Application of lactobacillus in the preparation of medicine for preventing and / or treating liver and / or kidney and / or bone damage caused by excessive intake of fluoride

Abstract

The present invention provides an application of lactobacillus in the preparation of a medicine for preventing and / or treating liver and / or kidney and / or bone damage caused by excessive intake of fluoride, and relates to the technical field of lactobacillus application. The present invention achieves the purpose of preventing and / or treating damage to the liver and / or kidney and / or bone by reducing the accumulation of fluoride in the liver and / or kidney and / or bone, and the lactobacillus is Lactobacillus johnsonii BS15. Lactobacillus johnsonii BS15 is used to reduce the accumulation of fluoride in the body, especially in the liver, kidney and bone during high fluoride exposure, improve the damage to organs caused by high fluoride exposure, promote the excretion of fluoride from the body, promote the excretion of fluoride from the kidney, promote the excretion of fluoride in feces and urine, and improve the damage to the liver, kidney, lung and nerve center of mice. It is safe and effective to use, has no toxic side effects, and has a wider application.

Description

Technical Field

[0001] The present invention relates to the technical field of application of lactobacillus, and in particular to application of lactobacillus in the preparation of a medicine for preventing and / or treating liver and / or kidney and / or bone damage caused by excessive intake of fluoride. Background Art

[0002] Fluorine exists in the human environment mainly in the form of fluoride ions. The daily intake of fluorine by the human body mainly comes from drinking water and food, of which drinking water accounts for a large proportion. Foods with high fluoride content include sardines and some dried seafood. In addition, there is a small amount of fluorine in the air. Using stone coal as fuel for cooking, heating, and baking food causes a large amount of fluorine in the coal to be emitted into the air, which significantly increases the fluorine content in the local microclimate air, contaminates food and vegetables, and becomes the main source of fluorine intake for local residents.

[0003] Excessive intake of fluoride can harm the health of the body and is called fluorosis. Skeletal fluorosis and dental fluorosis are common symptoms of fluorosis. In addition to causing damage to bones and teeth, excessive intake of fluoride can also cause damage to non-bone tissues such as the intestines, liver, lungs, kidneys, and nerve centers. The dose level of fluoride neurotoxicity is lower than that of dental fluorosis and skeletal fluorosis, so the neurotoxic effect of fluoride cannot be ignored.

[0004] There are two main treatments for fluorosis: one is to reduce the body's absorption of fluoride; the other is symptomatic treatment to relieve the patient's symptoms and improve physical signs. Calcium, magnesium, aluminum and boron are commonly used in clinical practice to reduce the accumulation of fluoride in the body. However, most of the drugs currently used to reduce the accumulation of fluoride in the body have toxic side effects:

[0005] 1. Calcium. Fluoride and calcium have a strong affinity. In the intestine, the two can form poorly soluble CaF2, which is excreted with feces, thus reducing fluoride absorption. Calcium is effective for the treatment of loose, softening and insufficient calcium absorption skeletal fluorosis, but long-term use of calcium can aggravate bone sclerosis and soft tissue calcification, which may be unfavorable for the treatment of sclerotic skeletal fluorosis.

[0006] 2. Magnesium supplements. The magnesium content in drinking water is inversely proportional to the absorption of fluoride in the body. Magnesium is an activator of certain enzymes. When fluoride in the body combines with magnesium to form a magnesium fluorophosphate complex, the amount of magnesium in the body decreases and the activity of the enzyme is inhibited. Increasing magnesium intake can reduce the inhibitory effect of magnesium. Serpentine is a naturally occurring magnesium silicate that can release fluoride from bones. Serpentine produced in different regions has different structures and different fluoride removal effects. Serpentine often contains asbestos, which is a human carcinogen and poses a safety hazard.

[0007] 3. Aluminum: Aluminum is a strong chelating agent for fluoride. 40-60% of fluoride in the intestine can be combined with aluminum. A small dose of aluminum hydroxide can increase the excretion of fecal fluoride. It is worth noting that the increase in aluminum in the body is not easy to be excreted, and it can affect calcium-phosphorus metabolism, leading to a decrease in blood phosphorus, bone phosphorus release, and osteomalacia.

[0008] 4. Boron: Boron and fluorine form a BF4 complex, which is easy to excrete. However, excessive intake can also lead to poisoning.

