleus pulposus cells caused by abnormal mechanical load.

[0010] Furthermore, TFEB agonists are used to promote matrix synthesis and metabolism of nucleus pulposus cells.

[0011] Furthermore, TFEB agonists are used to alleviate disc height loss.

[0012] Furthermore, the TFEB agonist is a curcumin analog, and the curcumin analog has the following structure: .

[0013] Furthermore, the drug for treating intervertebral disc degeneration is administered by at least one of oral administration, intravenous injection or local blocked injection.

[0014] Furthermore, when the curcumin analog is administered orally, it is administered in an amount of 1-20 mg per kg of the body weight of the subject.

[0015] The second technical solution provided by the present application is a composition for treating intervertebral disc degeneration, comprising an agonist that promotes the expression of TFEB transcription factor.

[0016] Furthermore, the agonist that promotes the expression of TFEB transcription factor is a curcumin analog, and the curcumin analog has the following structure: .

[0017] The beneficial effects of this application are: The key regulatory role of the transcription factor TFEB in intervertebral disc degeneration is provided. Through the study of intervertebral disc specimens with different degrees of degeneration and rat and mouse models, it was found that the expression level of TFEB decreased significantly with the increase of the degree of intervertebral disc degeneration. In addition, abnormal mechanical stress and other stimuli led to a significant decrease in TFEB expression in nucleus pulposus cells, accompanied by lipid metabolism disorders. This shows that TFEB plays a key role in maintaining the lipid metabolism homeostasis of nucleus pulposus cells. The loss of TFEB exacerbates abnormal mechanical load and age-related intervertebral disc degeneration and leads to disorders in the biological behavior of nucleus pulposus cells. On the contrary, overexpression of TFEB can significantly alleviate these degenerative phenomena and restore the normal function and lipid metabolism balance of nucleus pulposus cells. That is, TFEB can protect nucleus pulposus cells from abnormal mechanical loads by regulating nucleus pulposus cell lipid metabolism-related genes and lipid droplet autophagy.

[0018] On this basis, the present application discloses the application of curcumin monocarbonyl analogue TA as a TFEB agonist in the treatment of intervertebral disc degeneration. TA can promote TFEB entry into the nucleus and increase its expression level in nucleus pulposus cells, effectively alleviate lipid metabolism disorders and cell biological behavior disorders induced by abnormal mechanical stress, and ultimately reduce intervertebral disc degeneration. This shows that TA, as a drug with clinical application potential, can be used to reduce the pain and burden of patients with low back pain and improve the quality of life of patients.

[0019] In summary, this disclosure reveals the key role of TFEB in intervertebral disc degeneration and its mechanism of regulating lipid metabolism, verifies the value of TFEB agonist TA in the prevention and treatment of intervertebral disc degeneration, and provides a new theoretical basis and treatment idea for the treatment of intervertebral disc degeneration-related diseases, which is expected to reduce patients' disease burden and medical costs.

[0020] In addition, the present disclosure has important guiding significance for the research and treatment of other degenerative diseases. Due to the extensive role of TFEB in lipid metabolism, lysosomal function and cellular stress response, it may play a role in a variety of diseases. For example, TFEB may play an important regulatory role in diseases related to lipid metabolism disorders such as atherosclerosis, fatty liver, metabolic syndrome, etc. In addition, in neurodegenerative diseases, TFEB may help to clear the accumulation of pathological proteins in cells and slow down the degeneration of neurons by regulating lysosomal function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate embodiments of the present invention and are used together with the description to explain the principles of the present invention. The drawings described below are some embodiments of the present invention, but not all embodiments. For those of ordinary skill in the art, other drawings can be obtained from these drawings without creative work.

[0022] Figure 1 These are X-ray and MRI images of the rat coccygeal disc degeneration model (CMP group); Figure 2 These are the X-ray and MRI images of the lumbar instability model (LSI group) and the coccygeal puncture model (CSI group).

