Application of mangiferin in the preparation of drugs that improve glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics.

Abstract

This invention discloses the application of mangiferin in alleviating the multigenerational toxicity induced by polystyrene nanoplastics, primarily relating to the field of mangiferin application technology. The method includes the following steps: S1, culturing experimental fruit flies; S2, determining the fruit fly body weight (M); S3, determining glucose and triglyceride content; S4, qualitative and quantitative analysis of ROS in fruit fly hemolymph; S5, determining SOD, CAT enzyme activity, and MDA content; S6, determining the expression of genes related to glucose and lipid metabolism. Using fruit flies as a model organism, this invention evaluated the ability of mangiferin to alleviate metabolic damage induced by polystyrene nanoplastics, confirming that mangiferin has a positive effect on repairing metabolic damage caused by polystyrene nanoplastics and can improve the redox state of the fruit fly. This invention provides an important reference for further research on the toxicity of nanoplastics to organisms and for finding corresponding treatment strategies.

Description

Technical Field

[0001] This invention relates to the field of mangiferin application technology, specifically to the application of mangiferin in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics. Background Technology

[0002] Micro- and nanoplastics are a new type of persistent organic pollutant. Because they are distributed in various environmental media and diets, people inevitably ingest them through various routes. Therefore, the potential harm of micro- and nanoplastics to the environment and human health is receiving increasing attention. More importantly, the degradation process of plastics in the environment is continuous, leading to the long-term and widespread distribution of micro- and nanoplastics in the biosphere, and their toxic effects on humans and organisms will persist. Polystyrene is one of the five major high-yield plastics and is widely used in the processing of plastic tableware and food containers. Furthermore, polystyrene has a complex chemical structure, and ordinary microorganisms and external forces such as ultraviolet irradiation usually cannot completely degrade it. Some researchers estimate that it can persist in various environmental media for thousands of years or even longer; therefore, polystyrene nanoplastics have become a common type of nanoplastic.

[0003] Despite growing concern about the harmful effects of nanoplastics, there are currently almost no reports on defense mechanisms against nanoplastic-induced toxicity. Mangiferin (MGF) is a natural polyphenolic acid compound widely found in mango (Mango spp.), a plant in the Anacardiaceae family. Mangifera indicate The fruit, leaves, and bark of Anemarrhena asphodeloides (Liliaceae plant) AnemarrhenaasphodeloidesBge. The rhizomes, above-ground parts, and Iris tectorum (Iris family plant) Belamcandachinensis (L.) DC. Mangiferin has been shown to possess numerous physiological activities in the flowers, leaves, and other parts of plants, including antitumor, hypoglycemic, antioxidant, and immunomodulatory effects. However, there are no reports on its role in intervening in metabolic damage in Drosophila after multiple generations of exposure to polystyrene nanoplastics. The energy metabolism organs of Drosophila and mammals are highly conserved in biological evolution, and in recent years, Drosophila melanogaster has become a good model organism for studying metabolic diseases. Therefore, this invention utilizes the Drosophila melanogaster model to demonstrate the role of mangiferin in delaying the metabolic toxicity induced by polystyrene nanoplastics. Summary of the Invention

[0004] The purpose of this invention is to provide an application of mangiferin in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, in order to solve the above-mentioned technical problems.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This study describes the experimental method for the application of mangiferin in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics. Materials used included high-purity mangiferin standard reagent, conventional culture medium, polystyrene nanoplastic culture medium, mangiferin culture medium, and experimental fruit flies reared on these media. The high-purity mangiferin was a standard reagent with a purity ≥98%. The concentration of the polystyrene nanoplastic culture medium was 100 mg / L, and the concentration of the mangiferin culture medium was 80 mg / L. The initial rearing medium for the experimental fruit flies was cornmeal medium, with the following formula: 9.6 g cornmeal, 9.6 g sucrose, 3.2 g yeast extract, 0.8 g agar powder, 0.2 g methylparaben, 0.8 mL propionic acid, 2 mL anhydrous ethanol, and 100 mL distilled water. The culture environment was a constant temperature incubator at 25±1℃, relative humidity of 50%~60%, and a photoperiod of 12L:12D.

[0007] The experimental method includes the following steps:

[0008] S0: Collect unmated male and female fruit flies within 8 hours of emergence from the experimental fruit flies after rearing, and separate them into conventional fruit fly culture medium. The fruit flies at this time are recorded as generation F0. No polystyrene nanoplastics are added to generation F0.