[0009] After excessive fluoride enters the body, it accumulates in different tissues and organs through blood circulation, of which 90% enters the body's calcified tissues, while the remaining 10% is distributed in the body's soft tissues such as the kidneys, heart, liver, and brain. A large number of studies have shown that high fluoride exposure can seriously endanger the body's health, induce acute and chronic fluoride poisoning, lead to bone tissue damage such as fluorosis and dental caries, and can also harm other organs and body functions. Recent studies have shown that fluoride can affect brain development at doses below the level that causes fluorosis and fluorosis, and fluoride has been listed as a developmental neurotoxin. Studies from the United States, Canada, and Mexico have shown that fluoride exposure is positively correlated with the risk of attention deficit and hyperactivity disorder in adolescents and Alzheimer's disease in the elderly. Through association analysis with 27 epidemiological studies, researchers found that high fluoride in drinking water is strongly correlated with a decline in children's intelligence. Studies have shown that high fluoride exposure is accompanied by intestinal flora disorders, and intestinal flora disorders are closely related to body damage caused by high fluoride exposure.

[0010] At present, more and more studies have shown that probiotics can regulate intestinal flora disorders and improve the resulting damage to body functions. There are more than 1,000 types of bacteria in the intestines of adults, with more than 7,000 different strains, totaling 10 13 -10 14 That’s 10 times more than the total number of cells in the human body. High fluoride exposure can destroy the structure of the intestinal flora and reduce the proportion of Lactobacillus. Lactobacillus is a common probiotic used to regulate intestinal flora and improve the intestinal barrier. At present, there are no reports on the use of probiotics, especially Lactobacillus, to prevent or treat liver, kidney or bone tissue damage caused by excessive fluoride intake, nor have there been any reports on reducing the accumulation of fluoride in the liver, kidney or bone tissue to achieve treatment or prevention purposes. There are also no reports that Lactobacillus can reduce fluoride accumulation in the body.

[0011] Based on this, the present invention is proposed. Summary of the invention

[0012] One of the purposes of the present invention is to provide the use of lactobacillus in the preparation of a medicament for preventing and / or treating liver and / or kidney and / or bone damage caused by excessive intake of fluoride.

[0013] The second purpose of the present invention is to provide the use of lactobacillus in reducing the accumulation of fluoride in the body. It improves the damage to organs caused by high fluoride exposure, promotes the excretion of fluoride from the body, promotes the excretion of fluoride from the kidneys, promotes the excretion of fluoride in feces and urine, reduces gastrointestinal absorption, improves the accumulation of fluoride in the liver, kidneys, and bones, and improves the damage to the liver, kidneys, lungs, and nerve centers of mice. It is safe and effective to use, has no toxic side effects, and has a wider application.

[0014] The third object of the present invention is to provide the use of lactobacillus in the preparation of a medicine for preventing and / or treating damage to the lungs and / or spleen caused by fluoride poisoning or excessive intake of fluoride.

[0015] In order to achieve the above object, the present invention adopts the following technical solutions:

[0016] In a first aspect, the present invention provides the use of lactobacillus in the preparation of a medicament for preventing and / or treating liver and / or kidney and / or bone damage caused by excessive intake of fluoride, wherein the lactobacillus is Lactobacillus johnsonii BS15.

[0017] In the application of the first aspect of the present invention, further, the drug achieves the purpose of prevention and / or treatment by reducing the accumulation of fluoride in the liver and / or kidneys and / or bones.

[0018] The second aspect of the present invention provides the use of Lactobacillus in preparing a product for reducing the accumulation of fluoride in the body, wherein the Lactobacillus is Lactobacillus johnsonii BS15.

[0019] In the application of the second aspect of the present invention, further, the product includes a medicine.

[0020] In the application of the second aspect of the present invention, further, reducing the accumulation of fluoride in the body includes reducing the accumulation of fluoride in internal organs, blood and bone tissue.

[0021] In the application of the second aspect of the present invention, further, the organ is selected from the group consisting of liver and kidney.

[0022] In the application of the second aspect of the present invention, further, the method of reducing the accumulation of fluoride in the body includes excreting it from the body through feces and urine.

[0023] The third aspect of the present invention provides the use of Lactobacillus in the preparation of a medicament for preventing and / or treating damage to the lungs and / or spleen caused by fluoride poisoning or excessive intake of fluoride, wherein the Lactobacillus is Lactobacillus johnsonii BS15.

[0024] In the first to third aspects of the present invention, the Lactobacillus johnsonii BS15 is deposited in China Center for Type Culture Collection with the deposit number of CCTCC M2013663.

[0025] In the first to third aspects of the present invention, further, the product or medicine is a bacterial liquid of Lactobacillus johnsonii BS15.

