Application of 4-PBA in preparation of anti-renal fibrosis preparation

Abstract

The invention belongs to the technical field of biomedicine, and discloses application of 4-PBA in preparation of an anti-renal fibrosis preparation. Specifically, 4-PBA acts on the renal fibrosis resistance by inhibiting the expression of GRP78 and the expression of alpha-SMA protein. The anti-renal fibrosis preparation containing 4-PBA can effectively inhibit endoplasmic reticulum stress, reduce the fibrosis degree and improve renal fibrosis. The invention has a wide application prospect in the treatment of renal fibrosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of 4-PBA in the preparation of an anti-renal fibrosis preparation. Background Art

[0002] Renal fibrosis is the final pathological process common to any ongoing chronic kidney injury or maladaptive repair. It is considered the underlying pathological process of chronic kidney disease (CKD), which affects more than 10% of the world's population. [1] However, the current treatment for renal fibrosis is still mainly focused on remission, and its underlying mechanism has not been fully elucidated, so patients cannot receive fundamental treatment.

[0003] Studies have shown that endoplasmic reticulum stress (ERS) is closely related to renal fibrosis [2] Glucose-regulated protein 78 (GRP78) is a major chaperone and regulator of endoplasmic reticulum (ER) homeostasis[ 3] Under steady-state conditions, GRP78 binds to unfolded protein response (UPR) sensors to prevent UPR signaling [4] However, GRP78 binds to misfolded proteins accumulated in the ER under ERS conditions, keeping the proteins in a foldable state and releasing three UPR sensors, thereby activating the UPR signaling pathway aimed at alleviating ER stress. [5] 4-PBA (sodium 4-phenylbutyrate) is a small molecule chemical chaperone and histone deacetylase inhibitor that stabilizes misfolded proteins, promotes their correct conformation, and inhibits overactivation of the UPR, thereby alleviating endoplasmic reticulum stress. However, 4-PBA has not yet been used in the clinical treatment of renal fibrosis. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides the use of 4-PBA in the preparation of an anti-renal fibrosis preparation, which can effectively inhibit endoplasmic reticulum stress and reduce the expression of fibrosis markers, thereby achieving the purpose of improving renal fibrosis.

[0005] The purpose of the present invention is to protect the use of 4-PBA in the preparation of anti-renal fibrosis preparations, wherein the chemical structural formula of the 4-PBA is:

[0006]

[0007] Furthermore, 4-PBA is used in the preparation of anti-renal fibrosis preparations, specifically, 4-PBA acts on anti-renal fibrosis by inhibiting the expression of GRP78 and α-SMA protein.

[0008] Another object of the present invention is to protect an anti-renal fibrosis preparation, the ingredients of which include 4-PBA and excipients.

[0009] Furthermore, the auxiliary materials include one or more of purified water, surfactants, chelating agents, antioxidants, preservatives, and pH regulators.

[0010] Furthermore, the anti-renal fibrosis preparation is in the form of a solution or a powder.

[0011] The present invention offers significant advantages over existing technologies: 4-PBA inhibits GRP78 expression, significantly suppressing α-SMA protein expression, thereby combating renal fibrosis. Anti-renal fibrosis preparations containing 4-PBA can effectively inhibit endoplasmic reticulum stress, reduce the degree of fibrosis, and improve renal fibrosis. This invention has broad application prospects in the treatment of renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0013] Figure 1 is the correlation analysis diagram of Klotho, GRP78 and ɑ-SMA;

[0014] Figure 2 is the Western blot image of GRP78 and ɑ-SMA after adding 4-PBA;

[0015] Figure 3 It is a semi-quantitative statistical graph of GRP78 and α-SMA after adding 4-PBA. DETAILED DESCRIPTION

[0016] The present invention is described in detail below by specific examples, but the scope of protection of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0017] Example 1

[0018] The present invention uses Klotho transgenic mice. Klotho transgenic mice are divided into wild-type (WT), heterozygous (HE) and knockout (KO) groups. Immunoblotting analysis (Western blot) is used to detect the protein expression levels of Klotho, endoplasmic reticulum stress marker GRP78, and fibrosis marker α-SMA in the kidney tissue of each group of mice. At the same time, statistical analysis and correlation analysis are performed on the three. In addition, to further verify the mechanism of Klotho and endoplasmic reticulum stress on renal fibrosis, two primary renal tubular epithelial cells, WT and HE, are extracted in the present invention and added with 4-PBA. The protein expression level of α-SMA in each group of mice is detected by Western blot.

[0019] Western blot analysis of the three revealed a significant negative correlation between Klotho, GRP78, and ɑ-SMA, while GRP78 and ɑ-SMA showed a significant positive correlation (p < 0.05). Therefore, the present invention extracted primary renal tubular epithelial cells and added 4-PBA, and found that the protein expression level of the fibrosis-related protein ɑ-SMA was significantly reduced.

