Application of venenum bufonis active molecule in preparation of cGAS-STING pathway inhibitor

Abstract

The invention provides application of venenum bufonis active molecules in preparation of cGAS-STING pathway inhibitors, and particularly belongs to the technical field of protein inhibitors. Starting from a downstream interferon regulatory factor of a cGAS-STING pathway, the venenum bufonis active molecule is found to have relatively strong inhibitory activity on the cGAS-STING pathway through screening. The ulcerative colitis resisting activity evaluation on the active molecule arenobufagin of the venenum bufonis finds that the arenobufagin has a good relieving effect on the ulcerative colitis, the colon length can be obviously improved, and release of serum inflammatory factors of mice with the ulcerative colitis can also be inhibited. Finally, the venenum bufonis active molecule can be used as a cGAS-STING pathway inhibitor, plays a role in resisting ulcerative colitis, and lays a foundation for clinically developing medicines for treating ulcerative colitis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pathway inhibitors, and particularly relates to an application of an active molecule of toad venom in preparation of a cGAS-STING pathway inhibitor. BACKGROUND

[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease mainly involving the mucosa of the colon and rectum, with typical symptoms of recurrent diarrhea, mucopurulent bloody stool, abdominal pain, and tenesmus. Its etiology is not fully understood, and it may be related to immune abnormalities, genetics, intestinal flora imbalance, and environmental factors. Diagnosis mainly relies on colonoscopy and biopsy, and continuous mucosal inflammation, erosion, and ulceration can be seen. The treatment goal is to control inflammation and induce remission, and common drugs include aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biological agents. Severe cases may require surgical resection of the colon.

[0003] Inflammation is the core pathological feature of ulcerative colitis, mainly caused by abnormal activation of the immune system. In patients with ulcerative colitis, the intestinal mucosal barrier is damaged, allowing intestinal bacteria and antigens to be exposed to immune cells, triggering innate and adaptive immune responses. Immune cells (such as macrophages, neutrophils, and T cells) are overactivated, releasing large amounts of pro-inflammatory factors (such as TNF-α, IL-6, IL-1β, and IL-23), while anti-inflammatory factors (such as IL-10) are reduced, leading to the persistence of chronic inflammation. This inflammatory response can damage intestinal epithelial cells, forming ulcers, and causing typical symptoms such as diarrhea, bloody stool, and abdominal pain. Long-term inflammation also increases the risk of cancer. Therefore, the treatment of ulcerative colitis mainly focuses on inhibiting excessive immune responses and regulating inflammatory pathways.

[0004] The cGAS-STING pathway may play a role in ulcerative colitis by regulating innate immune responses and intestinal inflammation. When cell damage or infection leads to DNA release, cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthetase) senses cytosolic DNA and catalyzes the production of the second messenger cGAMP, which in turn activates STING (stimulator of interferon genes), triggering the release of type I interferons (IFN-I) and pro-inflammatory factors (such as TNF-α and IL-6). In ulcerative colitis, intestinal barrier disruption and microbial DNA infiltration can overactivate this pathway, exacerbating mucosal immune responses and chronic inflammation. Studies have shown that cGAS deletion or inhibitors can reduce inflammatory damage in ulcerative colitis, suggesting that targeting the cGAS-STING pathway may be a new strategy for the treatment of ulcerative colitis. SUMMARY

[0005] The application aims to provide an application of active molecules of Venenum Bufonis in preparation of cGAS-STING pathway inhibitors.

[0006] The application provides an application of active molecules of Venenum Bufonis in preparation of cGAS-STING pathway inhibitors; the active molecules of Venenum Bufonis include bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin or resibufogenin.

[0007] The application also provides an application of active molecules of Venenum Bufonis in preparation of drugs for treating intestinal inflammation; the active molecules of Venenum Bufonis include bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin or resibufogenin.

[0008] The application also provides an application of active molecules of Venenum Bufonis in preparation of drugs for treating intestinal inflammation; the active molecules of Venenum Bufonis include bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin or resibufogenin.

[0009] The application also provides an application of active molecules of Venenum Bufonis in preparation of drugs for treating intestinal inflammation; the active molecules of Venenum Bufonis include bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin or resibufogenin.

[0010] Preferably, the dosage form of the drug includes injection, powder, granule, powder, pill, oral liquid or tablet.

[0011] Preferably, the drug also includes a pharmaceutically acceptable excipient.

