Phytobacterium plantarum CCFM242 with function of relieving intrahepatic cholestasis and application of phytobacterium plantarum CCFM242

Abstract

The invention discloses a plant lactobacillus CCFM242 with a function of relieving intrahepatic cholestasis and application of the plant lactobacillus CCFM242, and belongs to the technical field of microorganisms and the technical field of medicines. The lactobacillus plantarum CCFM242 and the active ingredients of the lactobacillus plantarum CCFM242 disclosed by the invention have good safety, and can be used for relieving the symptoms of the cholestasis type liver diseases. Therefore, the lactobacillus plantarum CCFM242 has a huge application prospect in preparation of drugs for preventing and / or treating intrahepatic cholestasis type liver diseases.

Description

Technical Field

[0001] The present invention relates to a Lactiplantibacillus plantarum CCFM242 with the function of relieving intrahepatic cholestasis and its application, belonging to the technical fields of microbiology and medicine. Background Art

[0002] Cholestasis refers to the pathological state in which the flow of bile from the liver or biliary system is blocked, resulting in the accumulation of bile components, especially bile acids (BAs), and further causing a series of physiological and metabolic disorders. Common symptoms of cholestasis include jaundice, pruritus, abnormal liver function, etc. Its etiology is complex, including congenital diseases, biliary diseases, liver diseases, drug induction and other factors. Among these factors, the excessive accumulation of BAs in the liver is the key to its pathogenesis. Excessive cytotoxic BAs in the liver may not only induce liver fibrosis and cirrhosis, but may even increase the risk of liver cancer. Therefore, at the stage of intrahepatic cholestasis, inhibiting BA synthesis and promoting BA excretion are protective mechanisms to prevent the accumulation of BAs in the liver and cell poisoning.

[0003] In the past decade, the important role of gut microbiota in host health has been established. Gut microbiota acts as a bridge between the gut and other tissues, receiving and releasing signals to various parts of the body, and affecting basic functions, including digestion, energy metabolism and host inflammation. It is well known that in the "gut-liver axis" dialogue, the microbiota encodes many biochemical activities related to host health and disease. In recent years, the chemical modification of BAs by the microbiota and the "crosstalk" between gut microbiota and BAs have attracted extensive attention. The high-concentration accumulation of BAs will affect the structure and function of the gut microbiota. Under normal circumstances, BAs will enter the liver through intestinal reflux, but in the case of cholestasis, the abnormal accumulation of BAs will change the pH value of the intestine, affect the balance of the intestinal microecology, lead to the growth of harmful bacteria and the reduction of beneficial bacteria, thus further aggravating liver damage. At the same time, BAs are metabolites synthesized by the host and derived from microorganisms. Host-encoded BA receptors recognize BAs as signaling molecules that regulate host immunity, metabolism and circadian rhythm. The effect of BAs on the microbiota echoes the microbial transformation of BAs, thus establishing the chemical complexity of the intestinal BA pool.

[0004] Numerous studies have shown that various probiotics have therapeutic potential in animal models of liver diseases, including cholestasis, acute liver injury, non-alcoholic fatty liver disease, cirrhosis, and primary sclerosing cholangitis. Lactiplantibacillus plantarum, as a dietary supplement, is also a probiotic that can regulate the intestinal flora and intestinal homeostasis, but its functions are strain-specific, which further affects the regulatory effect of Lactiplantibacillus plantarum on diseases related to abnormal BA metabolism. At present, studies have pointed out that Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus helveticus can prevent / treat / relieve cholestatic liver disease. However, no researcher has paid attention to the preventive and relieving effects of Lactiplantibacillus plantarum on cholestatic mice. Therefore, it is necessary to explore the protective effect of Lactiplantibacillus plantarum on cholestatic mice and develop Lactiplantibacillus plantarum that can effectively relieve cholestatic mice. Summary of the Invention

[0005] The present invention provides the use of Lactiplantibacillus plantarum CCFM242 in the preparation of a medicament for preventing and / or treating cholestatic liver disease.

[0006] In one embodiment of the present invention, the amount of Lactiplantibacillus plantarum CCFM242 in the medicament is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0007] In one embodiment of the present invention, the medicament is a microbial preparation, and the microbial preparation contains wet cells or freeze-dried cells of Lactiplantibacillus plantarum CCFM242.

[0008] In one embodiment of the present invention, the preparation method of the microbial preparation is: obtained by mixing the cultured Lactiplantibacillus plantarum CCFM242 cells with a cryoprotectant.

