CN115418332A - Lactobacillus plantarum capable of preventing and improving chemical liver injury - Google Patents
Lactobacillus plantarum capable of preventing and improving chemical liver injury Download PDFInfo
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- CN115418332A CN115418332A CN202211073589.9A CN202211073589A CN115418332A CN 115418332 A CN115418332 A CN 115418332A CN 202211073589 A CN202211073589 A CN 202211073589A CN 115418332 A CN115418332 A CN 115418332A
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Abstract
The invention provides lactobacillus plantarum capable of preventing and improving chemical liver injury, and belongs to the technical field of probiotics. The lactobacillus plantarum (Lactobacillus plantarum) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.16648 and the preservation date of 2018, 10 months and 29 days. According to the invention, the Lactobacillus plantarum HFY05 separated from the naturally fermented yak milk is prepared into freeze-dried powder or other preparations, and preliminary research results show that the probiotic preparation has the effects of preventing and assisting in treating chemical liver injury (such as alcoholic hepatitis and toxic hepatitis), chronic hepatitis, fatty liver and autoimmune hepatitis.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to lactobacillus plantarum capable of preventing and improving chemical liver injury.
Background
The liver has multiple functions of detoxification, metabolism, synthesis and the like, and plays an important role in maintaining physical health. At the same time, the liver is also vulnerable. Various harmful substances such as drugs, viruses, alcohol and the like may cause liver inflammation, necrosis, fibrosis and cirrhosis, and finally liver cancer. Various liver diseases are usually caused by acute liver injury caused by harmful substances. The pathogenesis of acute liver injury is known to involve the interaction of processes such as apoptosis, oxidative stress, inflammatory response, and the like. Currently, the exact mechanism of liver injury is still under study, and the treatment of liver injury is still a big problem in human beings. Although many drugs are available for treating liver damage, there are side effects of drug therapy that can also cause damage to the body. Therefore, there is an urgent need to find a new method for preventing liver damage.
Probiotics refer to dietary supplements containing live microbial strains that improve host health by amplifying the beneficial effects of the normal gut flora of exogenous microorganisms from food. Lactobacillus plantarum is a microbial species widely used as a probiotic in the food industry, and has been confirmed to have various functions such as regulating skin health, alleviating memory impairment in mice, and inhibiting enterovirus coxsackievirus B4 (CV-B4). In addition, the research results show that lactobacillus fermentum LA12 can be used for preventing and treating alcoholic liver injury, lactobacillus plantarum C88 can prevent aflatoxin B1-induced liver injury of mice, and lactobacillus plantarum DSMZ 2017 protects the liver through an antioxidation effect, and the results of the research show that the function of lactobacillus as a functional food for protecting the liver is proved.
The yak yoghourt is fermented milk, is usually found in Qinghai-Tibet plateau areas, contains more abundant proteins, essential amino acids, fat and the like than common cow milk, and therefore has extremely high nutritional value. Due to the unique conditions of climate, altitude, technology and the like of the Qinghai-Tibet plateau, the taste of the yak yoghourt and the microbial community in the yak yoghourt are also very rich. The health care function of the yak yogurt is closely related to the predominant lactic acid bacteria contained in the yak yogurt, and experiments show that the predominant bacteria in the yak milk are lactobacillus.
Disclosure of Invention
The invention aims to provide Lactobacillus plantarum capable of preventing and improving chemical liver injury, which is prepared by separating Lactobacillus plantarum HFY05 from naturally fermented yak milk and preparing the Lactobacillus plantarum HFY05 into freeze-dried powder or other preparations, and preliminary research results show that the probiotic preparation has the effects of preventing and assisting in treating chemical liver injury (such as alcoholic hepatitis and toxic hepatitis), chronic hepatitis, fatty liver and autoimmune hepatitis.
The technical scheme of the invention is realized as follows:
the invention provides Lactobacillus plantarum (Lactobacillus plantarum) HFY05, which is classified and named Lactobacillus plantarum HFY05 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation number is CGMCC NO.16648, the preservation date is 2018, 10 and 29 days, and the preservation address is Beijing City Chaoyang district North Chen Wen Lu No. 1.
