Application of chiparolone in preparation of medicine for relieving renal fibrosis induced by unilateral ureteral ligation

Abstract

The invention discloses an application of chiparolone in preparation of a medicine for relieving renal fibrosis induced by unilateral ureteral ligation. The application discovers that the KLF4 activity in the kidney is inhibited, so that the activation of kidney fibroblasts and the generation of an extracellular matrix (ECM) are inhibited, then the renal fibrosis induced by unilateral ureteral ligation is slowed down, and the KLF4 can become an effective clinical medicine for preventing and treating the renal fibrosis.

Description

Technical Field

[0001] The invention relates to a new drug application of Kenpaullone, and in particular to application of Kenpaullone in the preparation of a drug for alleviating renal fibrosis caused by unilateral ureteral ligation. Background Art

[0002] Renal fibrosis is the main cause of the progression of CKD (Chronic kidney disease) and a common result of various chronic kidney diseases. Its main characteristics are: inflammatory cell infiltration, myofibroblast activation and a large amount of extracellular matrix deposition, which eventually lead to the destruction of the normal structure of the kidney and the formation of scar tissue. For a long time, the occurrence and development mechanism of renal fibrosis has not been fully clarified. Recent studies have shown that the main source of extracellular matrix is ​​myofibroblasts in fibrotic kidneys. Then, lineage tracing and single-cell sequencing technology showed that activated fibroblasts are the main source of myofibroblasts. Therefore, how to block the activation of fibroblasts is the key to preventing and treating renal fibrosis. At present, the treatment of renal fibrosis in clinical practice is relatively limited, mainly for primary diseases or symptoms, and renal transplantation, but there are still problems such as lack of specific targeted drugs, large side effects of drugs, and uncertain efficacy. Therefore, it is of great clinical significance to develop new and effective drugs that can improve renal fibrosis.

[0003] KLF4 is a transcription factor with a CYS2-His2 zinc finger structure. As a major regulator of chromatin reorganization, it plays an important role in the reprogramming mixture of fibroblasts into induced pluripotent stem cells (iPSCs). The involvement of KLF4 can be observed in many diseases, especially in cancer. Apart from this, its role in the progression of chronic kidney disease is still unclear.

[0004] KLF4 inhibitor Kenpaullone is a class of small molecule compounds with the following structural formula:

[0005]

[0006] At present, there is no public literature reporting that Kenpaullone can inhibit the expression of KLF4 in renal fibroblasts under pathological conditions. Summary of the invention

[0007] Purpose of the invention: The present invention provides the use of Kenpaullone in the preparation of a medicament for alleviating renal fibrosis induced by unilateral ureteral ligation.

[0008] The present application unexpectedly discovered that the KLF4 protein level was significantly increased in the renal tissue of mice with renal fibrosis induced by unilateral ureteral ligation, suggesting that the use of related small molecule compounds to intervene in KLF4 function may improve renal fibrosis induced by unilateral ureteral ligation, and further verified the above speculation through experiments.

[0009] Specifically, the present invention provides a use of a KLF4 protein inhibitor Kenpaullone in the preparation of a drug for renal fibrosis, which solves the technical problem in the prior art that there is no suitable drug for treating renal structural and functional damage caused by unilateral ureteral ligation.

[0010] Specifically, the present application proposes that Kenpaullone inhibits the activation of fibroblasts by inhibiting the activity of the transcription factor KLF4, thereby alleviating renal fibrosis induced by unilateral ureteral ligation.

[0011] The present invention uses Kenpaullone on a mouse model of renal fibrosis induced by unilateral ureteral ligation and an in vitro cell model to explore the use of Kenpaullone as a KLF4 inhibitor in a drug for alleviating unilateral ureteral ligation-induced renal fibrosis-related diseases. The results showed that after intervention with Kenpaullone on a mouse model of unilateral ureteral ligation and in vitro cells, the renal structure and function of renal fibrosis can be significantly improved and the activation of renal fibroblasts can be inhibited. Our discovery may provide an effective clinical drug for the prevention and treatment of renal fibrosis.