[0026] Furthermore, the concentration of the Lactobacillus johnsonii BS15 bacterial solution is 1×10 8 CFU / mL to 1×10 10 CFU / mL.

[0027] Furthermore, the concentration of the Lactobacillus johnsonii BS15 bacterial solution is 1×10 9 CFU / mL.

[0028] Furthermore, the preparation method of the Lactobacillus johnsonii BS15 bacterial liquid comprises the following steps:

[0029] S1: Activate the Lactobacillus johnsonii BS15 strain at least once using MRS medium;

[0030] S2: inoculating the activated bacterial solution into a sterile MRS liquid culture medium for culturing, and counting the live bacteria in the cultured bacterial solution;

[0031] S3: centrifuging the cultured bacterial solution to obtain bacterial cells;

[0032] S4: According to the live bacteria count results, the bacteria are prepared into a Lactobacillus johnsonii BS15 bacterial liquid of corresponding concentration using a buffer solution.

[0033] The present invention has the following beneficial effects:

[0034] 1. The present invention utilizes Lactobacillus johnsonii BS15 to reduce the accumulation of fluoride in the body, especially in the liver, kidneys and bones during high fluoride exposure, thereby improving the damage of high fluoride exposure to these organs.

[0035] 2. The inventors also found that during high fluoride exposure, Lactobacillus johnsonii BS15 can promote the excretion of fluoride from the body, promote the excretion of fluoride from the kidneys, promote the excretion of fluoride in feces and urine, reduce the concentration of fluoride in the blood, and avoid irreversible damage to internal organs, nervous tissue, bones, teeth, etc.

[0036] 3. In addition to improving liver, kidney and bone damage in mice, Lactobacillus johnsonii BS15 can also have a certain therapeutic effect on damage to the lungs, spleen and nerve centers. It is safe and effective to use, has no toxic side effects, and is more widely used.

[0037] In conclusion, there is no research and application of probiotics in preventing and treating high fluoride accumulation in the body. The present invention is the first to study the ability of Lactobacillus johnsonii BS15 to reduce the accumulation of fluoride in the body. The test results show that Lactobacillus johnsonii BS15 reduces the accumulation of fluoride in the body during high fluoride exposure and has a significant effect on improving the damage of high fluoride exposure to organs and bone tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 A schematic diagram of the experimental process provided for the experimental example of the present invention;

[0040] Figure 2 A graph showing changes in fluoride ions in mouse urine in the 20th week provided in the test example of the present invention;

[0041] Figure 3 A graph showing changes in fluoride ions in mouse feces at the 20th week provided in the test example of the present invention;

[0042] Figure 4 A graph showing changes in fluoride ions in mouse serum at the 20th week provided in the test example of the present invention;

[0043] Figure 5 A graph showing changes in fluoride ions in the spleen of mice at the 20th week provided in the test example of the present invention;

[0044] Figure 6 A graph showing changes in fluoride ions in the lungs of mice at the 20th week provided in the test examples of the present invention;

[0045] Figure 7 A graph showing changes in fluoride ions in the liver of mice at the 20th week provided in the test example of the present invention;

[0046] Figure 8 A graph showing changes in fluoride ions in mouse kidneys at the 20th week provided in the test example of the present invention;

[0047] Fig. 9 A graph showing changes in fluoride ions in mouse muscle at the 20th week provided in the test example of the present invention;

[0048] Fig.10 A graph showing changes in fluoride ions in the femur of mice at the 20th week provided in the test example of the present invention;

[0049] Fig.11 A graph showing changes in fluoride ions in mouse ileum tissue at the 20th week provided in the test example of the present invention;

[0050] Fig.12 The mouse lung pathology results provided for the test examples of the present invention;

[0051] Fig.13 The mouse liver pathology results provided for the test examples of the present invention;

[0052] Fig.14 Mouse spleen pathology results provided for the test examples of the present invention;

[0053] Fig.15 The mouse kidney pathology results provided for the test examples of the present invention;

[0054] Fig.16 The histopathological results of mouse ileum provided in the test examples of the present invention;

[0055] in, Figure 2-Figure 11 In the table, * indicates that there is a significant difference between the two groups, more * indicates that there is a more significant difference between the two groups, and ns indicates that there is no significant difference between the two groups. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. The process parameters of the following embodiments that do not specify specific conditions are usually based on conventional conditions.

[0057] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0058] The first aspect of the present invention provides the use of Lactobacillus in the preparation of a medicament for preventing and / or treating liver and / or kidney and / or bone damage caused by excessive intake of fluoride, wherein the Lactobacillus is Lactobacillus johnsonii BS15.

