Application of neuregulin 4 in preparation of podocyte apoptosis resisting medicine

Abstract

The invention provides application of neuregulin 4 in preparation of podocyte apoptosis resisting drugs, belongs to the technical field of biological medicine and cell protection, and discloses that Nrg4 can be combined with an ErbB4 receptor in podocytes to start an AKT / GSK-3beta signal channel, so that the anti-apoptosis ability of the podocytes is enhanced, and the podocyte apoptosis resisting drugs can be used for preparing podocyte apoptosis resisting drugs. The foot cell injury is repaired, the normal function is recovered, and the obvious foot cell protection effect is achieved. Nrg4 not only can reduce podocyte apoptosis caused by various factors, but also can remarkably enhance expression of anti-apoptotic protein Bcl-2 and inhibit activity of pro-apoptotic protein Bax and caspase-3, so that the podocyte can continuously exert physiological functions in a pathological environment. The invention provides a basis for application of Nrg4 as a podocyte apoptosis resisting drug, particularly has wide potential in podocyte injury model and apoptosis mechanism research, and is suitable for podocyte protection requirements in various diseases or experiments.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and cell protection, and more specifically, relates to an application of neuregulin 4 in the preparation of an anti-podocyte apoptosis drug. Background Art

[0002] Podocytes are important cells that maintain multiple tissues, especially the glomerular filtration barrier. Podocyte injury and apoptosis are key factors in the progression of multiple diseases and are associated with multiple pathological and physiological states. Existing research focuses on controlling external factors that cause podocyte injury, such as high glucose, high pressure, or inflammation. However, there is a lack of effective intervention measures specifically targeting podocyte injury, especially anti-apoptotic agents used to explore podocyte protection mechanisms in experiments have not been fully developed.

[0003] Podocytes are an important component of tissue filtration and barrier function. Their apoptosis not only affects the function of local tissues, but also aggravates the progression of the disease. However, existing treatments have failed to effectively solve the problem of podocyte apoptosis. Therefore, an effective drug agent that can directly protect podocytes and inhibit their apoptosis is urgently needed.

[0004] Studies on neuregulin 4 (Nrg4) have shown that it has metabolic regulation, anti-inflammatory and antioxidant functions, and regulates a variety of physiological processes through brown adipose tissue secretion. However, the prior art has not yet studied the role of Nrg4 in inhibiting podocyte apoptosis. Summary of the invention

[0005] The purpose of the present invention is to provide an application of neuregulin 4 in the preparation of an anti-podocyte apoptosis drug, so as to effectively solve the problem of podocyte damage and apoptosis and protect podocytes.

[0006] To achieve the above object, the present invention provides a use of neuregulin 4 in the preparation of an anti-podocyte apoptosis drug. Neuregulin 4 (Nrg4) can inhibit podocyte apoptosis under various injury conditions and restore its normal function by activating the ErbB4 receptor on the surface of podocytes.

[0007] Nrg4 activates the AKT / GSK-3β signaling pathway, enhances the expression of anti-apoptotic protein Bcl-2 in podocytes, and inhibits the activity of pro-apoptotic proteins Bax and caspase-3, effectively reducing podocyte apoptosis.

[0008] Compared with the prior art, the present invention has the following technical effects:

[0009] The present invention reveals for the first time that Nrg4 can activate the ErbB4 receptor in podocytes and start the AKT / GSK-3β signaling pathway, thereby enhancing the anti-apoptotic ability of podocytes, repairing podocyte damage and restoring their normal functions, and playing a significant role in protecting podocytes. Nrg4 can not only reduce podocyte apoptosis caused by various factors, but also significantly enhance the expression of anti-apoptotic protein Bcl-2, inhibit the activity of pro-apoptotic proteins Bax and caspase-3, so that podocytes can continue to play their physiological functions under different pathological environments.

[0010] The present invention provides a basis for the application of Nrg4 as an anti-podocyte apoptosis agent (drug), especially in the study of podocyte injury models and apoptosis mechanisms, and has broad potential, and is suitable for the needs of podocyte protection in various diseases or experiments. Nrg4 is particularly suitable for the application of drugs related to podocyte apoptosis inhibition, and can also be used as an experimental anti-podocyte apoptosis agent to explore podocyte injury and apoptosis mechanisms, that is, Nrg4 can be used for the treatment of podocyte-related diseases and can also be used for experimental research. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0012] Figure 1 The animal experiment flow chart provided in Example 1 of the present invention;

[0013] Figure 2 The detection results of PAS, TEM, WT1-TUNEL double fluorescence staining, and co-localization of ErbB4 and Nrg4 proteins by immunofluorescence of podocytes of each group of mice provided in Example 1 of the present invention are shown;

[0014] Figure 3 This is a graph showing the test results of the expression of apoptosis-related proteins and signaling pathway proteins in each group of mice provided in Example 1 of the present invention;

[0015] Figure 4 This is a diagram showing the detection results of immunofluorescence co-localization of ErbB4 and Nrg4 proteins in various groups of podocytes provided in Example 2 of the present invention;

[0016] Figure 5 This is a graph showing the test results of the expression of apoptosis-related proteins and signal pathway proteins in each group of podocytes provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0019] Podocyte injury is a key issue in a variety of pathological environments, especially damage related to apoptosis is often difficult to reverse. The present invention proposes Nrg4 as an anti-podocyte apoptosis agent or drug, which is suitable for the research and treatment of podocyte-related diseases, especially the protection of podocyte injury or apoptosis, filling the gap in the prior art.

