Method for relieving intestinal barrier injury caused by fluorine overexposure by tea polyphenol

Abstract

The invention provides a method for relieving intestinal barrier injury caused by fluorine overexposure by using tea polyphenol. The method for relieving intestinal barrier injury caused by fluorine overexposure by using tea polyphenol comprises the following steps: carrying out an in-vitro test on porcine jejunum epithelial cells (IPEC-J2 cells) and establishing a NaF induction model by using the IPEC-J2 cells, and specifically comprises the following steps: S1, preparing test materials: the IPEC-J2 cells, a DMEM culture medium, a sterile phosphate buffer solution, fetal calf serum, mycillin and a cell freezing medium; s2, carrying out cell resuscitation, and establishing a NaF-induced cell model. The method for relieving intestinal barrier injury caused by fluorine overexposure by tea polyphenol has the advantages that the integrity of the intestinal barrier is increased and the intestinal microecology is repaired by increasing the activity of antioxidant enzyme, reducing the permeability of the intestinal tract, reducing the production of reactive oxygen species (ROS) and relieving oxidative stress and intestinal cell apoptosis caused by NaF.

Description

Technical Field

[0001] The invention belongs to the technical field of tea polyphenols alleviating fluoride stress, and in particular relates to a method for tea polyphenols to alleviate intestinal barrier damage caused by fluoride overexposure. Background Art

[0002] Humans and animals can be exposed to fluoride through drinking water, air, and food supplements. However, long-term excessive intake of fluoride can cause fluorosis, which interferes with oxygen metabolism, leads to an increase in oxygen free radicals, and causes significant oxidative damage to bones, teeth, gastrointestinal tract, etc. Many studies have shown that excessive fluoride can damage the antioxidant defense system, promote the production of reactive oxygen species (ROS) in the body, and reduce the body's antioxidant capacity. However, there is still no effective non-oxidative treatment for fluorosis, which has become a global public health issue in many countries including China.

[0003] It is well known that the use of antioxidants can be used as a therapeutic strategy to restore the normal redox state of the intestine and maintain intestinal health. Tea polyphenols, as the main active ingredient in tea, mainly remove reactive oxygen and inhibit DNA oxidative damage by increasing the activity of endogenous antioxidant enzymes. In addition, tea polyphenols are also an effective stress mediator that can inhibit some heavy metal-induced oxidative stress and damage. Fluoride is absorbed by the gastrointestinal tract and transported to various tissues of the body. As the organ that is mainly exposed to nutrients and toxic food contaminants, the intestine has a poor defense ability to resist intestinal damage and even lesions caused by long-term excessive fluoride exposure. Therefore, the present invention aims to explore the effect of tea polyphenols on alleviating intestinal barrier damage caused by fluoride exposure under fluoride-induced conditions.

[0004] Therefore, it is necessary to provide a new method for tea polyphenols to alleviate intestinal barrier damage caused by excessive fluoride exposure to solve the above technical problems. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for alleviating intestinal barrier damage caused by excessive exposure to fluoride by increasing the activity of antioxidant enzymes, reducing intestinal permeability, reducing the generation of reactive oxygen species (ROS), alleviating oxidative stress and intestinal cell apoptosis caused by NaF, thereby increasing the integrity of the intestinal barrier and repairing the intestinal microecology of tea polyphenols.

[0006] In order to solve the above technical problems, the method provided by the present invention for tea polyphenols to alleviate intestinal barrier damage caused by excessive exposure to fluoride includes: conducting in vitro experiments with porcine jejunal epithelial cells (IPEC-J2 cells) to establish a NaF-induced model, and the specific method is as follows:

[0007] S1. Experimental materials: IPEC-J2 cells, DMEM medium, sterile phosphate buffered saline, fetal bovine serum, penicillin-streptomycin and cell freezing solution;

[0008] S2. Cell recovery. The method for establishing the NaF-induced cell model is as follows:

[0009] (1) Take the cryovial out of liquid nitrogen, place it in a -80℃ refrigerator overnight, and then place it in a 37℃ water bath for 1 min to dissolve;

[0010] (2) The cell suspension was aspirated and placed in a 15 ml centrifuge tube, and then an appropriate amount of culture medium was added. The tube was centrifuged at 1.0 rpm and 0.2 rcf for 4 min to discard the culture medium containing dimethyl sulfoxide;

[0011] (3) Resuspend the cells in 5 ml of DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, pipette the cell suspension into a cell culture dish, shake gently, and observe the cell morphology and whether the cells are evenly dispersed under a microscope;

[0012] (4) Place the cells in a 5% CO2 cell culture incubator at 37°C, then observe the cell status under an inverted microscope. When the cells are well attached to the wall and cover 80% of the bottom of the cell plate, they can be subcultured. After 2-3 subcultures, the experiment can be carried out when the cell status is stable;

[0013] S3. The method for selecting the sodium fluoride treatment concentration is as follows:

[0014] (1) IPEC-J2 cells grown to the logarithmic growth phase were inoculated into a 96-well cell culture plate and cultured for 48-60 hours. When the cell confluence reached about 80%, the original culture medium was removed and the cells were rinsed twice with PBS;

[0015] (2) Discard PBS, add 200 μL of NaF diluted with serum-free DMEM medium to each well to obtain different concentrations, and add the same amount of serum-free medium to the control group and culture for 24 h;

[0016] (3) Based on the Japanese Tongren Cell Counting Kit-8 cell proliferation toxicity detection kit to detect cell viability, select the appropriate NaF treatment time to establish a cell fluoride exposure model

[0017] S4. The method for selecting the tea polyphenols treatment concentration is as follows:

[0018] (1) After establishing the NaF-induced fluoride exposure model of IPEC-J2 cells, tea polyphenols diluted with serum-free DMEM were added to different concentrations while being treated with NaF;

