Application of nicotinamide in preparation of preparation for protecting intestinal barrier

Abstract

The invention belongs to the technical field of animal breeding, and particularly relates to application of nicotinamide in preparation of a preparation for protecting intestinal barriers. Tests prove that nicotinamide can improve expression of tight junction protein of small intestine epithelial cells, enhance expression of antioxidant enzyme and reduce oxidative damage; the blood fat level of oxidative stress mice can be reduced, the oxidation resistance of the body is enhanced, and oxidative stress injury is reduced; the intestinal permeability of oxidative stress mice can be reduced, the expression of tight junction protein between intestinal epithelium can be improved, the oxidative damage of intestinal tracts can be relieved, and the health of the intestinal tracts can be protected. The additive amount of the nicotinamide is 100 to 200 mg / kg / d.

Description

Technical Field

[0001] The invention belongs to the technical field of animal breeding, and particularly relates to application of nicotinamide in the preparation of a preparation for protecting intestinal barrier. Background Art

[0002] Vitamins play an important role in regulating animal growth and health. Nicotinamide is the amide form of vitamin B3, which can be used as a component of coenzyme I (NAD) and coenzyme II (NADP) and participate in the body's redox reaction. When animals are in a special physiological metabolic stage or are subjected to external stress, the body is often in a state of high metabolic peroxidation and is prone to oxidative stress. The intestine is a target organ prone to oxidative stress. Intestinal oxidative stress not only affects the digestion and absorption of nutrients, but also causes intestinal barrier dysfunction and increased permeability. Bacteria, toxins and harmful metabolites pass through the intestine into the blood, triggering an inflammatory response and inducing various metabolic diseases. It is known that nicotinamide has been used to treat pellagra and has an antioxidant effect. However, there are currently no reports on the effect of nicotinamide on intestinal barrier function. Summary of the invention

[0003] The purpose of the present invention is to provide an application of nicotinamide in the preparation of a preparation for protecting the intestinal barrier. Nicotinamide can enhance antioxidant capacity, reduce the increase in permeability induced by oxidative stress, promote the expression of intestinal tight junction proteins, and protect the intestinal mucosal barrier.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] An application of nicotinamide in the preparation of an intestinal barrier protection preparation. Experiments have shown that nicotinamide effectively reduces the degree of oxidative damage to intestinal epithelial cells in dairy cows. Combining the results of cell survival rate, lactate dehydrogenase activity and total antioxidant capacity, the appropriate concentration of nicotinamide for oxidative damage to intestinal epithelial cells in dairy cows is 2mM-4mM. Nicotinamide can reduce intestinal oxidative stress and alleviate intestinal damage. In addition, 2mM-4mM nicotinamide can regulate the autophagy pathway of intestinal epithelial cells in oxidatively stressed dairy cows, playing a key role in alleviating oxidative damage. Moreover, this experiment unexpectedly found that 100-200mg / kg / d of nicotinamide can significantly alleviate the oxidative stress of mice caused by diquat, increase the weight of mice, and protect the spleen of mice, further indicating that nicotinamide mainly acts on the small intestine and improves intestinal health.

[0006] Preferably, the preparation is a medicine, a health product, a food or a feed additive.

[0007] Preferably, the protection of the intestinal barrier includes the protection against intestinal tissue damage, the protection against the mucus barrier and the protection against the integrity of the intestinal mucosal mechanical barrier.

[0008] Preferably, the intestinal tract is the small intestine.

[0009] Preferably, when nicotinamide is used as a feed additive, the applicable animals are cattle or sheep, especially dairy cows.

[0010] Preferably, the daily feeding amount of nicotinamide is 100-200 mg / kg / d, which can effectively repair oxidative damage in the jejunum and ileum, protect intestinal morphology and structure, promote the absorption of nutrients, and upregulate the expression of tight junction proteins in intestinal damage caused by oxidative stress.

[0011] The beneficial effects of the present invention are:

[0012] 1. Nicotinamide can increase the expression of tight junction proteins in small intestinal epithelial cells, enhance the expression of antioxidant enzymes, and reduce oxidative damage;

[0013] 2. Nicotinamide can reduce the blood lipid levels of mice with oxidative stress, enhance the body's antioxidant capacity, and reduce oxidative stress damage.

[0014] 3. Nicotinamide can reduce the intestinal permeability of mice with oxidative stress, increase the expression of tight junction proteins between intestinal epithelial cells, alleviate intestinal oxidative damage, and protect intestinal health. The dosage of nicotinamide is 100-200 mg / kg / d. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the effect of nicotinamide on oxidative damage of intestinal epithelial cells of dairy cows induced by hydrogen peroxide, A is the survival rate of intestinal epithelial cells of dairy cows treated with different concentrations of nicotinamide, B is the survival rate of intestinal epithelial cells of dairy cows induced by hydrogen peroxide treated with different concentrations of nicotinamide, C is the lactate dehydrogenase activity of intestinal epithelial cells of dairy cows induced by hydrogen peroxide treated with different concentrations of nicotinamide, and D is the total antioxidant capacity of intestinal epithelial cells of dairy cows induced by hydrogen peroxide treated with different concentrations of nicotinamide;

[0016] Figure 2 The effect of nicotinamide on the expression of antioxidant enzyme proteins in intestinal epithelial cells of dairy cows induced by hydrogen peroxide. A is the protein expression graph, B is the expression level of catalase protein, and C is the expression level of superoxide dismutase protein.

