Application of L.massiosenegalense and phenyllactic acid produced by L.massiosenegalense in preparation of medicine for preventing and treating acute pancreatitis

Abstract

The invention provides application of L.massiosenegalense and phenyllactic acid generated by the L.massiosenegalense in preparation of a medicine for preventing and treating acute pancreatitis, and belongs to the technical field of biological medicine. The L.massiosenegalense and the phenyllactic acid produced by the L.massiosenegalense can prevent and treat acute pancreatitis, relieve alveolar cell injury and inflammatory cell infiltration caused by acute pancreatitis, and reduce amylase concentration in serum and transcription and expression of inflammatory cytokines in pancreas and serum.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of L. massiliosenegalense and the phenyllactic acid produced therefrom in the preparation of drugs for the prevention and treatment of acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is one of the most common diseases of the digestive system, and its incidence is increasing year by year. AP can be divided into mild acute pancreatitis (MAP) and severe acute pancreatitis (SAP) according to the degree of inflammation. MAP is mainly characterized by pancreatic interstitial edema and rarely results in death, although 15-25% of MAP patients will further develop into SAP. SAP, on the other hand, is characterized by persistent organ failure and has a mortality rate as high as 36-50%. Currently, there are no effective clinical drugs for its treatment.

[0003] *Lachnoclostridium massiliosenegalense* (L. massiliosenegalense) was isolated from the feces of a healthy 38-month-old girl. Since its isolation and identification, research on its biological functions has been limited, and its specific functions remain unclear. Phenyllactic acid (PLA) is a microbial metabolite with broad-spectrum biological activity. It is mainly synthesized by probiotics such as *Lactobacillus* and *Pediococcus* through the phenylalanine metabolic pathway and is an important active molecule in the intestinal microbiome. Recent studies have found that PLA has broad-spectrum antibacterial properties, effectively inhibiting foodborne pathogens such as *Listeria monocytogenes*, *Salmonella*, and *Escherichia coli* O157:H7, and also showing good inhibitory effects on fungi such as *Penicillium* and *Aspergillus*.

[0004] Currently, there are no reports on the effects of phenyllactic acid derived from gut microbiota on acute pancreatitis, nor are there any studies on the immunological effects and systemic inflammatory responses of phenyllactic acid derived from gut microbiota. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the use of L. massiliosenegalense and the phenyllactic acid produced therefrom in the preparation of medicaments for the prevention and treatment of acute pancreatitis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the use of L. massiliosenegalense in the preparation of medicaments for the prevention / treatment of acute pancreatitis.

[0008] The present invention also provides the use of phenyllactic acid in the preparation of medicaments for the prevention / treatment of acute pancreatitis.

[0009] Preferably, the phenyllactic acid is a metabolite of L. massiliosenegalense.

[0010] Preferably, the prevention / treatment of acute pancreatitis includes at least one of the following:

[0011] (1) Relieves acinar cell damage and inflammatory cell infiltration;

[0012] (2) Reduce serum amylase concentration;

[0013] (3) Reduce the transcription and expression of inflammatory cytokines in the pancreas and serum.

[0014] Preferably, the inflammatory cytokines include IL-1β and IL-6.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention is the first to discover and confirm the role of L. massiliosenegalense in the prevention and treatment of acute pancreatitis, and the role of phenyllactic acid produced by it in the prevention and treatment of acute pancreatitis.

[0017] This invention investigated the role of *L. massiliosenegalense* in preventing acute pancreatitis by priming mice with pancreatitis after 48 hours of intestinal bacterial clearance using quadruple antibiotics and colonization with *L. massiliosenegalense* via intraperitoneal injection. The results showed that, compared to the control group, mice in the *L. massiliosenegalense* gavage group exhibited significantly lower serum amylase concentrations, reduced pancreatic acinar cell damage and inflammatory infiltration, and decreased levels of inflammatory cytokines in the pancreas and serum. This indicates that *L. massiliosenegalense* can prevent the occurrence and development of acute pancreatitis.

[0018] This invention investigated the therapeutic effect of *L. massiliosenegalense* on acute pancreatitis by intraperitoneal injection of L-arginine into mice, followed by gavage administration of *L. massiliosenegalense* on days 1, 2, and 3 post-modeling. The results showed that, compared to the control group, the *L. massiliosenegalense* gavage group exhibited significantly lower serum amylase concentration, reduced pancreatic acinar cell damage and inflammatory infiltration, and decreased levels of inflammatory cytokines in the pancreas and serum. This indicates that *L. massiliosenegalense* can treat the occurrence and progression of acute pancreatitis.

[0019] This invention, through non-targeted metabolomics analysis of L. massiliosenegalense culture supernatant, found that compared with the culture medium control, the content of phenyllactic acid (PLA) and 2-hydroxy-4-methylvaleric acid ((S)-Leucic acid, SLA) in L. massiliosenegalense culture supernatant was significantly increased.

