CN115137837A - Application of polyvinylpyrrolidone modified palladium nanoparticles - Google Patents
Application of polyvinylpyrrolidone modified palladium nanoparticles Download PDFInfo
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- CN115137837A CN115137837A CN202211075886.7A CN202211075886A CN115137837A CN 115137837 A CN115137837 A CN 115137837A CN 202211075886 A CN202211075886 A CN 202211075886A CN 115137837 A CN115137837 A CN 115137837A
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Abstract
The invention relates to application of palladium nanoparticles, and discloses application of palladium nanoparticles modified by polyvinylpyrrolidone, which comprises application of palladium nanoparticles modified by polyvinylpyrrolidone in preparation of a product for resisting aging and/or enhancing heat stress resistance, application of palladium nanoparticles modified by polyvinylpyrrolidone in preparation of a product for improving mitochondrial respiration function, and application of palladium nanoparticles modified by polyvinylpyrrolidone in a product for enhancing expression of mitochondrial metabolic enzyme. The palladium nano-particles modified by the polyvinylpyrrolidone can reverse the reduction of mitochondrial functions caused by aging so as to achieve the effects of delaying aging and resisting aging.
Description
Technical Field
The invention relates to application of palladium nanoparticles, in particular to application of palladium nanoparticles modified by polyvinylpyrrolidone in preparing products for resisting aging and/or enhancing heat stress resistance, application of palladium nanoparticles modified by polyvinylpyrrolidone in preparing products for improving mitochondrial respiration function, and application of palladium nanoparticles modified by polyvinylpyrrolidone in preparing products for enhancing expression of mitochondrial metabolic enzymes.
Background
The health and longevity are the targets of people unchanged from the ancient times. Nowadays, with the continuous improvement of the average life span of human beings, the aging problem is increasingly prominent. Aging has become a medical problem and a social problem which are becoming more and more serious, and is considered to be a major factor in the onset of neurodegenerative diseases, tumors, cardiovascular diseases and diabetes, which are characterized by degenerative phenomena occurring at the molecular, cellular organelle, tissue and even whole organism level. The research and development of anti-aging drugs and the elucidation of the regulation mechanism thereof have important practical significance for relieving the increasingly serious aging phenomenon and the pressure of the sustainable development of economy and society.
Cytochrome c oxidase (mitochondrial complex IV) is an enzyme complex of respiratory chain enzyme, is a key enzyme in respiratory chain enzyme, participates in oxidative phosphorylation and many reactions in tricarboxylic acid cycle, is an important cofactor for mitochondrial function exertion and biosynthesis, and the change of enzyme activity directly affects the function change of the whole respiratory chain. However, cytochrome c oxidase is unstable and fragile, and is vulnerable to free radical oxidation damage, and the destruction of its protein subunit can cause electron loss during electron transfer, the efficiency of electron transfer is reduced, which further affects the function of respiratory chain and the insufficient supply of cell energy, and the metabolic capability of the organism is reduced, resulting in aging and aging-related diseases. Research finds that various diseases related to aging are related to structural and functional disorder of cytochrome c oxidase in tissues rich in mitochondria such as heart, brain and the like; similarly, mitochondrial enzymatic activity is also reduced in neurodegenerative disorders.
Previous researches show that the mitochondrial respiratory chain enzyme is reconstructed, expressed and purified by an enzyme engineering method, but the natural enzyme has the problems of insufficient stability, complicated reconstruction and purification, high preparation cost and the like. Therefore, the research on the anti-aging nano enzyme with high physical and chemical stability, simple preparation process and low cost has great significance.
Disclosure of Invention
The invention aims to solve the problems of poor stability, complex synthesis process and high cost of natural enzymes in the prior art, and provides the application of the palladium nanoparticle modified by polyvinylpyrrolidone.
In order to achieve the above objects, the present invention provides, in a first aspect, the use of polyvinylpyrrolidone-modified palladium nanoparticles for the preparation of a product for anti-aging and/or enhancing heat stress resistance.
Preferably, the particle size of the polyvinylpyrrolidone-modified palladium nanoparticle is 15-25nm.
Preferably, in the palladium nanoparticle modified by polyvinylpyrrolidone, the molar ratio of polyvinylpyrrolidone to palladium is 1-9:1.
Preferably, the anti-aging and/or anti-heat stress enhancing product is selected from at least one of a drug, a health product and a food additive.
In a second aspect, the invention provides the use of polyvinylpyrrolidone-modified palladium nanoparticles in the preparation of a product for improving mitochondrial respiratory function.
Preferably, the particle size of the polyvinylpyrrolidone-modified palladium nanoparticle is 15-25nm.
Preferably, in the polyvinylpyrrolidone-modified palladium nanoparticle, the molar ratio of the polyvinylpyrrolidone to the palladium is 1-9:1.
Preferably, the mitochondrial respiration function improving product is selected from at least one of a drug, a nutraceutical, and a food additive.
In a third aspect, the invention provides the use of polyvinylpyrrolidone-modified palladium nanoparticles in the preparation of a product for enhancing expression of a mitochondrial metabolic enzyme.
Preferably, the particle size of the polyvinylpyrrolidone-modified palladium nanoparticle is 15-25nm.
Preferably, in the polyvinylpyrrolidone-modified palladium nanoparticle, the molar ratio of the polyvinylpyrrolidone to the palladium is 1-9:1.
Preferably, the product for enhancing the expression of mitochondrial metabolic enzyme is selected from at least one of a drug, a health product and a food additive.
Preferably, the metabolic enzyme is selected from citrate synthasects-1Hexokinasehxk-1And pyruvate carboxylasepyc-1At least one of (a).