[0017] Figure 3 The effect of nicotinamide on the expression of proteins related to the barrier function of intestinal epithelial cells of dairy cows induced by hydrogen peroxide. A is the protein expression graph, B is the expression level of Occludin protein, C is the expression level of Claudin-2 protein, and D is the expression level of ZO-1 protein.

[0018] Figure 4is the effect of nicotinamide on hydrogen peroxide-induced autophagy in dairy cow intestinal epithelial cells, A is the immunofluorescence expression of total cell autophagy; B is the map of autophagy-related proteins and their expression levels, and C is the immunofluorescence expression of autophagy protein LCII;

[0019] Figure 5 This is the effect of nicotinamide on the mitochondrial membrane potential of dairy cow intestinal epithelial cells induced by hydrogen peroxide. The left picture is the immunofluorescence image of mitochondrial membrane potential, and the right picture is the calculated value of the immunofluorescence image.

[0020] Figure 6 The figure shows the effects of autophagy blockers and nicotinamide on the expression level of autophagy proteins in intestinal epithelial cells of dairy cows induced by hydrogen peroxide. A is the protein expression graph, B is the expression level of LCII protein, C is the expression level of P62 protein, D is the lactate dehydrogenase activity of cell supernatant, and E is the total antioxidant capacity of cell supernatant.

[0021] Figure 7 The effect of different concentrations of nicotinamide on the body weight of mice induced by oxidative stress caused by diquat;

[0022] Figure 8 The effects of different concentrations of nicotinamide on organ indexes of mice induced by diquat-induced oxidative stress, A is the liver index, B is the kidney index, and C is the spleen index;

[0023] Fig. 9 The effect of different concentrations of nicotinamide on the jejunal morphology of mice with oxidative stress induced by diquat. The upper figure is a paraffin section, and the lower figure is the villus height, crypt depth and their ratio;

[0024] Fig.10 The effect of different concentrations of nicotinamide on the ileum morphology of mice with diquat-induced oxidative stress. The upper figure is a paraffin section, and the lower figure is the villus height, crypt depth and their ratio;

[0025] Fig.11 The effect of nicotinamide on serum antioxidant and stress-related indices in mice with oxidative stress induced by diquat;

[0026] Fig.12 is the effect of nicotinamide on the expression level of intestinal barrier function genes in mice with oxidative stress induced by diquat;

[0027] Fig.13 The effect of nicotinamide on the expression level of intestinal barrier function proteins in mice with oxidative stress induced by diquat. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0029] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0030] The reagents used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent stores.

[0031] Example 1 Effect of nicotinamide on alleviating oxidative damage of intestinal epithelial cells in dairy cows

[0032] The oxidative stress model of bovine intestinal epithelial cells was established by inducing 0.5 mM hydrogen peroxide. Different concentrations (0, 1, 2, 4 and 8 mM) of nicotinamide (NAM) were added to treat the cells for 12 hours, and the cell survival rate, lactate dehydrogenase (LDH) release and total antioxidant capacity (T-AOC) were measured.

[0033] Cell viability was assessed using the Cell Counting Kit-8 assay. Take cells in good growth condition, resuspend the cells in digestion medium and inoculate them in 96-well plates, 100 μL per well. Place in an incubator at 37°C and 5% CO2. When the cells are fused to 70-80%, discard the old culture medium, wash twice with PBS, add a fresh culture medium solution containing 10% CCK-8 to each well, and continue to culture in an incubator at 37°C and 5% CO2 for 2 hours. Finally, use an ELISA reader (BioTek, Winooski, VT, USA) to measure the absorbance of each well at 450nm (OD450). The experiment was divided into a blank group, a control group and an experimental group, with 8 parallel replicates in each group, and the experiment was repeated 3 times. Relative cell survival rate = (treatment well OD450-blank well OD450) / (control well OD450-blank well OD450)×100%.