[0012] Specifically, kemperolone improves renal damage in unilateral ureteral ligation-induced renal fibrosis and can effectively improve renal pathological damage such as tubular dilatation, tubular epithelial cell shedding, brush border atrophy, inflammatory cell infiltration, and tubular casts.

[0013] At the same time, Kenpaullone improved the deposition of collagen fibers in renal fibrosis induced by unilateral ureteral ligation. Specifically, Kenpaullone could significantly reduce the expression of renal interstitial FN, α-SMA, and Col-I in fibrotic kidneys.

[0014] Kenparotone alleviates TGFβ1-induced fibroblast activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0016] Figure 1 It was shown that in the renal fibrosis model induced by unilateral ureteral ligation, the level of KLF4 protein in mouse kidney tissue was significantly increased;

[0017] Figure 2 Kenpaullone significantly improves renal injury in unilateral ureteral ligation-induced renal fibrosis;

[0018] Figure 3 It was shown that in the unilateral ureteral ligation-induced renal fibrosis model, Kenpaullone downregulated the levels of FN, Col-I, and α-SMA proteins in renal tissue;

[0019] Figure 4 Kenpaullone significantly inhibited the expression of KLF4 protein in a-SMA+ myofibroblasts and the mRNA expression of KLF4 in the kidney in a unilateral ureteral ligation-induced renal fibrosis model.

[0020] Figure 5 Kenpaullone significantly alleviated TGFβ1-induced fibroblast activation in an in vitro renal fibroblast model. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments. The embodiments will help to understand the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0022] The materials and methods used in the following examples are as follows:

[0023] 1. Materials and reagents:

[0024] Normal rat kidney fibroblasts (NRK-49F) were purchased from American Type Culture Collection; Kenpaullone was purchased from American MCE; TGFβ1 was purchased from R&D Systems; KLF4 antibody was purchased from Santa Cruz; α-SMA and Fibronectin antibodies were purchased from Sigma; Col-I antibody was purchased from Southernbiotech; GAPDH antibody was purchased from Proteintech; fluorescent secondary antibody was purchased from Invitrogen;

[0025] (II) Cell culture and treatment:

[0026] Normal rat renal fibroblasts (NRK-49F) were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37°C, 5% carbon dioxide and 95% air. To study the role of Kenpaullone in the activation of renal fibroblasts induced by TGFβ1, when NRK-49F grew to about 70% density, the medium was changed to DMEM medium without fetal bovine serum and starved for 10-12 hours, then Kenpaullone (dissolved in DMSO) was added for pretreatment for 30 minutes or treated with DMSO in the same way, and TGFβ1 was added, and the cells were incubated in the incubator for 24 hours, and finally the cells were collected for Western Blot detection and immunofluorescence analysis.

[0027] (III) Mouse model of renal fibrosis induced by unilateral ureteral ligation and experimental groups:

[0028] In the renal fibrosis model induced by unilateral ureteral ligation, 8-12 week old CD-1 mice were randomly divided into four groups, 7 mice in each group, namely unilateral ureteral ligation model group (UUO) and unilateral ureteral ligation + Kenpaullone group (UUO + Kenpaullone). Kenpaullone was administered by intraperitoneal injection at a dose of 5 mg / kg. Mice in the control group or Kenpaullone treatment group were given solvent or Kenpaullone by intraperitoneal injection every other day starting from 1 day before UUO surgery. After 7 days, mice in all groups were euthanized and kidney specimens were collected. All experimental animals complied with the Regulations on the Management and Use of Laboratory Animals in China.

[0029] (IV) Scoring of renal tubular injury and collagen fiber deposition:

[0030] The degree of tubular damage was observed in renal PAS pathological staining, and the degree of collagen fiber deposition was observed in Masson and picrosirius red pathological staining. Image J software was used to perform semi-quantitative scoring of collagen fiber deposition.