[0059] Specifically, the drug achieves the purpose of prevention and / or treatment by reducing the accumulation of fluoride in the liver and / or kidneys and / or bones. Under normal circumstances, fluoride will accumulate in the liver, kidneys, and bones, causing damage to them. The Lactobacillus johnsonii BS15 used in the present invention can help the fluoride in the liver, kidneys, and bones to be discharged or reduce the accumulation of fluoride in the liver, kidneys, and bones, thereby achieving the repair of liver, kidney, and bone damage or fundamentally preventing or reducing liver, kidney, and bone damage.

[0060] The excessive intake of fluoride in the present invention refers to the amount of fluoride taken into the human body, animal body or microorganism through the external environment being at or above the minimum intake amount that causes health problems in the corresponding body. The minimum intake amount can be referred to in relevant literature or other public data reports, which is well known and will not be elaborated here.

[0061] The second aspect of the present invention provides the use of Lactobacillus johnsonii BS15 in the preparation of a product for reducing the accumulation of fluoride in the body.

[0062] Specifically, the present invention intervenes in the treatment of a mouse model exposed to high fluoride by oral administration of probiotics. The results show that oral administration of the probiotic Lactobacillus johnsonii BS15 reduces the concentration of fluoride in the blood, reduces the concentration of fluoride in the liver, kidney and bone tissue, promotes the excretion of fluoride from the body, promotes the excretion of fluoride from the kidneys, and promotes the excretion of fluoride in feces and urine, thereby avoiding irreversible damage to internal organs, nervous tissue, bones and teeth.

[0063] The Lactobacillus johnsonii BS15 is a strain isolated by the Animal Microecology Research Center of Sichuan Agricultural University, with a deposit number of CCTCC M2013663 in the China Center for Type Culture Collection and a deposit date of December 13, 2013. Lactobacillus johnsonii BS15 is recorded in the published patent document CN 113018320A.

[0064] The fluoride described in the present invention includes fluoride derived from the environment, food and water, such as sodium fluoride, silicon tetrafluoride, boron trifluoride and sodium fluorosilicate.

[0065] The degree of accumulation of fluoride in the body of the present invention can be a degree that does not cause obvious pathological changes or a degree that causes obvious pathological changes, and can be chronic fluorosis or acute fluorosis. The product of the present invention has no particular limitation on the concentration of fluoride in the body.

[0066] The in vivo may be the human body, the animal body or the body of other microorganisms, and the present invention does not make any particular limitation on the in vivo.

[0067] In the application of the second aspect, the product can be a product in any usable form, preferably a medicine. Specifically, Lactobacillus johnsonii BS15 can be prepared into a medicine, and when prepared into a medicine, in addition to Lactobacillus johnsonii BS15, other necessary auxiliary materials are also included.

[0068] In the application of the second aspect, as an optional manner, reducing the accumulation of fluoride in the body includes reducing the accumulation of fluoride in internal organs, blood and bone tissue. The internal organs are preferably selected from the liver and kidney.

[0069] In the application of the second aspect above, as an optional manner, the method of reducing the accumulation of fluoride in the body includes excreting it from the body through feces and urine.

[0070] The third aspect of the present invention provides the use of Lactobacillus johnsonii BS15 in the preparation of a medicament for preventing and / or treating damage to the lungs and / or spleen caused by fluoride poisoning or excessive intake of fluoride.

[0071] Experiments have shown that although Lactobacillus johnsonii BS15 did not significantly reduce the fluoride concentration in the lungs and / or spleen, it could also significantly improve the damage to the lungs and / or spleen caused by fluoride accumulation in the body.

[0072] In the applications of the first to third aspects above, as an optional manner, the product or medicine is a bacterial liquid of Lactobacillus johnsonii BS15.

[0073] In the applications of the first to third aspects, as an optional method, the concentration of the Lactobacillus johnsonii BS15 bacterial solution is 1×10 8 CFU / mL to 1×10 10 CFU / mL. Preferably, the concentration of the Lactobacillus johnsonii BS15 bacterial solution is 1×10 9 CFU / mL.