[0034] Lactate dehydrogenase detection: Inoculate cells in a 6-well plate, and when the cells are fused to 70-80%, treat the cells according to the above experimental design. After treating the cells, carefully aspirate the cell culture supernatant into a 1.5mL EP tube, and the measurement steps are carried out according to the lactate dehydrogenase assay kit of Nanjing Jiancheng Bioengineering Institute. Finally, use an ELISA reader to measure the absorbance of each well at 450nm (OD450). Lactate dehydrogenase activity (U / L) = (measured OD450-control OD450) ÷ (standard OD450-blank OD450) × 0.2μmol / L × 1000

[0035] Total antioxidant capacity detection: The cells were plated in 6-well plates. After 12 hours of different treatments, the cell culture supernatant was discarded, washed twice with PBS, and an appropriate amount of PBS was added. The cells were scraped with a cell scraper and collected in a 1.5mL EP tube. The cells were ultrasonically broken to release antioxidant substances, centrifuged at 4°C, 12000rmp / min for 5min, and the supernatant was taken for determination. The determination steps were carried out according to the instructions of the total antioxidant capacity test kit of Nanjing Jiancheng Bioengineering Institute. The absorbance value of each well was measured at 405nm by an enzyme reader, and the total antioxidant capacity of the sample was calculated according to the standard curve formula. Finally, the protein concentration of each sample was determined and the ratio was made. The results of the total antioxidant capacity of the cells were expressed in mmoL / g.

[0036] Statistical analysis The data of this experiment were first processed using Excel, and then IBM SPSS Statistics26 was used to perform one-way analysis of variance on the data, and the Duncan method was used for multiple comparisons.

[0037] Results The data on the effect of nicotinamide on hydrogen peroxide-induced oxidative damage in dairy cow small intestinal epithelial cells are shown in Figure 1 A is the survival rate of intestinal epithelial cells of dairy cows treated with different concentrations of nicotinamide. Figure 1 A shows that, compared with the control group, different concentrations of nicotinamide significantly increased cell survival rate (P < 0.05), among which 2mM nicotinamide was the most significant (P < 0.05). The survival rate of cow intestinal epithelial cells induced by hydrogen peroxide treated with different concentrations of nicotinamide is shown in Figure 1 As shown in Figure B, compared with the control group, the addition of 0.5mM hydrogen peroxide significantly reduced the survival rate of cells (P < 0.05); the addition of 2mM nicotinamide significantly increased the survival rate of hydrogen peroxide-damaged cells (P < 0.05), and other concentrations of nicotinamide had no effect on the results of hydrogen peroxide-damaged cells. In addition, compared with the control group, hydrogen peroxide significantly increased the activity of lactate dehydrogenase (P < 0.05), while the addition of 1, 2, 4, and 8mM nicotinamide (NAM) significantly reduced the effect of hydrogen peroxide on the activity of cell lactate dehydrogenase (P < 0.05), and the effect of 2mM and 4mM nicotinamide was the most significant ( Figure 1 C, P<0.05). Adding 0.5mM hydrogen peroxide significantly reduced the total antioxidant capacity of the cells (P<0.05), and adding 1, 2, 4, and 8mM nicotinamide significantly increased the total antioxidant capacity of the cells induced by hydrogen peroxide ( Figure 1 D, P<0.05).

[0038] Conclusion: In this experiment, the addition of hydrogen peroxide can significantly induce cell damage, while different concentrations of nicotinamide reduced lactate dehydrogenase activity and increased total antioxidant capacity. This shows that nicotinamide effectively alleviates the degree of cell damage. Combining the results of cell survival rate, lactate dehydrogenase activity and total antioxidant capacity, the appropriate concentrations of nicotinamide to alleviate oxidative damage in dairy cow small intestinal epithelial cells are 2 and 4 mM.

[0039] Example 2 Effect of nicotinamide on antioxidant enzyme activity and barrier function of intestinal epithelial cells of dairy cows

[0040] An oxidative stress model was established in bovine intestinal epithelial cells induced by 0.5 mM hydrogen peroxide (H2O2). 2 mM and 4 mM nicotinamide (NAM) were selected to co-treat the cells for 12 hours. The experiment was divided into control group, oxidative stress group (OS), treatment group 1 (OS2N, H2O2+2 mM NAM) and treatment group 2 (OS4N, H2O2+4 mM NAM). The activities of antioxidant enzymes and the expressions of tight junction proteins were determined.

[0041] RNA extraction and real-time fluorescence quantitative PCR Total RNA was extracted from treated cells using Total RNA extraction reagent (Takara, Beijing, China) and operated according to the instructions provided by the manufacturer. The concentration and purity of RNA were determined by measuring the absorbance at 260 nm and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Science Inc.). Reverse transcription was then performed using the Prime SCRIPT RT kit (Takara). Quantitative PCR was performed on a CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA) using the SYBR PreMix Ex Taq II kit (Takara). The PCR cycle program included a 20 μL reaction system (containing 6.4 μL of enzyme-free water, 0.8 μL of forward primer, 0.8 μL of reverse primer, 2 μL of cDNA, and 10 μL of SYBR premix Ex Taq). First, denaturation was performed at 95°C for 1 min, followed by 40 cycles of amplification at 95°C for 30 s and 58°C for 60 s. The results were normalized to the expression level of β-actin and the expression of 2 -ΔΔCT Method to calculate.

