Application of angiotensin 1-7 in preparation of medicine for activating AMPK / mTOR pathway to enhance autophagy

Abstract

The invention discloses an application of angiotensin 1-7 in preparation of a medicine for activating an AMPK / mTOR pathway to enhance autophagy, and an application of angiotensin 1-7 as an active component in preparation of a septicopyemia renal tissue cell autophagy promoter medicine. The research proves that the polypeptide Ang-(1-7) has the autophagy influence on mice suffering from septicopyemia acute kidney injury and the effect of relieving kidney injury and inflammation level, has obvious curative effects on improving renal functions and pathological injury and inhibiting inflammation and cell apoptosis, and has no obvious toxicity on the mice. The invention provides a new treatment strategy for septicopyemia acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of angiotensin 1-7 in preparing a drug for activating AMPK / mTOR pathway to enhance autophagy. Background Art

[0002] Sepsis is an organ dysfunction syndrome caused by the host's dysregulated response to infection, which is mainly manifested by symptoms such as chills, fever (or hypothermia), palpitations, shortness of breath, and changes in mental status. Sepsis can develop into severe sepsis and septic shock, leading to septic acute kidney injury, which has a high mortality rate. Renal tubular epithelial cell damage is an important cause of septic acute kidney injury. Studies have confirmed that renal tubular epithelial cell autophagy is involved in the occurrence and development of septic acute kidney injury.

[0003] Angiotensin 1-7 [Ang-(1-7)] is a heptapeptide, which is mainly synthesized by ACE2 hydrolysis of angiotensin II and participates in regulating autophagy in tissues such as the heart, brain, lung, and liver. Many studies have confirmed that Ang-(1-7) can activate AMPK and there may be a positive correlation between AMPK and AMPK. The AMPK / mTOR signaling pathway jointly regulates cell metabolism and growth. After AMPK is activated, it inhibits mTOR and enhances autophagy.

[0004] There is no report on the role of Ang-(1-7) in protecting against acute kidney injury by promoting autophagy through the AMPK / mTOR pathway. Summary of the invention

[0005] The purpose of the present invention is to address the above problems and provide an application of angiotensin 1-7 in the preparation of a drug for activating the AMPK / mTOR pathway to enhance autophagy.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of angiotensin 1-7 in the preparation of a drug for activating the AMPK / mTOR pathway to enhance autophagy.

[0008] In the above-mentioned application technology scheme, angiotensin 1-7 enhances autophagy by activating the AMPK / mTOR pathway, thereby improving or alleviating sepsis-induced acute kidney injury, and is used in the preparation of a drug for treating sepsis-induced acute kidney injury.

[0009] In the above-mentioned application technology scheme, angiotensin 1-7 enhances autophagy in sepsis renal tissue.

[0010] In the above-mentioned application technology scheme, angiotensin 1-7 reduces the levels of serum creatinine and urea nitrogen in subjects with sepsis, reduces the levels of serum inflammatory factors, and inhibits apoptosis of renal tubular epithelial cells in sepsis renal tissue.

[0011] In the above-mentioned application technology scheme, angiotensin 1-7 regulates the expression of AMPK and mTOR, reversing the trend of decreased AMPK expression and increased mTOR expression caused by acute kidney injury in sepsis.

[0012] The second aspect of the present invention provides the use of angiotensin 1-7 as an active ingredient in the preparation of a drug for promoting autophagy of sepsis renal tissue cells, wherein the drug enhances the autophagy of sepsis renal tissue cells.

[0013] In the above-mentioned application technology scheme, the drug regulates the expression of AMPK and mTOR, and enhances autophagy by activating the AMPK / mTOR pathway.

[0014] In the above-mentioned application technology scheme, the drug reverses the trend of decreased AMPK expression and increased mTOR expression caused by acute kidney injury in sepsis.