[0009] Subsequently, fertilized eggs of fruit flies were selected and transferred to polystyrene nanoplastic experimental medium and standard conventional medium without polystyrene nanoplastic. The fruit flies at this time were recorded as F1 generation.

[0010] After the F1 generation fruit flies emerged, they were immediately transferred to their respective polystyrene nanoplastic experimental culture media. After the F1 generation fruit flies laid eggs, the adult flies were discarded. The fruit flies in the culture media at this time were recorded as the F2 generation.

[0011] The subsequent F3, F4 and F5 were cultured using the same method.

[0012] S1. Experimental fruit flies were cultured and divided into three groups: a negative control group, which was cultured in a conventional culture medium without exposure to polystyrene nanoplastics; a positive control group, which was cultured in a polystyrene nanoplastic medium with polystyrene nanoplastic exposure; and an experimental group, which was cultured in a polystyrene nanoplastic medium with polystyrene nanoplastic exposure for 5 generations, and then cultured in mangiferin medium.

[0013] S2, Determination of fruit fly body weight M: Weigh an empty 1.5 mL centrifuge tube using an analytical balance and record the weight as m1 (mg). Randomly place 10 third-instar fruit fly larvae into the centrifuge tube, wash off the surface food with PBS, blot dry with filter paper, and weigh again as m2 (mg). Repeat each experiment 6 times. The fruit fly body weight M is calculated using the following formula:

[0014]

[0015] S3, Determination of glucose and triglyceride content: Weigh 50 mg of third-instar fruit fly larvae, add 0.45 mL of physiological saline, homogenize mechanically, centrifuge at 3500 rpm for 10 min, collect the supernatant, and determine the glucose concentration using a glucose detection kit; Weigh 50 mg of third-instar fruit fly larvae, add 9 times the volume of anhydrous ethanol, homogenize mechanically under ice-water bath conditions, centrifuge at 3000 rpm for 10 min, collect the supernatant, and determine the triglyceride content using a triglyceride detection kit. The triglyceride content in the sample was homogenized using protein concentration.

[0016] S4, Qualitative and quantitative analysis of ROS in Drosophila hemolymph; 100 mg of Drosophila was weighed, washed with PBS buffer, blotted dry with filter paper, placed in a 1.5 mL centrifuge tube, 1 mL of PBS buffer was added, and the mixture was homogenized thoroughly using a bead mill. The homogenate was centrifuged at 4000 rpm and 4℃ for 6 min. After centrifugation, 190 μL of the homogenate supernatant diluted 5-10 times and 10 μL of O12 fluorescent probe were added to a black 96-well fluorescent plate. The plate was incubated at 37℃ in the dark for 25 min, and then placed in a continuous wavelength multi-functional microplate detection platform. Quantitative analysis was performed using an excitation wavelength of 488 nm and an emission wavelength of 526 nm. The tissue ROS intensity was expressed as fluorescence intensity / protein concentration.

[0017] The ROS in the hemolymph of third-instar larvae of Drosophila melanogaster were determined using the DCFH-DA fluorescent probe. The specific method is as follows: 100 third-instar larvae of Drosophila were taken, and under a stereomicroscope, the bodies of the larvae were punctured in a 1.5 mL centrifuge tube with dissecting forceps to release the hemolymph. The hemolymph was centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant was the hemolymph of the Drosophila. The hemolymph was incubated with 5 μmol / L DCFH-DA fluorescent probe at room temperature in the dark for 30 min to label cells containing ROS. The ROS in the hemolymph of the Drosophila was qualitatively analyzed using an inverted fluorescence microscope.

[0018] S5, determine SOD, CAT enzyme activity and MDA content; weigh 50 mg of third instar fruit fly larvae, add 1.35 mL of physiological saline for mechanical homogenization, centrifuge at 3000 rpm for 10 min, take the supernatant, and determine SOD, CAT enzyme activity and MDA content. Each indicator is standardized by the absorbance of the sample protein.

