Application of selenium-enriched yeast combined with lactobacillus plantarum in improvement of liver injury caused by cadmium

Abstract

According to the application of the selenium-enriched yeast combined with the lactobacillus plantarum to improvement of the liver injury caused by cadmium, in a cadmium chloride induced mouse liver injury model, the selenium-enriched yeast combined with the lactobacillus plantarum can effectively relieve the injury to the liver through the anti-oxidation effect and the inhibition of inflammation and apoptosis. The invention shows the possibility of combining the selenium-enriched yeast with the lactobacillus plantarum as the medicine for treating the liver injury, and provides a new application field for the selenium-enriched yeast and the lactobacillus plantarum.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and particularly relates to an application of selenium-enriched yeast combined with Lactobacillus plantarum to improve liver damage in mice through intestinal flora, and particularly to an application in improving liver damage caused by cadmium. Background Art

[0002] Cadmium is a toxic heavy metal. With the intensification of industrialization and urbanization, as well as the migration of human activities, the human body is exposed to cadmium from different sources, which has caused great harm to human health. For example, exposure to cadmium from industrial and agricultural sources can cause breast cancer, lung cancer, prostate cancer, nasopharyngeal cancer, pancreatic cancer and kidney cancer. Studies have shown that cadmium can induce various epigenetic changes in mammalian cells both in vivo and in vitro, including chemical modifications of DNA and histones, mainly by affecting DNA methyltransferase, histone acetyltransferase, histone deacetylase, histone methyltransferase and microRNA to participate in epigenetic changes. Other studies have shown that after injecting mice with different doses of cadmium, it has a significant effect on the physiological functions of mice, which will reduce the activity of superoxide dismutase in mice, increase brain lipid peroxidase, and cause liver damage in mice. In addition, the administration of cadmium causes behavioral, biochemical and neurochemical dysfunction in a dose-dependent manner. The presence of heavy metal cadmium from different sources has brought great harm to the human body.

[0003] As a natural food additive, selenium-enriched yeast has been widely used in health care products due to its powerful nutritional function. It can stimulate a variety of immune cells in the body, promote the high level of immune proteins in the blood, improve the body's defense ability, and thus achieve immunomodulatory effects. It has been reported in the literature that biological selenium (i.e., selenoproteins and selenium-containing amino acids, such as selenoprotein P and selenocysteine) in selenium-enriched yeast can reduce oxidative stress damage, maintain the balance of intracellular oxidation and anti-oxidation, and play a key regulatory role in human life and health. As disclosed in patent application 202311206874.8, a freckle removal and whitening product and its preparation method and use, through the compounding of selenium-enriched yeast and other ingredients, synergistically achieve good freckle removal and whitening effects. Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. Lactobacillus plantarum is a widely distributed and widely used lactic acid bacterium. Studies have shown that Lactobacillus plantarum can affect the physiological functions of the body by affecting the composition of intestinal microorganisms.

[0004] Studies have shown that cadmium can change the functional structure, quantity, and diversity of intestinal microorganisms, leading to the occurrence and development of various diseases. Selenium, as an important immune enhancer, is known to have an antagonistic effect on cadmium and can alleviate cadmium poisoning to a certain extent. However, the impact of cadmium poisoning on liver damage and intestinal microorganisms in mice is not yet clear, and further research is necessary. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an application of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium, and discovers new uses of selenium-enriched yeast and Lactobacillus plantarum.

[0006] Another object of the present invention is to provide an application of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium. The selenium-enriched yeast combined with Lactobacillus plantarum can improve the relevant symptoms of liver damage in mice, so it can be used in therapeutic drugs for liver damage.

[0007] The applicant found that selenium-enriched yeast has the effects of increasing sperm and preventing cancer, and has significant repair functions on the reproductive cells, liver, kidneys, etc. of mice poisoned by cadmium. Based on this, cadmium-poisoned mice treated with selenium-enriched yeast and Lactobacillus plantarum were used as research objects, and the results were characterized by microbial diversity and multiple physiological indicators of mice. The effects of the two on the intestinal bacterial diversity and metabolism of mice under cadmium poisoning conditions were studied, which can help the application of using selenium-enriched yeast and Lactobacillus plantarum to alleviate the intestinal flora disorder caused by heavy metals and the harm to the human body.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] The invention discloses an application of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium. The application of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium can reduce the accumulation of cadmium in the liver and colon.

