Method for evaluating water using aquaporin and application thereof

Abstract

The application provides a method for evaluating water by using water channel proteins, and the water is evaluated by obtaining the absorption efficiency information of different water by cells expressing water channel proteins. The physiological function of water molecules and the cell absorption and utilization efficiency are scientifically evaluated, and the evaluation standard of healthy drinking water is established. By using the enrichment characteristics of different types of water channel proteins in tissues and organs, the detection means of individualized drinking water which is easy to cross the blood-brain barrier is established for special groups, and a new method and standard for individualized rehabilitation drinking water for special groups is established.

Description

Technical Field

[0001] This invention belongs to the fields of bioinformatics and functional genomics, and specifically relates to a method for evaluating water using aquaporins and its applications. Background Technology

[0002] Water is the source of life and the foundation of health; 80% of human tissue is composed of water. According to a survey report on drinking water and health by the United Nations Environment Programme and the World Health Organization, 80% of diseases and 33% of deaths worldwide are caused by drinking unsafe water. On March 15, 2022, the National Health Commission, the State Administration for Market Regulation, and the Standardization Administration of China jointly formulated and released the latest "Standards for Drinking Water Quality" (GB 5749-2022), which stipulates the requirements for drinking water quality, product hygiene requirements for drinking water safety, and water quality testing methods. The document sets clear limits for microbial, toxicological, radioactive, and disinfectant indicators, as well as sensory indicators in drinking water. Corresponding to each indicator, the current national standard "Test Methods for Drinking Water" (GB 5750) uses spectrophotometry, gas chromatography, and in vitro microbial culture and counting to test the limits for various indicators of sensory, physicochemical properties, and microbial content of water. These water quality testing methods are limited to testing the basic indicators of drinking water safety (toxicity), that is, as long as there are no toxic side effects within a certain period of time, it is considered qualified drinking water.

[0003] However, modern medicine has proposed the concept of healthy drinking water. In addition to focusing on the basic requirements of water quality such as microbiology, toxicology and physicochemical properties, it has also proposed higher standards for the physiological functions or activities of healthy drinking water, such as physiological indicators such as water permeability, solubility and metabolic capacity, whether water molecules are easily absorbed and utilized by human cells, whether they can pass smoothly through different tissues and organs (such as the blood-brain barrier), and the water quality indicators and characteristics of personalized drinking water for special populations. At present, there are no corresponding effective detection methods and means for these.

[0004] The 2003 Nobel Prize in Chemistry revealed the mechanism by which human cells "drink" or absorb and utilize water, discovering aquaporins (AQPs) that mediate or assist in the transport and distribution of water molecules in the body. Subsequently, it was found that aquaporins are widely distributed in almost all living organisms. There are 13 members of the human aquaporin family, mainly expressed in different tissues and organs such as the kidneys, lacrimal glands, nervous system, and brain tissue. For example, AQP4 is located in human brain tissue. They possess different transmembrane water permeability activities and play important roles in maintaining osmotic homeostasis, water transport across the blood-brain barrier, fluid secretion, nutrient metabolism, and metabolite excretion, among many other aspects. They are also molecular targets for many diseases.

[0005] The safety and health of drinking water are crucial to people's quality of life and disease control. "Water nutrition" and healthy, high-quality drinking water have become issues of social and market concern. my country's current "Standards for Drinking Water Quality" (GB 5749-2022) and "Standard Test Methods for Drinking Water" (GB 5750) set clear limits for microbial, toxicological, radioactive, and disinfectant indicators, as well as sensory evaluation of drinking water. The corresponding testing methods mainly rely on physicochemical property data analysis and in vitro microbial culture, which are limited to the safety aspects of drinking water. While products marketed as "healthy water" and "water quality from longevity areas" exist, current testing methods cannot accurately and scientifically assess and detect the physiological functions of water molecules and their absorption and utilization efficiency by cells. Summary of the Invention

[0006] To address the above technical problems, this invention proposes a biological method for detecting the physiological function and absorption and utilization efficiency of water molecules in living cells using aquaporins. Specifically, the technical solution of this invention is as follows:

[0007] A first aspect of this invention provides a method for evaluating water using aquaporins, which evaluates different types of water by obtaining information on the absorption efficiency of aquaporins for different types of water. Specifically, this invention evaluates different types of water by obtaining information on the absorption efficiency of cells expressing aquaporins for different types of water.