[0019] S6, Expression determination of genes related to glucose and lipid metabolism; collect third-instar larvae of Drosophila, wash three times with PBS buffer, add 1 mL of trizol and grind twice; centrifuge at 12000 g, 4℃ for 5 min, aspirate the supernatant into a centrifuge tube, place on ice for 5 min, add 200 μL of chloroform, vortex for 15 s, place on ice for 3 min; centrifuge at 12000 g, 4℃ for 15 min, accurately aspirate the colorless aqueous phase containing total RNA into another centrifuge tube, add an equal volume of pre-chilled isopropanol, slowly invert and mix, place for 10 min; centrifuge at 12000 g, 4℃ for 10 min, discard the supernatant to obtain RNA precipitate; add 1 mL of 75% ethanol to the precipitate, invert and mix; centrifuge at 7500 g, 4℃ for 5 min, discard the supernatant, and after the RNA has dried, add 20-50 μL of... The RNA was dissolved in DEPC water; its concentration was measured using a NanoDrop nucleic acid and protein quantification instrument, and the RNA was diluted appropriately according to the concentration. Finally, the RNA was reverse transcribed into cDNA using reverse transcriptase, and analyzed by quantitative real-time PCR. Rp49 , Dilp2 , Dilp3 , Dilp5 The mRNA expression status.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention describes the application of mangiferin in alleviating the metabolic toxicity induced by polystyrene nanoplastics. Specifically, it proposes a novel method for intervening in metabolic disorders in Drosophila under multiple generations of polystyrene nanoplastic exposure. Using Drosophila as a model organism, the effects of mangiferin on body weight, glucose content in hemolymph, triglyceride content, ROS levels in vivo and in hemolymph, antioxidant enzyme activity, MDA levels, and mRNA expression levels of glucose and lipid metabolism-related genes in Drosophila under multiple generations of polystyrene nanoplastic exposure were measured. The results showed that mangiferin at a concentration of 80 mg / L improved glucose and lipid metabolism disorders in Drosophila under multiple generations of polystyrene nanoplastic exposure. This effect was related to antioxidative stress and regulation of glucose and lipid metabolism-related genes. Dilp2 , Dilp3 and Dilp5 It is related to mRNA expression. Attached Figure Description

[0022] Figure 1 Effects of mangiferin on body weight in Drosophila subjected to multigenerational exposure to polystyrene nanoplastics;

[0023] Figure 2 Effects of mangiferin on glucose and triglycerides in the hemolymph of Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics;

[0024] Figure 3Effects of mangiferin on ROS levels in tissues and hemolymph of Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics;

[0025] Figure 4 Effects of mangiferin on antioxidant enzyme activity and MDA content in Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics;

[0026] Figure 5 Effects of mangiferin on the expression of glucose and lipid metabolism-related genes mRNA in Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics. Detailed Implementation

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0028] This invention describes the application of mangiferin in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics. The materials used include high-purity mangiferin standard reagent, conventional culture medium, polystyrene nanoplastic culture medium, mangiferin culture medium, and experimental fruit flies reared on these media. The high-purity mangiferin is a standard reagent with a purity ≥98%. The concentration of polystyrene nanoplastics in the polystyrene nanoplastic culture medium is 100 mg / L, and the concentration of mangiferin in the mangiferin culture medium is 80 mg / L. The initial rearing medium for the experimental fruit flies is corn flour medium, with the following formula: 9.6 g corn flour, 9.6 g sucrose, 3.2 g yeast powder, 0.8 g agar powder, 0.2 g methylparaben, 0.8 mL propionic acid, 2 mL anhydrous ethanol, and 100 mL distilled water. The culture environment is a constant temperature incubator at 25±1℃, relative humidity of 50%~60%, and a photoperiod of 12L:12D.

[0029] The experimental method includes the following steps:

[0030] S0: Unmated male and female fruit flies within 8 hours of emergence were collected from the reared experimental fruit flies and aliquoted into conventional fruit fly culture medium. These fruit flies were designated as generation F0, without the addition of polystyrene nanoplastics. Three days later, fertilized eggs were selected under a stereomicroscope and transferred to experimental medium containing 100 mg / L polystyrene nanoplastics and standard conventional medium without polystyrene nanoplastics. These fruit flies were designated as generation F1. After emergence, the F1 generation fruit flies were immediately transferred to their respective corresponding polystyrene nanoplastics experimental medium. After the F1 generation fruit flies laid eggs, the adult flies were discarded. The fruit flies in the medium at this time were designated as generation F2. Subsequent generations F3, F4, and F5 were cultured using the same method.

[0031] S1. Experimental fruit flies were cultured and divided into three groups: a negative control group, which was cultured in conventional culture medium without exposure to polystyrene nanoplastics; a positive control group, which was cultured in 100 mg / L polystyrene nanoplastic medium for 5 consecutive generations; and an experimental group, which was cultured in 100 mg / L polystyrene nanoplastic medium for 5 consecutive generations, and then cultured in mangiferin medium.

