阅读说明：本技术 异鼠李素作为抗肾脏纤维化药物的应用 (Application of isorhamnetin as kidney fibrosis resisting medicine ) 是由 张珍 唐志书 宋忠兴 李艺文 于 2020-09-01 设计创作，主要内容包括：本发明属于医药领域,涉及一种异鼠李素作为抗肾脏纤维化药物的应用,尤其是异鼠李素在抑制上皮细胞-间充质转化EMT形成时作为抗肾脏纤维化药物的应用,本发明提供一种以细胞间隙连接蛋白Cx43为治疗肾脏纤维化新靶点以及效果显著的异鼠李素作为抗肾脏纤维化药物的应用。(The invention belongs to the field of medicines, relates to application of isorhamnetin as an anti-kidney fibrosis medicine, and particularly provides application of isorhamnetin as an anti-kidney fibrosis medicine when inhibiting formation of epithelial cell-mesenchymal transition EMT (endothelial cell-mesenchymal transition T).)

1. Use of isorhamnetin as kidney fibrosis resisting medicine is provided.

2. The isorhamnetin is used as an anti-renal fibrosis drug when the formation of the epithelial cell-mesenchymal transition EMT is inhibited.

3. Use according to claim 2, characterized in that: the concentration of isorhamnetin is not lower than 25 μ M.

4. The isorhamnetin is used as an anti-renal fibrosis drug when inhibiting the proliferation of the NRK-52E of the renal tubular epithelial cells of rats.

5. Use according to claim 4, characterized in that: the rat tubular epithelial cells NRK-52E were induced with 10% FBS.

6. Use according to claim 4 or 5, characterized in that: the concentration of isorhamnetin is not lower than 15 mu M.

7. The use of isorhamnetin for inhibiting the expression of intercellular gap junction protein Cx43 is provided.

8. The isorhamnetin is used as an anti-renal fibrosis drug when the isorhamnetin inhibits the expression of the intercellular gap junction protein Cx 43.

Technical Field

The invention belongs to the field of medicines, and relates to application of isorhamnetin as an anti-kidney fibrosis medicine.

Background

Chronic Kidney Disease (CKD) has become a public health problem worldwide. According to survey data published by king swallow and the like on the lancets, the incidence of CKD in adults in our country is as high as 10.8% and the number of CKD patients is as high as 1.2 hundred million by 2012. The kidney fibrosis is the main pathological feature of CKD, and currently, only angiotensin enzyme inhibitors and angiotensin receptor antagonists are used for clinically treating the kidney fibrosis, so that the treatment medicines are very limited, the development of the kidney fibrosis cannot be effectively prevented, a series of adverse reactions such as cough caused by ACEI and severe hyperkalemia can also occur, and the bottleneck of clinical treatment of CKD is formed. The key to overcoming this problem is the search for drugs that effectively prevent renal fibrosis. The traditional Chinese medicine has the characteristics of high efficiency and low toxicity, and the search of active ingredients which can effectively slow down the kidney fibrosis process from the traditional Chinese medicine becomes one of the hot directions for the research and development of the anti-kidney fibrosis medicine.

