Application of bile acid in preparation of medicine for preventing and treating ulcerative colitis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of bile acid in preparation of medicine for preventing and treating ulcerative colitis, and provides novel application of cholic acid CA, namely the cholic acid CA has prevention and treatment effects on colitis. And the content of proinflammatory cytokines in serum, ileum and colon is reduced, the expression of tight junction proteins in intestinal tracts is promoted, the expression of pro-apoptotic genes such as Bax and Caspase3 is inhibited, and the gene expression of anti-apoptotic protein Bcl-2 is up-regulated. On the basis, the cholic acid CA can obviously relieve systemic inflammatory injury of mice, improve immune regulation and barrier functions of intestinal tracts and inhibit apoptosis of intestinal tracts, and can be applied to preparation of medicines for preventing / treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of bile acid in the preparation of a medicine for preventing and treating ulcerative colitis. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease with an unclear etiology, including ulcerative colitis (UC) and Crohn's disease (CD). The prevalence of IBD is increasing significantly worldwide, with UC having a high incidence (11.6 / 100,000). It is a continuous inflammation of the colon mucosa and submucosa, usually affecting the rectum first and gradually spreading to the entire colon, becoming an important cause of colon cancer. Clinically, UC patients usually present with symptoms such as left lower abdominal pain and mucus, pus and blood in the stool. Diagnostic tests can observe lesions such as intestinal mucosal congestion, edema, and ulcers, and indicators such as inflammatory factors are significantly elevated.

[0003] At present, the commonly used drugs for the treatment of UC are mainly aminosalicylic acids, steroid hormones, immune preparations and biological preparations. Although these drugs are relatively effective, they are prone to cause serious side effects and increased drug tolerance.

[0004] Given the limitations of existing UC therapeutics, the development of new, safe and effective UC therapeutics is particularly urgent. Bile acids, as a class of small molecule metabolites in the human body, play a key role in digesting and absorbing lipids, maintaining cholesterol balance, and participating in the overall metabolic process. There are many types of bile acids in the human body with different functions, including primary bile acids (such as cholic acid CA and chenodeoxycholic acid CDCA) and secondary bile acids (such as deoxycholic acid DCA and lithocholic acid LCA), and the synthesis of secondary bile acids depends on intestinal flora. In recent years, although some progress has been made in the study of bile acids, its wide variety and complex functions still need further in-depth exploration. In particular, the potential role of bile acids in the prevention and treatment of ulcerative colitis has not been fully explored and utilized. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide an application of bile acid in the preparation of a drug for preventing and treating ulcerative colitis.

[0006] To achieve the above purpose, the present invention uses bile acid in the preparation of a drug for preventing and treating ulcerative colitis, using bile acid as an ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients to prepare a preparation for use.

[0007] Wherein, the bile acid is bile acid CA.

[0008] The prepared preparation is used to improve the body weight loss, colon shortening and increased serum cytokine release in colitis patients.

[0009] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly alleviate the weight loss and colon shortening of mice, and reduce the levels of serum proinflammatory cytokines IL-1β, IL-6 and TNF-α.

[0010] Among them, the prepared preparation is used to reduce the release of intestinal inflammatory factors in colitis bodies.

[0011] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly reduce the levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in the ileum and colon of mice and improve the immune regulation function of mice.

[0012] Among them, the prepared preparation is used to increase the expression of intestinal tight junction proteins in colitis bodies.

[0013] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly increase the levels of ZO-1, Occludin and Claudin1 protein and gene expression in the ileum and colon of mice, and maintain the intestinal barrier function of mice.

[0014] The prepared preparation is used to reduce intestinal apoptosis in colitis bodies.

[0015] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly inhibit the expression of Bax and Caspase3 genes in the intestine of mice, promote the expression of Bcl-2 gene, and inhibit the apoptosis of intestinal cells of mice.

[0016] The use of bile acid in the present invention in the preparation of a drug for preventing and treating ulcerative colitis has the advantage over the prior art that the present invention provides a new use of bile acid CA, namely, it has preventive and therapeutic effects on colitis.