[0010] Furthermore, the application of selenium-enriched yeast combined with Lactobacillus plantarum in improving cadmium-induced liver damage can regulate the species diversity of intestinal flora and improve species composition.

[0011] Furthermore, the application of selenium-enriched yeast combined with Lactobacillus plantarum in improving cadmium-induced liver damage can reduce the degree of liver damage.

[0012] Furthermore, the application of selenium-enriched yeast combined with Lactobacillus plantarum in improving cadmium-induced liver damage can inhibit liver oxidative stress.

[0013] Furthermore, the application of selenium-enriched yeast combined with Lactobacillus plantarum in improving cadmium-induced liver damage can inhibit liver inflammatory response.

[0014] Furthermore, the application of selenium-enriched yeast combined with Lactobacillus plantarum in improving cadmium-induced liver damage can inhibit liver apoptosis.

[0015] The application of the selenium-enriched yeast combined with Lactobacillus plantarum in the preparation of a product for liver damage caused by cadmium; the product is a medicine.

[0016] The drug contains selenium-enriched yeast and Lactobacillus plantarum, wherein the dosage of selenium-enriched yeast is 100 mg / kg, and the concentration and dosage of Lactobacillus plantarum are 1.0×10 9 CFU / mL, 10mL / kg.

[0017] Specifically, the medicine comprises one or more of the fermentation liquid, fermentation liquid supernatant, fermentation liquid precipitation, live bacteria, dead bacteria, freeze-dried powder and cell lysate of Lactobacillus plantarum.

[0018] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0019] The liver disease is a liver damage disease, and the model is a mouse liver damage model caused by cadmium chloride.

[0020] The research results of the present invention show that selenium-enriched yeast combined with Lactobacillus plantarum can improve the relevant symptoms of liver damage in mice, so it can be used in the treatment of liver damage.

[0021] Specifically, selenium-enriched yeast combined with Lactobacillus plantarum can increase the expression level of CAT protein, which indicates that selenium-enriched yeast combined with Lactobacillus plantarum can effectively reduce the oxidative stress level of the liver. Among the liver inflammatory factors, the expression levels of TNF-α and IL-6 proteins decreased, indicating that selenium-enriched yeast combined with Lactobacillus plantarum can effectively inhibit liver inflammatory factors. In the liver apoptosis effect, the expression of Bcl-2 protein increased significantly, indicating that the anti-apoptosis ability was significantly enhanced; the expression levels of BAX, Caspase-3 and Caspase-9 proteins decreased significantly, indicating that selenium-enriched yeast combined with Lactobacillus plantarum can effectively reduce the occurrence of liver cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

[0023] Figure 1 This is a schematic diagram of the effect of the selenium-enriched yeast combined with Lactobacillus plantarum on reducing the accumulation of cadmium in the liver implemented in the present invention.

[0024] Figure 2 This is a schematic diagram of the effect of the selenium-enriched yeast combined with Lactobacillus plantarum on reducing the accumulation of cadmium in the colon implemented in the present invention.

[0025] Figure 3 This is a schematic diagram of the effect of selenium-enriched yeast combined with Lactobacillus plantarum on the antioxidant activity of the liver implemented in the present invention.

[0026] Figure 4 This is a schematic diagram of the effect of selenium-enriched yeast combined with Lactobacillus plantarum on liver inflammatory factors implemented in the present invention.

[0027] Figure 5 This is a schematic diagram of the effect of selenium-enriched yeast combined with Lactobacillus plantarum on liver apoptosis implemented in the present invention. DETAILED DESCRIPTION

[0028] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0029] Example 1.

[0030] 1. Materials and methods.

[0031] 1. Test materials.

[0032] 1.1 Experimental animals

[0033] 70 male SPF mice, 10-12 weeks old, weighing 25-35 g, were provided by the Experimental Animal Center of Jiangxi Yichun University.

[0034] 1.2 Drugs and reagents.

[0035] Cadmium chloride (19269) was purchased from Thermo Fisher Scientific Inc.;

[0036] 30% Acr-Bis (ST003), ammonium persulfate (ST005), SDS-PAGE protein loading buffer (P0015L), Tween-20 (ST828), DMSO (ST038), and Tris (ST760) were all purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0037] Glycine (A502065-0500), methanol (A601617), formaldehyde (A500684), anhydrous ethanol (A500737), and xylene (A530011) were purchased from Shanghai Bioengineering Co., Ltd.;

[0038] CAT antibody (AF0084), GSH-PX antibody (AF7017), SOD1 antibody (AF8028), P65 antibody (AF0246), Bcl2 antibody (AF6285), BAX antibody (AF5120), Caspase-3 antibody (AF0081), Caspase-9 antibody (AF1264), GAPDH antibody (AF0006), BeyoColor TMColor prestained protein (P0072) and horseradish enzyme-labeled goat anti-rabbit / mouse IgG were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0039] TNF-α antibody (11948) and Nrf2 antibody (12721) were purchased from cell signaling technology company in the United States.

