Application of indole-3-lactic acid in preparation of medicine for resisting acute pancreatitis

Abstract

The invention relates to application of indole-3-lactic acid in preparation of an acute pancreatitis resisting medicine, and belongs to the technical field of medicines. The invention provides an application of indole-3-lactic acid in preparation of an acute pancreatitis resisting medicine. An antibiotic mixture is used for conducting intragastric administration on a mouse, intestinal bacteria are removed, ILA and STS are subjected to intragastric administration on the mouse, leptofrog and arginine are injected into the abdominal cavity of the mouse for acute pancreatitis modeling, the acute pancreatitis resisting effect of ILA is studied, and the result shows that the amylase concentration in serum of the mouse subjected to intragastric administration of ILA is remarkably reduced; compared with STS, the ILA has the advantages that pancreatic tissue alveolar cell damage and inflammatory infiltration are obviously reduced, inflammatory cytokines in pancreatic tissue and serum are obviously reduced, and compared with STS, the ILA has a better protection effect, so that the ILA has the effect of resisting acute pancreatitis, and a new treatment way is provided for preventing and treating acute pancreatitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of indole-3-lactic acid in the preparation of drugs for 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, has a high mortality rate, and currently there are no effective clinical treatments.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide the application of indole-3-lactic acid in the preparation of drugs for treating acute pancreatitis, in order to solve the problem of the lack of drugs for treating acute pancreatitis in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of indole-3-lactic acid in the preparation of drugs for treating acute pancreatitis.

[0006] Preferably, the indole-3-lactic acid achieves its anti-acute pancreatitis effect through at least one of the following: (1) Relieves damage to acinar cells and inflammatory cell infiltration in pancreatic tissue; (2) Reduce serum amylase concentration; (3) Reduce the transcription and expression of inflammatory cytokines in pancreatic tissue and / or serum.

[0007] The present invention has the following technical effects and advantages: This invention uses a mixture of antibiotics to gavage mice to eliminate intestinal bacteria. Mice were then gavaged with ILA and STS, and acute pancreatitis was modeled by intraperitoneal injection of vitexin and arginine, respectively. The effect of ILA on acute pancreatitis was investigated. The results showed that mice gavaged with ILA had significantly lower serum amylase concentration, significantly reduced pancreatic acinar cell damage and inflammatory infiltration, and significantly reduced inflammatory cytokines in pancreatic tissue and serum. Furthermore, ILA showed better protective effects compared to STS, indicating that ILA has an anti-acute pancreatitis effect and provides a new therapeutic approach for the prevention and treatment of acute pancreatitis. Attached Figure Description

[0008] Figure 1 Results of serum amylase content detection in mice with different treatment groups induced by taurine; Figure 2 The relative expression levels of inflammatory factors in the pancreatic tissue of mice with different treatment groups induced by taurine; Figure 3 The levels of inflammatory factors in the serum of mice with different treatment groups induced by taurine were determined. Figure 4 Results of pancreatic tissue lesions in mice with different treatment groups induced by taurine; Figure 5 The relative expression levels of inflammatory factors in the pancreatic tissue of arginine-induced model mice in different treatment groups; Figure 6 The levels of inflammatory factors in the serum of arginine-induced model mice in different treatment groups; Figure 7 Results of pancreatic tissue lesions in mice with different treatment groups induced by taurine. Detailed Implementation

[0009] This invention provides the application of indole-3-lactic acid in the preparation of drugs for treating acute pancreatitis.

[0010] In this invention, the indole-3-lactic acid achieves its anti-acute pancreatitis effect through at least one of the following: (1) Relieves damage to acinar cells and inflammatory cell infiltration in pancreatic tissue; (2) Reduce serum amylase concentration; (3) Reduce the transcription and expression of inflammatory cytokines in pancreatic tissue and / or serum.

[0011] 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.

[0012] In the examples, indole-3-lactic acid (ILA) was purchased from MedChemexpress; sodium thiosulfate (STS) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; the antibiotic mixture was obtained by mixing 10 mg of ampicillin, 10 mg of neomycin, 10 mg of metronidazole and 16.7 mg of vancomycin.

[0013] Example 1

[0014] 1. Experimental Grouping

[0015] Twenty healthy C57BL / 6J mice were divided into four groups of five each: a blank control group, a dimethyl sulfoxide group, an indole-3-lactic acid group, and a sodium thiosulfate group.