[0012] The application also provides an application of active molecules of Venenum Bufonis in preparation of products for reducing DAI scores; the active molecules of Venenum Bufonis include bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin or resibufogenin.

[0013] The application also provides an application of active molecules of Venenum Bufonis in preparation of products for reducing levels of inflammatory factors IL-1beta, IL-6, TNF-alpha and IFN-gamma; the active molecules of Venenum Bufonis include bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin, bufalin, resibufogenin or resibufogenin.

[0014] The application also provides application of active molecules of toad venom in preparation of products for prolonging colon length and reducing spleen size; the active molecules of toad venom include cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufonidine or saurufin.

[0015] The application provides application of active molecules of toad venom in preparation of cGAS-STING pathway inhibitors, the active molecules of toad venom including cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufonidine or saurufin. The application starts from the mechanism of cGAS-STING pathway and downstream target gene combination, finds that eight active molecules of toad venom have inhibitory activity on the cGAS-STING pathway, and saurufin is the strongest. The application further finds that saurufin has a good improvement effect on mouse ulcerative colitis through ulcerative colitis experiment of saurufin, can obviously improve colon length, and can inhibit the release of serum inflammatory factors of intestinal inflammation mice. In conclusion, the active molecules of toad venom can be used as cGAS-STING inhibitors to play a role in treating intestinal inflammation, and lay a foundation for clinical development of intestinal inflammation targeted therapy drugs. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0017] Figure 1 The IRF-Luc-THP-1 cell model result graph provided by the present application is shown in the following figure; A is the phorbol ester induced IRF-Luc-THP-1 cell graph; B is the HT-DNA induced IRF-Luc-THP-1 cell fluorescence value quantification graph; Figure 2 The IRF-Luc-THP-1 cell model screening result graph provided by the present application is shown in the following figure; Figure 3 The 48 active compound rescreening result and 31 active compound concentration reduction screening result graph provided by the present application is shown in the following figure; A is the 48 active compound rescreening result graph; B is the 31 active compound concentration reduction screening result graph; Figure 4 The 12 active molecule of toad venom activity screening and 8 active molecule of toad venom concentration reduction screening result graph provided by the present application is shown in the following figure; A is the 12 active molecule of toad venom activity screening result graph; B is the 8 active molecule of toad venom concentration reduction screening result graph; Figure 5The Caspase-3 activity screening diagram of eight active molecules of Venenum Bufonis provided in the application in HT-29 cells is as follows: Figure 6 The flow cytometry diagram and quantification of apoptosis of eight active molecules of Venenum Bufonis provided in the application in HT-29 cells are as follows; wherein, A is a flow cytometry diagram; and B is a quantification result diagram of apoptosis rate; Figure 7 The animal experiment flowchart provided in the application is as follows: Figure 8 The influence result diagram of three different doses of active molecules of Venenum Bufonis on DAI scores of C57 mice after UC model is as follows: Figure 9 The influence result diagram of three different doses of active molecules of Venenum Bufonis on the body weight of mice after UC model of C57 mice is as follows: Figure 10 The colon representative result diagram and quantification statistical diagram of three different doses of active molecules of Venenum Bufonis after UC model of C57 mice are as follows; wherein, A is a colon representative result diagram of three different doses of active molecules of Venenum Bufonis after UC model of C57 mice; and B is a colon quantification statistical result diagram; Figure 11 The colon HE staining, PAS staining and colon histology score result diagram of three different doses of active molecules of Venenum Bufonis after UC model of C57 mice is as follows; wherein, A is a colon HE staining, PAS staining representative result diagram of three different doses of active molecules of Venenum Bufonis after UC model of C57 mice; and B is a colon histology score quantification statistical result diagram; Figure 12 The influence result diagram of different doses of bufonidine on DAI scores of C57 mice after UC model is as follows: Figure 13 The influence result diagram of different doses of bufonidine on the body weight of mice after UC model of C57 mice is as follows: Figure 14 The colon representative result diagram and quantification statistical diagram of different doses of bufonidine after UC model of C57 mice are as follows; wherein, A is a colon representative result diagram of bufonidine after UC model of C57 mice; and B is a colon quantification statistical result diagram; Figure 15 The spleen representative result diagram and quantification statistical diagram of different doses of bufonidine after UC model of C57 mice are as follows; wherein, A is a spleen representative result diagram of bufonidine after UC model of C57 mice; and B is a spleen quantification statistical result diagram; Figure 16The different dose of bufalin provided by the present application on C57 mouse UC model after colon HE staining, PAS staining and colon histology score result graph; wherein, A is the representative result graph of the colon HE staining, PAS staining of the different dose of bufalin on C57 mouse UC model after; B is the quantitative statistical result graph of colon histology score; Figure 17 The different dose of bufalin provided by the present application on C57 mouse UC model after the expression level statistical result graph of mouse serum inflammatory factors IL-1β, IL-6, IFN-γ and TNF-α; wherein, A is the expression level statistical result graph of IL-1β; B is the expression level statistical result graph of IL-6; C is the expression level statistical result graph of IFN-γ; D is the expression level statistical result graph of TNF-α; Figure 18 The different dose of bufalin provided by the present application on C57 mouse UC model after the expression level statistical result graph of mouse colon inflammatory factors IL-1β, IL-6 and TNF-α; wherein, A is the mRNA expression level statistical result graph of TNF-α; B is the mRNA expression level statistical result graph of IL-1β; C is the mRNA expression level statistical result graph of IL-6; In the above figures, * represents P <0.05, compared with the model group, ** represents P <0.01, compared with the model group; ## represents P <0.001, compared with the control group. DETAILED DESCRIPTION