[0015] In the above-mentioned application technology scheme, the drug improves or alleviates sepsis-induced acute kidney injury by regulating the AMPK-mTOR signaling pathway to enhance autophagy, reduces serum creatinine and urea nitrogen levels in septic subjects, reduces serum inflammatory factor levels, inhibits apoptosis of renal tubular epithelial cells in septic renal tissue, and alleviates sepsis-induced acute kidney injury.

[0016] In the above-mentioned application technology solution, the drug also includes pharmaceutically acceptable excipients, and is preferably an injection.

[0017] The beneficial effects of the present invention are:

[0018] The present study established a mouse model of acute kidney injury in sepsis by intraperitoneal injection of LPS, intervened with Ang-(1-7), and detected the effect of Ang-(1-7) on improving renal function, pathological damage and reducing inflammation levels in mice with acute kidney injury in sepsis by regulating the activation of the AMPK / mTOR pathway and enhancing autophagy.

[0019] The present invention studies have confirmed that the polypeptide Ang-(1-7) has an effect on autophagy in mice with acute kidney injury caused by sepsis, and has an effect on reducing kidney injury and inflammation levels, and has a significant therapeutic effect on improving renal function and pathological damage, inhibiting inflammation and apoptosis, and has no obvious toxicity to mice. The present invention provides a new treatment strategy for acute kidney injury caused by sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1These are the results of serum creatinine and serum urea nitrogen level tests in mice treated with Ang-(1-7).

[0021] Figure 2 Representative images of HE staining of renal tissues treated with Ang-(1-7).

[0022] Figure 3 These are the results of the detection of TNF-a and IL-6 protein expression levels in renal tissue after Ang-(1-7) intervention.

[0023] Figure 4 These are the results of detecting the expression levels of P62 and LC3-Ⅱ proteins in renal tissues intervened by Ang-(1-7).

[0024] Figure 5 Representative transmission electron microscopic images and statistical analysis results of renal cortical tubular epithelial cells in septic mice treated with Ang-(1-7).

[0025] Figure 6 The results of serum creatinine and serum urea nitrogen levels in septic mice treated with Ang-(1-7) and the autophagy inhibitor 3-MA.

[0026] Figure 7 These are the results of the detection of serum inflammatory factors TNF-a and IL-6 levels in septic mice treated with Ang-(1-7) and the autophagy inhibitor 3-MA.

[0027] Figure 8 It was shown that 3-MA attenuated the inhibition of Ang-(1-7) on apoptosis of renal tubular epithelial cells in septic mice.

[0028] Fig. 9 The expression of autophagy-related proteins increased due to Ang-(1-7), and the expression levels of P62 and LC3-Ⅱ proteins in renal tissue after 3-MA treatment were detected.

[0029] Fig.10 Representative images and statistical analysis of the increase in the number of autophagosomes and autolysosomes caused by Ang-(1-7) under transmission electron microscopy in the number of autophagosomes and autolysosomes in renal cortical tubular epithelial cells of septic mice after 3-MA treatment.

[0030] Fig.11 The results showed that AMPK and mTOR protein expression and phosphorylation levels in renal tissue after Ang-(1-7) intervention.

[0031] Fig.12 These are the results of serum creatinine and urea nitrogen level tests in mice after intervention with Ang-(1-7)+AMPK inhibitor Compound C.

[0032] Fig.13 These are the test results of the levels of inflammatory factors TNF-a and IL-6 in mouse serum after intervention with Ang-(1-7)+AMPK inhibitor Compound C.

[0033] Fig.14 These are the results of detecting the expression levels of P62 and LC3-Ⅱ proteins in renal tissue after the intervention of Ang-(1-7)+AMPK inhibitor Compound C.

[0034] Fig.15 Representative transmission electron microscopic images and statistical analysis results of renal cortical tubular epithelial cells in septic mice after intervention with Ang-(1-7)+AMPK inhibitor Compound C. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0037] Example 1

[0038] 1. Sources of main reagents and materials:

[0039] Lipopolysaccharide was purchased from Sigma-Aldrich (USA); Ang-(1-7) was purchased from APExBIO (USA); AMPK, p-AMPK, and LC3B antibodies were purchased from CST (USA); TNF-a antibody was purchased from Wuhan Tri-Eagle Biotechnology (China); IL-6 antibody was purchased from Abiwei Biotech (China); p-mTOR and mTOR antibodies were purchased from Abmart (China); autophagy inhibitor 3-MA and AMPK inhibitor Compound C were purchased from Selleck (USA).

