Application of Lactobacillus plantarum P9 in the preparation of a drug for treating NAFLD

Abstract

The present invention discloses an application of Lactobacillus plantarum P9 in the preparation of a drug for treating NAFLD, which relates to the field of biotechnology. The Lactobacillus plantarum P9 provided by the present invention can play the role of probiotics, maintain the structure of a healthy intestinal microbial community to protect intestinal homeostasis, relieve non-alcoholic fatty liver disease by protecting the intestinal barrier function and improving the liver's tolerance to intestinal-derived factors through the gut-liver axis, maintain liver function, and ultimately reduce the risk of metabolic disorders. Therefore, Lactobacillus plantarum P9 has the application prospect of preparing a drug for preventing and / or treating non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to the application of Lactobacillus plantarum P9 in the preparation of drugs for treating NAFLD. Background Art

[0002] Unhealthy nutritional diets can stimulate normal innate immune responses and metabolic patterns, potentially triggering chronic low-grade systemic inflammation, also known as metabolic inflammation. Excessive calorie intake not only stimulates but also accelerates metabolic inflammation, thereby increasing the risk of developing metabolic disorders, including obesity, type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). As a hepatic manifestation of metabolic syndrome, the incidence of NAFLD has risen sharply worldwide, attracting research attention.

[0003] Patients with NAFLD have a relatively high risk of developing non-alcoholic steatohepatitis (NASH), which is characterized by liver inflammation and associated tissue damage, and ultimately progresses to cirrhosis. The "multiple-hit" hypothesis has confirmed multiple key factors involved in the pathogenesis of NASH, including hepatocyte dyslipidemia, gut microbiota translocation, and mitochondrial dysfunction. The high prevalence and complex pathogenesis of NAFLD have hindered the development of effective treatment methods and applications.

[0004] From the perspective of the "multiple-hit" theory, intestinal barrier dysfunction and its associated gut ecological dysbiosis are potential driving factors in the occurrence and development of NAFLD. The loss of intestinal integrity disrupts the balance between the gut microbiota and the host immune response, thereby promoting local inflammation, and further increasing the entry of inflammatory molecules, including endotoxin, into the liver through the portal vein. For example, lipopolysaccharide (LPS) produced by Gram-negative bacteria meets Kupffer cells in the liver, stimulating polarization into activated macrophages, causing chronic low-grade inflammation, inducing hepatocyte damage, and ultimately fibrosis. Increasing evidence shows that the gut microbiota promotes the immune defense system against the invasion of foreign organisms and pathogens. Enhancing gut microbiota balance helps maintain the intestinal microenvironment and is beneficial for maintaining the crosstalk of the immune, metabolic, and endocrine networks.

[0005] In recent years, more and more scientific evidence has supported that probiotics can restore the integrity of the intestinal barrier by upregulating the expression of tight junction (TJ) proteins, regulating the commensal flora structure, and producing metabolites such as short chain fatty acids (SCFAs), thereby reducing intestinal inflammation and preventing endotoxemia-mediated liver injury. Observations in a NASH mouse model supplemented with the VSL#3 probiotic mixture composed of Streptococcus, Thermophilus, Lactobacillus, and Bifidobacterium have shown that VSL#3 can effectively regulate liver inflammation and fibrosis. Additionally, Lactobacillus johnsonii BS15 can inhibit apoptosis of hepatocytes in obese mice by improving the antioxidant status of liver mitochondria.

[0006] However, the existing probiotics have limited effects on preventing and alleviating NAFLD / NASH, and no related strains have been approved for clinical use in this direction.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The object of the present invention is to provide an application of Lactobacillus plantarum P9 in the preparation of a drug for treating NAFLD to solve the above technical problems.

[0009] The present invention is implemented as follows:

[0010] An application of Lactobacillus plantarum P9 in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease, wherein the drug is used to restore the diversity of the intestinal flora, and the restoration of the diversity of the intestinal flora by the drug includes at least one of the following uses: changing the ratio of the intestinal flora of Firmicutes to Bacteroidetes, reducing the abundances of Faecalibaculum and Erysipelatoclostridium in the intestinal flora, and restoring the abundances of the inhibited Desulfovibrio and Colidextribacter.