[0020] This invention investigated the effects of PLA or SLA produced by *L. massiliosenegalense* on the prevention of acute pancreatitis by administering 100 mg / kg of PLA or SLA via gavage for one week to mice. Following intraperitoneal injection of *L. massiliosenegalense* to establish a pancreatitis model, the results showed that compared to the control group, the PLA gavage group exhibited significantly lower serum amylase concentration, reduced pancreatic acinar cell damage and inflammatory infiltration, and decreased levels of inflammatory cytokines in the pancreas and serum. Furthermore, PLA demonstrated a better protective effect than SLA, indicating that PLA can prevent the occurrence and development of acute pancreatitis. Attached Figure Description

[0021] Figure 1 The effect of L. massiliosenegalense treatment on mice 12 hours after acute pancreatitis was established using basilin (wherein, Figure 1 In this context, A represents the concentration of mouse serum amylase. Figure 1 In this context, B represents the nucleic acid transcription level of inflammatory factors in pancreatic tissue. Figure 1 (C in the figure represents the expression level of serum interleukin-6);

[0022] Figure 2 This study analyzed pathological sections of the pancreas from mice treated with *L. massiliosenegalense* 12 hours after acute pancreatitis was established using bacitracin.

[0023] Figure 3The effect of L. massiliosenegalense treatment on mice 72 hours after acute pancreatitis was established using arginine (wherein, Figure 3 In this context, A represents the concentration of mouse serum amylase. Figure 3 In this context, B represents the nucleic acid transcription level of inflammatory factors in pancreatic tissue. Figure 3 (C in the figure represents the expression level of serum interleukin-6);

[0024] Figure 4 This study analyzed pathological sections of the pancreas from mice treated with L. massiliosenegalense 72 hours after acute pancreatitis, using arginine as the starting material.

[0025] Figure 5 This is a volcano plot analysis of differential metabolites in the supernatant and culture medium of L. massiliosenegalense bacteria detected by non-targeted metabolomics.

[0026] Figure 6 The effect of phenyllactic acid treatment on mice 12 hours after acute pancreatitis was established using spirulina extract (wherein, Figure 6 In this context, A represents the concentration of mouse serum amylase. Figure 6 In this context, B represents the nucleic acid transcription level of inflammatory factors in pancreatic tissue. Figure 6 (C in the figure represents the expression level of serum interleukin-6);

[0027] Figure 7 This study used taurine to establish a pathological section analysis of the pancreas of mice treated with phenyllactic acid 12 hours after acute pancreatitis. Detailed Implementation

[0028] This invention provides the use of L. massiliosenegalense in the preparation of medicaments for the prevention / treatment of acute pancreatitis.

[0029] This invention also provides the use of phenyllactic acid in the preparation of medicaments for the prevention / treatment of acute pancreatitis. In this invention, the phenyllactic acid is a metabolite of *L. massiliosenegalense*.

[0030] In this invention, the prevention / treatment of acute pancreatitis includes at least one of the following:

[0031] (1) Relieves acinar cell damage and inflammatory cell infiltration;

[0032] (2) Reduce serum amylase concentration;

[0033] (3) Reduce the transcription and expression of inflammatory cytokines in the pancreas and serum.

[0034] In this invention, the inflammatory cytokines include IL-1β and IL-6.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] In this embodiment, L. massiliosenegalense was purchased from North Nacional Biotechnology Co., Ltd.

[0038] Twenty-five healthy C57BL / 6J mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., divided into five groups of five: control group (NC), model group (Vehical + SAP), LM treatment group (L. massiliosenegalense + SAP), PL.M treatment group (pasteurized L. massiliosenegalense + SAP), and SL.M treatment group (L. massiliosenegalense bacterial supernatant + SAP). The mice were treated as follows:

[0039] The model group (Vehical+SAP) and the L. massiliosenegalense+SAP group were administered a mixture of antibiotics consisting of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg) and vancomycin (16.7 mg) by gavage daily for 5 consecutive days to exclude interference from the intestinal flora.

[0040] Following combined antibiotic treatment, L. massiliosenegalense was treated with: 10 gavage. 9 CUF / day L. massiliosenegalense, administered by gavage for 2 consecutive days; PLM treatment: L. massiliosenegalense bacteria were treated in a 70℃ water bath for 30 minutes, centrifuged at 6000x g for 10 minutes, and the bacterial cells were collected and administered by gavage for 10 days. 9 CUF / day pL. massiliosenegalense, administered by gavage for 2 consecutive days; SL.M treatment: L. massiliosenegalense bacteria were centrifuged at 6000x g for 10 minutes, and the collected supernatant was filtered through a 0.22μm membrane and administered by gavage at 200μL / day SL. massiliosenegalense for one week.