The Lactobacillus plantarum HFY05 is derived from traditional naturally fermented yak yogurt in herdsmen in the region of Qiang of the Notopterygium prefecture of the Ataca, sichuan, the strain is subjected to sequencing analysis, the conserved Gene sequence of the strain is shown as SEQ ID No.1, comparison analysis of a BLAST program shows that the strain is Lactobacillus plantarum, the homology of the strain and known Lactobacillus in a Gene Bank database is 100%, and therefore the strain is named as Lactobacillus plantarum HFY05 (see Table 1).
Table 1 results of 16S rDNA sequence analysis of strains
The Lactobacillus plantarum (Lactobacillus plantarum) HFY05 colony is mostly white or milky white, round in shape, neat in edge and wet and smooth in surface.
The Lactobacillus plantarum (Lactobacillus plantarum) HFY05 cell morphology is long-rod, short-rod and spherical, and budding reproduction does not exist.
The Lactobacillus plantarum (Lactobacillus plantarum) HFY05 is a gram-positive bacterium.
The invention also provides a preparation for preventing and improving chemical liver injury, which comprises the Lactobacillus plantarum HFY05.
As a further improvement of the invention, the preparation contains all pharmaceutically acceptable carriers.
As a further improvement of the invention, the preparation comprises freeze-dried powder, capsules, tablets, granules and oral liquid.
As a further improvement of the invention, the viable count of the Lactobacillus plantarum HFY05 in the preparation is not less than 1 x 10 8 cfu/g。
The invention further provides a preparation method of the preparation for preventing and improving the chemical liver injury, which comprises the steps of inoculating Lactobacillus plantarum HFY05 into a fermentation medium for fermentation, wherein the obtained fermentation product is the preparation for preventing and improving the chemical liver injury, and the fermentation medium is added with yak milk.
As a further improvement of the invention, the obtained fermentation product is centrifuged, and the precipitate is added with a freeze-drying protective agent for vacuum freeze-drying to obtain a dry powder preparation for preventing and improving chemical liver injury.
As a further improvement of the invention, the fermentation temperature is 35-40 ℃, the fermentation time is 24-120h, and the content of yak milk in the culture medium is 15-20g/L.
The formula of the fermentation medium is as follows: 12.00g/L of casein peptone, 2.00g/L of ammonium nitrate, 3g/L of tyrosine, 5g/L of methionine, 5.00g/L of sodium acetate, 5.00g/L of yeast extract, 0.50g/L of magnesium sulfate, 20.00g/L of glucose, 5g/L of sucrose, 0.1g/L of zinc sulfate, 0.1g/L of ferric sulfate, 0.1g/L of manganese sulfate, 3.00g/L of potassium dihydrogen phosphate, 1.00g/L of tween-80 and 15-20g/L of yak milk.
The invention further protects the application of the Lactobacillus plantarum HFY05 and/or the preparation for preventing and improving chemical liver injury in preparing a medicament for preventing or treating liver diseases.
As a further improvement of the invention, the liver diseases comprise alcoholic hepatitis, toxic hepatitis, chronic hepatitis, fatty liver and autoimmune hepatitis.
The invention has the following beneficial effects: lactic acid bacteria, as a class of edible probiotics, can regulate the imbalance of the intestinal microbiota composition by increasing the number of bacteria, improving the barrier function of the intestinal epithelium and promoting the production of cytokines, thereby preventing various diseases such as obesity, cancer, liver injury and the like. The Lactobacillus plantarum HFY05 separated from the naturally fermented yak milk reduces the TG content and the CCl content by adjusting the ALT and AST levels of mouse livers 4 Damage to mouse liver, simultaneously, can obviously increase SOD and CAT activity in mouse body and reduce MDA level, thereby reducing CCl 4 The lactobacillus plantarum HFY15 can also obviously reduce the levels of IL-6, TNF-alpha and IFN-gamma in the oxidative damage caused to the liver, and has a good relieving effect on inflammatory response of mice induced by CCl 4.
When the lactobacillus is ingested by food or oral administration, the lactobacillus can firstly endure the acid environment of the gastrointestinal tract and then reaches the intestine, and the bile salt in the small intestine can destroy the cell membrane of the lactobacillus, so that the lactobacillus has certain gastric juice resistance and good tolerance of the bile salt, can reach the intestinal tract with higher viable count, and is fixedly planted on the intestinal tract to play the probiotic effect. The survival rate of the Lactobacillus plantarum HFY15 artificial gastric juice treated by the invention is 91.2%, the growth efficiency of the Lactobacillus plantarum HFY is 57.9% under 0.3% bile salt, the Lactobacillus plantarum HFY has excellent acid resistance, is not easily destroyed and inactivated by gastric acid after being taken orally, and can enter the intestinal tract to play a good role in protecting the liver.