[0011] Lactobacillus plantarum P9 is a strain with independent intellectual property rights isolated from naturally fermented sour porridge by Jiangzhong Pharmaceutical Group Co., Ltd. and Inner Mongolia Agricultural University. The strain information refers to Lactobacillus plantarum P9 disclosed in patent CN113558244A. The preservation number of Lactobacillus plantarum P9 is CGMCC No. 16662, and the preservation time is June 22, 2018.

[0012] The inventors found that Lactobacillus plantarum P9 can play the role of probiotics, maintain the structure of a healthy gut microbiota, and protect gut homeostasis; it can relieve non-alcoholic fatty liver disease by protecting gut barrier function and enhancing the liver's tolerance to gut-derived factors through the gut-liver axis, maintain liver function, and ultimately reduce the risk of metabolic disorders. Therefore, Lactobacillus plantarum P9 can be used to prepare a medicament for preventing and / or treating non-alcoholic fatty liver disease.

[0013] The fundamental reason why Lactobacillus plantarum P9 offsets the defects in gut barrier function is that supplementing P9 plays the role of probiotics, maintains the structure of a healthy gut microbiota, and protects gut homeostasis.

[0014] In a preferred embodiment of the application of the present invention, the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.

[0015] In a preferred embodiment of the application of the present invention, the medicament is used for at least one of the following purposes:

[0016] (1) Inhibiting colon length;

[0017] (2) Improving crypt distortion and lymphocyte infiltration in the colon;

[0018] (3) Protecting the integrity of the colon barrier;

[0019] (4) Inhibiting the increase in plasma triglyceride (TG) levels;

[0020] (5) Inhibiting the increase in plasma low-density lipoprotein (LDL) levels;

[0021] (6) Increasing the level of high-density lipoprotein (HDL);

[0022] (7) Reducing the levels of liver function biochemical parameters;

[0023] (8) Regulating the immune response and inhibiting Th1 and Th2 responses;

[0024] (9) Reducing fat accumulation in liver tissue;

[0025] (10) Upregulating the levels of Fas-associated death domain protein (FADD) and tumor necrosis factor receptor-associated death domain protein (TRADD) in liver tissue;

[0026] (11) Maintaining liver lipid metabolism homeostasis through the autophagy-lipophagy pathway.

[0027] The above-mentioned uses are all relative to animals with symptoms of non-alcoholic fatty liver disease (including non-alcoholic steatohepatitis).

[0028] The preventive effect of Lactobacillus plantarum P9 is manifested as follows: inhibiting the lipid profile changes caused by NAFLD (i.e., increased TG and LDL levels and decreased HDL level) and abnormal liver function (i.e., increased ALT and AST levels). In addition, P9 supplement can regulate the immune response and inhibit Th1 and Th2 responses, indicating a promising immunomodulatory effect to restore immune homeostasis against inflammation.

[0029] In a preferred embodiment of the application of the present invention, the drug regulates the immune response and inhibits Th1 and Th2 responses, including the following uses:

[0030] Inhibiting serum inflammatory factors.

[0031] In a preferred embodiment of the application of the present invention, the serum inflammatory factors are selected from at least one of MCP-1, IL-6, IL-10, and TNF-α.

[0032] In a preferred embodiment of the application of the present invention, the drug up-regulates the expression of Fas-associated death domain protein (FADD) and tumor necrosis factor receptor-associated death domain protein (TRADD) in liver tissue, including at least one of the following uses: increasing the expression level of tumor protein p53 in liver tissue.

[0033] In a preferred embodiment of the application of the present invention, the drug maintains the lipid metabolism homeostasis in the liver through the autophagy-lipophagy pathway, including at least one of the following uses:

[0034] Increasing the ratio of microtubule-associated protein light chain 3II (LC3II) to microtubule-associated protein light chain 3I (LC3I) in liver tissue;

[0035] Decreasing the expression level of peroxisome proliferator-activated receptor (PPAR)-α in liver tissue.

