Application of (6R, 9S)-4-macrosven-9, 10-dihydroxy-3-ketone in preparation of medicine for treating ulcerative colitis

Abstract

The invention provides an application of (6R, 9S)-4-giganteen-9, 10-dihydroxy-3-ketone in preparation of a medicine for treating ulcerative colitis, and relates to the technical field of medicines. According to the invention, (6R, 9S)-4-gigante-9, 10-dihydroxy-3-ketone is utilized, so that weight loss and colonic atrophy caused by ulcerative colitis can be effectively relieved, and the conditions of hematochezia and diarrhea can be improved. The (6R, 9S)-4-gigansis-9, 10-dihydroxy-3-ketone can be used for obviously improving the pathological change of colon tissue caused by the ulcerative colitis. The (6R, 9S)-4-gigante-9, 10-dihydroxy-3-ketone can be used for remarkably activating FXR bile acid regulation signal channels of IEC-6 cells, so that the transport of bile acid is regulated, and the homeostasis of the bile acid in the intestinal tract is maintained.

Description

Technical Field

[0001] The present invention provides the use of (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one in the preparation of a medicament for treating ulcerative colitis, belonging to the technical field of medicine. Background Art

[0002] Ulcerative Colitis (UC) is an inflammatory process confined to the colonic mucosa and submucosa. The disease is mostly located in the sigmoid colon and rectum, and can also extend to the descending colon or even the entire colon. The pathological process is long and often recurs. At present, conservative treatment methods are often used for ulcerative colitis, which have the problems of long treatment cycles and difficulty in effective cure. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide the use of (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one in the preparation of a medicament for treating ulcerative colitis.

[0004] (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one (CAS No. 1417726-57-8, hereinafter referred to as dihydroxy ketone), English name: (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one. Its structural formula is as follows:

[0005]

[0006] Compared with the prior art, the present invention has the following beneficial effects:

[0007] The present invention utilizes (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one to effectively relieve weight loss and colon atrophy caused by ulcerative colitis, and improve blood in the stool and diarrhea. (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one can significantly improve the histopathological changes of the colon caused by ulcerative colitis. (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one can significantly reduce the concentration of serum inflammatory factors and inhibit the expression of inflammatory proteins in colon tissues and cells. (6R,9S)-4-megastigmen-9,10-dihydroxy-3-one can significantly activate the FXR bile acid regulatory signaling pathway in IEC-6 cells, thereby regulating the transport of bile acids and maintaining intestinal bile acid homeostasis.

[0008] (6R,9S)-4-Gucisdemen-9,10-dihydroxy-3-one reduces intestinal bile acid accumulation by activating ileal FXR. The mechanism is to up-regulate the expression of ileal bile acid transporters, promote intestinal bile acid reabsorption, and at the same time promote the secretion of FGF15 and activate FGFR4 as a signaling molecule to reduce hepatic bile acid synthesis. When the Fxr gene in the body is knocked out, the bile acid signaling pathway regulated by FXR is disrupted, and its therapeutic effect on ulcerative colitis also disappears. Description of the Drawings

[0009] Figure 1 Improvement effect of dihydroxy ketone on DSS-induced ulcerative colitis in mice

[0010] Figure 2 Pathological analysis of colon tissue of DSS-induced ulcerative colitis mice by dihydroxy ketone;

[0011] Figure 3 Improvement effect of dihydroxy ketone on serum inflammatory factors of DSS-induced ulcerative colitis mice;

[0012] Figure 4 Effect of dihydroxy ketone on the expression levels of colon inflammation-related proteins in DSS-induced ulcerative colitis mice;

[0013] Figure 5 Improvement effect of dihydroxy ketone on bile acids in DSS-induced ulcerative colitis mice;

[0014] Figure 6 Effect of dihydroxy ketone on the expression of ileal FXR-related proteins in DSS-induced ulcerative colitis mice;

[0015] Figure 7 Effect of dihydroxy ketone on hepatic bile acid synthesis proteins in DSS-induced ulcerative colitis mice;

[0016] Figure 8 Effect of dihydroxy ketone on the FXR signaling pathway in TUDCA-treated IEC-6 cells;

[0017] Figure 9 Effect of dihydroxy ketone on inflammatory proteins in DCA-induced NCM460 cells;

[0018] Figure 10 Effect of dihydroxy ketone on DSS-induced wild-type and Fxr - / - ulcerative colitis in mice;

[0019] Figure 11 Effect of dihydroxy ketone on DSS-induced wild-type and Fxr - / - pathological analysis of ulcerative colitis in mice;

[0020] Figure 12 Effect of dihydroxy ketone on wild-type and Fxr - / - Expression of proteins related to colonic inflammation in mice with ulcerative colitis;

