Application of paeoniflorin in preparation of medicine for treating nano-zinc oxide induced renal injury

Abstract

The invention discloses paeoniflorin serving as an SIRT1 agonist and application of the paeoniflorin. Test results prove that paeoniflorin can be stably combined with SIRT1 at an agonist site so as to promote the activity of deacetylase SIRT1. The deacetylase SIRT1 agonist disclosed by the invention has a good activation effect on a deacetylation reaction of SIRT1 and a p53 substrate on a molecular level. The application proves that paeoniflorin can significantly improve nano-zinc oxide-induced mouse kidney injury in vivo, and can be used as a potential medicine for treating nano-zinc oxide-induced kidney injury by improving renal dysfunction, activating kidney SIRT1 and increasing antioxidant enzyme activity. The new function of paeoniflorin found in the invention provides a new strategy for research and development of SIRT1 agonist drugs, and provides a new direction for development of nano-zinc oxide protective agents.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmacy, and specifically relates to the application of paeoniflorin in the preparation of a drug for treating nano-zinc oxide-induced kidney injury. Background Art

[0002] SIRT1 belongs to the NAD+-dependent histone deacetylase family, that is, the most conserved member of the sirtuins family. SIRT1 participates in the deacetylation modification of various mammalian transcription factors such as p53, FOXOs, and NF-κB, and is a key metabolic regulator, participating in various disease-related cellular processes. For example, SIRT1 may inhibit renal cell apoptosis, inflammation, and fibrosis in the kidney, and may regulate lipid metabolism, autophagy, blood pressure, and sodium balance. SIRT1 plays a renoprotective role in acute kidney injury caused by different reasons, improves the resistance of the kidney to acute stress, and participates in the regulation of energy metabolism. Therefore, the activation of SIRT1 in the kidney may be a new therapeutic target.

[0003] Paeoniflorin (PF) is a bioactive component in the roots of plants of the Paeoniaceae family. PF was first discovered by isolating it from the roots of Paeonia lactiflora. Paeonia lactiflora has been used in traditional Chinese medicine for thousands of years to treat neurological diseases, liver diseases, and cardiovascular and cerebrovascular diseases. In 1963, PF was identified as a monoterpene glycoside compound. Subsequent studies have found that PF has anti-inflammatory and antioxidant biological effects, and has potential pharmacological effects on various liver diseases and injuries, especially has a protective effect on a variety of kidney diseases, including diabetic nephropathy, mesangial proliferative glomerulonephritis, and acute kidney injury (AKI) caused by acute pancreatitis. Research shows that paeoniflorin can reduce acute kidney injury caused by ischemia-reperfusion and reduce kidney injury by inhibiting Slc7a11-mediated ferroptosis. The protective effect and therapeutic potential of PF have been confirmed.

[0004] Zinc (Zn) is an important trace mineral element that needs to be ingested through diet to exert its physiological functions. In addition, Zn is an important constituent element of various enzymes in biological systems and plays important roles in genetic information expression, chromatin structure, protein synthesis, and cell membrane homeostasis. It is crucial for the growth and development, immunity, bacterial defense, reproduction, and antioxidant capacity of humans and animals. Zinc oxide (ZnO), as a Zn-source chemical additive, is not only widely used in industrial and daily chemical fields but has also been used as a feed additive in livestock production for a long time. ZnO in the diet can improve the growth performance of weaned piglets and inhibit intestinal inflammation. Since the 1990s, with the development of nanotechnology, zinc oxide nanoparticles (ZnO NPs), as a substitute for ZnO, have gradually been used in cosmetics, biomedical technology, food additives, and feed additives due to their high bioavailability and good antibacterial properties. As a feed supplement, ZnO NPs can promote animal growth, improve feed conversion rate, and inhibit diarrhea. Compared with ordinary ZnO, ZnO NPs have a smaller particle size, a higher specific surface area, and more surface atoms, as well as unsaturated characteristics and strong biological activity. In recent years, the global annual output of nano-zinc oxide has exceeded 1 million tons and continues to grow. However, the rapid development of nanotechnology industrialization has led to the easier entry of heavy metal nanomaterials such as nano-zinc oxide into the human body directly or indirectly through the environment and the food chain during production and use. For example, evidence shows that nano-zinc oxide can invade the human body and animals through various routes such as the digestive tract, respiratory tract, skin, and vein, causing kidney toxicity damage. Long-term high-dose exposure to nano-zinc oxide can inhibit animal growth and even cause animal death, resulting in economic losses to the livestock industry. The safety of the environment and the human body has also received great attention, and reducing the kidney damage caused by nano-zinc oxide is also an urgent problem to be solved. Summary of the Invention