[0018] The present application provides the application of active molecules of Chansu in the preparation of cGAS-STING pathway inhibitors; the active molecules of Chansu include cinobufagin, resibufogenin, bufalin, day bufadienolide, far bufadienolide, chinobufagin, bufadienolide or bufalin. In the present application, the active molecules of Chansu are all derived from traditional Chinese medicine Chansu, and the cGAS-STING pathway inhibitors are screened on a wide range of macrophages. In the present application, the macrophages are THP-1 cells.

[0019] The present application also provides the application of active molecules of Chansu in the preparation of drugs for treating intestinal inflammation; the active molecules of Chansu include cinobufagin, resibufogenin, bufalin, day bufadienolide, far bufadienolide, chinobufagin, bufadienolide or bufalin. In the present application, the bufalin can inhibit the cGAS-STING pathway of macrophages, thereby inhibiting the expression of downstream target genes and exerting the anti-intestinal inflammation effect. In specific embodiments, the dosage form of the drug includes injection, powder, granule, powder, pill, oral liquid or tablet. In specific embodiments, the drug also includes pharmaceutically acceptable excipients.

[0020] The application also provides use of active molecules of toad venom in preparation of a medicine for treating colitis; the active molecules of toad venom include cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufadienolide or gamabufagin. In the application, the gamabufagin can inhibit the cGAS-STING pathway of macrophages, thereby inhibiting the expression of downstream target genes, and can play an anti-colitis role. In specific embodiments, the dosage form of the medicine includes injection, powder, granules, powder, pills, oral liquid or tablets. In specific embodiments, the medicine also includes a pharmaceutically acceptable excipient.

[0021] The application also provides use of active molecules of toad venom in preparation of a medicine for treating colitis; the active molecules of toad venom include cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufadienolide or gamabufagin. In the application, the gamabufagin can inhibit the cGAS-STING pathway of macrophages, thereby inhibiting the expression of downstream target genes, and can play an anti-colitis role. In specific embodiments, the dosage form of the medicine includes injection, powder, granules, powder, pills, oral liquid or tablets. In specific embodiments, the medicine also includes a pharmaceutically acceptable excipient.

[0022] The application also provides use of active molecules of toad venom in preparation of a product for reducing DAI score; the active molecules of toad venom include cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufadienolide or gamabufagin.

[0023] The application also provides use of active molecules of toad venom in preparation of a product for reducing the levels of inflammatory factors IL-1β, IL-6, TNF-α and IFN-γ; the active molecules of toad venom include cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufadienolide or gamabufagin.

[0024] The application also provides use of active molecules of toad venom in preparation of a product for prolonging the length of the colon and reducing the size of the spleen; the active molecules of toad venom include cinobufagin, resibufogenin, bufalin, day bufadienolide, distil bufadienolide, chinobufagin, bufadienolide or gamabufagin.

[0025] In order to further illustrate the application, the use of active molecules of toad venom in preparation of a cGAS-STING pathway inhibitor provided by the application is described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the application.