Through the technical scheme, the invention has the beneficial effects that:
the invention provides a new function and a new application of palladium nanoparticles modified by polyvinylpyrrolidone, and the inventor discovers for the first time that the palladium nanoparticles modified by polyvinylpyrrolidone can increase the basic respiration rate, the maximum respiration rate and the ATP generation of organism mitochondria so as to enhance the respiration function of the organism mitochondria, and can be effectively applied to the preparation of products for improving the respiration function of the mitochondria; the polyvinylpyrrolidone modified palladium nanoparticles can prolong the life of organisms, increase the pharyngeal exercise (feeding) capacity and the head swing capacity of the old organisms, reduce the lipofuscin accumulation amount, and can be effectively applied to the preparation of products for resisting aging and/or enhancing the heat stress resistance; in addition, the palladium nano-particles modified by polyvinylpyrrolidone also have the function of enhancing metabolic enzyme citrate synthase in organism mitochondriacts-1Hexokinase, and a process for producing the samehxk-1And pyruvate carboxylasepyc-1The method can be effectively applied to the preparation of products for enhancing the expression of mitochondrial metabolic enzymes.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
FIG. 1 is a graph of UV-VIS absorption spectra of PVP-Pd NPs at various concentrations in example 1 after interaction with cytochrome c;
FIG. 2 is a graph of UV-VIS absorption spectra of PVP-Pd NPs in example 1 after exposure to cytochrome c for various periods of time;
FIG. 3 is a graph showing the basal oxygen consumption and maximum oxygen consumption of nematodes in the PVP-Pd NPs-treated group and the control group in example 2, wherein A is a graph showing the basal oxygen consumption of nematodes in the PVP-Pd NPs-treated group and the control group, and B is a graph showing the maximum oxygen consumption of nematodes in the PVP-Pd NPs-treated group and the control group;
FIG. 4 is a graph of the ATP content of nematodes in the PVP-Pd NPs treated group and the control group of example 3;
FIG. 5 is a graph of mRNA levels of various metabolic enzymes in mitochondria of nematodes in the PVP-Pd NPs-treated group and the control group in example 4;
FIG. 6 is a graph of mean nematode life time for the PVP-Pd NPs treated group and the control group of example 5;
FIG. 7 is a graph of nematode survival for the PVP-Pd NPs treated group and the control group of example 5;
FIG. 8 is fluorescence image of nematode lipofuscin of PVP-Pd NPs treated group and control group of example 6;
FIG. 9 is a graph of quantitative data on lipofuscin of nematodes in the PVP-Pd NPs treated group and the control group of example 6;
FIG. 10 is a graph of the number of head swings of nematodes in the group to which PVP-Pd NPs were administered and the control group in example 7;
FIG. 11 is a graph of nematode pharyngeal pumping frequency for the PVP-Pd NPs administration group and the control group of example 8;
FIG. 12 is a graph showing the survival rate of nematodes under heat stress and the number of body turns in the PVP-Pd NPs-administered group and the control group in example 9.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
The invention provides application of palladium nano particles modified by polyvinylpyrrolidone in preparing products for resisting aging and/or enhancing heat stress resistance.
In the research process, by observing the influences of the life, aging-related physiology and behavior indexes of organisms (such as nematodes and human bodies) treated by the palladium nanoparticles modified by polyvinylpyrrolidone, the inventor of the invention proves that the palladium nanoparticles modified by polyvinylpyrrolidone can prolong the life of the organisms, increase the pharyngeal exercise (eating) ability and the body bending ability of the aged organisms, reduce the lipofuscin accumulation amount, and have the effects of delaying aging, resisting aging and reversing the function reduction caused by aging.
In the present invention, the polyvinylpyrrolidone-modified palladium nanoparticles may be commercially available or may be prepared by a method disclosed in the prior art.
Illustratively, the method for preparing polyvinylpyrrolidone-modified palladium nanoparticles comprises the following steps: in a reaction solvent I, sodium tetrachloropalladate (Na) 2 PdCl 4 ) Mixing the palladium nanoparticle with polyvinylpyrrolidone (PVP), heating to 50-70 ℃, mixing the PVP with ascorbic acid for reaction to obtain a reaction solution, and separating and purifying the reaction solution to obtain the polyvinylpyrrolidone modified palladium nanoparticle.
In the present invention, sodium tetrachloropalladate is commercially available, or can be obtained by mixing palladium chloride with sodium chloride.
According to the invention, the sodium tetrachloropalladate and the polyvinylpyrrolidone can be mixed in any proportion. In order to further improve the effect of polyvinylpyrrolidone on palladium nanoparticle modification and better control the particle size of polyvinylpyrrolidone-modified palladium nanoparticle, the molar ratio of sodium tetrachloropalladate to polyvinylpyrrolidone is preferably 1 to 10-15.
In the invention, the reaction solvent I can adopt deionized water, and specifically, the process of mixing the sodium tetrachloropalladate and the polyvinylpyrrolidone is as follows: preparing the sodium tetrachloropalladate into a solution with the concentration of 0.04-0.06mol/L by using deionized water, preparing the polyvinylpyrrolidone into a solution with the concentration of 0.5-0.7mol/L by using the deionized water, and then mixing in the form of an aqueous solution.
According to the invention, the mixing with ascorbic acid is carried out by: adding an aqueous solution of ascorbic acid in a dropwise manner; the reaction conditions include: the temperature is 50-70 ℃, the time is 50-70min.
In the present invention, the separation and purification processes may adopt solid-liquid separation and purification methods which are conventional in the art, for example, solid-liquid separation may be performed by filtration and centrifugation, and purification may be performed by washing, membrane filtration, and the like.