[0020] Nrg4 can be applied to models or patients with podocyte injury by injection, local delivery or other appropriate means to achieve direct protection of podocytes. As an experimental reagent, it can be used to explore the molecular mechanism of podocyte apoptosis.

[0021] Nrg4 not only shows anti-podocyte apoptosis effects in specific diseases, but can also be widely used in experiments as a podocyte protector. Under different pathological and stress conditions, Nrg4 can significantly delay the process of podocyte injury through its unique mechanism of action, providing new possibilities for future treatment and research strategies for podocyte injury.

[0022] The following is an example of the application of Nrg4 in preventing podocyte apoptosis with reference to specific examples.

[0023] Example 1

[0024] In order to verify the protective effect of Nrg4 on podocytes in mouse experiments, the following animal experiment process was designed:

[0025] Step 1: Animal husbandry: 4-week-old SPF-grade C57BL / 6 male mice (wild type) were selected as WT mice, and 4-week-old SPF-grade C57BL / 6 male mice with complete knockout of the Nrg4 gene purchased from the Shanghai Southern Model Center were selected as KO mice. The breeding environment is the SPF-grade animal room of Tongji Medical College: temperature 24-26°C, dry and ventilated, humidity 55-65%, and light 12 hours per day. Breeding mice are housed together, and experimental mice are housed in separate cages. Adaptive feeding is required for 2 weeks before the start of the experiment. It is divided into normal diet NCD and high-fat diet HFD. High-fat feed: 41% kcal fat + 42% kcal carbohydrates + 17% kcal protein.

[0026] Preparation of Nrg4 gene knockout model mice: Southern Model Organisms Technology Co., Ltd. is responsible for the preparation of Nrg4 gene knockout model mice. Preparation process Prototype mice: C57BL / 6 mice, half male and half female. Preparation technology: Using non-homologous recombination repair technology. Wild-type mice and Nrg4- / - mice (KO mice) of the same litter were mated and raised with Nrg4+ / - mice to obtain a stable Nrg4 gene knockout animal model.

[0027] To make a podocyte injury mouse model: add streptozotocin (STZ) powder to citric acid buffer to prepare a 1% STZ solution, which was used immediately and kept on ice and away from light. The 1% STZ solution was intraperitoneally injected for 5 consecutive days (50 mg.kg-1.day-1, i.e., 50 mg of STZ was injected per kg of mouse per day), and the mice in this group were fed a high-fat diet HFD.

[0028] Step 2: 4-week-old C57BL / 6 male mice (18 4-week-old WT mice; 12 4-week-old KO Nrg4- / - mice) were randomly divided into five groups, with 6 mice in each group. The specific grouping is as follows:

[0029] WT+AAV-GFP group (i.e., WT-CON): wild-type mice were injected with AAV-GFP virus solution; WT podocyte injury+AAV-GFP group (i.e., WT-GFP): wild-type and podocyte injury mice were injected with AAV-GFP virus solution;

[0030] WT podocyte injury + AAV-Nrg4 group (i.e., WT-Nrg4): wild-type mice with podocyte injury were injected with AAV-Nrg4 virus solution;

[0031] Nrg4 gene knockout podocyte injury + AAV-GFP group (i.e., KO-GFP): Nrg4 gene knockout and podocyte injury mice were injected with AAV-GFP virus solution;

[0032] Nrg4 gene complete knockout podocyte injury + AAV-Nrg4 group (ie KO-Nrg4): Nrg4 gene knockout and podocyte injury mice were injected with AAV-Nrg4 virus solution.

[0033] Adenovirus injection: AAV-GFP virus solution or AAV-Nrg4 virus solution was injected into the scapular fat area of ​​each group of mice according to the above grouping. AAV-GFP virus solution is a solution of adenovirus containing GFP, which is prepared by diluting adenovirus with PBS. AAV-Nrg4 virus solution is a solution of adenovirus containing Nrg4. Among them, WT-CON, WT-GFP, and KO-GFP groups were injected with 2×10 11vg of AAV-GFP; WT-Nrg4 and KO-Nrg4 groups were injected with 2×10 11 vg of AAV-Nrg4.