[0026] The materials and methods used in the embodiments of the application are as follows: Materials are shown in Tables 1 and 2: Table 1 Reagents

[0027] Table 2 Instruments and equipment

[0028] Animal model: Example C57 mice were selected, and the age was controlled at about 8 weeks old. The mice were randomly divided into 6 groups: normal control group, model group, sulfasalazine group, RU.521 group, low-dose saponaria officinalis group, and high-dose saponaria officinalis group. In this experiment, 4% DSS was used to make the mice drink water every day to make the model, and intraperitoneal injection of drugs was started on the 4th day of modeling, which lasted for 7 days. The model group received intraperitoneal injection of the same volume of normal saline as a control. The mice were sacrificed on the 11th day, and the mouse spleen, mesenteric lymph nodes, and colon tissue were collected for subsequent experiments.

[0029] DAI score (see Table 3): Table 3 DAI score

[0030] Cell model: Example IRF-LUC-THP-1 cell model was selected. IRF-LUC-THP-1 cells were inoculated into a 10 cm dish, PMA (100 ng / ml) was added for induction for 24 h, and then trypsin digestion was performed for 1-2 min. The cells were collected and centrifuged, inoculated into a 96-well plate (2*10 4 cells per well), and cultured in serum-containing modified 1640 medium for 24 h. The original culture medium in the 96-well plate was discarded, and 1640 culture solution containing drugs was added. After 1 h, HT-DNA was added to stimulate the cells and cultured for 12 h. Then the original culture medium in the 96-well plate was discarded, and D-luciferin potassium salt (final concentration 100 μg / mL) was added, 100 μl per well. The 96-well plate with luciferin was placed in a fluorescence plate reader for measurement.

[0031] Statistical analysis: The evaluation indexes were statistically analyzed after the experiment using GraphPad Prism 8.0 software. The DAI score data were expressed as median (Median) ± interquartile range (IQR), and the C57 mouse body weight percentage and C57 mouse colon data were expressed as mean (Mean) ± standard deviation (SD). The statistical difference of the data was analyzed by One-way analysis of variance (ANOVA), Two-way ANOVA, and T-test. P <0.05, **P<0.01; #P <0.05, ## P< 0.01 indicates a statistically significant difference. # indicates a difference compared to the control group, and * indicates a difference compared to the model group. ns: P A value >0.05 indicates that there is no significant difference.

[0032] Medicinal materials and sample setup: CBG represents bufotoxin monomer, RBG represents bufotoxin ester monomer, BFL represents bufotoxin monomer, GBT represents bufotoxin monomer, TBG represents bufotoxin monomer, CBT represents bufotoxin monomer, BTD represents bufotoxin monomer, ABG represents bufotoxin monomer, 5-HTM represents 5-hydroxytryptamine monomer, N-MST represents N-methyl-5-hydroxytryptamine monomer, BFT represents bufotoxin monomer, and BFD represents bufotoxin monomer. Positive controls were sulfasalazine (SASP), a drug currently used to treat ulcerative colitis, and the cGAS inhibitor RU.521.

[0033] Example 1 Establishment of the IRF-LUC-THP-1 cell line screening model: (1) Constructing the IRF-LUC-THP-1 cell line M0 model According to the literature, PMA can induce IRF-LUC-THP-1 cells into M0 macrophages. In this example, IRF-LUC-THP-1 cells were induced with PMA (100 ng / ml) for 24 h, and the results are as follows... Figure 1 As shown in A, Figure 1 In the diagram, A represents the induced IRF-LUC-THP-1 cell line. According to the literature, HT-DNA, as an agonist of the cGAS-STING signaling pathway, can significantly activate the cGAS-STING signaling pathway. Figure 1 B in the figure represents the fluorescence activity detection of the IRF-LUC-THP-1 cell line after HT-DNA stimulation; the results show that the IRF-LUC-THP-1 cell line has significant fluorescence activity after HT-DNA stimulation. Figure 1 The results of the IRF-Luc-THP-1 cell model are shown in Figure A; where A is the IRF-Luc-THP-1 cell model induced by phorbol ester; and B is the quantitative fluorescence value of IRF-Luc-THP-1 cells induced by HT-DNA.

[0034] (2) Screening method for IRF-LUC-THP-1 cell line Cell lines were incubated with 10 μM of the compound in 96-well white plates for 12 h. The inhibitory effect of the compound on the cGAS-STING signaling pathway was then determined by detecting the fluorescence expression in the cell lines. Luciferase is expressed only in cells where IRF protein is present. During the assay, luciferase substrate luciferase potassium salt (final concentration 100 μg / mL) was added, and the effect of the compound on IRF protein expression in cells was determined based on the fluorescence intensity.