[0074] Furthermore, the preparation method of the Lactobacillus johnsonii BS15 bacterial liquid comprises the following steps:

[0075] S1: Activate the Lactobacillus johnsonii BS15 strain at least once using MRS medium;

[0076] S2: inoculating the activated bacterial solution into a sterile MRS liquid culture medium for culturing, and counting the live bacteria in the cultured bacterial solution;

[0077] S3: centrifuging the cultured bacterial solution to obtain bacterial cells;

[0078] S4: according to the live bacteria count result, the bacteria are prepared into a Lactobacillus johnsonii BS15 bacterial liquid of corresponding concentration using a buffer solution;

[0079] Specifically, the preparation method of the Lactobacillus johnsonii BS15 bacterial liquid comprises the following steps:

[0080] S1: The Lactobacillus johnsonii BS15 strain stored at -80°C was activated with MRS and cultured at 37°C for 24 hours to obtain the activated first-generation strain;

[0081] S2: Continue to activate the first-generation activated strain until the third-generation activated strain is obtained, inoculate it into sterile MRS liquid culture medium at a volume ratio of 2%, and culture it at 37°C for 24 hours before counting the viable bacteria;

[0082] S3: Centrifuge the culture medium at 3000 r / min for 15 min, discard the supernatant, and obtain the bacterial cells;

[0083] S4: According to the viable bacteria count results, the bacteria were made into 2×10 8 CFU / 0.2 mL of bacterial solution was stored at 4°C for future use, and fresh bacterial solution was prepared once a week.

[0084] The present invention is further described in detail below in conjunction with specific test examples.

[0085] Test example

[0086] 1. Preparation of bacterial liquid: Take out the Lactobacillus johnsonii BS15 strain (provided by Sichuan Agricultural University) stored at -80℃, activate it with MRS medium, and culture it at 37℃ for 24 hours as the first generation of activation. Inoculate the BS15 after activation for 3 generations into sterile MRS liquid medium at a ratio of 2% (volume ratio), culture it at 37℃ for 24 hours, and then count the viable bacteria. Centrifuge the culture at 3000 r / min for 15 minutes, discard the supernatant, and make 2×10 8 CFU / 0.2 mL of bacterial solution. Store at 4℃ for future use and prepare fresh bacterial solution once a week.

[0087] 2. Animal grouping and drug administration

[0088] The experimental animals were SPF mice of the breed C57BL / 6J, three weeks old, weighing 8±1g, male, a total of 45 mice, divided into the following three groups for treatment, each group of 15 experimental animals.

[0089] (1) Blank control group (Ctrl group or C group): gavage 0.2 mL of 1X PBS solution once a day for 20 weeks. During the 20 weeks, the mice were continuously fed with normal drinking water and feed;

[0090] (2) High fluoride exposure group (Group F): Oral administration of 0.2 mL of 1X PBS solution once a day for 20 weeks. During the 20 weeks, the mice were continuously fed with fluoride-containing drinking water (concentration 100 mg NaF / L) and ordinary feed;

[0091] (3) BS15 treatment group (FB group or F+BS15 group): 0.2 mL of prepared Lactobacillus johnsonii BS15 bacterial solution (concentration 1×10 9 CFU / mL), once a day, by gavage for 20 weeks. During the 20 weeks, the mice were continuously fed with fluoride-containing drinking water (concentration 100 mg NaF / L) and ordinary feed.

[0092] The specific process of the experiment is as follows Figure 1 As shown by Figure 1 It can be seen that the adaptation period of mice was 7 days, and the main test period was 140 days (i.e., 20 weeks). Within 20 weeks after the start of the main test period, mice in the BS15 treatment group were gavaged with Lactobacillus johnsonii BS15 solution (0.2 mL, concentration 1×10 9 CFU / mL), the control group and the high fluoride exposure group were gavaged with PBS buffer (pH 7.0); from the beginning to the end of the experiment, the control group was fed with pure water, and the high fluoride exposure group and the BS15 treatment group were fed with fluoride-containing drinking water (concentration 100 mg NaF / L), and other conditions remained unchanged.

[0093] 3. Sample collection

[0094] (1) At week 20, blood was collected from the retro-orbital venous plexus (i.e., medial canthal vein) and the blood was allowed to stand at room temperature for 30 min before centrifugation at 4°C at 3000 rpm for 15 min to separate the serum, which was then stored at -80°C for later use.

[0095] (2) At week 20, place the mice individually in clean cages, collect the mouse feces and store them at -80°C for later use;

[0096] (3) Collect urine from mice at week 20 and store at -80°C for future use;

[0097] (4) After 20 weeks, the mice were killed and their lungs, livers, kidneys, spleens, femurs and muscles were collected. Some of the lung, liver, kidney and spleen tissues were stored in 4% paraformaldehyde, and some were stored at -80°C for future use.

