Application of guanosine in medicine for treating inflammatory injury of digestive system

Abstract

The invention relates to the field of biological medicines, in particular to application of guanosine in medicines for treating inflammatory injury of a digestive system. The guanosine can be used for improving the problems of mucosa edema and damage, crypt atrophy and disappearance and the like of tissues by inhibiting the proliferation capacity reduction of digestive system cells caused by capsaicin or radiation, reducing the DNA damage of the cells and inhibiting cell apoptosis, so that the effect of treating inflammatory injury is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biological medicines, and in particular to application of guanosine in medicines for treating inflammatory damage of the digestive system. Background Art

[0002] Digestive inflammatory injury refers to an inflammatory response to the digestive tract caused by various factors, leading to tissue damage and dysfunction of digestive organs. Digestive inflammatory injury is a common and frequently occurring disease worldwide, characterized by high morbidity, recurrent course, and the tendency to become chronic. This condition not only severely impacts patients' quality of life but can also lead to fatal complications such as bleeding, perforation, and cancer.

[0003] At present, the main treatments for inflammatory damage to the digestive system are glucocorticoids and immunosuppressants. Glucocorticoid treatment has a high therapeutic efficiency and can be administered in a variety of ways, making it highly practical. However, glucocorticoids are prone to serious side effects, are dependent on the treatment of inflammation, and sudden discontinuation may lead to recurrence of inflammation. They are only suitable for inducing remission in the acute phase and cannot be maintained for a long time. In contrast, immunosuppressants can control inflammation in the long term and can be used in combination with glucocorticoids to reduce the dose and course of glucocorticoids, reduce side effects, and have a high selectivity for lymphocytes, resulting in higher biosafety and fewer side effects. However, immunosuppressants are slow to take effect and can lead to a certain amount of toxicity accumulation. Therefore, there is still a lot of room for optimization of drugs for the treatment of inflammatory damage to the digestive system. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel active ingredient capable of inhibiting inflammatory damage to the digestive system.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] Application of guanosine in medicine for treating inflammatory damage of the digestive system.

[0007] Optionally, the digestive system inflammatory injury includes one or more of esophagitis, gastritis and enteritis.

[0008] Optionally, the inflammatory damage to the digestive system is caused by capsaicin or radiation.

[0009] Optionally, the guanosine is administered by intraperitoneal injection.

[0010] Optionally, the dosage of guanosine is any value between 5 mg / kg and 10 mg / kg.

[0011] Optionally, when the inflammatory injury of the digestive system is enteritis, the administration concentration of guanosine is any value between 0.01 μM and 100 μM.

[0012] Optionally, when the inflammatory injury of the digestive system is gastritis, the administration concentration of guanosine is any value between 10 μM and 50 μM.

[0013] Optionally, the guanosine is used to restore the proliferation ability of cells.

[0014] Optionally, the guanosine is used to inhibit cell apoptosis.

[0015] Optionally, the guanosine is used to restore DNA damage in cells.

[0016] The beneficial effects of the present invention are as follows: guanosine inhibits the decreased proliferation capacity of digestive system cells caused by capsaicin or radiation, reduces cell DNA damage, and inhibits cell apoptosis, thereby improving the edema and destruction of the mucosal layer of the tissue, the atrophy and disappearance of the crypts, and other problems, thereby achieving the effect of treating inflammatory damage.

[0017] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a quantitative analysis of the effects of different concentrations of guanosine on capsaicin-induced IEC-6 cell death in Example 1 of the present invention;

[0019] Figure 2 This is a graph showing the cycle detection analysis of each group of IEC-6 cell samples after propidium iodide staining in Example 1 of the present invention;

[0020] Figure 3 Graphs showing the Edu fluorescence labeling analysis and clone formation assay results for each group of IEC-6 cell samples in Example 1 of the present invention;

[0021] Figure 4 This is a quantitative analysis chart of the ROS levels and cell proliferation protein expressions of each group of IEC-6 cell samples in Example 1 of the present invention;