[0008] In this invention, "different waters" refers to waters with different conventional interpretations. Different waters include waters from different sources, such as different mineral waters, waters collected from different regions, waters of different brands, and waters obtained through different methods. Different waters also include waters with different compositions; different waters can be pure water or water solutions. Pure water includes conventionally distilled water, multi-distilled water, or water filtered through a reverse osmosis membrane. Pure water also includes unavoidable impurities. Water solutions contain some common salts or acids and bases, such as mineral water. Salts include silicates, carbonates, or salts of alkali metals or alkaline earth metals. Besides differences in source and composition, different waters also differ in the hydrogen bonds between water molecules and the aggregation patterns of water molecules.

[0009] To more clearly define "different," in this invention, "different," or "a certain kind," or "this kind," is distinguished based on marking. The marking is used to indicate whether a certain characteristic of multiple marked items is the same, and the marked items are identified as different because of differences in this characteristic. For example, based on the source of water as a characteristic, marking "different" indicates that the water comes from different sources.

[0010] In this invention, the different markings are also used to distinguish cells expressing aquaporins. For example, for different cells expressing different aquaporins, the aquaporin can be understood as a marker; the aquaporin marks the cells expressing that aquaporin. Cells expressing different aquaporins are thus considered different cells due to their different markings.

[0011] In this invention, the mark can be not only a mark of a single feature, but also a mark of a combination of features.

[0012] For the purposes of this invention, in order to express the effect of different labels on the water absorption capacity of aquaporins and to ensure the comparability of these effects, when the identified labels are different, other characteristics besides the label should be the same or substantially the same. For example, when the water label is the source of the water, different labels are considered to indicate different water sources. In this case, apart from the different water sources, other characteristics of the water samples should be the same or substantially the same. For example, the water temperature should be the same, and the experimental conditions in the experiments on the water absorption capacity of aquaporins should be the same.

[0013] In this invention, the absorption efficiency information includes the rate and amount of water absorption by aquaporins. Specifically, the absorption efficiency information includes the rate at which different types of water enter cells expressing aquaporins or the time taken for cells expressing aquaporins to burst in water.

[0014] The rate at which water enters cells expressing aquaporins can be expressed by obtaining the rate of expansion of the cell's outer diameter in water, for example by measuring the rate of change of the cell's outer diameter over time.

[0015] In this invention, the absorption efficiency information further includes a comparative relationship between the absorption efficiencies of aquaporins expressed by cells for different types of water, obtained by comparing the speed or time.

[0016] This invention, through specific embodiments, reveals differences in the absorption efficiency of cells expressing aquaporins to different types of water. More specifically, this manifests as the rate at which different types of water enter cells expressing aquaporins, the time it takes for these cells to burst in water, or the amount of water absorbed by these cells, particularly the amount absorbed before cell bursting. This invention, by comparing these rates or times, establishes a comparative relationship between the absorption efficiency of aquaporins expressed by cells to different types of water. This allows for the identification of which types of water are absorbed faster and better for a specific aquaporin, or which types of water are absorbed slower for a specific aquaporin, thus enabling the evaluation of water quality.

[0017] In this invention, the cells expressing aquaporins are further defined as cells expressing different types of aquaporins. In this case, the absorption efficiency information refers to the rate at which different types of water enter cells expressing different types of aquaporins or the time consumed by cells expressing different types of aquaporins to burst in different types of water. This invention further compares these rates or times to obtain a comparative relationship between the absorption capacity and absorption efficiency of different types of aquaporins for different types of water.

[0018] In this method, different types of aquaporins are further distinguished between cells. For example, cells expressing AQP1 and AQP4 are labeled. Through labeling, a comparative relationship is obtained between different types of aquaporins and their corresponding water absorption efficiencies. This relationship further identifies the water with the best or relatively good absorption efficiency and the water with the worst or relatively poor absorption efficiency for AQP1 and AQP4 water flux proteins, respectively.

[0019] In this method, the cells expressing aquaporins are cells that quantitatively express water flux proteins. In this invention, the cells expressing aquaporins are oocytes.