[0098] 4. Observation items and test contents

[0099] (1) Detection of fluoride ion content in organs, serum, urine and feces;

[0100] The fluoride content determination method is carried out in accordance with national standards (WS / T 89-2015, WS / T 212-2001). The specific steps are as follows:

[0101] ① Preparation of sodium fluoride standard stock solution: Sodium fluoride is dried at 120℃ for 2 h, cooled to room temperature, accurately weighed 0.2210 g, dissolved in water, transferred to a 100 mL volumetric flask, diluted to scale with water, mixed, and poured into a polyethylene bottle for later use. This solution is 1000F ¯ μg / mL.

[0102] ② Preparation of sodium fluoride standard working solution: Pipette 10 mL of sodium fluoride standard stock solution, transfer to a 100 mL volumetric flask, and dilute to the mark with deionized water. At this point, the concentration is 100 μgF ¯ / mL, recorded as sodium fluoride standard working solution I. Pipette 10 mL of sodium fluoride standard working solution I, transfer it to a 100 mL volumetric flask, and dilute to the mark with deionized water. At this time, the concentration is 10 μgF ¯ / mL, recorded as sodium fluoride standard working solution II. Pipette 10 mL of sodium fluoride standard working solution II, transfer it to a 100 mL volumetric flask, and dilute to the mark with deionized water. At this time, the concentration is 1 μgF ¯ / mL, recorded as standard working solution III.

[0103] ③ Preparation of total ionic strength buffer (TISAB): weigh 58 g sodium chloride, 0.4 g trisodium citrate (Na 3 C 6 H 5 O 7 ·2H 2 O), add 57 mL of glacial acetic acid to about 500 mL of deionized water, adjust the pH to 5-5.2 with 5 mol / L sodium hydroxide, and make up to 1 L with deionized water.

[0104] ④ Pseudo urine solution: weigh 11.6 g sodium chloride and 2.0 g diammonium hydrogen phosphate, dissolve in 800 mL deionized water, add 1 mL concentrated sulfuric acid, and dilute to 1 L with water.

[0105] (2) Determination of urinary fluoride

[0106] Preparation of urine fluoride standard series: Take 11 50 mL volumetric flasks and prepare the standard series according to Table 1:

[0107] Table 1 Standard series of fluoride for preparing urine

[0108]

[0109] Determination of urine fluoride standard series: Take 6 mL of the above-mentioned fluoride standard solution of each concentration in a micro-dissolving cell, place a stir bar, move to a magnetic stirrer, insert a fluorine electrode and a reference electrode, and measure the potential value of the solution from low solubility to high concentration one by one. After the reading is stable (and the electrode potential change per minute is less than 1 mV), read the potential value, read the potential value and record the temperature during the measurement. Use logarithmic coordinate paper to represent the potential value with equal distances, and the logarithmic coordinates to represent the fluoride ion concentration, and draw a standard curve. The standard curve should be drawn at the same time as the sample is measured.

[0110] Urine sample determination: Mouse urine samples were collected at the 5th, 10th, 15th, and 20th weeks of the experiment. 0.4 mL of urine was accurately pipetted into a micro-solubility pool, and 5.6 mL of a mixed solution of TISAB and pseudo urine solution (1:1) was added. The potential value was determined according to the detection method of the fluorine standard series.

[0111] Calculation of urine samples: According to the measured potential value, find the corresponding fluorine concentration A (µg / mL) on the standard curve. The F content in the urine sample (µg / mL) = A*15.

[0112] (3) Determination of fluoride in serum, feces, tissues and femur

[0113] Preparation of fecal, tissue and femoral fluoride standard series: Take 8 25 mL volumetric flasks and prepare the standard series according to Table 2:

[0114] Table 2 Preparation of feces, tissue and femur fluoride standard series

[0115]

[0116] Determination of the standard series of fluoride in serum, feces, tissues and femurs: Take 8 mL of the above-mentioned fluoride standard solution of each concentration in a micro-dissolving cell, place a stirrer, move to a magnetic stirrer, insert a fluorine electrode and a reference electrode, and measure the potential value of the solution from low solubility to high concentration one by one. After the reading is stable (and the electrode potential change per minute is less than 1 mV), read the potential value, read the potential value and record the temperature during the measurement. Use logarithmic coordinate paper to represent the potential value with equal distances, and the logarithmic coordinates to represent the fluoride ion concentration, and draw the standard curve. The standard curve should be drawn at the same time as the sample is measured.

[0117] Determination of serum fluoride content: serum samples of mice were collected at the 5th, 10th, 15th and 20th weeks of the experiment. 0.2 mL of serum was accurately pipetted into a micro-dissolution pool, and 3.8 mL of deionized water and 4 mL of TISAB were added. The potential value was determined according to the detection method of the fluoride standard series.