[0040] 1.3 Experimental instruments.

[0041] TGL-16A high-speed low-temperature centrifuge, Hunan Pingfan Technology Co., Ltd.; E200MV microscope, Nikon, Japan; MultiskanFC microplate reader (Thermo Fisher, USA); protein electrophoresis transfer system (Bio Rad, USA); fluorescence quantitative PCR instrument (Eppendorf, Germany).

[0042] 2. Animal grouping and drug administration.

[0043] Seventy SPF mice were randomly divided into seven groups, with 10 mice in each group, namely the control group (Con), the control group + selenium-enriched yeast (Con+Se), the control group + Lactobacillus plantarum (Con+Lp), the model group (Cd), the model group + selenium-enriched yeast (Cd+Se), the model group + Lactobacillus plantarum (Cd+Lp), and the model group + selenium-enriched yeast + Lactobacillus plantarum (Cd+Se+Lp). The mice in the model group were intragastrically administered with cadmium chloride every day for 4 consecutive weeks at a dose of 5 mg / kg. The selenium-enriched yeast group and the Lactobacillus plantarum group were intragastrically administered with doses of 100 mg / kg and 1.0×10 9 CFU / mL, 10mL / kg, the model group + selenium-enriched yeast + Lactobacillus plantarum group was gavaged with selenium-enriched yeast and Lactobacillus plantarum at the same dose as the selenium-enriched yeast group and Lactobacillus plantarum group, and the drug was administered continuously for 4 weeks, which was carried out at the same time as the modeling of mice in the model group.

[0044] 3. Experimental methods.

[0045] 3.1 Determination of cadmium content in liver and colon.

[0046] Accurately weigh 0.1g tissue sample into a 1.5mL plastic EP tube, then add 300μL concentrated nitric acid (analytical grade) to the tube, then add 100μL perchloric acid (analytical grade), place it open on a floating plate, and transfer it to a 95℃ water bath for water bath digestion for 2h. Prepare a 10mL plastic EP tube, mark the sample order, and add 4mL deionized water in advance. After the digestion is completed, use a 1mL pipette to transfer all the liquid in the 1.5mL EP tube to the 10mL EP tube, mix it thoroughly with deionized water, and repeatedly rinse the digestion EP tube. Finally, fill the system to 5mL with deionized water. Number them according to the order of the groups, and use ICP-MS to detect the concentration of metallic cadmium in the samples.

[0047] 3.2 Western Blot method was used to determine protein levels.

[0048] The liver tissue homogenate was centrifuged at low temperature and high speed, and the supernatant was aspirated. The protein concentration was determined using a BCA protein quantification kit, and 5× protein loading buffer was added. After shaking and mixing, the sample was obtained by water bath at 95°C for 5 minutes. After the cells were lysed, the protein was extracted, loaded, electrophoresed, transferred to the membrane, blocked with milk, and washed with TBST. Then, primary antibodies such as SOD1, Nrf2, GSH-Px, CAT, TNF-α, P65, BAX, Caspase-3, and Caspase-9 were added for overnight incubation, washed 3 times with TBST, and incubated for 1 hour with secondary antibodies, and washed 3 times with TBST. After soaking in ultrasensitive ECL chemiluminescent solution for 2 minutes, the cells were developed, and the grayscale value was calculated using Image J software to compare the protein level expression between the groups.

[0049] 2. Conclusion

[0050] 1. Selenium-enriched yeast combined with Lactobacillus plantarum can reduce the cadmium content in the liver and colon

[0051] like Figure 1 , Figure 2 As shown in the figure, selenium-enriched yeast combined with plant lactobacillus can reduce the accumulation of cadmium in the liver and colon. Due to the lack of cadmium intake, the cadmium content in the colon and liver of the blank group was extremely low; after cadmium infusion in the model group, the cadmium content in the body increased significantly. After treatment with selenium-enriched yeast and plant lactobacillus, the cadmium content decreased to varying degrees. When the selenium-enriched yeast group and plant lactobacillus were used together, the cadmium content decreased significantly, and the effect was better.