[0032] S2, Determination of fruit fly body weight M: Weigh an empty 1.5 mL centrifuge tube using an analytical balance and record the weight as m1 (mg). Randomly place 10 third-instar fruit fly larvae into the centrifuge tube, wash off the surface food with PBS, blot dry with filter paper, and weigh again as m2 (mg). Repeat each experiment 6 times. The fruit fly body weight M is calculated using the following formula:

[0033]

[0034] S3, Determination of glucose and triglyceride content: Weigh 50 mg of third-instar fruit fly larvae, add 0.45 mL of physiological saline, homogenize mechanically, centrifuge at 3500 rpm for 10 min, collect the supernatant, and determine the glucose concentration using a glucose detection kit; Weigh 50 mg of third-instar fruit fly larvae, add 9 times the volume of anhydrous ethanol, homogenize mechanically under ice-water bath conditions, centrifuge at 3000 rpm for 10 min, collect the supernatant, and determine the triglyceride content using a triglyceride detection kit. The triglyceride content in the sample was homogenized using protein concentration.

[0035] S4. Qualitative and quantitative analysis of ROS in Drosophila hemolymph; 100 mg of Drosophila was weighed, washed with PBS buffer, blotted dry with filter paper, and placed in a 1.5 mL centrifuge tube. 1 mL of PBS buffer was added, and the mixture was homogenized thoroughly using a bead mill. The homogenate was centrifuged at 4000 rpm and 4℃ for 6 min. After centrifugation, 190 μL of the homogenate supernatant diluted 5-10 times and 10 μL of O12 fluorescent probe were added to a black 96-well fluorescent plate. The plate was incubated at 37℃ in the dark for 25 min, and then placed in a continuous wavelength multi-functional microplate detection platform. Quantitative analysis was performed using an excitation wavelength of 488 nm and an emission wavelength of 526 nm. The tissue ROS intensity was expressed as fluorescence intensity / protein concentration.

[0036] The ROS in the hemolymph of third-instar larvae of Drosophila melanogaster were determined using the DCFH-DA fluorescent probe. The specific method is as follows: 100 third-instar larvae of Drosophila were taken, and under a stereomicroscope, the bodies of the larvae were punctured in a 1.5 mL centrifuge tube with dissecting forceps to release the hemolymph. The hemolymph was centrifuged at 8000 rpm for 10 min at 4℃, and the supernatant was the hemolymph of the larvae. The hemolymph was incubated with 5 μmol / L DCFH-DA fluorescent probe at room temperature in the dark for 30 min, and the qualitative analysis of ROS in the hemolymph of the larvae was performed using an inverted fluorescence microscope.

[0037] S5, determine SOD, CAT enzyme activity and MDA content; weigh 50 mg of third instar fruit fly larvae, add 1.35 mL of physiological saline for mechanical homogenization, centrifuge at 3000 rpm for 10 min, take the supernatant, and determine SOD, CAT enzyme activity and MDA content. Each indicator is standardized by the absorbance of the sample protein.

[0038] S6, Expression determination of genes related to glucose and lipid metabolism; collect third-instar larvae of Drosophila, wash three times with PBS buffer, add 1 mL of trizol and grind twice on ice; centrifuge at 12000 g, 4℃ for 5 min, aspirate the supernatant into a centrifuge tube, place on ice for 5 min, add 200 μL of chloroform, vortex for 15 s, place on ice for 3 min; centrifuge at 12000 g, 4℃ for 15 min, accurately aspirate the colorless aqueous phase containing total RNA into another centrifuge tube, add an equal volume of pre-chilled isopropanol, slowly invert and mix, place on ice for 10 min; centrifuge at 12000 g, 4℃ for 10 min, discard the supernatant to obtain RNA precipitate; add 1 mL of 75% ethanol to the precipitate, invert and mix; centrifuge at 7500 g, 4℃ for 5 min, discard the supernatant, and after the RNA has dried, add 20-50 μL of... DEPC was dissolved in water; the concentration of extracted RNA was determined using a NanoDrop nucleic acid and protein quantification instrument, and the RNA was appropriately diluted according to the concentration. Finally, the RNA was reverse transcribed into cDNA using reverse transcriptase, and analyzed by real-time quantitative PCR. Rp49 , Dilp2 , Dilp3 , Dilp5 The mRNA expression status.