[0035] As can be seen from the above examples, the IRF-LUC-THP-1 cells selected in this patent can be used for subsequent screening experiments.

[0036] Example 2 Screening of active molecules: (1) Screening results of compounds in cell and protein screening models: a. Screening results of the IRF-LUC-THP-1 cell model IRF-LUC-THP-1 cells were used at a rate of 2*10 4 Cells were seeded per well in 96-well white plates and allowed to adhere overnight. The supernatant was then discarded, and 10 μM of a compound diluted in 1640 medium was added. Cells were treated with the compound for 12 h, and then the fluorescence intensity of the cells was detected using a fluorescence microplate reader. Results are as follows: Figure 2 As shown. Figure 2 The image shows the screening results of the IRF-Luc-THP-1 cell model. Figure 2 It can be seen that 48 molecules in the compound library (3496 compounds) have a greater fluorescence inhibition rate against IRF-LUC-THP-1 cells than the positive compound RU.521 (red dashed line).

[0037] b. Results of rescreening of 48 positive compounds and results of screening with reduced concentrations of 31 positive compounds Figure 3 Figures show the results of secondary screening of 48 active compounds and the results of reduced-concentration screening of 31 active compounds; where A represents the results of secondary screening of 48 active compounds; and B represents the results of reduced-concentration screening of 31 active compounds. The 48 compounds were added to the IRF-LUC-THP-1 cell model system at a concentration of 10 μM, and the results are as follows. Figure 3 As shown in A in the diagram. From Figure 3 As shown in Figure A, among the 48 positive compounds, 31 compounds had a lower relative fluorescence intensity than the positive control RU.521 (red dashed line) (the higher the pathway inhibition effect of a compound, the lower its relative fluorescence intensity). These 31 compounds were then screened at lower concentrations, and the results are as follows... Figure 3 As shown in Figure B. (From...) Figure 3As shown in FIG. B in the figure, 15 compounds had a relative fluorescence intensity lower than that of the positive control RU.521 by screening 31 compounds at a concentration of 5 μM. Among the 15 compounds, most were toad venom derivatives. Therefore, 12 toad venom derivatives were screened for cGAS-STING pathway inhibition activity, and the results are shown in Figure 4 Figure 4 FIGS. 12 and 8 are diagrams of activity screening of 12 toad venom active molecules and screening results of 8 toad venom active molecules at a reduced concentration. As shown in FIG. A, among the 12 toad venom derivatives, 8 compounds had an inhibition rate higher than that of the positive control. The 8 compounds were CBG, RBG, BFL, GBT, TBG, CBT, BTD, and ABG. Figure 4

[0038] Example 3 Effects of CBG, RBG, BFL, GBT, TBG, CBT, BTD, and ABG on the apoptosis rate of HT-29 cells induced by the supernatant of mouse primary macrophages and on the Caspase-3 activity in HT-29 cells.

[0039] (1) Caspase-3 activity screening Mouse primary macrophages were induced by LPS (100 ng / ml) for 24 h, and the supernatant of mouse primary macrophages was extracted. The supernatant was added to HT-29 cells and 8 compounds were given, respectively. After 12 h, the compounds were screened by detecting the Caspase-3 activity, and the results are shown in Figure 5 Figure 5 FIG. 8 is a diagram of Caspase-3 activity screening of 8 toad venom active molecules in HT-29 cells. The results show that CBG, RBG, and ABG most obviously inhibited the Caspase-3 activity of HT-29 cells among the 8 toad venom active molecules.

[0040] (2) Flow cytometry detection of the apoptosis rate of HT-29 cells Mouse primary macrophages were induced by LPS (100 ng / ml) for 24 h, and the supernatant of mouse primary macrophages was extracted. The supernatant was added to HT-29 cells and 8 compounds were given, respectively. After 12 h, the compounds were screened by detecting the apoptosis rate of HT-29 cells, and the results are shown in Figure 6 Figure 6 FIG. 8 is a diagram of Caspase-3 activity screening of 8 toad venom active molecules in HT-29 cells. The results show that CBG, RBG, and ABG most obviously inhibited the Caspase-3 activity of HT-29 cells among the 8 toad venom active molecules.