[0020] A second aspect of the present invention provides a method for evaluating water quality, which evaluates the water by obtaining information on the absorption efficiency of different types of aquaporins for a certain type of water. Specifically, the absorption information of different types of aquaporins for a certain type of water includes information on the absorption efficiency of cells expressing different aquaporins for a certain type of water.

[0021] In this invention, obtaining information on the absorption efficiency of cells expressing different types of aquaporins for a certain type of water can specifically involve obtaining information such as the water absorption rate, cell expansion rate, and water absorption volume of cells expressing different types of aquaporins for a certain type of water, and using the above information to reflect the absorption efficiency of different types of aquaporins for that type of water.

[0022] Furthermore, information correlations can be established between the absorption efficiencies of different waters in different aquaporins by comparison. For example, by comparing the absorption efficiencies of a certain type of water in different aquaporins, information can be obtained regarding whether the absorption rates of different aquaporins for a particular type of water are high or low.

[0023] In one application, because aquaporins accumulate to varying degrees in different tissues or organs, information on the absorption rate of a particular type of water for different aquaporins is valuable in medicine. Taking patients with cerebral edema or increased intracranial pressure as an example, during hydration, it's necessary to meet the water needs of various tissues and organs as much as possible, while simultaneously inhibiting excessive absorption of ingested water by the brain. Since AQP4 protein is enriched in the human brain, water with higher absorption efficiency from AQP4 protein needs to be excluded. Selecting water with lower AQP4 aquaporin absorption efficiency but higher absorption efficiency for aquaporins enriched in other parts of the body is the preferred approach for hydrating patients with cerebral edema or increased intracranial pressure. Therefore, water with a high absorption rate for a particular aquaporin can be used in hydration products or the preparation of hydration products for tissues or organs enriched with that aquaporin, or in the preparation of hydration products for patients with lesions in tissues or organs enriched with that aquaporin.

[0024] In another application example, it should be noted that when the types of water are limited, excluding certain types of water is also a method of water selection.

[0025] Water with low aquaporin absorption rate can also be used in the preparation of hydration products for tissues or organs rich in the aquaporin, or in the preparation of hydration products for patients with lesions in tissues or organs rich in the aquaporin.

[0026] The method described in this invention further includes comparing the obtained absorption efficiency information with a standard absorption efficiency.

[0027] In practical applications, a standard absorption efficiency value can be set. More specifically, this can be achieved by setting a standard value based on the rate of water absorption by cells expressing a specific aquaporin or the time taken for cells expressing different types of aquaporins to burst in different types of water. The measured values ​​are then compared with the standard value to evaluate or judge the water quality. For example, the standard value can be based on the absorption efficiency information obtained from water that meets national drinking water standards through the absorption of water by cells expressing different types of aquaporins. Alternatively, it can be based on water that meets a specific purpose, such as medical use, and the absorption efficiency information obtained from the absorption of water by cells expressing different types of aquaporins is used as the standard value.

[0028] A third aspect of the present invention is to provide a cell expressing aquaporin, which is obtained by genetic engineering techniques and has the ability to express aquaporin.

[0029] In this invention, the cell capable of expressing aquaporin is a cell capable of quantitatively expressing aquaporin. Specifically, the cell capable of expressing aquaporin is obtained by cloning aquaporin into an oocyte expression vector, synthesizing cRNA extracellularly, and further expressing the cRNA in cells. More specifically, the expression of cRNA in cells is quantitative.

[0030] In a specific embodiment of the present invention, the cell is an egg cell, particularly a non-human egg cell, and more particularly a non-human mammalian egg cell.

[0031] A fourth part of the invention is to provide a product for evaluating water, comprising cells expressing the water channel protein.

[0032] In this invention, the product for evaluating water may further include a sensor for obtaining data on the rate at which the cells absorb water, the rate at which the cells expand in size, or the time taken for the cells to burst.

[0033] Those skilled in the art can process the data obtained from the sensors and evaluate water quality using the method of this invention. The data processing implicitly includes using a computer or processing system containing a processor to process the data to obtain parameters such as cell water absorption rate, cell expansion rate, or the time taken for cells to burst.

[0034] The fifth part of this invention relates to the application of water obtained by screening aquaporins in the preparation of corresponding hydration products.

[0035] In this invention, the water obtained through aquaporin screening is the water screened using the method for evaluating water using aquaporins according to the present invention. More specifically, the screening includes selecting water based on a comparative relationship between the absorption efficiency of different waters by aquaporins expressed by cells.