[0036] In a preferred embodiment of the application of the present invention, the drug reducing the levels of liver function biochemical parameters has at least one of the following uses: reducing the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0037] In a preferred embodiment of the application of the present invention, the drug further comprises a pharmaceutically acceptable additive or auxiliary component.

[0038] In an alternative embodiment, the pharmaceutically acceptable additive or excipient is selected from one or more of the following: solvent, buffer, emulsifier, suspending agent, decomposing agent, disintegrating agent, dispersing agent, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, flavoring agent, sweetening agent, pigment, and liposome.

[0039] In a preferred embodiment of the application of the present invention, the dosage form of the above-mentioned drug is tablet, capsule, suspension, solution, emulsion, powder, granule, injection, lyophilized powder injection, liniment, paint, film-forming agent, ointment, lotion, suppository, aerosol, spray, powder inhaler, ointment, plaster, cataplasm or patch.

[0040] The present invention has the following beneficial effects:

[0041] The inventors found that Lactobacillus plantarum P9 can play the role of probiotics, maintain the structure of a healthy gut microbiota, and protect gut homeostasis; it can relieve non-alcoholic fatty liver disease by protecting gut barrier function and improving the liver's tolerance to enterogenic factors through the gut-liver axis, maintain liver function, and ultimately reduce the risk of metabolic disorders. Therefore, Lactobacillus plantarum P9 has the application prospect of preparing drugs for preventing and / or treating non-alcoholic fatty liver disease. Description of the Drawings

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0043] Figure 1 Experimental result graph of the effect of Lactobacillus plantarum P9 on the colon length of HFD+DSS-induced NALFD model mice ((A) Colon length of each group of mice, (B) Comparison of significant differences in colon length of each group of mice);

[0044] Figure 2 HE staining (200x) graph of the colon tissues of each group of mice;

[0045] Figure 3 Immunofluorescence staining of tight junction proteins and mucins in the colon tissues of each group of mice;

[0046] Figure 4 Statistical graph of mannitol concentration in the blood of each group of mice;

[0047] Figure 5 Graph of serum biochemical indexes of each group of mice;

[0048] Figure 6 Graph of serum inflammatory factor levels of each group of mice;

[0049] Figure 7 Result graph of the effect of Lactobacillus plantarum P9 on the α-diversity of the gut microbiota of mice;

[0050] Figure 8The figure shows the effect of Lactobacillus plantarum P9 on the species composition of the intestinal flora in mice;

[0051] Figure 9 The figure shows the result of the species difference analysis of the intestinal flora in mice by Lactobacillus plantarum P9;

[0052] Figure 10 The figure shows the effect of Lactobacillus plantarum P9 on the liver tissue of NAFLD model mice induced by HFD+DSS;

[0053] Figure 11 The figure shows the result of the transcriptome analysis of the expression of enriched signaling pathways related to innate immune response and inflammation in the NAFLD mouse model provided by the present invention;

[0054] Figure 12 The figure shows the effect of Lactobacillus plantarum P9 on the protein expression in each group;

[0055] Figure 13 The figure shows the flow cytometry of the effect of Lactobacillus plantarum P9 on Th1 and Th2 helper cells in the non-alcoholic fatty liver model;

[0056] Figure 14 For Figure 13 The statistical result figure of Th1 and Th2 helper cells;

[0057] Figure 15 The figure shows the results of liver tissue pathology and oil red staining experiments after gavage modeling of mice in the Lactobacillus plantarum P9 group and the Lactobacillus plantarum ACCC11095 group;

[0058] Figure 16 The figure shows the statistical chart of serum liver biochemical indexes after gavage modeling of mice in the Lactobacillus plantarum P9 group and the Lactobacillus plantarum ACCC11095 group;

[0059] Figure 17 The figure shows the analysis result of Lactobacillus plantarum P9 regulating pathways such as tumor protein p53 and PPAR-α;

[0060] In the attached drawings: NC represents the normal group; PC represents the model group; P9L represents the low-dose group of Lactobacillus plantarum P9; P9H represents the high-dose group of Lactobacillus plantarum P9. Detailed Description of the Invention