[0022] Figure 5 This is a quantitative analysis of the effects of different concentrations of guanosine on radiation-induced apoptosis in GES-1 cells in Example 1 of the present invention;

[0023] Figure 6 This is a quantitative analysis graph of ROS levels in each group of GES-1 cell samples in Example 1 of the present invention;

[0024] Figure 7 This is a graph showing protein content in each group of GES-1 cell samples in Example 1 of the present invention;

[0025] Figure 8 These are H&E staining images of the colon tissues of each group of mice in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0028] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0029] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] The present invention is applied to protect the use of guanosine in medicine for treating inflammatory damage of the digestive system.

[0031] Guanosine inhibits the decreased proliferation of digestive system cells caused by capsaicin or radiation, reduces cell DNA damage, and inhibits cell apoptosis, thereby improving the edema and destruction of the tissue's mucosal layer, crypt atrophy and disappearance, and achieves the effect of treating inflammatory damage.

[0032] In some embodiments, inflammatory damage to the digestive system includes one or more of esophagitis, gastritis, and enteritis.

[0033] In some embodiments, the inflammatory injury to the digestive system is caused by capsaicin or radiation.

[0034] In some embodiments, guanosine is administered by intraperitoneal injection.

[0035] In some embodiments, the dosage of guanosine is any value between 5 mg / kg and 10 mg / kg, for example, any value between 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg and 10 mg / kg.

[0036] In some embodiments, when the inflammatory injury of the digestive system is enteritis, the administration concentration of guanosine is any value between 0.01 μM and 100 μM, for example, any value between 0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM.

[0037] In some embodiments, when the inflammatory injury of the digestive system is gastritis, the administration concentration of guanosine is any value between 10 μM and 50 μM, for example, any value between 10 μM, 20 μM, 30 μM, 40 μM and 50 μM.

[0038] In some embodiments, guanosine is used to restore the proliferative capacity of cells.

[0039] In some embodiments, guanosine is used to inhibit apoptosis.

[0040] In some embodiments, guanosine is used to repair DNA damage in cells.

[0041] Please refer to the following examples for details.

[0042] Example 1:

[0043] IEC-6 cells were used for in vitro cell experiments. IEC-6 cells are an epithelial cell line derived from the small intestine of male rats. Capsaicin (CAP) at a concentration of 200 μM and guanosine (GUA) at varying concentrations were added to the cell culture medium inoculated with IEC-6 cells. After 48 hours of culture, cell viability was measured using a CCK8 kit, and the concentration of lactate dehydrogenase (LDH) in the supernatant of the culture medium was measured using an ELISA kit to characterize the amount of LDH released from the cells. For experimental results, see Figure 1, in which neither CAP nor GUA was added to the control group. Previous studies have shown that CAP at a concentration of 50μM to 100μM can significantly induce cell death in vitro. In this experiment, the concentration of CAP was as high as 200μM, resulting in a significant decrease in cell viability. As the concentration of added GUA increased, cell viability rebounded, indicating that GUA can inhibit capsaicin-induced IEC-6 cell death, and has a significant inhibitory effect when GUA is at any value between 0.01μM and 100μM. The release of LDH supports the above experimental results and proves that when GUA is 0.1μM, its inhibitory effect on cell damage is the strongest.