[0040] 2 Implementation methods

[0041] 2.1 Establishment and intervention of sepsis mouse model

[0042] ① The first batch: 18 male C57 mice aged 6-8 weeks were randomly divided into A (control group), B (LPS group), and C (LPS+Ang-(1-7) group), with 6 mice in each group. First, groups A, B, and C were given saline, saline, and Ang-(1-7) intraperitoneal injections for 2 consecutive days, respectively. Then on the third day, groups B and C were given saline and Ang-(1-7) intraperitoneal injections 30 minutes in advance of the intraperitoneal injection of LPS 15 mg / kg, respectively, and the mice were killed 24 hours after modeling. Blood and kidney tissues of mice were collected for subsequent experiments. The injection dose of Ang-(1-7) was 2 mg / kg each time.

[0043] ② The second batch: 24 male C57 mice aged 6-8 weeks were randomly divided into A (control group), B (LPS group), C (LPS+Ang-(1-7 group)), and D (LPS+Ang-(1-7)+3MA group), with 6 mice in each group. First, groups A, B, C, and D were given saline, saline and Ang-(1-7), and Ang-(1-7) intraperitoneal injections for 2 consecutive days. Then on the third day, groups B, C, and D were given saline, Ang-(1-7), and Ang-(1-7) intraperitoneal injections 30 minutes in advance of LPS 15 mg / kg, and group D was injected with autophagy inhibitor 3MA (20 mg / kg) 1 hour before LPS intraperitoneal injection. Mice were killed 24 hours after modeling. Blood and kidney tissues of mice were collected for subsequent experiments. The injection dose of Ang-(1-7) was 2 mg / kg each time.

[0044] ③ The third batch: 24 male C57 mice aged 6-8 weeks were randomly divided into A (control group), B (LPS group), C (LPS+Ang-(1-7 group)), and D (LPS+Ang-(1-7)+CC group), with 6 mice in each group. First, groups A, B, C, and D were given saline, saline and Ang-(1-7), and Ang-(1-7) intraperitoneal injections for 2 consecutive days. Then on the third day, groups B, C, and D were given saline, Ang-(1-7), and Ang-(1-7) intraperitoneal injections 30 minutes in advance of LPS 15 mg / kg, and group D was injected with AMPK inhibitor Compound C (15 mg / kg) 1 hour before LPS intraperitoneal injection. Mice were killed 24 hours after modeling. Blood and kidney tissues of mice were collected for subsequent experiments. The injection dose of Ang-(1-7) was 2 mg / kg each time.

[0045] 2.2 Renal function test

[0046] The collected mouse blood samples were tested using the mindray biochemical detection kit and a fully automatic biochemical analyzer (TOSHIBA TBA-120FR).

[0047] 2.3ELISA

[0048] First, take out the ELISA kit from the refrigerator and put it to room temperature. Pipette the serum into a centrifuge tube for later use, and mark the tube wall. Prepare the washing buffer: prepare it in a ratio of 1:19 by volume of washing buffer and distilled water, and use it immediately. Take out the microplate, set up standard wells and sample wells, and add 50μL of standards of different concentration gradients to the standard wells according to the instructions; add 10μL of the sample to be tested to the sample wells, and then add the sample diluent to make up to 50μL. Add 100μL of the labeled antibody in the kit to each well, seal the wells, and incubate in a 37℃ constant temperature incubator for 60 minutes. Wash the microplate with the pre-prepared washing buffer, pat dry, and repeat 5 times. Add 50μL of substrate each, and incubate in a 37℃ constant temperature incubator away from light for 30 minutes. Add 50μL of stop solution to stop the reaction. Measure the absorbance at a wavelength of 450nm with an enzyme reader. Analysis: Calculate the concentration of the sample based on the standard curve and absorbance.