[0061] Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0063] Experimental materials

[0064] Animals: Male C57BL / 6 mice (6 - 8 weeks old) were provided by Vital River Laboratory Animal Technology Co., Ltd. (Beijing). The breeding environment was 22 - 25°C, humidity 55% ± 15%, light / dark cycle 12 h, and they were not restricted by food and water. Animal research followed the regulations of the Animal Care Committee of Jiangxi University of Traditional Chinese Medicine and complied with Chinese laws and regulations on the use and care of laboratory animals (protocol number: JZLLSC20220006).

[0065] Reagents: Lactobacillus plantarum P9 (250 billion live bacteria / g) was provided by Zhejiang Jinhua Galaxy Biotechnology Co., Ltd. Dextran sulfate sodium salt (DSS, molecular weight 36 - 50 kDa) was obtained from MP Biomedicals (Irvine, CA, USA). The D-mannitol enzyme-linked immunosorbent assay kit was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. The endotoxin quantification kit (Thermo Fisher Scientific) was used to measure the plasma endotoxin concentration. Kits for total triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were all from Mindray (Shenzhen, China). Kits for tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-10 (IL-10), and adiponectin ELISA were purchased from Boster (Wuhan, China). TRIzol reagent, FastKing RT kit (containing gDNase), and SuperRealPreMix Plus (SYBR Green) were all purchased from Tiangen (Beijing, China). Antibodies against phosphatidylinositol 3-kinase (PI3K), p53, ubiquitin-binding protein p62, and microtubule-associated protein light chain (LC)-3 series were provided by Service Biotechnology Co., Ltd. (China), and peroxisome proliferator-activated receptor γ (PPAR-α) was purchased from Affinity Biotechnology Co., Ltd. (China).

[0066] Unless otherwise specified, in the figures of this embodiment, NC represents the normal group; PC represents the model group; P9L represents the low-dose group of Lactobacillus plantarum p9; P9H represents the high-dose group of Lactobacillus plantarum P9.

[0067] The features and performance of the present invention will be further described in detail below in conjunction with embodiments.

[0068] Example 1

[0069] Experiment on the effects of Lactobacillus plantarum P9 on the colon length, pathological indexes and intestinal permeability of NALFD model mice induced by HFD+DSS, and experiment on regulating immune function.

[0070] The methods involved in the experiment are as follows:

[0071] (1) Establishment of non-alcoholic fatty liver model: After 1 week of adaptive feeding of mice, 12 mice were randomly selected as the normal group and given normal diet, and the remaining mice were fed with high-fat diet for 13 weeks, and water containing 1.5% DSS was given every other week during this period.

[0072] (2) Animal grouping and administration: Subsequently, it was divided into 4 groups (n = 12): normal control group (NC), high-fat diet (HFD)+DSS treatment group (PC), HFD+DSS+P9 low-dose group (0.034 g / Kg BW), and HFD+DSS+P9 high-dose group (0.067 g / Kg BW).

[0073] (3) Observation indexes and detection methods:

[0074] At the end of the experiment, the animals were sacrificed. Blood, colon, intestinal contents, and liver tissues were collected and stored at -80 °C for biochemical, immunofluorescence and other analyses.

[0075] The colon length of mice is as Figure 1 shown in A and B below, mean ± standard deviation (n = 12), * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. NC represents the normal group; PC represents the model group; P9L represents the low-dose group of Lactobacillus plantarum p9; P9H represents the high-dose group of Lactobacillus plantarum P9.

[0076] Compared with the normal mice in the NC group, the HFD+DSS treatment significantly shortened the colon length first. However, in the mice supplemented with P9, the reduction in colon length caused by HFD plus DSS was significantly inhibited (p < 0.01, p < 0.0001).

