Application of resveratrol in improving and / or treating bladder fibrosis

Abstract

The invention discloses application of resveratrol in preparation of a medicine for improving and / or treating bladder fibrosis and the medicine for improving and / or treating bladder fibrosis, and the active ingredient of the medicine is resveratrol. Researches prove that resveratrol inhibits apoptosis of bladder epithelial cells, improves bladder functions, improves neurogenic bladder fibrosis, provides more therapeutic drug choices for patients, can be applied to treatment of neurogenic bladder through a variety of administration routes, and has a wide application prospect. Meanwhile, personalized treatment can be conveniently carried out according to the specific conditions of the patient.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of resveratrol in improving and / or treating bladder fibrosis. Background Art

[0002] Neurogenic bladder (NB) refers to bladder or urethral dysfunction caused by central or peripheral nervous system lesions, which in turn produces a series of lower urinary tract symptoms and complications. NB leads to high pressure in the bladder, decreased compliance, bladder fibrosis and vesicoureteral reflux, and ultimately leads to renal failure. As the duration of the disease increases, the degree of bladder fibrosis in most NB patients becomes more and more serious. Previous studies have shown that bladder fibrosis is one of the main causes of bladder dysfunction and upper urinary tract damage, and is an important factor affecting the treatment effect of NB.

[0003] Bladder fibrosis is a pathological change in which excessive extracellular matrix is ​​deposited in the bladder wall, leading to bladder storage and urination dysfunction. Collagen fibers are deposited in large quantities in the bladder interstitium or muscle, resulting in reduced bladder compliance and detrusor dysfunction. The pathological process of NB fibrosis involves a variety of bladder parenchyma and interstitial cells. It has been found that epithelial cells play an important role in the fibrosis of many tissues, and bladder epithelial cells, as an important component of the bladder, are one of the main cells responsible for synthesizing extracellular matrix. Bladder epithelial cells are also the main target of various injuries. Studies have shown that their continuous damage will promote the production and release of bioactive mediators that induce bladder fibrosis. After the bladder is damaged, the interaction between epithelial cells is gradually lost due to the inhibition of tight junction proteins, and then acquires the mesenchymal morphology of myofibroblasts, which has stronger migration ability and the ability to produce extracellular matrix, that is, the epithelial-mesenchymal transition (EMT) process. EMT is considered to be one of the main mechanisms of bladder fibrosis. Therefore, bladder epithelial cells have also become an effective therapeutic target for NB fibrosis.

[0004] Currently, the main treatment options for NB include clean intermittent catheterization (CIC), anticholinergic drug therapy, and surgical intervention. These treatment options can alleviate patient symptoms but cannot delay the progression of bladder fibrosis. Therefore, how to reverse or slow down bladder fibrosis and protect the patient's upper urinary tract is the focus of NB diagnosis and treatment.

[0005] Resveratrol is a non-flavonoid polyphenol organic compound with CAS registration number 501-36-0 and chemical formula C 14 H 12O3, mainly derived from natural plant extracts, such as grapes, knotweed and other natural plants, is a natural bioactive substance. Compared with synthetic drugs, it has better biocompatibility and safety, and reduces the risk of adverse drug reactions. The natural properties of resveratrol make its side effects on the human body relatively small during long-term use, and patients have better tolerance.

[0006] Currently, there are few studies on the application of resveratrol in neurogenic bladder fibrosis. Summary of the invention

[0007] The purpose of the present invention is to provide a use of resveratrol in improving and / or treating bladder fibrosis in view of the above problems.

[0008] In order to achieve its purpose, the present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides use of resveratrol in preparing a medicament for improving and / or treating bladder fibrosis.

[0010] In the application technology scheme, the resveratrol has at least one of the following effects:

[0011] (1) Inhibit apoptosis of bladder epithelial cells;

[0012] (2) reduce bladder barrier damage;

[0013] (3) Maintaining the integrity of the epithelial barrier structure;

[0014] (4) Prevent harmful substances in urine from penetrating into bladder tissue;

[0015] (5) reduce inflammatory responses and stimulation of fibrosis;

[0016] (6) Improve bladder function: improve bladder compliance and reduce intra-bladder pressure.