[0042] Protein extraction and Western blotting After cell incubation, the supernatant was removed and total protein was extracted from the cells using RIPA lysis buffer, protease inhibitor cocktail (100×) and phosphatase inhibitor cocktail (100×) (Beyotime, Shanghai, China), and nuclear protein was extracted using a cytoplasmic and nuclear protein extraction kit and phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). Protein concentration was determined using a biuret acid protein assay kit (Beyotime, Shanghai, China). Samples were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels for separation and then transferred to polyvinylidene difluoride (PVDF) membranes (Millikon, Burlington, MA, USA) using transfer buffer. Electrophoresis buffer, transfer buffer, and Tris-buffered saline containing Tween 20 were prepared according to standard methods. Western blotting equipment was provided by Bio-Rad. PVDF membranes were blocked in 5% bovine serum albumin (BSA, diluted 1:20, using Tris-buffered saline containing Tween 20) for 1 h at room temperature and then incubated with primary antibodies on a shaker at 4°C. The primary antibodies used were as follows: SOD2 (dilution 1:1000; CellSignaling Technology, Massachusetts, USA), CAT (dilution 1:1000; Abclonal, Wuhan, China), ZO-1 (dilution 1:5000, Proteintech, Wuhan, China), Occludin (dilution 1:2000, Proteintech), Claudin-2 (dilution 1:1000, Proteintech), LC3 (dilution 1:1000, Abmart, Shanghai, China), p62 (dilution 1:1000, CellSignaling Technology), Beclin1 (dilution 1:1000, Cell Signaling Technology), and β-actin (dilution 1:5000, Abclonal). After washing three times with washing buffer (Tris-buffered saline containing Tween 20) on a shaker for 10 min each time, the membrane was incubated for 1 h at room temperature on a shaker with goat anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody (dilution 1:10,000, Abclonal). The PVDF membrane was incubated with enhanced chemiluminescence substrate solution and visualized by an enhanced chemiluminescence (Beyotime, Shanghai, China) immunoblotting detection system. Images were acquired using a gel imaging system (Tanon, Shanghai, China). Finally, the relative amount of protein bands was quantified using Image J software 1.53 (NIH, Bethesda, MD, USA) and normalized to the β-actin protein in each sample.

[0043] Statistical analysis The data of this experiment were first processed using Excel, and then IBM SPSS Statistics26 was used to perform one-way analysis of variance on the data, and the Duncan method was used for multiple comparisons.

[0044] result

[0045] 1. Effects of nicotinamide on relative mRNA expression of antioxidant enzymes and tight junction proteins

[0046] The results of the effects of nicotinamide on the relative mRNA expressions of antioxidant enzymes and tight junction proteins are shown in Table 1 .

[0047] Table 1. Effects of nicotinamide on relative mRNA expression of antioxidant enzymes and tight junction proteins

[0048]

[0049] As shown in Table 1, compared with the control group, the addition of 0.5mM H2O2 significantly reduced the expression of the cell CAT gene (P<0.05), and the addition of 4mM NAM significantly increased the expression of this gene (P<0.05), and the addition of 2mM NAM had no effect on the expression of this gene; the addition of 0.5mM H2O2 had a trend of reducing the expression of the SOD2 gene (P=0.087), and the addition of 2 and 4mM NAM had a trend of increasing SOD2; the addition of 0.5mM H2O2 had no effect on the expression of GPX1, and the addition of NAM had no effect. The results of cell tight junction protein mRNA genes showed that compared with the control group, the addition of H2O2 in the OS group significantly downregulated the expression of the Claudin-2 gene (P<0.05), and the addition of 2 and 4mM NAM significantly upregulated the expression of the gene (P<0.05); it had no effect on the expression of the ZO-1 and Occludin genes.

[0050] 2. Effect of nicotinamide on the expression of antioxidant enzyme proteins in bovine intestinal epithelial cells induced by hydrogen peroxide.

[0051] Effects of nicotinamide on the expression of antioxidant enzymes in intestinal epithelial cells of dairy cows induced by hydrogen peroxide Figure 2 As shown, A is the protein expression graph, B is the catalase protein expression level, and C is the superoxide dismutase protein expression level. Figure 2 It can be seen that compared with the control group, the addition of 0.5mM H2O2 significantly reduced the expression levels of cell CAT and SOD2 proteins, 4mM NAM significantly increased the expression levels of CAT and SOD2 proteins (P<0.05), and the addition of 2mM NAM had no effect.

[0052] 3. Effect of nicotinamide on the expression of barrier function-related proteins in intestinal epithelial cells of dairy cows induced by hydrogen peroxide

[0053] Effects of nicotinamide on the expression of barrier function-related proteins in intestinal epithelial cells of dairy cows induced by hydrogen peroxide Figure 3 As shown, compared with the control group, the addition of H2O2 in the OS group significantly downregulated the amounts of Occludin, Claudin and ZO-1 proteins (P<0.05), and the addition of 2 and 4 mM NAM upregulated the expression of Occludin and Claudin proteins (P<0.05); while the addition of 2 mM NAM upregulated the expression of ZO-1 protein (P<0.05), and the addition of 4 mM NAM had no effect.