[0036] In this invention, by utilizing aquaporins to evaluate water, a comparative relationship can be obtained regarding the absorption efficiency of aquaporins for different types of water. Furthermore, since the enrichment levels of different aquaporins vary in different tissues and organs, the relationship between the absorption efficiency of different waters and different aquaporins can be extended to the relationship between the water absorption efficiency of different waters in tissues and organs enriched with different aquaporins. In this invention, the preparation of corresponding hydration products, where "corresponding" means preparing hydration products for corresponding tissues or organs based on the relationship between the water absorption efficiency of different waters in tissues and organs enriched with different aquaporins. Here, hydration products for corresponding tissues or organs can be interpreted as hydrating agents specifically targeting a particular tissue or organ, or, for example, hydrating agents prepared to avoid overhydration of a specific tissue or organ, as in the aforementioned example of hydration for patients with cerebral edema or increased intracranial pressure.

[0037] For example, by using cells expressing the AQP4 protein to evaluate the quality of different types of water, the optimal absorption efficiency of a particular type of water for the AQP4 protein can be determined. This type of water can then be selected for preparing hydration agents for AQP4 protein-rich areas, such as those targeting the brain. Similarly, by using cells expressing the AQP1 protein to evaluate the quality of different types of water, the optimal absorption efficiency of a particular type of water for the AQP1 protein can be determined. This type of water can then be selected for preparing hydration agents for AQP1 protein-rich areas, such as those targeting the nervous system, kidneys, and blood vessels.

[0038] This invention uses genetic engineering techniques to clone aquaporins with different characteristics into oocyte expression vectors, synthesize cRNA in vitro, and express it in oocytes via microinjection. The bursting time of cells after incubation with different water molecules is then recorded. The time required for water molecules in different microbiochemical states to be transported into the cell by aquaporins is compared and analyzed to evaluate the physiological function and absorption efficiency of water molecules. This invention scientifically evaluates the physiological function and cellular absorption efficiency of water molecules, establishing a new standard for healthy drinking water quality from a novel perspective. Furthermore, this invention utilizes the characteristics of human AQP4 to establish a personalized drinking water quality testing method that easily crosses the blood-brain barrier for specific populations (such as those with cerebral infarction), also establishing a new method and standard for personalized rehabilitation drinking water for special groups. Attached Figure Description

[0039] Figure 1 Schematic diagram of water molecules entering cells mediated by human water channels AQP1 and AQP4;

[0040] Among them: a. Observation of oocytes expressing AQP1 and AQP4 absorbing water and swelling in ultrapure water (Scale bar = 0.5 mm); b. Statistical graph of oocyte swelling time during water absorption; c. Statistical graph of cell diameter increase during oocyte water absorption (Errorbars, sd, n = 10) (Two-tails with Student t-test).

[0041] Figure 2 Schematic diagram showing that oocytes expressing AQP1 exhibit different absorption efficiencies for different brands of drinking water;

[0042] Among them: a. Observation of the swelling of oocytes expressing AQP1 in different brands of drinking water (Scale bar = 0.5 mm); b. Statistical graph of oocyte swelling and bursting time during water absorption; c. Statistical graph of cell diameter increase during oocyte water absorption (Errorbars, sd, n = 10) (Two-tails with Student t-test).

[0043] Figure 3 Schematic diagram showing that oocytes expressing AQP4 exhibit different absorption efficiencies for different brands of drinking water;

[0044] Among them: a. Observation of the swelling of oocytes expressing AQP4 in different brands of drinking water (Scale bar = 0.5 mm); b. Statistical graph of oocyte swelling and bursting time during water absorption; c. Statistical graph of cell diameter increase during oocyte water absorption (Errorbars, sd, n = 10) (Two-tails with Student t-test). Detailed Implementation

[0045] Example 1: Molecular cloning of AQP1 and AQP4 and verification of their water channel function

[0046] The target genes AQP1 and AQP4 were cloned and constructed into the pGEMHE vector using a double enzyme digestion method. Positive colonies were selected by colony PCR and sequenced to obtain recombinant vectors with correct sequences. Related cRNAs were prepared using an in vitro transcription kit. Subsequently, the AQP1 and AQP4 cRNAs were microinjected into oocytes and incubated at 18°C ​​for 3 days. The oocytes were then transferred to ultrapure water, and the time required for oocyte rupture was recorded under a stereomicroscope.