[0017] The present invention has found through experiments that bile acid CA can restore the body weight and colon length of colitis mice, reduce the content of pro-inflammatory cytokines in serum, ileum and colon, promote the expression of tight junction proteins in the intestine, inhibit the expression of pro-apoptotic genes such as Bax and Caspase3, and upregulate the gene expression of anti-apoptotic protein Bcl-2. Based on the above, bile acid CA can significantly alleviate systemic inflammatory damage in mice, improve intestinal immune regulation and barrier function, and inhibit intestinal cell apoptosis, and can be used to prepare drugs for preventing / treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The effect of the bile acid CA of the present invention on the body weight change of colitis mice ( Figure 1 A) Colon length ( Figure 1 B) and serum cytokines ( Figure 1 C) Schematic diagram of the results.

[0020] Figure 2 The bile acid CA of the present invention has an effect on the ileum of colitis mice ( Figure 2 A) and colon ( Figure 2 B) Schematic representation of the results of pro-inflammatory cytokines.

[0021] Figure 3 The bile acid CA of the present invention has an effect on the ileum of colitis mice ( Figure 3 AB) and colon ( Figure 3 CD) Schematic diagram of tight junction protein expression results.

[0022] Figure 4 It is a schematic diagram of the results of the bile acid CA of the present invention on the expression of intestinal apoptosis-related genes in colitis mice. DETAILED DESCRIPTION

[0023] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0024] The present invention provides an application of bile acid in the preparation of a medicine for preventing and treating ulcerative colitis. The bile acid is used as a component and is combined with pharmaceutically acceptable excipients or auxiliary components to prepare a preparation for use.

[0025] Wherein, the bile acid is bile acid CA.

[0026] The prepared preparation is used to improve the body weight loss, colon shortening and increased serum cytokine release in colitis patients.

[0027] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly alleviate the weight loss and colon shortening of mice, and reduce the levels of serum proinflammatory cytokines IL-1β, IL-6 and TNF-α.

[0028] Among them, the prepared preparation is used to reduce the release of intestinal inflammatory factors in colitis bodies.

[0029] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly reduce the levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in the ileum and colon of mice and improve the immune regulation function of mice.

[0030] Among them, the prepared preparation is used to increase the expression of intestinal tight junction proteins in colitis bodies.

[0031] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly increase the levels of ZO-1, Occludin and Claudin1 protein and gene expression in the ileum and colon of mice, and maintain the intestinal barrier function of mice.

[0032] The prepared preparation is used to reduce intestinal apoptosis in colitis bodies.

[0033] In the DSS ulcerative colitis model experiment, oral administration of bile acid CA can significantly inhibit the expression of Bax and Caspase3 genes in the intestine of mice, promote the expression of Bcl-2 gene, and inhibit the apoptosis of intestinal cells of mice.

[0034] Specific experimental process: Daily oral administration of bile acid CA prevented DSS-induced ulcerative colitis in mice.

[0035] During the experiment,

[0036] The experimental materials are as follows:

[0037] 1. The experimental animals were 8-week-old SPF male C57BL / 6J mice purchased from Hangzhou Medical College and kept in SPF independent ventilation cages, 3 mice / cage. They were fed with standard feed, free access to water and food, and kept at a temperature of 20-22°C, a humidity of 50-60%, good ventilation, and a daily light and dark time of 12h each.

[0038] 2. Reagents and consumables: Cholic acid CA was purchased from J&K Technology Co., Ltd.; dextran sulfate sodium (DSS) was purchased from MP Biomedicals; ELISA kits, anti-Occludin, anti-Claudin1 and anti-ZO1 were purchased from Proteintech.

[0039] The experimental design and grouping are as follows:

[0040] C57BL / 6J mice were used as experimental subjects. A mouse ulcerative colitis model was established by supplementing 2% DSS solution in the drinking water of mice to study the preventive effect of bile acid CA on colitis. Experimental groups: 30 male mice were randomly divided into 3 groups after 1 week of adaptation, with 10 mice in each group, namely: normal control group (abbreviated as CON group), DSS-model group (abbreviated as DSS group) and bile acid CA prevention group (abbreviated as CA+DSS group), and the experimental period was 4 weeks. All mice were fed with a normal basal diet and free drinking water every day. The bile acid CA prevention group was gavaged with 100 μL CA (30 mg / kg body weight) every day, and the other groups were gavaged with 100 μL PBS every day for a total of 4 weeks; starting from the 4th week, the drinking water of the other two groups except the normal control group was replaced with 2% DSS solution for colitis modeling, and the normal control group continued to drink double distilled water.