[0019] (2) The control group was added with an equal amount of serum-free medium and cultured for 24 h. The cell viability at different tea polyphenol concentrations was detected according to the kit instructions, and the optimal tea polyphenol treatment concentration was determined by the cell viability curve;

[0020] S5. The group design method is as follows:

[0021] (1) In a 12-well cell culture plate, IPEC-J2 cells with a cell confluence of 80% were randomly divided into three groups (n=3), namely, a normal group (Control), a model group (NaF), and a test group (NaF+TP);

[0022] (2) The normal group (Control) was cultured with serum-free DMEM for 24 h;

[0023] (3) Model group (NaF) was treated with serum-free DMEM supplemented with 4 mM NaF for 24 h;

[0024] (4) The experimental group (NaF+TP) was treated with serum-free DMEM containing 4 mM NaF and 200 mg·L-1 tea polyphenols for 24 h;

[0025] S6. The steps of indicator determination and method are as follows:

[0026] (1) Biochemical index detection: After trypsin digestion, cells were centrifuged and collected into 1.5 mL centrifuge tubes. According to the instructions of the biochemical kit, appropriate extracts were added in proportion. The cells were ultrasonically disrupted at 4°C and 300W for 3 min. The cells were centrifuged at 4°C and 8000g for 10 min. The supernatant was placed on ice for testing. The activities of SOD, CAT, GPX, GSH and GSSG in the cells were then tested.

[0027] (2) Cell monolayer permeability assay: using the Cytotoxicity LDH Assay from Japan The kit measures the LDH content in the culture medium to assess the permeability of the cell monolayer;

[0028] (3) Detection of reactive oxygen species (ROS) levels: ROS detection was performed using the Bio-Tech Reactive Oxygen Species Detection Kit, and then the fluorescence level was observed and photographed using a Feica DM61 microscope;

[0029] (4) Cell apoptosis assay: According to the instructions of the Bio-Tech One-Step In Situ Terminal Transferase Labeling Cell Apoptosis Detection Kit, the cell apoptosis level was detected. The Mitochondrial Membrane Potential Detection Kit (JC-1) was used for MMP detection, and the operation was carried out strictly according to the instructions;

[0030] (5) Detection of cell mRNA expression levels: Detect the mRNA expression levels of oxidative stress and endoplasmic reticulum stress-related proteins CAT, SOD, GPX1, GPX2, GPX3, GPX4, ATF4 and CHOP, and the mRNA expression levels of tight junction proteins Claudin-1 and Occludin, and then collect the cells for detection;

[0031] S7. Data analysis and processing: Image J v.1.8.0.345 was used to quantify the fluorescence intensity for subsequent analysis.

[0032] As a further embodiment of the present invention, the effect of tea polyphenols on the viability of fluorine-exposed IPEC-J2 cells is as follows:

[0033] (1) IPEC-J2 cells were selected for cell experiments. Fluoride at higher concentrations can induce cytotoxicity, oxidative stress and programmed cell death. NaF can significantly reduce cell viability (P<0.01) (e.g. Figure 3 -A), under 4 mM NaF treatment, cell viability decreased to 50-70% (P < 0.001);

[0034] (2) Tea polyphenols and the various active ingredients they contain have been proven to have a restorative effect on cell viability. Tea polyphenols can alleviate the decline in cell viability caused by excessive fluoride exposure.

[0035] As a further embodiment of the present invention, the effects of tea polyphenols on biochemical indices related to oxidative stress in IPEC-J2 cells exposed to fluoride are as follows:

[0036] (1). Fluorine has strong oxidizing properties and can induce oxidative stress by reducing the levels of GSH and various antioxidants and the activities of GPX and antioxidant enzymes. The activities of cellular oxidative stress-related enzymes and the changes in the contents of GSH and GSSG were detected. NaF significantly reduced the activities of CAT, SOD and GPX (P<0.01), while the addition of tea polyphenols significantly increased the activities of CAT and GPX (P<0.001), and had no significant effect on SOD activity. NaF did not significantly affect the content of GSH, but the addition of tea polyphenols reduced the content of GSH (P<0.001), resulting in an increase in the GSSG / GSH ratio (P<0.001);

[0037] (2) Tea polyphenols are one of the main active ingredients of tea. They have anti-inflammatory and antioxidant properties and can protect cells from damage by exogenous toxins by reducing oxidative stress and regulating the endoplasmic reticulum signaling pathway. The results showed that tea polyphenols increased the activity of CAT and GPX in IPEC-J2 cells exposed to fluoride, indicating that tea polyphenols can alleviate the oxidative stress caused by fluoride poisoning by activating the activity of antioxidant enzymes.

[0038] As a further embodiment of the present invention, the effect of tea polyphenols on the permeability of fluorine-exposed IPEC-J2 cell monolayer is as follows:

[0039] (1).NaF(4mM·L -1) treated the cells for 24 h, the level of lactate dehydrogenase (LDH) in the culture medium increased and the cell morphology changed. The results showed that 4 mM·L -1 NaF can increase the level of LDH in the culture medium, indicating that NaF overexposure leads to increased cell damage rate. After NaF treatment, the LDH content in the culture medium was significantly higher than that in the control group (P<0.01), while tea polyphenols can reduce the LDH content (P<0.05) and reduce the permeability of the cell membrane.

[0040] (2) Tea polyphenols can reduce the LDH content in the culture medium, reduce the permeability of IPEC-J2 cell membrane, and enhance the intestinal mechanical barrier.

[0041] As a further embodiment of the present invention, the effects of tea polyphenols on the ROS level of fluorine-exposed IPEC-J2 cells are as follows:

[0042] (1) ROS plays a key role in the development of oxidative stress. Excessive ROS can induce lipid peroxidation, DNA oxidative damage and protein modification, leading to oxidative stress and even cell apoptosis. In this study, fluoride exposure led to a significant increase in the ROS level of IPEC-J2 cells.

[0043] (2) The addition of tea polyphenols significantly reduced the ROS content in fluoride-induced IPEC-J2 cells, indicating that tea polyphenols have the function of alleviating fluoride-induced oxidative stress damage.