Lactobacillus plantarum HFY15 has high gastric acid resistance and good bile salt viability, can regulate the release of antioxidant related enzymes, controls the oxidation of liver fat, and avoids the occurrence of peroxidation. Lactobacillus plantarum HFY15 can also inhibit the production of proinflammatory factors and improve the anti-inflammatory ability of the liver. Moreover, lactobacillus plantarum HFY15 can promote expression of Bcl-2 by inhibiting expression of Caspase 3 and Bax, avoid apoptosis of liver cells, and finally can maintain normal morphology of liver tissues and liver cells.
According to the invention, the Lactobacillus plantarum HFY05 separated from the naturally fermented yak milk is prepared into freeze-dried powder or other preparations, and preliminary research results show that the probiotic preparation has the effects of preventing and assisting in treating chemical liver injury (such as alcoholic hepatitis and toxic hepatitis), chronic hepatitis, fatty liver and autoimmune hepatitis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a diagram of the agarose gel electrophoresis of the 16S rDNA amplified product of PCR in example 1 of the present invention;
FIG. 2 is a colony morphology of the P strain in example 2 of the present invention;
FIG. 3 is a graph showing the gram staining results of the strain in example 2 of the present invention;
FIG. 4 is a graph comparing the serum ALT activity of mice in each group in example 4 of the present invention;
FIG. 5 is a graph showing the comparison of AST activity in serum of mice of each group in example 4 of the present invention;
FIG. 6 is a graph showing the comparison of TG content in serum of each group of mice in example 4 of the present invention;
FIG. 7 is a graph comparing the serum MDA levels of various groups of mice in example 4 of the present invention;
FIG. 8 is a graph comparing the serum SOD levels of mice in each group in example 4 of the present invention;
FIG. 9 is a graph comparing the levels of CAT in the serum of mice in each group in example 4 of the present invention;
FIG. 10 is a graph showing a comparison of the expression levels of IL-6 in serum of each group of mice in example 4 of the present invention;
FIG. 11 is a graph comparing the serum TNF- α expression levels of various groups of mice in example 4 of the present invention;
FIG. 12 is a graph showing a comparison of the expression levels of IFN-. Gamma.in serum of each group of mice in example 4 of the present invention;
FIG. 13 is H & E staining pathology of liver of each group of mice in example 4 of the present invention;
FIG. 14 is an mRNA expression pattern of liver-related genes of each group of mice in example 4 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out according to conventional conditions or conditions recommended by the manufacturers. The test materials used in the present invention are commercially available products unless otherwise specified.
Example 1 isolation and characterization of Lactobacillus plantarum HFY15
1. Separation and purification of Lactobacillus plantarum (Lactobacillus plantarum) HFY15
Respectively taking 1mL of yoghourt samples, and carrying out gradient dilution by 10 times to 10 by using sterile normal saline -6 Then take 10 out -4 、10 -5 、10 -6 The 3 gradients of bacteria solution 100 u L plate coating, 37 degrees C culture 24-48h, observed and recorded colony morphology. And selecting colonies with different forms on the plate for streaking separation, culturing at 37 ℃ for 48h, selecting single colonies with different forms on the plate again for streaking separation, and repeating the steps for multiple times until pure single colonies with consistent forms are obtained.
2. DNA extraction of Lactobacillus plantarum HFY15
Inoculating the pure suspected target strain into MRS broth, culturing at 37 ℃ for 18-24h, and extracting DNA by using a bacterial genome DNA extraction kit. The extracted DNA is numbered and stored in a freezer at the temperature of minus 20 ℃ for later use.
3. Genomic DNAPCR amplification and agarose gel electrophoresis detection
The extracted DNA was subjected to PCR amplification using 1. Mu.L of the forward primer 27F (5'-AGA GTT TGA TCC TGGCTCAG-3'), 1. Mu.L of the reverse primer 1495R (5'-CTACGG CTACCTTGT TAC GA-3'), 12.5. Mu.L of 2 XTaqplus Buffer, 1. Mu.L of the template DNA, sterile dd H 2 O make up the system to 25. Mu.L. And sterile ultrapure water was used as a negative control instead of the template DNA. The amplification conditions were: 5min at 94 ℃; 30s at 94 deg.C, 30s at 55 deg.C, 1min at 72 deg.C for 29 cycles, and finally 5min at 72 deg.C.