[0077] The results of the effects of Lactobacillus plantarum P9 on the colonic pathological indexes of NALFD model mice induced by HFD+DSS are shown in reference to Figure 2 As shown, the crypts in the PC group were distorted, as indicated by the arrows, and the lamina propria was dilated. The dilation of the lamina propria was caused by lymphocyte infiltration, suggesting colonic inflammatory damage. Compared with the PC group, both high-dose and low-dose P9 could improve the crypt deformation and lymphocyte infiltration induced by HFD+DSS.

[0078] The inventors detected intestinal TJ proteins, including the expression of zonula occludens (ZO)-1 and mucin (MUC)-2, by immunofluorescence histology. As Figure 3 shown, the intensity of ZO-1 was lower relative to the PC group, while it was higher in the P9L and P9H groups. At the same time, compared with the NC group, the expression of MUC-2 disappeared in the PC group, but the expression of MUC-2 recovered after supplementation with low-dose or high-dose P9. The results showed that HFD+DSS had a negative impact on the integrity of the mucosal barrier.

[0079] Therefore, the experimental results of this example show that supplementing P9 can protect the integrity of the colonic epithelial barrier, suggesting that P9, as a probiotic, may have therapeutic potential for intestinal barrier function.

[0080] Experiment on the effect of Lactobacillus plantarum P9 on intestinal permeability of NASH model mice induced by HFD+DSS:

[0081] After fasting for 6 h, mice in different treatment groups were anesthetized with isoflurane, and serum was collected to measure the plasma D-mannitol concentration.

[0082] The results are shown in Figure 4 , compared with the normal group, the mannitol levels detected in the PC group and the P9-supplemented group were significantly increased (P<0.0001), indicating an increase in intestinal barrier permeability in mice treated with HFD+DSS. The presence of D-mannitol in the blood as a macromolecule indicates increased epithelial permeability and impaired intestinal barrier function, but the P9-supplemented group had no obvious effect on protecting intestinal permeability.

[0083] Experiment on regulating immune function:

[0084] Peripheral whole blood of each mouse was collected into a heparin-containing centrifuge tube by cardiac puncture, and red blood cells were depleted using red blood cell lysis buffer. According to the instructions of the mouse Th1 / Th2 staining kit, cells were stained with an antibody combination against cell markers, data was collected using a CytoFlex flow cytometer (Beckman), and analysis was performed using Flow JO and GraphPad software to determine the indicated cell proportions.

[0085] The results are shown in Figure 13 and Figure 14As shown, the immunomodulatory effect of P9 is manifested as the inhibition of Th1 and Th2 responses, indicating the immunomodulatory effect of P9. Compared with the PC group, the Th1 and Th2 in the high-dose P9 group (P9H) and the low-dose P9 group (P9L) were significantly lower than those in the PC group.

[0086] Example 2

[0087] The mice were modeled and grouped according to the experimental method of Example 1, and the serum biochemical indexes of different treatment groups were measured. As Figure 5 , compared with the NC group, the plasma triglyceride (TG) of the mice in the PC group was significantly increased, while the supplementation of P9 could effectively inhibit the increase of plasma triglyceride (TG) induced by HFD and DSS in mice (P < 0.001), but no similar results were found in the total plasma cholesterol (TC). Compared with the NC group, the plasma LDL of the mice in the PC group was significantly increased, and P9 had an inhibitory effect on LDL, but the inhibitory effect of low-dose P9 on LDL was significant (P < 0.05). Compared with the PC group, the addition of P9 could significantly increase the content of high-density lipoprotein (HDL) in HFD+DSS-induced mice (P < 0.001). Compared with the PC group, the supplementation of high-dose and low-dose P9 also showed a significant decrease in the levels of liver function biochemical parameters, including alanine aminotransferase ALT and AST (P < 0.05, P < 0.001).

[0088] The levels of serum inflammatory factors in mice of different treatment groups were further measured, and the results were as Figure 6 shown. The results of the inflammatory factor levels in each group of mice showed that compared with the normal group, the levels of MCP-1, IL-6, IL-10, and TNF-α in the model group were all increased, and among them, MCP-1, IL-10, and TNF-α were significant (p < 0.01, p < 0.001). Compared with the model group, the serum inflammatory factors in each drug administration group were all decreased, and among them, IL-10 and TNF-α had significant differences (p < 0.05, p < 0.01, p < 0.001).