[0041] The experiment was terminated after one week of drinking the DSS solution. The weight of mice was recorded in groups during the experiment. After the experiment, blood was collected from the eye sockets of the mice. The mice were killed and dissected, the colon was collected and the colon length was recorded, and the ileum and colon tissues were collected and quickly frozen in liquid nitrogen and transferred to -80℃ for storage.

[0042] The experimental method is as follows:

[0043] 1. Enzyme-linked immunosorbent assay (ELISA)

[0044] The blood was allowed to stand at room temperature for 3 h, centrifuged at room temperature for 10 min at 3000 rpm, and the resulting supernatant was serum. Ileal and colon tissue homogenates were prepared using a grinder, protein concentrations were determined using the BCA method, and IL-1β (Wuhan Proteintech, cat KE10003), IL-6 (Wuhan Proteintech, cat KE10007), and TNF-α (Wuhan Proteintech, cat KE10002) were determined according to the instructions of the ELISA kit.

[0045] 2. Protein immunoblot detection (Western blot)

[0046] (1) Preparation of total tissue protein: The protein in the intestinal tissue was extracted using the Keygen total protein extraction kit. The specific steps are as follows: first, add 600 μL of pre-cooled protein lysis buffer and 3 steel beads, place the intestinal tissue in it and homogenize it in a tissue homogenizer until the tissue blocks are invisible to the naked eye; then place the sample on ice for 30 min and centrifuge it at 4°C, 12,000 rpm for 15 min; finally, the supernatant obtained by centrifugation is the tissue protein.

[0047] (2) Protein sample quantification and deformation treatment: The tissue protein concentration was determined using the Keygen BCA protein content detection kit. Then, based on the measured protein concentration of each sample, the sample was diluted to the same concentration using protein lysis buffer, and 5× protein loading buffer was added. The sample was digested in a metal bath for 10 min, cooled on ice, and stored in aliquots at -80°C.

[0048] (3) Protein separation and transfer: Place the prepared gel into the Bio-rad electrophoresis instrument, add 1× electrophoresis buffer, and remove the gel comb; add protein samples (20-30 μg) and 4 μL pre-stained protein markers into the gel wells; electrophoresed the concentrated gel at 80V for 30 minutes, and then electrophoresed the separation gel at 110V for 1-1.5 hours until bromophenol blue is about to run out. Activate the PVDF membrane with methanol for 1 minute, and soak it in 1× transfer buffer for later use. Remove the gel and cut the gel strip according to the protein marker band and the molecular weight of the target protein. Then form a "sandwich" structure in the order of sponge pad-filter paper-membrane-gel-filter paper-sponge pad, install the PVDF membrane in the electrophoresis tank corresponding to the positive electrode, and transfer the membrane for 50-60 minutes at 260 mA; during this period, ice packs can be used to prevent excessive heat from affecting the transfer process. After the transfer is completed, block it in 5% skim milk for 60 minutes.

[0049] (4) Antibody incubation: After blocking, incubate the membrane with the primary antibody at 4°C overnight or at room temperature for 1 h; wash the membrane three times with 1× TBST, each time for 15 min; then incubate in the corresponding secondary antibody at room temperature for 1 h; then wash the membrane three times with 1× TBST, each time for 15 min.

[0050] (5) ECL development: Prepare fresh ECL luminescent liquid in proportion, drip the luminescent liquid evenly on the surface of the PVDF membrane, and then expose and develop it.

[0051] 3. Fluorescence quantitative PCR (q-PCR)

[0052] (1) RNA extraction: Total RNA from intestinal tissue was extracted using Trizol Reagent, and the concentration and purity of the obtained RNA were determined using NanoDrop 2000. If OD260 / 280 was between 1.8-2.0 and OD260 / 230 was between 1.7-2.0, the RNA sample passed the quality inspection. Otherwise, the RNA sample needs to be re-extracted. Samples that passed the quality inspection can be directly used for the next step of reverse transcription or stored at -80°C for future use.

[0053] (2) Reverse transcription to synthesize cDNA: The total volume of reverse transcription was 20 μL, and 2 μg of RNA sample was used for reverse transcription to synthesize cDNA. The required RNA volume V μL was calculated based on the RNA concentration, and the following components were added in a 0.2 mL RNase free centrifuge tube in sequence: RNase free water (12-V μL), Random Primer (1 μL) and RNA (V μL). After thorough mixing, the mixture was incubated at 65°C for 5 min and immediately placed on ice for cooling. Subsequently, the following mixture was added: 5× Reaction Buffer (4 μL), dNTPs (10 mM) (2 μL) and reverse transcriptase (1 μL). After vortex mixing and centrifugation, the mixture was incubated at 25°C for 10 min, 42°C for 60 min, and 72°C for 10 min. The mixture was immediately placed on ice for cooling. The resulting product was cDNA, which was diluted 10 times and stored in aliquots at -80°C for later use.