[0047] See results Figure 1 As shown, specifically, in Figure 1In the -a model, oocytes expressing AQP1 showed no significant changes in the first 30 seconds, but a noticeable protrusion appeared around 40 seconds, indicating that AQP1 mediated water molecule entry into the cell, causing the oocyte to absorb water and burst. Oocytes expressing AQP4 showed no significant changes in the first 20 seconds, but a noticeable protrusion appeared around 30 seconds, indicating that AQP4 mediated water molecule entry into the cell, causing the oocyte to absorb water and burst. Multiple experiments showed that both AQP1 and AQP4 were successfully expressed in oocytes, possessing biological activity and mediating the rapid entry of water molecules into the oocyte. Further comparison of the time taken for AQP1 and AQP4 to mediate water molecule entry into the cell (Tables 1 and 2) can be found in [reference needed]. Figure 1 -b, c, obtain the efficiency data of different aquaporins for water molecule absorption and utilization, and select the ultrapure water to absorb the above differences for hydration of the corresponding protein-rich organs and preparation of hydration agents.

[0048] Table 1 shows the average cell rupture time for cells expressing aquaporins 1 and 4, respectively.

[0049]

[0050] Table 2 shows the increase in cell diameter (mean) for cells expressing aquaporins 1 and 4, respectively.

[0051]

[0052] Example 2: Analyzing the absorption and utilization efficiency of different brands of mineral water on the market using AQP1 in live cells.

[0053] See Figure 2 As shown, oocytes expressing AQP1 were transferred to distilled water of brand A (a type of drinking water prepared by distillation) and used as a control group instead of ultrapure water in subsequent experiments. Incubation with six commonly available drinking waters revealed that oocytes expressing AQP1 absorbed water and burst in approximately 30 seconds in brands C, D, and F, significantly earlier than those in brands B, E, G (mineral water), and brand A. Statistical analysis from multiple experiments (see Tables 3 and 4) further supports this finding. Figure 2 -b, c, C brand water, D brand water and F brand mineral water can quickly enter cells through the AQP1 water channel, with high absorption and utilization efficiency.

[0054] Table 3. Burst time (average) of cells expressing aquaporin 1 in different brands of drinking water.

[0055]

[0056] Table 4 shows the increase in cell diameter (mean) of cells expressing aquaporin 1 in different brands of drinking water.

[0057]

[0058] Example 3: Analyzing the permeability of different brands of drinking water on the market using AQP4 in live cells.

[0059] See Figure 3 As shown, oocytes expressing AQP4 were transferred to six different brands of drinking water in the above experiment for incubation and observation. It was found that oocytes in brand B and brand F water absorbed water and burst in about 40 seconds; in distilled water and brand G mineral water, they absorbed water and burst in about 60 seconds. The bursting time of oocytes in brand B and brand F mineral water was significantly earlier than in brand A distilled water; the bursting time in brand E, brand D, and brand C mineral water was in the middle, at about 50 seconds. Statistical analysis through multiple experiments (see Tables 5 and 6) shows that brand B and brand F mineral water allows AQP4 to enter cells more quickly through the aquaporin channel, while brand G mineral water allows for a slower entry rate.

[0060] Table 5. Burst time (average) of cells expressing aquaporin 4 in different brands of drinking water.

[0061]

[0062] Table 6 shows the increase in cell diameter (mean) of cells expressing aquaporin 4 in different brands of drinking water.

[0063]

[0064] Analysis of results from Examples 1, 2, and 3

[0065] Both AQP1 and AQP4 water channels are expressed in oocytes, mediating rapid water entry into the oocyte, and exhibit different permeabilities to water molecules. The expression locations of AQP1 (nervous system, kidneys, and blood vessels, etc.) and AQP4 (brain tissue) differ in human tissue cells, leading to variations in their absorption and utilization of mineral water with different compositions. Physiologically, this satisfies the different characteristics of tissues requiring rapid or slow water absorption, thereby maintaining fluid and cellular osmotic balance and metabolic homeostasis. Measuring the differences in transport activity of specific water channels in mineral water with different compositions not only allows for the establishment of a new drinking water evaluation system but also facilitates precise hydration of specific human cells, ensuring that people drink water that meets their individual needs and truly benefits their health. Through Examples 2 and 3, water types with high AQP1 and AQP4 absorption rates can be screened. Based on the enrichment of AQP1 in the nervous system, kidneys, and blood vessels, and AQP4 in brain tissue, and considering the differences in expression locations in human tissue cells, hydration agents targeting specific organs and tissues can be prepared.