Then 5 mul of amplification product is taken to carry out agarose gel electrophoresis detection, the agarose concentration is 1.2%, the electrophoresis condition is 110V, and 45min.
The result of 1.2% agarose gel detection of the 16S rDNA amplification product is shown in FIG. 1, and the lane with sterile ultrapure water as a negative control has no band, indicating that the PCR amplification process is not contaminated. Wherein, 0 is a negative control group; 1-15 represent the strains numbered HFY01-HFY15, respectively. 15 is Lactobacillus plantarum HFY15, the length of the lane-specific amplified fragment is about 1500bp, which is consistent with the expected amplified fragment length.
The PCR product successfully detected is sent to Beijing Optimalaceae biotechnology limited for sequencing, the conserved gene sequence is shown as SEQ ID NO.1, and the sequence successfully sequenced is compared and analyzed by using BLAST (Basic Localalignment Search Tool) program in NCBI.
Example 2 cultivation of Lactobacillus plantarum (Lactobacillus plantarum) HFY15
The Lactobacillus plantarum (Lactobacillus plantarum) HFY15 pure colony on the plate of example 1 was selected, inoculated into 5 mM MRS liquid medium, and cultured at 37 ℃ for 24 hours. And (3) taking 1mL of the culture medium containing the bacteria in a sterile centrifuge tube, centrifuging at 4000r/min for 10min, removing an upper culture medium, re-suspending the bacterial precipitates in sterile normal saline, performing gram stain microscopic examination, and preliminarily identifying the bacterial precipitates as positive bacteria by the gram stain microscopic examination.
15 strains of lactic acid bacteria are separated and purified from a yak yoghourt sample, and the gram staining is positive. The strain is lactobacillus with the number of HFY15 according to morphological observation and 16S rDNA species analysis.
The colony morphology of the strain is shown in figure 2. As shown in FIG. 2, the colony of lactic acid bacteria is mostly white or milky white, and has a circular shape, regular edges and a moist and smooth surface.
The cell morphology results of the lactobacillus strains are shown in figure 3, and under a 100-fold oil lens, the cell morphology is long rod, short rod and spherical as shown in figure 3, and budding does not exist.
Example 3 Ex-resistance selection of Lactobacillus plantarum (Lactobacillus plantarum) HFY15
1. Lactobacillus plantarum HFY15 ability to tolerate 0.3% bile salts
Adding pig bile salt into MRS-THIO culture medium (MRS broth containing 0.2% sodium thioglycolate) to make its concentration be 0.3%, sterilizing at 121 deg.C for 15min, inoculating activated 5mL Lactobacillus plantarum (Lactobacillus plantarum) HFY15 strain with 2% (v/v) inoculation amount into MRS-THIO culture medium containing no bile salt (0.0%) and MRS-THIO culture medium containing 0.3% bile salt, respectively, using blank culture medium (unsterilized MRS-THIO culture medium) as control, culturing at 37 deg.C for 24h, and respectively determining OD of the culture medium with different concentrations 600nm The tolerance of the strain to bile salts was calculated according to equation (1):
2. simulated gastric fluid resistance test
Preparing artificial gastric juice: the artificial gastric juice is prepared by 0.2 percent of NaCl and 0.35 percent of pepsin according to the corresponding mass-volume ratio, respectively weighing the NaCl and the pepsin required by the test, adjusting the pH of the prepared artificial gastric juice to 3.0 by using 1mol/L of HCl, and filtering and sterilizing the artificial gastric juice by using a 0.22 mu m filter membrane for later use.
Sucking 5mL of cultured culture medium containing Lactobacillus plantarum (Lactobacillus plantarum) HFY15 in a super-clean workbench, placing the culture medium in a 10mL sterile centrifuge tube, centrifuging the culture medium for 10min at 4000r/min, removing the upper culture medium, collecting thalli, adding equal volume (5 mL) of sterile physiological saline, uniformly mixing the sterile physiological saline to prepare bacterial suspension, then uniformly mixing 1mL of the bacterial suspension with 9mLpH 3.0 artificial gastric juice, treating 1mL of the mixed solution as the artificial gastric juice for 0h, and placing the rest 9mL of the mixed solution in a constant-temperature water bath shaker (37 ℃,150 r/min) for culturing for 3h. The samples of 0h and 3h are respectively diluted by 10 times of gradient, the viable count is determined by selecting proper gradient and adopting a plate coating method, the samples are cultured for 48h at 37 ℃ on an MRS solid culture medium, and the survival rate (%) is calculated according to a formula 2.