Renal fibrosis is the last common pathway for the progression of all CKD lesions to End-Stage Renal Disease (ESRD), the major pathological basis for CKD. Currently, kidney fibrosis develops primarily through the TGF-. beta.1/Smads signaling pathway. In general, the kidney under various causes (infection, injury, chemicals, radiation, etc.) causes the overproduction of ROS (reactive Oxygen species), and excessive ROS induces the secretion and release of various cytokines, including mainly the Transforming Growth Factor TGF- β 1 (TGF- β 1), which further activates TGF- β 1/Smads signaling pathway. When the TGF-beta 1/Smads signal path is activated, Smad2/3 and Smad4 form trimer to enter the nucleus, and combine with corresponding transcription factors to mediate the formation of Epithelial-Mesenchymal Transition (EMT), wherein EMT is an important process in the generation and development of kidney fibrosis and is formed as the expression of Epithelial marker E-cadherin (E-cadherin) is reduced, the expression of intermediate product Vimentin (Vimentin) of Epithelial cell to myofibroblast is increased, the expression of myofibroblast marker alpha-Smooth Muscle Actin (alpha-Smooth Muscle Actin, alpha-SMA) is increased, mesangial cells, tubular Epithelial cells, glomerular Epithelial cells and the like are converted into myofibroblasts, the myofibroblasts are in a continuous activated proliferation state, and the Fibronectin (Fibronectin), Fibrin expression, such as type I Collagen (Collagen I) and type III Collagen (Collagen III), increases and enters the renal interstitium, resulting in progressive accumulation of extracellular Matrix (ECM), while degradation of ECM decreases, resulting in glomerulosclerosis, tubulointerstitial fibrosis, and ultimately loss of renal function.

As shown in fig. 1, Gap Junction (GJ), which is one of cell junctions, is widely present in various animal tissues. The "cross-talk" between gap junctions, also known as Gap Junction Intercellular Communication (GJIC), allows communication of small molecules, ions and electrical signals between cells, thereby affecting cellular metabolism, proliferation, migration and survival, and playing an important role in maintaining tissue integrity. The intercellular gap junction is a transmembrane channel structure formed by hexamers of intercellular gap junction proteins (Connexin, Cx), 6 identical or similar transmembrane Cx proteins surround to form a cylindrical structure called a linker (Connexon), and 2 linkers respectively extend out of a membrane on the cell membrane of adjacent cells and are butted to form a hydrophilic intercellular gap junction channel (Hemichannel). The Cx43 protein is the major Cx constitutive protein and is distributed in a variety of cells.

Beyer et al first discovered Cx43 in rat kidney in 1989. Barajas et al then provided a more complete study and concluded that: cx43 is localized to renal vessels, mesangial cells and collecting ducts. By researching the expression of Cx43 in biopsy tissues of patients with different inflammatory glomerular diseases, Hillis et al find that Cx43 has strong expression in both infiltrating cells and renal tubular epithelial cells and has high expression in damaged interstitial parts. In recent years, researches prove that the expression of Cx43 is increased in the early stage of hypertensive nephropathy and obstructive nephropathy, the inhibition of the expression of Cx43 reduces inflammatory cell infiltration and renal fibrosis, obviously improves the structure and the function of the kidney, and suggests that Cx43 may be a new target point for treating the CKD development. Experts in the field of kidney pathology have made tremendous efforts to explore the avenues for new and more effective CKD therapeutic targets, and the emergence of new Cx43 targets has brought hopes to patients and experts. Targeted therapies that prevent Cx43 signaling have potential clinical application value, but the search for suitable Cx43 inhibitors to treat kidney fibrosis remains an urgent problem to be solved.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides the application of isorhamnetin as an anti-renal fibrosis medicament, wherein the isorhamnetin has a novel target for treating renal fibrosis by using the intercellular gap junction protein Cx43 and has a remarkable effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

use of isorhamnetin as kidney fibrosis resisting medicine is provided.

The isorhamnetin is used as an anti-renal fibrosis drug when the formation of the epithelial cell-mesenchymal transition EMT is inhibited.

The concentration of isorhamnetin is not less than 25 μ M.

The isorhamnetin is used as an anti-renal fibrosis drug when inhibiting the proliferation of the NRK-52E of the renal tubular epithelial cells of rats.

The rat tubular epithelial cells NRK-52E were induced by 10% FBS.

The concentration of isorhamnetin is not less than 15 μ M.

The use of isorhamnetin for inhibiting the expression of intercellular gap junction protein Cx43 is provided.

The isorhamnetin is used as an anti-renal fibrosis drug when the isorhamnetin inhibits the expression of the intercellular gap junction protein Cx 43.