[0023] Figure 3 HE and SOFG staining images of intervertebral disc specimens in the LSI and CSI groups (left) and immunofluorescence detection of TFEB expression and localization changes (right).

[0024] Figure 4 These are MRI images of the CMP group rats 2 weeks after the TFEB overexpression adenovirus Ad-TFEB virus was injected into the intervertebral disc.

[0025] Figure 5 This is the HE / SOFG staining result 2 weeks after the TFEB overexpressing adenovirus Ad-TFEB virus was injected into the intervertebral disc of CMP group rats.

[0026] Figure 6 Representative immunofluorescence staining results of ACAN, COL2, ADAMTS4 and MMP3 were performed on normal nucleus pulposus cells, abnormal mechanical load-induced nucleus pulposus cells, and abnormal mechanical load-induced nucleus pulposus cells transfected with TFEB overexpression adenovirus.

[0027] Figure 7 This is a graph showing the results of a CCK8-cell viability assay on TA-treated human and rat nucleus pulposus cells.

[0028] Figure 8 The results of the Annexin V / PI staining experiment on TA-treated human and rat nucleus pulposus cells.

[0029] Fig. 9 The figure shows the X-ray examination results of mice in the LSI and CSI groups after oral administration of TA at different doses (0 mg / kg, 10 mg / kg, and 25 mg / kg).

[0030] Fig.10 These are the results of X-ray (left), MRI (middle), and HE / SOFG staining (right) examinations of rats in the CMP group that were treated with oral gavage of TA at different doses (0 mg / kg, 10 mg / kg, and 25 mg / kg). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present application provides a first technical solution which is the use of a TFEB agonist in the preparation of a drug for treating intervertebral disc degeneration.

[0033] In some embodiments, TFEB agonists treat intervertebral disc degeneration by activating TFEB to regulate the pathway of lipid metabolism.

[0034] In some embodiments, TFEB agonists are used to improve lipid metabolism disorders in nucleus pulposus cells caused by abnormal mechanical loading.

[0035] In some embodiments, TFEB agonists are used to alleviate decreased viability and / or apoptosis of nucleus pulposus cells caused by abnormal mechanical loading.

[0036] In some embodiments, TFEB agonists are used to promote matrix synthesis and metabolism in nucleus pulposus cells.

[0037] In some embodiments, TFEB agonists are used to alleviate disc height loss.

[0038] In some embodiments, the TFEB agonist is a curcumin analog having the following structure: .

[0039] In some embodiments, the drug for treating intervertebral disc degeneration is administered by at least one of oral administration, intravenous injection, or local blocked injection.

[0040] In some embodiments, when the curcumin analog is administered orally, it is administered in an amount of 1-20 mg / kg body weight of the subject.

[0041] The second technical solution provided by the present application is a composition for treating intervertebral disc degeneration, comprising an agonist that promotes the expression of TFEB transcription factor.

[0042] In some embodiments, the agonist that promotes the expression of TFEB transcription factor is a curcumin analog, and the curcumin analog has the following structure: .

[0043] Preparation of experimental animals: Based on the model designed by Japanese scholars Takashi Yurube et al., the rats' coccygeal intervertebral disc degeneration models (CMP group) with different degrees of degeneration were established by applying 1.3Mpa continuous pressure to the intervertebral discs of the C8 / 9 and C9 / 10 segments of the rats' coccygeal vertebrae for 1, 2, 3, and 4 weeks. X-ray and MRI imaging tests were performed on the rats' coccygeal vertebrae at the corresponding time points. The results were as follows: Figure 1 , showing that: as the pressurization time increases, the intervertebral disc height of the pressurized segment gradually decreases, and the water content of the intervertebral disc also decreases significantly. As the degree of degeneration increases, the nucleus pulposus becomes irregular in shape and its area decreases. The number of nucleus pulposus cells decreases, the morphology becomes clustered, and the vacuoles disappear. The nucleus pulposus and annulus fibrosus are unclear, the annulus fibrosus lamellar structure is disordered, partially torn, and local ossification occurs in the end plate. At the same time, TFEB immunofluorescence staining found that as the pressurization time continues, the degree of disc degeneration increases, and the TFEB expression level decreases, especially the nuclear positive ratio decreases.