[0031] (V) Histology and immunofluorescence staining:

[0032] Kidney tissue was fixed with 4% paraformaldehyde, wrapped in paraffin, and then sliced ​​for histology and immunofluorescence staining. The specific steps are as follows: paraffin-embedded sections, dewaxed to water, rinsed with distilled water, and rinsed with phosphate buffer saline (PBS) three times. Microwave repair with Tris-EDTA alkaline antigen repair solution for 20 minutes, then naturally cooled to room temperature. Rinse with PBS three times, block with Biyuntian QuickBlock immunostaining blocking solution for one hour, pour off, add the corresponding primary antibody and incubate overnight at 4°C. After rinsing with PBS three times, add the corresponding secondary antibody, incubate at room temperature for 1 hour, and rinse with PBS three times. For immunohistochemistry, the subsequent steps are: add DAB to color at room temperature to the appropriate depth, counterstain the nucleus with hematoxylin, rinse with running water to reverse blue, dehydrate and make it transparent, and finally seal the slide. For immunofluorescence, the subsequent steps are: add DAPI + anti-fluorescence quenching sealing agent integrated reagent to seal the slide, observe with a fluorescence microscope, and take pictures. For the corresponding primary antibody concentration, the FN concentration was 1:200 and the Col-I concentration was 1:100. NRK-49F was fixed with paraformaldehyde and then subjected to immunofluorescence staining. The primary antibody concentrations of FN and α-SMA were all 1:200, and the fluorescent secondary antibody concentration was 1:200. DAPI staining was performed according to the instructions.

[0033] (VI) Protein immunoblotting:

[0034] Total protein of kidney tissue was extracted by RIPA lysis buffer, and protein concentration was determined by BCA method. 20ug protein was loaded, 10% polyacrylamide gel electrophoresis (SDS.PAGE), and the protein was wet transferred to PVDF membrane by 400mA×2h fast transfer buffer, blocked with 5% skim milk at room temperature for 2h, eluted with TBST, and incubated overnight at 4℃. The membrane was washed 3 times with TBST, 15min each time, incubated with corresponding secondary antibody at room temperature for 2h, and then washed 3 times. Antigen-antibody complex was displayed by enhanced chemiluminescence (ECL), exposed and scanned by darkroom X-ray film, and protein quantification was performed by gray value analysis of the target band, and the relative expression of the target protein was expressed by the gray value of the target band / GAPDH gray value. The results of protein immunoblotting (Western blot) were analyzed by grayscale using ImageJ software.

[0035] 7. Statistical analysis:

[0036] Data were expressed as mean ± SEM. T-test was used to compare data between the two groups. P < 0.05 was considered statistically significant.

[0037] Example 1 Kenpaullone significantly improves renal injury in unilateral ureteral ligation-induced renal fibrosis.

[0038] In the renal fibrosis model induced by unilateral ureteral ligation, the level of KLF4 protein in the renal tissue of mice was significantly increased ( Figure 1 A, 1B). To evaluate the protective effect of Kenpaullone in renal fibrosis, we used unilateral ureteral ligation to induce chronic renal fibrosis. Glycogen staining (PAS) showed that 7 days after unilateral ureteral ligation, renal pathological damage such as tubular dilatation, tubular epithelial cell shedding, brush border atrophy, inflammatory cell infiltration, and tubular casts were more severe. After Kenpaullone treatment, the corresponding renal damage was significantly reduced ( Figure 2 A, 2B). These results suggest that Kenpaullone can protect against renal fibrosis induced by unilateral ureteral ligation without causing significant toxic side effects.

[0039] Example 2 Kenpaullone significantly improves the deposition of collagen fibers in renal fibrosis induced by unilateral ureteral ligation.