[0021] Figure 13 Effect of dihydroxy ketone on wild-type and Fxr - / - Effect of dihydroxy ketone on the intestinal FXR signaling pathway in mice with ulcerative colitis;

[0022] Figure 14 Effect of dihydroxy ketone on wild-type and Fxr - / - Effect of dihydroxy ketone on proteins involved in bile acid synthesis in the liver of mice with ulcerative colitis. Detailed implementation manners

[0023] To make the objectives, technical solutions and advantages of the present invention clearer, the preferred implementation manners of the present invention will be further described in detail below in conjunction with embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0024] First part: Animal experiments

[0025] 1. Experimental animals and solutions

[0026] Male C57BL / 6J mice (SPF level, 7 - 8 weeks old, 20 - 22 g), purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK(Xiang)2019 - 0004. They were placed in the SPF-class mouse room of the Animal Experiment Center (license number: SYXK(Yu)2020 - 0006) for adaptive feeding. The mouse room was maintained at a constant temperature and humidity, with a 12-hour light-dark cycle. The mice were fed with sufficient feed, allowed free access to water, and the bedding was changed regularly. They were adaptively fed for one week to adapt to the environment.

[0027] PBS buffer: 2×PBS phosphate buffer powder was completely dissolved in 2000 mL of pure water.

[0028] PBST buffer: 500 μL of Tween 20 was added to 1 L of PBS phosphate buffer and shaken well.

[0029] 0.5% sodium carboxymethylcellulose (CMC-Na) solution: Weighed 2.5 g of CMC-Na powder, added 500 mL of pure water, and stirred while heating to fully dissolve the CMC-Na powder. After cooling, it was stored in the refrigerator at 4 °C for later use.

[0030] 5×Running buffer (electrophoresis buffer): Measured 200 mL of 5×Running buffer solution and added pure water to a final volume of 1000 mL and shaken well. It was placed at room temperature.

[0031] 5× Transfer buffer: Measure 200 mL of 5× Transfer buffer solution, add 200 mL of methanol, and make up to 1000 mL with pure water. Store at 4°C.

[0032] Dihydroxy ketone (1 mg / mL, 3 mg / mL) solution: Weigh 14 mg and 42 mg of dihydroxy ketone powder respectively, and ultrasonically disperse them evenly in 14 mL of 0.5% CMC-Na solution.

[0033] Dextran sulfate sodium DSS (3%) solution: Weigh 12 g of dextran sulfate sodium DSS and dissolve it in 400 mL of pure water. Prepare fresh daily.

[0034] Sulfasalazine SASP (20 mg / mL) solution: Weigh 280 mg of sulfasalazine SASP, add it to 14 mL of 0.5% CMC-Na solution, and ultrasonically disperse it evenly.

[0035] 2. Experimental methods

[0036] 2.1 Animal grouping and administration

[0037] After the adaptive feeding period, the mice were randomly divided into five groups of 8 mice each. They were the normal control group, the DSS model group, the positive control group (SASP, 200 mg / kg), the low-dose dihydroxy ketone group (10 mg / kg), and the high-dose dihydroxy ketone group (30 mg / kg). Except for the normal group, the other groups were given 3% DSS in the drinking water to establish the model, and they had free access to food and water. The normal group and the model group mice were intragastrically administered the corresponding volume (100 μL / 10 g) of the vehicle, that is, 0.5% CMC-Na solution, according to their body weights. The positive control group and the dihydroxy ketone group mice were intragastrically administered SASP and dihydroxy ketone solutions at different concentrations. The intragastric administration was continued for 7 days. Serum, colon, ileum, and liver tissues of each group of mice were collected (the mice were fasted for 8 h before sampling, and had free access to water).

[0038] 2.3 Determination of serum biochemical indexes in mice

[0039] The determination of inflammatory factors in mouse serum was carried out according to the instructions of the detection kit. The absorbance of each well was read at different absorption wavelengths using an enzyme-linked immunosorbent assay reader, and the content was calculated.

[0040] 2.4 Histopathological analysis of colon tissue

[0041] Mouse colon tissue was fixed with 4% paraformaldehyde, and after dehydration, embedding, sectioning, staining and other operations, the histopathological changes of the colon tissue were observed under an inverted microscope.