[0017] In the application, resveratrol inhibits the Akt / mTOR signaling pathway, reduces the synthesis and deposition of collagen, and thus effectively reverses bladder fibrosis.

[0018] In the application, resveratrol inhibits the epithelial-mesenchymal transition process of bladder epithelial cells and reduces the production of extracellular matrix.

[0019] In the application, the bladder fibrosis is neurogenic bladder fibrosis.

[0020] The second aspect of the present invention provides a drug for improving and / or treating bladder fibrosis, wherein the active ingredient is resveratrol.

[0021] Preferably, the medicament is a pharmaceutical composition.

[0022] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0023] Preferably, in the pharmaceutical technical scheme, the bladder fibrosis is neurogenic bladder fibrosis.

[0024] The beneficial effects of the present invention are:

[0025] The present invention study confirms that resveratrol can specifically regulate the signaling pathways associated with neurogenic bladder fibrosis, such as inhibiting the Akt / mTOR signaling pathway, reducing the synthesis and deposition of collagen, thereby effectively reversing bladder fibrosis, and resveratrol inhibits the EMT process of bladder epithelial cells and reduces the production of extracellular matrix. The present invention study verifies that resveratrol inhibits bladder epithelial cell apoptosis: resveratrol can inhibit bladder epithelial cell apoptosis, reduce bladder barrier damage, maintain the integrity of the epithelial barrier structure, prevent harmful substances in urine from penetrating into bladder tissue, reduce inflammatory response and stimulation of fibrosis; verifies that resveratrol improves bladder function: resveratrol can improve bladder compliance and reduce bladder cavity pressure.

[0026] Resveratrol can be used in the treatment of neurogenic bladder through a variety of administration routes, including oral administration, local administration (such as bladder instillation), intraperitoneal injection, etc. This provides patients with more treatment options and also facilitates personalized treatment based on the patient's specific situation.

[0027] Resveratrol is mainly extracted from natural plants, such as grapes, Japanese knotweed and other natural plants. It is a natural bioactive substance. Compared with synthetic drugs, resveratrol has better biocompatibility and safety, and reduces the risk of adverse drug reactions. In the process of improving neurogenic bladder fibrosis and function, long-term medication may be required. The natural properties of resveratrol make its side effects on the human body relatively small during long-term use, and patients have better tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the analysis of urodynamic test results and indicators.

[0029] Figure 2 These are the results of morphological analysis of rat bladder: A. Changes in the gross morphology of bladder tissue; B. Changes in the weight of bladder tissue; C. HE and Masson staining of bladder tissue; D. Ratio of collagen fibers in bladder tissue.

[0030] Figure 3The expression levels of proteins related to fibrosis and EMT in rat bladder tissue are shown: A. Immunofluorescence expression of fibrosis proteins Fibronectin and α-SMA, and EMT proteins E-cadherin and Vimentin in bladder tissue; B. Western blot expression of fibrosis proteins Fibronectin and α-SMA, and EMT proteins E-cadherin and Vimentin in bladder tissue.

[0031] Figure 4 This is the Western blot expression result of Akt / mTOR signaling pathway related proteins in rat bladder tissue.

[0032] Figure 5 The expression levels of apoptosis in rat bladder tissue are shown: A. Immunohistochemical expression of apoptosis-related proteins BAX and Bcl-2 in bladder tissue; B. Western blot expression of apoptosis-related proteins BAX, Bcl-2 and cleaved caspase-3 in bladder tissue.