[0016] 2. Experimental Methods

[0017] Mice in each group were fed normally. The blank control group received no treatment. The dimethyl sulfoxide (DMSO) group, indole-3-lactic acid (ILA) group, and sodium thiosulfate (NSL) group were administered an antibiotic mixture by gavage for 5 consecutive days to exclude interference from the intestinal flora. The daily gavage dose was 50 mg / kg (body weight of the mouse). In addition to the antibiotic mixture, the indole-3-lactic acid (ILA) group was also administered 50 mg / kg (body weight of the mouse) of ILA by gavage for 7 consecutive days. The NSL group was also administered 50 mg / kg (body weight of the mouse) of STS by gavage for 7 consecutive days. The DMSO group was also administered 50 mg / kg (body weight of the mouse) of PBS buffer containing 5% DMSO by gavage for 7 consecutive days. Seven days later, the dimethyl sulfoxide group, indole-3-lactic acid group, and sodium thiosulfate group underwent laminarin modeling. Specifically, laminarin was dissolved in sterile saline, and 50 μg / kg (body weight of the mouse) of laminarin was injected intraperitoneally every hour for seven consecutive hours. At the same time, LPS (lipopolysaccharide) 10 mg / kg (body weight of the mouse) was injected intraperitoneally. Timing started 12 hours after the first laminarin injection, and all mice in the treatment groups were sacrificed. Pancreatic tissue and peripheral blood samples were collected from each group of mice.

[0018] 3. Results Analysis

[0019] 3.1 Determination of serum amylase content in mice with different treatment groups induced by taurine modeling

[0020] Peripheral blood samples were incubated at room temperature for 30 min, centrifuged at 3000 rpm for 10 min, and serum samples were transferred to new EP tubes. The serum amylase content was measured using an amylase assay kit (EPS substrate method, purchased from Sinopharm Biotech Co., Ltd.) according to the standard procedure in the manufacturer's instructions. The results of serum amylase content detection in different treatment groups of mice with the taeniacin-induced model are shown below. Figure 1 As shown in the figure, the horizontal axis represents different treatment groups, and the vertical axis represents the amylase content in the serum of mice with the taeniacin-induced model (enzyme activity units / L10). 3 ).

[0021] according to Figure 1 It was found that, compared with the dimethyl sulfoxide group, the serum amylase content of mice in the indole-3-lactic acid group and the sodium thiosulfate group was reduced, and the serum amylase content of mice in the indole-3-lactic acid group was significantly reduced.

[0022] 3.2 Relative expression levels of inflammatory factors in pancreatic tissue of mice with different treatment groups induced by taurine

[0023] Total RNA was extracted from pancreatic tissue using RNAiso Plus (purchased from Baori Biotechnology (Beijing) Co., Ltd.). The nucleic acid transcription levels of inflammatory factors were measured using the HiScript II One Step qRT-PCR SYBR Green kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The relative expression levels of inflammatory factors in the pancreatic tissue of rats induced by taurine in different treatment groups are as follows: Figure 2 As shown in the figure, the horizontal axis represents different treatment groups, and the vertical axis represents the relative expression levels of inflammatory factors in the pancreatic tissue of mice with taurine-induced modeling. Figure A shows the relative expression level of the inflammatory factor IL-6 (interleukin-6) in the pancreatic tissue of mice with taurine-induced modeling in different treatment groups, and Figure B shows the relative expression level of the inflammatory factor TNF-α (tumor necrosis factor-α) in the pancreatic tissue of mice with taurine-induced modeling in different treatment groups.

[0024] according to Figure 2 It was found that, compared with the dimethyl sulfoxide group, the relative expression levels of inflammatory factors IL-6 and TNF-α in the pancreatic tissue of mice in the indole-3-lactic acid group and the sodium thiosulfate group were reduced, and the relative expression levels of inflammatory factors IL-6 and TNF-α in the pancreatic tissue of mice in the indole-3-lactic acid group were significantly reduced.

[0025] 3.3 Serum levels of inflammatory factors in mice with different treatment groups induced by taurine-induced inflammatory factors

[0026] The levels of inflammatory factors IL-6 and TNF-α in serum were measured using an interleukin-6 (IL-6) ELISA kit and a tumor necrosis factor (TNF-α) ELISA kit (purchased from Hangzhou Lianke Biotechnology Co., Ltd.) according to the standard procedures in the instructions. The results of the levels of inflammatory factors in the serum of mice with the leptospirin model in different treatment groups are as follows: Figure 3 As shown in the figure, the horizontal axis represents different treatment groups, and the vertical axis represents the content of inflammatory factors in the pancreatic tissue of mice with taurine-induced modeling. Figure A shows the content of IL-6 (Ng / mL) in the serum of mice with taurine-induced modeling in different treatment groups, and Figure B shows the content of TNF-α (Ng / mL) in the serum of mice with taurine-induced modeling in different treatment groups.