[0049] 2.4 HE staining

[0050] First, fix the kidney with 4% paraformaldehyde, remove the fixed kidney, and dehydrate it with alcohol from low to high concentrations (70%, 80%, 90%\95%, 100%). Then put the dehydrated tissue in xylene for 10 minutes to make it transparent, repeat twice. Then soak it in melted paraffin and embed it for 1-2 hours. Then cut it into slices of appropriate thickness, place it on a slide, and dry it. Next, place the slices in a high-temperature water bath, and after the paraffin melts, put it in xylene for dewaxing, repeat 3 times, 10 minutes each time. Then put it in different concentrations of alcohol (100%, 100%, 90%, 80%, 70%, 50%) for dewaxing. Put the slices in hematoxylin stain for 5-10 minutes, rinse with running water; then differentiate with hydrochloric acid ethanol for 30 seconds, rinse with running water; then use ammonia water to return to blue for 1 minute, rinse with running water. The slices were sequentially immersed in different concentrations of alcohol (75%, 85%, 100%, 100%) for dehydration. Then, they were transparentized with xylene for 5 minutes. The slices were sealed with neutral gum and placed under a slice scanner after drying.

[0051] 2.5 Transmission electron microscopy

[0052] First, the kidneys were sampled and the renal cortex was cut into 1 mm3 slices with a sterile blade and placed in an electron microscope fixative to fix the renal tissue in time. Then, the fixed slices were stained with 1% osmium tetroxide. Dehydrated in increasing acetone concentrations. Next, the slices were embedded in epoxy resin and cut and stained with uranyl acetate and lead citrate. Finally, the ultrastructure of the renal cortical tubular epithelial cells was observed under an electron microscope and images were collected.

[0053] 2.6 Western blot

[0054] Total protein was extracted from HUVECs using RIPA lysis buffer containing protease inhibitors, phosphatase inhibitors, and PMSF, and the protein concentration was determined using a BCA protein concentration assay kit. Samples were boiled in SDS loading buffer. Equal amounts of protein were separated using SDS-PAGE and transferred to PVDF membranes. Next, the PVDF membrane was blocked for 1 hour at room temperature and stained with primary antibodies overnight at 4°C. The next day, the corresponding species-specific secondary antibodies were incubated for 60 minutes. Finally, the protein-antibody complex was visualized using an ECL enhancement kit.

[0055] 2.7 Tunnel staining

[0056] First, dewax the paraffin sections to water: sequentially place the sections in xylene I for 10 min, xylene II for 10 min, and xylene II for 10 min.

[0057] Ⅱ10min-anhydrous ethanolⅠ5min-anhydrous ethanolⅡ5min-anhydrous ethanolIII5min-distilled water washing. Then proteinase K repair: After the slices are slightly dried, use a tissue pen to draw a circle around the tissue (to prevent the liquid from flowing away), add proteinase K working solution (20μg / ml) in the circle to cover the tissue, and incubate in a 37-degree incubator for 22min. Place the slide in PBS (PH7.4) and shake and wash on a decolorizing shaker 3 times, each time for 5min. (Protease K working solution preparation method, original solution: PBS = 1:9). Re-permeabilization: After the slices are slightly dried, add permeabilization working solution in the circle to cover the tissue, incubate at room temperature for 20min, place the slide in PBS (PH7.4) and shake and wash on a decolorizing shaker 3 times, each time for 5min. (The membrane permeabilization solution is 0.1% triton. The configuration method is triron stock solution: PBS = 1:1000. Then add the reaction solution: according to the number of slides and tissue size, take an appropriate amount of TDT enzyme in the tunel kit, mix dUTP in a ratio of 1:50, add it to the circle to cover the tissue, and lay the slices flat in a humidified box and incubate them in a 37°C constant temperature box for 1-2 hours. Add a small amount of water to the humidified box to maintain humidity. DAPI counterstaining of cell nuclei: Wash the slices with PBS (PH7.4) 3 times, 5 minutes each time. After removing PBS, add DAPI staining solution to the circle and incubate at room temperature away from light for 10 minutes. Finally, seal the slides: Place the slides in PBS (PH7.4) and shake them on a decolorizing shaker to wash 3 times, 5 minutes each time. Shake the slices slightly dry and seal them with anti-fluorescence quenching sealing agents.

