CN116019831A - Application of gold nanospheres in preparation of drugs for regulating trimethylamine oxide metabolic disorder - Google Patents

Abstract

The invention relates to an application of gold nanospheres in preparing medicines for regulating trimethylamine oxide metabolic disorder. In order to solve the problem of chronic inflammation under the menopausal condition, the applicant finds out through experiments that the cash nanospheres can regulate the metabolic process of the trimethylamine oxide under the menopausal condition and inhibit the inflammatory factors from entering the blood, so that the malignant progress of the chronic inflammation under the menopausal condition is relieved, and the effect of relieving the inflammatory complications related to the menopause is achieved.

Description

Application of gold nanospheres in the preparation of drugs regulating trimethylamine oxide metabolic disorders

technical field

The invention belongs to the technical field of nanomedicine, and relates to the application of gold nanospheres in the preparation of drugs for regulating the metabolic disorder of trimethylamine oxide.

Background technique

Trimethylamine oxide (TMAO) is an active metabolite that is involved in the synthesis of intestinal flora. It is produced by the oxidation reaction of trimethylamine (TMA) by hepatic flavin monooxygenase (FMO1 and FMO3), which has been confirmed to be associated with cardiovascular diseases. , cancer and kidney disease and many other diseases are closely related. TMA can be obtained directly from food, or it can be metabolized from precursors such as carnitine, phosphatidylcholine/choline, and betaine. Many species of gut flora such as Clostridia, Proteus, and Shigella produce large amounts of TMA. Dietary changes, gut microbiota dysbiosis, and altered gut permeability can all lead to elevated concentrations of TMAO in the body. Large cohort studies have shown that increased plasma TMAO concentration is associated with increased risk of multiple malignant cardiovascular and cerebrovascular events such as hypertension, myocardial infarction, heart failure and stroke. Further studies have confirmed that elevated plasma TMAO interferes with the metabolism of cholesterol and lipoproteins, hinders the reverse transport of cholesterol; up-regulates the number of scavenger receptors on the surface of macrophages, and promotes the formation of foam cells; at the same time, it promotes IL-6, ICAM-1 and COX- 2 and other inflammatory factors, activate NF-κB signal to generate inflammatory response. In addition, TMAO is closely related to the occurrence and development of diabetes, aggravates impaired glucose tolerance, and reduces insulin sensitivity in patients. The mechanism may be related to TMAO blocking insulin-related signaling pathways and increasing the release of pro-inflammatory factors. Stanley L Hazen and others found that high levels of TMAO are related to the development and progression of chronic kidney disease through follow-up, which can cause renal artery atherosclerosis and promote renal tissue fibrosis. Associated with vascular inflammatory response. Drugs to alleviate TMAO metabolic disorders mainly include probiotics, prebiotics, antibiotics, and FMO3 enzyme inhibitors. Among them, the combined treatment of probiotics and prebiotics can inhibit the growth of TMA-producing flora, but its safety and colonization effect are still unclear. Antibiotic treatment can clear the TMA-producing flora and significantly inhibit the plasma TMAO level, but the beneficial flora in the intestinal tract will also be cleared, and long-term use has more side effects. FMO3 enzyme inhibitors can prevent the conversion of TMA into TMAO and reduce the production of TMAO, but the accumulation of TMA in the body can also induce inflammation and aggravate the disease. Menopausal conditions can cause many chronic inflammatory diseases, and the increased expression of inflammatory cytokines such as IL-1, TNF-α, and IL-6 is an important reason for its pathogenesis.

Nanomedicine refers to the design of new drugs that are superior to traditional drugs in pharmacokinetics and pharmacodynamics for disease treatment by using the particularity of nanomaterials in terms of spatial dimensions, such as surface effects and small size effects. Gold nanospheres have excellent optical properties, biocompatibility, surface adsorption capacity, and low biotoxicity, and are widely used in the fields of cell induction, drug delivery, clinical diagnosis, and antibody labeling. Relevant studies have confirmed that gold nanospheres have significant anti-angiogenic and anti-inflammatory activities, and have a good curative effect in inflammatory diseases such as rheumatoid arthritis. At present, the research on gold nanospheres is mainly focused on the treatment of cancer. There is little research on its role in regulating the endocrine metabolic system.

Contents of the invention

In view of this, the object of the present invention is to provide the application of gold nanospheres in the preparation of drugs for regulating the metabolic disorder of trimethylamine oxide, especially the application in the preparation of drugs for the treatment of chronic inflammation caused by the metabolic disorder of trimethylamine oxide after menopause. It provides a new way for the occurrence and development of sex-related diseases.

To achieve the above object, the present invention provides the following technical solutions:

1. The application of gold nanospheres in the preparation of drugs for regulating the metabolic disorder of trimethylamine oxide.

Preferably, the particle size of the gold nanospheres is 60nm.