[0005] The object of the present invention is the application of paeoniflorin as a SIRT1 agonist in improving kidney injury induced by nano-zinc oxide. The nano-zinc oxide mentioned is an irregular zinc oxide nano-feed additive with an average particle size of less than 50 nm. The paeoniflorin mainly refers to the monomeric compound produced from the roots of Paeonia lactiflora. The present invention discloses the application of paeoniflorin as a SIRT1 agonist in improving kidney injury induced by nano-zinc oxide.

[0006] The present invention clarifies that paeoniflorin stably binds to the agonist site of SIRT1, promoting the activity of the deacetylase SIRT1. It further proves that paeoniflorin can significantly reduce kidney injury induced by nano-zinc oxide, improve renal function disorders, activate renal SIRT1, and increase the activity of antioxidant enzymes. The present invention demonstrates that paeoniflorin can be used both as a drug developed from a SIRT1 agonist and as a protective agent against nano-zinc oxide-induced kidney injury.

[0007] The technical content of the present invention is as follows: The application of paeoniflorin in the preparation of a drug for treating nano-zinc oxide-induced kidney injury; the nano-zinc oxide-induced kidney injury refers to an irregular zinc oxide nano-feed additive with an average particle size of 50 nm. Experimental results show that paeoniflorin can stably bind to SIRT1 at the agonist site, thereby promoting the activity of the deacetylase SIRT1. At the same time, paeoniflorin can significantly improve nano-zinc oxide-induced kidney injury in mice by improving renal function disorders, activating renal SIRT1, and increasing antioxidant enzyme activity.

[0008] Paeoniflorin can stably bind to SIRT1 at the agonist site, thereby promoting the activity of the deacetylase SIRT1. The deacetylase SIRT1 agonist described in the present invention has a good activating effect on the deacetylation reaction of SIRT1 and the p53 substrate at the molecular level. The present invention proves that paeoniflorin can significantly improve nano-zinc oxide-induced kidney injury, improve renal function disorders, activate renal SIRT1, and increase antioxidant enzyme activity. Thus, it can be used as a potential drug for treating kidney injury caused by nano-zinc oxide. The newly discovered function of paeoniflorin in the present invention provides a new strategy for the research and development of SIRT1 agonist drugs and a new direction for the development of nano-zinc oxide kidney injury protectants.

[0009] The present invention uses molecular docking, cellular thermal shift assay, and surface plasmon resonance techniques to prove that paeoniflorin can stably bind to SIRT1 at the agonist site.

[0010] Verified by an in vitro SIRT1 enzyme-coupled reaction experiment, the paeoniflorin described in the present invention has a good activating effect as a deacetylase SIRT1 agonist.

[0011] (3) Through experiments in mice, it is found that paeoniflorin can activate the expression of the renal deacetylase SIRT1 protein in vivo.

[0012] (4) Using paeoniflorin to improve nano-zinc oxide-induced kidney injury, the addition method of paeoniflorin in this application is to intragastrically administer 75 mg / mL of paeoniflorin 2 hours in advance, and then intragastrically administer 360 mg / kg of nano-zinc oxide continuously for 28 days. Kidney pathological sections, serum urea nitrogen and creatinine, and renal antioxidant enzyme activity are used as detection indicators.

[0013] The present invention mainly investigates the use of paeoniflorin as a SIRT1 agonist and its application, with the focus on solving the lack of research and development of SIRT1 agonists and the lack of research on the mechanism of nano-zinc oxide nephrotoxicity and the need for the development of protectants. The difficulty of the invention lies in proving the stable binding of paeoniflorin to SIRT1 and promoting its deacetylase activity; the establishment of a nano-zinc oxide kidney injury model, the setting of the intervention route and dose of paeoniflorin; the detection of kidney injury protection indicators, etc.