[0027] according to Figure 3 It was found that, compared with the dimethyl sulfoxide group, the expression of inflammatory factors IL-6 and TNF-α in the serum of mice in the indole-3-lactic acid group and the sodium thiosulfate group was reduced, and the expression of inflammatory factors IL-6 and TNF-α in the serum of mice in the indole-3-lactic acid group was significantly reduced.

[0028] 3.4 Results of pancreatic tissue lesions in mice with different treatment groups induced by taurine

[0029] Pancreatic tissue was placed in 4% paraformaldehyde solution overnight, dehydrated stepwise with ethanol solutions of different concentrations, cleared with xylene, and then placed in paraffin for embedding using an embedding machine. The embedded tissue blocks were sectioned using a microtome. The paraffin sections were dewaxed and placed in water, stained with hematoxylin and eosin, and finally dehydrated and mounted to obtain tissue lesion sections. The tissue lesion sections were observed under a microscope, and the severity of acute pancreatitis was scored on the pathological sections. The score was divided into three aspects: edema (0 points, no edema; 1 point, focal widening of interlobular spaces; 2 points, diffuse widening of interlobular spaces; 3 points, acin rupture and separation), inflammatory cell infiltration (0 points, no inflammatory infiltration; 1 point, inflammatory infiltration in or at the edge of the lumen; 2 points, less than 50% of the parenchyma is infiltrated; 3 points, more than 50% of the parenchyma is infiltrated), and necrosis (0 points, no necrosis; 1 point, periluminal necrosis or necrosis area less than or equal to 5%; 2 points, local necrosis or necrosis area of ​​5% to 20%; 3 points, diffuse necrosis or necrosis area greater than 20%). The sum of the scores of the three aspects was taken as the total score. Results of pancreatic tissue lesions in mice with different treatment groups induced by taurine: Figure 4 As shown in the figure, Figure A represents the observation results of pancreatic tissue sections of mice with taurine modeling in different treatment groups, and Figure B represents the pathological scores of pancreatic tissue sections of mice with taurine modeling in different treatment groups. The horizontal axis in the figure represents different treatment groups, and the vertical axis represents the pathological scores of pancreatic tissue sections of mice with taurine modeling.

[0030] according to Figure 4 It can be seen that, compared with the dimethyl sulfoxide group, the indole-3-lactic acid group significantly reduced pancreatic acinar cell edema and inflammatory cell infiltration, and its mouse pancreatic tissue pathological section score was the lowest.

[0031] In summary, ILA significantly reduced serum amylase concentration in mice with acute pancreatitis induced by taurine, reduced acinar cell damage and inflammatory infiltration in mouse pancreatic tissue, and also reduced inflammatory cytokines in mouse pancreatic tissue and serum, showing better protective effects than STS, indicating that ILA has an anti-taurine-induced acute pancreatitis effect.

[0032] Example 2

[0033] 1. Experimental Grouping

[0034] Twenty healthy C57BL / 6J mice were divided into four groups of five each: a blank control group, a dimethyl sulfoxide group, an indole-3-lactic acid group, and a sodium thiosulfate group.

[0035] 2. Experimental Methods

[0036] Mice in each group were fed normally. The blank control group received no treatment. The dimethyl sulfoxide (DMSO) group, indole-3-lactic acid (ILA) group, and sodium thiosulfate (NSL) group were administered an antibiotic mixture by gavage for 5 consecutive days to exclude interference from the intestinal flora. The daily gavage dose was 50 mg / kg (body weight of the mouse). In addition to the antibiotic mixture, the indole-3-lactic acid (ILA) group was also administered 50 mg / kg (body weight of the mouse) of ILA by gavage for 7 consecutive days. The NSL group was also administered 50 mg / kg (body weight of the mouse) of STS by gavage for 7 consecutive days. The DMSO group was also administered 50 mg / kg (body weight of the mouse) of PBS buffer containing 5% DMSO by gavage for 7 consecutive days. Seven days later, arginine modeling was performed on the dimethyl sulfoxide group, indole-3-lactic acid group, and sodium thiosulfate group. Specifically, arginine was dissolved in sterile saline, and arginine was injected intraperitoneally once every hour at a dose of 4 g / kg (the dose based on the mouse's body weight). Two injections were given consecutively, with the timing starting from the first arginine injection. At 68 hours, LPS was injected intraperitoneally at a dose of 10 mg / kg (the dose based on the mouse's body weight). After 72 hours, the mice in each treatment group were sacrificed, and pancreatic tissue and peripheral blood samples were collected from each group.