[0034] Step 3: After 6 weeks, blood and kidney tissue samples were collected from each group of mice to detect Nrg4 levels, cell apoptosis rate and related protein expressions, and podocyte structural changes were observed by pathological means.

[0035] The whole animal experiment flow chart is as follows Figure 1 shown.

[0036] Experimental results: The results of PAS, TEM, WT1-TUNEL, co-localization of ErbB4 and Nrg4 proteins in podocytes of each group of mice, and the expression of apoptosis-related proteins and signaling pathway proteins were shown in Figure 2. Figure 2 , Figure 3 shown.

[0037] exist Figure 2 In the experiment, the PAS staining results of the glomeruli of each group of mice showed that the abnormal glomerular morphology of the AAV-Nrg4-treated group (WT-Nrg4 and KO-Nrg4) was alleviated compared with the control group (WT-GFP and KO-GFP), and the scale bar in the PAS staining image is 50μm; the transmission electron microscopy (TEM) images of podocytes showed that the foot process structure of podocytes in AAV-Nrg4-treated mice was improved, and the scale bar in the TEM image was 1μm; the apoptotic podocytes were quantitatively analyzed by double immunofluorescence staining, and the co-localization immunofluorescence staining of ErbB4 and Nrg4 showed that the apoptosis rate of the AAV-Nrg4-treated group was lower. Compared with other groups, the expression levels of these two proteins in the WT-Nrg4 and KO-Nrg4 group mice were higher, supporting the role of Nrg4 in podocyte protection, and the scale bar in the co-localization immunofluorescence staining of ErbB4+Nrg4 was 20μm; the immunofluorescence staining of the ErbB4 receptor expression in the glomerulus confirmed that it was localized on the podocyte membrane. The colocalization of ErbB4 and Nrg4 was significantly increased, and the expression levels in the WT-Nrg4 and KO-Nrg4 groups were significantly higher than those in the other groups, highlighting the interaction between Nrg4 and its receptor in the glomerulus.

[0038] exist Figure 3In the figure, B is the TEM analysis result of the glomerular basement membrane (GBM) thickness of mice in each group, indicating that the GBM thickness of mice in the AAV-Nrg4 treatment group was reduced, which was associated with reduced kidney damage; C is the quantitative analysis result of apoptotic podocytes in each group of mice by double immunofluorescence staining, indicating that the apoptosis rate in the AAV-Nrg4 treatment group was lower; D, E, F, G, and H are the RT-PCR detection results of apoptosis-related proteins and signaling pathway proteins in mice in each group, showing that the levels of pro-apoptotic proteins Bax and caspase-3 in mice with podocyte injury were increased, and P-AKT and P-GSK-3β The levels of P-AKT(S473) / AKT, P-AKT(T308) / AKT and P-GSK-3β / GSK-3β in podocytes of mice with podocyte injury were upregulated by Nrg4 in WT-Nrg4 and KO-Nrg4 groups, indicating that AAV-Nrg4 treatment restored the phosphorylation levels of AKT and GSK-3β in podocytes of mice with podocyte injury. I is the quantitative analysis result of immunofluorescence staining of ErbB4 receptor expression in glomeruli of mice in each group.

[0039] The above experimental results show that: Nrg4 gene knockout will aggravate streptozotocin + high-fat diet HFD-induced glomerular injury and podocyte injury apoptosis, Nrg4 binds to podocyte ErbB4 receptor, enhances the expression of anti-apoptotic protein Bcl-2, inhibits the activity of pro-apoptotic proteins Bax and caspase-3, activates AKT / GSK-3β signaling pathway, and significantly reduces podocyte apoptosis in podocyte injury model group mice. This result proves the unique effect of Nrg4 in podocyte protection.

[0040] Example 2

[0041] In in vitro experiments, the mouse podocyte cell line MPC5 was used to simulate podocyte injury under different sugar concentrations, and the protective effect of Nrg4 on podocytes was studied.

[0042] The experimental process is as follows:

[0043] Step 1: MPC5 podocytes were divided into control group, hyperosmotic group, injury group, and Nrg4 treatment group.

[0044] Step 2: The control group (NG) used 5.5mM glucose to treat podocytes for 48 hours, the hypertonic group (MA) used 25.0mM glucose + 8.3mM mannitol to treat podocytes for 48 hours, the injury group (HG) used 33.3mM glucose to treat podocytes for 48 hours, and the Nrg4 treatment group (HG+Nrg4) added Nrg4 recombinant protein to the injury group to treat podocytes for 24 hours. The concentration of Nrg4 recombinant protein in the treatment group was 50ng / ml.

