Evaluation method using zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate

Abstract

The application discloses an evaluation method of a zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate (BBP) environment, a verification method for rapidly evaluating the change of zebrafish neurobehavior caused by butyl benzyl phthalate by using a series of behavior tests, and further explores the application potential of the method in constructing a zebrafish anxiety neurotoxicity model. The application uses neurobehavioral science to measure the changes of motion parameters such as cognitive behavior, social behavior and exploration behavior of aquatic organisms zebrafish, to find out whether the existence of BBP in the aquatic environment constitutes a neurotoxicity risk to aquatic organisms zebrafish. The application studies the influence difference of different concentrations of BBP on the neurobehavior of zebrafish, and therefore introduces a zebrafish neurodegenerative disease behavior model to construct a reliable related experiment model for the research of nervous system diseases, and to evaluate the risk of BBP to aquatic organisms, thereby providing a reference for toxicity evaluation.

Description

Technical Field

[0001] This invention belongs to the field of environmental neuroscience technology, specifically relating to an evaluation method for a zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate. Background Technology

[0002] Endocrine disruptors (EDCs) are widespread in the environment, interfering with the synthesis, release, transport, and metabolism of normal endocrine substances, thereby affecting stability and regulatory capacity, and posing a serious threat to ecosystems and human health. Phthalate is one of the most common EDCs, widely used in industrial chemicals such as plasticizers, solvents, lubricants, and stabilizers, as well as commercial products such as personal care products, food packaging, medical devices, pharmaceuticals, building materials, and household goods. Phthalate is not chemically bonded to these products and is easily released into the environment through exposure to air, soil, and water. This means that humans inevitably come into contact with phthalates and their derivatives through ingestion of food and water, inhalation of polluted air, skin contact, and gastrointestinal contact with phthalate-containing plastic products, medical devices, etc., and are thus absorbed by the body. Phthalate exposure can lead to negative health effects, including reproductive toxicity, teratogenicity, cardiotoxicity, type II diabetes, obesity, asthma, and cancer. Phthalate and its metabolites have also been found in the urine of pregnant women, amniotic fluid, breast milk, umbilical cord blood of newborns, and urine of newborns. Fetuses and adolescents are more susceptible to the environmental risk factors of phthalates than adults. Phthalate esters and their metabolites can have negative effects on development, particularly developmental toxicities, which are of increasing concern to embryos, fetuses, newborns, infants, and pre-adolescents. Butylbenzyl phthalate (BBP) is one of the most widely used phthalates, ubiquitous in the environment, and has become a major global pollutant, seriously threatening human health.

[0003] Studies have shown that zebrafish are highly similar to mammals and are very sensitive to changes in pharmacological environment. Currently, the toxicity evaluation of zebrafish derivatives mainly relies on reproductive and developmental toxicity studies. BBP causes developmental toxicity in zebrafish embryos, and these behavioral manifestations can be detected in both larvae and adults. However, studies on neurotoxic behavior evaluation are relatively few. In addition, there is a clear correlation between zebrafish neuroanatomical and physiological characteristics and behavior. Given the potential harm of BBP to the central nervous system (CNS), we found that BBP can induce neurodegenerative disease-like behavior in adult zebrafish, affecting higher cognitive behaviors. Through comprehensive and detailed analysis of numerical and spatial changes in motor behavior, we induced neurodegenerative disease behavior in zebrafish and explored the effects of different concentrations of BBP on zebrafish neurobehavioral behavior. Our innovative research goal is to use neurobehavioral science to measure changes in cognitive, behavioral, and motor parameters in aquatic organisms (zebrafish) to determine whether the presence of BBP in the aquatic environment poses a neurotoxic risk to aquatic organisms (zebrafish). Specifically, this study investigated the differences in the effects of different concentrations of BBP on the neurobehavioral behavior of zebrafish. Therefore, a behavioral model of zebrafish neurodegenerative diseases was introduced to construct a reliable experimental model for the study of nervous system diseases, assess the risk of BBP to aquatic organisms, and provide a reference for toxicity assessment. Summary of the Invention