[0042] 2.5 Immunoblotting

[0043] 2.5.1 Tissue protein extraction

[0044] 2.5.1.1 Colon Protein Extraction

[0045] Take out the colon tissue from the -80°C refrigerator. Take an appropriate amount of tissue and rinse it with PBS to remove impurities and blood stains. Blot dry the residual PBS with filter paper, weigh it and cut it into pieces. Add RIPA lysis buffer (1% PMSF) at a ratio of 1:10, add 3 mm steel beads, grind it with a grinder at 60 HZ for 20 s, stop for 10 s, repeat three times, and grind it into a homogenate without tissue fragments. Lyse on ice for 5 min, centrifuge at 4°C and 10,000 rpm for 10 min, aspirate the supernatant. Take an appropriate amount of the supernatant for BCA protein quantification, and use PBS and protein loading buffer to adjust the protein concentration of each group to 2 μg / μl, mix well, boil at 100°C for 10 min to fully denature the protein, and store at -80°C after cooling.

[0046] 2.5.1.2 Ileum Protein Extraction

[0047] Take out the ileum tissue from the -80°C refrigerator. Take an appropriate amount of tissue and rinse it with PBS to remove impurities and blood stains. Blot dry the residual PBS with filter paper, weigh it and cut it into pieces. Add RIPA lysis buffer (1% PMSF) at a ratio of 1:10, add 3 mm steel beads, grind it with a grinder at 60 HZ for 20 s, stop for 10 s, repeat three times, and grind it into a homogenate without tissue fragments. Lyse on ice for 5 min, centrifuge at 4°C and 10,000 rpm for 10 min, aspirate the supernatant. Take an appropriate amount of the supernatant for BCA protein quantification, and use PBS and protein loading buffer to adjust the protein concentration of each group to 2 μg / μl, mix well. To prevent the formation of polymers by membrane proteins at high temperature, boil at 98°C for 3 min to denature the protein, and store at -80°C after cooling.

[0048] 2.5.1.3 Liver Protein Extraction

[0049] Take out the liver tissue from the -80°C refrigerator, cut it into pieces and mix evenly. Weigh about 100 mg of the sample and place it in an EP tube. Add RIPA lysis buffer (1% PMSF) at a ratio of 1:10, add 3 mm steel beads, grind it with a grinder at 60 HZ for 20 s, stop for 10 s, repeat three times, and grind it into a homogenate without tissue fragments. Lyse on ice for 5 min, centrifuge at 4°C and 10,000 rpm for 10 min, aspirate the supernatant. Take an appropriate amount of the supernatant for BCA protein quantification, and use PBS and protein loading buffer to adjust the protein concentration of each group to 2 μg / μl, mix well. To prevent the formation of polymers by membrane proteins at high temperature, boil at 98°C for 3 min to denature the protein, and store at -80°C after cooling.

[0050] 2.5.2 Western blot

[0051] Select separating gel and stacking gel with appropriate concentrations according to the molecular weight of the target protein to ensure sufficient separation of the target protein. Prepare 12% and 10% separating gels according to the kit from Wuhan Sevier Biotechnology Co., Ltd.

[0052] Loading: Vortex the samples before loading, perform intermittent ultrasonic treatment in an ice bath for 3 min, load 10 μl per well, and add 2.5 μl of colored pre-stained protein marker to both sides.

[0053] Electrophoresis: After pressing the samples out of the stacking gel at a constant voltage of 80 V, change to a constant voltage of 120 V and continue electrophoresis to fully separate the samples until the bromophenol blue band is close to the bottom of the gel to end the electrophoresis.

[0054] Transfer: Cool the transfer buffer in the refrigerator at 4 °C in advance. Soak the transfer sponge and filter paper in the transfer buffer until fully wet. Activate the PVDF membrane in methanol for 1 - 2 min in advance. Cut the gel according to the colored pre-stained protein marker and the location of the target protein. Lay the activated PVDF membrane flat on the gel according to the sandwich transfer method, being careful not to generate bubbles. Fasten the transfer cassette, place it in the transfer apparatus, and add transfer buffer to cover the transfer cassette. Transfer at a constant current of 200 mA in an ice bath by wet transfer method. Adjust the transfer time according to the molecular weight of the target protein.

[0055] Blocking: After the transfer is completed, take out the PVDF membrane from the transfer cassette and place it in an incubation box, and block it with a rapid blocking solution for 10 min.

[0056] Primary antibody incubation: Place the PVDF membrane in the corresponding primary antibody incubation bag for the target protein and incubate overnight at 4 °C.

[0057] Secondary antibody incubation: After the primary antibody incubation is completed, wash the PVDF membrane three times with PBST, 10 min each time, to wash away the residual primary antibody. Dilute the HRP-labeled secondary antibody with 5% BSA (1:2000). Immerse the PVDF membrane in the goat anti-rabbit or goat anti-mouse secondary antibody incubation box according to the animal origin of the primary antibody for the target protein and incubate on a shaker at room temperature for 2 h. After the incubation is completed, wash three times with PBST, 10 min each time.