[0044] As a further embodiment of the present invention, the effects of tea polyphenols on apoptosis of IPEC-J2 cells exposed to fluoride are as follows: the results of cell TUNEL and MMP detection show that tea polyphenols can reduce the generation of ROS, increase mitochondrial membrane potential, and alleviate NaF-induced IPEC-J2 cell damage, thereby reducing cell apoptosis.

[0045] As a further embodiment of the present invention, the effect of tea polyphenols on the relative expression level of protein mRNA in fluorine-exposed IPEC-J2 cells is as follows:

[0046] (1). NaF treatment down-regulated the relative mRNA expression of antioxidant-related genes GPX2, GPX3, GPX4, CAT, and SOD in cells, and up-regulated the relative mRNA expression of ATF4 and CHOP in the endoplasmic reticulum stress-related pathway, indicating that fluoride accumulation can activate the endoplasmic reticulum stress pathway and cause oxidative stress in cells;

[0047] (2) The addition of tea polyphenols increased the relative expression of fluoride-induced cellular antioxidant enzyme mRNA, decreased the relative expression of endoplasmic reticulum stress-related protein mRNA, and alleviated the cell damage induced by NaF.

[0048] As a further embodiment of the present invention, the effect of tea polyphenols on the relative expression level of tight junction protein mRNA in fluoride-exposed IPEC-J2 cells is as follows:

[0049] NaF induced the inhibition of the mRNA expression level of the tight junction protein Claudin-1 in IPEC-J2 cells, indicating that excessive fluoride exposure has the effect of destroying the barrier function of intestinal epithelial cells, while the addition of tea polyphenols increased the mRNA expression of Occludin in fluoride-exposed cells, indicating that it has the function of repairing the intestinal barrier.

[0050] As a further scheme of the present invention, the fluoride exposure can cause excessive oxidative damage to intestinal cells, reduce the activity of antioxidant enzymes, and increase ROS production, thereby leading to increased intestinal cell damage rate and apoptosis rate. The addition of tea polyphenols can improve the antioxidant capacity of the body and cells, reduce ROS production and cell apoptosis, and alleviate intestinal cell damage caused by fluoride exposure.

[0051] As a further embodiment of the present invention, the tea polyphenols can improve the activity of antioxidant enzymes, reduce intestinal permeability, and reduce ROS production to relieve the oxidative stress and cell apoptosis caused by NaF, thereby repairing the intestinal barrier.

[0052] Compared with the related art, the method of using tea polyphenols to alleviate intestinal barrier damage caused by excessive fluoride exposure provided by the present invention has the following beneficial effects:

[0053] 1. The present invention has the advantages of increasing the integrity of the intestinal barrier and repairing the intestinal microecology by increasing the activity of antioxidant enzymes, reducing intestinal permeability, and reducing the generation of reactive oxygen species (ROS), thereby alleviating the oxidative stress and intestinal cell apoptosis caused by NaF. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0055] Figure 1 Schematic diagram of endoplasmic reticulum stress-related protein of the present invention;

[0056] Figure 2 This is a schematic diagram of the tight junction protein of the present invention;

[0057] Figure 3 Schematic diagram of the effect of NaF and tea polyphenols on IPEC-J2 cell viability determined by the CCK8 method of the present invention;

[0058] Figure 4 Schematic diagram of the effect of tea polyphenols of the present invention on oxidative stress-related factors of IPEC-J2 cells treated with NaF;

[0059] Figure 5Schematic diagram of the effect of tea polyphenols of the present invention on the LDH content in the culture medium of IPEC-J2 cells treated with NaF;

[0060] Figure 6 Schematic diagram of the effect of tea polyphenols of the present invention on the ROS fluorescence intensity of IPEC-J2 cells treated with NaF;

[0061] Figure 7 Schematic diagram of the effect of tea polyphenols of the present invention on the TUNEL fluorescence intensity of IPEC-J2 cells treated with NaF;

[0062] Figure 8 Schematic diagram of the effect of tea polyphenols of the present invention on MMP of IPEC-J2 cells treated with NaF;

[0063] Fig. 9 Schematic diagram of the effect of tea polyphenols of the present invention on protein mRNA expression level of IPEC-J2 cells treated with NaF;

[0064] Fig.10 This is a schematic diagram of the observation of mouse jejunum tissue morphology of the present invention;

[0065] Fig.11 Schematic diagram of the oxidative stress-related enzyme activities and factor contents in the serum of mice of the present invention;

[0066] Fig.12 Schematic diagram of DAO activity in serum and jejunum of mice of the present invention;

[0067] Fig.13 This is a schematic diagram of the relative expression level of mouse intestinal protein mRNA of the present invention. DETAILED DESCRIPTION

[0068] Please refer to Figures 1 to 13 The method of tea polyphenols alleviating intestinal barrier damage caused by excessive fluoride exposure includes: pig jejunal epithelial cells (IPEC-J2 cells), in vitro experiments were carried out with IPEC-J2 cells to establish a NaF-induced model. The specific methods are as follows:

[0069] S1. Experimental materials: IPEC-J2 cells, DMEM medium, sterile phosphate buffered saline, fetal bovine serum, penicillin-streptomycin and cell freezing solution;

[0070] S2. Cell recovery. The method for establishing the NaF-induced cell model is as follows:

[0071] (1) Take the cryovial out of liquid nitrogen, place it in a -80℃ refrigerator overnight, and then place it in a 37℃ water bath for 1 min to dissolve;

[0072] (2) The cell suspension was aspirated and placed in a 15 ml centrifuge tube, and then an appropriate amount of culture medium was added. The tube was centrifuged at 1.0 rpm and 0.2 rcf for 4 min to discard the culture medium containing dimethyl sulfoxide;

[0073] (3) Resuspend the cells in 5 ml of DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, pipette the cell suspension into a cell culture dish, shake gently, and observe the cell morphology and whether the cells are evenly dispersed under a microscope;