[0044] The stability of the L2 / 3-L4 / 5 lumbar vertebrae of the mouse intervertebral disc was destroyed to obtain a lumbar instability model (LSI group), and the stability of the C5 / 6-C7 / 8 coccygeal vertebrae was destroyed to obtain a coccygeal puncture model (CSI group), and the mouse intervertebral disc degeneration model was established. At the corresponding time points, the mouse intervertebral disc was examined by X-ray and MRI imaging. The degree of degeneration was as follows: Figure 2 .

[0045] The intervertebral disc specimens of the LSI and CSI groups were collected and stained with HE and SOFG, and the expression and localization of TFEB were detected by immunofluorescence. Figure 3 The results showed that compared with the sham group, the intervertebral disc height in the LSI and CSI groups decreased, and typical degenerative changes such as a decrease in the number of nucleus pulposus cells appeared. The TFEB expression level was significantly decreased, and the proportion of nuclear positive cells was significantly decreased.

[0046] Quantitative metabololipidomics analysis of the intervertebral disc cells of rats in the CMP group revealed that the contents of various lipid subclasses in the intervertebral disc cells changed significantly, among which cholesterol ester (TC) and triglyceride (TG) increased significantly.

[0047] Example 1 The present invention designs and constructs adenovirus overexpressing TFEB (Ad-TFEB), and injects Ad-TFEB virus into the intervertebral disc of rats in the CMP group. The MRI results after 2 weeks are as follows: Figure 4 TFEB overexpression significantly reduced the grade of degeneration induced by abnormal mechanical loading. The specimens were collected and stained with HE / SOFG. The results were as follows Figure 5 It can be found that TFEB overexpression significantly reduces tissue degeneration induced by abnormal mechanical loading. These results indicate that TFEB overexpression significantly alleviates intervertebral disc degeneration induced by abnormal mechanical loading.

[0048] Example 2 To further explore the role of TFEB in the biological behavior of nucleus pulposus cells, the biological behavior of nucleus pulposus cells transfected with TFEB overexpression adenovirus (Ad-Tfeb) was detected. CCK8-cell viability assay showed that TFEB overexpression alleviated the decrease in nucleus pulposus cell viability induced by abnormal mechanical load. Annexin V / PI staining experiments showed that nucleus pulposus cell apoptosis induced by abnormal mechanical load could be significantly reduced by Ad-TFEB treatment. The results showed that TFEB overexpression significantly alleviated the disorder of nucleus pulposus cell biological behavior induced by abnormal mechanical load.

[0049] Example 3 Biochemical detection of triglyceride (TG), cholesterol ester (CE), and free fatty acid (FFA) as well as Oil Red O and Nile Red staining were performed on nucleus pulposus cells transfected with TFEB overexpression adenovirus, showing that abnormal mechanical load induced an increase in TG and CE levels and a decrease in FFA levels in nucleus pulposus cells. TFEB overexpression (Ad-Tfeb) effectively alleviated the accumulation of TG and CE and the decrease in FFA caused by abnormal mechanical load. Ad-Tfeb treatment significantly alleviated the accumulation of lipid droplets induced by abnormal mechanical load. These results show that TFEB overexpression alleviates the disorder of lipid metabolism indicators caused by abnormal mechanical load.