[0058] Development: Mix the high-sensitivity ECL luminescent developing solution A and B in a volume ratio of 1:1 and place it in the dark. Spread the developing solution evenly on the PVDF membrane for exposure, and adjust the exposure time according to the molecular weight of the target protein. Use Image J software to perform gray value statistics on the proteins in each group.

[0059] 2.6 Statistical analysis

[0060] The experimental data were expressed as "mean ± standard deviation (SD)", and one-way ANOVA was used for pairwise comparison of means between data. SPSS 26 statistical software was used to analyze the data of each group, and the P value was calculated. P < 0.05 was considered a significant difference, P < 0.01 was considered a highly significant difference, and P > 0.05 was considered no significant difference.

[0061] 3 Experimental Results

[0062] 3.1 Effects of Dihydroxyketone on Disease Activity Index of DSS-Induced Ulcerative Colitis Mice

[0063] Figure 1 To show the improvement effect of dihydroxyketone on DSS-induced ulcerative colitis in mice, where Figure 1 A is the body weight change curve; B is the blood in feces state; C is the colon state; D is the colon length statistics; E is the DAI score. The data were expressed as mean ± standard deviation (n = 8); +++ P < 0.001, ++ P < 0.01, compared with the control group; ***P < 0.001, **P < 0.01, *P < 0.05, compared with the model group.

[0064] As Figure 1 shown, the body weight of normal mice showed an upward trend, without blood in feces and diarrhea. In the first 4 days after DSS induction, the body weight of mice did not change significantly. On the 5th day, the body weight of mice began to decrease rapidly, and diarrhea and blood in feces occurred. The symptoms gradually worsened with the increase of the modeling time. After dissection, the colon length of mice was significantly shorter than that of the normal group. It was initially judged that the mouse colitis model was successfully established, and DSS-induced ulcerative colitis mice, referred to as UC mice, were obtained. Compared with the model group, the percentage of body weight loss in the positive control group (SASP) mice was significantly reduced, the conditions of blood in feces and diarrhea were also significantly improved, the colon length was significantly longer than that of the model group, and the disease activity score was significantly reduced (P < 0.001, P < 0.01). There was no significant difference in body weight change between the low-dose dihydroxyketone group (10 mg / kg) and the model group. The blood in feces was improved, but the diarrhea was severe, showing watery diarrhea. In the high-dose dihydroxyketone group (30 mg / kg), the body weight loss, blood in feces and diarrhea of mice were all significantly improved. The colon length and disease activity score were close to those of the positive drug, and the shortening of the colon length after dissection was also significantly improved (P < 0.001, P < 0.05). The results showed that dihydroxyketone had a good effect on relieving the phenotype of ulcerative colitis.

[0065] 3.2 Effects of Dihydroxyketone on Histopathology of DSS-Induced Ulcerative Colitis Mice

[0066] As Figure 2As shown, the colonic tissue folds of the control group mice were clear, the intestinal epithelial structure of the mucosal layer was intact, the morphological structure of the epithelial cells was normal, the number of intestinal glands in the lamina propria was abundant, a large number of goblet cells were visible, and there were no signs of inflammatory infiltration. In the model group mice, ulcers were visible in the colonic mucosal layer, the local mucosal epithelium was damaged, a large number of crypt necroses and dissolutions were visible in the lamina propria, and a large number of inflammatory cells infiltrated, and some inflammatory cells infiltrated into the submucosa. In the low-dose dihydroxyketone group (10 mg / kg), the base of the crypts was thickened and atrophied, and goblet cells were lost. In the high-dose dihydroxyketone group (30 mg / kg) and the SASP group, the crypt structure was intact, the number of goblet cells increased, and the inflammatory infiltration decreased. The results showed that dihydroxyketone significantly improved the histopathological changes of the colon tissue caused by DSS.

[0067] 3.3 Expression of serum inflammatory factors TNF-α, IL-1β, and IL-18 in mice of each group

[0068] Inflammatory factors are involved in the development process of UC. To further explore the effect of dihydroxyketone on the inflammatory process, ELISA was used to measure the expression of serum inflammatory factors TNF-α, IL-1β, and IL-18 in mice. As Figure 3 shown, compared with the normal group, the serum inflammatory factors of DSS-induced colitis mice increased significantly (P < 0.001, P < 0.05). Compared with the model group, in the dihydroxyketone (30 mg / kg) and SASP groups, the expression of serum inflammatory factors decreased significantly (P < 0.001, P < 0.05). The results showed that dihydroxyketone had a good anti-inflammatory effect.