[0054] (3) Real-time fluorescence quantitative PCR: The 10 μL reaction system was: SYBR green mastermix (5 μL), upper / lower primers (0.5 μL each) and cDNA (4 μL); the reaction conditions were set as follows: pre-deformation: 95°C, 10 min; PCR reaction: 95°C, 5 s; 60°C, 35 s; 40 cycles in total; melting curve analysis: in each cycle, 60°C, 1 min; 95°C, 15 s, automatic fluorescence collection.

[0055] 4. The experimental results are as follows:

[0056] (1) Macroscopic effects of bile acid CA on colitis model mice

[0057] See also Figure 1 , Figure 1 The effect of the bile acid CA of the present invention on the body weight change of colitis mice ( Figure 1 A) Colon length ( Figure 1 B) and serum cytokines ( Figure 1 C) Schematic diagram of the results. The CON group was normal wild-type control mice; the DSS group was colitis model mice; the CA+DSS group was DSS mice gavaged with 30 mg / kg bile acid CA (* indicates compared with the CON group, # indicates compared with the DSS group; * / #P<0.05, ** / ##P<0.01, *** / ###P<0.001).

[0058] like Figure 1 As shown in A, starting from the 5th day of colitis modeling, the body weight of mice in the DSS group decreased significantly compared with the CON group, and the body weight decreased extremely significantly on the 7th day, indicating that the DSS-induced ulcerative colitis model was successful. Compared with the DSS group, intragastric administration of bile acid CA significantly prevented the weight loss of mice. Figure 1As shown in B, the results of the mouse colon length showed that the colon of the DSS group was significantly shorter than that of the CON group; and the colon length of the CA+DSS group was significantly longer than that of the DSS group. Figure 1 As shown in C, the results of serum proinflammatory cytokine content showed that compared with the CON group, the levels of IL-1β, IL-6 and TNF-α in the serum of the DSS group were significantly increased; compared with the DSS group, the cytokine content in the serum of the CA+DSS group was significantly downregulated.

[0059] The above results suggest that DSS reduces the body weight and colon length of mice, promotes the release of serum cytokines, and leads to ulcerative colitis; bile acid CA can alleviate the weight loss and colon shortening of mice, inhibit the release of serum cytokines, and ultimately alleviate the progression of colitis in mice.

[0060] (2) Bile acid CA improves intestinal immune function in mice with colitis

[0061] See also Figure 2 , Figure 2 The bile acid CA of the present invention has an effect on the ileum of colitis mice ( Figure 2 A) and colon ( Figure 2 B) Schematic diagram of the results of pro-inflammatory cytokines. The CON group was normal wild-type control mice; the DSS group was colitis model mice; and the CA+DSS group was DSS mice gavaged with 30 mg / kg bile acid CA (* indicates compared with the CON group, # indicates compared with the DSS group; * / #P<0.05, ** / ##P<0.01, *** / ###P<0.001).

[0062] like Figure 2 As shown in AB, the results of the proinflammatory cytokine content in the ileum and colon tissues of mice showed that compared with the CON group, the levels of IL-1β, IL-6 and TNF-α in the ileum and colon of the DSS group were significantly increased; compared with the DSS group, the cytokine content in the ileum and colon of the CA+DSS group was significantly downregulated.

[0063] The above results suggest that DSS causes the release of cytokines in the ileum and colon of mice, leading to ulcerative colitis; bile acid CA pretreatment can alleviate inflammation and regulate the intestinal immune function of mice.

[0064] (3) Bile acid CA enhances intestinal barrier function in colitis mice

[0065] See also Figure 3 , Figure 3 The bile acid CA of the present invention has an effect on the ileum of colitis mice ( Figure 3 AB) and colon ( Figure 3CD) Schematic diagram of tight junction protein expression. The CON group was normal wild-type control mice; the DSS group was colitis model mice; the CA+DSS group was DSS mice gavaged with 30 mg / kg bile acid CA (* indicates compared with the CON group, # indicates compared with the DSS group; * / #P<0.05, ** / ##P<0.01, *** / ###P<0.001).