Preferably, the trimethylamine oxide metabolism is intestinal trimethylamine oxide metabolic disorder, and its mediator is selected from any one or more of the following: trimethylamine oxide (TMAO) metabolic cycle disorder, trimethylamine oxide (TMAO) in intestinal metabolites TMAO) content increased, and the content of inflammatory factors in peripheral blood serum increased.

2. Application of gold nanospheres in the preparation of drugs for treating chronic inflammation caused by postmenopausal trimethylamine oxide metabolic disorder.

3. Application of gold nanospheres in the preparation of medicines for reducing the content of inflammatory factor IL-6 in peripheral blood serum.

4. Application of gold nanospheres in the preparation of medicines for reducing the content of inflammatory factor TNF-α in peripheral blood serum.

5. Application of gold nanospheres in the preparation of medicines for reducing the content of inflammatory factor G-CSF in peripheral blood serum.

The beneficial effects of the present invention are:

In order to solve the chronic inflammation under menopausal conditions, the metabolic disorder of intestinal trimethylamine oxide leads to the release of a large number of inflammatory factors into the blood and aggravates the occurrence and development of inflammation. Through experiments, the applicant found that gold nanospheres can regulate trimethylamine oxide under menopausal conditions. Metabolic process, and inhibit inflammatory factors into the blood, thereby alleviating the malignant progression of chronic inflammation under menopausal conditions, and achieve the effect of alleviating postmenopausal related inflammatory complications.

Description of drawings

In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1 is a micrograph of GNS under a scanning electron microscope.

Figure 2 is a heat map of GNS intervention to reduce the content of trimethylamine oxide (TMAO) metabolites under menopausal conditions, specifically the content of TMAO-related metabolites in the intestinal metabolites of mice.

Figure 3 is GNS intervention to reduce the content of trimethylamine oxide (TMAO) metabolites under menopausal conditions, specifically the content bar chart of six TMAO-related metabolites, where (A) is Betaine betaine (P>0.05), (B) Choline (P>0.05), (C) Creatinine (P<0.05), (D) TMA trimethylamine (P>0.05), (E) TMAO trimethylamine oxide (P<0.05), ( F) For L-Carnitine: L-carnitine (P<0.05).

Figure 4 is GNS intervention to reduce the content of trimethylamine oxide (TMAO) metabolites under menopausal conditions, where A is a two-dimensional display of the difference results of potential structure discriminant analysis (OPLS-DA), and B is the difference result of principal component analysis (PCA) 2D representation of .

Fig. 5 shows that GNS intervention reduces the content of inflammatory factors in peripheral blood serum under menopausal conditions (volcano graph obtained by detecting the levels of different inflammatory factors by cytokine chip).

Figure 6 shows that GNS intervention reduces the content of inflammatory factors in peripheral blood serum under menopausal conditions (the heat map obtained by detecting the levels of different inflammatory factors by the cytokine chip).

Figure 7 is a schematic diagram of the mechanism related to the regulation of postmenopausal TMA metabolism by gold nanospheres.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

1) Gold nanospheres

Gold nanosphere stock solution with a concentration of 1mg/ml was purchased from Xiamen numantechnology Co.Ltd. The particle size of gold nanospheres (GNS) contained therein was 60nm. The scanning electron micrograph is shown in Figure 1.

Figure 1 shows the scanning electron microscope image of the gold nanospheres used in this experiment, with a particle size of 60nm.

2) Model construction of ovariectomized (OVX) mice (under menopausal conditions)

Ten 8-week-old C57BL/6J female wild-type mice (Speyford Animal Experiment Center, Beijing, China) with a body weight of 18-22 g were taken. Pentobarbital (50mg/kg) anesthetized, OVX operation was carried out, administered continuously for 8 weeks, the mice were randomly divided into two groups, 5 in each group;

OVX group: 0.01ml/g (body weight) was administered by intragastric administration, and the administration was gold nanosphere stock solution;

Control group: each rat was given the same amount of normal saline by intragastric administration;

3) GNS significantly reduces the production of trimethylamine oxide (TMAO)-related metabolites

Quantitative Metabolomics Analysis of Intestinal Metabolite TMAO

50 mg of intestinal metabolite samples (mouse feces of different groups) were placed in EP tubes, and 1 ml of extraction solution (acetonitrile:methanol:water=2:2:1 (volume ratio)) was added. After the mixture was centrifuged (1000rpm, 5min), the resulting supernatant was transferred to a fresh glass bottle for analysis. Prepare quality control samples by mixing equal volumes of supernatant from each sample. LC-MS/MS analysis was performed using UHPLC system (Vanquish, Thermo Fisher Scientific), UPLC BEH Amide column (2.1mm×100mm, 1.7μm) and Q Exactive HFX mass spectrometry (Orbitrap MS, Thermo). Use ProteoWizard to convert raw data into mzXML format for peak detection, extraction, alignment and integration. Metabolites were then annotated using the in-house MS2 database (Biotree DB). The cutoff for annotation was set at 0.3. The above experiments and data analysis were completed with the support of Biotree Biomedical Technology Company (Shanghai, China). Use Compound Discoverer2.1 (Thermo Fisher Scientific, Waltham, MA, USA) to correct the data, and import SIMCA16.0.2 software package (Sartorius Stedim data Analytics AB, Umea, Sweden) for principal component analysis (PCA) and potential structure Orthogonal projection for discriminant analysis (OPLS-DA). Then perform 7-fold cross-validation to calculate the values of R2 and Q2.