[0041] ​​​​Eight kinds of compounds were screened by Caspase-3 activity screening and flow cytometry. The results showed that eight kinds of compounds had inhibitory effect on HT-29 cell apoptosis, and CBG, RBG and ABG had the most obvious effect.

[0042] Example 4 Therapeutic effect of CBG, RBG and ABG on C57 mouse UC model: Example C57 mice were selected, and the age was controlled at about 8 weeks old. The mice were randomly divided into 6 groups: normal control group, model group, sulfasalazine group, CBG group, RBG group and ABG group. In this experiment, 4% DSS was used to make the mice drink water every day to make the model, and intraperitoneal injection of drugs was started on the 4th day of modeling, which lasted for 7 days. The model group received intraperitoneal injection of the same volume of normal saline as a control. The mice were sacrificed on the 11th day, and the mouse spleen and colon tissues were collected for subsequent experiments. The animal experiment flow chart is shown in Figure 7 , note: after the C57 mouse UC model, the mice were intraperitoneally injected with drugs for 7 consecutive days, and the C57 mice were sacrificed after isoflurane anesthesia to take the colon tissue.

[0043] Figure 8 The results of the effect of three different doses of active molecules of toad venom on the DAI score of C57 mouse UC model are shown in the figure. Figure 9 The results of the effect of three different doses of active molecules of toad venom on the body weight of C57 mouse UC model are shown in the figure. Figure 10 The results of the effect of three different doses of active molecules of toad venom on the DAI score of C57 mouse UC model are shown in the figure. Figure 8 ), and the body weight percentage decreased significantly ( Figure 9 ). Compared with the model group, intraperitoneal injection of CBG (2 mg / kg), RBG (4 mg / kg) and ABG (4 mg / kg) in mice could reduce the DAI score ( Figure 8 ), increase the body weight percentage ( Figure 9 ), and ABG (4 mg / kg) had the most obvious effect. Compared with the normal group, the colon of the model group was significantly shortened ( Figure 10 ). Compared with the model group, the colon length of the drug administration group was significantly prolonged ( Figure 10 ).

[0044] Figure 11Figure 1 is a diagram of HE staining, PAS staining and histological score results of C57 mice UC model after treatment with active molecules of different doses of toad venom; wherein, A is a representative result diagram of HE staining, PAS staining of C57 mice UC model after treatment with active molecules of different doses of toad venom; B is a quantitative statistical result diagram of histological score. Compared with the normal group, the HE staining and PAS staining of the model group showed obvious inflammatory infiltration, disappearance of crypt structure and columnar epithelial cells (P < 0.05) Figure 11 ). Compared with the model group, intraperitoneal injection of CBG (2 mg / kg), RBG (4 mg / kg) and ABG (4 mg / kg) can significantly reduce the inflammatory symptoms of mice colon, which shows that CBG (2 mg / kg), RBG (4 mg / kg) and ABG (4 mg / kg) can improve the symptoms of ulcerative colitis in UC model mice at 7 days after administration (P < 0.05) Figure 11

[0045] In summary, compared with the UC model group, the administration group can reduce the inflammatory symptoms of ulcerative colitis in mice and reduce the DAI score.

[0046] Example 5 Therapeutic effect of ABG on C57 mouse UC model In this example, C57 mice aged about 8 weeks were randomly divided into 6 groups: normal control group, model group, sulfasalazine group, RU.521 group, ABG low-dose group and ABG high-dose group. In this experiment, 4% DSS was used to make the mice drink water every day to make the model, and intraperitoneal injection of drugs was started at the 4th day of modeling and lasted for 7 days. The model group received intraperitoneal injection of the same volume of normal saline as a control. The mice were sacrificed on the 11th day, and the mouse spleen, mesenteric lymph nodes and colon tissue were collected for subsequent experiments. The animal experiment process is shown in Figure 7