[0118] Calculation for serum samples: Based on the measured potential value, find the corresponding fluorine concentration A (µg / mL) on the standard curve. The F content in the serum sample (µg / mL) = A*40.

[0119] Fecal and tissue sample determination: Fecal samples of mice were collected at the 5th, 10th, 15th and 20th weeks of the experiment, and organ, ileum and muscle samples were collected after the 20th week. Accurately weigh 0.2-0.5g of fresh sample and record it, then transfer it to a sterile grinding tube, add 1mL of deionized water to the tube, use a homogenizer to fully homogenize the tissue, centrifuge at 2500 rpm for 20 minutes, accurately pipette 0.5mL of the supernatant of the extract into a micro-dissolver, add 3.5mL of deionized water and 4mL of TISAB, and determine the potential value according to the detection method of the fluorine standard series.

[0120] Calculation for feces and tissue samples: Based on the measured potential value, find the corresponding fluoride concentration A (µg / mL) on the standard curve, record the weighed sample weight as B g, and the F content in feces and tissue samples (µg / g) = A*8*(1+B) / B.

[0121] (4) Femoral sample measurement:

[0122] After the mice were killed at the 21st week of the experiment, the right long bones were taken, the attached muscles and fat were removed, the weight was weighed and accurately recorded, and then dried in an oven at 105°C. The bones were crushed, passed through a 40-mesh sieve, placed in a nickel crucible, carbonized on an electric furnace until smokeless, placed in a muffle furnace, and incinerated at 650°C for 4 hours. The ashes were white powder. The total weight of the ashes was accurately weighed and recorded. 0.025g was taken in a 25mL small beaker, 5 ml of 0.25mol / L hydrochloric acid was added to completely dissolve it, and a drop of 0.05% bromophenol blue was added. It was neutralized with 1mol / L sodium hydroxide until it just showed azure blue (pH=4.0). 12.5mL TISAB was accurately added, diluted to the scale with water, and mixed. A magnetic rod was placed in it, and the electrode was inserted. The determination method was the same as the standard curve.

[0123] Calculation of femur sample results: Based on the measured potential value, find the corresponding fluoride concentration A (µg / mL) on the standard curve, the ratio of the actual weight of dry bone and bone ash C, and the F content in feces and tissue samples (µg / g) = 500*A*C.

[0124] (5) Comparison of pathological changes in intestine, liver and kidney among the groups;

[0125] Detection method: Take part of the lung, liver, kidney, spleen and ileum and fix them with 4% paraformaldehyde solution at room temperature for more than 24 hours, embed them in paraffin and do HE staining, and observe the tissue pathological changes under a light microscope.

[0126] 5. Test results

[0127] (1) Serum, urine and feces fluoride content test results

[0128] like Figure 2 and Figure 3 As shown in the figure, the results of the fluoride content test in the urine and feces of mice exposed to high fluoride showed that the fluoride content in the urine and feces of mice exposed to high fluoride was significantly higher than that in the control group. The fluoride content in the urine and feces of mice in the Lactobacillus johnsonii BS15 group was higher than that in the F group, and the difference in fluoride content in urine between the two groups was significant, indicating that the supplementation of BS15 promoted the excretion of fluoride in urine and feces. Figure 4 As shown, after 20 weeks of high fluoride exposure, the fluoride content in the serum of mice in group F was significantly higher than that in the control group, but the fluoride ion content in the serum of mice in the Lactobacillus johnsonii BS15 supplementation group was significantly lower than that in group F.

[0129] (2) Results of fluoride content test in organs

[0130] like Figure 5-6 As shown in the data, after 20 weeks of high fluoride exposure, there was no significant increase in fluoride accumulation in the lungs and spleen of group F compared with the control group, indicating that fluoride would not accumulate significantly in the lungs and spleen, and the supplementation of probiotic BS15 had no significant effect on the fluoride content in the lungs and spleen. It can be seen that the therapeutic or repair effect of probiotic BS15 on lung and spleen damage caused by high fluoride exposure is not achieved by reducing the accumulation of fluoride in these organs.

[0131] like Figure 7-8 As shown, after 20 weeks of high fluoride exposure, the fluoride accumulation in the liver and kidneys of group F was significantly increased compared with the control group, indicating that fluoride would accumulate significantly in the liver and kidneys, while the supplementation of probiotic BS15 significantly reduced the accumulation of high fluoride in the liver and kidneys, thereby reducing the organ damage caused by the accumulation of fluoride in the liver and kidneys.