[0009] In one embodiment of the present invention, the cryoprotectant includes one or more of skim milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate.

[0010] In one embodiment of the present invention, the medicament contains Lactiplantibacillus plantarum CCFM242, a pharmaceutical carrier, and / or a pharmaceutical excipient.

[0011] In one embodiment of the present invention, the pharmaceutical carrier includes microcapsules, microspheres, nanoparticles, and / or liposomes.

[0012] In one embodiment of the present invention, the pharmaceutical excipient includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0013] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0014] In one embodiment of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0015] In one embodiment of the present invention, the additive comprises a solubilizer, a cosolvent, a cosolvent and / or a preservative.

[0016] In one embodiment of the present invention, the dosage form of the drug is a granule, a capsule, a tablet, a pill or an oral liquid.

[0017] In one embodiment of the present invention, the drug can regulate the flora structure, increase the relative abundance of Firmicutes with high BSH activity, change the BAs profile, and activate the enterohepatic axis FXR-FGF-15 signaling pathway to relieve intrahepatic cholestasis.

[0018] In one embodiment of the present invention, the prevention and / or treatment of intrahepatic cholestasis includes at least one of the following effects:

[0019] (1) Reducing the levels of total bile acids (TBAs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in the serum of cholestatic individuals;

[0020] (2) Reducing the disease symptoms of cholestatic individuals and relieving liver tissue damage;

[0021] (3) Reducing the liver and colon inflammation levels in cholestatic individuals;

[0022] (4) Activating the enterohepatic axis FXR-FGF-15 signaling pathway in cholestatic individuals to promote bile acid excretion;

[0023] (5) Regulating the flora structure of cholestatic individuals and increasing the relative abundance of Firmicutes with high BSH activity;

[0024] (6) Regulating the bile acid profiles in the liver and ileum of cholestatic individuals.

[0025] Beneficial effects

[0026] 1. The present invention provides the application of a Lactiplantibacillus plantarum CCFM242 in intrahepatic cholestatic liver disease, specifically manifested as:

[0027] (1) Reducing total bile acids (TBAs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in the serum of cholestatic mammals;

[0028] (2) Reduce the disease symptoms of cholestatic mammals and relieve liver tissue damage;

[0029] (3) Reduce the inflammatory levels in the livers and colons of cholestatic mammals;

[0030] (4) Activate the FXR-FGF-15 signaling pathway of the enterohepatic axis in cholestatic mammals to promote bile acid excretion;

[0031] (5) Regulate the microbiota structure of cholestatic mammals and increase the relative abundance of Firmicutes with high BSH activity;

[0032] (6) Regulate the bile acid profiles in the livers and ileums of cholestatic mammals.

[0033] 2. The Lactiplantibacillus plantarum CCFM242 of the present invention and its active ingredients have good safety and possess therapeutic uses, and can be used for developing probiotic products. Therefore, Lactiplantibacillus plantarum CCFM242 has great application prospects in the preparation of preventing and / or treating intrahepatic cholestatic liver disease. Description of the Drawings

[0034] Figure 1 : Blood biochemical indexes of mice in different groups. Among them, A is alanine aminotransferase (ALT); B is aspartate aminotransferase (AST); C is alkaline phosphatase (ALP); D is total bile acids (TBAs).

[0035] Figure 2 : H&E staining images of the livers of mice in different groups.

[0036] Figure 3 : Contents of total bile acids (TBAs) in the livers and fecal tissues of mice in different groups. Among them, A is the content of TBAs in liver tissues, and B is the content of TBAs in fecal tissues.

[0037] Figure 4 : Protein levels of inflammatory factors in the livers and colons of mice in each group.

[0038] Figure 5 : Transcription and protein levels of receptors related to BAs synthesis and transport in the liver tissues of mice in each group. Among them, A-F are the transcription levels of FXR, CYP7A1, BSEP, MRP-2, OATP, and NTCP respectively; G-I are the protein levels of FXR, CYP7A1, and BSEP respectively.

[0039] Figure 6: Transcription and protein levels of BA transporters-related receptors in the liver tissues of mice in each group. Among them, A - C are the transcription levels of FXR, FGF-15, and ASBT respectively; D - F are the protein levels of FXR, FGF-15, and ASBT respectively.

[0040] Figure 7 : BSH levels in the feces of mice in each group.