[0039] The primer sequences used are shown in Table 1:

[0040] Table 1 Gene Primer Sequences

[0041]

[0042] The qRT-PCR protocol used was as follows: denaturation program (95℃, 5 min), followed by 40 cycles of 95℃ for 10 s and 60℃ for 30 s. Melting curve analysis was performed at the end of the amplification phase; the specificity of the qRT-PCR product was tested by cycling at 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s. Rp49 As an internal reference, the expression of each gene at the mRNA level was determined, and 2- ΔΔCt The method calculates the relative expression level of mRNA.

[0043] The statistical analysis steps are as follows:

[0044] Data were plotted and analyzed using GraphPad Prism 9 and SPSS 16.0 software. All experimental results are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to assess differences between groups. P <0.05, P <0.01 and P <0.001 indicates a statistically significant difference between groups. Each experiment should be repeated at least 3 times.

[0045] Specifically:

[0046] The initial feeding medium for the experimental fruit flies was corn flour medium, and the preparation process is as follows:

[0047] Step 1: The corn flour culture medium formula is as follows: taking 100 mL of food as an example, 100 mL of distilled water, 9.6 g of corn flour, 9.6 g of sucrose, 3.2 g of yeast powder, 0.8 g of agar powder, 0.2 g of methylparaben, 0.8 mL of propionic acid, and 2 mL of anhydrous ethanol;

[0048] Step 2: Mix corn flour, sucrose, yeast powder and agar powder in a pot, add appropriate amount of distilled water, stir thoroughly to dissolve and then heat;

[0049] Step 3: Stir constantly during heating until boiling, and continue boiling for 10-15 minutes until the culture medium becomes viscous.

[0050] Step 4: When the temperature is cooled to about 60°C at room temperature, add methylparaben dissolved in propionic acid and anhydrous ethanol. Stir well while hot and dispense into fruit fly culture tubes. After cooling to room temperature, solidify and set for later use.

[0051] Specifically:

[0052] The statistical analysis steps are as follows:

[0053] Based on the above, the following data were plotted and analyzed using GraphPad Prism 9 and SPSS 16.0 software. All experimental results are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used to assess differences between groups. P <0.05, P <0.01 and P <0.001 indicates a statistically significant difference between groups.

[0054] The experimental data analysis is as follows:

[0055] Notes: 1. Negative control group: Control; 2. Positive control group: PS-NPs; 3. Experimental group: PS-NPs+MGF.

[0056] Figure 1 The effect of mangiferin on the body weight of Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics was investigated. Data are expressed as mean ± standard deviation, N = 6. This indicates a significant difference between the two sample groups. P <0.05). By Figure 1 It can be seen that, compared with the untreated negative control group (Control), after 5 generations of exposure to 100 mg / L polystyrene nanoplastics, the body weight of fruit flies decreased by 12.21%. However, after treatment with 80 mg / L mangiferin, the body weight of fruit flies increased significantly by 1.06 times compared with the group exposed to polystyrene nanoplastics for 5 consecutive generations. P <0.05). The results showed that continuous exposure to polystyrene nanoplastics for multiple generations reduced the weight of fruit flies, while mangiferin could slow down the trend of weight loss in fruit flies.

[0057] Figure 2 The effects of mangiferin on glucose (A) and triglycerides (B) in the hemolymph of Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics were investigated. Data are expressed as mean ± standard deviation, N = 3. This indicates a significant difference between the two sample groups. P <0.05), This indicates a highly significant difference between the two sample groups. P <0.01). By Figure 2 As shown in Figure A, compared with the untreated negative control group (Control), exposure to 100 mg / L polystyrene nanoplastics for 5 generations resulted in a 27.15% decrease in glucose content in the hemolymph of fruit flies. However, treatment with 80 mg / L mangiferin significantly increased the glucose content in the hemolymph of fruit flies by 1.12 times compared with the group exposed to polystyrene nanoplastics for 5 consecutive generations. P <0.01). By Figure 2 As shown in Figure B, compared with the untreated negative control group (Control), exposure to 100 mg / L polystyrene nanoplastics for 5 generations reduced triglyceride levels in Drosophila by 42.61%. However, treatment with 80 mg / L mangiferin significantly increased triglyceride levels in Drosophila by 1.60 times compared to the group exposed to polystyrene nanoplastics for 5 generations. P <0.01). The results showed that continuous exposure to polystyrene nanoplastics for multiple generations induced glucose and lipid metabolism disorders in Drosophila, while mangiferin had a protective effect against the glucose and lipid metabolism disorders induced by continuous exposure to polystyrene nanoplastics for multiple generations.