[0132] comprehensive Figure 5-8 It can be seen that the treatment and repair mechanism of probiotic BS15 on liver and kidney damage caused by high fluoride exposure is different from that on lung and spleen damage caused by high fluoride exposure.

[0133] (3) Results of fluoride content test in muscles and bones

[0134] like Figure 9-10 As shown in the figure, compared with the control group, high fluoride exposure did not affect the fluoride content in muscle, and there was no difference in muscle fluoride content between the groups. As the tissue with the most fluoride accumulation, the accumulation of fluoride in bone was significantly affected by high fluoride exposure. After 20 weeks of high fluoride exposure, the fluoride content in the bone of group F was significantly higher than that of the control group, and the supplementation of probiotic BS15 significantly reduced the accumulation of fluoride in bone.

[0135] (4) Results of fluoride content test in ileum

[0136] like Fig.11 As shown in the data, after 20 weeks of high fluoride exposure, there was no significant increase in fluoride accumulation in the ileum of group F compared with the control group, indicating that fluoride would not accumulate significantly in the ileum. Although probiotic BS15 was added to the F+BS15 group, the fluoride content in the ileum did not change significantly. It can be seen that the therapeutic or repair effect of probiotic BS15 on ileal damage caused by high fluoride exposure is not achieved by reducing the accumulation of fluoride in the ileum.

[0137] (5) Results of pathological examination of organs and ileum

[0138] like Fig.12 As shown in the figure, compared with the control group, the alveolar septa in the lung tissue of mice exposed to high fluoride were thickened, a large number of inflammatory cells infiltrated the lung septa, and the alveoli were congested. Supplementation of Lactobacillus johnsonii BS15 improved the pathological changes in lung tissue induced by high fluoride exposure.

[0139] like Fig.13 As shown, high fluoride exposure caused liver hemorrhage and vacuolation of liver cells in mice in group F compared with the control group. Supplementation of Lactobacillus johnsonii BS15 improved liver hemorrhage and vacuolation of stem cells induced by high fluoride exposure.

[0140] like Fig.14 As shown, high fluoride exposure caused hyalinization of the central arterioles of the spleen in group F, and Lactobacillus johnsonii BS15 significantly improved the hyalinization of the central arterioles caused by high fluoride exposure.

[0141] like Fig.15 As shown, high fluoride exposure caused the enlargement of the glomeruli in group F, the significant thickening of the glomerular basement membrane, and obvious cell proliferation in the glomerular capillaries. Lactobacillus johnsonii BS15 improved the renal pathological damage caused by high fluoride exposure.

[0142] like Fig.16 As shown, the intestinal epithelial structure of the control group was intact and no pathological changes were observed; the intestinal villi of the F group were sparse, the villi were atrophied and shortened, the mucosa and submucosa were edematous, the Paneth cells were fused, and the dense granules of the Paneth cells showed hyaline degeneration; the F+BS15 group improved the damage to intestinal tissue caused by high fluoride exposure.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The invention discloses an application of lactobacillus in the preparation of a medicine for preventing and / or treating liver and / or bone damage caused by excessive intake of fluoride, wherein the medicine reaches the prevention and / or treatment purpose by reducing the accumulation of fluoride in the liver and / or bone, and the lactobacillus is Lactobacillus johnsonii BS15, which is deposited in China Center for Type Culture Collection, and the deposit number is CCTCC M2013663.

2. The use according to claim 1, characterized in that: The medicine is Lactobacillus johnsonii BS15 bacterial liquid.

3. The use according to claim 2, characterized in that: The concentration of the Lactobacillus johnsonii BS15 bacterial solution is 1×10 8 CFU / mL to 1×10 10 CFU / mL.

4. The use according to claim 3, characterized in that: The concentration of the Lactobacillus johnsonii BS15 bacterial solution is 1×10 9 CFU / mL.

5. The use according to claim 2, characterized in that: The preparation method of the Lactobacillus johnsonii BS15 bacterial liquid comprises the following steps: S1: Activate the Lactobacillus johnsonii BS15 strain at least once using MRS medium; S2: inoculating the activated bacterial solution into a sterile MRS liquid culture medium for culturing, and counting the live bacteria in the cultured bacterial solution; S3: centrifuging the cultured bacterial solution to obtain bacterial cells; S4: According to the live bacteria count results, the bacteria are prepared into a Lactobacillus johnsonii BS15 bacterial liquid of corresponding concentration using a buffer solution.

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