The results are shown in Table 2.
TABLE 2 survival in artificial gastric juice at pH3.0 and 0.3% bile salts
| Strain numbering | Name of Chinese | Survival in artificial gastric juice (%). At pH3.0 | Growth efficiency in 0.3% bile salt (%) |
| HFY15 | Lactobacillus plantarum | 86.26±10.78 | 53.45±2.74 |
As can be seen from Table 2, the survival rate of Lactobacillus plantarum (Lactobacillus plantarum) HFY15 in artificial gastric juice with pH3.0 is close to 90%, and Lactobacillus can grow in 0.3% of bile salt to 53.45%, indicating that Lactobacillus plantarum (Lactobacillus plantarum) HFY15 has strong bile salt tolerance.
Example 4 on CCl 4 Rat chemogenesisAction of liver damage
50 male Kunming mice of 6 weeks old, weighing (20. + -.5 g), purchased at the university of Chongqing pharmaceutical laboratory animal center (No. SYXK 2018-0003), all fed standard feed and water under constant conditions in a12 h light/dark cycle at a temperature of 25. + -. 2 ℃ and under constant conditions. One week after acclimation feeding, mice were randomly divided into 5 groups, respectively: normal, model, silymarin, HFY15 and LDSB groups, 10 mice per group. Different groups of mice are treated every day, and the mice of the normal group and the model group are gavaged with 10mL kg of gastric lavage -1 The normal saline and silymarin group mice are injected with 50mg kg -1 Hepadilus silymarin, HFY15 group mice gavage 10 9 CFU kg -1 Lactobacillus plantarum (Lactobacillus plantarum) HFY15, LDSB group mice intragastric administration 10 9 CFU kg -1 Lactobacillus bulgaricus subspecies delbrueckii, and all mouse body weights were measured and recorded for two weeks. All groups of mice except the normal group were intraperitoneally injected with 0.8% of CCl on the fourteenth day 4 (10mL kg -1 ). After all mice were fasted for 16 hours without water deprivation, the mice were sacrificed and whole blood was centrifuged to separate serum, and frozen at-80 ℃. The livers were isolated and weighed, and then stored frozen at-80 ℃ or fixed with 4% formaldehyde solution.
The mouse body weight, liver weight and liver index results are shown in table 3.
TABLE 3
| Group of | Normal group | CCl 4 Induction group | Silymarin group | HFY15 group | LDSB group |
| Body weight (g) | 35.54±1.50 a | 27.75±1.90 d | 32.25±1.64 b | 33.98±1.81 a | 29.98±1.50 c |
| Liver weight (mg) | 1.53±0.24 b | 1.99±0.37 a | 1.62±0.14 b | 1.61±0.14 b | 1.74±0.17 b |
| Liver index (%) | 4.30 d | 7.17 a | 5.02 c | 4.74 d | 5.80 b |
Note that: a-d indicate significant differences between letters (p < 0.05).
Liver organ index is the ratio of mouse liver tissue weight to its body weight. As shown in table 3, the average liver weight and liver index were lowest in the normal group. CCl 4 After treatment, the average liver weight and liver index of the mice were highest. HFY15 group mice reduced liver weight and liver index, and livers of silymarin group and LDSB groupIndex and CCl 4 The comparison also tended to be decreasing, but the effect was not as pronounced as in the HFY15 group.
1. Measurement of ALT, AST, TG, MDA, SOD and CAT levels in mouse serum
The kit and the specification produced by Nanjing institute of bioengineering are used for detecting the levels of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), triglyceride (TG), malondialdehyde (MDA), superoxide dismutase (SOD) and Catalase (CAT) in serum.