[0058] Figure 3 The effect of mangiferin on ROS levels in Drosophila tissues (A) and hemolymph (B) after multiple generations of exposure to polystyrene nanoplastics was investigated. Data are expressed as mean ± standard deviation, N = 3. This indicates a highly significant difference between the two sample groups. P <0.001). By Figure 3As can be seen from A, after 5 generations of exposure to 100 mg / L polystyrene nanoplastics, the ROS level in fruit flies was 1.76 times that of the negative control group (Control). However, after treatment with 80 mg / L mangiferin, the ROS level in fruit flies decreased from 1.76 times that of the negative control group to 1.26 times.

[0059] The effects of mangiferin on ROS levels in the hemolymph of fruit flies were investigated, and the results are as follows: Figure 3 As shown in Figure B, compared with the negative control group (Control), after five consecutive generations of treatment with polystyrene nanoplastics, the intensity of green fluorescence in the field of view increased, and the number of fluorescent particles increased. This indicates that continuous exposure to polystyrene nanoplastics for multiple generations can induce the production of ROS in fruit flies. However, after treatment with polystyrene nanoplastics, the fluorescence intensity in the field of view decreased, and the number of fluorescent particles decreased. Therefore, mangiferin can effectively scavenge excess ROS in fruit flies, exerting an antioxidant effect and thus preventing oxidative damage to cells.

[0060] Figure 4 The effects of mangiferin on the activity of antioxidant enzymes (AB) and the content of MDA (C) in Drosophila subjected to multiple generations of exposure to polystyrene nanoplastics were investigated. Data are expressed as mean ± standard deviation, N = 3. This indicates a highly significant difference between the two sample groups. P <0.01), This indicates a highly significant difference between the two sample groups. P <0.001). By Figure 4 As shown in Figure A, compared with the untreated negative control group (Control), after five generations of exposure to 100 mg / L polystyrene nanoplastics, the SOD antioxidant enzyme activity in Drosophila increased by 1.04 times. Furthermore, after treatment with 80 mg / L mangiferin, the SOD antioxidant enzyme activity in Drosophila significantly increased to 1.11 times that of the negative control group (Control). P <0.01). By Figure 4 B shows that, compared with the untreated negative control group (Control), after 5 generations of exposure to 100 mg / L polystyrene nanoplastics, the CAT enzyme activity in Drosophila decreased by 21.06%, while after treatment with 80 mg / L mangiferin, the CAT enzyme activity in Drosophila increased significantly by 1.80 times compared with the polystyrene nanoplastics continuous 5-generation exposure group. P <0.001). By Figure 4As can be seen from C, compared with the untreated negative control group (Control), after 5 generations of exposure to 100 mg / L polystyrene nanoplastics, the MDA content in Drosophila increased by 1.26 times. However, after treatment with 80 mg / L mangiferin, the MDA content in Drosophila was significantly reduced by 7.15% compared with the group exposed to polystyrene nanoplastics for 5 consecutive generations. P <0.01), the above results indicate that continuous multi-generation exposure to polystyrene nanoplastics can increase the activity of SOD enzyme in Drosophila in response to oxidative stress, converting ROS free radicals into H2O2. However, continuous multi-generation exposure to polystyrene nanoplastics can also reduce the activity of intracellular CAT antioxidant enzyme, leading to H2O2 accumulation, disrupting the intracellular oxidation-antioxidant balance, and reducing antioxidant activity, thus causing damage to the Drosophila organism. The addition of mangiferin can enhance the activity of SOD and CAT enzymes, enabling timely scavenging of ROS free radicals and protecting the Drosophila organism from oxidative damage. Simultaneously, continuous multi-generation exposure to polystyrene nanoplastics causes redox imbalance in the Drosophila organism, resulting in a large accumulation of ROS, inducing lipid peroxidation, and producing a large amount of MDA, further aggravating oxidative damage. Mangiferin can effectively reduce cellular lipid peroxidation and decrease MDA content, providing protection against metabolic disorders and oxidative damage in Drosophila organisms induced by continuous multi-generation exposure to polystyrene nanoplastics.