[0033] All statistical results were expressed as mean ± SD. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0036] Example 1

[0037] 1 Experimental animals and groups

[0038] This study was approved by the Ethics Committee of the Children's Hospital Affiliated to Chongqing Medical University (CHCMU-IACUC20210625002). All experiments were conducted in accordance with relevant regulations on experimental animal welfare and ethics. Rats were purchased from the Experimental Animal Center of Chongqing Medical University. All rats were housed under SPF conditions with a temperature of 25°C, a relative humidity of 50%, and an artificial light / dark cycle of 12 h. Rats had free access to water and food. After 1 week of adaptive feeding, 24 purchased female SD rats were randomly divided into two groups: (1) sham operation group (n = 8); (2) NB model group (n = 16). The model group was constructed by transection of the L6-S1 spinal nerve. Our experiment adopted a completely randomized design method. After modeling, the model group rats were randomly divided into NB model group (n = 8) and resveratrol treatment group (n = 8). The treatment group was given resveratrol by gavage at 20 mg / kg / d for 8 weeks, and the other groups were given corresponding doses of normal saline.

[0039] 2 Experimental methods

[0040] 2.1 Determination of urodynamic indicators:

[0041] Five rats were randomly selected from each group 8 weeks after surgery. After anesthesia was induced by intraperitoneal injection of 1% sodium pentobarbital solution (0.4 ml / 100 g), one end of the epidural catheter was inserted into the bladder. The other end of the epidural catheter was connected to a microinfusion pump and an RM-6240 signal acquisition system through a three-way connector for infusion and pressure recording. Normal saline was injected into the bladder at a rate of 20 ml / h, and parameters such as bladder capacity BV, intravesical pressure difference ΔP, and bladder compliance BC of each rat were recorded and calculated. Each rat underwent bladder measurement every 30 minutes for three consecutive times to ensure the consistency of bladder activity.

[0042] 2.2 HE and Masson staining

[0043] HE staining: Precool the embedded bladder tissue wax block in a 4°C refrigerator, and then use a tissue slicer to slice the precooled tissue wax block continuously with a slice thickness of 4μm. Put the continuous tissue slices into 37°C warm water and fully flatten them, then use anti-shedding slides to mount them, put the prepared slices into a 37°C oven for at least 24 hours, and dry them for subsequent experiments. After dewaxing, rehydration, staining, and hematoxylin and eosin staining, make them transparent and seal them.

[0044] Masson staining: Mix Weigert solution A and Weigert solution B in the purchased kit in a ratio of 1:1 to prepare Weigert iron hematoxylin staining solution; drip the prepared Weigert staining solution on the slices, differentiate with acidic ethanol for 3s after 5 minutes; wash the slices with water, add Masson blueing solution to turn blue, rinse under running tap water for 1 minute after 1 minute; add Ponceau fuchsin staining solution, wash with weak acid working solution (ddH2O: weak acid solution = 2:1) for 1 minute after 7 minutes; add phosphomolybdic acid solution to wash, wash with weak acid working solution for 1 minute after 1 minute; add aniline blue staining solution to stain, wash with weak acid working solution for 1 minute, observe the staining under a microscope and make adjustments; dehydrate the slices in gradient alcohol, make them transparent with xylene, add neutral gum to seal the slices; finally observe under a microscope and take pictures to record.

[0045] 2.3 Immunofluorescence staining

[0046] The tissue paraffin sections were dewaxed, rehydrated, and antigen repaired; the tissue sections were blocked and incubated with primary antibodies overnight; the next day, the wet box was placed at room temperature for 1 hour, and the fluorescent secondary antibody was added to the dried sections and incubated in the dark for 1 hour; Hoechst33342 stain was added to the sections and incubated in the dark for 0.5 hour; finally, the sections were sealed with anti-fluorescence quencher, observed and photographed under a confocal microscope, and finally analyzed using NIS Viewer software.