[0004] The purpose of this invention is to provide an evaluation method for a zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate (BBP). This method evaluates the biotoxicity and preliminary activity screening caused by BBP through a series of behavioral characteristic changes, and elucidates the neurotoxic effects related to BBP.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: an evaluation method for a zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate, characterized by the following specific steps: Step S1: Select 64 healthy, wild-type adult zebrafish of the same batch with equal male and female ratios and raise them in a breeding tank. Maintain the circulating water temperature at 28.5℃ and the stocking density at 5 fish per liter of water. Use a light cycle of 14 hours of light and 10 hours of darkness. Turn on the lights at 07:00 and turn them off at 21:00. Keep the water circulating in the zebrafish breeding system. Feed the fish twice a day with fresh brine shrimp eggs, ensuring that the fish in the tank finish feeding within 3-5 minutes. In step S2, zebrafish were randomly assigned to four groups and transferred to 2 L aquariums. Each group contained 16 zebrafish. Three concentrations of butyl benzyl phthalate (BBP) were set up as experimental groups: 0.02 mg / L, 0.1 mg / L, and 0.2 mg / L. A blank control group was also set up. In the three experimental groups, the zebrafish were placed in containers containing the corresponding concentrations of BBP solution. The blank control group was raised in standard aquaculture water as a reference. To ensure good water quality and the accuracy of the BBP solution concentration, the water in each sample tank was changed daily and replaced with BBP solution of the same concentration as before. After two weeks of continuous treatment, behavioral phenotype assessment experiments were conducted on all zebrafish. Step S3: Test and evaluate the behavioral phenotypes of zebrafish. After assembling the light / dark box experimental device tank, the new diving experimental device tank, the group interaction experimental device tank, and the open mine experimental device tank, place them in the experimental area. Debug and assemble the video data acquisition camera. According to the different behavioral experimental requirements, place the camera directly above or to one side of the experimental device tank. Adjust the background of the experimental area and the distance between the camera and the camera to achieve the best experimental effect. Add sufficient circulating water from the breeding system to the experimental device tank and ensure that the water temperature of the zebrafish is consistent before and after the experiment. Each time, use a fishnet to move a single zebrafish to the experimental device tank. After swimming freely for 2 minutes to adapt to the experimental device tank, record the video for 3 minutes. Record each zebrafish in all groups individually. After recording, return the zebrafish to the corresponding concentration breeding tank. Then, perform statistical analysis on the behavioral trajectory and movement parameters of each group of zebrafish. Step S4: Import the video recordings of zebrafish behavior, such as movement time, movement distance, latency, rotation angle, slow movement time, and stationary period data, into Excel 2016. Compare the experimental group data with the control group data. Use GraphPadPrism 9.5.1 software for unpaired t-test and 2-way ANOVA analysis to generate graphs. Results are expressed as mean ± standard error. Significance is determined by... * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0006] This invention offers the following advantages and benefits: It relates to the effects of long-term exposure to phthalate derivatives (BBPs), commonly used plasticizers in industrial production, on the neurobehavioral behavior of zebrafish. It utilizes a series of behavioral tests to rapidly evaluate the ability of butyl benzyl phthalate (BBP) to alter zebrafish neurobehavioral behavior and further explores its potential application in constructing a zebrafish anxiety neurotoxicity model. This invention leverages neurobehavioral science to determine whether the presence of BBPs in the aquatic environment poses a neurotoxic risk to zebrafish by measuring changes in cognitive, behavioral, and motor parameters. By studying the differences in the effects of different concentrations of BBPs on zebrafish neurobehavioral behavior, a zebrafish neurodegenerative disease behavioral model is introduced. This leads to the construction of a reliable experimental model for the study of neurological diseases, assessing the risk of BBPs to aquatic organisms, and providing a reference for toxicity assessment. Attached Figure Description

[0007] Figure 1 Figure A shows the swimming time heatmap of each group of zebrafish in the test tank at the mine, and Figure B shows the data of the time zebrafish spent in the Inner and Outer regions.