[0089] Example 3

[0090] In this example, an experiment on the effect of Lactobacillus plantarum P9 on the intestinal flora of NAFLD model mice induced by HFD+DSS was carried out.

[0091] High-throughput sequencing of the 16S rRNA gene of the intestinal microbiota in mice. For the determination, 0.2 g of mouse cecal contents was taken, and total microbial community DNA was extracted and the V3-V4 region of the 16S rRNA gene was amplified by PCR. Sequencing was performed using the Illumina MiseqPE300 platform. The raw data was quality controlled and optimized sequences were obtained. The optimized sequences were clustered into OTUs for non-redundant sequences according to 97% similarity, and chimeras were removed during the clustering process to obtain the representative sequences of the OTUs; all optimized sequences were mapped to the representative sequences of the OTUs, and sequences with similarity above 97% to the representative sequences were selected to generate an OTU table. Then, based on the OTU table, species classification was performed, a phylogenetic tree was generated, and the relative abundances at each level of the samples were divided; diversity analysis of each library was performed based on the OTU table data, and box plots of the Chao1 index and Shannon index were drawn using R software (Version 3.4.2).

[0092] 1. Analysis of species diversity of intestinal flora

[0093] The Chao1 and Shannon indices reflect the species richness and diversity of a single sample. As can be observed from Figure 7 it, compared with normal mice, HFD and DSS significantly reduced species diversity (p < 0.01). However, in this example, no significant enrichment of the intestinal flora species by P9 supplementation was observed. However, according to the Shannon index, a recovery of the intestinal flora diversity was detected in the high-dose P9 treatment group.

[0094] 2. Further analysis of the species composition of the intestinal flora.

[0095] As Figure 8 shown, the intestinal flora at the phylum level was mainly composed of Bacteroidetes, Firmicutes, and Verrucomicrobia. The results showed that the Firmicutes / Bacteroidetes (F / B) ratio was higher in the NC group and the low-dose P9 treatment group, and there was a statistically significant difference between the PC group and the low-dose P9 treatment group (p < 0.05), indicating that P9 could change the intestinal structure composition of the model group mice.

[0096] 3. Analysis of species differences in intestinal flora

[0097] Through Figure 9Species difference analysis showed that the core microbiota of mice in the PC group were mainly Faecalibaculum, Erysipelatoclostridium, Dubosiella, and Parasutterella. Notably, higher abundances of Faecalibaculum and Erysipelatoclostridium were found in the NAFLD mouse model. Adding P9 could regulate the abundances of Faecalibaculum and Erysipelatoclostridium in a dose-dependent manner, and the high-dose P9 treatment group showed a significant decrease (p<0.05).

[0098] Example 4

[0099] In this example, liver tissue experiments were conducted.

[0100] 1. Effects of Lactobacillus plantarum P9 on liver tissues of mice with HFD+DSS-induced NAFLD model

[0101] From Figure 10 in A, the HE staining results of liver tissues showed that the hepatic lobule structure of liver tissues of mice in the normal group was clear, the hepatic cords were arranged neatly, the cytoplasm of hepatocytes was abundant, the structure was normal, the hepatic sinusoids were not significantly dilated or compressed, and no obvious lipid droplets were seen. In the liver tissues of mice in the model group, obvious fatty degeneration occurred, and more round vacuoles could be seen in the cytoplasm. The cell nuclei were shrunk and became smaller, and the boundaries between cells were unclear. Compared with the model group, the hepatic lobule structure of the high-dose and low-dose P9 administration groups was clear, the hepatic cords were arranged neatly, the cytoplasm of hepatocytes was abundant, and the morphological structure was normal. The above results indicated that the high-dose and low-dose P9 administration groups could reduce fat accumulation in the livers of mice and prevent the formation of fatty degeneration of liver tissues and secondary non-alcoholic fatty liver.