[0041] Figure 8 : Intestinal flora diversity of mice in each group. Among them: A is the α-diversity of the intestinal flora of mice; B is the β-diversity of the intestinal flora of mice.

[0042] Figure 9 : Intestinal flora structure and composition of mice in each group. A is the stacked bar chart of the phylum level of the intestinal flora of mice; B is the relative abundance of Firmicutes in the intestinal flora of mice; C is the relative abundance of Bacteroidetes in the intestinal flora of mice.

[0043] Figure 10 : BA composition and content in the liver of mice in each group. Among them, A is the content of conjugated bile acids in the liver; B is the content of free bile acids in the liver.

[0044] Figure 11 : BA composition and content in the feces of mice in each group. A is the content of conjugated bile acids in the feces; B is the content of free bile acids in the feces. Specific implementation methods

[0045] The α-isothiocyanate (ANIT) involved in the following examples was purchased from Shanghai Macklin Biochemical Co., Ltd.; the chemicals such as tryptone and yeast powder involved in the following examples were purchased from Sinopharm Group; the mice involved in the following examples were 6 - 8-week-old male SPF (Specific pathogen free) grade C57BL / 6J mice purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.; the Fast DNA Spin Kit for Feces kit involved in the following examples was purchased from MP Biomedicals; the paraformaldehyde involved in the following examples was purchased from Wuhan Sevier Biotechnology Co., Ltd. The primers involved in the following examples were purchased from Shanghai Sangon Biotech Co., Ltd.

[0046] The Lactiplantibacillus plantarum CCFM242 involved in the following examples has been published in the literature "Research on the Antioxidant Evaluation and Antioxidant Mechanism of Lactiplantibacillus plantarum".

[0047] The culture media involved in the following examples are as follows:

[0048] MRS liquid medium: Peptone 10 g / L, Beef extract 10 g / L, Yeast extract 5 g / L, Glucose 20 g / L, Sodium acetate anhydrous 2 g / L, Diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Cysteine hydrochloride 1 g / L, Tween-80 1 g / L, Distilled water 1000 g / L.

[0049] MRS solid medium: Peptone 10 g / L, Beef extract 10 g / L, Yeast extract 5 g / L, Glucose 20 g / L, Sodium acetate anhydrous 2 g / L, Diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Cysteine hydrochloride 1 g / L, Tween-80 1 g / L, Agar 20 g / L, Distilled water 1000 g / L.

[0050] The strains involved in the following examples are as follows:

[0051] Table 1 Strains used in this experiment

[0052]

[0053] Example 1: Evaluation of the alleviating effect of Lactiplantibacillus plantarum on intrahepatic cholestasis

[0054] 1. Effect of Lactiplantibacillus plantarum on serum indexes of mice with intrahepatic cholestasis

[0055] Inbred C57BL / 6J male mice with small individual differences, 6 - 8 weeks old, were raised in the Animal Experiment Center of Jiangnan University (25 ± 2°C, 12 / 12 h light / dark period). The mice had free access to food and water. After one week of the adaptation period, the mice were randomly divided into 6 groups: NC group, ANIT model group, Lactiplantibacillus plantarum CCFM242 intervention group, Lactiplantibacillus plantarum FGSYC225L2 intervention group, Lactiplantibacillus plantarum FJLHD37M1 intervention group, and Lactiplantibacillus plantarum FCQNA34M6 intervention group, with 6 mice in each group. All groups were administered once a day for 21 consecutive days during the experiment. On the 14th day of the experiment, except for the NC group which was given the vehicle olive oil, the model group and the bacteria-administered intervention groups were all single-dose gavaged with ANIT (65 mg / kg, dissolved in olive oil). After gavaging the corresponding drugs to each group on the 21st day of the experiment, the mice were fasted for 4 h, anesthetized with isoflurane, blood was collected from the eye socket, and the mice were sacrificed by cervical dislocation for sampling.

[0056] The treatment methods for each group of animals are as follows, and normal feed was maintained during the treatment period.

[0057] Blank control (NC) group: Mice were gavaged with normal saline for 21 days at a volume of 0.2 mL. On the 14th day, 4 hours after gavaging with normal saline, olive oil was gavaged once. On the 21st day, 4 hours after gavaging with normal saline, the mice were sacrificed under anesthesia.