The invention has the advantages that:

the invention provides an application of isorhamnetin as an anti-kidney fibrosis drug, and by establishing a TGF-beta 1 induced NRK-52E cell fibrosis model and a rat UUO kidney fibrosis model, ISO is found to have the effect of inhibiting intercellular gap connection; by establishing a 10% FBS-induced NRK-52E cell proliferation model and a TGF-beta 1-induced NRK-52E cell fibrosis model, ISO was found to inhibit the proliferation of NRK-52E cells, the formation of EMT and the deposition of ECM; establishing a rat UUO kidney fibrosis model, and remarkably improving the thickness, length and weight of the kidney cortex in the UUO model by ISO; the Masson trichrome dyeing result shows that ISO inhibits the deposition of kidney fibrosis protein, and the ISO has the function of resisting the kidney fibrosis and has better effect than the positive drug enalapril. In vivo, Western blots demonstrated that ISO inhibited the formation of EMT and deposition of ECM. The invention discovers for the first time that ISO inhibits EMT formation and ECM deposition, has the effect of resisting renal fibrosis, and the effect of resisting renal fibrosis of ISO is related to the regulation of intercellular junction protein Cx43, and Cx43 is a new target point for treating renal fibrosis in recent years, so ISO is likely to become a potential therapeutic drug for resisting renal fibrosis.

Drawings

FIG. 1 is a schematic view of intercellular space junctions;

FIG. 2 is a molecular structural formula of isorhamnetin;

FIG. 3 is the effect of ISO on intercellular junctions;

FIG. 4 is the effect of ISO on the fibrosis of NRK-52E cells;

figure 5 is the effect of ISO on kidney fibrosis in UUO rats.

Detailed Description

The parts of seabuckthorn fruit usually used for disease treatment mainly comprise seabuckthorn oil, seabuckthorn fruit paste, seabuckthorn flavone and the like. The application of Isorhamnetin (ISO) (the molecular structural formula of isorhamnetin is shown in figure 2) as the main component in seabuckthorn flavone is not reported, and research shows that quercetin in seabuckthorn flavone can up-regulate factor H₂O₂The expression of Cx43 is reduced, and a fluorescence yellow diffusion experiment shows that quercetin can restore the opening function of intercellular gap junction, so that the bystander effect of the intercellular gap junction is enhanced, and the expression of Cx43 protein is up-regulated, and ISO and quercetin have very similar structures, so that the regulation effect of ISO on the intercellular gap junction Cx43 is presumed to exist.

The rat Unilateral ureteral ligation (UUO) model is the most widely used experimental animal model for studying renal interstitial fibrosis. Since 1950, more than 10000 publications were published for related articles applying the UUO model. The UUO model has high success rate, small damage to animals and low mortality. The method is simple and convenient, and has better repeatability. In the UUO model, ligation of the unilateral ureter causes obstruction of the renal drainage system, sustained urinary tract pressure increases, renal blood flow decreases, venous drainage is blocked, mononuclear macrophages are activated and generate Ang II, triggering activation of a cascade of signaling pathways such as TGF- β 1/Smads signaling pathway, leading to progressive renal interstitial fibrosis. The UUO model has uniform pathological changes, and does not have endotoxin and uremia environments. The contralateral kidney can be used as a control and does not need to take into account its cellular, metabolic, compensatory functional changes. Multiple lines of evidence suggest that some of the findings of the UUO model are similar to those observed in patients with obstructive nephropathy, and that rodent models reflect human obstructive nephropathy and fibrotic renal disease processes. The UUO model has early renal fibrosis changes 3 days after surgery, and many quantifiable pathological changes occur about one week after UUO surgery, mainly including tubular epithelial EMT formation, ECM production, and the like. Therefore, the rat UUO model is a fast and reliable animal model for researching the renal interstitial fibrosis.