[0054] Conclusion: Nicotinamide can significantly increase the expression of tight junction proteins Occludin, Claudin and ZO-1 and the protein expression of antioxidant enzymes CAT and SOD2 in intestinal damage caused by oxidative stress, relieve intestinal oxidative stress, and reduce intestinal damage. The appropriate concentration of nicotinamide to relieve oxidative damage in small intestinal epithelial cells of dairy cows is 4mM.

[0055] Example 3 Effect of nicotinamide on autophagy of intestinal epithelial cells in dairy cows

[0056] The experimental design and treatment were the same as in Example 2. The MDC method was used to detect the level of cellular autophagy flow, the expression of autophagy-related proteins was determined, the fluorescence degree of LC3 at the level of cellular autophagy, and the mitochondrial membrane potential were detected by immunofluorescence. The autophagy inhibitor chloroquine (CQ, 100 μm) was used to study the effect of autophagy inhibition on NAM alleviating oxidative damage.

[0057] Detection of autophagy level and mitochondrial membrane potential The autophagy staining detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. After the treated cells were stained, they were incubated at 37°C in the dark for 1 hour and then washed with detection buffer. The mitochondrial membrane potential detection kit was purchased from Shanghai Aibixin Biotechnology Co., Ltd. (Shanghai, China). The collected cells were stained with JC-1 dye, incubated at 37°C for 40 minutes, and then the red and green fluorescence were observed using a fluorescence microscope.

[0058] After immunofluorescence treatment, cells were fixed in 4% paraformaldehyde for 40 minutes, washed twice, and then permeabilized with 0.3% TritonX-100 for 10 minutes at room temperature. Next, cells were blocked with 5% blocking solution for 2 hours at room temperature, and then incubated with 1:50 diluted primary antibodies overnight at 4°C in the dark, followed by washing. After that, fluorescent secondary antibodies were added at a dilution of 1:1000, incubated at room temperature for 1 hour, and then stained with DAPI solution at room temperature for 10 minutes. Samples were mounted and fluorescent images were taken using a fluorescence microscope.

[0059] Autophagy inhibition 100 μM chloroquine (CQ, Sigma-Aldrich) was used as an autophagy inhibitor to block cellular autophagy. The LDH concentration in the cell supernatant, the total antioxidant capacity (T-AOC) in the cells, and the expression of autophagy-related proteins were then measured.

[0060] Statistical analysis The data of this experiment were first processed using Excel, and then IBM SPSS Statistics26 was used to perform one-way analysis of variance on the data, and the Duncan method was used for multiple comparisons.

[0061] result

[0062] 1. Effect of nicotinamide on hydrogen peroxide-induced autophagy in intestinal epithelial cells of dairy cows

[0063] Effects of nicotinamide on hydrogen peroxide-induced autophagy in intestinal epithelial cells of dairy cows Figure 4 ,Depend on Figure 4 A shows that compared with the control group, the level of green fluorescence in the cells of the OS group was significantly increased (P<0.05), while the 4mM NAM treatment group was significantly decreased compared with the OS group (P<0.05), and the 2mM NAM group had no significant effect. In addition, compared with the control group, the expression levels of Beclin1, P62 and LC3 proteins in the OS group were significantly increased (P<0.05); compared with the OS group, the 4mM NAM group significantly reduced the expression levels of Beclin1, P62 and LC3 proteins (P<0.05); the 2mM NAM group only reduced the expression level of LC3 protein (P<0.05), and had no effect on the expression levels of Beclin1 and P62 proteins ( Figure 4 B). The results of immunofluorescence showed that compared with the control group, the green fluorescence area of ​​LC3 in each cell in the OS group was significantly increased (P<0.05); the green fluorescence area in the 2 and 4 mM NAM groups was significantly decreased compared with the OS group ( Figure 4 C, P<0.05).

[0064] 2. Effect of nicotinamide on hydrogen peroxide-induced mitochondrial membrane potential of intestinal epithelial cells in dairy cows

[0065] Figure 5 The results are from the effect of nicotinamide on hydrogen peroxide-induced mitochondrial membrane potential of cow intestinal epithelial cells. Figure 5 It can be seen that compared with the control group, the red / green ratio of the positive control group (CCCP) and OS group was significantly reduced (P<0.05), and the red / green ratio of the 2 and 4 mM NAM groups was significantly increased compared with the positive control group (CCCP) and OS group (P<0.05). This result shows that hydrogen peroxide reduces the mitochondrial membrane potential, and nicotinamide can effectively alleviate it.

[0066] 3. Effects of autophagy inhibitors and nicotinamide on the expression level of autophagy proteins in intestinal epithelial cells of dairy cows treated with hydrogen peroxide

[0067] Figure 6 The effects of autophagy blockers and nicotinamide on the expression levels of autophagy proteins in intestinal epithelial cells of dairy cows treated with hydrogen peroxide, such as Figure 6 As shown in the figure, compared with the control group, the effect of adding nicotinamide alone on the activity of lactate dehydrogenase and total antioxidant capacity of cells and the expression of autophagy proteins was consistent with the previous results. CQ significantly upregulated the expression of autophagy protein LC3 (P<0.05), and NAM significantly downregulated the expression of LC3 and P62 (P<0.05). In addition, after CQ blocked autophagy, the content of lactate dehydrogenase activity decreased significantly (P<0.05), and the content of total antioxidant capacity increased significantly (P<0.05).