[0058] ANIT-induced model group: Mice were gavaged with normal saline for 21 days at a volume of 0.2 mL. On the 14th day, 4 hours after gavaging with normal saline, ANIT (65 mg / kg, dissolved in olive oil) was gavaged once to construct a cholestasis model. On the 21st day, 4 hours after gavaging with normal saline, the mice were sacrificed under anesthesia.

[0059] Lactiplantibacillus plantarum intervention group: Mice were gavaged with Lactiplantibacillus plantarum for 21 days (1×10 9 CFU / day) at a volume of 0.2 mL. On the 14th day, 4 hours after gavaging with Lactiplantibacillus plantarum, ANIT (65 mg / kg, dissolved in olive oil) was gavaged once to construct a cholestasis model. On the 21st day, 4 hours after treatment with Lactiplantibacillus plantarum, the mice were sacrificed under anesthesia.

[0060] Method for biochemical analysis of mouse serum: After the experiment, blood was collected from the mouse eyeballs into 1.5 mL EP tubes and allowed to stand at room temperature for 2 h, then centrifuged at 3000 r / min at 4°C for 15 min. The upper serum was carefully aspirated, aliquoted, and stored in a -80°C refrigerator for detection of blood biochemical indices. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in the mouse serum liver function indices were detected and analyzed using an automatic biochemical analyzer. The determination of the total bile acid (TBAs) content in the serum was carried out according to the operation guide of the kit from Nanjing Jiancheng Institute.

[0061] As Figure 1As shown, the contents of ALT, AST, and ALP in the sera of the model group mice were respectively: 819.38±246.27 (U / L), 666.75±214.89 (U / L), 505.00±57.08 (U / L), which were significantly increased compared with the model group (p<0.01). After the intervention of Lactiplantibacillus plantarum CCFM242, the increase in the levels of liver function-related enzymes was significantly inhibited (p<0.05), while the treatments with the other three strains of Lactiplantibacillus plantarum had no significant effect on the serum liver enzyme levels. In addition, the content of serum TBAs also increased significantly after ANIT modeling (p<0.001), and the content was 127.25±29.12 (μmol / L). Lactiplantibacillus plantarum CCFM242 significantly inhibited the increase in TBAs (p<0.05); after the intervention with the other three strains of Lactiplantibacillus plantarum, the recovery effect of serum TBAs levels was poor. Among them, Lactiplantibacillus plantarum CCFM242 had the best alleviating effect. Compared with the model group, the contents of ALT, AST, ALP, and TBAs decreased by 59.87%, 60.67%, 36.67%, and 68.61% respectively. The above results indicate that Lactiplantibacillus plantarum can significantly improve the liver function of ANIT-induced cholestatic mice.

[0062] 2. Effects of Lactiplantibacillus plantarum on the liver tissues of intrahepatic cholestasis mice

[0063] The specific implementation method is the same as 1 in Example 1.

[0064] Detection method for the histopathological characteristics of liver tissues: Fix the liver tissues in 4% (m / V) paraformaldehyde fixative for 24 h, send them to Wuhan Sevier Biotechnology Co., Ltd. to complete the H&E staining of liver tissues and prepare sections, scan the prepared H&E liver sections with a Pannoramic MIDI digital slide scanner, take pictures, and observe the liver tissue damage.

[0065] It can be seen from Figure 2 that ANIT induced severe liver injury and inflammation. It was found that hepatocyte degeneration, necrosis, and inflammatory cell infiltration occurred in the portal vein area and around the central vein, which were not observed in the NC group. Lactiplantibacillus plantarum CCFM242 alleviated the severe damage to the liver tissue structure, improved the infiltration of inflammatory granulocytes, and weakened the degree of hepatocyte necrosis. The degrees of liver tissue damage in the mice treated with Lactiplantibacillus plantarum FGSYC225L2, FJLHD37M1, and FCQNA34M6 were similar to those observed in the ANIT group, with only a slight alleviating effect. The above results indicate that Lactiplantibacillus plantarum CCFM242 can significantly improve the pathological damage of the liver tissues of cholestatic mice.

[0066] 3. Effects of Lactiplantibacillus plantarum on the total bile acids in the liver and feces of intrahepatic cholestasis mice

[0067] The specific implementation method is the same as that in Example 1.

[0068] Method for measuring total bile acids in mouse liver and fecal tissues: Weigh liver and fecal tissue samples, add 0.9% normal saline and 3 bead mill beads according to the ratio of weight (g) / volume (mL) equal to 1:9, grind them into homogenate, centrifuge at 6000×g for 10 min, take the supernatant, and measure according to the operation guide of the kit from Nanjing Jiancheng Company.