In the invention, specifically, the preparation process of the palladium nanoparticle modified by polyvinylpyrrolidone comprises the following steps: mixing palladium chloride and sodium chloride aqueous solution to prepare Na with the concentration of 0.05mol/L 2 PdCl 4 Solution, 10mL Na 2 PdCl 4 The solution was mixed with 10mL of deionized water, and 10mL of a 0.6mol/L aqueous solution of polyvinylpyrrolidone was added to Na with magnetic stirring 2 PdCl 4 Heating the solution and keeping the temperature in a water bath at 60 ℃, then dropwise adding 10mL of ascorbic acid aqueous solution with the concentration of 0.06mol/L into the solution within 15min, continuously stirring the solution for 60min at the temperature after the dropwise adding is finished to obtain reaction liquid, carrying out solid-liquid separation on the reaction liquid to obtain separation and precipitation, and purifying the separation and precipitation to obtain the polyvinylpyrrolidone modified palladium nano-particles (hereinafter referred to as PVP-Pd NPs).
According to the present invention, the particle size of the polyvinylpyrrolidone modified palladium nanoparticle is preferably 15 to 25nm, specifically 15 nm, 17 nm, 19 nm, 21 nm, 23 nm, 25nm, or any value in a range formed by any two of the above values. The inventor finds that under the preferred embodiment, the absorption of the nano particles by organisms is favorably improved, and the anti-aging effect and the anti-heat stress capability of the nano particles are further improved.
According to the present invention, preferably, in the polyvinylpyrrolidone-modified palladium nanoparticle, the molar ratio of polyvinylpyrrolidone to palladium is 1 to 9:1.
In the invention, the anti-aging product can be a product for prolonging the life of an organism, can also be a product for improving the motion ability of the organism in the aspects of pharyngeal motion ability, body bending ability, head swinging ability and the like, or a product for reducing the lipofuscin accumulation amount in the organism; the product for enhancing the heat stress resistance can be a product for enhancing the resistance of an organism to external heat stress (namely the heat stress resistance), wherein the external heat stress of the organism refers to a phenomenon of generating metabolism and growth development stress on the organism at a temperature higher than the highest point of a temperature range suitable for the growth of the organism, and for example, the external heat stress temperature of nematodes can be 32-40 ℃.
According to the present invention, preferably, the anti-aging and/or anti-heat stress enhancing product is at least one selected from the group consisting of a pharmaceutical, a health product and a food additive. The dosage form of the medicine or the health-care product is oral liquid, capsules, tablets, powder, granules or pills, and the medicine or the health-care product of various dosage forms can be prepared according to the conventional method in the field and can also comprise a carrier acceptable in pharmacy or health-care products. The content of the polyvinylpyrrolidone-modified palladium nanoparticles in the anti-aging and/or anti-heat stress enhancing product can be designed and adjusted according to dosage.
In a second aspect, the invention provides the use of polyvinylpyrrolidone-modified palladium nanoparticles in the preparation of a product for improving mitochondrial respiratory function.
The inventor of the invention further discovers, through research and observation, that the palladium nanoparticles modified by polyvinylpyrrolidone can increase the basal respiration rate, the maximum respiration rate and the ATP production of organism mitochondria, further enhance the respiratory function of organism mitochondria, and can be effectively applied to the preparation of products for promoting the mitochondrial respiration.
The polyvinylpyrrolidone-modified palladium nanoparticles according to the present invention may be commercially available or may be prepared by methods disclosed in the prior art. Preferably, the particle size of the polyvinylpyrrolidone-modified palladium nanoparticle is 15-25nm. The inventors have found that, in the preferred embodiment, it is advantageous to increase the nanoparticle absorption by the living body, and thereby to improve the effect of the nanoparticle on improving and promoting the mitochondrial respiratory function of the living body.
According to the present invention, preferably, in the polyvinylpyrrolidone-modified palladium nanoparticle, the molar ratio of polyvinylpyrrolidone to palladium is 1 to 9:1.
In the present invention, the product for improving mitochondrial respiratory function may be a product for regulating mitochondrial electron transport chain disorders, a product for regulating mitochondrial metabolic enzyme disorders, or a product for increasing cellular ATP synthesis.
According to the present invention, preferably, the mitochondrial respiratory function improving product is selected from at least one of a drug, a health product and a food additive. The dosage form of the medicine or the health-care product is oral liquid, capsules, tablets, powder, granules or pills, and the medicine or the health-care product of various dosage forms can be prepared according to the conventional method in the field and can also comprise a carrier acceptable in pharmacy or health-care products. The content of the polyvinylpyrrolidone-modified palladium nanoparticles in the product for improving the mitochondrial respiratory function can be designed and adjusted according to the dosage.
In a third aspect, the invention provides the use of polyvinylpyrrolidone-modified palladium nanoparticles in the preparation of a product for enhancing expression of a mitochondrial metabolic enzyme.
The inventors of the present invention have also surprisingly found that polyvinylpyrrolidone-modified palladium nanoparticles have the ability to enhance citrate synthase in mitochondria of organismscts-1Hexokinasehxk-1And pyruvate carboxylasepyc-1The method can be effectively applied to the preparation of products for enhancing the expression of mitochondrial metabolic enzymes.
According to the present invention, the polyvinylpyrrolidone-modified palladium nanoparticles may be commercially available or may be prepared by a method disclosed in the prior art. Preferably, the particle size of the polyvinylpyrrolidone-modified palladium nanoparticle is 15-25nm. The inventors have found that in this preferred embodiment, it is advantageous to increase the uptake of nanoparticles by the organism, and thus to increase the enhancement of mitochondrial metabolic enzymes by the nanoparticles.
According to the present invention, preferably, in the polyvinylpyrrolidone-modified palladium nanoparticle, the molar ratio of polyvinylpyrrolidone to palladium is 1 to 9:1.