[0066] This invention proposes a biological method for detecting water suitable for absorption by living organisms in living cells using human aquaporins, scientifically evaluating the physiological functions of water molecules and the efficiency of cellular absorption and utilization, and establishing a novel approach to the detection and analysis of healthy water.

[0067] In the example, the technical solution of the present invention was used to test mineral water of different brands. The results showed that mineral water of different brands exhibited different physiological functions of water molecules. The method successfully detected drinking water that is conducive to cell absorption and utilization at the physiological and cellular levels, providing a scientific means of testing healthy water quality.

Claims

1. A method for evaluating water using aquaporins, characterized in that, By obtaining information on the absorption efficiency of aquaporins for different types of water, and using this information to evaluate the water quality, the aquaporins are either AQP1 or AQP4 aquaporins.

2. The method for evaluating water using aquaporins according to claim 1, characterized in that, The absorption efficiency information includes the rate at which different amounts of water enter cells expressing aquaporins, the time it takes for cells expressing aquaporins to burst in water, and / or the amount of water absorbed by cells expressing aquaporins before they burst.

3. The method for evaluating water using aquaporins according to claim 2, characterized in that, The absorption efficiency information further includes a comparative relationship between the absorption efficiency of aquaporins expressed by cells for different types of water, obtained by comparing the amount of water absorbed based on the speed, time, or amount absorbed until the burst.

4. The method for evaluating water using aquaporins according to claim 2, characterized in that, The cells expressing aquaporins are different types of aquaporins expressed in different cells.

5. The method for evaluating water using aquaporins according to claim 4, characterized in that, The absorption efficiency information refers to the rate at which different types of water enter cells expressing different types of aquaporins, the time consumed by cells expressing different types of aquaporins to burst in different types of water, and / or the amount of water absorbed by cells expressing aquaporins before they burst.

6. The method for evaluating water using aquaporins according to claim 5, characterized in that, The absorption efficiency information further includes a comparative relationship between different types of aquaporins and their water absorption efficiencies, obtained by comparing the speed or time.

7. The method for evaluating water using aquaporins according to claim 2, characterized in that, The cells expressing aquaporins are cells that quantitatively express water flux proteins.

8. The method for evaluating water using aquaporins according to claim 2, characterized in that, The cells expressing the aquaporin are oocytes.

9. A method for evaluating water quality, characterized in that, The water quality is evaluated by obtaining information on the absorption efficiency of a certain type of water expressed by different types of aquaporins, wherein the aquaporins are AQP1 or AQP4 aquaporins.

10. A method for evaluating water quality, characterized in that, By obtaining information on the absorption efficiency of a certain aquaporin for a certain type of water, the obtained absorption efficiency information is compared with the standard absorption efficiency. The aquaporin is either AQP1 or AQP4 aquaporin.

11. An application of water obtained through aquaporin screening in the preparation of corresponding hydration products, characterized in that, The water obtained by the aquaporin screening is the water obtained by the method for evaluating water quality using aquaporins according to any one of claims 1-8.

12. The application of water obtained by screening aquaporins according to claim 11 in the preparation of corresponding hydration products, characterized in that, The screening process includes selecting water based on a comparison of the absorption efficiency of different waters by aquaporins expressed by cells, and the hydration product is a hydration product corresponding to the tissues or organs where the aquaporins are abundant.

13. The application of water obtained by screening aquaporins according to claim 11 in the preparation of corresponding hydration products, characterized in that, The aquaporin sieve is AQP1 aquaporin, and the aquaporin is enriched in the nervous system, kidneys, and blood vessels.

14. The application of water obtained by screening aquaporins according to claim 11 in the preparation of corresponding hydration products, characterized in that, The aquaporin sieve is AQP4 aquaporin, and the corresponding aquaporin-rich tissue or organ is brain tissue.

Images