[0069] As Figure 3 shown, the contents of total bile acids in the liver and fecal tissues of the ANIT-induced model group were 70.60±3.11 (μmol / L) and 32.05±4.44 (μmol / L), respectively, indicating that the modeling significantly increased the level of liver TBAs (p<0.001) and decreased the excretion of fecal TBAs (p<0.0001). Treatment with Lactiplantibacillus plantarum CCFM242 and FGSYC225L2 significantly restored the level of TBAs in the liver (p<0.01), while Lactiplantibacillus plantarum FJLHD37M1 and FCQNA34M6 had no effect on liver TBAs. It is worth noting that only Lactiplantibacillus plantarum CCFM242 greatly promoted fecal BAs excretion (p<0.01), and the other strains only showed a trend of promotion but no significance. Among them, the overall effect of Lactiplantibacillus plantarum CCFM242 was better. Compared with the model group, liver TBAs decreased by 39.23%, and fecal TBAs increased by about one time. In summary, Lactiplantibacillus plantarum can significantly promote the excretion of BAs in ANIT-induced cholestatic mice and reduce the accumulation of BAs in the liver.

[0070] Example 2: Analysis of the mode of action of Lactiplantibacillus plantarum CCFM242 in relieving intrahepatic cholestasis

[0071] 1. Lactiplantibacillus plantarum CCFM242 reduces the inflammatory levels in the liver and ileum tissues of intrahepatic cholestatic mice

[0072] The specific implementation method is the same as that in Example 1.

[0073] Method for determining cytokine content in mouse liver and ileum tissues: Weigh liver and ileum tissue samples, add 0.9% normal saline and 3 grinding beads according to the ratio of weight (g) / volume (mL) equal to 1:9, grind into homogenate, centrifuge at 6000×g for 10 min, and take the supernatant to measure tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-10 (IL-10) in mouse liver and ileum tissues with reference to the instructions of the relevant kit of Senbeijia Company.

[0074] The accumulation of BAs in the liver can exacerbate the inflammatory response by promoting macrophage accumulation and cytokine production, such as IL-6, TNF-α, IL-1β, etc. The persistence of inflammation, in turn, exacerbates hepatocyte necrosis and fibrosis during intrahepatic cholestasis. Figure 4 As can be seen, ANIT led to a significant increase in the protein levels of IL-6, TNF-α, and IL-1β in the liver (p<0.05), and a significant decrease in the protein level of IL-10 (p<0.05). Under the treatment of Lactiplantibacillus plantarum CCFM242, the protein levels of IL-6 and TNF-α decreased significantly, and the protein level of IL-10 was restored. In addition to the inflammation in liver tissues, the study also detected an increase in the levels of pro-inflammatory cytokines in intestinal tissues. Figure 4 As shown, compared with the NC group, several pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1β, showed a significant increase in the ANIT group (p<0.01). Treatment with Lactiplantibacillus plantarum CCFM242 significantly reduced the levels of these pro-inflammatory cytokines and significantly increased the level of IL-10 (p<0.05). The above research results indicate that Lactiplantibacillus plantarum CCFM242 plays an important role in alleviating intrahepatic cholestasis and can reduce ANIT toxicity through its anti-inflammatory activity to cope with liver injury and the subsequent inflammation.

[0075] 2. Effect of Lactiplantibacillus plantarum CCFM242 on the expression of receptors related to BA synthesis and transport in the liver tissues of mice with intrahepatic cholestasis

[0076] The specific implementation method is the same as 1 in Example 1.

[0077] Method for determining receptors related to BA synthesis and transport in mouse liver tissue: Use TRIzol reagent to extract RNA from mouse liver and ileum tissues, and use an RNA reverse transcription kit to reverse transcribe it into cDNA. The RT-qPCR technique was used to determine the expression levels of FXR, CYP7A1, BSEP, NTCP, MRP-2, and OATP receptor genes in liver tissue. GADPH was used as an internal reference gene, and the 2 -ΔΔCT method was used to quantitatively analyze the target gene. The sequences of related primers are listed in Table 2. The enzyme-linked immunosorbent assay was used to analyze the protein contents of FXR, CYP7A1, BSEP in liver tissue and FXR, FGF-15, ASBT in ileum tissue. The specific method refers to the instructions of the relevant kit of Senbeijia Company.