According to the present invention, preferably, the product for enhancing the expression of mitochondrial metabolism enzyme is selected from at least one of pharmaceuticals, nutraceuticals, and food additives. The dosage form of the medicine or the health-care product is oral liquid, capsules, tablets, powder, granules or pills, and the medicine or the health-care product of various dosage forms can be prepared according to the conventional method in the field and can also comprise a carrier acceptable in pharmacy or health-care products. The content of polyvinylpyrrolidone-modified palladium nanoparticles in the product for enhancing the expression of mitochondrial metabolic enzymes can be designed and adjusted according to dosage.
According to the invention, the metabolic enzyme may be any enzyme of an organism involved in mitochondrial metabolic processes. Preferably, the metabolic enzyme is selected from citrate synthasects-1Hexokinase, and a process for producing the samehxk-1And pyruvate carboxylasepyc-1At least one of (1).
The present invention will be described in detail below by way of examples.
In the following examples and comparative examples, polyvinylpyrrolidone-modified palladium nanoparticles (PVP-Pd NPs) were obtained from Jiangsu Xiancheng nanomaterial science and technology Co., ltd, product No. XFJ, particle size 15-25nm, molar ratio of polyvinylpyrrolidone to palladium 5:1, cytochrome c was obtained from Yuanye (Shanghai) Co., ltd, product No. 9007-43-6, mitochondrial oxidative phosphorylation release FCCP was obtained from Selleck, product No. S8276, palladium chloride PdCl 2 From Allantin reagent (Shanghai) Inc., with product number 7647-10-1, FUDR for inhibiting nematode oviposition, from Allantin reagent (Shanghai) Inc., with product number 50-91-9; the mitochondrial ATP detection kit is purchased from Saimer Feishale science and technology (China) Co., ltd., and the product number is A22066;cts-1, hxk-1, pyc-1 andreference geneOf actinThe q-PCR primer is prepared by Shanghai Biotechnology Limited company, and the specific primer is a document: the NAD + /Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling，Laurent Mouchiroud，et alCell, volume 154, issue 2, 18 July 2013, pages 430-441; the q-PCR reverse transcription kit is purchased from Promega reagent company Limited, and the product number is A5001; taq Pro Universal SYBR aPCR Master mix available from Promega reagent Inc., product number Q712-02; the mitochondrial ATP detection kit is purchased from Biyunnan reagent Co., ltd, and the product number is S0026B; the rest chemical reagents and raw materials are conventional commercial products.
In the following examples and comparative examples, a confocal laser microscope, a luminescence detector, a mitochondrial function assay system, seahorse XF24, and an ultraclean bench were purchased from Nikon, japan, promega, usa, seahorse Bioscience, usa, respectively.
In the following examples and comparative examples, wild-type N2 C.elegans was obtained from the genetic center of C.elegans (university of Minnesota, USA); escherichia coli OP50 is purchased from NTCC type culture collection center and is numbered as NTCC445651, and the culture process of the bacterial liquid is as follows: escherichia coli OP50 was inoculated into LB medium and cultured in a shaking incubator at a rotation speed of 220rpm and a temperature of 37 ℃.
In the following examples and comparative examples, the procedure for configuring the NGM plate was: 3g of NaCl, 17g of agar and 2.5g of peptone, adding double distilled water to 1000mL, adjusting the pH to 6.0, and sterilizing to obtain a basic culture medium; the water bath at 55 ℃ is balanced for 15min, so that the solidification of a basic culture medium caused by too low temperature is prevented. In a super clean bench, after ultraviolet sterilization of a basic culture dish, a magnetic stirrer, gloves and substances to be added, firstly, a conical flask filled with a basic culture medium is placed on the magnetic stirrer for stirring, the rotation speed is based on no generation of bubbles, then, alcohol cotton balls are used for wiping the gloves clean, a culture plate package and a bottleneck sealing film (the gloves do not touch the bottleneck) are disassembled, and 25mL KH is sequentially added into the basic culture medium 2 PO 4 Solution (mother liquor concentration: 1M), 1mL CaCl 2 Solution (mother liquor concentration: 1M), 1mL MgSO 4 The solution (mother liquor concentration: 1M) was then slowly added dropwise along the wall of the flask with stirring to 1mL of cholesterol solution (mother liquor concentration: 5 mg/mL) and thoroughly mixed with the solution in the flask until the surface of the solution appearedObtaining NGM culture medium without visible oil drop on the surface; pouring the NGM culture medium on a culture dish with the diameter of 9cm, making the NGM culture medium have no visible air bubbles as much as possible, standing the culture dish at room temperature for one day after plate pouring, and solidifying the culture medium for later use.
In the following examples and comparative examples, the synchronization process for caenorhabditis elegans was:
collecting caenorhabditis elegans adults in a good growth state into a centrifuge tube, and adding 10mL M9 buffer solution to wash away redundant escherichia coli OP50; and (3) reserving 4mL of supernatant in a centrifugal tube, adding 500 mu L of 5M NaOH solution and 500 mu L of 5% sodium hypochlorite solution, oscillating for about 3min on a vortex oscillator, detecting under an electron microscope while oscillating, cleaning 3 times by using M9 buffer solution as soon as possible when a nymph body is broken, centrifuging by using a miniature centrifuge after cleaning, removing redundant solution and leaving ova, adding 500 mu L of ova to a blank NGM culture plate, and culturing for 12-16h in an incubator at 20 ℃ so that the ova are incubated into synchronized L1 larvae.
Example 1
To test the cytochrome c oxidase mimetic activity of PVP-Pd NPs, 60. Mu.L of a 10mg/mL cytochrome c solution (using PBS buffer as a solvent) and 400. Mu.L of different concentrations of PVP-Pd NPs (0.1. Mu.g/mL, 0.2. Mu.g/mL, 1. Mu.g/mL, 10. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL of PVP-Pd NPs) were added in sequence to a 5mL EP tube containing 4mL of a PBS buffer solution (phosphate concentration of 10mM, pH 7.4), and UV-visible absorption measurement was performed after 60min of reaction, and PVP-Pd NPs concentration of 0 was used as a control (control), as shown in FIG. 1.