[0058] 2.8 Statistical analysis

[0059] Statistical analysis was performed using commercial software (GraphPadPrism 10.0.0; GraphPad Software, San Diego, CA, USA). Each experiment was repeated at least 3 times. Data are presented as mean ± standard deviation (SD). The comparison between two groups was performed using t-test, and the comparison between multiple groups was performed using one-way analysis of variance. A p value of 0.05 was considered statistically significant.

[0060] (P.Value<0.05 is indicated by *; P.Value<0.01 is indicated by **; P.Value<0.001 is indicated by ***)

[0061] 3 Results

[0062] 3.1 Protective effect of Ang-(1-7) on acute kidney injury in sepsis and its effect on autophagy

[0063] 1. Ang-(1-7) reduces sepsis-induced acute kidney injury

[0064] Ang-(1-7) reduces renal dysfunction in sepsis Figure 1 ); HE staining can improve the pathological changes of kidney tissue ( Figure 2 ); reduce the expression level of inflammatory factor proteins in renal tissue ( Figure 3 ), thereby protecting against sepsis-induced acute kidney injury.

[0065] 2. Ang-(1-7) enhances autophagy in kidney tissue of septic mice

[0066] The results of immunoblotting show that when Ang-(1-7) intervened in mice, the LC3-Ⅱ value increased significantly compared with the LPS group, and the p62 protein level decreased significantly compared with the LPS group ( Figure 4 ); Transmission electron microscopy showed that the number of autophagosomes and autophagolysosomes in the renal cortical tubular epithelial cells of mice in the Ang-(1-7) intervention group increased significantly ( Figure 5 ); indicating that Ang-(1-7) treatment enhances autophagy in the kidney tissue of septic mice.

[0067] Autophagy significantly increased in the kidney tissue of septic mice treated with Ang-(1-7), indicating that Ang-(1-7) has a protective effect on sepsis-induced acute kidney injury, which may be related to enhanced autophagy. Therefore, it is still unclear whether the protective effect of Ang-(1-7) on sepsis-induced acute kidney injury depends on the regulation of autophagy. In this study, the autophagy inhibitor 3-MA was used to explore whether autophagy is involved in the protective effect of Ang-(1-7) on sepsis-induced acute kidney injury mice.

[0068] 3.2 The protective effect of Ang-(1-7) on acute kidney injury in sepsis depends on the regulation of autophagy

[0069] 1. When Ang-(1-7)-treated septic mice were given the autophagy inhibitor 3-MA at the same time, 3-MA treatment eliminated the protective effects of Ang-(1-7) on improving renal function, reducing inflammatory factors, and reducing apoptosis of renal tubular epithelial cells ( Figure 6 , 7 , 8).

[0070] 2. The autophagy inhibitor 3-MA abolished the effect of Ang-(1-7) on autophagy in mice with acute kidney injury caused by sepsis

[0071] The increase of LC3II caused by Ang-(1-7) was significantly reduced after 3-MA treatment, the downregulation of P62 was significantly increased, and the autophagosomes were significantly reduced ( Fig. 9 , 10 ).

[0072] These results indicate that Ang-(1-7) plays a role in sepsis-induced acute kidney injury by enhancing autophagy.

[0073] It is not clear how Ang-(1-7) regulates autophagy and participates in the occurrence of acute kidney injury in sepsis. Whether Ang-(1-7) regulates autophagy by affecting the AMPK-mTOR pathway needs further exploration. Next, this study used the AMPK inhibitor Compound C to explore whether Ang-(1-7) regulates autophagy by affecting the AMPK-mTOR pathway and participates in the protective effect on mice with acute kidney injury in sepsis.