[0020] 1. Western blot detection of protein expression: (1) Lysis: Take kidney tissues from mice of different genotypes, add these tissues to tissue protein lysis buffer, and cut them into pieces. Discard the supernatant and add RIPA:PMSF = 100:1. Place it on ice and let it stand. Next, use an ultrasonic disruptor to fully lyse the tissue blocks. Centrifuge the mixture and store it. (2) Protein quantification: Follow the instructions for using BCA reagent. Use an enzyme-linked microplate reader to measure the absorbance of the sample and calculate the corresponding data based on the absorbance of the standard well and the known concentration. Draw a standard curve and deduce the concentration of the sample to be tested. (3) Sample preparation: Mix the sample of known concentration with the protein loading buffer in proportion, place it in a metal bath, and heat it at 100℃ for 10 minutes to prepare a protein electrophoresis sample. After cooling the sample to room temperature, store it in a -20℃ refrigerator for later use. (4) Gel preparation: Prepare a 5% upper layer of concentrated gel and a 10% lower layer of separation gel. (5) Loading. (6) Electrophoresis: In the experiment, first perform electrophoresis at 80V, 360mA and constant current for 30 minutes. Then adjust the voltage to 120V, 360mA and continue running at constant current. (7) Cut PVDF membranes of equal size according to the size of the target protein gel and place these membranes in methanol for activation. (8) Place the transfer tank in an ice water tank. Set the current to 360mA and the voltage to 300V, and select an appropriate transfer time according to the molecular weight under the condition of constant voltage. (9) Blocking: Pour an appropriate amount of TBST solution into the incubation box, place the membrane to be treated into the box, rinse off the excess transfer solution and pour it out, then add the prepared 5% BSA blocking solution to the incubation box and block it on a shaker for 2 hours. (10) Primary antibody incubation and washing. (11) Secondary antibody incubation and washing. (12) Development. (13) Result analysis.

[0021] Western blot was used to detect the protein expression of Klotho, GRP78, and ɑ-SMA in the renal tissues of the above experimental groups. The results showed that Klotho was significantly negatively correlated with GRP78 and ɑ-SMA, while GRP78 was significantly positively correlated with ɑ-SMA. This suggests that Klotho and GRP78 are closely related to renal fibrosis ( Figure 1 ).

[0022] In the present invention, two primary renal tubular epithelial cells (WT and HE) were extracted and added with 4-PBA, and the protein expression levels of α-SMA in each group of mice were detected by Western blot.

[0023] Primary renal tubular epithelial cell culture: (1) Select mice, kill them by cervical dislocation, and soak them in 75% alcohol for 10 minutes. (2) Place the mice in a culture dish, separate the left and right kidneys, and remove the fascia tissue. Wash them in PBS solution. (3) Mince the renal cortex and place it in an EP tube containing 0.25% trypsin, then place it in a CO2 incubator for digestion. After the end, add 10% DMEM to stop the digestion. (4) Pass the cell through a cell sieve to filter out the tissue blocks and collect the liquid. (5) Add an appropriate amount of 20% DMEM to the EP tube and mix it evenly. (6) Transfer the mixed cell suspension to a new culture dish and then culture it in an incubator at 5% CO2 and 37°C. When the cells grow to a density of more than 80% of the culture dish, add 4-PBA and incubate in a cell incubator for 8 hours. Then prepare protein samples for subsequent Western blot experiments.

[0024] The results showed that after adding 4-PBA, the expression of GRP78 was inhibited. At the same time, the expression of α-SMA protein was also significantly inhibited (p<0.05) ( Figure 2 and Figure 3 ). This suggests that 4-PBA has anti-fibrotic effects.

[0025] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

[0026] References

[0027] [1]DJUDJAJ S,BOOR P.Cellular and molecular mechanisms of kidneyfibrosis[J].MolAspects Med,2019,65:16-36.

[0028] [2] TANIGUCHI M, YOSHIDA H. Endoplasmic reticulum stress in kidney function and disease [J]. Curr Opin Nephrol Hypertens, 2015, 24(4): 345-50.

[0029] [3] M,KAUFMAN R J.ER stress and the unfolded proteinresponse[J].Mutat Res,2005,569(1-2):29-63.

[0030] [4]NI M,LEE A S.ER chaperones in mammalian development and humandiseases[J].FEBS Lett,2007,581(19):3641-51.

[0031] [5]KAUFMAN R J.Orchestrating the unfolded protein response in healthand disease[J].J Clin Invest,2002,110(10):1389-98。

Claims

The use of 1.4-PBA in the preparation of an anti-renal fibrosis preparation is characterized in that: The chemical structural formula of the 4-PBA is:

2. The use of 4-PBA in the preparation of an anti-renal fibrosis preparation according to claim 1, characterized in that: Specifically, 4-PBA acts against renal fibrosis by inhibiting the expression of GRP78 and α-SMA protein.

3. An anti-renal fibrosis preparation, characterized in that: The ingredients include 4-PBA and auxiliary materials.

4. The anti-renal fibrosis preparation according to claim 3, characterized in that The auxiliary materials include one or more of purified water, surfactants, chelating agents, antioxidants, preservatives, and pH regulators.

5. The anti-renal fibrosis preparation according to claim 3, characterized in that The anti-renal fibrosis preparation is in the form of a solution or a powder.

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