[0102] As Figure 10 in B, compared with the NC group, obvious lipid aggregation was detected in the PC group, and the lipid droplets increased sharply, indicating the appearance of fatty liver characteristics in the NAFLD mouse model of the present invention. Both the high-dose and low-dose P9 groups had obvious inhibitory effects on hepatic tissue lipid aggregation.

[0103] 2. Control experiment:

[0104] Lactobacillus plantarum P9 and Lactobacillus plantarum ACCC11095 (strain preservation number: ACCC11095) were intragastrically administered to mice with intermittent DSS treatment and high-fat diet-induced secondary non-alcoholic fatty liver to compare the curative effects.

[0105] Experimental method: The live preparations of Lactobacillus plantarum P9 and Lactobacillus plantarum ACCC11095 were used to intragastrically administer to the modeled mice, corresponding to the Lactobacillus plantarum P9 group and the Lactobacillus plantarum ACCC11095 group, respectively. It was administered once a day, 200 μL each time, and the intragastric administration dose was 1×10 10 CFU / mouse, and intragastric administration was continuously performed for 13 weeks. After the experiment, the Lactobacillus plantarum P9 group and the Lactobacillus plantarum ACCC11095 group were taken for detection of relevant liver pathology and biochemical indexes.

[0106] As Figure 15 , Figure 16 shown by the results, Lactobacillus plantarum P9 has a more obvious effect in improving non-alcoholic fatty liver lesions compared with Lactobacillus plantarum ACCC11095, which is manifested in that the liver tissue pathology and oil red staining are better than those of the ACCC11095 group, and the P9 group is better than the ACCC11095 group in terms of serum liver biochemical indexes. Therefore, the live preparation of Lactobacillus plantarum P9 has a significantly better improvement effect on non-alcoholic fatty liver mice than the live preparation of Lactobacillus plantarum ACCC11095.

[0107] Example 5

[0108] RNA-seq analysis of mouse liver tissue

[0109] RNA-seq analysis was performed on mouse liver tissue samples to screen out the gene profiles with differential expression in the liver RNA-seq analysis supplemented with P9. Comparative analysis of the liver transcriptome profiles of the NC group and the PC group showed that there were 3,277 differentially expressed genes (DEGs) in the PC group compared with the NC group, including 1,842 up-regulated genes and 1,345 down-regulated genes [|log2(FC)|>2, P<0.05]. Comparative analysis of the liver transcriptome profiles showed that there were approximately 987 DEGs between the PC group and the high-dose P9 treatment group, including 310 up-regulated genes and 677 down-regulated genes [|log2(FC)|>2, P<0.05]. In the NAFLD mouse model provided by the present invention, rich signaling pathways related to innate immune response and inflammation were up-regulated in the liver ( Figure 11 ). Interestingly, in the NAFLD mouse model, the characteristic pathways regulated by high-dose supplementation of P9 include the tumor protein p53 and PPAR-α pathways, which are more than their respective immune pathways ( Figure 17 ).

[0110] Example 6

[0111] Western blotting was used to study the molecular mechanism of the liver function protection effect of Lactobacillus plantarum P9 on HFD+DSS-induced NAFLD model mice.

[0112] In the liver tissues obtained in this example, selected biomarkers in metabolic and autophagy signaling events were evaluated, including phosphatidylinositol 3-kinase (PI3K), p53, ubiquitin-binding protein p62, microtubule-associated protein light chain (LC)-3, and peroxisome proliferator-activated receptor (PPAR)-α. The PI3K / protein kinase B (AKT) signaling pathway in hepatocytes is crucial for increasing fatty acid synthesis, and the dysregulation of this pathway increases the risk of developing metabolic dysfunctions such as non-alcoholic fatty liver disease (NAFLD).

[0113] However, in this example, no significant differences in the expression of PI3K in the liver were detected among the NC group, PC group, and high-dose P9 supplementation group ( Figure 12 in B). The expression level of p53 in the high-dose P9 treatment group was slightly higher compared with that in the PC group. The expression of p62 in the HFD+DSS-treated mice decreased slightly compared with that in normal mice; while in the HFD+DSS-treated mice, the supplementation of P9 could restore the abundance of p62 protein ( Figure 12 in B). Meanwhile, the LCII / LCI ratio in the P9 supplementation group was significantly higher than that in the HFD+DSS treatment group, and LCII / LCI is a marker of autophagy induction.