[0040] Masson staining and picrosirius red staining showed that 7 days after unilateral ureteral ligation, there was obvious deposition of collagen fibers in the renal interstitium, while after Kenpaullone intervention, the deposition of collagen fibers in the renal interstitium was significantly reduced ( Figure 2 A, 2B). To further demonstrate the protective effect of Kenpaullone in renal fibrosis, we detected the specific markers of extracellular matrix, FN and Col-I, and the specific marker of myofibroblasts, α-SMA. Figure 3 Immunofluorescence showed that after unilateral ureteral ligation, FN, α-SMA, and Col-I were highly expressed in the kidneys of unilateral ureteral ligation-induced renal fibrosis. Kenpaullone could significantly reduce the expression of FN, α-SMA, and Col-I in the renal interstitium of fibrotic kidneys ( Figure 3 A, 3B). Western Blot also showed that in kidneys with unilateral ureteral ligation-induced renal fibrosis, the expression levels of FN, α-SMA, and Col-I were significantly decreased after Kenpaullone treatment ( Figure 3 C). The above results indicate that Kenpaullone can improve renal fibrosis induced by unilateral ureteral ligation.

[0041] Example 3 Kenpaullone significantly inhibited the KLF4 protein level in the unilateral ureteral ligation-induced renal fibrosis model.

[0042] We further explored the possible mechanism of action of Kenpaullone. We found in the literature that Kenpaullone can specifically inhibit the expression of KLF4, a transcription factor with a zinc finger structure, a major regulator of chromatin reorganization, and plays an important role in renal fibrosis. Our experimental results showed that treatment with Kenpaullone can significantly downregulate the expression of KLF4 protein in unilateral ureteral ligation-induced fibrotic kidneys ( Figure 4 A, 4B). Example 4 In the in vitro renal fibroblast TGFβ1 model, Kenpaullone intervention improves TGFβ1-induced fibroblast activation.

[0043] Furthermore, we cultured normal rat kidney fibroblasts (NRK-49F) in vitro, pretreated them with Kenpaullone at concentrations of 0.1, 0.5, and 5uM for 30min, then added 2ng / ml TGFβ1 for 24h, and collected the cells to detect the KLF4 level of fibroblasts and the production of extracellular matrix ( Figure 5 A), after TGFβ1 intervention, the KLF4 level of fibroblasts, the fibroblast activation marker α-SMA, and the production of extracellular matrix FN and Col-1 increased significantly, while after Kenpaullone intervention, the expression levels of the above proteins decreased significantly, and showed a dose-dependent decrease. Further, for the intervention concentration of Kenpaullone, we selected 0.5uM. Immunofluorescence showed that after TGFβ1 intervention, the production of fibroblast activation marker α-SMA and extracellular matrix FN increased significantly, while after Kenpaullone intervention, the expression levels of the above proteins decreased significantly ( Figure 5 B).

[0044] In summary, the present invention provides a use of Kenpaullone, a transcription factor KLF4 inhibitor, in the preparation of a drug for alleviating renal fibrosis-related diseases induced by unilateral ureteral ligation. The drug is administered by intraperitoneal injection, and the mouse dose is 5 mg / kg / d. It inhibits the protein synthesis of fibroblast KLF4, the activation of fibroblasts, and the production of extracellular matrix, thereby improving renal fibrosis.

[0045] The present invention provides an application idea and method of kemperolone in the preparation of a drug for reducing renal fibrosis induced by unilateral ureteral ligation. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. Use of kemperolone in the preparation of a drug for alleviating renal fibrosis induced by unilateral ureteral ligation, wherein: The molecular formula of kenparotone is: 。 2. The use according to claim 1, characterized in that: Kenparotone ameliorates renal injury in unilateral ureteral ligation-induced renal fibrosis.

3. The use according to claim 1, characterized in that: Kenparotone improves collagen fiber deposition in unilateral ureteral ligation-induced renal fibrosis.

4. The use according to claim 3, characterized in that: Kenparotone reduces the expression of renal interstitial FN, α-SMA, and Col-I in fibrotic kidneys.

5. The use according to claim 3, characterized in that: Kenparotone alleviates TGFβ1-induced fibroblast activation.

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