[0052] 2. Selenium-rich yeast combined with Lactobacillus plantarum effectively inhibits liver peroxidation.

[0053] like Figure 3The figure shows the effect of selenium-enriched yeast combined with Lactobacillus plantarum on the antioxidant activity of the liver. As shown in the figure, the SOD1, Nrf2, GSH-Px and CAT protein expressions in the model group were significantly decreased compared with the blank group, indicating an increase in the oxidative stress level in the mice in the model group. Compared with the model group, the expression levels of the above proteins increased after the administration of selenium-enriched yeast and Lactobacillus plantarum respectively (P<0.05), and the increase was greater after combined administration, indicating that the selenium-enriched yeast combined with Lactobacillus plantarum can effectively reduce the oxidative stress level of the liver.

[0054] 3. Selenium-enriched yeast combined with Lactobacillus plantarum effectively inhibits the expression of inflammatory factors.

[0055] like Figure 4 As shown, the situation of the selenium-enriched yeast combined with plant lactobacillus on liver inflammatory factors implemented by the present invention. As shown in the figure, compared with the blank group, due to the influence of cadmium, the expression levels of TNF-α and P65 proteins in the model group increased significantly. Compared with the model group, after the administration of selenium-enriched yeast and plant lactobacillus, the expression levels of TNF-α and IL-6 proteins decreased (P<0.05), especially when the combination of drugs was used, the protein expression level decreased more, indicating that selenium-enriched yeast combined with plant lactobacillus can effectively inhibit liver inflammatory factors.

[0056] 4. Selenium-enriched yeast combined with Lactobacillus plantarum has anti-apoptosis effects.

[0057] like Figure 5 As shown, Figure 5 Schematic diagram of the effect of selenium-enriched yeast combined with plant lactobacillus on liver apoptosis implemented by the present invention. Compared with the blank group, the Bcl-2 protein expression in the model group increased, indicating the weakening of anti-apoptosis ability. After treatment with selenium-enriched yeast combined with plant lactobacillus, the anti-apoptosis ability was significantly enhanced, almost the same as normal. The expression levels of BAX, Caspase-3 and Caspase-9 proteins in the model group increased significantly, indicating the increase in the incidence of liver cell apoptosis. Compared with the model group, the protein expression levels of selenium-enriched yeast and plant lactobacillus treatment group decreased significantly (P<0.05), indicating that selenium-enriched yeast combined with plant lactobacillus can effectively reduce the occurrence of liver cell apoptosis.

[0058] In summary, the selenium-enriched yeast combined with Lactobacillus plantarum of the present invention can improve the relevant symptoms of liver injury in mice, so it can be used in the treatment of liver injury.

[0059] At the same time, the selenium-enriched yeast is combined with Lactobacillus plantarum to improve liver damage caused by cadmium; and the product is a medicine.

[0060] Furthermore, the drug comprises selenium-enriched yeast and Lactobacillus plantarum, wherein the dosage of selenium-enriched yeast is 100 mg / kg, and the concentration and dosage of Lactobacillus plantarum are 1.0×10 9 CFU / mL, 10mL / kg.

[0061] Specifically, the medicine comprises one or more of the fermentation liquid, fermentation liquid supernatant, fermentation liquid precipitation, live bacteria, dead bacteria, freeze-dried powder and cell lysate of Lactobacillus plantarum.

[0062] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium, characterized in that: The application of the selenium-enriched yeast combined with Lactobacillus plantarum in the preparation of a product for liver damage caused by cadmium; the product is a medicine.

2. The use of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium according to claim 1, characterized in that: The drug contains selenium-enriched yeast and Lactobacillus plantarum, wherein the dosage of selenium-enriched yeast is 100 mg / kg, and the concentration and dosage of Lactobacillus plantarum are 1.0×10 9 CFU / mL, 10mL / kg.

3. The use of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium according to claim 1, characterized in that: The medicine comprises one or more of the fermentation liquid, fermentation liquid supernatant, fermentation liquid precipitation, live bacteria, dead bacteria, freeze-dried powder and cell lysis liquid of plant lactobacillus.

4. The use of selenium-enriched yeast combined with Lactobacillus plantarum in improving liver damage caused by cadmium according to claim 1, characterized in that: The medicine also includes pharmaceutical excipients.

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