[0078] Table 2 Primer sequences for RT-qPCR

[0079]

[0080]

[0081] It can be seen from Figure 5 that ANIT significantly reduced the mRNA expression levels of liver FXR, BSEP, and MRP-2 (p<0.05). Lactiplantibacillus plantarum CCFM242 significantly restored the expression of FXR, BSEP, and MRP-2 genes (p<0.05), indicating that Lactiplantibacillus plantarum CCFM242 can increase the efflux of BAs through the FXR pathway. As key transporters responsible for the reabsorption of BAs from the blood into hepatocytes, after ANIT treatment, the expressions of OATP and NTCP were significantly decreased (p<0.0001), and Lactiplantibacillus plantarum CCFM242 significantly up-regulated the expression of the NTCP gene (p<0.01). The intervention of ANIT and Lactiplantibacillus plantarum did not affect the expression of CYP7A1. In terms of protein level expression, Lactiplantibacillus plantarum CCFM242 significantly restored the decrease in the protein levels of FXR and BSEP caused by ANIT (p<0.05), but did not change the protein level of CYP7A1.

[0082] The FXR receptor is highly expressed in hepatocytes and the intestine. It is a bile acid sensor that regulates bile acid absorption, metabolism, and transport, and is an important target for the treatment of cholestasis and other liver diseases. BSEP and MRP-2 are two major bile acid efflux transporters and are FXR target genes. A decrease in the expression levels of these genes indicates impaired pathways for bile acids to be transported to the intestine. Lactiplantibacillus plantarum CCFM242 can significantly upregulate the protein levels of FXR and BSEP. These data suggest that under intrahepatic cholestasis conditions, the effect of Lactiplantibacillus plantarum CCFM242 on ANIT-induced intrahepatic bile acid accumulation is not the result of bile acid synthesis inhibition, but rather the result of upregulation of FXR-mediated bile acid efflux proteins.

[0083] 3. Effect of Lactiplantibacillus plantarum CCFM242 on the expression of bile acid transport-related receptors in the ileum tissue of intrahepatic cholestasis mice

[0084] The specific implementation method is the same as 1 in Example 1.

[0085] Method for measuring bile acid transport-related receptors in mouse ileum tissue: The specific implementation method is the same as 2 in Example 2, and the primer sequences involved are shown in Table 2.

[0086] As Figure 6 shown, Lactiplantibacillus plantarum CCFM242 significantly alleviated the decrease in ileal FXR and FGF-15 gene expression caused by ANIT (p < 0.05). For the protein concentrations of ileal FXR and FGF-15, the results showed that FXR and FGF-15 were significantly decreased in the ANIT group (p < 0.05). After treatment with Lactiplantibacillus plantarum CCFM242, the protein levels of FXR and FGF-15 were restored to the NC group, which was consistent with the gene expression results. At the same time, a significant increase in the protein level of ASBT was also observed in the ANIT group (p < 0.01), and Lactiplantibacillus plantarum CCFM242 significantly downregulated the ileal ASBT content (p < 0.01).

[0087] In the ileum, activation of FXR can inhibit bile acid synthesis through the FXR-FGF-15-FGFR4 pathway, in which FXR induces the expression of the FGF-15 hormone, which is transported to the liver through portal venous blood and then binds to the FGFR4 and β-klotho complex, ultimately regulating the expression of related receptor genes in the liver. Therefore, hepatic FXR and intestinal FXR coordinately regulate bile acid synthesis, transport, reabsorption, and metabolic balance. These results together indicate that Lactiplantibacillus plantarum CCFM242 alleviates ANIT-induced intrahepatic cholestasis in mice by activating the enterohepatic axis FXR-FGF-15 signaling pathway.

[0088] 4. Effect of Lactiplantibacillus plantarum CCFM242 on the BSH level in the feces of intrahepatic cholestasis mice

[0089] The specific implementation manner is the same as that in 1 of Example 1.

[0090] Method for measuring the BSH level in mouse feces: The specific measurement method refers to the instruction manual of the microbial BSH ELISA detection kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.).

[0091] As Figure 7 shown, after ANIT-induced liver injury, the fecal BSH level decreased significantly, while the intervention with Lactiplantibacillus plantarum CCFM242 could significantly increase the fecal BSH level (p < 0.05). Under cholestasis, the fecal BA content was also positively correlated with the BSH activity of intestinal bacteria, because BSH bacteria with high activity could catabolize conjugated BAs into free BAs, and free BAs had lower polarity and lower transport efficiency through ASBT, making them easier to excrete from the intestine. These results indicate that Lactiplantibacillus plantarum CCFM242 can effectively improve ANIT-induced intrahepatic cholestasis by inducing the deconjugation of intestinal conjugated BAs and increasing the excretion of fecal BAs.