The results are shown in FIGS. 4-9. Note that: a-e indicates that the mean values of different letters in different bars differ significantly according to Duncan's multipolar difference test (p)<0.05)。
ALT, AST and TG levels in mouse serum are shown in FIGS. 4-6. ALT and AST levels are often used as indicators to assess liver function impairment. ALT, AST and TG levels were significantly increased in the model group mice compared with the normal group. Silymarin is widely used in the treatment of liver damage as a positive control. Compared with the model group, the ALT, AST and TG levels of the HFY15 group are all significantly reduced, and the silymarin group and the LDSB group also have the same trend, but the silymarin group and the HFY15 group have the levels close to the normal group and have significant difference with the LDSB group.
Serum MDA, SOD and CAT levels (FIGS. 7-9). MDA, SOD and CAT are important indicators of oxidation. Compared with the normal group, the MDA content of the model group is obviously increased, and the SOD and CAT activities are obviously reduced. After HFY15 treatment, the trend is opposite to that of the model group, the MDA content is reduced, and the SOD activity and the CAT activity are improved. The silymarin group and the LDSB group showed the same trend as the HFY15 group, with the silymarin group and the HFY15 group having values closer to those of the normal group.
2. Serum cytokine IL-6, TNF-alpha and IFN-gamma level measurement
Serum cytokine levels were detected according to interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-gamma) cytokine detection kits produced by Shanghai enzyme-linked Biotechnology, inc.
The results are shown in FIGS. 10-12. And (3) annotation: a-e indicates that the mean values of different letters in different bars differ significantly according to Duncan's multipolar difference test (p)<0.05)。
Serum cytokine levels are shown in FIGS. 10-12. Serum cytokines are often used as indicators of liver inflammation. The IL-6, TNF-alpha and IFN-gamma levels in the model group were significantly higher than those in the normal group. While the HFY15 group, LDSB group and silymarin group significantly reduced IL-6, TNF- α and IFN- γ levels compared to the model group. The silymarin group was closest to the normal group, next to the HFY15 group, and the expression level of LDSB group was significantly higher than those of silymarin group and HFY15 group.
3. Preparation of H & E stained liver tissue section
Mouse liver tissue was taken, fixed by immersion in 10% neutral formalin, and then dehydrated in 95% ethanol for 24 hours. Paraffin embedding, sectioning, staining with hematoxylin and eosin (H & E), and histopathological analysis. Histopathological changes were observed under an optical microscope and images were taken.
The results are shown in FIG. 13. As can be seen from the H & E stained section of mouse liver in FIG. 13, the hepatocytes in the normal group were intact, the nuclei were aligned, the structure of liver lobules was clear, and the distribution was uniform. The model group showed severe hepatocyte degeneration, lobular disorders, nuclear aggregation and massive inflammatory cell infiltration. In the silymarin group, HFY15 group, and LDSB group, there was reduced hepatocyte degeneration, more well-aligned nuclei, and mild inflammatory cell infiltration.
4. Measurement of mRNA expression level in liver tissue of mouse (qPCR measurement)
50-100mg of liver tissue was placed in a homogenizer tube containing a small steel ball, and 1ml of a trizol reagent was added to separate and extract total RNA from the liver homogenate. Total RNA concentration and purity were determined using a microspectrophotometer. Total RNA is used as a template, and cDNA is synthesized through reverse transcription. mu.L of cDNA, 2. Mu.L of the upstream and downstream primers (Table 3), 10. Mu.L of the premix and 7. Mu.L of sterile ultrapure water were added to an eight-tube, and then denatured at 95 ℃ for 3min, annealed at 60 ℃ for 2 s, and extended at 95 ℃ for 1min, and the whole process was carried out for 40 cycles. GAPDH is used as an internal reference gene and is expressed by a formula 2 -△△Ct The relative expression level of mRNA of each target gene was calculated.
TABLE 1 primer sequence Listing
The results are shown in FIG. 14. Note that: a-e indicates that the mean values of different letters in different bars differ significantly according to Duncan's multipolar difference test (p)<0.05)。
The qPCR result indicates that CCl 4 Reduces the expression of CAT, SOD1, SOD2 and Bcl-2 genes and increases the expression of TNF-alpha, IFN-gamma, IL-6, bax and Caspase-3 genes. Compared with a model group, the lactobacillus plantarum HFY15 can obviously improve the expression of CAT, SOD1, SOD2 and Bcl-2 and reduce the expression of TNF-alpha, IFN-gamma, IL-6, bax and Caspase-3 genes. The silymarin and LDSB groups showed similar results to the HFY15 group, but the related gene expression was closer to the normal group after silymarin and HFY15 treatment.