[0014] The innovation points of the present invention are as follows: (1) For the first time, the in vitro binding of paeoniflorin to SIRT1 and the promotion of the deacetylase rate of SIRT1 were verified; (2) For the first time, the application of paeoniflorin as a protective agent against nano-zinc oxide-induced kidney injury was developed, and the protective mechanism of paeoniflorin was explored in an in vivo mouse model. By activating the SIRT1 expression pathway, the effects of reducing nano-zinc oxide-induced kidney injury and oxidative stress were achieved.

[0015] The application of paeoniflorin in activating SIRT1 and protecting against nano-zinc oxide-induced kidney injury disclosed by the present invention has the following positive effects compared with the prior art: Paeoniflorin was analyzed as a candidate drug for SIRT1 agonists; Paeoniflorin was developed as a protective agent against nano-zinc oxide-induced kidney injury, providing a new strategy for solving nano-zinc oxide-induced kidney injury. Brief Description of the Drawings

[0016] Figure 1 It is the interaction mode of PF-SIRT1 at the agonist site Note: (A) Molecular docking of the binding mode of PF and SIRT1; (B) Details of the action of PF-SIRT1; The results show that: Molecular docking of PF-SIRT1 (PDB: 4ZZJ) was carried out between PF and the STAC binding domain (SBD) in the N-terminal region of SIRT1. The binding energy was -5.228 kcal / mol. Hydrogen bonds and hydrophobic interactions are the keys to stable binding.

[0017] Figure 2 It is the analysis of the thermal stability of SIRT1 protein by thermal shift experiment The results show that: PF can bind to SIRT1 protein to improve the protein stability.

[0018] Figure 3 Surface plasmon resonance analysis of the binding affinity of PF-SIRT1 The results show that: PF and SIRT1 showed a typical binding-dissociation curve, and the affinity constant (KD) was 45.34 μM, indicating that PF can directly bind to SIRT1.

[0019] Figure 4 The effect of PF on the deacetylase rate of SIRT1 The results show that PF can concentration-dependently increase the deacetylase rate of SIRT1 on the substrate, and PF has the targeting property of specifically binding and activating SIRT1.

[0020] Figure 5 The effect of PF on kidney tissue pathology and renal function indexes Note: (A) Kidney pathological section; (B) Cre creatinine, BUN blood urea nitrogen. Compared with the control group, ** P <0.01; compared with the group treated with ZnO NPs alone, # P <0.05, ## P <0.01; The results showed that PF significantly reduced the kidney injury and renal function disorder induced by zinc oxide nanoparticles; Figure 6 Effects of PF on oxidative stress indexes Note: MDA malondialdehyde, CAT catalase, SOD superoxide dismutase, GSH-PX glutathione peroxidase. Compared with the control group, * P <0.05, ** P <0.01; compared with the group treated with ZnO NPs alone, # P <0.05, ## P <0.01; The results showed that PF significantly reduced the level of oxidative stress in the kidneys induced by zinc oxide nanoparticles, Figure 7 Effects of PF on the expression of SIRT1 in the kidneys; The results showed that PF significantly activated the activity of the deacetylase SIRT1. Detailed implementation manners

[0021] The present invention will be described below through specific implementation manners. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the implementation manners should be understood as illustrative rather than limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications to the material components and dosages in these implementation manners also fall within the protection scope of the present invention. The raw material paeoniflorin and reagents used in the present invention are all commercially available. Examples

[0022] Download the 3D pdb structure of SIRT1 from the RCSB PDB database with the ID 4ZZJ. Open AutoDock vina with AutoDock tools, remove the ligand, remove water molecules, add hydrogen, and save it as a pdbqt format receptor file. Download the mol2 format structure of PF from the TCMSP database, optimize the molecular configuration with ChemOffice 2019, and output the lowest energy. Open the mol2 format file of PF with AutoDock vina, add hydrogen, set it as the ligand, detect the center of rotation and rotatable chemical bonds, and export the pdbgt format ligand file. Open the receptor and ligand with AutoDock vina. Adjust the center coordinates of the docking box to Center x = -0.827, Center y = 45.618, and Center z = -1.076; set the box size to 20×20×20, and perform the docking 100 times to obtain the binding energy, and export the complex for further visualization with Pymol.