As shown in Figure 2, the heat map of the content of TMAO-related metabolites in the intestinal metabolites of mice showed that GNS significantly reduced the content of TMAO-related metabolites. Figure 3 shows that GNS intervention reduces the content of trimethylamine oxide (TMAO) metabolites in intestinal metabolites under menopausal conditions, where (A) is Betaine (P>0.05), (B) is Choline choline (P>0.05) ), (C) is Creatinine (P<0.05), (D) is TMA trimethylamine (P>0.05), (E) is TMAO trimethylamine oxide (P<0.05), (F) is L-Carnitine: left-handed The contents of carnitine (P<0.05), creatinine (Creatinine), trimethylamine oxide (TMAO) and L-carnitine (L-Carnitine) in the intestinal metabolites of the OVX+GNS group were significantly lower than those of the OVX group (P< 0.05). Betaine (Betaine), choline (Choline) and trimethylamine (TMA) content did not change significantly (P>0.05). Figure 4 shows that GNS intervention can reduce the content of trimethylamine oxide (TMAO) metabolites under menopausal conditions, where A is a two-dimensional display of the difference results of potential structure discriminant analysis (OPLS-DA), and B is the difference of principal component analysis (PCA) The two-dimensional display graph of the results shows that there are significant differences in TMAO metabolism under GNS intervention conditions. Therefore, it was concluded that GNS can significantly reduce the content of TMAO-related metabolites.

4) Gold nanospheres significantly reduced the content of inflammatory factors in peripheral blood serum under menopausal conditions

Cytokine Chip Detection

The specific experimental process is as follows:

1. Sample incubation:

1) The microbeads were shaken at 1,400rpm for 30s, and then the microbeads were diluted with Assay Buffer (Abcam, USA);

2) The diluted microbeads were shaken again at 1,400rpm for 30s, and 50 μL of microbeads were added to each well of a 96-well plate, and then the plate was washed 3 times;

3) Take 50 μL of standard substance and sample and put them into 96-well plate respectively, and then the plate is covered with parafilm, and shaken at 850 rpm at room temperature for 30 min under light-proof conditions.

2. Antibody detection:

1) Discard the sample from the 96-well plate after incubation, and then the plate is washed 3 times;

2) According to the instructions, the Detection Antibody was diluted with Antibody Diluent;

3) Add 25 μL of the diluted Detection Antibody to each well, and then the plate is covered with a parafilm and shaken at 850 rpm for 30 minutes at room temperature under dark conditions.

3. Data collection:

1) The detection antibody was discarded from the incubated 96-well plate, and then the plate was washed 3 times;

2) According to the instructions, Streptavidin-PE was diluted with Antibody Diluent;

3) Add 50 μL of diluted Streptavidin-PE to each well, attach a parafilm, and shake at room temperature for 10 minutes at 850 rpm in the dark;

4) The plate was washed 3 times;

5) Add 125 μL Assay Buffer to each well to resuspend the microbeads, cover the plate with parafilm, and shake at 850 rpm for 30 seconds at room temperature under dark conditions;

6) Finally, a BioPlex 200 machine was used for data acquisition.

As shown in Figures 5 and 6, gold nanospheres significantly reduced the levels of inflammatory cytokines IL-6, TNF-α, and G-CSF in the peripheral blood of OVX mice (P<0.05).

Figure 7 is a schematic diagram of the mechanism related to the regulation of postmenopausal TMA metabolism by gold nanospheres.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.

Claims (7)

1. The application of gold nanospheres in preparing medicines for regulating trimethylamine oxide metabolic disorder is provided.

2. The use according to claim 1, characterized in that the gold nanospheres have a particle size of 60nm.

3. Use according to claim 1, characterized in that the trimethylamine oxide is metabolized into intestinal trimethylamine oxide metabolic disorders, the mediating factors of which are selected from any one or several of the following: the metabolic loop of trimethylamine oxide is disturbed, the content of trimethylamine oxide in intestinal metabolites is increased, and the content of inflammatory factors in peripheral blood serum is increased.

4. The application of the gold nanospheres in preparing medicines for treating chronic inflammation caused by postmenopausal trimethylamine oxide metabolic disorder.

5. Application of gold nanospheres in preparing medicines for reducing inflammatory factor IL-6 content in peripheral blood serum.

6. The application of gold nanospheres in preparing medicaments for reducing the content of inflammatory factors TNF-alpha in peripheral blood serum.

7. The application of gold nanospheres in preparing medicaments for reducing the content of inflammatory factor G-CSF in peripheral blood serum.

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