[0045] Step 3: CCK8 was used to detect the apoptosis rate of podocytes. The localization and expression of ErbB4 receptor and Nrg4 protein in podocytes were analyzed by immunofluorescence staining of ErbB4 and Nrg4 co-localization. PCR was used to analyze the expression levels of anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax, caspase-3, and AKT / GSK-3β cell signaling pathway protein.

[0046] Experimental results: The apoptosis rate of podocytes in each group, the co-localization of ErbB4 and Nrg4 proteins by immunofluorescence, and the expression level of each protein were tested. Figure 4 , Figure 5 shown.

[0047] exist Figure 4 In Figure 2, double immunofluorescence staining of ErbB4 and Nrg4 on MPC5 cells (400×) showed the colocalization of the two. The protein expression levels in the NG and MA groups were low, and the expression in the HG group was the lowest. However, the colocalization of ErbB4 and Nrg4 in the HG+Nrg4 group was significantly higher than that in the HG group.

[0048] exist Figure 5 In the figure, B and C are the results of CCK8 proliferation experiment of podocytes in each group. The results show that under high glucose conditions, the activity of podocytes in the Nrg4-treated group is enhanced; the results of the NG and MA groups show that the proliferation rate of MPC5 podocytes under 5.5mM and 25.0mM glucose conditions are similar. 25.0mM is the glucose concentration in the DEME medium for culturing MPC5, and 33.3mM glucose is the high glucose condition that induces apoptosis of MPC5 podocytes. Among the four groups of NG, MA, HG, and HG+Nrg4, the apoptosis rate of podocytes in the HG group was the highest, significantly higher than that in other groups. The apoptosis rate of the HG+Nrg4 group was slightly higher than that of the NG and MA groups, but significantly lower than that of the HG group.

[0049] D, E, F, G, and H are the results of RT-PCR detection of podocytes in each group, and the Bcl-2 / Bax ratio and Cleaved-caspase3 protein expression level in different groups were analyzed. The Bcl-2 / Bax ratio in the HG+Nrg4 group was significantly higher than that in the HG group, while the Cleaved-caspase3 protein expression level was significantly lower than that in the HG group. The levels of P-AKT(S473) / AKT, P-AKT(T308) / AKT, and P-GSK-3β / GSK-3β in MPC5 podocytes in the HG group were lower than those in the NG and MA groups, and Nrg4 significantly upregulated the levels of P-AKT(S473) / AKT, P-AKT(T308) / AKT, and P-GSK-3β / GSK-3β in the HG+Nrg4 group.

[0050] The experimental results of the above podocyte in vitro studies showed that the phosphorylation levels of AKT at serine 473 (P-AKT(S473) / AKT), threonine 308 (P-AKT(T308) / AKT) and GSK-3β (P-GSK-3β / GSK-3β) in podocytes treated with high glucose (HG) decreased, and Nrg4 treatment (50ng / mL, for 24 hours) reversed these defects. Nrg4 significantly increased the level of P-AKT, phosphorylated GSK-3β can inhibit podocyte apoptosis, and Nrg4 significantly increased the level of P-GSK-3β in podocytes induced by high glucose. Nrg4 reversed the downward trend of P-GSK-3β levels observed in podocytes treated with high glucose.

[0051] The experimental results showed that Nrg4 could bind to the ErbB4 receptor of MPC5 podocytes, downregulate the expression of pro-apoptotic proteins Bax and caspase-3 in podocytes, upregulate the expression of significant anti-apoptotic proteins Bcl-2 and phosphorylation levels of AKT and GSK-3β, reduce high glucose-induced podocyte apoptosis, effectively maintain the normal structure and function of podocytes, and maintain their functional integrity. This discovery further confirms the potential for the wide application of Nrg4 in the protection of podocyte injury, proving that it is not only suitable for the treatment of podocyte-related diseases, but also provides an important tool for studying the mechanism of podocyte apoptosis.

[0052] The results of in vivo and in vitro experiments showed that Nrg4 improved podocyte survival through the AKT / GSK-3β-dependent signaling pathway, and the AKT / GSK-3β signaling pathway was indispensable for Nrg4 to improve mouse podocyte apoptosis.

[0053] The embodiments of the present invention have experimentally found that: Nrg4 has a significant protective effect on podocytes in a high-glucose environment. Nrg4 activates the AKT / GSK-3β signaling pathway, enhances the expression of the anti-apoptotic protein Bcl-2, and inhibits the activity of the pro-apoptotic proteins Bax and caspase-3, thereby effectively reducing the apoptosis of podocytes under high-glucose conditions. This direct podocyte protection effect has been verified in in vivo and in vitro experiments, proving that Nrg4 can significantly repair podocyte damage and protect its physiological functions. The effect of Nrg4 in inhibiting podocyte apoptosis provides new tools and directions for the field of podocyte damage and repair, and has broad application prospects.

[0054] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.

Claims

1. An application of neuregulin 4 in the preparation of anti-podocyte apoptosis drugs.

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