[0068] Conclusion: NAM can play a key role in alleviating oxidative damage of intestinal epithelial cells in dairy cows by regulating the autophagy pathway. The appropriate concentrations of nicotinamide to alleviate oxidative damage of intestinal epithelial cells in dairy cows are 2 and 4 mM.

[0069] Example 4 Effects of different concentrations of nicotinamide on diquat-induced oxidative stress in mice

[0070] Sixty 7-week-old ICR female mice (BW 24g, purchased from Hangzhou Medical College, acclimation period of 7d, free access to food and water) were selected and divided into 5 groups (n=12) according to the principle of similar body weight by completely random design, namely control group (CON group), OS group, OS+100mg / kg / dNAM group, OS+200mg / kg / dNAM group, OS+500mg / kg / dNAM group. All groups of mice were fed the same basic diet. On the first and fourth days of the formal start of the experiment, the control group was intraperitoneally injected with 0.2mL of normal saline, and the other groups were intraperitoneally injected with an equal volume of diquat (CAS No.6385-62-2, Aladdin) at a dose of 10mg / kg BW to establish an oxidative stress model. Nicotinamide (CAS No.98-92-0, purity ≥ 99.5%, Sigma) was prepared into an aqueous solution with 0.9% saline and administered by regular intragastric administration. The control group was replaced with 0.9% saline, and the intragastric volume was 0.2 mL. The experimental period was 14 days, and body weight was measured every day. After fasting for 12 hours at the end of the experiment, the mice were killed by cervical dislocation and dissected.

[0071] Organ development determination: The liver, spleen and kidney were collected, cleaned with saline and weighed, and the index of each organ was calculated according to the formula: organ index (%) = organ weight / live weight before slaughter × 100.

[0072] Preparation and observation of intestinal tissue sections 1 cm of clean jejunal and ileal tissue was fixed in 4% paraformaldehyde and left to stand for 24 hours. The mouse jejunum and ileum were removed, cut into small sections of about 4 mm and placed in an embedding box, washed overnight, and paraffin sections of jejunal and ileal tissues were prepared through a series of steps including tissue gradient alcohol dehydration, transparency, wax immersion, embedding, and finally H&E staining. The intestinal morphology was observed using an inverted integrated fluorescence microscope, and the villus height (VH) and crypt depth (CD) were measured, and the ratio of villus height to crypt depth (VH / CD) was calculated.

[0073] Statistical analysis The data of this test were first processed with Excel, and then IBM SPSS Statistics26 was used to perform one-way analysis of variance on each data, and Duncan's method was used for multiple comparisons, and Graphpad prism was used for image drawing. The results were expressed as mean and standard error (SEM), P < 0.05 was considered significant, and 0.05 ≤ P < 0.10 was considered a trend.

[0074] result

[0075] 1. Effects of different concentrations of nicotinamide on the body weight of mice with oxidative stress induced by diquat

[0076] Figure 7 The effects of different concentrations of nicotinamide on the body weight of mice induced by diquat oxidative stress were analyzed. Figure 7 It can be seen that compared with the OS group, the body weight of mice in the 200NAM+OS group increased significantly on days 7-9 and 14 (P<0.05), and there was an increasing trend on day 12 (0.05≤P<0.10); the body weight of mice in the 500NAM+OS group increased significantly on days 7-8 and 14 (P<0.05), and there was an increasing trend on day 9 (0.05≤P<0.10). The average body weight of the 200NAM+OS group and the 500NAM+OS group exceeded that of the control group on day 6, and the average body weight of mice in the 200NAM+OS group was the highest.

[0077] 2. Effects of different concentrations of nicotinamide on organ indexes of mice with oxidative stress induced by diquat

[0078] Figure 8 The effects of different concentrations of nicotinamide on organ indexes of mice induced by diquat oxidative stress were analyzed. Figure 8As shown, the liver index and kidney index of the five groups of mice were not significantly different (P>0.05), while the spleen index was significantly different (P<0.05). Among them, compared with the OS group, the spleen index of the 200NAM+OS group and the 100NAM+OS group decreased significantly (P<0.05), while there was no significant difference in the 500NAM+OS group (P<0.05). The spleen index of the OS group was higher than that of the control group, with no significant difference (P>0.05), while the spleen index of the 200NAM+OS group was lower than that of the control group (P<0.05).