[0008] Figure 2 Figure A shows various representative movement trajectories, while Figure B shows the percentage of movement distance and dwell time of zebrafish in the near and far regions of the test chamber.

[0009] Figure 3 Figure A shows the movement time heatmap of representative zebrafish in each group in the light and dark test chamber, and Figure B shows the data of zebrafish staying in the light and dark areas.

[0010] Figure 4 Figure A shows the heatmap of the movement time of representative zebrafish in the new type of underwater test tank. Figure B shows the data of relevant movement parameters of zebrafish, such as the time spent in the top and bottom areas, the distance traveled, the average time spent in the top area, and the time spent in slow movement. Detailed Implementation

[0011] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Example 1

[0012] Open mine experimental exploration test (1) Sixty-four healthy adult zebrafish (4-5 months old, male and female in equal proportion) from the same batch were selected and randomly divided into four groups of tanks, with 16 fish in each group. Three groups of zebrafish were cultured with three different concentrations of BBP solution: 0.02 mg / L, 0.1 mg / L, and 0.2 mg / L. The solutions were prepared using water from the culture system, while the control group was cultured using the system water. To ensure good water quality and the accuracy of the BBP solution concentration, the water in each sample tank was changed daily with BBP solution of the same concentration. After two weeks of continuous treatment, behavioral tests were conducted on all zebrafish.

[0013] (2) The mine experiment is a classic test method for evaluating anxiety and depression in rodents. We used the exploratory behavior of zebrafish in an open mine experiment to verify the effect of long-term exposure to BBP on the anxiety and depression behavior of zebrafish. A circular open area exploration device was constructed. The circular transparent breeding tank with a radius of 10 cm and a height of 10 cm was filled with circulating water from the breeding system to about 2 / 3 of the tank height to ensure that the water temperature was consistent before and after the zebrafish were introduced, thus eliminating the influence of temperature.

[0014] (3) A video data acquisition camera is fixed directly above the device to observe and record the two-dimensional planar movement trajectory of the zebras. The area with a radius of 5 cm from the center of the tank bottom is designated as the inner zone, and the remaining area is designated as the outer zone. The zebrafish are transferred into the round tank using a net. In the test tank, each zebrafish needs to adapt to the environment for 2 minutes. Subsequently, each zebrafish is recorded for 3 minutes. After recording, the zebrafish are carefully transferred back to their original rearing tank. This process is repeated for all zebrafish to ensure the comprehensiveness and consistency of the experiment.

[0015] (4) The movement data was imported into Excel 2016, and the time spent by zebrafish in the Inner and Outer regions was statistically analyzed using GraphPad Prism 9.5.1 software. The heatmap of the swimming time of each group of zebrafish in the test tank is shown below. Figure 1 As shown in Figure A, BBP exposure can alter the exploratory behavior of zebrafish in a mining test tank. Specific experimental results indicate that, in zebrafish with long-term BBP exposure, the exploration time in the inner region decreases with increasing BBP concentration, exhibiting a dose-dependent effect. Figure 2As shown in Figure B, the BBP 0.1 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.05), and the BBP 0.2 mg / L experimental group also showed a statistically significant difference compared to the control group (P < 0.05), indicating that BBP can reduce the exploration time of zebrafish in the inner area of ​​the open mine experiment. However, the BBP-exposed zebrafish showed an increased exploration time in the outer area, exhibiting a concentration-dependent effect. The BBP 0.1 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.05), and the BBP 0.2 mg / L experimental group also showed a statistically significant difference compared to the control group (P < 0.01). These results indicate that long-term exposure to BBP can alter the exploration behavior of zebrafish in the open mine experiment and increase the exploration time in the outer area. Studies have reported that in open mine experiments, anxious and depressed mice in the mine test chamber showed increased exploration behavior in the outer edge area. Therefore, the open mine experiment results show that BBP exposure can also induce anxious and depressed behavior in zebrafish. Example 2

[0016] Zebrafish social behavior test (1) Sixty-four healthy wild-type adult zebrafish (4-5 months old, male and female in equal proportion) from the same batch were randomly divided into four groups of tanks, with 16 zebrafish in each group. Three groups of zebrafish were cultured with three different concentrations of BBP solution: 0.02 mg / L, 0.1 mg / L, and 0.2 mg / L. The solutions were prepared using water from the culture system, while the control group was cultured using the system water. To ensure good water quality and the accuracy of the BBP solution concentration, the water in each sample tank was changed daily with BBP solution of the same concentration. After two weeks of continuous treatment, behavioral tests were conducted on all zebrafish.