[0044] IEC-6 cells cultured for 48 hours served as the control group, IEC-6 cells co-cultured with 200 μM capsaicin for 48 hours as the capsaicin group, IEC-6 cells co-cultured with 0.1 μM GUA for 48 hours as the GUA group, and IEC-6 cells co-cultured with 200 μM capsaicin and 0.1 μM GUA for 48 hours as the capsaicin+GUA group. Cells in each group were stained with PI, and cell parameters were analyzed by flow cytometry. After collection, fixation, washing, and resuspending, RNase A was added to a final concentration of 50 μg / mL and incubated at 37°C for 30 minutes to remove RNA interference. Propidium iodide (PI) staining solution was then added to a final concentration of 50 μg / mL. Incubation was performed at room temperature in the dark for 30 minutes before analysis by flow cytometry. PI staining solution can specifically bind to DNA. By detecting the fluorescent signal carried by PI, the DNA content of cells is measured. Based on the distribution of DNA content, the cell cycle is analyzed using the cycle fitting software Modifit. The content of cell populations in the resting phase (G0) or diploid DNA state (G1 phase), cell populations in the DNA synthesis phase (S phase), and cell populations in the late DNA synthesis phase (G2 phase) or mitosis phase (M phase) is obtained. The content of cell subpopulations with DNA content below 2N is recorded as SubG1, and the content of dead cells or cell fragments is recorded as Dead. Please refer to the test results for details. Figure 2 It can be seen that after the addition of GUA, the decrease in the content of cells in the G2 or M phase caused by Capsaicin was significantly inhibited, proving that GUA helps to restore the decreased proliferation ability of cells caused by Capsaicin, that is, GUA can treat inflammatory damage to the digestive system.

[0045] The EdU cell proliferation detection kit was used to stain the four samples mentioned above. Cells were seeded in culture dishes and cultured until adherent. The culture medium was then replaced with 50 μM EdU. After incubation for 2 hours, the medium was discarded and the cells were washed with phosphate-buffered saline (PBS) to remove free EdU. EdU replaces thymidine and is incorporated into newly synthesized DNA during DNA replication. Cells containing EdU are considered proliferating cells. The cells were fixed and incubated with 4% paraformaldehyde for 20 minutes at room temperature. The supernatant was discarded and the cells were washed three times with PBS. The cells were then permeabilized by adding 0.1% PBS-Tween solution and incubated at room temperature for 15 minutes. The prepared Apollo staining solution was then added and incubated on a shaker at room temperature in the dark for 30 minutes. The supernatant was discarded and the cells were washed with PBS. After resuspending, the cells were stained with Hoechst 33258 working solution and incubated at room temperature in the dark for 10 minutes. The cells were then washed to remove free dye. Hoechst dye emits blue fluorescence by binding to the minor groove of the DNA double helix. Apollo staining solution binds to the acetylene group of EdU to form a stable red fluorescent compound. Under a fluorescence microscope, all cell nuclei emit blue fluorescence, while the nuclei of proliferating cells containing EdU emit red fluorescence. Four groups of samples were used for clonal proliferation experiments. IEC-6 cells were seeded at a density of 5,000 cells / well in six-well plates and cultured until adherent. Drug treatment was performed using the same method as the above experiment, and then cultured in a 37°C incubator for 7 days to allow cell colonies to form. After washing with PBS, 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 30 minutes. After washing again, 1 mL of crystal violet stain was added to each well for 20 minutes. Samples were labeled as the control group, capsaicin group, GUA group, and capsaicin + GUA group according to the different drug treatments. The cells' clonogenic ability was observed under a microscope. Please refer to Figure 3 Quantitative analysis based on the fluorescence distribution diagram confirmed that GUA helps to improve the proliferation ability of cells. The results of the clone formation experiment proved that GUA also helps to improve the clone formation ability of cells.

[0046] Cell samples from each group were labeled with a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe. DCFH-DA itself is non-fluorescent and can freely penetrate the cell membrane. Once inside the cell, it is hydrolyzed by esterases into DCFH, which cannot penetrate the cell membrane. DCFH is then oxidized by ROS to the green fluorescent substance DCF. The fluorescence intensity is measured to indicate the level of ROS. Western blotting was used to detect the expression of cell proliferation proteins PCNA and MCM2 in the cell samples from each group. Figure 4, it can be seen that GUA helps to reduce ROS levels and promotes the expression of MCM2 and PCNA. ROS, as a "redox messenger", is involved in cell proliferation, apoptosis, gene expression regulation and stress response. The reduction in ROS levels indicates that cell damage has been alleviated. PCNA is a key auxiliary factor in the DNA replication process. It is directly involved in the elongation of DNA polymerase and is specifically highly expressed in the S phase (DNA synthesis phase) of the cell cycle. The increase in PCNA expression level reflects that the current proliferation state of the cell is relatively active. MCM2 is responsible for the unwinding of the double helix at the beginning of DNA replication. It is expressed and assembled to the replication origin from the late G1 phase to the S phase of the cell cycle. The expression of MCM2 and the formation of the complex are the "permission" signs for cells to enter the S phase. The increase in MCM2 expression reflects that the potential for cell proliferation has increased.