[0041] The Vehical group was given an equal volume of culture medium by gavage daily.

[0042] Then, the above 5 groups of mice were used to establish a model of hymenoplasmosis: hymenoplasmosis was dissolved in sterile saline, and hymenoplasmosis (50 μg / kg mouse body weight) was injected intraperitoneally every hour for 7 consecutive hours, and LPS (10 mg / kg body weight) was injected intraperitoneally.

[0043] The timing began 12 hours after the first injection of basilin, and the mice were sacrificed.

[0044] Pancreatic tissue and peripheral blood samples were collected from mice in each group. Blood samples were placed at room temperature for 30 min, centrifuged at 3000 rpm for 10 min, and serum samples were transferred to new EP tubes. The amylase content in serum was determined using an amylase assay kit (EPS substrate method, Zhong Sheng Bei Kong Biotechnology Co., Ltd.) according to the standard procedure in the manufacturer's instructions. Pancreatic tissue was incubated overnight in 4% paraformaldehyde solution, trimmed, dehydrated stepwise with ethanol solutions of different concentrations, cleared with xylene, and then embedded in paraffin using an embedding machine. The embedded tissue blocks were sectioned using a microtome, the paraffin sections were dewaxed to water, stained with hematoxylin and eosin, and finally dehydrated and mounted. Tissue lesions were observed under a microscope. The serum IL-6 content was determined using an interleukin-6 (IL-6) ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd.) according to the standard procedure in the manufacturer's instructions. Total RNA was extracted from pancreatic tissue using RNAiso Plus (Takara) reagent, and the nucleic acid transcription levels of inflammatory factors were measured using the HiScript II One Stepq RT-PCR SYBR Greenkit kit (Vazyme). Peripheral blood lymphocytes were isolated from mouse peripheral anticoagulated blood according to the standard procedure specified in the Peripheral Blood Lymphocyte Isolation Kit (TDB).

[0045] Experimental results: such as Figures 1-2 As shown.

[0046] like Figure 1 As shown, compared with SAP mice, the serum amylase concentration in mice treated with L. massiliosenegalense and S-LM was significantly reduced. Figure 1 In A), and significantly reduced the expression of interleukin-1β (IL-1β) and IL-6 in the pancreas. Figure 1 B in the middle), and the expression of IL-6 in systemic circulation ( Figure 1 (C in the middle).

[0047] like Figure 2 As shown, compared with SAP mice, L. massiliosenegalense treatment and S-LM treatment significantly reduced pancreatic edema and inflammatory cell infiltration.

[0048] Example 2

[0049] In this embodiment, L. massiliosenegalense was purchased from North Nacional Biotechnology Co., Ltd.

[0050] Fifteen healthy C57BL / 6J mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., with five mice in each group. They were divided into three groups: control group (NC), model group (Vehical + SAP), and treatment group (L. massiliosenegalense + SAP). The mice underwent the following treatments:

[0051] Arginine modeling was performed on the above three groups of mice: arginine was dissolved in sterile saline and injected intraperitoneally once every hour (4g / kg mouse body weight) for two consecutive hours. At 68 hours after the start of arginine modeling, LPS (10mg / kg body weight) was injected intraperitoneally.

[0052] L. massiliosenegalense treatment: On days 1, 2, and 3 after the start of arginine modeling, administer 10 mg of L. massiliosenegalense via gavage. 9 CUF / day L. massiliosenegalense;

[0053] The Vehical group was given an equal volume of culture medium by gavage daily.

[0054] The mice were euthanized 72 hours after the first arginine injection.

[0055] Pancreatic tissue and peripheral blood samples were collected from mice in each group. The relevant detection indicators and methods are described in Example 1.

[0056] Experimental results: such as Figures 3-4 As shown. Figure 3 As shown, compared with SAP mice, the serum amylase concentration in mice treated with L. massiliosenegalense was significantly reduced. Figure 3 In A), and significantly reduced the expression of interleukin-1β (IL-1β) and IL-6 in the pancreas. Figure 3 B in the middle), and the expression of IL-6 in systemic circulation ( Figure 3 (C in the middle).

[0057] like Figure 4 As shown, compared with SAP mice, L. massiliosenegalense treatment significantly reduced pancreatic edema and inflammatory cell infiltration.

[0058] Example 3

[0059] In this embodiment, L. massiliosenegalense was purchased from North Nacional Biotechnology Co., Ltd.