[0017] (2) Zebrafish are highly social animals and exhibit gregarious behavior when swimming. Utilizing this natural characteristic of zebrafish, a gregarious interaction device test tank was assembled. The tank is 30 cm long, 10 cm wide, and 10 cm high, made of 0.5 mm thick food-grade transparent acrylic sheet. The tank is divided into a test chamber (20 cm × 10 cm × 10 cm) and a zebrafish school chamber (10 cm × 10 cm × 10 cm) by a transparent partition at 1 / 3 of the tank length. The test chamber is further divided into two areas: a proximal area (close to the school chamber) and a distal area, both measuring 10 cm × 10 cm × 10 cm (length × width × height), used to test the effect of long-term exposure to the BBP environment on the gregarious behavior of zebrafish.

[0018] (3) A video data acquisition camera is fixed directly above the device to observe and record the two-dimensional planar movement trajectory of the zebra. Seven to eight zebrafish are placed in the fish enclosure, and one test zebrafish is placed in the test chamber at a time. The end of the test chamber closest to the fish enclosure is marked as the near end, and the other end is marked as the far end. The zebrafish are moved into the test tank using a net. After the zebrafish have adapted to the tank for 2 minutes, a 3-minute video recording is made of each zebrafish. After recording, the zebrafish are returned to the rearing tank. The above experiment is repeated for all zebrafish.

[0019] (4) Import the movement data into Excel 2016, and use GraphPad Prism 9.5.1 software to statistically analyze the proportion of movement distance and dwell time of zebrafish in the proximal and distal regions of the test chamber. Various representative movement trajectories are shown below. Figure 2 As shown in Figure A, the movement trajectories of zebrafish in the BBP-exposed group were significantly altered compared to the normal group, with increased exploratory behavior at distal points. In the proximal region, as BBP concentration increased, the swimming distance and time of zebrafish in the proximal region decreased, exhibiting a concentration-dependent relationship. Figure 2 As shown in Figure B, and with statistically significant differences, compared with the control group, the BBP 0.1 mg / L experimental group showed a significant difference (P < 0.05); the BBP 0.2 mg / L experimental group also showed a significant difference (P < 0.05). In the distal region, with increasing BBP concentration, the swimming distance and time of zebrafish in the distal region increased, exhibiting a concentration gradient dependence, and with statistically significant differences. The BBP 0.1 mg / L experimental group showed a significant difference compared with the control group (P < 0.05); the BBP 0.2 mg / L experimental group showed a significant difference compared with the control group (P < 0.01). These test results indicate that BBP can alter the gregarious characteristics of zebrafish, leading to isolated and withdrawn behaviors. These experimental results also suggest that BBP can induce anxiety and depression in zebrafish. Example 3

[0020] Light and dark box preference test (1) Sixty-four healthy adult zebrafish (4-5 months old, male and female in equal proportion) from the same batch were selected and randomly divided into four groups of tanks, with 16 zebrafish in each tank. Three different concentrations of BBP solution (0.02 mg / L, 0.1 mg / L, and 0.2 mg / L) were used to raise zebrafish in three groups respectively. The solutions were prepared using water from the culture system, while the control group was raised using the system water. To ensure good water quality and the accuracy of the BBP solution concentration, the water in each sample tank was changed daily with BBP solution of the same concentration. After two weeks of continuous treatment, behavioral tests were conducted on all zebrafish.

[0021] (2) Utilizing the animal’s tendency to seek light and avoid darkness, assemble a transparent food-grade acrylic test tank with a length of 20 cm, a width of 10 cm, and a height of 10 cm. The test tank is covered with black opaque wallpaper to create a dark environment, while the other half allows natural light to pass through to create a bright environment. Fill the tank with about 2 / 3 of the height of the aquaculture circulation system water.