[0069] 3.4 Effect of dihydroxyketone on colonic inflammation in DSS-induced ulcerative colitis mice

[0070] Colonic mucosal inflammatory reaction is an important link in the progression of ulcerative colitis. As Figure 4 shown, compared with the normal group, the expression of inflammatory proteins such as TNF-α, IL-6, IL-18, and IL-1β in the colonic tissue of UC mice increased significantly (P < 0.001, P < 0.01, P < 0.05). After administration of dihydroxyketone (10 mg / kg), the expression of TNF-α and IL-18 decreased, but there was no significant difference, and the expression of IL-6 decreased significantly (P < 0.01). After administration of dihydroxyketone (30 mg / kg) or SASP, the expression of TNF-α, IL-6, IL-18, and IL-1β decreased significantly (P < 0.001, P < 0.01, P < 0.05). The results showed that dihydroxyketone could inhibit the expression of colonic inflammatory proteins, and showed a certain dose-dependence with the increase of dose. The inhibitory effect of the dihydroxyketone (30 mg / kg) group was close to that of the positive drug.

[0071] 3.5 Bile acid contents in serum, ileum, and feces of mice in each group

[0072] Bile acids are closely related to the occurrence and development of colitis. To explore the changes in bile acids during colitis, we used a bile acid detection kit to measure the bile acid content in the serum, ileum, and feces of mice. As Figure 5 shown, compared with the normal group, DSS-induced mice showed obvious bile acid disorders, with significantly increased bile acids in the ileum and feces (P<0.01, P<0.05), and bile acids accumulated in the intestine. In the dihydroxy ketone (10 mg / kg) group, there was no significant difference in ileal bile acids compared with the model group, and fecal bile acids decreased (P<0.001). In the dihydroxy ketone (30 mg / kg) group, serum bile acids did not increase, but significantly decreased the bile acid content in the ileum and feces (P<0.001, P<0.05). The results showed that dihydroxy ketone significantly reduced the accumulation of intestinal bile acids. 3.6 Effect of dihydroxy ketone on the FXR signaling pathway in the ileum of DSS-induced ulcerative colitis mice

[0073] To further explore the relationship between the effect of dihydroxy ketone in relieving ulcerative colitis and intestinal bile acid metabolism, immunoblotting was performed on FXR and related proteins regulating the bile acid signaling pathway in the ileum tissue of mice. As Figure 6 shown, compared with the normal group, the expression level of FXR in the ileum of DSS-induced colitis mice decreased (P<0.01), and the expression of downstream bile acid reabsorption transporters OSTα, OSTβ, FABP6, and FGF15 decreased (P<0.01, P<0.05). After administration of SASP, there was no significant change in the expression of FXR and downstream transporters. After administration of dihydroxy ketone (30 mg / kg), the expression of FXR and downstream bile acid transporters in the ileum increased significantly (P<0.01, P<0.05). The results showed that dihydroxy ketone could significantly activate intestinal FXR, up-regulate the expression of ileal bile acid transporters, promote bile acid reabsorption, reduce the accumulation of bile acids in the intestine, and the positive drug did not exert its effect through FXR.

[0074] 3.7 Effect of dihydroxy ketone on the bile acid synthesis signaling pathway in the liver of DSS-induced ulcerative colitis mice

[0075] After activation, intestinal FXR secretes the signaling molecule FGF15 into the liver to regulate bile acid synthesis. Therefore, the expression of proteins related to liver bile acid synthesis was further detected. As Figure 7As shown, compared with the normal group, the FGFR4 in the liver of DSS-induced colitis mice decreased (P<0.01), and the rate-limiting enzyme CYP7A1 for bile acid synthesis increased (P<0.05). After administration of SASP, there was no significant difference in FGFR4 and CYP7A1 compared with the model group. After administration of dihydroxy ketone, FGFR4 increased (P<0.05), and the expression of CYP7A1 decreased (P<0.05). The results showed that after activation of intestinal FXR by dihydroxy ketone, FGFR4 was activated through the FGF15 signaling molecule to reduce liver bile acid synthesis. The second part: Anti-inflammatory activity of dihydroxy ketone in vitro and the effect of activating FXR

[0076] 1. Experimental cells

[0077] The human normal colon cell line (NCM460) and the rat small intestinal epithelial cell line (IEC-6) were both from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection.

[0078] 2 Experimental methods

[0079] 2.1 Preparation of main solutions

[0080] Tauroursodeoxycholic acid TUDCA stock solution (400 mmol / L): Dissolve 10 mg of tauroursodeoxycholic acid TUDCA in 50 μL of DMSO, filter and sterilize, and store at -20 after aliquoting.

[0081] Deoxycholic acid DCA (400 mmol / L): Weigh 5 mg of deoxycholic acid DCA powder, add 63.7 μL of DMSO to dissolve, and filter and sterilize.