[0050] Immunoblotting and lysosome-lipid droplet confocal analysis of lipid droplet metabolism-related proteins were performed on nucleus pulposus cells of CMP group rats transfected with TFEB overexpression adenovirus. Immunoblotting experiments showed that Ad-Tfeb could alleviate the decrease of LAMP2 and the upregulation of PLIN2 induced by abnormal mechanical load, and could significantly increase the levels of lipid metabolism-related molecules LAL and ATGL, and upregulate lipid metabolism-related transcription factor PPARα. This indicates that TFEB overexpression can alleviate the lipid droplet-related metabolic molecule disorder induced by abnormal mechanical load. Furthermore, Ad-Tfeb alleviates lipid accumulation induced by abnormal mechanical load and increases the co-localization level of lysosomes and lipid droplets. This indicates that TFEB overexpression can alleviate the lipid droplet accumulation induced by abnormal mechanical load by upregulating lipophagy.

[0051] Example 4 Representative immunofluorescence staining experiments of ACAN, COL2, ADAMTS4 and MMP3 were performed on normal nucleus pulposus cells, nucleus pulposus cells induced by abnormal mechanical load, and nucleus pulposus cells transfected with TFEB overexpression adenovirus. The results are shown in Figure 6 , showing that abnormal mechanical load induced a decrease in ACAN and COL2 levels and an increase in ADAMTS4 and MMP3 levels in nucleus pulposus cells. Abnormal mechanical load induced ACAN, COL2, ADAMTS4 and MMP3 levels in nucleus pulposus cells transfected with TFEB overexpression adenovirus to be close to those in normal nucleus pulposus cells, indicating that TFEB overexpression can improve cell matrix synthesis and metabolism.

[0052] Example 5 The present application also provides a TFEB-specific agonist, namely a curcumin analog: 1,5-bis(2-methoxyphenyl)penta-1,4-diene-3-one, referred to as TA, having the following structure: .

[0053] Human (HNP) and rat nucleus pulposus cells (RNP) were treated with different doses of TA (0.1 / 0.3 / 0.6 / 1.2 μM), and their nuclear proteins were extracted and subjected to immunoblotting experiments. It was found that TA treatment could increase their TFEB nuclear protein levels in a dose-dependent manner, and reached the maximum at a dose of 1.2 μM. Immunofluorescence experiments confirmed that TA treatment could increase its TFEB protein level and nuclear localization level in a dose-dependent manner. These results indicate that TA is a specific agonist that effectively promotes TFEB nuclear entry and expression.

[0054] Example 6 In order to verify the protective effect of TA on nucleus pulposus cells, the present disclosure conducted lipid metabolism-related tests on human and rat nucleus pulposus cells after TA treatment. Biochemical detection experiments of triglycerides TG, cholesterol esters CE and free fatty acids FFA showed that abnormal mechanical load induced a significant increase in TG and CE levels in nucleus pulposus cells and a significant decrease in FFA levels, while TG / CE levels in nucleus pulposus cells treated with TA were significantly downregulated and FFA returned to normal. At the same time, Oil Red O / Nile Red staining showed that TA treatment can significantly improve the number of lipid droplets increased due to abnormal mechanical load.

[0055] Example 7 To further explore the effect of TA on lipid droplet metabolism in nucleus pulposus cells, immunoblotting and lysosome-lipid droplet confocal analysis of lipid droplet metabolism-related proteins were performed on nucleus pulposus cells treated with TA. Immunoblotting experiments showed that TA treatment could alleviate the decrease of LAMP2 and the upregulation of PLIN2 induced by abnormal mechanical load, and could significantly increase the levels of lipid metabolism-related molecules LAL and ATGL, and upregulate lipid metabolism-related transcription factor PPARα. Further, fluorescence confocal analysis showed that TA treatment alleviated lipid accumulation induced by abnormal mechanical load and increased the number of lysosomes. This indicates that TA can promote TFEB-mediated lipophagy to alleviate lipid droplet accumulation induced by abnormal mechanical load.