[0114] In addition, the results of this example showed that the expression of PPAR-α in the NAFLD mouse model was decreased by HFD+DSS treatment. However, compared with the PC group, the abundance of PPAR-α was significantly restored after high-dose supplementation of P9 in the P9H group ( Figure 12 in B).

[0115] In summary, based on the characteristics of promoting exogenous metabolism, Lactobacillus plantarum P9 has the potential to promote health in the liver. Therefore, the experimental results of the present invention show that in the HFD+DSS-induced mouse model, the protective effect of supplementing P9 on intestinal barrier integrity can maintain liver function alone. First, the present invention determined that the preventive effect of P9 supplements the lipid profile changes caused by NAFLD (i.e., elevated TG and LDL levels and decreased HDL levels) and abnormal liver function (i.e., elevated ALT and AST levels). In the NAFLD mouse model, it was found that P9 supplements can regulate the immune response and inhibit Th1 and Th2 responses, indicating a promising immunomodulatory effect that can restore immune homeostasis against inflammation. Second, the present invention observed that HFD plus DSS led to changes in intestinal morphology, disappearance of ZO-1 and muc2, and elevated d-mannitol concentration, which was due to damage to the integrity of the intestinal barrier. This caused systemic inflammation by significantly increasing plasma LPS and pro-inflammatory cytokines (such as MCP-1 and TNF-α). However, the experimental results of the present invention show that supplementing P9 can offset the defects in intestinal barrier function. The possible underlying reason is that supplementing P9 exerts probiotic effects to maintain a healthy intestinal microbiota structure to protect intestinal homeostasis. Although no significant enrichment of the intestinal flora was found in the group supplemented with P9, P9 was shown to inhibit the growth of NAFLD-specific intestinal microbiota, including Faecalibaculum and Erysipelatoclostridium, and restore the abundances of Desulfovibrio and Colidextribacter, which are particularly inhibited in NAFLD. In addition, since the disordered luminal environment changes the NAFLD intestinal microbiota structure and causes mucosal immune responses, the present invention explored the relevant differentially expressed gene profiles. The experimental results of the present invention found that adding P9 can upregulate TRADD and FADD in HFD+DSS-induced mice, suggesting that P9 may maintain intestinal-derived immune homeostasis by regulating functional intestinal autophagy and apoptosis. It can be concluded from the present invention that the effective lipid-aggregation-improving Lactobacillus plantarum P9 can supplement the NAFLD mouse model in the liver. The results of RNA-seq and WB analysis showed that the characteristic pathways regulated by high-dose P9 were the p53 and PPAR-α pathways. In addition, elevated P53, LC3-II / LC3-I ratio, and PPAR-α expression were found in the NAFLD mouse model supplemented with P9, suggesting that adding P9 can maintain liver lipid metabolism homeostasis through the autophagy-lipophagy pathway.

[0116] In conclusion, the present invention provides in-depth understanding of the interaction between the probiotic P9 in the intestinal microbiota and the host immune response, thereby maintaining intestinal integrity and liver function through the gut-liver axis and ultimately reducing the risk of developing metabolic disorders.

[0117] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of Lactobacillus plantarum P9 in the preparation of a drug for preventing and / or treating and restoring the diversity of intestinal flora, characterized in that, The drug restores the diversity of the intestinal flora by changing the proportion of the intestinal flora of Firmicutes and Bacteroidetes. The preservation number of Lactobacillus plantarum P9 is CGMCC No. 16662, and the preservation time is June 22, 2018.

2. The application according to claim 1, wherein The drug is used for at least one of the following purposes: (1) Restoring the colon length; (2) Improving the crypt deformation and lymphocyte infiltration of the colon; (3) Protecting the integrity of the colonic epithelial barrier.

3. The application according to any one of claims 1-2, characterized in that, The drug also includes pharmaceutically acceptable additives or auxiliary components.

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