[0047] Combined with the above experimental results, it can be seen that guanosine reduces capsaicin-induced cell death at the cellular level by promoting cell proliferation, and plays a role in the biological process of cell mitosis.

[0048] GES-1 cells, derived from fetal gastric epithelial cells, were cultured with GUA at final concentrations of 0μM, 10μM, 20μM, 30μM, 40μM, and 50μM, yielding multiple cell samples. After irradiation with 12Gy of X-rays for 3 days, the cells were stained with AVP1 and the apoptosis level of GES-1 cells was determined by flow cytometry. Figure 5 The scatter plots of cell survival status of each group were drawn, and the fluorescence area parameter of PE channel (PE-A) reflected the membrane eversion marked by Annexin V, that is, the total number of early apoptotic cells and necrotic cells, and the fluorescence area parameter of FITC channel (FITC-A) reflected the membrane permeability marked by PI, that is, the number of late apoptotic cells or necrotic cells. The apoptosis and necrosis of each group of samples were quantitatively analyzed. It can be seen from the figure that the apoptosis rate of cells without GUA was (35.71±0.507)%, and the apoptosis rate of cells with 10μM GUA was (24.73±1.3 The apoptosis rate of cells was (23)%, (24.65±0.327)% when 20μM GUA was added, (22.94±1.070)% when 30μM GUA was added, (20.48±0.963)% when 40μM GUA was added, and (25.69±0.308)% when 50μM GUA was added. Compared with the group without GUA, the apoptosis rate of cells in the group with GUA was significantly decreased, P<0.0001, indicating that guanosine can effectively reduce the apoptosis behavior of GES-1 cells caused by radiation damage.

[0049] Multiple groups of GES-1 cell samples were irradiated with 12Gy of X-rays for 3 hours, then stained with DCFH-DA. The mean fluorescence intensity of each group of cells was measured by flow cytometry to characterize the ROS level of the samples. Figure 6 The average fluorescence intensity of the cells was (5136±39.63) when no GUA was added, (4363±18.48) when 10μM GUA was added, (3543±161.0) when 20μM GUA was added, (2477±40.25) when 30μM GUA was added, (2497±60.48) when 40μM GUA was added, and (2432±31.34) when 50μM GUA was added. ). Compared with the group without GUA, the ROS level in the group with 10 μM GUA was significantly decreased (P < 0.0001). The ROS level in the group with 20 μM GUA was significantly decreased compared with the 10 μM GUA group, and the ROS level in the group with 30 μM GUA was significantly decreased compared with the 20 μM GUA group. However, there was no significant difference in ROS levels between the groups with 30 μM GUA, 40 μM GUA, and 50 μM GUA (P > 0.05). This indicates that within the 0 μM to 30 μM concentration range, as the GUA concentration increases, the radiation-induced ROS level in GES-1 cells gradually decreases. Within the 30 μM to 50 μM concentration range, GUA concentration has no significant effect on the radiation-induced ROS level in GES-1 cells.