[0092] 5. Lactiplantibacillus plantarum CCFM242 affects the intestinal flora diversity in mice with intrahepatic cholestasis

[0093] The specific implementation manner is the same as that in 1 of Example 1.

[0094] Method for measuring the intestinal flora of mice: After the experiment, mouse colonic contents were collected. After extracting the fecal bacterial metagenome of each group of mice using the Fast DNA Spin Kit for Feces, the 16s V3-V4 region sequences were amplified by PCR, and all the obtained PCR products were subjected to 1.5% agarose gel electrophoresis. The target gene was recovered and purified from the agarose gel containing the target band using a gel extraction kit and sequenced on the Illumina Miseq PE300 platform. The downloaded data was analyzed using the QIIMEII software.

[0095] As Figure 8As shown in the figure, in terms of α-diversity, compared with the NC group, ANIT intervention tended to increase the Shannon index and significantly increased the Simpson index. After intervention with Lactiplantibacillus plantarum CCFM242, the Shannon and Simpson indices were decreased, making them closer to the NC group. For the Pielou and Chao1 indices, neither ANIT nor Lactiplantibacillus plantarum intervention had a significant effect. In terms of β-diversity, the intestinal microbiota of the NC group and the ANIT group of mice were well separated, indicating differences in the microbiota structures of the two groups. ANIT intervention caused significant changes in the intestinal microbiota of mice; there was also a certain degree of dispersion between the Lactiplantibacillus plantarum CCFM242 intervention group and the ANIT group, and the intestinal microbiota structure of mice was closer to that of the NC group. This indicates that the supplementation of Lactiplantibacillus plantarum CCFM242 can regulate the imbalance of intestinal microorganisms and achieve a new homeostasis of the intestinal microbiota structure.

[0096] 6. Lactiplantibacillus plantarum CCFM242 affects the intestinal microbiota structure and composition of mice with intrahepatic cholestasis

[0097] The specific implementation method is the same as item 1 in Example 1.

[0098] Method for measuring the intestinal microbiota of mice: The specific implementation method is the same as item 5 in Example 2.

[0099] As Figure 9 As shown in the figure, the community stacked bar chart at the phylum level shows that Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria together constitute the main phyla in the mouse intestine. Among them, Firmicutes and Bacteroidetes are the two phyla with the highest relative abundances, accounting for up to 80% of the total abundance of the identifiable microbiota. Moreover, Firmicutes contains bacteria with high BSH activity. Under ANIT intervention, the level of Firmicutes decreased significantly (p < 0.01), while the relative abundance of Firmicutes in the mice of the Lactiplantibacillus plantarum CCFM242 group increased significantly by 20% (p < 0.01); on the contrary, Bacteroidetes contains bacteria with low BSH activity. Compared with the NC group, the relative abundance of Bacteroidetes increased significantly in the ANIT group, and the intervention of Lactiplantibacillus plantarum CCFM242 could correct the above changes (p < 0.05). This indicates that Lactiplantibacillus plantarum CCFM242 can relieve intrahepatic cholestasis by regulating the relative abundances of phyla with BSH activity.

[0100] 7. Lactiplantibacillus plantarum CCFM242 changes the bile acid profile in the liver of mice with intrahepatic cholestasis

[0101] The specific implementation method is the same as item 1 in Example 1.

[0102] Method for determining bile acid profile in mouse liver: Weigh and record liver and fecal tissues respectively and place them in 1.5 mL centrifuge tubes. Add 1 mL of methanol, grind them into a uniform paste, and precipitate at room temperature for 1 h. Centrifuge at 13400×g for 15 min at 4 °C, and collect the supernatant; for the precipitate, add 1 mL of methanol again, shake and mix evenly, precipitate at room temperature for 20 min and then centrifuge, and collect the supernatant. Repeat this process once to ensure sufficient extraction. Combine all the obtained supernatants, and use vacuum centrifugal concentration technology to evaporate the solution to a completely dry state, and store it at -20 °C. Before measurement, re-dissolve with 400 μL of methanol and centrifuge, and take the supernatant into the injection vial liner. ACQUITY The HSS T3 1.8 μm (2.1×100 mm) chromatographic column was used for the quantitative analysis of BAs, and 2 μL of sample was injected for gradient elution.