[0047] 2.4 Western blot experiment

[0048] Select a PAGE gel rapid preparation kit of appropriate concentration according to the molecular weight of the target protein; place the prepared gel in the electrophoresis tank, then inject 1100ml of the freshly prepared electrophoresis solution between the two glass plates, pull out the comb, add the protein sample into the well according to the amount of protein to be loaded, and perform electrophoresis; according to the molecular weight of the target protein and the length of the loading well, select PVDF membranes of different pore sizes, use the molecular weight displayed by the protein marker as a reference, cut the gel at the location of the target protein, and put the cut gel into the transfer clip for transfer; after the transfer, incubate with rapid blocking solution for 10 minutes; add an antigen solution to the back-to-back strips for incubation, and place them on a shaker at 4°C overnight; after the incubation is completed on the second day, select a secondary antibody corresponding to the species of the primary antibody, and incubate at room temperature on a shaker for 1 hour; prepare ECL developer (A solution: B solution = 1:1) under light-proof conditions, add it dropwise to the strips during development, and place them in a Bio-RAD image analyzer for development; use Image Lab software was used to analyze the grayscale values ​​of the bands. Each group of samples needed to repeat the effective experiment at least 3 times to ensure the authenticity of the results.

[0049] 2.5 Immunohistochemistry

[0050] The tissue paraffin sections were dewaxed, rehydrated, and antigen repaired; endogenous peroxidase blocker was added to the dried sections and incubated for 10 minutes; after the sections were washed, 0.5% BSA was added for blocking for 1 hour and placed at room temperature; the primary antibody was dropped on the dried sections and placed in a 4°C refrigerator for overnight incubation; the sections were rewarmed the next day, placed at room temperature for 30 minutes, and then washed with PBS and dropped with reaction enhancement solution for 20 minutes; the secondary antibody corresponding to the species of the primary antibody was added to the sections and placed at room temperature for incubation for 20 minutes; under microscope observation, the color development solution was added to the sections and the timing was started. The time when the positive group showed brown positive results was counted as the standard time, and the reaction was immediately terminated with double distilled water. After that, other sections were color developed according to this time; finally, the sections were placed in hematoxylin for nuclear staining for 3 minutes, rinsed with tap water for 1 minute, placed in hydrochloric acid alcohol for differentiation for 1 second, rinsed with tap water for 1 minute, and then placed in saturated lithium carbonate for blueing. The staining was observed under a microscope at each step; finally, the sections were sealed with neutral gum and photographed after the gum dried.

[0051] 3 Experimental results

[0052] 3.1 Resveratrol increases bladder compliance and improves bladder function

[0053] like Figure 1 As shown in the results of urodynamics, the sham group rats had bladder filling and urination periods, and an obvious detrusor contraction peak could be observed. However, the bladder of the NB group rats did not have an obvious urination period, and urinary incontinence occurred. In addition, the bladder capacity BV and intravesical pressure difference ΔP of the NB group rats increased significantly 8 weeks after surgery compared with the sham group, while the bladder compliance BC decreased significantly, indicating severe bladder dysfunction. After treatment with resveratrol, the bladder capacity BV increased, the intravesical pressure difference ΔP decreased, and the bladder compliance BC increased, improving bladder function.

[0054] 3.2 Resveratrol improves bladder fibrosis in NB rats

[0055] According to the results of HE and Masson staining of bladder tissue ( Figure 2 ) It can be observed that the bladder tissue structure and morphology of the sham group were normal, while the bladder tissue muscle bundles of the NB group were thickened and arranged in disorder, and a large number of collagen fibers were deposited in the interstitium. After Masson staining, the collagen fibers were blue and the bladder smooth muscle was red. Compared with the control group, the bladder fibrosis area of ​​the NB group was significantly increased, indicating that the bladder tissue of the NB group had obvious fibrosis. After treatment with resveratrol, it can be observed that the bladder muscle bundles were arranged neatly, the interstitium was reduced, and the fibrosis area was reduced.