[0060] The sequencing and analysis of the untargeted metabolome were performed by Beijing Novogene Technology Co., Ltd. The main methods are as follows:

[0061] The *L. massiliosenegalense* culture was centrifuged at 6000 x g for 10 min. The collected supernatant was filtered through a 0.22 μm membrane. 100 μL of the *L. massiliosenegalense* culture supernatant sample was added to 500 μL of 80% methanol solution, thoroughly vortexed, and incubated on ice for 5 min. The mixture was then centrifuged at 13000 rpm for 15 min at 4 °C. The supernatant was then injected into an LC-MS / MS system for metabolite analysis. The raw data file generated by LC-MS / MS was processed using Compound Discoverer 3.1 (CD3.1, ThermoFisher) for peak alignment, peak extraction, and quantification of each metabolite. These metabolites were annotated using the KEGG database (https: / / www.genome.jp / kegg / pathway.html), the HMDB database (https: / / hmdb.ca / metabolites), and the LIPIDMaps database (http: / / www.lipidmaps.org / ). Principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) were performed using MetaX software.

[0062] Experimental results: such as Figure 5 As shown, compared with the culture medium control, the contents of phenyllactic acid (PLA) and 2-hydroxy-4-methylvaleric acid ((S)-Leucic acid, SLA) in the culture supernatant of L. massiliosenegalense were significantly increased.

[0063] Example 4

[0064] Since current technology cannot purify phenyllactic acid and 2-hydroxy-4-methylvaleric acid metabolized by L. massiliosenegalense, commercially available phenyllactic acid and 2-hydroxy-4-methylvaleric acid were used as substitutes. D-(+)-3-phenyllactic acid (PLA) was purchased from Merck KGaA, Darmstadt, Germany, and 2-hydroxy-4-methylvaleric acid ((S)-Leucicacid, SLA) was purchased from MedChemexpress (MCE).

[0065] Twenty healthy C57BL / 6J mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., with five mice in each group. They were divided into four groups: control group (NC), model group (Vehical+SAP), SLA treatment group (SLA+SAP group), and PLA treatment group (PLA+SAP group). The mice underwent the following treatments:

[0066] The model group (Vehical+SAP), SLA treatment group (SLA+SAP group), and PLA treatment group (PLA+SAP group) were administered an antibiotic mixture consisting of ampicillin (10 mg), neomycin (10 mg), metronidazole (10 mg), and vancomycin (16.7 mg) by gavage daily for 5 consecutive days to exclude interference from the intestinal flora.

[0067] In addition to combined antibiotic treatment, the SLA treatment group (SLA+SAP group) and PLA treatment group (PLA+SAP group) were treated by gavage with 100 mg / kg SLA or PLA for one week.

[0068] The vehical group was given an equal volume of PBS by gavage daily.

[0069] Then, the above three groups of mice were used to establish a hymenoplasmosis model: hymenoplasmosis was dissolved in sterile saline, and hymenoplasmosis (50 μg / kg mouse body weight) was injected intraperitoneally every hour for 7 consecutive hours, and LPS (10 mg / kg body weight) was injected intraperitoneally.

[0070] The timing began 12 hours after the first injection of basilin, and the mice were sacrificed.

[0071] Pancreatic tissue and peripheral blood samples were collected from mice in each group. The relevant detection indicators and methods are described in Example 1.

[0072] Experimental results: such as Figures 6-7 As shown. Figure 6 As shown, compared with SAP mice, the concentration of amylase in the serum of PLA-treated mice was significantly reduced. Figure 6 In A), and significantly reduced the expression of interleukin-1β (IL-1β) and IL-6 in the pancreas. Figure 6 B in the middle), and the expression of IL-6 in systemic circulation ( Figure 6 (C in the text). Furthermore, PLA exhibits better protective performance than SLA.

[0073] like Figure 7 As shown, compared with SAP mice, PLA treatment significantly reduced pancreatic edema and inflammatory cell infiltration.

[0074] As can be seen from the above embodiments, L. massiliosenegalense and the phenyllactic acid produced therefrom can prevent and treat acute pancreatitis, and alleviate acinar cell damage and inflammatory cell infiltration caused by acute pancreatitis, reduce serum amylase concentration and the transcription and expression of inflammatory cytokines in the pancreas and serum.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of L. massiliosenegalense in the preparation of drugs for the prevention / treatment of acute pancreatitis.

2. Application of phenyllactic acid in the preparation of drugs for the prevention / treatment of acute pancreatitis.

3. The application according to claim 2, characterized in that, The phenyllactic acid is a metabolite of L. massiliosenegalense.

4. The application according to any one of claims 1 to 3, characterized in that, The prevention / treatment of acute pancreatitis includes at least one of the following: (1) Relieves acinar cell damage and inflammatory cell infiltration; (2) Reduce serum amylase concentration; (3) Reduce the transcription and expression of inflammatory cytokines in the pancreas and serum.

5. The application according to claim 4, characterized in that, The inflammatory cytokines include IL-1β and IL-6.

Images