[0061] Figure 5 Genes related to glucose and lipid metabolism in Drosophila suffering from multigenerational exposure to polystyrene nanoplastics and mangiferin. Dilp2 (A) Dilp3 (B) and Dilp5 (C) Effect of mRNA expression, data are expressed as mean ± standard deviation, N = 3. This indicates a significant difference between the two sample groups. P <0.05), This indicates a highly significant difference between the two sample groups. P <0.01). By Figure 5 It can be seen that, compared with the untreated negative control group (Control), after five generations of exposure to 100 mg / L polystyrene nanoplastics, the number of fruit flies in vivo decreased significantly. Dilp2 , Dilp3 and Dilp5 The relative expression levels of mRNA decreased by 90.07%, 50.69%, and 23.49%, respectively, while after treatment with 80 mg / L mangiferin, the expression levels in fruit flies decreased significantly. Dilp2 , Dilp3 and Dilp5 The relative expression level of mRNA was upregulated by 2.16% compared to the polystyrene nanoplastic continuous five-generation exposure group. P <0.05), 1.09 ( P <0.01) and 1.20 (P <0.05 times. The results indicate that mangiferin may alleviate metabolic damage induced by continuous multi-generational exposure to polystyrene nanoplastics by upregulating the expression of mRNAs of genes related to glucose and lipid metabolism.

[0062] This study investigated the novel application of mangiferin in intervening in metabolic disorders in Drosophila under multiple generations of polystyrene nanoplastic exposure. Using Drosophila melanogaster as a model organism, the effects of mangiferin on body weight, glucose content in hemolymph, triglyceride levels, ROS levels in vivo and in hemolymph, antioxidant enzyme activity, MDA levels, and expression levels of glucose and lipid metabolism-related genes in Drosophila under multiple generations of polystyrene nanoplastic exposure were determined. The results showed that 80 mg / L mangiferin effectively improved glucose and lipid metabolism disorders in Drosophila under multiple generations of polystyrene nanoplastic exposure, and this effect was related to anti-oxidative stress and regulation of glucose and lipid metabolism-related genes. Dilp2 , Dilp3 and Dilp5 It is related to mRNA expression.

[0063] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. The application of mangiferin in the preparation of drugs that improve glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The materials used included high-purity mangiferin standard reagent, conventional culture medium, polystyrene nanoplastic culture medium, mangiferin culture medium, and experimental fruit flies raised on the culture medium. Includes the following steps: S1. Experimental fruit flies were cultured and divided into three groups: negative control group, cultured in conventional culture medium without polystyrene nanoplastic exposure; positive control group, cultured in polystyrene nanoplastic culture medium with polystyrene nanoplastic exposure; and experimental group, cultured in polystyrene nanoplastic culture medium, and after polystyrene nanoplastic exposure, cultured in mangiferin culture medium. S2, determination of fruit fly body weight M; S3, determination of glucose and triglyceride content; S4, Qualitative and quantitative analysis of ROS in Drosophila hemolymph; S5, to determine SOD, CAT enzyme activity and MDA content; S6, expression of genes related to glucose and lipid metabolism.

2. The application of mangiferin according to claim 1 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: Step S0 is included before step S1; S0: Collect unmated male and female fruit flies within 8 hours of emergence from the experimental fruit flies after rearing, and separate them into conventional fruit fly culture medium. The fruit flies at this time are recorded as F0 generation. F0 generation does not contain polystyrene nanoplastics. Subsequently, fertilized eggs of fruit flies were selected and transferred to polystyrene nanoplastic experimental medium and standard conventional medium without polystyrene nanoplastic. The fruit flies at this time were recorded as F1 generation. After the F1 generation fruit flies emerged, they were immediately transferred to their respective polystyrene nanoplastic experimental culture media. After the F1 generation fruit flies laid eggs, the adult flies were discarded. The fruit flies in the culture media at this time were recorded as the F2 generation. The subsequent F3, F4 and F5 were cultured using the same method.

3. The application of mangiferin according to claim 2 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The specific measurement steps for step S3 above are as follows: Weigh 50 mg of third-instar fruit fly larvae, add 0.45 mL of physiological saline and homogenize mechanically. Then centrifuge at 3500 rpm for 10 min, collect the supernatant, and determine the glucose concentration using a glucose assay kit. Determine the triglyceride content using the GPO-PAP enzymatic method. Prepare a 10%–20% tissue homogenate of third-instar fruit fly larvae, centrifuge at 3000 rpm for 10 min, collect the supernatant, and determine the triglyceride content using a triglyceride assay kit. The triglyceride content in the sample is homogenized using protein concentration.