[0022] (3) The video data acquisition camera is fixed directly above the device to observe and record the two-dimensional planar movement trajectory of the zebra. The zebrafish are moved into the test tank with a net and allowed to adapt in the tank for 2 minutes. The behavior trajectory of each zebrafish is recorded for 3 minutes. After recording, the zebrafish are moved back to the breeding tank. The above experiment is repeated for all zebrafish.

[0023] (4) The test results of the light and dark chamber experiment are as follows: Figure 3 As shown in sections A and B, Figure 3 Figure A shows the heatmap of the movement time of representative zebrafish in the light and dark test chambers for each group. It can be seen that BBP exposure significantly alters the exploratory behavior of zebrafish in both light and dark chambers. The movement parameter data were imported into Excel 2016, and GraphPad Prism 9.5.1 software was used for statistical analysis of the zebrafish's residence time in the light and dark areas. In the light area, as the BBP concentration increased, the swimming time and distance of the zebrafish decreased. The BBP 0.02 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.0001); the BBP 0.1 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.0001); and the BBP 0.2 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.05). Within the dark zone, as the BBP concentration increased, the swimming time and distance of zebrafish in the dark zone increased with concentration, exhibiting a certain concentration gradient dependence. The BBP 0.02 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.0001); the BBP 0.1 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.0001); and the BBP 0.2 mg / L experimental group showed a statistically significant difference compared to the control group (P < 0.05). Compared to the control group, all three BBP concentration groups affected the zebrafish's residence time in the light / dark zones to varying degrees, and also affected their preference behavior between light and dark zones to varying degrees. The light / dark chamber test results indicate that long-term exposure to a BBP environment can alter the light / dark preference of zebrafish and induce anxiety and depression behaviors. Example 4

[0024] New type of diving test (1) Sixty-four healthy adult zebrafish (4-5 months old, male and female in equal proportion) from the same batch were selected and randomly divided into four groups of tanks, with 16 zebrafish in each tank. Three different concentrations of BBP solution (0.02 mg / L, 0.1 mg / L, and 0.2 mg / L) were used to raise zebrafish in three groups respectively. The solutions were prepared using water from the culture system, while the control group was raised using the system water. To ensure good water quality and the accuracy of the BBP solution concentration, the water in each sample tank was changed daily with BBP solution of the same concentration. After two weeks of continuous treatment, behavioral tests were conducted on all zebrafish.

[0025] (2) Based on the exploratory behavior of zebrafish in a new environment, zebrafish tend to stay closer to the bottom of the tank when they feel anxious. Therefore, a novel diving experiment was set up to assess the degree of anxiety-induced behavior in zebrafish. Based on this principle, we designed a novel diving device to assess the degree of anxiety-induced behavior in zebrafish of different BBP concentration groups. An inverted trapezoidal diving tank was constructed, with a height of 15.2 cm, a top of 27.9 cm, a bottom of 22.5 cm, and a width of 7.1 cm. It was vertically divided into an upper top area and a lower bottom area. Circulating water from the aquaculture system was added to ensure that the temperature of the zebrafish was consistent before and after the experiment, thus eliminating the influence of temperature.

[0026] (3) The video data acquisition camera was fixed 50 cm outside the same horizontal line as the water tank to observe and record the two-dimensional vertical movement trajectory of the zebra. Each time, a single zebrafish to be tested was moved into the test tank with a fishnet, and the behavior trajectory of the zebrafish was immediately recorded for 3 minutes. After recording, the zebrafish was moved back to the breeding tank. The above experiment was repeated for all zebrafish.