[0037] 3. Results Analysis

[0038] 3.1 Relative expression levels of inflammatory factors in pancreatic tissue of arginine-induced model mice under different treatment groups

[0039] The relative expression levels of inflammatory factors in the pancreatic tissue of arginine-induced model mice in different treatment groups were determined using the method described in 3.2 of Example 1. The results are as follows: Figure 5 As shown in the figure, the horizontal axis represents different treatment groups, and the vertical axis represents the relative expression levels of inflammatory factors in the pancreatic tissue of arginine-induced model mice. Figure A shows the relative expression level of inflammatory factor IL-6 in the pancreatic tissue of arginine-induced model mice in different treatment groups, and Figure B shows the relative expression level of inflammatory factor TNF-α in the pancreatic tissue of arginine-induced model mice in different treatment groups.

[0040] according to Figure 5 It was found that, compared with the dimethyl sulfoxide group, the relative expression levels of inflammatory factors IL-6 and TNF-α in the serum of mice in the indole-3-lactic acid group and the sodium thiosulfate group were reduced, and the relative expression levels of inflammatory factors IL-6 and TNF-α in the serum of mice in the indole-3-lactic acid group were significantly reduced.

[0041] 3.2 Serum levels of inflammatory factors in arginine-induced model mice under different treatment groups

[0042] The levels of inflammatory factors in the serum of arginine-induced model mice in different treatment groups were determined using the method described in 3.3 of Example 1. The results are as follows: Figure 6As shown in the figure, the horizontal axis represents different treatment groups, and the vertical axis represents the content of inflammatory factors in the pancreatic tissue of arginine-induced model mice. Figure A shows the content of inflammatory factor IL-6 (Ng / mL) in the serum of arginine-induced model mice in different treatment groups, and Figure B shows the content of inflammatory factor TNF-α (Ng / mL) in the serum of arginine-induced model mice in different treatment groups.

[0043] according to Figure 6 It was found that, compared with the dimethyl sulfoxide group, the levels of inflammatory factors IL-6 and TNF-α in the serum of mice in the indole-3-lactic acid group and the sodium thiosulfate group were reduced, and the levels of inflammatory factors IL-6 and TNF-α in the serum of mice in the indole-3-lactic acid group were significantly reduced.

[0044] 3.3 Results of pancreatic tissue lesions in arginine-induced model mice under different treatment groups

[0045] Tissue lesion sections were obtained using the method described in Example 1, section 3.4. The sections were observed under a microscope, and the severity of acute pancreatitis was scored. The results of pancreatic tissue lesions in arginine-induced model mice in different treatment groups are as follows: Figure 7 As shown in the figure, Figure A represents the observation results of pancreatic tissue sections of arginine-induced model mice in different treatment groups, and Figure B represents the pathological scores of pancreatic tissue sections of arginine-induced model mice in different treatment groups. The horizontal axis in the figure represents different treatment groups, and the vertical axis represents the pathological scores of pancreatic tissue sections of arginine-induced model mice.

[0046] according to Figure 7 It can be seen that, compared with the dimethyl sulfoxide group, the I-indole-3-lactic acid group significantly reduced pancreatic edema and inflammatory cell infiltration, and its mouse pancreatic tissue pathological section score was the lowest.

[0047] In summary, ILA significantly reduced acinar cell damage and inflammatory infiltration in the pancreatic tissue of mice with arginine-induced acute pancreatitis, and also reduced inflammatory cytokines in the pancreatic tissue and serum of mice, showing better protective effects than STS, indicating that ILA has an anti-arginine-induced acute pancreatitis effect.

[0048] As shown in the above embodiments, this invention provides the application of indole-3-lactic acid in the preparation of drugs for treating acute pancreatitis. This invention used an antibiotic mixture to gavage mice to eliminate intestinal bacteria, and then gavaged mice with ILA and STS. Acute pancreatitis was modeled by intraperitoneal injection of vitexin and arginine, respectively. The effect of ILA on acute pancreatitis was studied. The results showed that mice gavaged with ILA had significantly lower serum amylase concentration, significantly reduced pancreatic acinar cell damage and inflammatory infiltration, and significantly reduced inflammatory cytokines in pancreatic tissue and serum. Furthermore, ILA showed better protective effects compared to STS, indicating that ILA has an anti-acute pancreatitis effect, providing a new therapeutic approach for the prevention and treatment of acute pancreatitis.

[0049] 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. Use of indole-3-lactic acid in the preparation of a drug for resisting acute pancreatitis.

2. Use according to claim 1, characterized in that, The indole-3-lactic acid realizes the effect of resisting acute pancreatitis by at least one of the following: (1) relieving the damage of acinar cells of pancreatic tissue and inflammatory cell infiltration; (2) reducing the concentration of amylase in serum; (3) reducing the transcription and expression of inflammatory cytokines in pancreatic tissue and / or serum.

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