[0082] Dihydroxy ketone stock solution (18 mmol / L): Weigh 3 mg of dihydroxy ketone powder, add 737.5 μL of DMSO to dissolve, and filter and sterilize.

[0083] GW4064 (6 mmol / L): Weigh 2 mg of GW4064 powder, add 614 μL of DMSO to dissolve, and filter and sterilize.

[0084] 2.2 TUDCA inhibits the bile acid regulatory signaling pathway in IEC-6 cells

[0085] Observed under a microscope, when the density of IEC-6 cells in the culture dish reached about 80%, subculture was performed. The cells were blown into a single-cell suspension and seeded into a 6-well cell culture plate. After the cells adhered, 200 μmol / L of the FXR inhibitor TUDCA, 3 μmol / L of the FXR agonist GW4064, and 1 μmol / L, 3 μmol / L, and 9 μmol / L of dihydroxy ketone were simultaneously added to treat the cells for 24 h. The original culture medium was discarded, and RIPA lysis buffer was added to each well. The cells were lysed on ice for 5 min, scraped off, centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was aspirated. The absorbance values of each well were measured by the BCA method, the protein concentrations of each group of samples were calculated, and different volumes of 5× protein loading buffer and PBS were added to make the protein concentrations of each group of samples consistent. The protein was denatured by heating at 100°C for 3 min, and after cooling, the samples were aliquoted and stored at -20°C.

[0086] 2.3 DCA-induced inflammation in NCM460 cells

[0087] Observed under a microscope, when the density of NCM460 cells in the culture dish reached about 80%, subculture was performed. The cells were blown into a single-cell suspension and seeded into a 6-well cell culture plate. After the cells adhered, 200 μmol / L of DCA and 1 μmol / L, 3 μmol / L, and 9 μmol / L of dihydroxy ketone were simultaneously added to treat the cells for 24 h. The original culture medium was discarded, and RIPA lysis buffer was added to each well. The cells were lysed on ice for 5 min, scraped off, centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was aspirated. The absorbance values of each well were measured by the BCA method, the protein concentrations of each group of samples were calculated, and different volumes of 5× protein loading buffer and PBS were added to make the protein concentrations of each group of samples consistent. The protein was denatured by heating at 100°C for 10 min, and after cooling, the samples were aliquoted and stored at -20°C.

[0088] 2.4 Immunoblotting

[0089] The method was the same as that in subsection 2.5 of the first part.

[0090] 3 Experimental results

[0091] 3.1 Effect of dihydroxy ketone on the bile acid signaling pathway in IEC-6 cells

[0092] To understand the effect of dihydroxy ketone on the FXR-regulated bile acid signaling pathway in small intestinal cells, the FXR signaling pathway in IEC-6 cells was inhibited with TUDCA. As Figure 8As shown, after TUDCA administration, the expressions of FXR and bile acid transport-related proteins OSTβ and FABP6 were downregulated. Compared with the model group, after treating cells with GW4064 and dihydroxy ketone at different concentrations, the expressions of bile acid transport proteins were significantly upregulated, and the dihydroxy ketone group showed a certain dose-dependence. The results indicate that dihydroxy ketone can significantly activate the FXR bile acid regulatory signaling pathway in IEC-6 cells, thereby regulating bile acid transport and maintaining intestinal bile acid homeostasis.

[0093] 3.2 Anti-inflammatory effect of dihydroxy ketone on DCA-induced inflammation in NCM460 cells

[0094] To evaluate the effect of bile acids on inflammation and the anti-inflammatory effect of dihydroxy ketone. The inflammatory factors of NCM460 cells were measured by Western blot. As Figure 9 shown, after stimulating cells with DCA, the expressions of inflammatory proteins such as TNF-α, IL-18, and IL-1β in NCM460 cells were upregulated, while after giving dihydroxy ketone, the expressions of inflammatory proteins decreased in a dose-dependent manner. The results indicate that DCA can cause inflammation in colon cells in vitro, and dihydroxy ketone also has a good anti-inflammatory effect in vitro.

[0095] Part Three: Effect of dihydroxy ketone on DSS-induced Fxr - / - Effect on ulcerative colitis in mice

[0096] 1.3 Experimental animals

[0097] Wild-type male C57BL / 6J mice (SPF grade, 7 - 8 weeks old, 20 - 22 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with the license number: SCXK(Xiang)2019 - 0004. They were placed in the SPF-grade mouse room of the Animal Experiment Center (license number: SYXK(Yu)2020 - 0006) for adaptive feeding. The mouse room was kept at a constant temperature and humidity, with a 12-hour light-dark cycle. The mice were fed with sufficient feed, allowed to drink water freely, and the bedding was changed regularly. They were adaptively fed for one week to adapt to the environment.