[0056] 3.3 Resveratrol improves bladder fibrosis and EMT of bladder epithelial cells in NB rats

[0057] Immunofluorescence and Western blot were used to detect bladder tissue fibrosis related indicators. Figure 3 A) showed that the fibrosis-related proteins Fibronectin and α-SMA in the bladder tissue of the NB group increased, the expression of the epithelial cell EMT-related protein E-cadherin in immunofluorescence decreased, and the expression of Vimentin in immunofluorescence increased. After resveratrol treatment, the degree of fibrosis and EMT decreased. Western blot experimental results ( Figure 3 B) shows that the expression of fibrotic proteins Fibronectin and α-SMA, EMT protein E-cadherin in bladder tissue of NB group was reduced and Vimentin expression was increased compared with that of Sham group. After resveratrol treatment, the degree of fibrosis and EMT was reduced. In conclusion, these results indicate that resveratrol improves bladder fibrosis and EMT of bladder epithelial cells in NB rats.

[0058] 3.4 Resveratrol inhibits the activation of Akt / mTOR signaling pathway, a signaling pathway related to bladder fibrosis in NB rats

[0059] To investigate whether the Akt / mTOR signaling pathway, a fibrosis-related signaling pathway, is activated, we performed Western blot experiments on rat bladder tissue ( Figure 4 ). The results showed that the expression of phosphorylated protein mTOR and phosphorylated Akt in bladder tissue of NB group was increased, and phosphorylated protein was inhibited under the action of resveratrol. This indicates that Akt / mTOR signaling pathway was activated in bladder of NB group, and after resveratrol treatment, Akt / mTOR signaling pathway was inhibited.

[0060] 3.5 Resveratrol inhibits bladder epithelial cell apoptosis

[0061] Immunohistochemical experiments were performed on rat bladder tissues ( Figure 5 A), it was found that the expression of pro-apoptotic protein Bax increased in the bladder epithelium of rats in the NB group, and the expression of apoptosis inhibitor protein Bcl-2 decreased in the bladder epithelium of rats in the NB group. After resveratrol treatment, the expression of pro-apoptotic protein Bax decreased, and the expression of apoptosis inhibitor protein Bcl-2 increased. By Western blot experiment ( Figure 5 B) It can be found that the expression of apoptosis-related protein cleaved caspase-3 and pro-apoptosis protein Bax in the bladder of rats in the NB group increased, and the expression of apoptosis-inhibiting protein Bcl-2 in the bladder of rats in the NB group decreased. After treatment with resveratrol, apoptosis-related proteins decreased, indicating that apoptosis occurred in the bladder epithelial cells of rats in the NB group, and resveratrol reduced the apoptosis of epithelial cells.

Claims

1. Use of resveratrol in the preparation of a drug for improving and / or treating bladder fibrosis.

2. The use according to claim 1, characterized in that: The resveratrol has at least one of the following effects: (1) Inhibit apoptosis of bladder epithelial cells; (2) reduce bladder barrier damage; (3) Maintaining the integrity of the epithelial barrier structure; (4) Prevent harmful substances in urine from penetrating into bladder tissue; (5) reduce inflammatory responses and stimulation of fibrosis; (6) Improve bladder function: improve bladder compliance and reduce intra-bladder pressure.

3. The use according to claim 1, characterized in that: in, Resveratrol inhibits the Akt / mTOR signaling pathway, reduces the synthesis and deposition of collagen, and thus effectively reverses bladder fibrosis.

4. The use according to claim 1, characterized in that: in, Resveratrol inhibits the epithelial-mesenchymal transition process of bladder epithelial cells and reduces the production of extracellular matrix.

5. The use according to any one of claims 1 to 4, characterized in that: The bladder fibrosis is neurogenic bladder fibrosis.

6. A drug for improving and / or treating bladder fibrosis, characterized in that: Its active ingredient is resveratrol.

7. The drug according to claim 6, characterized in that: The medicine is a pharmaceutical composition.

8. The drug according to claim 7, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

9. The drug according to claim 6, characterized in that: The bladder fibrosis is neurogenic bladder fibrosis.

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