TGF-. beta.1 has been shown in a large body of literature to be the most important pro-renal fibrotic cytokine, which is abundantly expressed and secreted in a variety of chronic kidney diseases. Research shows that TGF-beta 1 can induce proliferation of renal tubular epithelial cells cultured in vitro at the concentration of 2-10 ng/mL, cause formation of EMT, synthesize fibrinectin, Collagen and Collagen III Collagen and the like through receptor signal transduction, cause excessive deposition of ECM, inhibit expression of ECM degrading enzyme, further inhibit degradation of ECM, and further cause generation and development of fibrosis. Currently, TGF-. beta.1 has been used in many studies as a classical inducer for studying the transdifferentiation of renal tubular epithelial cells into fibroblasts. The model adopts TGF-beta 12 ng/mL concentration to induce the transdifferentiation of rat renal tubular epithelial cells (NRK-52E) cultured in vitro to construct a renal interstitial fibrosis cell model.

Serum contains various cell factors and nutrients for promoting cell proliferation, and high-concentration serum is usually adopted to promote cell proliferation, and then medicines are given for intervention to detect the influence of the medicines on the cell proliferation. In the present invention, a cell proliferation model is established using 10% FBS (Fetal Bovine Serum (FBS)).

In order to research whether Isorhamnetin (ISO) has a regulating effect on intercellular Gap Junction (GJ), the invention establishes a TGF-beta 1 induced NRK-52E cell fibrosis model, and researches show that ISO has the effect of inhibiting intercellular Gap Junction for the first time. The fluorescent yellow diffusion experiment shows that ISO inhibits the opening function of the intercellular gap junction channel, so that the bystander effect is weakened (A and B in figure 3); the Western blot result shows that ISO down-regulates the expression (n is 4) of GJ protein Cx43 and has significant difference (C and D in figure 3), the invention establishes a rat UUO kidney fibrosis model, researches whether ISO influences the expression of Cx43, and the immunohistochemical result shows that ISO inhibits the expression (n is 4) of Cx43 and has significant difference.

Considering that ISO has the function of inhibiting the opening of a cell gap junction channel, and Cx43 is a main protein of cell gap junction and is a new target point for anti-renal fibrosis treatment, the invention establishes a 10% FBS-induced NRK-52E cell proliferation model and researches the influence of ISO on NRK-52E cell proliferation; establishing a TGF-beta 1 induced NRK-52E cell fibrosis model to research whether ISO has the effect of resisting renal fibrosis. The research result firstly discovers that: ISO inhibited 10% FBS-induced NRK-52E cell proliferation (FIG. 4A) at concentrations ranging from 15 μ M to 50 μ M; western blot results showed that ISO inhibited the expression of α -SMA, while up-regulating the expression of E-cadherin, indicating that ISO25 μ M concentration inhibited the formation of EMT (B-E in FIG. 4). The Western blot results showed that ISO inhibited fibrinectin expression, indicating that ISO25 μ M concentration inhibited ECM deposition (F and G in fig. 4); all have significant differences (n-4).

In order to determine whether ISO has the effect of resisting renal fibrosis in vivo, the rat UUO renal fibrosis model is established, whether ISO has the effect of resisting renal fibrosis is researched, and the result shows that: ISO significantly improved renal cortex thickness, length and renal weight in the UUO model (a-D in fig. 5); masson trichrome staining results indicate that ISO inhibits the deposition of kidney fibrotic proteins; the research firstly finds that ISO has the function of resisting renal fibrosis and has better effect than the positive drug enalapril (E and F in figure 5); all have significant differences (n-7-11).

In conclusion, the invention discovers for the first time that ISO inhibits the formation of EMT and the deposition of ECM, has the effect of resisting renal fibrosis, and the effect of resisting renal fibrosis of ISO is related to the regulation of intercellular junction protein Cx43, while Cx43 is a new target point for treating renal fibrosis in recent years, so ISO is likely to become a potential therapeutic drug for resisting renal fibrosis.