The results are shown in FIG. 2, in which 60. Mu.L of a 10mg/mL cytochrome c solution (using PBS buffer as a solvent) and 400. Mu.L of a 1. Mu.g/mL PVP-Pd NPs solution were added in this order to a 5mL EP tube containing 4mL of a PBS buffer solution (concentration: 10mM, pH 7.4) and reacted for 2min,7min, 111min, 15min,19min,22min,26min,30min, followed by UV-visible absorption measurement, and the reaction time was 0 as a control (control).
The results of FIGS. 1 and 2 demonstrate cytochrome c oxidase-like activity of PVP-Pd NPs. As can be seen from FIG. 1, the control group (control) readily detected a higher absorption peak at the alpha band (550 nm) of iron cytochrome c, indicating the presence of cytochrome c in the form of divalent iron. In contrast, after the PVP-Pd NPs with the concentration between 0.1 and 100 mu g/mL are treated, the absorption peak of cytochrome c in a ferrous form is reduced, and the concentration is dependent, and the PVP-Pd NPs with the concentration between 0.1 and 100 mu g/mL can convert cytochrome c in a ferrous form into cytochrome c in a ferric form, so that the cytochrome c oxidase catalytic function of the PVP-Pd NPs is proved. As can be seen from FIG. 2, after the PVP-Pd NPs are added into the reaction system, the characteristic absorption of the iron cytochrome c gradually shows time-dependent reduction within the time range of 2min to 30min, and the decrease of the absorption peak of the cytochrome c in the form of ferrous iron can be obviously detected after the reaction is carried out for 7min, thus confirming the reaction sensitivity of the catalytic function of the cytochrome c oxidase similar to the PVP-Pd NPs. The above results indicate that PVP-Pd NPs are able to mimic the activity of cytochrome c oxidase in vitro, catalyzing the oxidation of cytochrome c in the ferrous form.
Example 2
Mitochondrial function is particularly important for understanding energy metabolism of cells, and the oxygen consumption of cells can reflect mitochondrial function. Therefore, the effect of PVP-Pd NPs on improving the mitochondrial respiratory function can be researched by researching the oxygen consumption rate of mitochondria. Adding PVP-Pd NPs and FUDR into an escherichia coli OP50 bacterial solution, uniformly mixing, uniformly coating the mixture on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of the PVP-Pd NPs is 0.5 mu g/mL, the final concentration of the FUDR is 80 mu M, and the final concentration of the FUDR is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into the escherichia coli OP50 bacterial solution, uniformly mixing, and uniformly coating the mixture on the NGM flat plate to obtain an NGM flat plate (the final concentration of the FUDR is 80 mu M) containing only FUDR and not containing PVP-Pd NPs; carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to the L4 stage; transferring another part of L4 stage nematode strain onto NGM plate containing no PVP-Pd NPs and only FUDR as control group, culturing about 50-100 nematodes in each group at 20 deg.C incubator, collecting nematodes at 7 days of adult, and consumingAnd (4) measuring oxygen quantity. Specifically, the oxygen consumption of nematodes was measured using the mitochondrial function assay system Seahorse XF24, specifically, the test plate was hydrated overnight by adding 1mL of hydration solution per well one day in advance in a 37 ℃ incubator, and then the nematodes were placed in a 24-well standard Seahorse plate (18 nematodes per well). Using Seahorse XF-24 software, the assay template was loaded, mitochondrial oxidative phosphorylation uncoupler FCCP (final concentration: 20. Mu.M) was injected into each well to induce maximal respiration, and NaN was added 3 (final concentration: 40 mM) was used to distinguish non-mitochondrial respiration, and the maximum and basal oxygen consumption rates per nematode were calculated on average for the treatment and control groups, and the results are shown in FIG. 3.
As can be seen from FIG. 3, PVP-Pd NPs can enhance the mitochondrial respiration of senile nematodes; specifically, on adult day 7, the nematodes in the control group easily detected a certain basal oxygen consumption and maximum oxygen consumption. The basal oxygen consumption (shown as a in fig. 3) and the maximum oxygen consumption (shown as B in fig. 3) were significantly increased in the PVP-Pd NPs-treated group compared to the control group. The result shows that the PVP-Pd NPs can improve the respiratory function of mitochondria, so the PVP-Pd NPs can be used as an effective mitochondria activator and have important application prospect.
Example 3
Mitochondria are the main site for aerobic respiration and energy conversion of cells, and are self-understood as the capacity factory of cells, most energy required for cell life activities comes from mitochondria, and the transfer of electron transfer on the inner membrane of mitochondria promotes proton discharge to the mitochondrial membrane gap, and finally, ADP is converted into ATP through ATP synthase for energy supply. Therefore, the effect of PVP-Pd NPs on improving the mitochondrial respiratory function can be researched by researching the ATP production of mitochondria. Adding PVP-Pd NPs and FUDR into an escherichia coli OP50 bacterial solution, uniformly mixing, uniformly coating the mixture on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of the PVP-Pd NPs is 0.5 mu g/mL, the final concentration of the FUDR is 80 mu M, and the final concentration of the FUDR is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into the escherichia coli OP50 bacterial solution, uniformly mixing, and uniformly coating the mixture on the NGM flat plate to obtain an NGM flat plate (the final concentration of the FUDR is 80 mu M) containing only FUDR and not containing PVP-Pd NPs; carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to the L4 stage; another portion of the L4 phase nematode strains was transferred to NGM plates containing FUDR alone without PVP-Pd NPs as a control group, incubated at 20 ℃ in an incubator, collected at day 7 of the adult, and mitochondrial ATP production was measured, specifically, approximately 100-200 nematodes per group were collected and resuspended in M9 buffer, subjected to 5 freeze/thaw cycles (from liquid nitrogen to 40 ℃) and then boiled for 20min to release ATP and destroy ATPase activity prior to centrifugation, and the diluted supernatant was used for ATP measurement according to the instructions of the ATP detection kit, the results being shown in FIG. 4.