[0047] Figure 12 Figure 5 is a diagram of the effect of different doses of bufalin on the DAI score of C57 mouse UC model. Figure 13 Figure 6 is a diagram of the effect of different doses of bufalin on the body weight of C57 mouse UC model. Figure 14 Figure 7 is a diagram of the effect of different doses of bufalin on the colon of C57 mouse UC model; wherein, A is a representative result diagram of bufalin on C57 mouse UC model; B is a quantitative statistical result diagram of colon. Figure 15 ​​Figure 6 shows representative results of the spleen of C57 mice after UC model by different doses of bufonin A and quantitative statistical graphs; A shows representative results of the spleen of C57 mice after UC model by bufonin A; B shows quantitative statistical results of the spleen. The results show that compared with the normal group, the DAI score of the mice in the model group increased significantly (p<0.01) Figure 12 , and the percentage of body weight decreased significantly (p<0.01) Figure 13 . Compared with the model group, intraperitoneal injection of ABG (2 mg / kg and 4 mg / kg) in mice can significantly reduce the DAI score (p<0.01) Figure 12 , and increase the percentage of body weight (p<0.01) Figure 13 . Compared with the normal group, the colon of the model group was significantly shortened (p<0.01) Figure 14 , and the spleen of the model group was significantly enlarged (p<0.01) Figure 15 . Compared with the model group, the colon length of the drug group was significantly prolonged (p<0.01) Figure 14 , and the spleen was significantly reduced (p<0.01) Figure 15 .

[0048] Figure 16 Figure 7 shows HE staining, PAS staining and histological score results of the colon of C57 mice after UC model by different doses of bufonin A; A shows representative results of HE staining and PAS staining of the colon of C57 mice after UC model by bufonin A; B shows quantitative statistical results of the histological score of the colon. Compared with the normal group, HE staining and PAS staining of the model group showed obvious inflammatory infiltration, crypt structure and disappearance of columnar epithelial cells (p<0.01) Figure 16 . Compared with the model group, intraperitoneal injection of ABG (2 mg / kg and 4 mg / kg) in mice can significantly reduce the inflammatory symptoms of the colon, indicating that ABG (2 mg / kg and 4 mg / kg) can significantly improve the symptoms of ulcerative colitis in UC model mice at 7 days after administration (p<0.01) Figure 16 .

[0049] Compared with the normal group, the levels of inflammatory factors IL-1β, IL-6, TNF-α and IFN-γ in the serum of the model group increased significantly; compared with the model group, the levels of inflammatory factors IL-1β, IL-6, TNF-α and IFN-γ in the serum of the drug group decreased significantly (p<0.01) Figure 17Statistical result graph of expression levels of serum inflammatory factors IL-1β, IL-6, IFN-γ and TNF-α of C57 mice after different doses of bufalin on UC model of mice; wherein, A is the statistical result graph of IL-1β expression level; B is the statistical result graph of IL-6 expression level; C is the statistical result graph of IFN-γ expression level; D is the statistical result graph of TNF-α expression level). By detecting the mRNA level of colon-related inflammatory factors, the results show that compared with the normal group, the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the model group are significantly increased, and compared with the model group, the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the administration group are significantly decreased Figure 18 Statistical result graph of mRNA expression levels of colon inflammatory factors IL-1β, IL-6 and TNF-α of C57 mice after different doses of bufalin on UC model of mice; wherein, A is the statistical result graph of mRNA expression level of TNF-α; B is the statistical result graph of mRNA expression level of IL-1β; C is the statistical result graph of mRNA expression level of IL-6).

[0050] In summary, compared with the UC model group, ABG can reduce the inflammation of ulcerative colitis in mice, reduce the DAI score, and the effect is better than that of the positive control. Therefore, ABG can provide a candidate drug for the treatment of such diseases.

[0051] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. Application of toad venom active molecules in the preparation of cGAS-STING pathway inhibitors; the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

2. Application of toad venom active molecules in the preparation of drugs for treating enteritis; the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

3. Application of toad venom active molecules in the preparation of drugs for treating colitis; the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

4. Application of toad venom active molecules in the preparation of drugs for treating ulcerative colitis; wherein the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

5. The application according to any one of claims 2 to 4, characterized in that, The dosage forms of the drug include injections, powders, granules, powders, pills, oral liquids, or tablets.

6. The application according to any one of claims 2 to 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. Application of toad venom active molecules in the preparation of products that reduce DAI scores; the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

8. Application of toad venom active molecules in the preparation of products that reduce the levels of inflammatory factors IL-1β, IL-6, TNF-α, and IFN-γ; wherein the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

9. Application of toad venom active molecules in the preparation of products that prolong colon length and shrink spleen; the toad venom active molecules include bufotoxin, bufotoxin ester, bufotoxin, bufotoxin, bufotoxin, bufotoxin, bufotoxin, or bufotoxin.

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