Lactic acid bacteria, as a class of edible probiotics, can regulate the imbalance of the intestinal microbiota composition by increasing the number of bacteria, improving the barrier function of the intestinal epithelium and promoting the production of cytokines, thereby preventing various diseases such as obesity, cancer, liver injury and the like. In recent years, there has also been a use of lactic acid bacteria for CCl alleviation 4 Study of induced liver injury.
When the lactobacillus is ingested by food or oral administration, the lactobacillus can firstly endure the acid environment of the gastrointestinal tract and then reaches the intestine, and the bile salt in the small intestine can destroy the cell membrane of the lactobacillus, so that the lactobacillus has certain gastric juice resistance and good tolerance of the bile salt, can reach the intestinal tract with higher viable count, and is fixedly planted on the intestinal tract to play the probiotic effect. The results show that the survival rate of the Lactobacillus plantarum (Lactobacillus plantarum) HFY15 artificial gastric juice after treatment is 91.2%, and the growth efficiency under 0.3% bile salt is 57.9%. The survival rate of Lactobacillus fermentum Lee used in the experiment of Qian and the like in artificial gastric juice with pH3.0 is 87.99%, and the growth efficiency under 0.3% bile salt is 25.31, compared with the Lactobacillus plantarum HFY15, the Lactobacillus plantarum HFY has good gastric acid resistance and bile salt resistance.
CCl 4 Induction can cause intrahepatic injury and hepatic fibrosis in mice. Liver histopathology is an important clinical standard for diagnosing liver injury. The CCl is found through experiments 4 The liver cell swelling, the cell nucleus size irregularity and the lymphocyte infiltration of the mice are obvious after the treatment, the condition is better after the treatment of the Lactobacillus plantarum HFY15, and the liver cells are slightly swollen. Liver weight and liver index are commonly referred to as CCl 4 Indices of induced liver damage. The results show that the Lactobacillus plantarum (Lactobacillus plantarum) HFY15 treatment can reduce the liver weight and liver indexes of the model group, and the Lactobacillus plantarum (Lactobacillus plantarum) HFY15 plays a positive role in relieving the liver injury induced by CCl 4.
ALT and AST are catalysts which are necessary in the normal running process of the liver, ALT exists in liver cells, AST exists in mitochondria of the liver cells, when the liver cells are damaged, ALT enters blood firstly, and when the liver cells are seriously damaged, ALT also enters the blood, so that transaminase in the serum of mice is increased. Therefore, ALT and AST levels can be used to assess the degree of impairment of liver function, a value which correlates positively with the degree of liver cell damage. Liver damage can lead to fat in peripheral adipose tissue being transported to the liver and accumulating, raising the TG content in the liver. The result shows that the Lactobacillus plantarum HFY15 can regulate the liver ALT and AST levels of mice, reduce the TG content of a model group and reduce the damage of CCl4 to the liver.
Acute liver injury is associated with oxidative stress of the liver. Therefore, the study evaluates the oxidative stress capability of the liver by measuring antioxidant parameters such as SOD, CAT and MDA. SOD and CAT are antioxidase, SOD can catalyze superoxide anion to carry out disproportionation reaction and remove free radical, and CAT can eliminate hydrogen peroxide in vivo and enhance the effect of SOD on removing free radical. MDA is a metabolic end product of lipid peroxidation and is considered to be an important marker of oxidative stress. Experiments show that the Lactobacillus plantarum HFY15 can obviously increase SOD and CAT activities in mice and reduce MDA level, thereby reducing CCl 4 Oxidative damage to the liver.
Liver damage caused by inflammatory cytokinesInjury plays a key role. CCl 4 Induces liver oxidation and inflammation, releases various inflammation mediators during oxidative stress injury, and obviously increases the level of mouse serum inflammatory factors IL-6, TNF-alpha and IFN-gamma. IL-6 promotes the proliferation and differentiation of T lymphocytes and enhances the body's inflammatory response. TNF- α can aggregate inflammatory cells, leading to inflammatory cell infiltration and tissue edema. IFN-gamma increases the sensitivity of hepatocytes to TNF-alpha, further damaging the hepatocytes. Experiments show that the Lactobacillus plantarum (Lactobacillus plantarum) HFY15 can remarkably reduce the IL-6, TNF-alpha and IFN-gamma levels, which indicates that the Lactobacillus plantarum (Lactobacillus plantarum) HFY15 is used for CCl 4 The induced inflammatory reaction of the mice has good relieving effect.