[0023] As Figure 1 shown in A, according to the reported binding site of SIRT1 agonists in the literature, that is, the STAC binding domain (SBD) located in the N-terminal region, molecular docking of PF-SIRT1 (PDB: 4ZZJ) was carried out. The binding energy of PF-SIRT1 at the agonist site was calculated by AutoDock vina to be -5.228 kcal / mol, and the binding was relatively stable. The interaction mode of PF-SIRT1 is shown in Figure 1 A, and PF can bind to SIRT1. The details of the interaction were demonstrated using PyMOL 2.04 ( Figure 1 B), and it was found that the oxygen atom of PF near the carbonyl group of the benzene ring formed a hydrogen bond with the hydrogen atom on ASN226 of the SIRT1 protein, and PF formed hydrophobic interactions with the hydrophobic amino acids LEU206, ILE223, and ILE227 of SIRT1. It was shown that PF could bind to the SIRT1 agonist site, and hydrogen bonds and hydrophobic interactions were the key to stable binding.

[0024] An experiment to verify the binding affinity between a drug and an intracellular target protein based on the characteristic that the binding of a drug to a protein improves thermal stability. HepG2 cells were seeded in 6-well plates at 2×10 5 cells / well, and PF and DMEM full liquid intervention groups were set up. After the treatment was completed, the cells were washed twice with PBS, digested to obtain a single-cell suspension and transferred to a centrifuge tube, centrifuged at 1500 rpm for 5 min, and the precipitate was retained and resuspended with 200 μL of DMEM. The samples were heated and denatured at room temperature and temperature gradients of 45°C to 65°C for 5 min in a PCR instrument, then centrifuged at 1500 rpm for 5 min to discard the supernatant, 60 μL of RIPA lysis buffer was added, and the samples were lysed on ice for 10 min, and then Western blot experiments were performed.

[0025] It can be seen from Figure 2 that although the proteins in each group were gradually degraded under stepwise temperature, compared with the control group without PF intervention, the thermal stability of intracellular SIRT1 protein in the PF group was higher, suggesting that PF might bind to SIRT1 to enhance the structural stability of this receptor protein.

[0026] Surface plasmon resonance (SPR): (1) Chip preparation: A mixture of 50 mM NHS and 200 mM EDC was obtained using an amino coupling kit to activate the sensing chip. The SIRT1 target protein was diluted to 50 μg / mL with 10 mM sodium acetate (pH 4.5). The flow rate was set at 10 μL / min for 7 min. After immobilization, it was blocked with 1 M ethanolamine (pH 8.5) in the amino coupling kit. The final coupling amount of SIRT1 was 13053.0 RU. (2) Reaction conditions: PF was diluted to obtain 8 analytical concentrations of 0 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. The flow rate was set at 30 μL / min, the binding time was 90 s, and the dissociation time was 120 s. (3) Kinetic data determination: Kinetic parameters were obtained by fitting the data using BIAcore T200 software.

[0027] It can be seen from Figure 3 that SPR further verified the binding affinity between PF and SIRT1. The results showed that when the concentration of SIRT1 protein was 50 μg / mL, it could be stably immobilized. PF and SIRT1 presented a typical binding-dissociation curve, and the affinity constant (KD) was 45.34 μM, indicating that PF could directly bind to SIRT1 ( Figure 3 ) SIRT1 enzyme-coupled reaction: SIRT1 hydrolyzes NAD to generate nicotinamide, and nicotinamidase PncA converts nicotinamide to ammonia. Under the action of glutamate dehydrogenase, ammonia, α-ketoglutaric acid, and NAD(P)H are converted to NAD(P)+, which causes a change in absorbance at 340 nm. The reaction system is as follows:

[0028] After mixing the above reagents (without adding NAD), incubate at 37 °C for 10 min, add NAD, and read at 340 nm wavelength on an enzyme-labeling instrument once every 30 s for 30 times, and then take the stage with the fastest reading change. Calculate the reaction rate using the Lambert-Beer law, and use the formula A = kcd, where A = absorbance, k = molar absorption coefficient 6.22 mM-1cm-1, and d = optical path 0.3 cm. The change in c, that is, the change in the molar concentration of NADPH, was measured through the change in A. The deacetylation rate = the reaction rate of each group - the reaction rate of the control group without adding NAD (μM / s).

[0029] It can be seen from Figure 4 that PF can increase the deacetylase rate of SIRT1 on p53 substrates in a concentration-dependent manner, showing a good activation effect. Thus, PF has the targeting property of specifically binding to and activating SIRT1.

[0030] Conclusion: It shows that paeoniflorin stably binds to SIRT1 and promotes its enzyme activity.

[0031] Example 2 Paeoniflorin reduces nano-zinc oxide-induced kidney injury and renal function disorder: A total of 40 6-8-week-old male C57BL / 6 mice (body weight: 18-22 g) were raised under pathogen-free conditions (temperature 22 °C, 12-hour light-dark cycle), fed with standard feed during the experiment, and given free access to water. All mice were randomly divided into 4 groups (n = 10): control group, 75 mg / kg PF group, 360 mg / kg ZnO NPs group, 75 mg / kg PF + 360 mg / kg ZnO NPs group. The first control group was given sterile normal saline orally; the second group was given 75 mg / kg PF, the third group was given 360 mg / kg ZnO NPs by gavage; the fourth group was given 75 mg / kg PF by gavage 2 hours before ZnO NPs-intoxicated mice, and the administration was continued for 28 days. After all treatments were completed, all mice were fasted and water-deprived for 12 hours. The mice were anesthetized with 2.6 mg / kg ketamine and 0.16 mg / kg xylazine respectively, and blood was collected from the mice by eye enucleation method. After dissection, the kidney tissues of the mice were taken. The kidney tissues were quickly fixed in 10% formalin for 48 hours. Tissue section preparation and hematoxylin-eosin (HE) staining procedure: After fixation in formalin, the kidney tissues were rinsed with distilled water, then dehydrated with alcohol of gradient concentration, specimens were prepared by paraffin embedding method, and sections with a thickness of 4 μm were collected using a rotary microtome. The paraffin sections were stained with hematoxylin and eosin, and the method was to place the paraffin sections on glass slides and dry them at 37 °C for 24 hours. The sections were dewaxed and dehydrated with xylene and gradient ethanol respectively. The sections were rinsed with distilled water, then stained with hematoxylin for 10 minutes and differentiated with ethanol. After being immersed in water and rinsed again, the sections were stained with eosin for 10 minutes and rinsed again. Finally, the sections were dehydrated with alcohol, soaked in xylene, and then sealed with neutral gum and observed under an optical microscope.

[0032] It can be seen from Figure 5 that obvious damage occurred in the kidney tissues of the ZnO NPs single-treatment group, with pathological changes such as cytoplasmic vacuolization, interstitial edema, interstitial hemorrhage, and infiltration of inflammatory factors. While in the paeoniflorin treatment group, cytoplasmic vacuolization and interstitial edema, interstitial hemorrhage in the kidney were restored, and the density of inflammatory cells was reduced.Figure 5 It can be seen that, compared with the control group, the ZnO NPs alone treatment group significantly increased the levels of creatinine and blood urea nitrogen, while the paeoniflorin treatment group significantly decreased the levels of creatinine and blood urea nitrogen.