[0050] GES-1 cells were cultured with GUA at final concentrations of 0 μM and 30 μM, and then irradiated with 0 Gy, 6 Gy, and 12 Gy of X-rays. Protein samples were extracted 4 and 24 hours after irradiation and Western Blot analysis was performed. The protein expression levels of γ-H2AX and Bcl2 in each sample were detected. Figure 7After GUA treatment, γ-H2AX protein levels did not significantly differ in the 0 Gy irradiation group. However, γ-H2AX protein levels decreased in the 6 Gy and 12 Gy irradiation groups after GUA treatment. There was no significant difference in γ-H2AX between the 6 Gy GUA-treated group and the 0 Gy GUA-treated group. However, Bcl2 protein levels increased in the GUA-treated group compared with the non-GUA group under all 0 Gy, 6 Gy, and 12 Gy irradiation conditions. γ-H2AX protein is rapidly formed at DNA double-strand breaks. As a marker of DNA double-strand breaks, decreased γ-H2AX protein levels indicate a decrease in the DNA damage burden of the cell population. Bcl2 protein inhibits mitochondrial membrane permeability, preventing the release of cytochrome c and blocking the intrinsic apoptotic pathway. Increased Bcl2 protein levels indicate inhibition of apoptosis. These results indicate that GUA can reduce γ-H2AX protein levels and increase Bcl2 protein levels in radiation-damaged GES-1 cells. In other words, GUA treatment reduces radiation-induced DNA damage and inhibits apoptosis in GES-1 cells.

[0051] Combined with the above experiments, it can be seen that GUA treatment effectively inhibited the death of GES-1 cells induced by radiation.

[0052] Seven-week-old male C57 mice were taken and randomly divided into four groups, named control group, enteritis group, guanosine treatment group and enteritis + guanosine treatment group. The mice in the enteritis group and enteritis + guanosine treatment group were gavaged with capsaicin for 5 consecutive days. The gavage liquid was CAP solution in normal saline, and the gavage dose was 100 mg / kg each time. The mice in the control group and guanosine group were gavaged with the same volume of normal saline. After gavage, the treatment was carried out by intraperitoneal injection of the treatment solution for 5 consecutive days. The treatment solution used in each group was the same volume but different components. The treatment solution for the control group and enteritis group was normal saline, and the treatment solution for the guanosine treatment group and enteritis + guanosine treatment group was normal saline solution of GUA, and the dosage of GUA was 8 mg / kg each time. The mice were killed 24 hours after the end of administration, and the colon tissues were collected and stained with hematoxylin-eosin (H&E). For the staining results, please see Figure 8 The colon mucosa of the control mice was intact, while the enteritis group showed significant mucosal edema and destruction, with fewer glands and crypt atrophy and disappearance. However, the mucosal destruction and crypt atrophy and disappearance were alleviated in the enteritis + guanosine treatment group. This suggests that GUA has a significant inhibitory effect on capsaicin-induced enteritis.

[0053] The present invention provides the beneficial effect of using guanosine in a drug for treating inflammatory damage to the digestive system. Guanosine inhibits the decrease in the proliferation of digestive system cells induced by capsaicin or radiation, reduces cellular DNA damage, and inhibits cell apoptosis, thereby improving edema and damage to the mucosal layer of the tissue, as well as crypt atrophy and disappearance, thereby achieving the therapeutic effect of inflammatory damage.

[0054] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0055] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Application of guanosine in medicine for treating inflammatory damage of the digestive system.

2. The use according to claim 1, characterized in that The digestive system inflammatory injury includes one or more of esophagitis, gastritis and enteritis.

3. The use according to claim 1, characterized in that The inflammatory damage to the digestive system is caused by capsaicin or radiation.

4. The use according to any one of claims 1 to 3, characterized in that The guanosine is administered by intraperitoneal injection.

5. The use according to claim 4, characterized in that The dosage of guanosine is any value between 5 mg / kg and 10 mg / kg.

6. The use according to claim 4, characterized in that When the digestive system inflammatory injury is enteritis, the administration concentration of guanosine is any value between 0.01 μM and 100 μM.

7. The use according to claim 4, characterized in that When the digestive system inflammatory injury is gastritis, the administration concentration of guanosine is any value between 10 μM and 50 μM.

8. The use according to claim 1, wherein The guanosine is used to restore the proliferation ability of cells.

9. The use according to claim 1, wherein The guanosine is used to inhibit cell apoptosis.

10. The use according to claim 1, wherein The guanosine acts to repair DNA damage in cells.

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