Apoptosis is a form of programmed cell death, a major mechanism regulating cell death, and usually occurs during development or aging, also as a defense mechanism in the event of cell injury or stress, with orderly and efficient removal of damaged cells through multigenic control. Mitochondrial pathways regulated by Bcl-2 family pro-apoptotic and anti-apoptotic proteins have been demonstrated in CCl 4 Plays an important role in induced apoptosis. Bcl-2 is mainly located on the membrane of intracellular organelle and has anti-apoptosis effect. Bax protein is mainly distributed in cytoplasm, and when cells receive an apoptosis signal, bax can migrate from cytoplasm to mitochondrial membrane, so that the mitochondrial membrane is damaged, and the Bax protein has the effect of promoting apoptosis. Down-regulation of Bcl-2 and up-regulation of Bax lead to release of cytochrome c by mitochondria, which activates Caspase in cytoplasm, the most important of which is Caspase-3, ultimately leading to apoptosis. Experimental data show that Lactobacillus plantarum (Lactobacillus plantarum) HFY15 can remarkably up-regulate expression of Bcl-2 and down-regulate expression of Bax and Caspase-3, and the Lactobacillus plantarum (Lactobacillus plantarum) HFY15 can relieve CCl 4 The resulting hepatocyte apoptosis protects the normal physiological functions and procedures of hepatocytes.
In conclusion, lactobacillus plantarum HFY15 has high gastric acid resistance and good bile salt viability, can regulate the release of antioxidant-related enzymes, controls the oxidation of liver fat, and avoids the occurrence of peroxidation. Lactobacillus plantarum HFY15 can also inhibit the production of proinflammatory factors and improve the anti-inflammatory ability of the liver. Moreover, lactobacillus plantarum HFY15 can promote expression of Bcl-2 by inhibiting expression of Caspase 3 and Bax, avoid apoptosis of liver cells, and finally can maintain normal morphology of liver tissues and liver cells.
In conclusion, lactobacillus plantarum HFY15 relieves CCl by increasing mouse antioxidant capacity, reducing body inflammatory response and inhibiting hepatocyte apoptosis 4 Induced acute liver injury.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The Lactobacillus plantarum is characterized in that the Lactobacillus plantarum is preserved in China general microbiological culture collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.16648 and the preservation date of 2018, 10 months and 29 days.
2. A preparation for preventing and ameliorating chemical liver injury, which comprises the Lactobacillus plantarum (Lactobacillus plantarum) of claim 1.
3. The preparation for preventing and improving chemical liver injury according to claim 2, wherein the preparation contains all pharmaceutically acceptable carriers.
4. The preparation for preventing and improving chemical liver injury according to claim 2, wherein the preparation comprises lyophilized powder, capsules, tablets, granules and oral liquid.
5. The method according to claim 2 for preventing or amelioratingA preparation for treating chemical hepatic injury, wherein the viable count of Lactobacillus plantarum (Lactobacillus plantarum) in the preparation is not less than 1 × 10 8 cfu/g。
6. A preparation method of the preparation for preventing and improving chemical liver injury according to any one of claims 1-5, wherein Lactobacillus plantarum (Lactobacillus plantarum) is inoculated into a fermentation medium for fermentation, and the obtained fermentation product is the preparation for preventing and improving chemical liver injury, wherein yak milk is added into the fermentation medium.
7. The preparation method according to claim 6, wherein the obtained fermentation product is centrifuged, and the precipitate is added with a lyoprotectant for vacuum freeze-drying to obtain a dry powder preparation for preventing and improving chemical liver injury.
8. The preparation method of claim 6, wherein the fermentation temperature is 35-40 ℃, the fermentation time is 24-120h, and the content of yak milk in the culture medium is 15-20g/L.
9. Use of the Lactobacillus plantarum (Lactobacillus plantarum) according to claim 1 and/or the agent for preventing or ameliorating chemical liver injury according to any one of claims 2-5 in the preparation of a medicament for preventing or treating liver disease.
10. The use according to claim 9, wherein the liver disease comprises alcoholic hepatitis, toxic hepatitis, chronic hepatitis, fatty liver, autoimmune hepatitis.
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