The results in FIG. 4 show that PVP-Pd NPs increase mitochondrial productivity of senile nematodes; specifically, on day 7 of adult worms, the average ATP content of mitochondria of the nematode strains of the control group is 9597.8 RLU/mug protein, compared with the nematode control group, the ATP production level of mitochondria treated by PVP-Pd NPs is obviously increased, and the average ATP content is 68111.6 RLU/mug protein. The results show that PVP-Pd NPs can obviously improve the mitochondrial productivity of the aged individuals and improve the respiratory function of the mitochondria. Therefore, the PVP-Pd NPs can be used as an effective mitochondrion activator and have important application prospects.
Example 4
Citrate synthasects-1Hexokinasehxk-1And pyruvate carboxylasepyc-1Is a key enzyme involved in mitochondrial sugar synthesis or glycolytic metabolism, and is important for the exertion of respiratory function of mitochondria. Adding PVP-Pd NPs and FUDR into an escherichia coli OP50 bacterial solution, uniformly mixing, uniformly coating the mixture on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of the PVP-Pd NPs is 0.5 mu g/mL, the final concentration of the FUDR is 80 mu M, and the final concentration of the FUDR is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into the escherichia coli OP50 bacterial solution, uniformly mixing, and uniformly coating the mixture on the NGM flat plate to obtain an NGM flat plate (the final concentration of the FUDR is 80 mu M) containing only FUDR and not containing PVP-Pd NPs; adopting wild caenorhabditis elegans model, performing synchronous nematode treatment, and allowing stage I to grow to stage L4 after wild type caenorhabditis elegans in stage L1 grows to stage ITransferring part of L4-stage nematode strains to NGM plates containing PVP-Pd NPs and FUDR to serve as an administration group; transferring another part of L4-stage nematode strains to NGM plates which do not contain PVP-Pd NPs and only contain FUDR to serve as a control group, culturing 50-100 nematodes in an incubator at 20 ℃ for 48h, collecting the nematodes treated by the control group and the PVP-Pd NPs, washing the nematodes with M9 buffer solution for three times, adding 1mL of TRIZOL (purchased from Invitrogen biotechnology company) into each nematode tube, splitting the nematodes and extracting mRNA; cDNA was synthesized using M-MLV reverse transcriptase (purchased from Invitrogen Biotechnology Inc., product number # 28025013), and citrate synthase was determined by real-time PCR (q-PCR) using SYBR Green Supermix and q-PCR primer pairscts-1Hexokinase, and a process for producing the samehxk-1And pyruvate carboxylasepyc-1The level of mRNA transcription expressed.
PVP-Pd NPs on nematode mitochondrial citrate synthasects-1Hexokinasehxk-1And pyruvate carboxylasepyc- 1Relative reference geneactinThe results are shown in FIG. 5.
As can be seen from FIG. 5, feeding the nematodes with PVP-Pd NPs increased the level of mitochondrial expression of metabolic enzymes in the nematodes relative to the control group; the specific expression is that the PVP-Pd NPs treatment group can promote glucose metabolism enzyme after feeding the nematodes in the L4 stage for two dayscts-1Glucose metabolizing enzymehxk-1And glucose synthase pyc-1 were up-regulated 2-fold, 1.5-fold and 1.6-fold, respectively, relative to the reference gene (P)<0.05 This shows that PVP-Pd NPs fed nematode can effectively improve the metabolic function of mitochondria.
Example 5
Adding PVP-Pd NPs and FUDR into Escherichia coli OP50 bacterial liquid, mixing, uniformly coating on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of PVP-Pd NPs is 0.5 mu g/mL; the final concentration of FUDR is 80 mu M, and is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into Escherichia coli OP50 bacterial liquid, mixing, and uniformly coating on an NGM flat plate to obtain an NGM flat plate containing only FUDR and not containing PVP-Pd NPs (the final concentration of FUDR is 80 mu M); carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to L4 stage; transferring another part of L4-stage nematode strains to NGM plates which do not contain PVP-Pd NPs and only contain FUDR as a control group, culturing in an incubator at 20 ℃, counting the number of dead nematodes every other day by using an electron microscope until adult nematodes die, and removing the dead nematodes from the culture dish until all the nematodes die; summarizing data, drawing a survival curve by Graph Pad, calculating a significance difference (Log-rank test) and counting the life span of the nematodes. The life test data are shown in Table 1, the results of the mean life are shown in FIG. 6, and the results of the survival rate are shown in FIG. 7.
As can be seen from FIGS. 6 and 7, the mean life span of the nematodes in the control group was approximately 16.6 days, and the 0.5. Mu.g/mL PVP-Pd NPs-treated group significantly extended the life span of the nematodes, which was 19.7 days, up to 16.7% longer than the control group (P < 0.001). The number of days of nematode death in the PVP-Pd NPs-treated group was relatively delayed compared to the control group, and the longest life span of the nematodes was 30 days, while the longest life span of the nematodes in the control group was 24 days, and the longest life span of the nematodes in the PVP-Pd NPs-treated group was 6 days later than that in the control group. The result shows that the PVP-Pd NPs can obviously prolong the service life and have good anti-aging effect.