[0079] 3. Effects of different concentrations of nicotinamide on intestinal morphology of mice with diquat-induced oxidative stress

[0080] Fig. 9 The figure below shows the effect of different concentrations of nicotinamide on the jejunal morphology of mice with oxidative stress induced by diquat. The upper figure shows the paraffin section, and the lower figure shows the height of jejunal villi, crypt depth and their ratio. Fig. 9 It can be seen that compared with the CON group, the jejunal villus length and villus-crypt ratio of the OS group were significantly reduced, and the crypt depth was significantly increased (P<0.05). Compared with the OS group, the jejunal villus length and villus-crypt ratio of the 200NAM+OS group and the 100NAM+OS group were significantly increased, and the crypt depth was significantly decreased (P<0.05); while there was no significant difference between the 500NAM+OS group and the OS group (P>0.05).

[0081] Fig.10 The figure below shows the effect of different concentrations of nicotinamide on the ileum morphology of mice with oxidative stress induced by diquat. The upper figure shows the paraffin section, and the lower figure shows the height of ileal villi, crypt depth and their ratio. Fig.10 It can be seen that compared with the CON group, the length of ileal villi and the villus-crypt ratio of the OS group were significantly reduced, and the crypt depth was significantly increased (P<0.05). Compared with the OS group, the length of ileal villi in the 200NAM+OS group was significantly increased (P<0.05), and the villus-crypt ratio of the ileum was significantly increased (P<0.05); the depth of ileal crypts in the 200NAM+OS group and the 500NAM+OS group was significantly reduced (P<0.05); while there was no significant difference between the 100NAM+OS group and the OS group (P>0.05).

[0082] Conclusion: This experiment unexpectedly found that 100-200 mg / kg / d of nicotinamide can significantly alleviate the oxidative stress of mice caused by diquat, increase the weight of mice, and protect the spleen of mice, but there is no significant difference in the kidney and liver indexes between the groups, which further shows that nicotinamide mainly acts on the small intestine and improves intestinal health. In addition, 100-200 mg / kg / d of nicotinamide significantly alleviated the oxidative damage of the jejunum and ileum of mice, protected the morphology and structure of the small intestine, and promoted the absorption of nutrients. Therefore, the appropriate concentration of nicotinamide to alleviate organ and intestinal damage in mice with oxidative stress is 100-200 mg / kg / d.

[0083] Example 5 Effects of nicotinamide on serum antioxidant and biochemical indices in mice with oxidative stress induced by diquat

[0084] Thirty-six 7-week-old ICR female mice (BW 24g, purchased from Hangzhou Medical College, acclimation period of 7 days, free access to food and water) were selected and divided into three groups (n=12) according to the principle of similar body weight by completely random design, namely control group (CON), oxidative stress group (OS) and nicotinamide group (NAM). All groups of mice were fed the same basic diet. On the first and fourth days of the formal start of the experiment, the control group was intraperitoneally injected with 0.2mL of normal saline, and the other two groups were intraperitoneally injected with an equal volume of diquat (CAS No.6385-62-2, Aladdin) at a dose of 10mg / kg BW to establish an oxidative stress model. Nicotinamide (CAS No.98-92-0, purity≥99.5%, Sigma) was prepared into an aqueous solution with 0.9% normal saline at a concentration of 200mg / kg / d, and was administered by regular gavage, freshly prepared and used, and the control group was replaced with 0.9% normal saline, with a gavage volume of 0.2mL. The experimental period was 14 days. After fasting for 12 hours at the end of the experiment, the blood of the mice was collected by removing the eyeballs and put into EP tubes. The tubes were allowed to stand at 4°C for 2 hours and centrifuged at 3500 r / min for 20 minutes. The supernatant was collected for later use.

[0085] The serum antioxidant and biochemical indexes were determined using kits (purchased from Nanjing Jiancheng Bioengineering Institute): low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), total cholesterol (TC), glucose (GLu); total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reactive oxygen species (ROS), diamine oxidase (DAO), D-lactate (D-LA) and lactate dehydrogenase (LDH).

[0086] result

[0087] 1. Effects of nicotinamide on serum biochemical parameters of mice with oxidative stress induced by diquat

[0088] The effects of nicotinamide on serum biochemical parameters of mice with oxidative stress induced by diquat are shown in Table 2.

[0089] Table 2. Effects of nicotinamide on serum biochemical parameters of mice induced by diquat-induced oxidative stress

[0090]

[0091] As shown in Table 2, compared with the control group, the serum triglyceride, total cholesterol, and glucose levels of mice in the OS group were significantly increased (P<0.001), low-density lipoprotein was significantly increased (P<0.05), and high-density lipoprotein was not significantly affected (P>0.05). Compared with the OS group, the serum high-density lipoprotein, triglyceride, total cholesterol, and glucose levels of mice in the NAM group were significantly decreased (P<0.001), while high-density lipoprotein was significantly decreased (P=0.021). The above results show that nicotinamide can effectively reduce the blood lipid level of mice with oxidative stress, and improve the lipid metabolism disorder and liver function damage of mice with oxidative stress.