[0074] (4) Place the cells in a 5% CO2 cell culture incubator at 37°C, then observe the cell status under an inverted microscope. When the cells are well attached to the wall and cover 80% of the bottom of the cell plate, they can be subcultured. After 2-3 subcultures, the experiment can be carried out when the cell status is stable;

[0075] S3. The method for selecting the sodium fluoride treatment concentration is as follows:

[0076] (1) IPEC-J2 cells grown to the logarithmic growth phase were inoculated into a 96-well cell culture plate and cultured for 48-60 hours. When the cell confluence reached about 80%, the original culture medium was removed and the cells were rinsed twice with PBS;

[0077] (2) Discard PBS, add 200 μL of NaF diluted with serum-free DMEM medium to each well to obtain different concentrations, and add the same amount of serum-free medium to the control group and culture for 24 h;

[0078] (3) Based on the Japanese Tongren Cell Counting Kit-8 cell proliferation toxicity detection kit to detect cell viability, select the appropriate NaF treatment time to establish a cell fluoride exposure model

[0079] S4. The method for selecting the tea polyphenols treatment concentration is as follows:

[0080] (1) After establishing the NaF-induced fluoride exposure model of IPEC-J2 cells, tea polyphenols diluted with serum-free DMEM were added to different concentrations while being treated with NaF;

[0081] (2) The control group was added with an equal amount of serum-free medium and cultured for 24 h. The cell viability at different tea polyphenol concentrations was detected according to the kit instructions, and the optimal tea polyphenol treatment concentration was determined by the cell viability curve;

[0082] S5. The group design method is as follows:

[0083] (1) In a 12-well cell culture plate, IPEC-J2 cells with a cell confluence of 80% were randomly divided into three groups (n=3), namely, a normal group (Control), a model group (NaF), and a test group (NaF+TP);

[0084] (2) The normal group (Control) was cultured with serum-free DMEM for 24 h;

[0085] (3) Model group (NaF) was treated with serum-free DMEM supplemented with 4 mM NaF for 24 h;

[0086] (4) The experimental group (NaF+TP) was treated with serum-free DMEM containing 4 mM NaF and 200 mg·L-1 tea polyphenols for 24 h;

[0087] S6. The steps of indicator determination and method are as follows:

[0088] (1) Biochemical index detection: After trypsin digestion, cells were centrifuged and collected into 1.5 mL centrifuge tubes. According to the instructions of the biochemical kit, appropriate extracts were added in proportion. The cells were ultrasonically disrupted at 4°C and 300W for 3 min. The cells were centrifuged at 4°C and 8000g for 10 min. The supernatant was placed on ice for testing. The activities of SOD, CAT, GPX, GSH and GSSG in the cells were then tested.

[0089] (2) Cell monolayer permeability assay: using the Cytotoxicity LDH Assay from Japan The kit measures the LDH content in the culture medium to assess the permeability of the cell monolayer;

[0090] (3) Detection of reactive oxygen species (ROS) levels: ROS detection was performed using the Bio-Tech Reactive Oxygen Species Detection Kit, and then the fluorescence level was observed and photographed using a Feica DM61 microscope;

[0091] (4) Cell apoptosis assay: According to the instructions of the Bio-Tech One-Step In Situ Terminal Transferase Labeling Cell Apoptosis Detection Kit, the cell apoptosis level was detected. The Mitochondrial Membrane Potential Detection Kit (JC-1) was used for MMP detection, and the operation was carried out strictly according to the instructions;

[0092] (5) Detection of cell mRNA expression levels: Detect the mRNA expression levels of oxidative stress and endoplasmic reticulum stress-related proteins CAT, SOD, GPX1, GPX2, GPX3, GPX4, ATF4 and CHOP (such as Figure 1 ), the mRNA expression levels of tight junction proteins Claudin-1 and Occludin (as shown Figure 2 The cells were then collected and tested;

[0093] S7. Data analysis and processing: Image J v.1.8.0.345 was used to quantify the fluorescence intensity for subsequent analysis.

[0094] The effects of tea polyphenols on the viability of fluoride-exposed IPEC-J2 cells are as follows:

[0095] (1) IPEC-J2 cells were selected for cell experiments. Fluoride at higher concentrations can induce cytotoxicity, oxidative stress and programmed cell death. NaF can significantly reduce cell viability (P<0.01) (e.g. Figure 3 -A), under 4mMNaF treatment, cell viability decreased to 50-70% (P<0.001) ( Figure 3 -B indicates);

[0096] (2) Tea polyphenols and the various active ingredients they contain have been proven to have a restorative effect on cell viability. Tea polyphenols can alleviate the decline in cell viability caused by excessive fluoride exposure.

[0097] The effects of tea polyphenols on biochemical indices related to oxidative stress in IPEC-J2 cells exposed to fluoride are as follows:

[0098] (1) Fluorine has strong oxidizing properties and can induce oxidative stress by reducing the levels of GSH and various antioxidants and the activities of GPX and antioxidant enzymes. The activities of cellular oxidative stress-related enzymes and the changes in the contents of GSH and GSSG were tested. The results are as follows: Figure 4 As shown in the results, NaF significantly reduced the activities of CAT, SOD and GPX (P<0.01), while the addition of tea polyphenols significantly increased the activities of CAT and GPX (P<0.001), and had no significant effect on SOD activity. NaF did not significantly affect the GSH content, but the addition of tea polyphenols reduced the GSH content (P<0.001), resulting in an increase in the GSSG / GSH ratio (P<0.001);

[0099] (2) Tea polyphenols are one of the main active ingredients of tea. They have anti-inflammatory and antioxidant properties and can protect cells from damage by exogenous toxins by reducing oxidative stress and regulating the endoplasmic reticulum signaling pathway. The results showed that tea polyphenols increased the activity of CAT and GPX in IPEC-J2 cells exposed to fluoride, indicating that tea polyphenols can alleviate the oxidative stress caused by fluoride poisoning by activating the activity of antioxidant enzymes.