[0033] Conclusion: It shows that paeoniflorin restored the kidney injury and renal function disorders in mice caused by zinc oxide nanoparticles. Example

[0034] Application of paeoniflorin in reducing kidney oxidative stress indexes induced by zinc oxide nanoparticles: A total of 40 male C57BL / 6 mice aged 6 - 8 weeks (body weight: 18 - 22 g) were raised under pathogen-free conditions (temperature 22°C, 12-hour light-dark cycle), fed with standard feed during the experiment, and given free access to water. All mice were randomly divided into 4 groups (n = 10): control group, 75 mg / kg PF group, 360 mg / kg ZnO NPs group, 75 mg / kg PF + 360 mg / kg ZnO NPs. The first control group was given sterile normal saline orally; the second group was given 75 mg / kg PF, the third group was given 360 mg / kg ZnO NPs by gavage; the fourth group was given 75 mg / kg PF by gavage 2 hours before ZnO NPs intoxication in mice, and the administration was continued for 28 days. After all treatments were completed, all mice were fasted and water-deprived for 12 hours. The mice were anesthetized with 2.6 mg / kg ketamine and 0.16 mg / kg xylazine respectively, and the kidney tissues of the mice were taken after dissection. The kidney proteins were lysed, and the protein concentration was detected by Nanodrop ONE. The oxidative stress markers in the kidneys were detected according to the instructions of the CAT (catalase), SOD (superoxide dismutase), MDA (malondialdehyde) and GSH-PX (glutathione peroxidase) kits.

[0035] It can be seen from Figure 6 that, by detecting the oxidative stress indexes, the concentration of MDA in the total protein was restored from 3.96 nmol / mg in the ZnO NPs alone treatment group to 2.67 nmol / mg under PF intervention ( P <0.05); the concentrations of CAT and SOD were restored from 18.53 U / mg and 14.29 U / mg to 32.28 U / mg and 25.69 U / mg ( P <0.05); the concentration of GSH-PX was restored from 950.16 U / mg in the ZnO NPs alone treatment group to 1334.65 U / mg under PF intervention ( P <0.01). The results show that PF significantly reduces the kidney oxidative stress level induced by zinc oxide nanoparticles.

[0036] The activation effect of paeoniflorin on SIRT1 protein was evaluated by Western blot. Mouse kidneys were treated with lysis buffer containing 1% PMSF and 1% protease inhibitor on ice for 10 minutes, and then cell lysates were collected on ice using a cell scraper. Homogenates were obtained by a cell sonicator, and then the total protein concentration was detected using a BCA kit. A 4-20% SDS-PAGE gel was prepared, and 30 μg of the extracted protein from each group was separated by the gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The blank part of the membrane that was not transferred was blocked with a rapid blocking solution for 15 minutes (equivalent to 5% skim milk powder for 2 hours). After blocking, the PVDF membrane was washed three times in TBST buffer saline containing 0.05% Tween-20. Subsequently, the PVDF membrane was incubated with primary antibodies. The types and dilution multiples of the primary antibodies were anti-SIRT1 (1:1000) and β-actin (1:1000) respectively. After incubation for 24 hours, it was washed three times in TBST. The PVDF membrane was incubated with the corresponding secondary antibody at a dilution of 1:5000 for 1.5 hours and continued to be washed three times in TBST. Finally, it was briefly incubated with ECL chemiluminescence reagent, and the immunoblot reaction bands were imaged using Image lab software. The protein density was analyzed and compared using Image j software. Statistical analysis of the data was performed using SPSS 22.0 software. The results were expressed as "mean ± standard deviation", and One-Way ANOVA was used for significant difference analysis. The experimental results were repeated 3 times, and P < 0.05 indicated a significant difference.

[0037] It can be seen from Figure 7 that under PF intervention, compared with the treatment with ZnO NPs alone, 75 mg / kg PF significantly increased the protein expression level of SIRT1 (P < 0.05), indicating that PF can significantly activate the expression of SIRT1 protein in cells and improve the resistance of the kidneys to acute stress.

[0038] The above results indicate that PF plays a protective role against ZnO NPs-induced kidney injury by activating SIRT1, reduces kidney pathological injury and renal function disorder, and reduces the level of kidney oxidative stress induced by nano-zinc oxide.

Claims

1. Use of paeoniflorin in the preparation of a drug for treating kidney injury induced by nano-zinc oxide; the kidney injury induced by nano-zinc oxide refers to an irregular nano-zinc oxide feed additive with an average particle size of 50 nm.

Images