[0074] Ang-(1-7) affects the AMPK-mTOR pathway to regulate autophagy and protects mice from acute kidney injury caused by sepsis

[0075] 1. Effect of Ang-(1-7) on the expression of AMPK-mTOR pathway when regulating autophagy in acute kidney injury caused by sepsis

[0076] We used Western blot to detect the expression of AMPK / mTOR pathway-related proteins in the cortex of mouse renal tissue. Compared with the Control group, LPS and Ang-(1-7) intervention had no effect on the expression of AMPK and mTOR total proteins. However, after LPS intervention, the expression of p-AMPK showed a downward trend, and the expression of p-mTOR showed an upward trend. Ang-(1-7) significantly reversed the protein expression levels of p-AMPK and p-mTOR ( Fig.11 ). This indicates that AMPK and mTOR are involved in the occurrence of acute kidney injury in sepsis, and Ang-(1-7) can regulate the expression of AMPK and mTOR.

[0077] 2. Ang-(1-7) intervention can significantly reduce the levels of serum creatinine and urea nitrogen in septic mice, and reduce the levels of serum inflammatory factors TNF-a and IL-6. The above effects of Ang-(1-7) can be inhibited by AMPK inhibitor Compound C. Fig.12 , 13 ).

[0078] 3. Ang-(1-7) intervention can significantly enhance the autophagy level in septic mice, and this effect can be inhibited by AMPK inhibitor Compound C:

[0079] The increase of LC3II caused by Ang-(1-7) was significantly reduced after treatment with AMPK inhibitor Compound C, while the downregulation of P62 was significantly increased and the autophagosomes were significantly reduced ( Fig.14 , 15 ).

[0080] The above results demonstrate that Ang-(1-7) can alleviate sepsis-induced acute kidney injury by promoting autophagy, which may be achieved by regulating the AMPK-mTOR signaling pathway.

Claims

1. Application of angiotensin 1-7 in the preparation of drugs for activating AMPK / mTOR pathway to enhance autophagy.

2. The use according to claim 1, characterized in that: Angiotensin 1-7 enhances autophagy by activating the AMPK / mTOR pathway, thereby improving or alleviating sepsis-induced acute kidney injury, and is used in the preparation of drugs for treating sepsis-induced acute kidney injury.

3. The use according to claim 2, characterized in that: Angiotensin 1-7 enhances autophagy in sepsis renal tissue.

4. The use according to claim 2, characterized in that: Angiotensin 1-7 reduces serum creatinine and urea nitrogen levels in subjects with sepsis, reduces serum inflammatory factor levels, and inhibits apoptosis of renal tubular epithelial cells in sepsis renal tissue.

5. The use according to claim 2, characterized in that: Angiotensin 1-7 regulates the expression of AMPK and mTOR, reversing the trend of decreased AMPK expression and increased mTOR expression caused by acute kidney injury in sepsis.

6. Use of angiotensin 1-7 as an active ingredient in the preparation of a drug for promoting autophagy of renal tissue cells in sepsis, wherein the drug enhances the autophagy of renal tissue cells in sepsis.

7. The use according to claim 6, characterized in that: The drug regulates the expression of AMPK and mTOR and enhances autophagy by activating the AMPK / mTOR pathway.

8. The use according to claim 7, characterized in that: The drug reverses the trend of decreased AMPK expression and increased mTOR expression caused by acute kidney injury in sepsis.

9. The use according to claim 7, characterized in that: The drug improves or alleviates sepsis-induced acute kidney injury by regulating the AMPK-mTOR signaling pathway to enhance autophagy, reduces serum creatinine and urea nitrogen levels in sepsis subjects, reduces serum inflammatory factor levels, inhibits apoptosis of renal tubular epithelial cells in sepsis renal tissue, and alleviates sepsis-induced acute kidney injury.

10. The use according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients, An injection is preferred.

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