[0056] Example 8 To verify the effect of TA on the biological behavior of nucleus pulposus cells, biological behavior-related tests were performed on human and rat nucleus pulposus cells treated with TA. The results of the CCK8-cell viability test are summarized as follows Figure 7 TA treatment alleviated the abnormal mechanical load-induced decrease in nucleus pulposus cell viability in a dose-dependent manner. The results of Annexin V / PI staining experiments are summarized as follows Figure 8, showing that abnormal mechanical load-induced apoptosis of nucleus pulposus cells can be significantly reduced by TA treatment in a dose-dependent manner. Further, immunofluorescence staining was used to analyze the expression levels of matrix metabolism-related proteins in nucleus pulposus cells treated with TA, and the results confirmed that TA treatment alleviated the downregulation of matrix anabolic molecules Aggrecan / COL2A1 and upregulation of decomposition molecules MMP13 / ADAMTS5 induced by abnormal mechanical load. These results indicate that TA treatment significantly alleviates the biological behavior disorder of nucleus pulposus cells induced by abnormal mechanical load.

[0057] Example 9 The present invention uses the prepared TFEB specific agonist TA in an intervertebral disc degeneration model. After oral administration of TA at different doses to mice in the LSI and CSI groups, the X-ray results are as follows: Fig. 9 , showing that TA alleviated the decrease in intervertebral disc height and was comparable to the level of the sham operation group at 10 mg / kg.

[0058] After different doses of TA were given to rats in the CMP group through oral gavage, X-ray / MRI examinations showed that the decrease in intervertebral disc height and T2 signal intensity induced by abnormal mechanical load could be alleviated by TA, which was comparable to the level of the sham operation group at a dose of 25 mg / kg. Furthermore, HE / SOFG staining also showed that TA effectively alleviated the tissue morphological changes induced by abnormal mechanical load. The results of X-ray / MRI examinations and HE / SOFG staining are summarized as follows: Fig.10 , these results suggest that oral administration of TA effectively alleviates disc degeneration induced by abnormal mechanical loading.

[0059] According to the dosage conversion relationship between animals and humans, the effective human dosage of TA for oral administration is about 1 mg / kg (body weight) to 28 mg / kg (body weight) per day. Preferably, the effective human dosage of TA for oral administration is recommended to be 1 mg / kg (body weight) to 20 mg / kg (body weight) per day.

[0060] The contents described above may be implemented individually or in combination in various ways, and these variations are all within the protection scope of the present invention.

[0061] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the statement "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device comprising the elements.

[0062] The above contents are further detailed descriptions of the present invention in combination with specific preferred implementation modes, and it cannot be determined that the specific embodiments of the present invention are limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Claims

1. Use of TFEB agonists in the preparation of drugs for treating intervertebral disc degeneration.

2. The use according to claim 1, characterized in that: The TFEB agonist is used to improve lipid metabolism disorder in nucleus pulposus cells caused by abnormal mechanical load.

3. The use according to claim 1, characterized in that: The TFEB agonist is used to alleviate the decrease in nucleus pulposus cell activity and / or nucleus pulposus cell death caused by abnormal mechanical load.

4. The use according to claim 1, characterized in that: The TFEB agonist is used to promote matrix synthesis and metabolism of nucleus pulposus cells.

5. The use according to claim 1, characterized in that: The TFEB agonist is used to alleviate the loss of intervertebral disc height.

6. The use according to claim 1, characterized in that: The TFEB agonist is a curcumin analog, and the curcumin analog has the following structure: 。 7. The use according to claim 6, characterized in that: The drug for treating intervertebral disc degeneration is administered by at least one of oral administration, intravenous injection or local blocked injection.

8. The use according to claim 6, characterized in that: When the curcumin analog is administered orally, it is administered in an amount of 1-20 mg per kg of the body weight of the subject.

9. A composition for treating intervertebral disc degeneration, characterized in that: These include agonists that promote expression of the TFEB transcription factor.

10. The composition according to claim 9, characterized in that The agonist for promoting the expression of TFEB transcription factor is a curcumin analog, and the curcumin analog has the following structure: 。

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