[0092] 2. Effect of nicotinamide on serum antioxidant capacity of mice with diquat-induced oxidative stress

[0093] Fig.11 The effect of nicotinamide on serum antioxidant indexes in mice with oxidative stress induced by diquat. Fig.11 It can be seen that compared with the control group, the total antioxidant capacity of serum of mice in the OS group was significantly decreased, and the activities of glutathione peroxidase, superoxide dismutase, catalase and lactate dehydrogenase as well as the contents of ROS, DAO and D-LA in serum were significantly increased (P<0.05). Compared with the OS group, the serum T-AOC of mice in the NAM group was significantly increased, and the activities of GSH-Px, SOD, CAT and LDH as well as the contents of reactive oxygen, diamine oxidase and D-lactic acid in serum were significantly decreased (P<0.05).

[0094] In general, nicotinamide can reduce blood lipid levels in mice with oxidative stress, enhance the antioxidant capacity of mice, and alleviate oxidative damage in mice. The appropriate concentration of nicotinamide to alleviate lipid metabolism disorders and enhance antioxidant capacity in mice with oxidative stress is 200 mg / kg / d.

[0095] Example 6 Effect of Nicotinamide on Intestinal Barrier Function in Mice Subjected to Diquat-Induced Oxidative Stress

[0096] The experimental design and treatment were the same as those in Example 5. After fasting for 12 hours at the end of the experiment, the mice were killed by cervical dislocation, the abdominal cavity was opened to obtain 2 cm of jejunum, the contents were rinsed with sterile saline, and then placed in a sterile 2 mL cryotube, which was quickly frozen in liquid nitrogen; the cecum was cut with a sterile cotton rope and placed in a sterile 2 mL cryotube, which was quickly frozen in liquid nitrogen, and the samples were immediately transferred to a -80°C refrigerator for standby use.

[0097] mRNA expression of genes related to intestinal barrier function The total RNA of jejunal tissue was extracted using RNAios Plus (Total RNA extraction reagent, TaKaRa) kit, and the RNA concentration and quality were measured and then reverse transcribed to synthesize cDNA (PrimeScriptTMRTreagent Kit with gDNAEraser, TaKaRa). The relative expression of related genes was detected using TB Green Premix ExTaq kit (TaKaRa), the reaction system was 10 μL, and the experiment was performed on ice.

[0098] Expression of proteins related to intestinal barrier function Jejunal tissue samples were collected, and total protein was extracted using RIPA lysis buffer (P0013B, Beyotime), and protein concentration was determined (BCA method, Beyotime). Gel preparation was performed according to the rapid gel preparation kit, protein samples were separated, membrane was transferred (PVDF membrane), blocked, incubated with primary and secondary antibodies, exposed for color development, and grayscale values ​​of each group were calculated using Image J for data analysis.

[0099] result

[0100] 1. Effect of nicotinamide on the mRNA expression level of intestinal barrier function in mice with oxidative stress induced by diquat

[0101] Effects of nicotinamide on the expression level of intestinal barrier function genes in mice with diquat-induced oxidative stress Fig.12 As shown in the figure, compared with the control group, the relative mRNA expression levels of ZO-1, Occludin and Claudin-1 in the jejunum of mice in the OS group were significantly decreased (P<0.05). Compared with the OS group, the relative mRNA expression levels of ZO-1 and Claudin-1 in the jejunum of mice in the NAM group were significantly increased (P<0.05), and there was no significant difference in the relative mRNA expression level of Occludin (P>0.05).

[0102] 2. Effect of nicotinamide on the expression level of intestinal barrier function proteins in mice with oxidative stress induced by diquat

[0103] Effects of nicotinamide on the expression level of intestinal barrier function proteins in mice with diquat-induced oxidative stress Fig.13 As shown in the figure, compared with the control group, the protein expression level of ZO-1 in the jejunum of mice in the OS group was significantly decreased (P<0.05). Compared with the OS group, the protein expression level of ZO-1 in the jejunum of mice in the NAM group was significantly increased (P<0.05). There was no significant difference in the expression level of Occludin protein among the three groups (P>0.05).

[0104] In general, nicotinamide alleviated the oxidative damage of the mouse intestine, reduced the intestinal permeability of the mouse, increased the expression of tight junction proteins ZO-1 and Claudin-1 between the intestinal epithelium, and thus enhanced the function of the intestinal epithelial barrier. The appropriate concentration of nicotinamide to enhance the intestinal barrier function of oxidative stress mice is 200 mg / kg / d.

[0105] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

Claims

1. An application of nicotinamide in the preparation of a preparation for protecting the intestinal barrier.

2. The use according to claim 1, characterized in that: The preparation is a medicine, a health product, a food or a feed additive.

3. The use according to claim 1, characterized in that: The protection of the intestinal barrier includes the protection against intestinal tissue damage, the protection against the mucus barrier and the protection against the integrity of the intestinal mucosal mechanical barrier.

4. The use according to claim 1, characterized in that: The intestinal tract is the small intestine.

5. The use according to claim 1, characterized in that: When nicotinamide is used as a feed additive, the applicable animals are cattle or sheep.

6. The use according to claim 1 or 5, characterized in that: The daily feeding amount of nicotinamide is 100-200 mg / kg / d.

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