[0066] like Figure 3 As shown in AB, the protein and gene expression results of tight junction proteins in mouse ileum tissue showed that compared with the CON group, the protein expression of Claudin1, Occludin and ZO1 in the ileum of the DSS group was significantly reduced, and the gene expression of Claudin1 was significantly reduced; compared with the DSS group, the protein and gene expression of Claudin1, Occludin and ZO1 in the ileum of the CA+DSS group was significantly increased. Figure 3 As shown in CD, the protein and gene expression results of tight junction proteins in mouse colon tissue showed that compared with the CON group, the protein expressions of Claudin1, Occludin and ZO1 in the ileum of the DSS group were significantly decreased, and the ZO1 gene expression was significantly decreased; compared with the DSS group, the protein and gene expressions of Claudin1, Occludin and ZO1 in the ileum of the CA+DSS group were significantly increased.

[0067] The above results suggest that DSS destroys the expression of tight junction proteins in the intestine of mice, leading to ulcerative colitis; bile acid CA pretreatment can enhance the intestinal barrier function of mice and alleviate colitis.

[0068] (4) Bile acid CA inhibits intestinal apoptosis in colitis mice

[0069] See also Figure 4 , Figure 4 This is a schematic diagram of the results of the bile acid CA of the present invention on the expression of intestinal apoptosis-related genes in colitis mice. The CON group is normal wild-type control mice; the DSS group is colitis model mice, and the CA+DSS group is DSS mice gavaged with 30 mg / kg bile acid CA (* indicates compared with the CON group, # indicates compared with the DSS group; * / #P<0.05, ** / ##P<0.01, *** / ###P<0.001).

[0070] like Figure 4 As shown in the figure, the results of apoptosis-related gene expression in mouse colon tissue showed that compared with the CON group, the expression of pro-apoptotic genes Caspase3 and Bax in the colon of the DSS group was significantly upregulated; compared with the DSS group, the gene expression of Caspase3 and Bax in the colon of the CA+DSS group was significantly downregulated, and the expression of anti-apoptotic gene Bcl-2 was significantly upregulated.

[0071] The above results suggest that DSS causes intestinal cell apoptosis in mice, leading to ulcerative colitis; bile acid CA pretreatment can alleviate cell apoptosis and inhibit intestinal inflammation.

[0072] In summary, the following conclusions were drawn: bile acid CA alleviates weight loss and colon shortening in mice by reducing the release of proinflammatory cytokines IL-1β, IL-6 and TNF-α in serum, ileum and colon, promoting the protein and gene expression of Claudin1, Occludin and ZO1, inhibiting the expression of anti-apoptotic genes such as Caspase3 and Bax, and promoting the expression of Bcl-2 gene, so it can be used to prevent and / or treat ulcerative colitis.

[0073] The use of a bile acid in this embodiment in the preparation of a drug for preventing and treating ulcerative colitis has the advantage, compared with the prior art, that the present invention provides a new use of bile acid CA, namely, it has preventive and therapeutic effects on colitis.

[0074] The present invention has found through experiments that bile acid CA can restore the body weight and colon length of colitis mice, reduce the content of pro-inflammatory cytokines in serum, ileum and colon, promote the expression of tight junction proteins in the intestine, inhibit the expression of pro-apoptotic genes such as Bax and Caspase3, and upregulate the gene expression of anti-apoptotic protein Bcl-2. Based on the above, bile acid CA can significantly alleviate systemic inflammatory damage in mice, improve intestinal immune regulation and barrier function, and inhibit intestinal cell apoptosis, and can be used to prepare drugs for preventing / treating ulcerative colitis.

[0075] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A use of bile acid in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: Bile acid is used as an ingredient and is combined with pharmaceutically acceptable excipients or auxiliary ingredients to prepare a preparation for use.

2. The use of bile acid according to claim 1 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The bile acid is cholic acid CA.

3. The use of bile acid as claimed in claim 2 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The prepared preparation is used to improve the body weight loss, colon shortening and increased serum cytokine release in colitis patients.

4. The use of bile acid as claimed in claim 2 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The prepared preparation is used to reduce the release of intestinal inflammatory factors in colitis bodies.

5. The use of bile acid as claimed in claim 2 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The prepared preparation is used to increase the expression of intestinal tight junction proteins in colitis bodies.

6. The use of bile acid as claimed in claim 2 in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The prepared preparation is used to reduce intestinal apoptosis in colitis bodies.

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