[0103] As Figure 10 shown, after ANIT administration to mice, the concentrations of conjugated BAs in the liver: T-β-MCA, TUDCA, TCA, TCDCA, TDCA all increased significantly (p < 0.01). For free BAs, except for CA, the others did not change significantly, indicating that after ANIT modeling, bile accumulation in the liver was severe. Intervention with Lactiplantibacillus plantarum CCFM242 could significantly reduce the contents of T-β-MCA, TUDCA, TDCA (p < 0.05), and there was a tendency to increase the contents of the corresponding free BAs. These results suggest that Lactiplantibacillus plantarum CCFM242 can regulate the liver BAs pool and make its composition closer to that of the NC group, thus playing a role in alleviating intrahepatic cholestasis.

[0104] 8. Lactiplantibacillus plantarum CCFM242 alters the fecal bile acid profile in mice with intrahepatic cholestasis

[0105] The specific implementation method is the same as 1 in Example 1.

[0106] Method for determining bile acid profile in mouse liver: The specific implementation method is the same as 7 in Example 2.

[0107] As Figure 11 shown, after ANIT intervention, the intestinal BAs profile changed, specifically manifested as the increase of T-β-MCA, TCDCA (p < 0.05); the decrease of TDCA, TCA, β-MCA, UDCA, CDCA, DCA (p < 0.05). While Lactiplantibacillus plantarum CCFM242 could alleviate this change, significantly reduce the levels of T-β-MCA, TCA, TCDCA, up-regulate the contents of β-MCA, CDCA (p < 0.05), and there was a tendency to up-regulate UDCA, DCA.

[0108] In cholestasis, FXR can be activated by BAs with different structures and mediate the synthesis and secretion of BAs. T-β-MCA has been identified as an effective inhibitor of intestinal FXR and cannot be metabolized in the absence of bacteria. The microbiota can promote FXR signaling in mice by deconjugating T-β-MCA. CDCA is considered the most effective ligand for FXR, followed by DCA and then CA. This further explains the activation of hepatic and ileal FXR receptors after intervention with Lactiplantibacillus plantarum CCFM242. The above data indicate that Lactiplantibacillus plantarum CCFM242 can regulate the intestinal microbiota structure, alter the BA profile, and further activate the FXR signaling pathway to alleviate intrahepatic cholestasis.

[0109] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of Lactiplantibacillus plantarum CCFM242 in the preparation of a medicament for preventing and / or treating cholestatic liver disease.

2. The application according to claim 1, wherein The number of Lactiplantibacillus plantarum CCFM242 in the said drug is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

3. The application according to claim 1 or 2, characterized in that, The medicament is a microbial preparation, and the Lactiplantibacillus plantarum CCFM242 in the microbial preparation is wet cells or freeze-dried cells.

4. The application according to claim 3, characterized in that, The preparation method of the microbial preparation is: obtained by mixing the cultured Lactiplantibacillus plantarum CCFM242 cells with a cryoprotectant.

5. The application according to claim 4, characterized in that The cryoprotectant includes one or more of skim milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate.

6. The application according to claim 1 or 2, characterized in that, The medicament contains Lactiplantibacillus plantarum CCFM242, a drug carrier, and / or pharmaceutical excipients.

7. The application according to claim 6, wherein The drug carrier includes microcapsules, microspheres, nanoparticles, and / or liposomes.

8. The application according to claim 7, wherein The pharmaceutical excipients include one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

9. The application according to claim 8, wherein The dosage form of the medicament is granules, capsules, tablets, pills, or oral liquids.

10. The application according to any one of claims 1 to 9, characterized in that, The medicament has at least one of the following effects: (1) Reducing the levels of total bile acids, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in the serum of cholestatic individuals; (2) Reducing the disease symptoms of cholestatic individuals and alleviating liver tissue damage; (3) Reducing the liver and colon inflammation levels in cholestatic individuals; (4) Activating the enterohepatic axis FXR-FGF-15 signaling pathway in cholestatic individuals and promoting bile acid excretion; (5) Regulating the flora structure of cholestatic individuals and increasing the relative abundance of Firmicutes with high BSH activity; (6) Regulating the bile acid profiles in the liver and ileum of cholestatic individuals.

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