4. The application of mangiferin according to claim 3 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The specific measurement steps for step S4 above are as follows: Weigh 100 mg of Drosophila, wash with PBS, blot dry with filter paper, place in a 1.5 mL centrifuge tube, add 1 mL of PBS buffer, and homogenize thoroughly using a bead mill. Centrifuge the homogenate at 4000 rpm and 4℃ for 6 min. After centrifugation, add 190 μL of the homogenate supernatant diluted 5-10 times and 10 μL of O12 fluorescent probe to a black 96-well fluorescent plate. Incubate at 37℃ in the dark for 25 min, then place in a continuous wavelength multi-functional microplate detection platform for quantitative analysis using an excitation wavelength of 488 nm and an emission wavelength of 526 nm. Tissue ROS intensity is expressed as fluorescence intensity / protein concentration. The ROS in the hemolymph of third-instar larvae of Drosophila melanogaster were determined using the DCFH-DA fluorescent probe. The specific method is as follows: 100 third-instar larvae of Drosophila were taken, and under a stereomicroscope, the bodies of the larvae were punctured in a 1.5 mL centrifuge tube with dissecting forceps to release the hemolymph. The tubes were centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant was the hemolymph of the larvae. The hemolymph was incubated with 5 μmol / L DCFH-DA fluorescent probe at room temperature in the dark for 30 min. The ROS in the hemolymph of the larvae were qualitatively analyzed using an inverted fluorescence microscope.

5. The application of mangiferin according to claim 4 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The specific measurement steps for step S5 above are as follows: Weigh 50 mg of third-instar fruit fly larvae, add 1.35 mL of physiological saline and homogenize mechanically to prepare a 10%–20% tissue homogenate. After centrifugation at 3000 rpm for 10 min, collect the supernatant and measure the activities of SOD and CAT enzymes and the content of MDA. Each indicator was standardized by the absorbance of the sample protein. The protein concentration of each group of samples was determined according to the BCA protein quantification kit method.

6. The application of mangiferin according to claim 5 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The specific measurement steps for step S6 above are as follows: Third-instar larvae of Drosophila were collected, washed three times with PBS buffer, and then ground twice with 1 mL of trizol. The mixture was centrifuged at 12000 g and 4°C for 5 min, and the supernatant was transferred to a centrifuge tube. After incubating on ice for 5 min, 200 μL of chloroform was added, and the mixture was vortexed for 15 s. The mixture was then centrifuged at 12000 g and 4°C for 15 min, and the colorless aqueous phase containing total RNA was accurately transferred to another centrifuge tube. An equal volume of pre-chilled isopropanol was added, and the mixture was slowly inverted and mixed. After incubating on ice for 10 min, the mixture was centrifuged at 12000 g and 4°C for 10 min, and the supernatant was discarded to obtain the RNA precipitate. 1 mL of 75% ethanol was added to the precipitate, and the mixture was inverted and mixed. The mixture was centrifuged at 7500 g and 4°C for 5 min, and the supernatant was discarded. After the RNA had dried, 20–50 μL of ethanol was added to the precipitate. DEPC was dissolved in water; the concentration of extracted RNA was determined using a NanoDrop nucleic acid and protein quantification instrument, and the RNA was appropriately diluted according to the concentration. Finally, the RNA was reverse transcribed into cDNA using reverse transcriptase, and analyzed by real-time quantitative PCR. Rp49 , Dilp2 , Dilp3 , Dilp5 The mRNA expression status.

7. The use of mangiferin according to any one of claims 1-6 in the preparation of a drug having the effect of improving glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The initial feeding medium for the experimental fruit flies in the above steps was corn flour medium. The formula of corn flour medium was: 9.6 g corn flour, 9.6 g sucrose, 3.2 g yeast powder, 0.8 g agar powder, 0.2 g methylparaben, 0.8 mL propionic acid, 2 mL anhydrous ethanol, and 100 mL distilled water. The culture environment was: a constant temperature incubator with a temperature of 25±1℃, a relative humidity of 50%~60%, and a light cycle of 12L:12D.

8. The application of mangiferin according to claim 7 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The specific measurement steps for step S2 above are as follows: Weigh an empty 1.5 mL centrifuge tube using an analytical balance and record the weight as m1. Randomly place 10 third-instar fruit fly larvae into the centrifuge tube, wash off the surface food with PBS, blot dry with filter paper, and weigh again, recording the weight as m2. Repeat each experiment 6 times. The weight M of the fruit flies is calculated using the following formula: 。 9. The use of mangiferin according to claim 8 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The high-purity mangiferin standard reagent is a standard reagent with a purity of ≥98%.

10. The use of mangiferin according to claim 9 in the preparation of a drug that improves glucose and lipid metabolism disorders under multiple generations of exposure to polystyrene nanoplastics, characterized in that: The concentration of polystyrene nanoplastics in the culture medium was 100 mg / L, and the concentration of mangiferin was 80 mg / L.

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