[0100] The effects of tea polyphenols on the permeability of fluoride-exposed IPEC-J2 cell monolayers are as follows:

[0101] (1).NaF(4mM·L -1 ) treated the cells for 24 h, the level of lactate dehydrogenase (LDH) in the culture medium increased and the cell morphology changed. The results showed that 4 mM·L -1NaF can increase the level of LDH in the culture medium, indicating that NaF overexposure leads to increased cell damage rate. After NaF treatment, the LDH content in the culture medium was significantly higher than that in the control group (P<0.01), while tea polyphenols can reduce the LDH content (P<0.05) and reduce the permeability of the cell membrane. Figure 5 As shown;

[0102] (2) Tea polyphenols can reduce the LDH content in the culture medium, reduce the permeability of IPEC-J2 cell membrane, and enhance the intestinal mechanical barrier.

[0103] The effects of tea polyphenols on ROS levels in fluoride-exposed IPEC-J2 cells are as follows:

[0104] (1) ROS plays a key role in the development of oxidative stress. Excessive ROS can induce lipid peroxidation, DNA oxidative damage and protein modification, leading to oxidative stress and even cell apoptosis. In this study, fluoride exposure led to a significant increase in the ROS level of IPEC-J2 cells. Figure 6 As shown;

[0105] (2) The addition of tea polyphenols significantly reduced the ROS content in fluoride-induced IPEC-J2 cells, indicating that tea polyphenols have the ability to alleviate fluoride-induced oxidative stress damage.

[0106] The effects of tea polyphenols on apoptosis of IPEC-J2 cells exposed to fluoride are as follows: The results of cell TUNEL and MMP assays show that tea polyphenols can reduce the generation of ROS, increase mitochondrial membrane potential, and alleviate NaF-induced IPEC-J2 cell damage, thereby reducing cell apoptosis. Figure 7 and Figure 8 shown.

[0107] The effects of tea polyphenols on the relative expression levels of protein mRNA in IPEC-J2 cells exposed to fluorine are as follows:

[0108] (1) NaF treatment down-regulated the relative mRNA expression of antioxidant-related genes GPX2, GPX3, GPX4, CAT, and SOD in cells, and up-regulated the relative mRNA expression of ATF4 and CHOP in the endoplasmic reticulum stress-related pathway, indicating that fluoride accumulation can activate the endoplasmic reticulum stress pathway and cause oxidative stress in cells, such as Fig. 9 As shown;

[0109] (2) The addition of tea polyphenols increased the relative expression of fluoride-induced cellular antioxidant enzyme mRNA, decreased the relative expression of endoplasmic reticulum stress-related protein mRNA, and alleviated the cell damage induced by NaF.

[0110] The effects of tea polyphenols on the relative expression level of tight junction protein mRNA in fluoride-exposed IPEC-J2 cells are as follows:

[0111] NaF induced the inhibition of the mRNA expression level of the tight junction protein Claudin-1 in IPEC-J2 cells, indicating that excessive fluoride exposure has the effect of destroying the barrier function of intestinal epithelial cells, while the addition of tea polyphenols increased the mRNA expression of Occludin in fluoride-exposed cells, indicating that it has the function of repairing the intestinal barrier.

[0112] The fluoride exposure can cause excessive oxidative damage to intestinal cells, reduce the activity of antioxidant enzymes, and increase ROS production, thereby leading to increased intestinal cell damage rate and apoptosis rate. The addition of tea polyphenols can improve the antioxidant capacity of the body and cells, reduce ROS production and cell apoptosis, and alleviate intestinal cell damage caused by fluoride exposure.

[0113] The tea polyphenols can relieve the oxidative stress and cell apoptosis caused by NaF by increasing the activity of antioxidant enzymes, reducing intestinal permeability, and reducing ROS production, thereby repairing the intestinal barrier.

[0114] NaF can significantly reduce the viability of a variety of cells, indicating that the IPEC-J2 cell fluoride exposure model has been successfully established. Tea polyphenols and the various active ingredients they contain have been proven to have a restorative effect on cell viability. The research results show that tea polyphenols can restore the decreased cell viability caused by fluoride exposure.

[0115] Tea polyphenols restored the activities of CAT and GPX, indicating that they can resist oxidative stress by enhancing the activities of antioxidant enzymes.

[0116] 4mM·L -1 NaF can lead to an increase in the level of LDH in the culture medium, indicating that NaF exposure can damage the cell membrane, increase LDH release, and ultimately lead to cell death. Tea polyphenols can reduce the content of LDH in the culture medium and reduce the permeability of IPEC-J2 cell membranes, which may have the effect of enhancing the intestinal mechanical barrier.

[0117] NaF has the effect of inducing cellular oxidative stress. Adding tea polyphenols to the feed can significantly reduce the content of ROS in the animal body and improve the antioxidant capacity. After adding tea polyphenols, the content of ROS in the cells is significantly reduced, indicating that it has the ability to alleviate the oxidative stress damage caused by fluoride.

[0118] TUNEL and MMP detection confirmed that NaF induction could significantly increase the cell apoptosis rate, and tea polyphenols could alleviate oxidative stress by regulating the related pathways of ROS production, thereby reducing cell apoptosis. The research results further proved that tea polyphenols could restore cell viability, antioxidant enzyme activity and cell membrane permeability, while reducing the production of ROS, thereby alleviating NaF-induced IPEC-J2 cell damage and reducing cell apoptosis.

[0119] NaF treatment downregulated the relative expression of intracellular antioxidant-related genes GPX2, GPX3, GPX4, CAT and SOD mRNA, and upregulated the relative expression levels of ATF4 and CHOP mRNA in the endoplasmic reticulum stress-related pathway, indicating that NaF can activate the endoplasmic reticulum stress pathway and cause oxidative stress in cells. Studies have shown that tea polyphenols can reduce endoplasmic reticulum stress-induced cell apoptosis by inhibiting oxidative stress. The results show that tea polyphenols restore the relative expression of antioxidant enzyme mRNA, reduce the relative expression of endoplasmic reticulum stress-related protein mRNA, and alleviate the damage caused by NaF.