[0027] (4) Results of new diving tests, such as Figure 4 China A and Figure 4 As shown in B, through Figure 4 In study A, it was found that the zebrafish in the BBP-exposed group spent significantly less time exploring the top of the head. Specific motion parameters are as follows: Figure 4As shown in Figure B, the movement data were imported into Excel 2016, and GraphPad Prism 9.5.1 software was used to statistically analyze the zebrafish's dwell time, movement distance, average dwell time, and slow movement time in the top and bottom areas. In the top area, as the BBP concentration increased, the zebrafish's exploration time, movement distance, average dwell time, and slow movement time showed a decreasing trend in the line graph, exhibiting a certain dose-dependent relationship. In the bottom area, as the BBP concentration increased, the zebrafish's exploration time, movement distance, average dwell time, and slow movement time showed an increasing trend in the line graph. Based on the zebrafish's tendency to stay closer to the bottom of the tank in new environments and when feeling anxious, the effects of different BBP concentrations on the zebrafish's anxiety-related neurobehavior were assessed. The test results indicate that BBP can alter the movement parameters of zebrafish in a novel diving test experiment, suggesting that BBP can induce anxiety and depression behaviors in zebrafish.

[0028] In summary, this invention constructs an evaluation system for a zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate (BBP), providing an in-depth analysis of the potential impact of BBP on the neurobehavioral behavior of zebrafish. This achievement not only provides a solid basis for scientifically assessing the ecological risks and neurotoxicity of BBP and other environmental pollutants to aquatic organisms, but also establishes a neurotoxic anxiety animal model by utilizing the neuro-anxiety behavior exhibited by zebrafish under long-term BBP exposure. This model holds promise for further applications in the preliminary assessment of neuro-anxiety toxicity, high-throughput screening of anti-anxiety drug activity, and research on drug toxicity effects.

[0029] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. An evaluation method for a zebrafish anxiety model induced by long-term exposure to butyl benzyl phthalate, characterized in that... The specific steps are as follows: Step S1: Select 64 healthy, wild-type adult zebrafish of the same batch with equal male and female ratios and raise them in a breeding tank. Maintain the circulating water temperature at 28.5℃, with a stocking density of 5 fish per liter of water. Use a light cycle of 14 hours of light and 10 hours of darkness. Turn on the lights at 07:00 and turn them off at 21:

00. Keep the zebrafish breeding system circulating water. Feed them fresh brine shrimp eggs twice a day. The fish in the tank finish feeding in 3-5 minutes. In step S2, zebrafish were randomly assigned to four groups and transferred to 2 L aquariums. Each group contained 16 zebrafish. Three concentrations of butyl benzyl phthalate (BBP) were set up as experimental groups: 0.02 mg / L, 0.1 mg / L, and 0.2 mg / L. A blank control group was also set up. In the three experimental groups, the zebrafish were placed in containers containing the corresponding concentrations of BBP solution. The blank control group was raised in standard aquaculture water as a reference. To ensure good water quality and the accuracy of the BBP solution concentration, the water in each sample tank was changed daily and replaced with BBP solution of the same concentration as before. After two weeks of continuous treatment, behavioral phenotype assessment experiments were conducted on all zebrafish. Step S3: Test and evaluate the behavioral phenotypes of zebrafish. After assembling the light / dark box experimental device tank, the new diving experimental device tank, the group interaction experimental device tank, and the open mine experimental device tank, place them in the experimental area. Debug and assemble the video data acquisition camera. According to the different behavioral experimental requirements, place the camera directly above or to one side of the experimental device tank. Adjust the background of the experimental area and the distance between the camera and the camera to achieve the best experimental effect. Add sufficient circulating water from the breeding system to the experimental device tank and ensure that the water temperature of the zebrafish is consistent before and after the experiment. Each time, use a fishnet to move a single zebrafish to the experimental device tank. After swimming freely for 2 minutes to adapt to the experimental device tank, record the video for 3 minutes. Record each zebrafish in all groups individually. After recording, return the zebrafish to the corresponding concentration breeding tank. Then, perform statistical analysis on the behavioral trajectory and movement parameters of each group of zebrafish. Step S4: Import the video recordings of zebrafish behavior, such as movement time, movement distance, latency, rotation angle, slow movement time, and stationary period data, into Excel 2016. Compare the experimental group data with the control group data, and use GraphPadPrism 9.5.1 software for unpaired t-test and 2-way ANOVA analysis to generate graphs. Results are expressed as mean ± standard error, and significance is determined. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Images