[0098] Knockout parental mice (7 - 8 weeks old, 20 - 22 g, SPF grade) were purchased from Cyagen Biosciences (Taicang) Co., Ltd., with the license number: SCXK(Su)2018 - 0003. After being identified by the company, the genotype was heterozygous (Fxr - / )). The mice were housed in the SPF-grade mouse room of the Animal Experiment Center (license number: SYXK(Yu)2020 - 0006). The mouse room had a 12-hour light-dark cycle. The mice drank water freely, and the feed was added and the bedding was changed regularly. After one week of adaptive feeding, male and female mice were caged together for breeding. After the female mouse's abdomen bulged significantly, it was separated and housed alone.

[0099] 2. Experimental methods

[0100] 2.1 Preparation of Main Solutions

[0101] 2.1 Preparation of Main Solutions

[0102] PBS buffer: The powder of 2×PBS phosphate buffer is completely dissolved in 2000 mL of pure water.

[0103] PBST buffer: 500 μL of Tween 20 is added to 1 L of PBS phosphate buffer and shaken well.

[0104] 0.5% sodium carboxymethyl cellulose (CMC-Na) solution: Weigh 2.5 g of CMC-Na powder, add 500 mL of pure water, heat and stir to fully dissolve the CMC-Na powder, and store it in the refrigerator at 4 °C for later use after cooling.

[0105] 5×Running buffer (electrophoresis solution): Measure 200 mL of 5×Running buffer solution and add pure water to make up to 1000 mL, shake well, and place at room temperature.

[0106] 5×Transfer buffer (membrane transfer solution): Measure 200 mL of 5×Running buffer solution, add 200 mL of methanol, add pure water to make up to 1000 mL, and place at 4 °C.

[0107] Dihydroxyketone (1 mg / mL, 3 mg / mL) solution: Weigh 14 mg and 42 mg of dihydroxyketone powder respectively, and ultrasonically disperse them evenly in 14 mL of 0.5% CMC-Na solution.

[0108] 2.2 Animal Grouping and Drug Administration

[0109] After the adaptive feeding, the mice are randomly divided into 6 groups with 8 mice in each group. The wild-type mice are divided into a normal control group, a DSS model group, and a dihydroxyketone group (30 mg / kg). The Fxr - / - mice are also divided into a normal control group, a DSS model group, and a dihydroxyketone group (30 mg / kg). Except for the normal group, 3% DSS is added to the drinking water of the other groups for free drinking. The mice in the normal group and the model group are respectively gavaged with the corresponding volume (100 μL / 10 g) of the solvent, that is, 0.5% CMC-Na solution according to their body weights. The mice in the dihydroxyketone group are gavaged with armentol H solution. Gavage continuously for 7 days. Collect the serum, colon, ileum, and liver tissues of the mice in each group (the mice are fasted for 8 h before sampling, and drink water freely).

[0110] 2.3 Histopathological Analysis of Colon Tissues

[0111] Mouse colon tissues were fixed with 4% paraformaldehyde, and after dehydration, embedding, sectioning, staining and other operations, the pathological changes of colon tissues were observed under an inverted microscope.

[0112] 2.4 The immunoblotting method was the same as that in Subsection 2.5 of Chapter 1.

[0113] 3 Experimental results

[0114] 3.1 Effects of dihydroxy ketone on Fxr - / - Effect on the disease activity index of ulcerative colitis mice

[0115] Figure 10 For the effects of dihydroxy ketone on DSS-induced ulcerative colitis in wild-type and Fxr - / - mice, where A is the body weight change curve; B is the colon state; C is the DAI score; D is the blood in stool state; E is the colon length

[0116] As Figure 10 shown, after wild-type mice were induced by DSS, their body weight significantly decreased on the fifth day, and diarrhea and blood in stool occurred. The symptoms gradually worsened with the increase of the modeling time. After dissection, the colon length of the mice was significantly shortened compared with the normal group (P<0.001, P<0.01, P<0.05); after administration of dihydroxy ketone (30 mg / kg), the percentage of body weight loss was significantly reduced, the symptoms of blood in stool and diarrhea were significantly improved, the disease activity index was significantly decreased, and the shortening of the colon length after dissection was also significantly improved (P<0.01, P<0.05). However, when Fxr - / - mice were induced by DSS, their body weight significantly decreased on the fourth day, and diarrhea and blood in stool occurred. After dissection, the colon length of the mice was significantly shortened (P<0.001, P<0.01). Compared with wild-type mice, Fxr - / - mice showed colitis symptoms earlier, and the degrees of blood in stool, diarrhea and body weight loss were more severe; after administration of dihydroxy ketone (30 mg / kg), there were no obvious changes in indicators such as body weight change, blood in stool, diarrhea, shortening of colon length and disease activity index. The results showed that the therapeutic effect of dihydroxy ketone on ulcerative colitis in mice disappeared with the knockout of the Fxr gene.