TABLE 1
Example 6
Adding PVP-Pd NPs and FUDR into an escherichia coli OP50 bacterial solution, uniformly mixing, uniformly coating the mixture on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of the PVP-Pd NPs is 0.5 mu g/mL, the final concentration of the FUDR is 80 mu M, and the final concentration of the FUDR is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into the escherichia coli OP50 bacterial solution, uniformly mixing, and uniformly coating the mixture on the NGM flat plate to obtain an NGM flat plate (the final concentration of the FUDR is 80 mu M) containing only FUDR and not containing PVP-Pd NPs; carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to the L4 stage; transferring another part of L4-stage nematode strains to NGM plates which do not contain PVP-Pd NPs and only contain FUDR as a control group, culturing in an incubator at 20 ℃ until the 1 st day or the 7 th day of adults, detecting lipofuscin fluorescence images of the nematodes by using a laser confocal imaging method (excitation: 365 nm; emission: 420 nm), randomly shooting about 10 nematodes in each experimental group, and quantitatively analyzing lipofuscin in bodies of each nematode by using Image J software to obtain the lipofuscin fluorescence intensity of the nematodes, wherein the data are shown in FIG. 9.
Lipofuscin, an undegradable lysosome substance, is one of the useful biomarkers of caenorhabditis elegans physiological age, and its accumulation intensity is correlated with the age of the nematode. As shown in fig. 8, PVP-Pd NPs fed nematodes significantly decreased intestinal lipofuscin levels on days 1 and 7 of adult worms; specifically, in the control group, part of luminous lipofuscin is observed in the intestinal tract of the nematode on the first day of the adult, and the content of the lipofuscin in the intestinal tract of the adult on the 7 th day is obviously increased relative to the content of the lipofuscin on the first day of the adult; in the PVP-Pd NPs treatment group, the lipofuscin content of the adult insects at day 1 and day 7 is far less than that of the adult insects at day 1 and day 7 in the control group respectively. The relative quantitative data (as shown in fig. 9) also demonstrate that, in particular, the mean fluorescence intensity of the intestinal lipofuscin of the adult nematodes at day 1 was 20.4 and the mean fluorescence intensity of the intestinal lipofuscin of the adult nematodes at day 7 was 44.6 in the control group, indicating that the lipofuscin content increased with the age of the nematodes. While the average fluorescence intensity of intestinal lipofuscin of the nematodes on day 1 of the imagoes in the PVP-Pd NPs treatment group is 12.3, which is reduced by 40 percent compared with the contrast group (day 1 of the imagoes); on day 7 of PVP-Pd NPs treatment of the adult worms, the mean fluorescence intensity of intestinal lipofuscin in the nematodes was 37.1, which was a 17% reduction relative to the control (day 7 of adult worms). The results show that the PVP-Pd NPs can reduce the content of lipofuscin in the intestinal tracts of organisms of different age groups, and have a good anti-aging effect.
Example 7
The movement of caenorhabditis elegans is related to the strength of muscle tissues of the caenorhabditis elegans, and the influence of materials on the movement capability and the body function of the caenorhabditis elegans can be reflected through the detection of the swing behavior of the caenorhabditis elegans. To further evaluate the effect of PVP-Pd NPs on nematode health parameters, the nematode body swing ability was tested. Adding PVP-Pd NPs and FUDR into an escherichia coli OP50 bacterial solution, uniformly mixing, uniformly coating the mixture on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of the PVP-Pd NPs is 0.5 mu g/mL, the final concentration of the FUDR is 80 mu M, and the final concentration of the FUDR is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into the escherichia coli OP50 bacterial solution, uniformly mixing, and uniformly coating the mixture on the NGM flat plate to obtain an NGM flat plate (the final concentration of the FUDR is 80 mu M) containing only FUDR and not containing PVP-Pd NPs; carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to the L4 stage; transferring another part of L4-stage nematode strains to NGM plates which do not contain PVP-Pd NPs and only contain FUDR to serve as a control group, culturing about 50-100 nematodes in each group at 20 ℃, collecting caenorhabditis elegans on 1 st day, 7 th day and 10 th day of adult nematodes after continuous administration, and recording the head swing times of the nematodes within 20s; after counting the data, significant differences were calculated using Graph Pad (Two-way ANOVA, sidak multiple complexes test), the error bars identify the mean Standard Error (SEM), and the results are shown in fig. 10.
As can be seen from FIG. 10, after 2 days of administration, i.e., day 1 of the administration, the head swing phenomenon was easily detected by the nematodes of the control group, the average number of head swings within 20s was 16.7 for the nematodes of the control group, and the average number of pharyngeal pumping was 17.4 for the nematodes of the PVP-Pd NPs administration group; compared with the blank group, the PVP-Pd NPs administration group has no obvious difference, and the PVP-Pd NPs are proved to have biological safety. On the 7 th day and the 10 th day of the adult, the number of head swings of the nematodes in the control group is obviously reduced relative to that of the adult on the 1 st day, and the number of head swings is 10.5 times and 9.4 times respectively in average every 20s times; the decrease range of the head swing quantity of the nematodes in the PVP-Pd NPs administration group is smaller, and the average 20s times is 12.1 times and 11.8 times respectively; compared with a control group, the PVP-Pd NPs administration group improves the head swing capacity of the nematodes on the 7 th day and the 10 th day, and has a significant difference with the control group. The results show that the PVP-Pd NPs have better biological safety, are beneficial to improving the swing ability of the head and have the effect of delaying the decline of the exercise ability related to aging.
Example 8
The pharynx regresses with aging, and the feeding capacity of the pharynx is continuously reduced, and the feeding capacity of the pharynx can be usually judged by the movement frequency of a pharyngeal pump of the nematode. The process for detecting the swallowing frequency of the nematode comprises the following steps: adding PVP-Pd NPs and FUDR into an escherichia coli OP50 bacterial solution, uniformly mixing, uniformly coating the mixture on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of the PVP-Pd NPs is 0.5 mu g/mL, the final concentration of the FUDR is 80 mu M, and the final concentration of the FUDR is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into the escherichia coli OP50 bacterial solution, uniformly mixing, and uniformly coating the mixture on the NGM flat plate to obtain an NGM flat plate (the final concentration of the FUDR is 80 mu M) containing only FUDR and not containing PVP-Pd NPs; carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to the L4 stage; transferring another part of L4-stage nematode strains to NGM plates which do not contain PVP-Pd NPs and only contain FUDR to serve as a control group, culturing about 50-100 nematodes in each group at 20 ℃, collecting caenorhabditis elegans on the 1 st, 7 th and 10 th days of adult nematodes after continuous administration, observing nematode video under a stereoscopic microscope and counting the number of times of a 30-s endopharyngeal pumping, and recording 10 parallel times in each group. Data were analyzed by One-way ANOVAY statistics and data results are expressed as Mean + -SEM, as shown in FIG. 11.