[0120] Tight junction proteins constitute the main barrier in the paracellular pathway. They can narrow or close the space between cells according to the functional requirements of epithelial cells, establish semipermeable barriers, and reduce or prevent paracellular diffusion. Claudin-1 and Occludin are important components of tight junctions. NaF caused abnormal expression levels of Claudin-1 protein mRNA in IPEC-J2 cells, indicating that it has the effect of inducing intestinal epithelial cell barrier dysfunction. Studies have shown that tea extracts can inhibit the loss of cell junction proteins and restore the connection between intestinal cells. In the study, although tea polyphenols did not affect the mRNA expression of Claudin-1 in fluoride-exposed cells, it enhanced the mRNA expression of Occludin, indicating that it has the function of repairing the intestinal barrier.

[0121] In summary, fluoride exposure reduces IPEC-J2 cell activity and antioxidant enzyme activity, increases ROS levels and cell membrane permeability, and aggravates intestinal cell apoptosis, while tea polyphenols alleviate the fluoride-induced oxidative stress of intestinal cells by regulating the endoplasmic reticulum stress, apoptosis level, cell membrane permeability and expression of antioxidant-related genes in IPEC-J2 cells.

[0122] The present invention improves the integrity of the intestinal barrier and repairs the intestinal microecology by increasing the activity of antioxidant enzymes, reducing intestinal permeability, and reducing the generation of reactive oxygen species (ROS), thereby alleviating the oxidative stress and intestinal cell apoptosis caused by NaF.

[0123] To demonstrate the effects of tea polyphenols, the following is an example of the effects of tea polyphenols on mice.

[0124] Implemented 1

[0125] Effects of tea polyphenols on intestinal tissue morphology of fluoride-exposed mice:

[0126] like Fig.10 The results showed that the length of jejunal villi of mice treated with NaF was significantly shorter than that of normal mice, and the length of villi increased accordingly after adding tea polyphenols. The length of jejunal villi and the depth of crypts of the three groups of mice were measured, showing that the addition of NaF had a tendency to shorten the length of jejunal villi (P=0.063), and the value of jejunal villi length / crypt depth also had a corresponding downward trend, but it was not significant (P=0.093).

[0127] Note: Control means control group, mice were gavaged with normal saline and drank purified water; NaF means sodium fluoride group, mice were gavaged with normal saline and drank purified water containing 100 mg·L-1 fluoride; NaF+TP means sodium fluoride + tea polyphenols group, mice were gavaged with 200 mg·kg-1·bw tea polyphenols and drank purified water containing 100 mg·L-1 fluoride, *P<0.05, **P<0.01, ***P<0.001, n=3.

[0128] Implemented 2

[0129] Effects of tea polyphenols on serum oxidative stress-related factors in fluoride-exposed mice:

[0130] CAT, SOD and GPX are the main natural antioxidant enzymes in cells, which can remove hydrogen peroxide and other peroxides. Fig.11 The results showed that the CAT activity in the serum of mice was significantly increased after NaF treatment (P<0.05), and significantly decreased after tea polyphenol treatment (P<0.01), while GPX and SOD showed the opposite trend (P<0.001). There was no significant change in the GSH content among the three groups. In addition, the addition of tea polyphenols also significantly increased the NADPH content in the serum of fluoride-induced mice (P<0.05).

[0131] Note: Control means control group, mice were gavaged with normal saline and drank purified water; NaF means sodium fluoride group, mice were gavaged with normal saline and drank purified water containing 100 mg·L-1 fluoride; NaF+TP means sodium fluoride + tea polyphenols group, mice were gavaged with 200 mg·kg-1·bw tea polyphenols and drank purified water containing 100 mg·L-1 fluoride, *P<0.05, **P<0.01, ***P<0.001, n=6.

[0132] Implemented 3

[0133] Effects of tea polyphenols on serum and jejunal DAO activity in fluoride-exposed mice:

[0134] like Fig.12 As shown, the DAO activity in the serum of mice treated with NaF increased significantly (P<0.05), while that in the serum of mice treated with tea polyphenols decreased significantly (P<0.01), and the change trend of DAO activity in the jejunum of mice was consistent with that in the serum (P<0.05).

[0135] Note: Control means control group, mice were gavaged with normal saline and drank purified water; NaF means sodium fluoride group, mice were gavaged with normal saline and drank purified water containing 100 mg·L-1 fluoride; NaF+TP means sodium fluoride + tea polyphenols group, mice were gavaged with 200 mg·kg-1·bw tea polyphenols and drank purified water containing 100 mg·L-1 fluoride, *P<0.05, **P<0.01, ***P<0.001, n=6.

[0136] Implemented 4

[0137] Effects of tea polyphenols on relative expression levels of intestinal protein mRNA in fluoride-exposed mice:

[0138] CAT, SOD and GPX are common oxidative stress-related enzymes in animals, such as Fig.13 As shown in the results, the mRNA expression of SOD1, SOD2 and GPX1 in the jejunum was significantly increased during NaF exposure (P<0.05), while CAT showed an opposite trend (P<0.05). In contrast, tea polyphenols significantly decreased the mRNA expression levels of GPX1 (P<0.05) and SOD2 (P=0.069) in the jejunum exposed to NaF, while the expression level of CAT increased (P<0.05). ATF4-CHOP is the main apoptotic pathway of endoplasmic reticulum stress. Its expression level is low under physiological conditions, but it is strongly expressed during endoplasmic reticulum stress. This study found that after NaF exposure, the ATF4 gene The expression of ATF4 and CHOP genes in the jejunum was significantly increased (P<0.05). After adding tea polyphenols, the expression of ATF4 and CHOP genes was significantly decreased (P<0.05). Tight junction proteins are the main components of the intestinal mechanical barrier. The increase in Claudin-1 gene expression (P<0.01) and the decrease in Occludin gene expression (P<0.001) both indicate that NaF can cause disorder in the expression of intestinal tight junction proteins. After adding tea polyphenols, the expression of Claudin-1 decreased (P<0.01) and the expression of Occludin increased (P<0.01) to a level similar to that of healthy mice.