[0117] 3.2 Effects of dihydroxy ketone on Fxr - / - Effect on the histopathology of ulcerative colitis mice

[0118] Observation of pathological sections of colon tissues under an inverted microscope found that, as Figure 11 shown, in wild-type mice, ulcers were visible in the colonic mucosa layer induced by DSS, the local mucosal epithelium was damaged, a large number of crypts in the lamina propria were necrotic and dissolved, a large number of inflammatory cells infiltrated, and some inflammatory cells infiltrated into the submucosa. In the dihydroxy ketone group, the crypt structure was complete, the number of goblet cells increased, and the inflammatory infiltration decreased. In Fxr - / -In mice, crypt necrosis and dissolution occurred in the model group and the dihydroxy ketone group, with a large number of inflammatory cell infiltrations, and the inflammatory cells infiltrated into the submucosa. The results showed that dihydroxy ketone could significantly improve DSS-induced colitis, but the therapeutic effect of dihydroxy ketone on colitis disappeared with the knockout of Fxr.

[0119] 3.3 Effects of dihydroxy ketone on Fxr - / - Effects of dihydroxy ketone on colonic inflammation in ulcerative colitis mice

[0120] Immunoblotting was performed on wild-type and Fxr - / - mice respectively. As Figure 12 shown, in wild-type mice, the expression of inflammatory proteins such as TNF-α, IL-6, IL-18, and IL-1β in the colon tissue of DSS-induced mice increased significantly (P<0.001, P<0.01, P<0.05). After administration of dihydroxy ketone, the expression of inflammatory proteins decreased significantly (P<0.01, P<0.05), and the results were consistent with previous in vivo experiments. In Fxr - / - mice, the expression of inflammatory proteins in the colon tissue of DSS-induced mice increased significantly, and the inflammatory factors did not decrease after administration of dihydroxy ketone. The results showed that the anti-inflammatory effect of dihydroxy ketone in vivo disappeared with the knockout of FXR.

[0121] 3.4 Effects of dihydroxy ketone on Fxr - / - Effects of dihydroxy ketone on the FXR signaling pathway in the ileum of ulcerative colitis mice

[0122] Immunoblotting was performed on the related proteins of the FXR bile acid regulatory signaling pathway in the ileum tissues of wild-type mice and Fxr - / - mice respectively. As Figure 13 shown, in the experimental group of wild-type mice, the experimental results were consistent with the previous in vivo experiments; the expressions of the bile acid reabsorption transporters OSTα, OSTβ, FABP6, and FGF15 in the ileum of DSS-induced colitis mice decreased (P<0.01, P<0.05); after administration of dihydroxy ketone, the bile acid reabsorption transporters were significantly upregulated (P<0.01, P<0.05). In Fxr - / - mice, due to the deletion of the FXR protein, the bile acid transporters regulated by FXR were significantly reduced; there were no statistical differences in the OSTβ, FABP6, and FGF15 proteins between the model group and the dihydroxy ketone group. The results showed that dihydroxy ketone reduced the excessive accumulation of intestinal bile acids during the occurrence of ulcerative colitis by activating the expression of intestinal FXR-regulated bile acid transporters in vivo, thereby alleviating colitis. When the Fxr gene in vivo was knocked out, this regulatory effect also disappeared.

[0123] 3.5 Effects of dihydroxy ketone on Fxr - / - Effects of dihydroxy ketone on the bile acid synthesis signaling pathway in the liver of ulcerative colitis mice

[0124] Intestinal FXR activates FGFR4 through the FGF15 signal, inhibiting the expression of CYP7A1, the rate-limiting enzyme for bile acid synthesis. As Figure 14 shown, in wild-type mice, DSS-induced colitis decreased FGFR4 in the liver of mice (P<0.05) and increased CYP7A1 (P<0.05); after administration of dihydroxy ketone, FGFR4 increased (P<0.01) and the expression of CYP7A1 decreased (P<0.05). In Fxr - / - mice, there were no statistical differences in the FGFR4 and CYP7A1 proteins between the model group and the dihydroxy ketone group. The results indicate that dihydroxy ketone activates FXR and regulates the FGF15-FGFR4 signaling pathway, and this regulatory effect disappears when the Fxr gene is knocked out.

[0125] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts fall within the scope of protection of the present invention.

Claims

1. Application of (6R, 9S)-4-gustimide-9,10-dihydroxy-3-one in the preparation of drugs for treating ulcerative colitis.

2. The use according to claim 1, characterized in that: The medicament also includes medically acceptable adjuvants.

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