As can be seen from FIG. 11, the average number of pharyngeal pumping was 123 for the control group nematodes and 133 for the PVP-Pd NPs administration group on the first day of adult life; on the 7 th day of adult nematode, the average number of pharyngeal pumping of the nematode in the control group is 82, and the average number of pharyngeal pumping of the nematode in the PVP-Pd NPs administration group is 97; on day 10 of adult, the average number of pharyngeal pumping was 71 for the control group nematodes and 85 for the PVP-Pd NPs administration group. As can be seen, the PVP-Pd NPs administration group can obviously improve the pharyngeal swallowing frequency of the caenorhabditis elegans adults at 1 day, 7 days and 10 days. The results show that PVP-Pd NPs are beneficial to improving the pharyngeal exercise capacity and have the effect of delaying the decline of exercise capacity related to aging.
Example 9
Adding PVP-Pd NPs and FUDR into Escherichia coli OP50 bacterial liquid, mixing, uniformly coating on an NGM flat plate to obtain an NGM flat plate containing PVP-Pd NPs and FUDR (the final concentration of PVP-Pd NPs is 0.5 mu g/mL; the final concentration of FUDR is 80 mu M, and is used for inhibiting the oviposition of caenorhabditis elegans), adding FUDR into Escherichia coli OP50 bacterial liquid, mixing, and uniformly coating on an NGM flat plate to obtain an NGM flat plate containing only FUDR and not containing PVP-Pd NPs (the final concentration of FUDR is 80 mu M); carrying out nematode synchronization treatment by adopting a wild caenorhabditis elegans model, and transferring a part of L4-stage nematode strains to an NGM flat plate containing PVP-Pd NPs and FUDR as an administration group when L1-stage wild type N2 caenorhabditis elegans grows to the L4 stage; transferring another part of L4-stage nematode strains to an NGM plate which does not contain PVP-Pd NPs and only contains FUDR as a control group, culturing about 50-100 nematodes in each group at 20 ℃, collecting the nematodes on the 7 th day of adult nematodes, transferring to the NGM plate without PVP-Pd NPs to detect the acute oxidative stress capability of the caenorhabditis elegans: placing caenorhabditis elegans in a constant temperature incubator at 35 ℃ for 3h, transferring to a blank plate, placing on a constant temperature incubator at 20 ℃ for recovering for 1h, evaluating the survival rate of the caenorhabditis elegans, and judging the death standard in the same way as in example 5; evaluating the motor behavior ability of the survival nematodes, and recording the body bending times of the nematodes within 20s (the distance of one wavelength for the nematodes to climb forwards is recorded as one body bending); statistical data, significance differences were calculated using Graph Pad (Two-way ANOVA, sidak multiple complexes test), error bars identify the mean Standard Error (SEM), and the results are shown in fig. 12.
As can be seen from fig. 12, the results of the acute heat stress experiment showed that the survival rate of the nematodes in the control group was 20.4% after the adult nematodes were treated at 35 ℃ for 3 hours at the seventh day, and the body bending frequency of the surviving nematodes was 2.6 times/20 s; the survival rate of the nematodes in the PVP-Pd NPs administration group after the same treatment is 53.3%, and the body bending frequency of the surviving nematodes is 3.3 times/20 s, which have significant differences. The result shows that the PVP-Pd NPs can obviously improve the acute hot stress capability of the nematodes, and further the PVP-Pd NPs can be applied to the preparation of products for improving the acute hot stress capability of organisms.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. Application of palladium nanoparticles modified by polyvinylpyrrolidone in preparing products for resisting aging and/or enhancing heat stress resistance.
2. The use according to claim 1, wherein the anti-aging and/or anti-heat stress enhancing product is selected from at least one of a pharmaceutical, a nutraceutical and a food additive.
3. Application of palladium nanoparticles modified by polyvinylpyrrolidone in preparing products for improving mitochondrial respiration function.
4. The use according to claim 3, wherein the mitochondrial respiratory function improving product is selected from at least one of a pharmaceutical, a nutraceutical and a food additive.
5. Application of palladium nanoparticles modified by polyvinylpyrrolidone in preparing products for enhancing expression of mitochondrial metabolic enzymes.
6. The use according to claim 5, wherein the product that enhances expression of mitochondrial metabolic enzymes is selected from at least one of a pharmaceutical, a nutraceutical, and a food additive.
7. Use according to claim 5, characterized in that the mitochondrial metabolizing enzyme is selected from the group consisting of citrate synthasescts-1Hexokinasehxk-1And pyruvate carboxylasepyc-1At least one of (1).
8. Use according to any one of claims 1 to 7, wherein the polyvinylpyrrolidone-modified palladium nanoparticles have a particle size of 15-25nm.
9. The use according to any one of claims 1 to 7, wherein the polyvinylpyrrolidone-modified palladium nanoparticles have a molar ratio of polyvinylpyrrolidone to palladium of 1 to 9:1.
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| CN118649245A (en) * | 2024-08-16 | 2024-09-17 | 北京市疾病预防控制中心 | Use of polyvinylpyrrolidone-modified palladium nanoparticles for preparing products for treating huntington's disease |
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