[0139] Note: Control means the control group, mice were gavaged with normal saline and drank purified water; NaF means the sodium fluoride group, mice were gavaged with normal saline and drank purified water containing 100 mg·L-1 fluoride; NaF+TP means the sodium fluoride + tea polyphenols group, mice were gavaged with 200 mg·kg -1 ·bw tea polyphenols, and drink tea containing 100mg·L-1 Fluoride-free pure water, *P<0.05, **P<0.01, ***P<0.001, n=6.

[0140] In summary, excessive fluoride exposure reduces the activity of serum antioxidant enzymes in mice and increases intestinal permeability. Tea polyphenols can restore the redox homeostasis in the serum and intestine of fluoride-exposed mice, increase the expression level and activity of antioxidant enzymes, thereby alleviating intestinal oxidative stress caused by excessive fluoride exposure and enhancing the intestinal mechanical barrier.

[0141] The present invention improves the activity of antioxidant enzymes, reduces intestinal permeability, reduces the generation of reactive oxygen species (ROS), alleviates the oxidative stress and intestinal cell apoptosis caused by NaF, thereby increasing the integrity of the intestinal barrier and repairing the intestinal microecology.

[0142] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for using tea polyphenols to alleviate intestinal barrier damage caused by excessive fluoride exposure, characterized in that: include: Porcine jejunal epithelial cells (IPEC-J2 cells) were used to conduct in vitro experiments and establish a NaF-induced model. The specific method is as follows: S1. Experimental materials: IPEC-J2 cells, DMEM medium, sterile phosphate buffered saline, fetal bovine serum, penicillin-streptomycin and cell freezing solution; S2. Cell recovery. The method for establishing the NaF-induced cell model is as follows: (1) Take the cryovial out of liquid nitrogen, place it in a -80℃ refrigerator overnight, and then place it in a 37℃ water bath for 1 min to dissolve; (2) The cell suspension was aspirated and placed in a 15 ml centrifuge tube, and then an appropriate amount of culture medium was added. The tube was centrifuged at 1.0 rpm and 0.2 rcf for 4 min to discard the culture medium containing dimethyl sulfoxide; (3) Resuspend the cells in 5 ml of DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, pipette the cell suspension into a cell culture dish, shake gently, and observe the cell morphology and whether the cells are evenly dispersed under a microscope; (4) Place the cells in a 5% CO2 cell culture incubator at 37°C, then observe the cell status under an inverted microscope. When the cells are well attached to the wall and cover 80% of the bottom of the cell plate, they can be subcultured. After 2-3 subcultures, the experiment can be carried out when the cell status is stable; S3. The method for selecting the sodium fluoride treatment concentration is as follows: (1) IPEC-J2 cells grown to the logarithmic growth phase were inoculated into a 96-well cell culture plate and cultured for 48-60 hours. When the cell confluence reached about 80%, the original culture medium was removed and the cells were rinsed twice with PBS; (2) Discard PBS, add 200 μL of NaF diluted with serum-free DMEM medium to each well to obtain different concentrations, and add the same amount of serum-free medium to the control group and culture for 24 h; (3) Based on the Japanese Tongren Cell Counting Kit-8 cell proliferation toxicity detection kit to detect cell viability, select the appropriate NaF treatment time to establish a cell fluoride exposure model S4. The method for selecting the tea polyphenols treatment concentration is as follows: (1) After establishing the NaF-induced fluoride exposure model of IPEC-J2 cells, tea polyphenols diluted with serum-free DMEM were added to different concentrations while being treated with NaF; (2) The control group was added with an equal amount of serum-free medium and cultured for 24 h. The cell viability at different tea polyphenol concentrations was detected according to the kit instructions, and the optimal tea polyphenol treatment concentration was determined by the cell viability curve; S5. The group design method is as follows: (1) In a 12-well cell culture plate, IPEC-J2 cells with a cell confluence of 80% were randomly divided into three groups (n=3), namely, a normal group (Control), a model group (NaF), and a test group (NaF+TP); (2) The normal group (Control) was cultured with serum-free DMEM for 24 h; (3) Model group (NaF) was treated with serum-free DMEM supplemented with 4 mM NaF for 24 h; (4) The experimental group (NaF+TP) was treated with serum-free DMEM containing 4 mM NaF and 200 mg·L-1 tea polyphenols for 24 h; S6. The steps of indicator determination and method are as follows: (1) Biochemical index detection: After trypsin digestion, cells were centrifuged and collected into 1.5 mL centrifuge tubes. According to the instructions of the biochemical kit, appropriate extracts were added in proportion. The cells were ultrasonically disrupted at 4°C and 300W for 3 min. The cells were centrifuged at 4°C and 8000g for 10 min. The supernatant was placed on ice for testing. The activities of SOD, CAT, GPX, GSH and GSSG in the cells were then tested. (2) Cell monolayer permeability assay: using the Cytotoxicity LDH Assay from Japan The kit measures the LDH content in the culture medium to assess the permeability of the cell monolayer; (3) Detection of reactive oxygen species (ROS) levels: ROS detection was performed using the Bio-Tech Reactive Oxygen Species Detection Kit, and then the fluorescence level was observed and photographed using a FeicaDM61 microscope; (4) Cell apoptosis assay: According to the instructions of the Bio-Tech One-Step In Situ Terminal Transferase Labeling Cell Apoptosis Detection Kit, the cell apoptosis level was detected. The Mitochondrial Membrane Potential Detection Kit (JC-1) was used for MMP detection, and the operation was carried out strictly according to the instructions; (5) Detection of cell mRNA expression levels: Detect the mRNA expression levels of oxidative stress and endoplasmic reticulum stress-related proteins CAT, SOD, GPX1, GPX2, GPX3, GPX4, ATF4 and CHOP, and the mRNA expression levels of tight junction proteins Claudin-1 and Occludin, and then collect the cells for detection; S7. Data analysis and processing: Image J v.1.8.0.345 was used to quantify the fluorescence intensity for subsequent analysis.

2. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on the viability of fluoride-exposed IPEC-J2 cells are as follows: (1) IPEC-J2 cells were selected for cell experiments. Fluoride at higher concentrations can induce cytotoxicity, oxidative stress and programmed cell death. NaF can significantly reduce cell viability (P<0.01) (as shown in Figure 3-A). Under 4mM NaF treatment, cell viability decreased to 50-70% (P<0.001); (2) Tea polyphenols and the various active ingredients they contain have been proven to have a restorative effect on cell viability. Tea polyphenols can alleviate the decline in cell viability caused by excessive fluoride exposure.

3. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on biochemical indices related to oxidative stress in IPEC-J2 cells exposed to fluoride are as follows: (1). Fluorine has strong oxidizing properties and can induce oxidative stress by reducing the levels of GSH and various antioxidants and the activities of GPX and antioxidant enzymes. The activities of cellular oxidative stress-related enzymes and the changes in the contents of GSH and GSSG were detected. NaF significantly reduced the activities of CAT, SOD and GPX (P<0.01), while the addition of tea polyphenols significantly increased the activities of CAT and GPX (P<0.001), and had no significant effect on SOD activity. NaF did not significantly affect the content of GSH, but the addition of tea polyphenols reduced the content of GSH (P<0.001), resulting in an increase in the GSSG / GSH ratio (P<0.001); (2) Tea polyphenols are one of the main active ingredients of tea. They have anti-inflammatory and antioxidant properties and can protect cells from damage by exogenous toxins by reducing oxidative stress and regulating the endoplasmic reticulum signaling pathway. The results showed that tea polyphenols increased the activity of CAT and GPX in IPEC-J2 cells exposed to fluoride, indicating that tea polyphenols can alleviate the oxidative stress caused by fluoride poisoning by activating the activity of antioxidant enzymes.

4. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on the permeability of fluoride-exposed IPEC-J2 cell monolayers are as follows: (1).NaF(4mM·L -1 ) treated the cells for 24 h, the level of lactate dehydrogenase (LDH) in the culture medium increased and the cell morphology changed. The results showed that 4 mM·L -1 NaF can increase the level of LDH in the culture medium, indicating that NaF overexposure leads to increased cell damage rate. After NaF treatment, the LDH content in the culture medium was significantly higher than that in the control group (P<0.01), while tea polyphenols can reduce the LDH content (P<0.05) and reduce the permeability of the cell membrane. (2) Tea polyphenols can reduce the LDH content in the culture medium, reduce the permeability of IPEC-J2 cell membrane, and enhance the intestinal mechanical barrier.

5. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on ROS levels in fluoride-exposed IPEC-J2 cells are as follows: (1) ROS plays a key role in the development of oxidative stress. Excessive ROS can induce lipid peroxidation, DNA oxidative damage and protein modification, leading to oxidative stress and even cell apoptosis. In this study, fluoride exposure led to a significant increase in the ROS level of IPEC-J2 cells. (2) The addition of tea polyphenols significantly reduced the ROS content in fluoride-induced IPEC-J2 cells, indicating that tea polyphenols have the function of alleviating fluoride-induced oxidative stress.

6. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on apoptosis of IPEC-J2 cells exposed to fluoride are as follows: the results of cell TUNEL and MMP detection show that tea polyphenols can reduce the generation of ROS, increase mitochondrial membrane potential, and alleviate NaF-induced IPEC-J2 cell damage, thereby reducing cell apoptosis.

7. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on the relative expression levels of protein mRNA in IPEC-J2 cells exposed to fluorine are as follows: (1). NaF treatment down-regulated the relative mRNA expression of antioxidant-related genes GPX2, GPX3, GPX4, CAT, and SOD in cells, and up-regulated the relative mRNA expression of ATF4 and CHOP in the endoplasmic reticulum stress-related pathway, indicating that fluoride accumulation can activate the endoplasmic reticulum stress pathway and cause oxidative stress in cells; (2) The addition of tea polyphenols increased the relative expression of fluoride-induced cellular antioxidant enzyme mRNA, decreased the relative expression of endoplasmic reticulum stress-related protein mRNA, and alleviated the cell damage induced by NaF.

8. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The effects of tea polyphenols on the relative expression level of tight junction protein mRNA in fluoride-exposed IPEC-J2 cells are as follows: NaF induced the inhibition of the mRNA expression level of the tight junction protein Claudin-1 in IPEC-J2 cells, indicating that excessive fluoride exposure has the effect of destroying the barrier function of intestinal epithelial cells, while the addition of tea polyphenols increased the mRNA expression of Occludin in fluoride-exposed cells, indicating that it has the function of repairing the intestinal barrier.

9. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The fluoride exposure can cause excessive oxidative damage to intestinal cells, reduce the activity of antioxidant enzymes, and increase ROS production, thereby leading to increased intestinal cell damage rate and apoptosis rate. The addition of tea polyphenols can improve the antioxidant capacity of the body and cells, reduce ROS production and cell apoptosis, and alleviate intestinal cell damage caused by fluoride exposure.

10. The method of tea polyphenols for alleviating intestinal barrier damage caused by fluoride overexposure according to claim 1, characterized in that: The tea polyphenols repair the intestinal barrier by increasing the activity of antioxidant enzymes, reducing intestinal permeability, reducing ROS generation, and alleviating oxidative stress and cell apoptosis caused by NaF.

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